Category Archives: Cell Medicine

SAN DIEGO, April 13, 2022 (GLOBE NEWSWIRE) -- Oncternal TherapeuticsInc. (Nasdaq: ONCT), a clinical-stage biopharmaceutical company focused on the development of novel oncology therapies, today announced that it has deprioritized further development of ONCT-216 to reallocate resources to zilovertamab, the Companys investigational anti-ROR1 monoclonal antibody, and its Phase 3 registrational trial that the Company expects to initiate in Q3 2022. As such, the Company has discontinued enrollment in the Phase 1/2 study evaluating ONCT-216 in patients with relapsed or refractory Ewing sarcoma.

This asset prioritization allows us to further sharpen our focus on hematological malignancies and prostate cancer, while deploying our capital towards meaningful catalysts as we navigate this historically challenging pandemic, geopolitical and capital markets macroenvironment, said James Breitmeyer, MD, PhD, Oncternals President and CEO. We believe this focused approach, along with prudent cash management, will enable us to fund our operations well into the third quarter of 2023, as we continue to explore all potential sources of capital to enable us to reach our milestones.

The Company expects to initiate its global registrational Phase 3 Study ZILO-301 in the third quarter of 2022, taking into account the impact of geopolitical factors and COVID-19 related supply chain issues. The study will randomize patients with relapsed or refractory MCL who have experienced stable disease or a partial response after receiving four months of oral ibrutinib therapy to receive either blinded zilovertamab or placebo, and all patients will continue receiving oral ibrutinib. The novel ZILO-301 design is supported by encouraging data from the Companys ongoing Phase 1/2 clinical trial of zilovertamab plus ibrutinib for patients with MCL or CLL, as well as by a successful End-of-Phase-2 meeting with the U.S. Food and Drug Administration (FDA).

The Companys lead autologous ROR1-targeted CAR-T cell therapy program candidate, ONCT-808, is advancing according to plan towards an Investigational New Drug (IND) application submission expected in mid-2022, based on supportive manufacturing and preclinical data as well as a productive pre-IND meeting with the FDA earlier this year.

Finally, the Company continues to advance ONCT-534, its lead candidate in its DAARI program, and expects to initiate IND-enabling GLP toxicology studies and GMP manufacturing later this quarter. ONCT-534 has shown anti-tumor activity in preclinical studies relevant to multiple clinically important forms of resistance for patients with prostate cancer, including those involving overexpression of the androgen receptor, or expression of mutants of the androgen receptor, or splice variants such as AR-V7.

AboutZilovertamab (formerly Cirmtuzumab)Zilovertamab is an investigational, humanized, potentially first-in-class monoclonal antibody targeting Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1). Zilovertamab is currently being evaluated in a Phase 1/2 clinical trial in combination with ibrutinib for the treatment of patients with MCL or chronic lymphocytic leukemia (CLL), in a collaboration with theUniversity of California San Diego(UC San Diego) School of Medicine and theCalifornia Institute for Regenerative Medicine(CIRM). In addition, Oncternal is supporting two investigator-sponsored studies being conducted at the UC San Diego School of Medicine, a Phase 1b clinical trial for patients with metastatic castration-resistant prostate cancer (mCRPC), and a Phase 2 clinical trial of zilovertamab in combination with venetoclax, a Bcl-2 inhibitor, for patients with relapsed/refractory CLL. Both are open for enrollment.

ROR1 is a potentially attractive target for cancer therapy because it is an onco-embryonic antigen, not usually expressed on adult cells, but its expression confers a survival and fitness advantage when reactivated and expressed by tumor cells. Researchers at theUC San Diego School of Medicinediscovered that targeting a critical epitope on ROR1 was key to specifically inhibiting ROR1 expressing tumors. This led to the development of zilovertamab which binds this critical epitope of ROR1, highly expressed on many different cancers but not on normal tissues. Preclinical data showed that when zilovertamab bound to ROR1, it blocked Wnt5a signaling, inhibited tumor cell proliferation, migration and survival, and induced differentiation of the tumor cells. The FDA has granted Orphan Drug Designations to zilovertamab for the treatment of patients with MCL and CLL/small lymphocytic lymphoma. Zilovertamab is in clinical development and has not been approved by the FDA for any indication.

About Oncternal TherapeuticsOncternal Therapeuticsis a clinical-stage biopharmaceutical company focused on the development of novel oncology therapies for the treatment of patients with cancers that have critical unmet medical need. Oncternal pursues drug development targeting promising, yet untapped biological pathways implicated in cancer generation or progression, focusing on hematological malignancies and prostate cancer. The clinical pipeline includeszilovertamab, an investigational monoclonal antibody designed to inhibit ROR1, a type I tyrosine kinase-like orphan receptor. Zilovertamab is being evaluated in a Phase 1/2 clinical trial in combination with ibrutinib for the treatment of patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), in investigator-initiated studies, including a Phase 1b clinical trial in combination with paclitaxel for the treatment of women with HER2-negative metastatic or locally advanced, unresectable breast cancer, in a Phase 2 clinical trial of zilovertamab in combination with venetoclax, a Bcl-2 inhibitor, in patients with relapsed/refractory CLL, and in a Phase 1b study of zilovertamab in combination with docetaxel in patients with metastatic castration-resistant prostate cancer (mCRPC). Oncternal is also developingONCT-808, a chimeric antigen receptor T cell (CAR-T) therapy that targets ROR1, which is currently in preclinical development as a potential treatment for hematologic cancers and solid tumors. The early-stage pipeline also includesONCT-534, a dual-action androgen receptor inhibitor (DAARI), that is in preclinical development as a potential treatment for castration resistant prostate cancer and other androgen-receptor dependent diseases. More information is available athttps://oncternal.com/.

Forward-Looking Information

Oncternal cautions you that statements included in this press release that are not a description of historical facts are forward-looking statements. In some cases, you can identify forward-looking statements by terms such as may, will, should, expect, plan, anticipate, could, intend, target, project, contemplates, believes, estimates, predicts, potential or continue or the negatives of these terms or other similar expressions. These statements are based on Oncternals current beliefs and expectations. Forward-looking statements include statements regarding Oncternals development programs, including Oncternals estimated cash and cash equivalents as of March 31, 2022, the anticipated timing for announcing additional preclinical and clinical data; timing of reaching any milestones, including IND submissions; timing for regulatory communications; Oncternals expected cash runway; and the potential that Study ZILO-301 can serve as a registrational clinical trial; and the expected initiation of clinical trials, including Study ZILO-301. Forward-looking statements are subject to risks and uncertainties inherent in Oncternals business, including risks associated with the clinical development and process for obtaining regulatory approval of Oncternals product candidates, such as potential delays in the commencement, enrollment and completion of clinical trials; we have not conducted head-to-head studies of zilovertamab in combination with ibrutinib compared to ibrutinib monotherapy and data from separate studies may not be directly comparable due to the differences in study protocols, conditions and patient populations; the risk that interim results of a clinical trial do not predict final results and that one or more of the clinical outcomes may materially change as patient enrollment continues, following more comprehensive reviews of the data, as follow-up on the outcome of any particular patient continues, and as more patient data become available; later developments with the FDA may be inconsistent with the minutes from the completed end of Phase 2 meeting, including that the proposed Study ZILO-301 that may not support registration of zilovertamab in combination with ibrutinib which is a review issue with the FDA upon submission of a BLA; and other risks described in Oncternals filings with theU.S. Securities and Exchange Commission. All forward-looking statements in this press release are current only as of the date hereof and, except as required by applicable law, Oncternal undertakes no obligation to revise or update any forward-looking statement, or to make any other forward-looking statements, whether as a result of new information, future events or otherwise. All forward-looking statements are qualified in their entirety by this cautionary statement. This caution is made under the safe harbor provisions of the Private Securities Litigation Reform Act of 1995.

Contact Information:

InvestorsRichard Vincent858-434-1113rvincent@oncternal.com

MediaCorey Davis, Ph.D. LifeSci Advisors 212-915-2577 cdavis@lifesciadvisors.com

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Oncternal Therapeutics Deprioritizes Development of ONCT-216 to Focus Resources on Phase 3 Trial for Zilovertamab in the Treatment of Mantle Cell...

Autoimmune hepatitis (AIH) is a chronic progressive inflammatory disorder commonly identified in females with a tendency to occur during the teen years and the fourth to sixth decades of life. Its distinguishing features include the presence of serum-specific autoantibodies and histopathology features of interface hepatitis [1-3]. Etiopathogenesis involves a multitude of independent factors such as human leukocyte antigen (HLA) allelic variants, infections, drugs, and the prevalence of autoimmune tautology, which cause an immunoregulatory imbalance, leading to a resultant T-cell mediated inflammation [1]. Steroids and immunosuppressants form the primary pillars of treatment, along with close monitoring of liver function tests to assess therapeutic response. The recommended duration of treatment is approximately two years, after which withdrawal can be considered. However, given the high risk of relapse, lifelong immunosuppressive therapy may be essential in most cases [3].

Natural supplements have been in use for decades in conjunction with modern medicine by mankind to treat a variety of common ailments. The rationale behind the use of such supplements is their presumed regulatory effect on the immune system, although studies conducted over the years have had mixed results [4-6]. One of the commonly used botanical supplements of interest with immunomodulatory effects is Sambucus nigra, also commonly known as elderberry, frequently used for the treatment of the common cold. The effects of elderberry on the production of pro-inflammatory cytokines such as IL-6,IL-8, and TNF have been controversial, with studies indicating a stimulatory versus inhibitory effect, leading to inconsistent interpretation [4]. We present a case of autoimmune hepatitis in a middle-aged female who was on long-term elderberry-containing supplements and developed autoimmune hepatitis. This case report aims to identify the risk factors associated with autoimmune hepatitis, debate the effects of elderberry on the immune system, and consider a causal association between the two entities.

A 60-year-old Caucasian female with a medical history of Hashimoto thyroiditis and Medullary sponge kidneys presented to the hospital with nausea, decreased appetite, abdominal pain, and bloating for two days. She was on levothyroxine, indapamide, tamsulosin, potassium citrate supplements, and an over-the-counter elderberry supplement for several years. Her vital signs were stable. Physical examination findings were remarkable for scleral icterus, generalized jaundice, and abdominal distention without tenderness.

Initial laboratory studies revealed transaminitis with elevation of AST to 1821 IU/L (13-39 IU/L), ALT >2500 IU/L (7-52 IU/L), ALP of 232 IU/L (35-104 IU/L), total bilirubin of 10.5 mg/dL (0.0-1.0 mg/dL), and direct bilirubin of 5.66 mg/dL (0.00-0.20 mg/dL) (Table 1). Abdominal imaging studies, including ultrasound and CT scans, showed mild peri hepatic inflammation and a contracted gallbladder with no obvious stones or intrahepatic dilation. The acute hepatitis panel was negative;however, she was found to have a positive antinuclear antibody (ANA) screen and SSA (RO) Ab 1.4 (<1.0) (Table 1), which raised a concern for autoimmune hepatitis.

Subsequent evaluation with a liver biopsy revealed extensive interface hepatitis, lymphohistiocytic infiltrate, eosinophils and neutrophils in the portal areas, periportal and lobular liver cell damage with piecemeal necrosis (Figure 1, 2).

Based on histological findings of the portal tract, the differential diagnoses included autoimmune hepatitis versus drug-induced hepatitis. She was instructed to stop taking natural supplements, and immunosuppressive therapy with daily Prednisone 40 mg was started for four weeks.

At a follow-up visit four weeks later, her repeat liver function tests improved to baseline. As elderberry supplements were presumed causes of hepatitis, they were not rechallenged. Prednisone therapy was tapered over two weeks, after which she was started on long-term maintenance therapy with azathioprine.

Autoimmune hepatitis is a chronic inflammatory liver disease depicted by a combination of elevated liver enzymes, the presence of specific serum autoantibodies and histological features of interface hepatitis, infiltration of lobules with lymphocytes, and plasma cells [3]. AIH is further classified into two types depending on the presence of autoantibodies: type 1 is positive for ANAand/or anti-smooth muscle antibodies (SMA), while type 2 is positive for liver-kidney-microsomal-type-1 (LKM-1) antibodies [2,3].While studies have shown that it affects all ages, ethnicities, and genders, it has a bimodal distribution with peaks in the teens and the fourth to the sixth decadeof life, with a female preponderance [1-3]. Literature suggests that the presence of one autoimmune disorder can increase the risk of an individual developing other autoimmune disorders [7]. One such commonly studied association with AIH is the presence of autoimmune thyroiditis [8,9]. In a case-control study, it was found that 30% of patients with AIH have concurrent extrahepatic autoimmune manifestations, out of which 51.4% were found to be diagnosed with autoimmune thyroid disease [9]. Another similar study revealed that autoimmune thyroid disease was the most common concurrent extrahepatic autoimmune disease with an 18% prevalence in those with autoimmune hepatitis [8]. On the other hand, in another cohort study, autoimmune hepatitis was found in 8.69% of patients with Hashimoto thyroiditis [10]. Uncertainty lies within the issue of whether autoimmune thyroid disorder is a risk factor or a cause of autoimmune liver disease, or vice versa, warranting further exploration.

The mechanism behind the development of autoimmune hepatitis is still not completely clear. A complex interplay between genetic predisposition, molecular mimicry, and effector-regulatory immunity is thought to be involved in the pathogenesis of autoimmune hepatitis. The presentation of an autoantigenic peptide to nave CD4 T-helper cells (Th0) causes the secretion of proinflammatory cytokines like IL-12, IL-6, and TGF-B which leads to the development of Th1, Th2, and Th17 cells [11]. Th1 cells produce IL-2 and IFN-y, which activate cytotoxic T lymphocytes (CD8), thereby inducing the expression of HLA class I and II molecules on hepatocytes with further stimulation of macrophages. Th2 cells secrete IL-4, IL-10, and IL-14, leading to the maturation of B cells and plasma cells that produce autoantibodies [11]. Regulatory T cells (Treg) derived from Th0 cells are responsible for restricting hepatocellular injury by Th1, Th17, macrophages, complement, and natural killer cells [8,12]. Th17 cells produce cytokines that suppress Treg cells, thus amplifying liver damage along with the cascading effects of Th1 and Th2 cells, leading to the formation of characteristic lymphocytic and plasma cell infiltrates termed interface hepatitis [8].

As per another study, SPW 2, a neutral polysaccharide derived from Sambucus leaves, induced the secretion of IL-beta, IL-6, and TNF-alpha and increased the mRNA expression level of IL-6 and TNF-alpha in macrophages in vitro [6].

Sambucus species, commonly known as elderberry, is a traditional dietary supplement used to treat minor health conditions, particularly influenza [5]. While there are insufficient data to substantiate the effectiveness of elderberry, some studies have displayed its immunomodulatory effects. In a study done by Barak et al., Sambucol (elderberry) extract led to the production of inflammatory cytokines including IL-1 beta, TNF-alpha, IL-6, and IL-8 (2-45 fold) as compared to lipopolysaccharide, a known monocyte activator (3.6-10.7 fold) in vitro, thereby concluding that Sambucol extract activates the immune system by increasing inflammatory cytokine production [5]. Another comparable study revealed similar in vitro effects of Sambucus as well as its ability to stimulate the mRNA expression of inducible Nitric oxide synthase in macrophages, supporting the notion of elderberrys potential regulatory effects on the immune system [6]. Considering the aforementioned discussion, it is worth debating whether elderberry may be contributing to the amplification of the cytokine production in vivo, inciting autoimmunity in genetically predisposed individuals - parallel to the development of autoimmune hepatitis in our patient with a history of a known autoimmune condition (Hashimoto's thyroiditis) on long-term elderberry supplements.

In addition to playing a vital role in the diagnosis, the purpose of a liver biopsy in AIH is to determine the prognosis, severity of the disease, and therapeutic response. The characteristic histological features of AIH are depicted by the presence of interface hepatitis or piecemeal necrosis, which is the inflammation of hepatocytes at the junction of the portal tract and hepatic parenchyma consisting of lymphocytes and plasma cells [13]. However, there exists approximately 25% overlap between AIH and acute viral hepatitis, with the degree of plasmacytosis being the discriminating factor, although 33% of patients with AIH do not exhibit plasma cells in the portal tract, and thus diagnosis should be made in the context of a clinical and serological background [13]. The presence of eosinophils seen in Figure 1can correspond to a differential diagnosis of drug-induced hepatitis, although these findings can also be seen with AIH, making it difficult to differentiate between the two and establish a clear diagnosis. At the same time, our patient had a negative hepatitis serology, which helped in making the distinction.Another entity that is relevant to our case is a drug-induced liver injury with autoimmune features, which is a syndrome where one develops biochemical and histological features of autoimmune hepatitis after the ingestion of a drug or a herbal product [14,15]. It has a shorter incubation period with a gradual improvement observed after the inciting drug is stopped and can recur if it is re-introduced, whereas idiopathic AIH requires long-term immunosuppression [14,15]. In addition to the presence of a pre-existing autoimmune condition (Hashimotos thyroiditis) in our patient, there is a possibility that elderberry supplements could have initiated a self-propagating autoimmune process that could have caused the liver injury.

The cornerstone of treatment in AIH lies in limiting inflammation and preventing the progression to end-stage liver disease [11]. Debilitating symptoms like arthralgia and fatigue accompanied by hepatic inflammation, serum AST elevation 10-fold upper normal limit or AST elevation 5-fold upper normal limit concurrent with -globulin level 2-fold upper normal limit, and histological findings of bridging necrosis are absolute indications for treatment [16]. Treatment regimens include prednisone alone or in conjunction with azathioprine, which is continued until normalization of transaminases and IgG is achieved and is further maintained depending on the individuals response and tolerance to therapy [2,11,16]. Considering the possibility of drug-induced hepatitis, our patient was instructed to stop the elderberry supplement and not to reuse it again. In addition, she was given immunosuppressive therapy to treat the significant hepatic inflammation as evidenced by severely elevated transaminases and liver biopsy. A four-week course of prednisone resulted in the resolution of symptoms and normalization of liver function tests. She was started on azathioprine maintenance therapy with a plan to potentially discontinue it after sustained remission for 18 months. Follow-up over time regarding the development of flare-ups, should they arise, will shed light on the subject of autoimmune hepatitis triggered by elderberry, as opposed to elderberry-induced hepatic injury. This could aid in recognizing the significance of using elderberry judiciously in patients with a history or predisposition to autoimmune disorders.

Exposure to elderberry could be responsible for either the initiation or progression of autoimmune liver disease in the setting of genetic predisposition and molecular mimicry. Therefore, obtaining a meticulous history pertaining to medications is warranted, with an emphasis on over-the-counter supplements. A combination of serum-specific antibodies and distinct histological features in the liver biopsy can aid in formulating a diagnosis, while an initial course of steroids along with indefinite immunosuppressive therapy is essential to control inflammation as well as avert relapses.

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A Plausible Association Between the Use of Elderberry and Autoimmune Hepatitis - Cureus

Introduction

Staphylococcus aureus (S. aureus), a major pathogen of healthcare-associated infections, causes many infectious diseases in hospital and community (eg, pneumonia, endocarditis, and bacteremia).1 Due to its well-known exotoxins and ability to form biofilms,2 the S. aureus infections are particularly difficult to treat. Furthermore, S. aureus rapidly develops resistance to the -lactams antibiotics via the acquisition of the staphylococcal chromosome cassette carrying mecA gene.35 The Methicillin-resistance S. aureus (MRSA) has become one of the most lethal pathogen in bacteremia. The case fatality rate of MRSA bacteremia has been reported to be up to 27.3%, with a mortality rate approximately 23 times higher than those infected with methicillin-susceptible S. aureus.6 Although vancomycin remains the most effective antibiotic currently, the treatment of MRSA infections is still challenging due to the dramatic increase in MRSA isolation and the limited clinical treatment options.7,8 Thus, there is a strong need to develop novel antibiotics for the treatment of MRSA.

Berberine (BER) is an isoquinoline quaternary alkaloid (5,6-dihydrodibenzo quinolizinium derivative) isolated from many medicinal plants, such as Hydrastis canadensis, Berberis aristata, Coptis chinensis, Coptis rhizome, Coptis japonica, Phellodendron amurense, and Phellodendron chinense schneid.9 BER is used to treat diarrhea and bowel disorders in China, with the potential to be used for the treatment of many diseases.10 Furthermore, BER has received extensive attention for its role as an antimicrobial,1113 antifungal,14 anti-tumor,15,16 and anti-diabetes drug.17 However, the anti-MRSA mechanism of BER is still unclear.

Therefore, the current study evaluated the effects of BER on MRSA using the minimum inhibitory concentration (MIC) and time-killing test. Moreover, the potential mechanism of BERs activities on MRSA, including biofilm formation, cell membrane integrity, and cell morphology variation were investigated. We also explored the synergistic effect of BER combined with rifampicin and clindamycin against MRSA in vitro.

Fifteen MRSA isolates were collected from blood cultures between March and September 2021 at Beijing Friendship Hospital, affiliated with the Capital Medical University of China. After collection, the bacteria were labeled AO and stored in skim milk at 80C for further use. Subsequently, American Type Culture Collection (ATCC) 43300 was selected as the control strain. Bacteria were routinely grown in a Columbia blood plate (Thermo Fisher Scientific, Waltham, MA, USA) at 37C for 18h unless otherwise specified.

The minimum inhibitory concentration (MIC) of each antibacterial agents is defined as the lowest concentration of the drug that resulted in no visible growth.18 Berberine (BER), clindamycin (CLI), and rifampicin (RIF) were acquired from the National Institutes for Food and Drug Control (Beijing, China). The MICs of BER were determined based on the agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI, PA, USA). Mueller-Hinton agar (MHA, OXOID) was mixed with BER in dishes, where the final concentration of BER was 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048 mg*L.1 The MICs of antibiotics, including RIF and CLI, against MRSA strains were determined using a standard broth microdilution method recommended by the CLSI.19 Cation adjusted Mueller-Hinton broth (CAMHB, OXOID) was mixed with RIF and CLI separately, where the final concentration of RIF and CLI was twofold series dilution concentration from 0.015625 to 2048 mg*L1 in triplicate. Then, the tested bacterial were spotted on the medium and incubated at 37C for 18h. All experiments were conducted at least three times on different days, and the most consistent results were presented.

MLST was determined by the PCR amplification of seven housekeeper genes (aro, arc, glp, gmk, pta, tpi, and yqi), according to the conditions published in https://pubmlst.org/. The PCR products were bidirectionally sequenced, and all sequences in the online database were analyzed to determine the allele number and respective ST types.

Checkerboard assays were used to determine the interactions between BER and antibiotics on the tested MRSA strains, as recommended by the CLSI guidelines. The concentration gradients of BER and antibiotics were selected based on the MICs. After MRSA cells were cultured in CAMHB medium with continuous shaking at 37C, the cells were grown to 0.5McF (1.5108 colony forming unit*mL1, CFU*mL1); then, the cultures were diluted 32-fold with sterile broth (approximately 5106CFU*mL1). Delivering 10L aliquots of cultures to 96-well plates containing 100L of two-fold diluted BER and antibiotics in cation-adjusted Mueller Hinton broth, where the final concentration of bacterial culture was approximately 5105 CFU*mL1.20 The plates were incubated for 18 h at 37C. Finally, each well was examined for signs of MRSA growth. For each interaction, the fractional inhibitory concentration (FIC) was calculated for each agent: synergism, antagonism, and indifference correlated with FIC 0.5, FIC > 4, and 0.5 < FIC 4, respectively.21

Growth kinetics were used to assess the antimicrobial effect of BER against MRSA and sensitization to other antibiotics.22 The tested MRSA strain was selected based on MLST types. The experiments were performed using CAMHB in 10mL with an initial inoculum of 5105 CFU/mL. The cultures were transferred to tubes with individual compounds and in combination (2 MIC, 1 MIC, 1/2 MIC, 1/4 MIC of BER, 1/8 MIC, 1/32 MIC of RIF, or 1/4 MIC of CLI) treated and incubated. The concentration of BER and antibiotics were chosen based on the MIC assay and synergistic effects assay. A kinetic growth or inactivation curve was constructed for each treatment using the viable cell counts performed at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24h of incubation at 37C. The cultures containing 1% DMSO and bacterial inoculum served as the bacterial DMSO control (DC) and growth control (GC) separately.

The effect of BER on MRSA biofilm formation was tested. Static biofilm formation assay was performed in 96-well microtiter plates, as previously reported.23 Delivering approximately 5105 CFU*mL1 of MRSA cells to 96-well plates containing diluted BER and antibiotics in brain heart infusion broth (BHI, OXIOD) supplemented with 0.5% glucose.21 Wells containing only the BHI were used as the negative control, whereas wells containing bacteria grown in BHI were used as the positive control. The suspension was transferred to 96-well plates with individual compounds and combinations (4 MIC, 2 MIC, 1 MIC, 1/2 MIC, 1/4 MIC of BER, 1/16 MIC, 1/32 MIC of RIF, or 1/4 MIC of CLI) treated and incubated for 18 h at 37C. The concentrations of BER, RIF, and CLI were selected based on the previous MIC assays and synergistic effects assays. The tested MRSA strain was selected based on the MLST types, consistent with kinetics assay. After incubation, each well was washed three times with PBS to remove the planktonic cells, fixed for 15 min with methanol to stabilize biofilm, stained for 5 min with crystal violet (0.5% in methanol), and washed two times again with deionized water. The adhering dye was dissolved in 200L of 33% acetic acid and the absorbance was measured at OD600 to quantify biofilm formation.

Cell membrane permeability of MRSA cells was assessed by confocal laser scanning microscopy (CLSM) (TCS-SP5, Leica, GER), similar to the previous description.24 To test membrane integrity, fluorescence images were acquired using fluorochrome, which were carboxyfluorescein diacetate (CFDA SE; Sigma-Aldrich, St. Louis, MO, USA) and propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) at final concentrations of 10 mmol*L1 and 5 mg*L1, separately. CFDA SE is a nonfluorescent precursor that only passing through intact cell membranes, producing a fluorescent compound. When the cell membrane is damaged, PI can enter the cells and bind to the DNA to form a red fluorescent DNA-complex.25 S. aureus are gram-positive cocci with a cytoderm structure. However, cytomembrane was essential to maintain cell function and prevent cytoplasmic leakage. Fluorescent staining was used to examine cell membrane integrity as previously reported.24 After MRSA cells were grown to an approximately 1.5108CFU*mL1 in CAMHB at 37C, delivering 10 mL of volume cultures to tubes. Then, the cells were treated with BER at 0 MIC, 1 MIC, or 2 MIC for 4 h, centrifuged at 5000g for 5 min, and resuspended with equivalent volumes of 0.9% NaCl. After incubation with cFDA SE for 15 min and PI for 1 min at room temperature away from light, the suspension was fixed on a glass slide using agar. Finally, the bacteria were examined by confocal laser scanning microscopy (CLSM, OLYMPUS, FV1000, Japan) at the excitation-emission wavelengths of 530nm for PI and 488nm for CFDA SE.

Field emission scanning electron microscopy (FESEM, Joel JSM-7900F, Japan) was used to detect morphological changes after BER treatment, according to the published protocols.26 Subsequently, approximately 1.5108CFU*mL1 of MRSA cells were incubated in CAMHB of 10mL volume supplemented with BER at 0 MIC, 1 MIC, 2 MIC, and 4 MIC for 4 h at 37C. The treated cells were washed with PBS three times and fixed with 2.5% glutaraldehyde for 4 h and 1% osmic acid for 2 h. Then, the cells were washed with PBS again. After dehydrating with different concentrations of ethanol, the cells were fixed on silicon slices, sputter-coated with gold under vacuum, and observed using FESEM.

Assays were conducted with at least two biological replications. The KruskalWallis analysis was used to determine the differences between the multiple groups. Data are expressed as mean SD, and p-values <0.05 are considered statistically significant. No adjustments were made for the p-values reported in this study.

The MLST determination showed 15 MRSA clinical isolates and distinguished them into five subtypes. The MIC values of BER varying from 256 to 64 mg*L1 (Table 1); however, among ST5 subtypes, only one strain had a MIC significantly different from the others (ie, >2048 mg*L1). Excluding this strain, the highest MIC value was 256 mg*L1 for ATCC 43300, and the lowest MIC value was 64 mg*L1 for ST239 and ST5. Six clinical isolates were resistant to CLI, and eight strains were susceptible, whereas 11 isolates were resistant to RIF, and three isolates were susceptible. The MRSA control strain was resistant to CLI at MIC of 1024 mg*L1 and susceptible to RIF at MIC of 0.06 mg*L1 (Table 2). Using the checkerboard assay, BER in combination with RIF and CLI showed a significant effect against the MRSA strain, leading to a reduction in MIC values. Synergistic activity was observed for the combination of BER with CLI or RIF with FIC index values of 0.190.5 (Table 2). However, two isolates showed indifferent effects compared to others when BER was combined with CLI, and one isolate showed indifferent effects when BER was combined with RIF (Table 3).

Table 1 The MIC Value of Berberine Among Different MLST Types of MRSA

Table 2 MIC and FIC Index of CLI and RIF in the Presence of BER Against MRSA Strains

Table 3 The Antibacterial Activity of BER in Combination with CLI and RIF Against MRSA Strains (%, n/N)

The time-killing curves for BER alone and in combination with RIF or CLI against ATCC 43300 (control strain) and clinical isolate ST239 are presented in Figure 1. Growth cultures without BER treatment increased to 7lg (CFU*mL1) for the control strain and 9lg (CFU*mL1) at ST239 after 24 h of incubation. The DMSO control group showed similar results to the growth cultures, suggesting that DMSO does not affect the growth of cells. Furthermore, BER at its MIC value showed dramatic inhibition of bacterial growth during 24 h of incubation, and showed stronger inhibition at 2 MIC than at MIC after 12 h of incubation with similar bacterial growth compared to that at MIC after 24 h of incubation (reduction of 2lg (CFU*mL1) and 1lg (CFU*mL1) for ATCC 43300 and ST239, separately). BER at sub-MIC had a weak effect on the growth of the MRSA strain over 24 h; however, bacterial growth was positive (Figure 1A and D). Importantly, the combination of BER with CLI showed a synergistic effect against the MRSA control strain and clinical isolate ST239 with ~2lg (CFU*mL1) killing compared to the single drug applied during 24 h (Figure 1B and E). In addition, the combination of BER with RIF showed more remarkable synergistic effects against MRSA than those of BER and CLI. As such, the combination of BER with RIF resulted in a > 2L g (CFU*mL1) inhibition compared to the combination of BER with CLI at 24 h (Figure 1C and F).

Figure 1 The time-killing curves for BER alone and in combination with RIF or CLI against MRSA strains; (AC): Control (DF): ST239. The data are the mean SD of colony counts at least two biological replicates in each group.

According to the assay of biofilm formation, BER possessed an excellent inhibitory effect on the biofilm in a dose-dependent manner, especially at the MIC or higher concentrations, which was more prominent than that of the sub-MIC and control groups. Interestingly, biofilm formation sharply increased at sub-MIC in the ST 239 strain (Figure 2). In addition, the combination of BER with RIF or CLI at sub-MIC significantly inhibited biofilm formation compared to each drug separately in the control strain and clinical isolate (Figure 3). The percentages of biofilm reduction, which were compared with positive control in each group, are presented at Table 4.

Table 4 The Percentage of Biofilm Formation Inhibited

Figure 2 The effects of BER alone on biofilm formation of MRSA strains. (A) Control (B) ST 239; relative biofilm formation levels were represented as meanSD of at least two biological replicates; significant difference was determined at P < 0.0001 with comparison between the groups.

Figure 3 The effects of BER in combination with RIF or CLI on biofilm formation of MRSA strains. (A and B): Control (C and D): ST 239; relative biofilm formation levels were represented as meanSD of at least two biological replicates; significant difference was determined at P < 0.0001 with comparison between the groups.

According to the images, untreated MRSA cells emitted green fluorescence, showing that the cell membrane was intact. Conversely, BER-treated MRSA appeared red and the proportion of red fluorescence increased with increasing BER concentrations, suggesting a damaged cell membrane (Figure 4). Meanwhile, the total number of cells declined after BER treatment, and the reduction was proportional to the BER concentration.

Figure 4 The effect of BER on cell membrane integrity of MRSA strain by confocal laser scanning microscopy.

The FESEM images illustrated significant changes in the morphology of MRSA cells with BER (Figure 5) at low magnification (10,00015,000) and high magnification (45,00050,000). Untreated control S. aureus exhibited arrangement in clusters and smooth surface and spherical contour at 10,000 and 50,000, separately (Figure 5A and E). However, the number of MRSA cells in the field of vision decreased and the morphology of the clusters was changed with increasing BER concentrations (Figure 5BD). Remarkably, numerous flocculent substances were adhering to the cells that were not significantly cleaned by PBS. These flocculent substances increased with the increase in BER concentration compared to the untreated control. Furthermore, the cells were less uniform in size and had a rough surface (50,000) compared to the untreated cells. When treated with MIC or higher concentrations of BER, the cell morphology exhibited shrinkage and deformation (Figure 5F); some cells even had a defective and punched surface, suggesting that the content was released (Figure 5G and H).

Figure 5 The changes in the morphology of MRSA cells with BER by field emission scanning electron microscope. (A and E): 0MIC; (B and F): 1MIC; (C and G): 2MIC; (D and H): 4MIC. Shown by the arrow is flocculent substances.

The dramatic increase and global spread of MRSA strains pose a serious threat to human health since it was first identified in 1960.27 Vancomycin and daptomycin are recommended as the first-line antibiotics by the 2011 Infectious Diseases Society of America guidelines for MRSA.28 However, each antimicrobial agent has limitations29 related to its effectiveness against bacteria30 or its increasing resistance.31,32 What is more, the emergence of vancomycin-resistant S. aureus leaves physicians with limited therapeutic options. Therefore, there is an urgent need in the development of new therapeutics to aid in the treatment of infectious diseases caused by MRSA. BER, an alkaloid extracted from a traditional Chinese herb, has emerged as a promising antibacterial agent.

In the current study, we observed that BER had anti-MRSA activities at MIC of 64256 mg*L1, which related to the subtype of MRSA strains. As previously reported, subtype strains within the same bacterial species may have different clinical uses.2 In this study, 15 strains were divided into five subtypes. We found that the MIC value of BER differed among various MLST types, and many strains showed diverse BER MIC values even in the same subtype. The MIC showed three values for ST5 and two values for ST239, unlike what has been reported in previous studies.2 In this study, a subtype was matched with a MIC value. However, the MIC value of ST239 and ST39 in our study which was 64 or 128 mg*L1 and 256mg*L1, was different with this study (64 mg*L1 and 512 mg*L1).2 Therefore, we recommend that the types of MLST should be expanded to verify the results.

In the current investigation, a synergistic effect of BER with CLI and RIF was observed against MRSA. As such, BER can markedly reduce the MIC value of antibacterial agents against MRSA. A synergistic effect of BER has also been reported in combination with oxacillin,33 azithromycin,34 and levofloxacin,34 whereas an additive effect has been reported for BER in combination with ampicillin33,34 and cefazolin.34 This study is the first one to report the synergistic effects of BER in combination with CLI and RIF.

The time-killing assays showed similar results. The time-killing test of the control strains, the strain ST239 for BER alone, and the combination of BER with CLI or RIF separately showed significant growth inhibition. The inhibition of growth by BER alone strengthened with the increasing concentration. Notably, the test strain ST239 and the control strain showed attenuation of inhibition with the increase in curve after 12 h, suggesting that BER alone may not kill all the cells, but it maintains the bacteria at low levels when the BER gradually expands. When this occurs, the remaining cells are reduced, which is similar to the results of a previous study on the antimicrobial activity of a Chinese herb.35 Therefore, there is a significant effect of BER concentration on its antimicrobial activity, suggesting that BER must maintain a persistently high blood concentration. In addition, the combined treatment resulted in obvious reduction in the cell quantity within 824 h of incubation in the control strain and 1024 h of incubation in ST239, compared to CLI or RIF separately. These results suggest that BER has excellent anti-MRSA activity, which is enhanced when combined with other antibiotics, such as CLI and RIF.33,34,3639

Biofilm formation plays an essential role in the persistence of pathogens.34,40 Biofilms harbor bacteria that are irreversibly attached to cell surfaces and prevent antibiotic exposure.41 In light of this, BER showed excellent inhibition of biofilm formation with increasing concentrations, similar to previous studies.2,42 However, biofilm formation increased at sub-MIC in the ST 239 strain in our study, similar to other studies.2 The concentration of BER cannot increase slowly when treating MRSA infections, because low concentration of BER may promote biofilm formation. Importantly, BER, in combination with CLI or RIF separately, also inhibited biofilm formation better than either drug alone. These data are consistent with previous studies that suggest the existence of synergistic effects of BER with those of CLI and RIF against MRSA. Therefore, antibacterial activity and anti-biofilm formation character of BER can be used as the basis of clinical practice and the synergistic effect of BER with CLI and RIF can be used as the alternative treatment when the bacterial infection was difficult to treat. However, the specific mechanism underlying the effects of BER in combination with CLI or RIF separately remains unclear and should be explored in future studies.

Morphological analysis was used to explore the mechanism of the BER effect against MRSA. In the current study, we observed cell wall alteration and its punched appearance due to BER exposure at high magnification. Flocculent substances were observed to adhere to the cells. Zhang et al found that BER could damage the cell surface of MRSA.43 Therefore, it is reasonable to speculate that the substances may include the contents of punched bacterial cells due to BER treatment. This observation is consistent with the above findings, illustrating that BER can destroy MRSA cell walls in a concentration-dependent manner.

The cytoplasmic membrane is an important dynamic structure that allows cells to interact with other cells and environmental molecules.44 While the cytoplasmic membrane integrity of MRSA has not been studied previously, the function of the gram-positive bacterial membrane is essential for subsisting membrane proteins and transportation.45 As assessed by CLSM, our results showed that the integrity of the MRSA cytoplasmic membrane was compromised at increased BER concentrations. This finding showed that BER possessed anti-MRSA activities by damaging the cell wall and cytoplasmic membrane to perturb cell functions and release intracellular contents. Although BER could exert an influence on the cell wall and cytoplasmic membrane, the mechanism here is unclear and needs to be investigated further. Meanwhile, combination antibacterial assays would verify the hypothesis that BER could damage the cytoderm and cytoplasmic membrane to kill MRSA. Because CLI and RIF play antibacterial roles inside the cells,46,47 they may easily enter the cells in the presence of BER.

The current study demonstrated that BER has remarkable antimicrobial effects against MRSA with favorable MIC. It can also be used as an adjuvant to RIF or CLI to treat MRSA infections. BER alone and in combination with RIF or CLI reduces biofilm formation. In addition, BER significantly damages the cell wall and membrane of MRSA to kill the bacteria. This study provides novel mechanistic insight into supporting the development of BER as an antibacterial drug.

The datasets generated for this study are available from the corresponding author Miaorong Xie.

This study was approved by the Ethics Committee of Beijing Friendship hospital, Capital Medical University (20210208). Adult patients wrote the informed consent and a parent or legal guidance of patients under 18 years of age provided informed consent prior to the experiment described below. This study was conducted in accordance with the Declaration of Helsinki.

We thank Wei-dong Qian from Shaanxi University of Science and Technology providing the experimental consults.

This work was supported by the National Key New Drug Creation and Manufacturing Program, Ministry of Science and Technology (YFC1702605).

The authors declare no conflicts of interest for this study.

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32. Gomez Casanova N, Siller Ruiz M, Munoz Bellido JL. Mechanisms of resistance to daptomycin in Staphylococcus aureus. Rev Esp Quimioter. 2017;30(6):391396.

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37. Aksoy CS, Avci FG, Ugurel OM, Atas B, Sayar NA, Sariyar Akbulut B. Potentiating the activity of berberine for Staphylococcus aureus in a combinatorial treatment with thymol. Microb Pathog. 2020;149:104542. doi:10.1016/j.micpath.2020.104542

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In vitro antimicrobial activity and mechanism of berberine | IDR - Dove Medical Press

The National Comprehensive Cancer Network has announced their recommendation for the use of pacritinib in the first line and second line setting for treating patients with myeloproliferative neoplasms.

The novel kinase inhibitor pacritinib (Vonjo) was added to the latest National Comprehensive Cancer Network (NCCN) guidelines as a recommended treatment for patients with myeloproliferative neoplasms (MPN), according to a press release.1

"We are grateful that the NCCN acted quickly to include (pacritinib) with a Category 2A designation in its Clinical Practice Guidelines in Oncology as a first line treatment for high-risk patients with myelofibrosis with platelet counts less than 50 x 109/L who are not candidates for transplant, said Adam R. Craig, MD, PhD, president, and chief executive officer of CTI BioPharma, in a statement. This therapeutic option helps address an unmet medical need for patients who previously have no other treatment options. There is no other FDA-approved first line treatment for these patients with a 2A designation within the NCCN guidelines.

Pacritinib was approved by the FDA earlier this year for the treatment of patients with myelofibrosis and severe thrombocytopenia, defined as a platelet count less than 50x109/L. In addition to this designation from the FDA, the NCCN has recommended pacritinib as a second-line treatment in low-risk and high-risk patients with myelofibrosis who have a platelet count greater than 50 x 109/L who are also not candidates for transplant. According to Craig, this will allow for even more treatment options in patients with MPNs.

The JAK family of enzymes promotes normal blood cell growth in patients because JAK is central component in signal transaction pathways, meaning that there is a direct relationship between mutations in these pathways and the development of hematological cancers like MPNs. Thus, allowing pacritinib to impact the development of the disease.

In the US, there are approximately 21,000 patients with myelofibrosis, two-thirds of which have cytopenias (thrombocytopenia or anemia), commonly resulting from the toxicity of other approved therapies, Craig said in a press release when announcing the FDA approval of pacritinib.2 Severe thrombocytopenia, defined as a blood platelet count below 50 109/L, occurs in one-third of the overall myelofibrosis population, and has a particularly poor prognosis.

Results from the phase 3 PERSIST-1 (NCT01773187), phase 3 PERSIST-2 (NCT02055781), and phase2 dose-finding PAC203 trial led to the initial approval, and now recommendation by the NCCN, for the use of pacritinib in multiple lines of therapy for patients with MPNs. These findings demonstrated that the kinase inhibitor, with specificity for inhibiting JAK2 and IRAK1 without inhibiting JAK1, was associated with better outcomes in patients in comparison to the best available therapy.

After the PERSIST-1 trial met its primary endpoint at week 24 of the study when 19% of patients on pacritinib had a spleen reduction size by 35% or more compared to 5% in the control group, 311 patients enrolled in the PERSIST-2 trial saw better results on pacritinib given twice a day.3 Eighteen percent of patients in the twice daily pacritinib arm of the trial had a spleen reduction of 35% or greater vs 3% in the best available therapy arm.4

The most common adverse events (AEs) observed on twice-daily pacritinib at 200 mg, seen in 20% or more of paitents, was diarrhea, thrombocytopenia, nausea, anemia and peripheral edema. Serious AEs seen in 3% or more of patients on the same regimen were anemia, thrombocytopenia, pneumonia, cardiac failure, disease progression, pyrexia, and squamous cell carcinoma of skin.

The approval of pacritinib establishes a new standard of care for myelofibrosis patients suffering from cytopenic myelofibrosis," said John Mascarenhas, MD, associate professor, Medicine, Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, in a press release on the approval of the drug. "Myelofibrosis with severe thrombocytopenia, defined as blood platelet counts below 50 109/L, has been shown to result in poor survival outcomes coupled with debilitating symptoms. Limited treatment options have rendered this disease an area of urgent unmet medical need."

References

1. NCCN Guidelines Recommend VONJO (pacritinib) for the Treatment of Myeloproliferative Neoplasms. CTI BioPharma Corp. News release. April 14, 2022. Accessed April 14, 2022. https://prn.to/3EiDaN0

2. CTI BioPharma announces FDA accelerated approval of VONJO (pacritinib) for the treatment of adult patients with myelofibrosis and thrombocytopenia. CTI BioPharma Corp. News release. February 28, 2022. Accessed April 14, 2022. https://bit.ly/3vsDPsO

3. Mesa R, Vannuchhi A, Mead A, et al. Pacritinib versus best available therapy for the treatment of myelofibrosis irrespective of baseline cytopenias (PERSIST-1): an international, randomised, phase 3 trial. The Lan. Hem. Published: March 20, 2017. doi: https://doi.org/10.1016/S2352-3026(17)30027-3

4. Mascarenhas J, Hoffman R, Talpaz M, et al. Pacritinib vs Best Available Therapy, Including Ruxolitinib, in Patients With Myelofibrosis: A Randomized Clinical Trial. JAMA Oncol. 2018;4(5):652659. doi:10.1001/jamaoncol.2017.5818

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Pacritinib Added to NCCN Recommendation for Treatment in MPNs - Targeted Oncology

The study, published in the journal Nature Aging, lays the ground for research that explores the possibility of translating the findings into humans.

People have traditionally thought of aging as an inevitable part of life. But since the seminal work of Cynthia Kenyon in the 90s, researchers have also become aware that aging is under genetic control.

Scientists continue to be interested in finding out whether the negative effects of aging can be reduced or reversed entirely.

Currently, 16% of the United States population is 65 years or older. By 2050 this is expected to reach 22%.

The Centers for Disease Control and Prevention (CDC) note that aging increases a persons risk of various serious chronic illnesses, such as cancer, dementia, type 2 diabetes, and heart disease.

The National Institute on Aging points out that there are various things a person can do to help reduce the effects of aging.

These include staying physically active, eating a healthy diet with lots of vegetables, fruit, and whole grains, getting a good amount of quality sleep, avoiding smoking and drinking alcohol, and regularly seeing a doctor.

In 2020 the World Health Organization (WHO) published a baseline report for the Decade of Healthy Ageing, highlighting how countries can go about ensuring health and well-being as people age.

Dr. Tedros Adhanom Ghebreyesus, WHO Director-General, says that humans now live longer than at any time in history. But adding more years to life can be a mixed blessing if it is not accompanied by adding more life to years.

The Baseline Report for the Decade of Healthy Ageing has the potential to transform the way policy-makers and multiple service providers engage with older adults. We have to work together, to foster the abilities and well-being of our older generations, who continue to give us so much.

As well as lifestyle and policy changes, scientists are also exploring whether new types of medical interventions could reduce the physiological effects of aging.

The authors behind the present study have previously found that epigenetic markers in mice could be reprogrammed using the molecules Oct4, Sox2, Klf4, and cMyc. These molecules, known as Yamanaka factors, increased the lifespan and reduced the effects of aging in mice with premature aging.

Medical News Today spoke with Prof. Juan Carlos Izpisua Belmonte, of the Gene Expression Laboratory at the Salk Institute for Biological Studies, San Diego, CA, and a corresponding author of the present study.

In the 2016 paper, we developed a protocol and showed for the first time that Yamanaka factors could be expressed in mice safely without generating cancer. Moreover, in our previous study, we used a premature aging mouse model to demonstrate that Yamanaka factors can extend the lifespan of these mice by preventing the accumulation of aging phenotypes in cells and tissues.

However, we did not know if expressing the Yamanaka factors for an extended period of time in animals without any preexisting pathologies will work and whether it would be safe. The goal of the current study was to establish whether long-term partial reprogramming would have a positive or negative impact on a wild-type animals health, said Prof. Izpisua Belmonte.

To do this, Prof. Izpisua Belmonte and his colleagues split the mice into three groups. The first group received Yamanaka factors from 15 to 22 months or around 50 to 70 years in human terms.

The second group received the Yamanaka factors from 12 to 22 months or 35 to 70 in human years.

The third group was treated for a single month at 25 months or 80 years in human terms.

The researchers found that compared with mice that acted as a control, the mice who received the Yamanaka factors did not develop cancer or see any blood cell or neurological changes.

Further, the mice that received the Yamanaka factors for a number of months showed various reversals in the effects of aging.

The kidneys and skin of the mice resembled those of younger mice, their skin healed from wounds without producing as much scarring, and the scientists did not observe the usual metabolic changes in the blood typically seen in older animals.

The animals treated for just a single month late in life did not see these effects.

Prof. Izpisua Belmonte said there were still necessary steps before the research could be tested in humans.

The translation of our approach to humans requires developing ways to deliver the factors and controlling the levels and how long the factors are expressed. These steps will allow [us] to demonstrate the safe delivery of the factors, a critical aspect before we could start thinking in clinical trials.

Nonetheless, the findings provide exciting evidence that the technique could have benefits far beyond the reversal of the effects of aging.

After our initial 2016 study, our lab, as well as several other laboratories around the world, have used the same approach to demonstrate improvement in the regeneration of different tissues in mice and rejuvenation of human cells.

All these studies further prove that the controlled expression of Yamanaka factors for cell reprogramming could benefit diverse conditions and might be a general medicine approach in the future for various complications that arise during life, said Prof. Izpisua Belmonte.

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Aging safely reversed in mice by reprogramming cells - Medical News Today

image:Intensities of protein expression of markers are shown on viSNE map as spectrum colored dots, with low in blue, high in red. view more

Credit: Bachmaier, et al. ACS Nano

Using a protein nanoparticle they designed, scientists at the University of Illinois Chicago have identified two distinct subtypes of neutrophils and found that one of the subtypes can be used as a drug target for inflammatory diseases.

Neutrophils are a type of white blood cell that help fight infection, clear dead cell debris, and heal tissue injury. But for people with health conditions caused by chronic inflammation, like arthritis or Crohns disease, or excessive inflammation, like sepsis, the role of neutrophils may be deleterious. Neutrophils have been described in research as also contributing to tissue damage the double-edged sword of inflammation. Unfortunately, current drugs for inflammatory diseases that target neutrophils suppress all their effects, including their anti-infection and healing functions.

The UIC team is the first to characterize neutrophils into two subsets.

Understanding the differences between these neutrophil subsets opens the door for more research on treatments that address inflammatory diseases without increasing patients risks of infections, said study author Kurt Bachmaier, assistant professor in the department of pharmacology and regenerative medicine at the College of Medicine, who led the research.

Bachmaier and his colleagues first used the nanoparticle platform, formulated from a protein called albumin, to analyze how neutrophils from bone marrow, blood, and spleen and lung tissues interact with the nanoparticle. They found that some neutrophils brought the albumin nanoparticle into the cell through a process called endocytosis, while others didnt.

The scientists labeled the subtype that readily endocytosed the nanoparticle as ANP-high, for albumin nanoparticle high. The neutrophils that did not absorb the albumin nanoparticle were labeled as ANP-low.

Further investigation with the albumin nanoparticle showed that the subtypes have different cell surface receptors and that they are functionally distinct in their helpful capacities to kill bacteria and their harmful potential to promote inflammation. ANP-highneutrophils did not help to kill bacteria but produced inordinate amounts of reactive oxygen species and inflammatory chemokines and cytokines, which contribute to inflammatory disease.

Because the ANP-high neutrophils are also the ones that captured the nanoparticle, the scientists conducted clever experiments using the albumin nanoparticle to deliver drug treatments. They filled the nanoparticle with an anti-inflammatory drug and administered it to mice with sepsis. They found that the mice treated with the drug-loaded nanoparticle had reduced signs of tissue inflammation, but that the neutrophilic host-defense was preserved.

The albumin nanoparticle, which was filled with the drug, specifically bound to ANP-high neutrophils and unloaded their cargo into the cell, stopping it in its tracks, Bachmaier said.We found ANP-high neutrophils not only in mice but also in humans, opening the possibility of neutrophil subset-specific targeted therapy for human inflammatory diseases.

Science can be a bit like magic by targeting only the ANP-high neutrophils, we stopped the out-of-control inflammation while preserving the bacteria-fighting inflammation of these Janus-like cells, said senior author Asrar Malik, Schweppe Family Distinguished Professor and head of the department of pharmacology and regenerative medicine.

These findings are reported in the article Albumin Nanoparticle Endocytosing Subset of Neutrophils for Precision Therapeutic Targeting of Inflammatory Tissue Injury, which is published in ACS Nano, a scientific publication of the American Chemical Society and the primary nanotechnology journal.

Co-authors of the article are Andrew Stuart, Amitabha Mukhopadhyay, Sreeparna Chakraborty, Zhigang Hong, Li Wang, Yoshikazu Tsukasaki, Mark Maienschein-Cline, Balaji Ganesh, Prasad Kanteti and Jalees Rehman.

Experimental study

Animals

Albumin Nanoparticle Endocytosing Subset of Neutrophils for Precision Therapeutic Targeting of Inflammatory Tissue Injury

1-Mar-2022

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

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Newly identified neutrophil subset is a promising therapeutic target - EurekAlert

Bo Xie,1,* Yi Chen,2,* Yebei Hu,2 Yan Zhao,2 Haixin Luo,2 Jinhui Xu,1 Xiuzu Song1

1Department of Dermatology, Hangzhou Third Peoples Hospital, Affiliated Hangzhou Dermatology Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, Peoples Republic of China; 2Department of Dermatology, Hangzhou Third Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, 310009, Peoples Republic of China

Correspondence: Xiuzu Song, Department of Dermatology, Hangzhou Third Peoples Hospital, Affiliated Hangzhou Dermatology Hospital, Zhejiang University School of Medicine, West Lake Road 38, Hangzhou, 310009, Peoples Republic of China, Tel +86-571-87823102, Email [emailprotected]

Objective: The treatment of vitiligo is often challenging to dermatologists. There is ample evidence to suggest that hydroxychloroquine (HCQ) is effective for vitiligo treatment; nonetheless, the underlying mechanism remains unknown. In the present study, we sought to uncover the molecular targets of HCQ by an integrated network-based pharmacologic and transcriptomic approach.Methods: The potential targets of HCQ were retrieved from databases based on the crystal structure. Targets related to vitiligo were screened and intersected with potential targets of HCQ. A protein-protein interaction network of the intersected targets was generated. Interactions between the targets were verified by molecular docking. Moreover, human vitiligo immortalized melanocytes (PIG3V) were evaluated after treatment with HCQ (1g/mL) for 24h. The total RNA of PIG3V was extracted and determined by RNA-seq transcriptomics for differential gene expression analysis. Network pharmacology was then used to identify the relationships between putative targets of HCQ and differentially expressed genes.Results: Molecular docking analysis revealed four putative key targets (ACHE, PNMT, MC1R, and VDR) of HCQ played important roles in vitiligo treatment. According to the transcriptomic results, the melanosomal biogenesis-related gene BLOC1S5 was upregulated 138005.020 fold after HCQ treatment. Genes related to protein repair (MSRB3) and anti-ultraviolet (UV) effect (UVSSA) were upregulated 4.253 and 2.603 fold, respectively, after HCQ treatment.Conclusion: The expression of the BLOC1S5 gene is significantly upregulated, indicating upregulated melanosomal biogenesis after HCQ treatment. In addition, HCQ yields a protective effect on melanocytes by upregulating genes associated with damaged protein repair (MSRB3) and anti-UV effect (UVSSA). The protective effects of HCQ are mediated by binding to putative targets ACHE, PNMT, MC1R, and VDR according to network pharmacology and docking verification.

Keywords: vitiligo, hydroxychloroquine, treatment, pigmentation, melanocyte protection

Vitiligo is an autoimmune depigmentation disorder characterized by the loss of functional melanocytes and patchy skin pigmentation.1 It has been reported that at least 0.5% of the population suffers from vitiligo worldwide.2 Importantly, vitiligo can lead to stigma, shame and embarrassment in this patient population.3 Until now, the mechanisms that belie the pathogenesis of vitiligo remain unknown. An increasing body of evidence suggests that both the adaptive and innate immune systems are involved in the pathogenesis of vitiligo.4 Moreover, interferon (IFN) -inducible chemokines and CD8+ T cells can be initiated by external triggers such as ultraviolet (UV) and chemical stimuli.5 Mitochondria generate reactive oxygen species (ROS) in response to oxidative stimuli, disrupting the normal functions of organelles such as mitochondria, lysosome, endoplasmic reticulum (ER), etc.6 ER injury has been reported to lead to the generation of unfolded proteins.7 Besides, it has been established that damaged proteins and exosomes secreted by melanocytes can be recognized by antigen-presenting cells to stimulate autoreactive T cell maturation.8 The positive feedback of melanocyte-specific CD8+ T cells recruitment induced by chemokines can reportedly potentiate the autoimmune attack towards melanocytes.9 Notwithstanding that unprecedented progress has been achieved in understanding the pathophysiology, the specific mechanisms have not been clearly elucidated, accounting for the difficulty dermatologists face in treating this disease during clinical practice, hence emphasizing the need for future studies.10

According to current guidelines, topical glucocorticoids, calcineurin inhibitors, vitamin D3 derivatives, and phototherapy remain the mainstay of treatment for vitiligo. Systemic glucocorticoids are indicated with rapid progression of the lesions.14 Indeed, efficient vitiligo treatment is often difficult, with low repigmentation rates and high relapse rates. Currently, HCQ is recommended to treat rheumatic diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), antiphospholipid antibody syndrome, and Sjogrens syndrome, which are autoimmune-related.1113 In addition, HCQ is reportedly effective in treating vitiligo, chronic actinic dermatitis, chronic urticaria, dermatomyositis, vasculitis, lichen planus, etc.14 In RA and SLE patients presenting with vitiligo, it was observed that HCQ treatment also promotes pigmentation in vitiligo lesions.10,15 In addition, skin pigmentation after HCQ treatment was observed in RA and SLE patients without vitiligo.16,17 It is widely believed that HCQ exerts an anti-inflammatory effect that can disrupt T-cell receptor-related Ca2+ signaling and antigen processing.18 Importantly, Li DG et al19 showed that HCQ could protect melanocytes from autoantibody-induced damage by reducing the formation of antigen-antibody complexes.

At present, the mechanisms underlying the therapeutic effect of HCQ in vitiligo are unclear. Current evidence suggests that HCQ could be a multi-target drug given its wide range of effects. Indeed, the immunoregulatory effect of HCQ plays a key role in its pharmacological mechanisms. In recent years, the rapid development of computer technology and big data analytics has led to the advent of network pharmacology, which provides a new strategy for the research of multi-targets drugs. Network pharmacology integrates poly-pharmacology, bioinformatics and systems biology for multi-targets drug research and evaluation. Importantly, network pharmacology emphasizes the multi-targets network/drug pattern in contrast to the conventional one target/ one drug paradigm.2023 In the present study, human vitiligo immortalized melanocytes (PIG3V) were used to conduct differential gene expression analysis by RNA-sequencing after HCQ treatment. Transcriptomic, GO annotation, and KEGG enrichment analyses were conducted to elaborate changes in gene expression and biological functions after HCQ treatment. Finally, network pharmacology and transcriptomic results were integrated to uncover the mechanisms of HCQ in treating vitiligo, providing a foothold for future studies on its potential use for vitiligo treatment.

The predicted target proteins were retrieved from the database according to the crystal structure and chemical groups of HCQ. Then, vitiligo-related proteins were collected from disease databases. Data on these proteins were imported into Cytoscape software (v.3.8.2). The two groups of proteins were then intersected. A protein-protein interaction (PPI) network of the intersected proteins was constructed with the plugin Bisogenet of Cytoscape software. Subsequently, all intersected targets and hub proteins in the PPI network were chosen for molecular docking with HCQ. Then, gene expression in PIG3V was explored after treatment with HCQ. Transcriptomic, GO annotation and KEGG enrichment analyses were conducted. Finally, we integrated network pharmacology and transcriptomic results to assess the relationships between HCQ molecular docking targets and differentially expressed genes after HCQ treatment in PIG3V cells (Figure 1). According to the guidelines of Ethics Committee of Hangzhou Third Peoples Hospital, the human public databases we used in this research were exempted from approval, because only studies of identifiable human specimens or data need to be approved by ethics committee.

Figure 1 The flow chat of strategy layout. The mechanisms of HCQ in treating vitiligo were predicted by network pharmacology. Hub targets were verified by molecular docking. The efficacy of HCQ on vitiligo were then observed on PIG3V cell line. Differential gene expression was analyzed and linked to HCQ targets that verified by docking.

The chemical structure (Canonical SMILES) of HCQ was retrieved from PubChem database (https://pubchem.ncbi.nlm.nih.gov/): CCN(CCCC(C)NC1=C2C=CC(=CC2=NC=C1)Cl)CCO. The SMILES molecular formula of HCQ was imported into the SwissTargetPrediction database (http://www.swisstargetprediction.ch/index.php). Then predicted targets of HCQ were calculated and retrieved from the SwissTargetPrediction database based on the crystal structure. The species was set as Homo sapiens. The top 100 potential targets were selected according to the calculated parameters.24,25 Vitiligo-related proteins were searched using the keywords and species (vitiligo and Homo sapiens) across four databases, including Genecards (https://www.genecards.org/), OMIM (https://omim.org/), Drugbank (https://go.drugbank.com/) and DisGeNET (https://www.disgenet.org/). A total of 1185 vitiligo-related proteins were screened.26,27 The bisogenet plugin of Cytoscape was used for the PPI network construction. The intersected proteins between vitiligo and HCQ were entered into bisogenet. The topological features degree, betweenness and closeness were used to select the putative targets using the Cytoscape plugin CytoNCA.28

Along with the intersection targets between HCQ and vitiligo, the core targets screened in the previous step were chosen as candidates for molecular docking. The spatial interactions between target proteins and HCQ were analyzed by AutoDock (v.4.2) software. First, the 3-dimensional crystal structure of HCQ was retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) database. Then, modifications such as water and ligand removal, amino acid optimization and patching, and hydrogen addition were manipulated in AutoDock. ChemBioDraw 3D (v.15.1) software was used for 3-D visualization and energy minimizing. Finally, MolegroVirtualDocker software was used to compute docking targets by comparing the conformation with the existing 3-D crystal structure of HCQ.29

The Human vitiligo melanocyte cell line PIG3V was a gift from Professor Chunying Li (Xijing Hospital of Air Force Medical University, Xian, China) and cultured (2105 cells/mL) in a 6-well plate (2mL/well) supplemented with DMEM/F12, 10% FBS, basic fibroblast growth factor (10ng/mL), phorbol myristate acetate (10ng/mL), and penicillin/streptomycin (10000U/mL, 10000g/mL) at 37C in 5% CO2 for 24 hours.30 The cultured PIG3V cells were randomly divided into an HCQ group and a control group. Cells in the HCQ group were treated with 1g/mL HCQ (Sigma-Aldrich Corp., St. Louis, MO, USA) for 24 hours. The HCQ powder was dissolved in phosphate-buffered saline (PBS). In contrast, cells in the control group were treated with PBS for 24 hours. Finally, the total RNA of PIG3V cells was purified and extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA).

RNA samples extracted from PIG3V cells were quantified by NanoDrop (Wilmington, DE, USA) and Bioanalyzer (Agilent, CA, USA).31,32 Poly (A) RNA was extracted from total RNA by 2 rounds of purification with Dynabeads Oligo (Thermo Fisher, CA, USA). Small pieces of Poly A RNA were returned through Magnesium RNA Fragmentation Module (NEB, USA) under the condition of 94C for 5 minutes. Subsequently, SuperScript Reverse Transcriptase (Invitrogen, USA) was used to reverse-transcribe the cleaved RNA pieces into cDNA. Then, U-labeled second-stranded DNAs were synthesized. After addition of A-base to each strand, index read preparation, and heat-labile UDG enzyme treatment, PCR amplification was conducted. PCR consisted of preheating at 95C for 3 minutes; 8 cycles of 98C for 15 seconds, annealing at 60C for 15 seconds and extension at 72C for 5 minutes. Ultimately, RNA sequencing was performed according to the protocol of Illumina Novaseq 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China).33,34 Genes differential expression analysis was performed by DESeq2 software between two different groups. After calculation, differentially expressed genes were screened by fold change (FC)>2 or FC<0.5 and p value<0.05. These genes underwent GO and KEGG functional enrichment analyses. Hypergeometric test was used to calculate p value in GO and KEGG enrichment.35,36

According to the value of | log2(FC) |, the top 30 upregulated and downregulated genes in PIG3V cells were selected. To screen genes related to synthesis, transport, metabolism of melanin, melanocyte protection and other mechanisms associated with vitiligo, the functions of these genes/proteins and previous studies were retrieved from the databases of UniProt (https://www.uniprot.org/) and NCBI PubMed (https://www.ncbi.nlm.nih.gov/). Then these differentially expressed genes along with docking HCQ targets were input into Cytoscape software. A PPI network was constructed to explore the interactions between HCQ targets and vitiligo-related differentially expressed genes.37,38

The predicted targets of HCQ were computed and retrieved from databases by analyzing the 2 and 3-dimensional chemical structure (Figure 2A). The top 100 potential targets (Figure 2B) were retrieved according to the index of possibility. These predicted targets were mainly classified as G protein-coupled receptors (29%), kinases (26%) and surface antigens (13%) (Figure 2C).

Figure 2 Predicted targets of HCQ. (A) 3 dimensional and 2 dimensional structure of HCQ; (B) top 100 predicted targets of HCQ from databases; (C) categories of the predicted targets.

The intersection between vitiligo-related proteins (n=1185) and top 100 potential HCQ targets yielded 15 proteins (Table 1). A PPI network of the intersected targets was generated by Cytoscape using the plugin Bisogenet. Then, 1686 additional proteins closely linked to the 15 intersected proteins were retrieved from Bisogenet. Finally, a total of 42,371 interactions (edges) between 1686 targets (nodes) were identified (Figure 3A). A topological degree greater than 62 was used to screen vital nodes in these 1686 targets (Figure 3B). Then, 459 nodes and 18,251 edges were screened according to a betweenness value greater than 734.84 and closeness value greater than 0.50. The 78 hub proteins and their 1452 interactions which may play important roles in the therapeutic effects of HCQ in treating vitiligo, were retrieved (Figure 3C). The top 10 hub proteins are listed in Table 2.

Table 1 The Intersection of HCQ Predicted Targets and Vitiligo-Related Proteins

Table 2 The Hub Proteins Screened in PPI Network

Figure 3 The PPI network conducted with 15 mutual genes of vitiligo and HCQ potential targets. (A) PPI network of the enlarged 1686 nodes; (B) the nodes and edges after the first screening; (C) the nodes and edges after the second screening.

The intersected 15 proteins and the top 10 hub proteins of the PPI network were chosen as target candidates for molecular docking verification (Tables 1 and 2), providing them visual explanations of spatial interactions with HCQ. A docking score below 20 demonstrated that HCQ could effectively combine with target proteins. Acetylcholinesterase (ACHE), Phenylethanolamine N-methyltransferase (PNMT), Melanocortin receptor 1 (MC1R), and Vitamin D3 receptor (VDR) were identified by molecular docking analysis as the putative targets of HCQ during vitiligo treatment (Table 3). The 4 key targets exhibited the most solid chemical binding forces and spatial conjunctions with HCQ. Importantly, the docking results showed that ionic bonds, hydrogen bonds and - stacking interactions were the predominant chemical forces. For instance, the hydroxyl, carbonyl and amino groups within HCQ formed hydrogen bonds with target proteins. The benzene and aromatic rings of HCQ formed - stacking interactions with target proteins (Figure 4).

Table 3 The Docking Results of Target Candidates

Figure 4 Molecular docking verification. Target candidates (A) ACHE, (B) VDR, (C) MC1R, and (D) PNMT were shown interacting with HCQ molecule (represented by a green ball-and-stick model).

PIG3V is an immortalized cell line derived from human vitiligo melanocytes. To better understand the state of PIG3V cells after HCQ (1g/L) treatment for 24 hours, we analyzed the transcriptome of PIG3V. We found 108 and 97 DEGs were upregulated and downregulated by 2 fold or more in PIG3V cells, respectively, after HCQ treatment compared to the PIG3V control (p value<0.05). The top 30 upregulated and down-regulated genes in PIG3V cells were selected according to the value of | log2(FC) | and displayed in Table 4. As for the melanin synthesis pathway, BLOC1S5 was highly upregulated by 138005.020 fold after HCQ treatment compared to the control group, suggesting that melanin synthesis was significantly enhanced. In addition, two upregulated DEGs, MSRB3 and UVSSA, were enriched in melanocyte protection, which indicated an antioxidant effect in PIG3V cells after HCQ treatment. In addition, many upregulated genes were involved in modulating the activity of the immune system during acute-phase reactions such as ORM1, SAA1, SAA2, HP, FGA, FGB, FGG, CRP, AMBP, PTPRC, SERPINA1, and SERPINA3. These acute-phase genes may be related to the antibiotic effect of HCQ, acting as guards against the invasion of harmful microorganisms such as plasmodium.39 In contrast to acute-phase genes, some chronic-phase genes were downregulated, including COL1A1, DCN, and ELN. The downregulated genes were associated with decreased collagen synthesis and fibrosis, which are features of chronic inflammation. This finding may be a potential mechanism of HCQ in treating RA and other rheumatic diseases. Moreover, current evidence suggests that COL1A1 and ELN downregulation are associated with skin aging.40,41 KEGG pathway analysis showed that platelet activation, protein digestion, absorption, and herpes simplex virus 1 infection were significantly enriched pathways. GO enrichment analysis showed that GO terms, including acute-phase response, collagen-containing extracellular matrix, and extracellular matrix organization, were significantly enriched for molecular functions (Figure 5). However, it remains unclear whether these acute-phase genes are linked to vitiligo.

Table 4 Differential Gene Expression of PIG3V After HCQ Treatment

Figure 5 Transcriptomics analysis of PIG3V cells treated by HCQ. (A) number of up-regulated and down-regulated genes; (B) heat map of up-regulated and down- regulated genes; (C) KEGG enrichment of differential expressed genes; (D) volcano map of up-regulated and down-regulated genes; (E) GO enrichment of differential expressed genes.

Abbreviations: HCQ, hydroxychloroquine treated PIG3V cells group; NG, normal control group (PIG3V without hydroxychloroquine treatment).

To validate that ACHE, PNMT, MC1R, and VDR were targets of HCQ during vitiligo treatment, the differentially expressed genes in PIG3V cells were detected by RNA sequencing. Except for acute phase genes and other genes detected by RNA sequencing, genes potentially related to vitiligo or melanocyte (including BLOC1S5, MSRB3 and UVSSA) underwent PPI network analysis with docked HCQ targets, yielding a total of 163 nodes (proteins) along with 1019 edges (interactions) (Figure 6).

Figure 6 The network between HCQ targets and differential expressed genes of PIG3V cells after the treatment of HCQ. (A) PPI network between docked HCQ targets and differential expressed genes of PIG3V showed enlarged 163 nodes (proteins) and 1019 edges (interactions); (B) main connections of the PPI network.

In the present study, the transcriptomic results showed that BLOC1S5 gene expression exhibited the highest fold change (138005.020 fold) after PIG3V cells were treated with HCQ. It has been established that BLOC1S5 encodes a subunit of the biogenesis of the lysosome-related organelles complex (BLOC-1), which is involved in melanosomal biogenesis.42 Oculocutaneous depigmentation is widely acknowledged as one of the characteristics of Hermansky-Pudlak Syndrome (HPS). Recent studies have demonstrated the presence of pathogenic BLOC1S5 variants in HPS.43,44 Moreover, it has been shown that knockdown of BLOC1S5 in zebrafish led to retinal depigmentation.45 In addition, a significant association between single nucleotide polymorphism (SNP) of 3 genes, including BLOC1S5, involved in skin pigmentation and 25-(OH)D serum concentration has been found.46 In the present study, network pharmacology analysis demonstrated that Vitamin D3 receptor (VDR) was a predicted target of HCQ, which may play a role similar to 25-(OH)D. In addition, another docking target of HCQ, MC1R, is a documented G-protein-coupled receptor that plays a vital role in skin pigmentation. It has been reported that melanocyte-stimulating hormone (-MSH) could stimulate cAMP signaling and melanin production after combination with MC1R, enhancing the repair of damaged DNA and protein after UV exposure.45 The MC1R/cAMP/MITF signaling pathway is well-established to regulate UV-induced pigmentation.46 Although no direct relationship between MC1R/VDR and BLOC1S5 was found in our PPI network, the highly upregulated BLOC1S5 indicated modulation of this pathway (Figure 7). Further studies are warranted in the future to validate this finding.

Figure 7 Illustration of related genes involved in pigmentation pathway. BLOC1S5, PMEL, DTNBP1 and PLDN are genes involved in genesis of the melanosome. MC1R, MITF, DKK1, RAB27A, MLPH and MYO5A encode proteins in membrane or cytoplasm which are involved in signaling pathways of skin pigmentation. PAX3 and SOX10 are transcription factors in melanocyte. RACK1 is a regulator to the signal transduction in melanocyte. MSRB3 and UVSSA encode proteins that are involved in damaged proteins/DNA repair under UV. MC1R, VDR, PNMT, and ACHE was targeted by HCQ according to network pharmacology and molecular docking, which could further up-regulate gene expression of BLOC1S5, MSRB3, and UVSSA, showing the promotion effect of melanosome genesis and melanocyte protection under UV damage.

Abbreviations: MSH, melanocyte stimulating hormone; MC1R, melanocortin 1 receptor; BLOC1S5, biogenesis of lysosomal organelles complex-1 subunit 5; cAMP, 3,5-cyclic adenosine monophosphate; PMEL, premelanosome protein; DTNBP1, dystrobrevin binding protein 1; PLDN, biogenesis of lysosomal organelles complex-subunit 6; MITF, microphthalmia-associated transcription factor; PAX3, paired box 3; RACK1, guanine nucleotide binding protein; SOX10, SRY (sex determining region Y)-box 10; RAB27A, member RAS oncogene family; MLPH, melanophilin; MYO5A, Myosin VA (heavy chain 12, myoxin); UV, ultraviolet; PNMT, Phenylethanolamine N-methyltransferase; ACHE, Acetylcholinesterase; VDR, Vitamin D3 receptor; Ach, acetylcholine; AD, adrenaline; NA, noradrenaline; HCQ, hydroxychloroquine.

Current evidence suggests that in vitiligo patients, organelle functions of melanocytes are damaged in response to external triggers such as UV and chemical stimuli, leading to the attack of the bodys immune system.7 Our transcriptomic analysis showed that DEGs, including MSRB3 and UVSSA, were upregulated 2 fold or more after treatment with HCQ. These two genes are widely acknowledged for protein and DNA repair and maintaining organelle homeostasis. It has been shown that MSRB3 encodes methionine-R-sulfoxide reductase (MSR). In response to environmental stress, many reactive oxygen species (ROS) are produced. ROS can oxidize methionine (Met) residues in protein peptides to form methionine sulfoxide [Met(O)], which leads to protein function impairment. Importantly, MSR could catalyze the reduction of Met(O) to restore the function of proteins in response to oxidative damage. Accordingly, MSRB3 plays a pivotal role in cell protection under environmental stress.47 Moreover, it has been shown that UVSSA encodes for the UV-stimulated scaffold protein A, a transcription-coupled nucleotide excision repair (TCNER) factor, in response to UV damage. TCNER stabilizes gene ERCC6 by recruiting the enzyme USP7 into the TCNER complex, preventing UV-induced ERCC6 degradation by proteasomes.48 TCNER induces the removal of RNA polymerase II (RNA pol II) from the active genes for transcription. Subsequently, ubiquitination at UV-damaged sites can accelerate RNA pol II recalling to nucleotide excision repair machinery.49 According to the molecular docking and PPI network analysis results (Figure 6), the MC1R-MSRB3/UVSSA axis could be an essential mechanism of HCQ in the process of melanocyte protection against oxidative stress, and the MC1R agonist could serve as a cutaneous pigmentation promoter. An increasing body of evidence suggests that activation of MC1R alleviates oxidative stress and neuronal apoptosis through protein kinase R-like endoplasmic reticulum kinase (PERK)-nuclear factor erythroid 2-related factor 2 (NRF2) pathway, exhibiting an antioxidant role in the unfolded protein response (UPR) system. As the UPR system is initiated, impaired (unfolded, misfolded, etc.) proteins are restored by enzymes through a series of reactions.7,50

As molecular docking targets of HCQ, ACHE and PNMT potentially play important roles in vitiligo treatment. Non-neuronal acetylcholine (ACh) is well-recognized to regulate human keratinocyte (KCs) functions, including cell differentiation, cell-cell interaction, secretion, and mitosis. Besides, ACh plays an important role in immune regulation. It has been established that the Ach receptor is expressed in both KCs and melanocytes.51 A study reported that the average levels of ACh were higher in areas of skin depigmentation compared to controls. Interestingly, the level of ACh was significantly decreased after treatment, which showed a significant positive correlation with the severity of vitiligo.52 A study by Taieb et al53 substantiated the toxic effect of ACh on melanocytes. In addition to ACHE, PNMT, which catalyzes the synthesis of adrenaline (AD) from noradrenaline (NA), plays an important role in vitiligo. It has been shown that KCs could synthesize NA and AD. Current evidence suggests that KCs in the lesion area of vitiligo patients synthesized 4 times more NA than normal skin area, with low PNMT activity.54 Therefore, increasing the activity of ACHE and PNMT to reduce skin ACh and NA may be one of the mechanisms underlying the efficacy of HCQ in vitiligo treatment. Nonetheless, further studies are required to increase the robustness of our findings.

This present study uncovered HCQ targets (ACHE, PNMT, MC1R, and VDR) during vitiligo treatment after screening by network pharmacology and molecular docking. PIG3V cells were used to explore the mechanisms of HCQ treatment through transcriptomic analysis. The results of transcriptomic study further showed that through the above targets, HCQ significantly promoted the expression of genes related to melanin synthesis. In addition, the expression of the genes which are related to DNA and protein damage repair was also significantly up-regulated, indicating the protective effect of HCQ on vitiligo melanocytes. In silico methods were used to identify the relationships between HCQ targets and differentially expressed genes in PIG3V cells. These findings provided the theoretical basis for the mechanisms of HCQ in treating vitiligo (Figure 7). Nevertheless, there were some limitations in our study. The specific mechanisms and signal pathways of the MC1R/VDR-BLOC1S5 axis and MC1R-MSRB3/UVSSA axis involved in the pigmentation process and melanocytes protection effect were not explored. In addition, the effect of HCQ on ACHE and PNMT and further pathways were not assessed, warranting further studies.

This work was supported by [National Natural Science Foundation of China] under Grant [number 81872517].

The authors declare that they have no conflicts of interest.

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Pigmentation and cell protection effect in melanocytes | DDDT - Dove Medical Press

Newswise Robert Gallo, MD, The Homer & Martha Gudelsky Distinguished Professor in Medicine, co-founder and director of the Institute Human Virology at the University of Maryland School of Medicine and co-founder and Chair of the Scientific Leadership Board of the Global Virus Network, was awarded the Distinguished Alumni Award by the University of Chicago Medical & Biological Sciences Alumni Association (UChicago MBSAA) for his lifetime achievements. Honorees will participate in a panel discussion on May 10 and will be presented the award on May 21 at the Hyde Parke campus.

Being at the University of Chicago was a great inspiration for me by being so surrounded by excellence and by mentors who delighted in helping the beginning physician-scientist, said Robert C. Gallo, MD, 65.I will never forget those days which deeply impacted my entire career. Obviously, I am honored and grateful to receive this recognition.

The University of Chicago uniquely nurtured scientific discovery in Dr. Gallo's training in the practice of medicine, said I. David Goldman, MD, 62, Susan Fischer Chair, Albert Einstein College of Medicine and Director Emeritus, Albert Einstein Cancer Center.This was followed by the National Cancer Institute's recognition of the extraordinary talent and passion of a young physician-scientist providing Dr. Gallo with the freedom and resources to go on to make seminal discoveries on the biology of retroviruses and human T-cells that culminated in unraveling the causation of human T-cell leukemia and AIDS.Dr. Goldman is a former resident of the University of Chicago.

The UChicago MBSAA takes great pride in recognizing our alumni who, through their work, have made significant contributions to the biological sciences and medicine, said Mark R. Aschliman, MD'80, Chair, Alumni Awards Committee.

Dean E. Albert Reece, MD, PhD, MBA, Executive Vice President for Medical Affairs, University of Maryland Baltimore, and the John Z. and Akiko K. Bowers Distinguished Professor at the University of Maryland School of Medicine, said, My sincerest congratulations to Dr. Robert Gallo for receiving this prestigious award from one of our most preeminent academic medical institutions. Dr. Gallo is a world-renowned scientist whose breakthrough discoveries and scholarly contributions have made major contributions to global health for more than four decades. He is a visionary investigator who has unlocked many important mysteries of human viruses and diseases. He embodies all of the attributes of what it means to be a great scientist. We have been fortunate to have him as one of our most distinguished members of the University of Maryland School of Medicine faculty for many years. He has led our Institute of Human Virology, which has been transformative in its work to eradicate chronic and deadly viral and immune disorders. He is most deserving of this honor from the University of Chicago Medical Alumni Association.

Dr. Gallo graduated from Thomas Jefferson University School of Medicine before completing his medical training at the University of Chicago. After 30 years at the National Cancer Institute at the National Institutes of Health in Bethesda, Maryland, he became the co-founder of the Institute of Human Virology and the founding director and The Homer & Martha Gudelsky Distinguished Professor of Medicine and Microbiology and Immunology at the University of Maryland School of Medicine. In 2011, Dr. Gallo became the Co-Founder and Chair of the Scientific Leadership Board to the Global Virus Network.

Dr. Gallos career interests have focused on studying the basic biology of human blood cells, their normal and abnormal growth, and the involvement of viruses in these abnormalities.

Dr. Gallo and his co-workers pioneered human retrovirology, discovering the first human retrovirus (HTLV-1) and, along with others, showing it was a cause of a particular form of human leukemia. A year later, he and his group discovered the second known human retrovirus (HTLV-2). Dr. Gallo and his colleagues independently discovered HIV and provided the first results to show it was the cause of AIDS. They also developed a lifesaving HIV blood test. In 1986, he and his co-workers discovered the first new human herpes in more than 25 years, Human Herpes Virus-6 (HHV-6). Previously in 1978, Gallo discovered a variant of gibbon ape leukemia virusHalls Island strainwhich causes T-cell leukemia.

Dr. Gallo and his co-workers discovered Interleukin-2 in 1976, thus setting the stage for all groups to culture human T-cells. Gallo and his co-workers spent years developing detailed biochemical and immunological characteristics of human cellular DNA polymerases alpha, beta, and gamma and reverse transcriptase (RT) from several retroviruses to use RT as a sensitive and specific surrogate marker for retroviruses.

In 1995 he and his colleagues discovered the first natural (endogenous) inhibitors of HIV, which led to the discovery of the HIV co-receptor, CCR5, and opened new approaches to treatment. Currently, Dr. Gallo and his team have been working on a HIV preventive vaccine candidate.

Dr. Gallo has received 35 honorary doctorates from universities around the world. He was the most cited scientist from 1980 to 1990 and was ranked third in the world for scientific impact from 1983 to 2002, publishing nearly 1,300 papers.

Dr. Gallo is a member of the National Academy of Sciences and the National Academy of Medicine and has received several international prizes, including the U.S. Albert Lasker Award twice.

About the Institute of Human Virology

Formed in 1996 as a partnership between the State of Maryland, the City of Baltimore, the University System of Maryland, and the University of Maryland Medical System, the IHV is an institute of the University of Maryland School of Medicine and is home to some of the most globally-recognized and world-renowned experts in all of virology. The IHV combines the disciplines of basic research, epidemiology, and clinical research in a concerted effort to speed the discovery of diagnostics and therapeutics for a wide variety of chronic and deadly viral and immune disorders - most notably, HIV the virus that causes AIDS. For more information, visit http://www.ihv.org and follow us on Twitter @IHVmaryland.

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UM School of Medicine Institute of Human Virologys Robert Gallo Receives Distinguished Alumni Award by the University of Chicago Medical Association -...

OXFORD, England--(BUSINESS WIRE)--Neuro-Bio Ltd, a biotechnology company developing a first-in-class treatment for neurodegenerative disease, finds its new drug candidate effectively treats the signs of neurodegeneration in a mouse model of Alzheimers disease.

Publishing their work in Alzheimers & Dementia: Translational Research & Clinical Interventions (TRCI), Neuro-Bio researchers, in collaboration with the drug discovery company Evotec SE, UCLA, and Kings College London studied the ability of their patented drug, NBP14, to combat neurodegeneration in an established mouse model of Alzheimers disease.

Intranasal treatment for 6 weeks resulted in a marked decrease of brain amyloid and, after 14 weeks, improved cognitive performance comparable to that of normal mice. The results underscore the effectiveness of Neuro-Bios drug candidate and represent a remarkable step forward towards the treatment of Alzheimers disease in humans.

Baroness Professor Susan Greenfield, Founder and CEO of Neuro-Bio, says: By using basic neuroscientific knowledge we have identified what we believe is an underlying mechanism driving Alzheimers disease in the brain, and have developed a molecule (NBP14) to combat it. Our recent efficacy study in mouse models further validates previous work describing an erstwhile unidentified process in neurodegeneration and offers very exciting prospects for treating the disease in humans. This research should help position the drug intercepting this process, NBP14, for human clinical trials and hopefully create an entirely new era of Alzheimers therapeutics.

Professor Paul L Herrling, former Global Head of Research of Novartis Pharma and Non-Executive Director at Neuro-Bio, says: The results consistently indicate that NBP14 might interfere with the neurotoxic process that leads to neuronal degeneration in Alzheimers. This work has very exciting implications for treating Alzheimers because it is based on a strong scientific theory that hasnt yet been applied to treatment of the disease.

The UK regulator, the Medicines & Healthcare Products Regulatory Agency, has accredited NBP14 with one of their first Innovation Passports as part of a new licensing pathway that aims to reduce the time to market for innovative medicines.

NBP14 works by intercepting the process that Neuro-Bio believe could be a primary driver of neurodegeneration, the action of a brain chemical named T14 [1, 2]. In the last twenty years since it was first identified [3], evidence has become increasingly compelling that T14 plays an important role in early cell growth and normal development. However, this action can become toxic if triggered inappropriately in maturity [4] and ultimately could lead to Alzheimers disease where brain levels of T14 are shown in the current paper to reflect degree of degeneration.

Inactivation of T14 could potentially serve as a treatment for Alzheimers by halting the early advance of cell damage occurring first in primarily vulnerable cells deep the brain. Initially identified by the neurologist Martin Rossor back in 1981 [5], these primarily vulnerable cells form a kind of central hub in the brain, extending up from the top of the spinal cord. A key feature is that they are the first to display a pathology early in neurodegeneration [6].

Neuro-Bio believes that detection and measurement of T14 could be developed as a blood test or skin biopsy to identify the occurrence of the degenerative process during the window of ten to twenty years that typically occurs before symptoms start. If NBP14 proves effective in human trials, it could become a routine, home-administered nasal spray to halt neurodegeneration before any symptoms appear.

No harmful side effects at the active dose in the disease model were observed with NBP14 during the efficacy study. Neuro-Bio plan to take the drug to clinical Phase I trials as soon as possible.

Dr Gregory Cole, Professor of Medicine and Neurology at UCLA and Associate Director of the UCLA Alzheimer's Center, says: I've specialized in Alzheimers disease research and worked for real treatments in UCLAs Alzheimer program since 1994. Along with my colleagues around the world, weve all witnessed hundreds of failed drug trials based on existing theories and we are ready for truly new approaches. NBP14 has distinct advantages over other drug candidates and I am happy to work with this team at Neuro-Bio and share in their success.

ENDS

Notes to Editors

About Neuro-Bio

Neuro-Bio is a privately-owned biotech out of Oxford University with a focus on developing a first-in-class effective treatment for neurodegenerative disease. The company has discovered a novel 14 amino acid bioactive peptide (T14) derived from the C terminus of acetylcholinesterase (AChE). T14 is neurotoxic in the mature brain and published data shows it to be a potential key driver of neurodegeneration.

Based on almost 40 years of research by Professor Baroness Greenfield at Oxford University, Neuro-Bio Ltd was incorporated in 2013, when seed funding enabled the first patent filing on chemical composition of matter for neuroprotection.

For more information, visit, http://www.neuro-bio.com

About NBP14

NBP14 is a cyclated peptide, ie a structurally modified form of T14 itself, bent into a circle such that it is inactive at the target (an allosteric site on the alpha-7 receptor). By occupying this target, it will displace its naturally occurring, potentially toxic counterpart from the crucial site of action, thereby protecting against further degeneration. Hence, as reported in the paper, subsequent downstream effects will be blocked such as the production of amyloid in the hippocampus.

References

[1] Garcia-Rates S, Morrill P, Tu H, Pottiez G, Badin AS, Tormo-Garcia C, et al. (I) Pharmacological profiling of a novel modulator of the alpha7 nicotinic receptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains. Neuropharmacology. 2016;105:487-99.[2] Brai E, Simon F, Cogoni A, Greenfield SA. Modulatory Effects of a Novel Cyclized Peptide in Reducing the Expression of Markers Linked to Alzheimer's Disease. Front Neurosci. 2018;12:362.[3] Greenfield S, Vaux DJ. Parkinson's disease, Alzheimer's disease and motor neurone disease: identifying a common mechanism. Neuroscience. 2002;113:485-92.[4] Day T, Greenfield SA. Bioactivity of a peptide derived from acetylcholinesterase in hippocampal organotypic cultures. Exp Brain Res. 2004;155:500-8.[5] Rossor MN. Parkinson's disease and Alzheimer's disease as disorders of the isodendritic core. Br Med J (Clin Res Ed). 1981;283:1588-90.[6] Theofilas P, Dunlop S, Heinsen H, Grinberg LT. Turning on the Light Within: Subcortical Nuclei of the Isodentritic Core and their Role in Alzheimer's Disease Pathogenesis. J Alzheimers Dis. 2015;46:17-34.

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Novel Drug From Neuro-Bio Effective in a Mouse Model of Alzheimer's Disease - Business Wire

Dublin, April 05, 2022 (GLOBE NEWSWIRE) -- The "Global Circulating Tumor Cells (CTC) Market (2021-2026) by Technology, Application, Product, Specimen, End-User, and Geography, Competitive Analysis and the Impact of Covid-19 with Ansoff Analysis" report has been added to ResearchAndMarkets.com's offering.

The Global Circulating Tumor Cells (CTC) Market is estimated to be USD 9.52 Bn in 2021 and is expected to reach USD 18.41 Bn by 2026, growing at a CAGR of 14.1%.

Key factors such as the growing incidence of cancer followed by the increasing potential of CTCs in diagnosis and treatment have been a prominent driver for the Global Circulating Tumor Cells (CTC) Market.

Similarly, the shifting preference towards minimally invasive diagnostic methods and higher awareness about cancer has led to preventive initiatives taken by individuals in demand for preventive medicines.

However, factors such as lack of awareness and technical difficulties in detection are likely to restrain the market growth. Moreover, stringent government regulations and reluctance to adopt novel CTC technologies are posing to cause significant challenges for the market growth.

Market Segmentation

Company Profiles

Some of the companies covered in this report are Aviva Biosciences, Advanced Cell Diagnostics, Biocept, LungLife AI, Creatv Micro Tech, Miltenyi Biotec, Menarini Silicon Biosystems, Precision for Medicine, Qiagen, etc.

Key Topics Covered:

1 Report Description

2 Research Methodology

3 Executive Summary3.1 Introduction3.2 Market Size and Segmentation3.3 Market Outlook

4 Market Influencers4.1 Drivers4.1.1 Growing Incidence of Cancer and Potential Of CTC In Diagnosis and Treatment4.1.2 Increasing Demand for Preventive Medicine and Companion Diagnostics4.1.3 Increasing Preference for Non-Invasive Methods Cancer Diagnosis4.2 Restraints4.2.1 Stringent Government Regulations4.2.2 Technical Difficulties in Detection4.2.3 High Variability Among Patient Samples and Assays in Immuno-Oncology Trials4.3 Opportunities4.3.1 Increasing R&D Activities in CTC Analysis and Detection Practices4.3.2 Advancements in Chip Technology4.3.3 Emergence of New Single-Cell Technologies4.4 Challenges4.4.1 Lack Of Awareness4.4.2 Reluctance For the Adoption of Novel CTC Technologies

5 Market Analysis5.1 Regulatory Scenario5.2 Porter's Five Forces Analysis5.3 Impact of COVID-195.4 Ansoff Matrix Analysis

6 Global Circulating Tumor Cells (CTC) Market, By Technology6.1 Introduction6.2 CTC Enrichment6.2.1 Immunocapture/Label-Based6.2.1.1 Positive Selection6.2.1.2 Negative Selection6.2.2 Size-Based Separation/Label-Free6.2.2.1 Membrane-Based Size Separation (Label-Free)6.2.2.2 Microfluidic-Based Size Separation (Label-Free)6.2.3 Density-Based Separation (Label-Free)6.2.4 Combined Methods (Label-Free)6.3 CTC Direct Detection6.3.1 Microscopy6.3.2 SERS6.3.3 Immunocytochemical Technology6.3.4 Molecular (RNA)-Based Technology6.3.5 Others6.4 CTC Analysis & Downstream Assays

7 Global Circulating Tumor Cells (CTC) Market, By Application7.1 Introduction7.2 Multiple Chromosome Abnormalities7.3 RNA Profiling7.4 Protein Expression7.5 Cellular Communication

8 Global Circulating Tumor Cells (CTC) Market, By Product8.1 Introduction8.2 Devices or Systems8.3 Kits & Reagents8.4 Blood Collection Tubes

9 Global Circulating Tumor Cells (CTC) Market, By Specimen9.1 Introduction9.2 Blood9.3 Bone Marrow9.4 Other Body Fluids

10 Global Circulating Tumor Cells (CTC) Market, By End User10.1 Introduction10.2 Research & Academic Institutes10.3 Hospitals/ Clinics10.4 Diagnostic Centers

11 Global Circulating Tumor Cells (CTC) Market, By Geography11.1 Introduction

12 Competitive Landscape12.1 Competitive Quadrant12.2 Market Share Analysis12.3 Strategic Initiatives

13 Company Profiles

For more information about this report visit https://www.researchandmarkets.com/r/9nffnt

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The Global Circulating Tumor Cells (CTC) Market Will Grow to USD 18.41 Billion by 2026, at a CAGR of 14.1% - GlobeNewswire