Antitumor activities against breast cancers by an afucosylated anti-HER2 monoclonal antibody H2 Mab-77-mG2a -f.

Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H2 Mab-77 (mouse IgG1 , kappa). This was then altered to produce H2 Mab-77-mG2a -f, an afucosylated mouse IgG2a . In the present work, we examined the reactivity of H2 Mab-77-mG2a -f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressing breast cancer cell line, was identified by H2 Mab-77-mG2a -f with a strong binding affinity (a dissociation constant [KD ]: 5.0 × 10-9 M). H2 Mab-77-mG2a -f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in Western blot analysis. Furthermore, H2 Mab-77-mG2a -f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2 Mab-77-mG2a -f. Additionally, in the BT-474-bearing tumor xenograft model, H2 Mab-77-mG2a -f substantially suppressed tumor development when compared with the control mouse IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H2 Mab-77-mG2a -f. These findings point to the possibility of H2 Mab-77-mG2a -f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.

Role of PPARα in inflammatory response of C2C12 myotubes.

Recent studies have shown a role of inflammation in muscle atrophy and sarcopenia. However, no anti-inflammatory pharmacotherapy has been established for the treatment of sarcopenia. Here, we investigate the potential role of PPARα and its ligands on inflammatory response and PGC-1α gene expression in LPS-treated C2C12 myotubes. Knockdown of PPARα, whose expression was upregulated upon differentiation, augmented IL-6 or TNFα gene expression. Conversely, PPARα overexpression or its activation by ligands suppressed 2-h LPS-induced cytokine expression, with pemafibrate attenuating NF-κB or STAT3 phosphorylation. Of note, reduction of PGC-1α gene expression by LPS treatment for 24 hours was partially reversed by fenofibrate. Our data demonstrate a critical inhibitory role of PPARα in inflammatory response of C2C12 myotubes and suggest a future possibility of PPARα ligands as a candidate for anti-inflammatory therapy against sarcopenia.

Assessment of the 2021 AASLD Practice Guidance for Albumin Infusion in Elective Therapeutic Paracentesis: A Regression Discontinuity Design.

INTRODUCTION: The 2021 American Association for the Study of Liver Disease (AASLD) Practice Guidance recommends albumin infusion when removing ≥5 L of ascites to prevent post-paracentesis circulatory dysfunction. However, the optimal criteria and scenarios for initiating albumin infusion subsequent to therapeutic paracentesis (TP) have been subject to limited scientific inquiry. METHODS: We conducted a retrospective cohort study at a US academic healthcare center. Participants received elective, outpatient TP between July 2019 and December 2022. Patients with spontaneous bacterial peritonitis, post-TP clinical adjustments, and/or hospitalization were excluded. The institution strictly followed the AASLD Guidance. We used a sharp regression discontinuity (RD) design to estimate the effect of albumin infusion at the AASLD Guidance-recommended cutoff of 5 L on serum creatinine and sodium trajectory after TP. RESULTS: Over the study period, 1,457 elective TPs were performed on 235 unique patients. Albumin infusion at the threshold of 5 L of ascites removal reduced serum creatinine levels by 0.046 mg/dL/d (95% confidence interval 0.003-0.116, P = 0.037) and increased serum sodium levels by 0.35 mEq/L/d (95% confidence interval 0.15-0.55, P = 0.001) compared with those who did not receive albumin infusion. The RD plots indicated worsened serum creatine/sodium levels after draining 3 L of fluid, approaching levels similar to or worse than with albumin infusion at 5 L or more. DISCUSSION: Our RD models supported the 2021 AASLD Guidance with robust estimation of causal effect sizes at the cutoff level of 5 L. Nevertheless, the findings also highlight the need to further evaluate the efficacy of albumin infusion in patients who undergo elective TP and have 3-5 L of ascites removed.

Proceedings of the 28th Annual Congress of the International Liver Transplantation Society.

The 2023 Joint International Congress of the International Liver Transplantation Society (ILTS), the European Liver and Intestine Transplant Association (ELITA), and the Liver Intensive Care Group of Europe (LICAGE) held in Rotterdam, the Netherlands, marked a significant recovery milestone for the liver transplant community after COVID-19. With 1159 participants and a surge in abstract submissions, the event focused on "Liver Disorders and Transplantation: Innovations and Evolving Indications." This conference report provides a comprehensive overview of the key themes discussed during the event, encompassing Hepatology, Anesthesia and Critical Care, Acute Liver Failure, Infectious Disease, Immunosuppression, Pediatric Liver Transplantation, Living Donor Liver Transplantation, Transplant Oncology, Surgical Approaches, and Machine Perfusion. The congress provided a platform for extensive discussions on a wide range of topics, reflecting the continuous advancements and collaborative efforts within the liver transplant community.

Evolving trends and outcomes on the US liver transplant waitlist of alcohol-associated hepatitis patients with very high MELD scores.

Liver transplantation (LT) in patients with alcohol-associated hepatitis (AH) has rapidly increased following the coronavirus disease 2019 pandemic and the implementation of the Acuity Circle policy, raising questions of equity and utility. Waitlist mortality among high (≥37) Model for End-Stage Liver Disease LT candidates with AH and post-transplant survival were assessed with a semiparametric survival regression and a generalized linear mixed-effect model with LT centre- and listing date-level random intercepts. These models demonstrate a lower mortality for the candidates listed with AH (adjusted sub-hazard ratio .58_.72_.90 and odds ratio .44_.66_.99) when compared to other diagnoses (autoimmune hepatitis, metabolic dysfunction-associated fatty liver disease and primary biliary cholangitis). Post-LT survival was comparable. This study highlights the limitations of current tools in characterizing the risk of mortality, and thus need for the modifications in prioritizing LT candidates with AH. Policy revision may be needed to ensure equivalent access to LT regardless of diagnosis.

The landscape of inpatient admissions for alcoholic hepatitis in the era of early liver transplantation.

INTRODUCTION AND OBJECTIVES: Liver transplant (LT) is a recent option available in the United States (US) to treat those with severe, refractory alcoholic hepatitis (AH). We examined changes in clinical characteristics of patients admitted with AH and tracked hospital outcomes as practice changes involving LT have shifted. MATERIALS AND METHODS: Using the National Inpatient Sample, we performed a cross-sectional analysis of patients admitted with AH during the years 2016-2020 in the US. Differences in clinical characteristics over time were assessed. To compare outcomes between 2016-2017 (when LT was less common) and 2018-2020 (when LT was more common), we conducted linear and logistic regression. Propensity-score matching was used to compare outcomes between patients with and without LT. RESULTS: From 2016-2017 to 2018-2020, patients admitted with AH tended to have a higher frequency of infection (p = 0.006), hepatorenal syndrome (<0.001), and ascites (<0.001). Hospital costs and length of stay (LOS) were highest in transplant hospitals, and costs rose over time in both non-transplant (NT) teaching and non-teaching hospitals (p < 0.001). Mortality decreased in NT teaching hospitals [aOR 0.7 (95% CI: 0.6-0.8)] and slightly decreased in NT non-teaching hospitals [aOR 0.7 (95% CI: 0.5-1.0)]. In the propensity-matched cohort involving LT versus non-LT patients, there was a 10% absolute reduction in-hospital mortality, but this came at a higher cost (p < 0.001) and length of stay (p < 0.001). CONCLUSIONS: The severity of AH has been increasing over time, yet mortality has declined after adjusting for severity of disease. Patients who underwent LT survived; however, the healthcare burden of LT is substantial.

A Cancer-Specific Monoclonal Antibody against HER2 Exerts Antitumor Activities in Human Breast Cancer Xenograft Models.

Monoclonal antibody (mAb)-based and/or cell-based immunotherapies provide innovative approaches to cancer treatments. However, safety concerns over targeting normal cells expressing reactive antigens still exist. Therefore, the development of cancer-specific mAbs (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy is required to minimize the adverse effects. We previously screened anti-human epidermal growth factor receptor 2 (HER2) mAbs and successfully established a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (IgG1, kappa). In this study, we showed that H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells in flow cytometry. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, recognized both breast cancer and normal epithelial cells. We further compared the affinity, effector activation, and antitumor effect of H2Mab-250 with trastuzumab. The results showed that H2Mab-250 exerted a comparable antitumor effect with trastuzumab in the mouse xenograft models of BT-474 and SK-BR-3, although H2Mab-250 possessed a lower affinity and effector activation than trastuzumab in vitro. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

Development of a Novel Anti-CD44 Variant 4 Monoclonal Antibody C44Mab-108 for Immunohistochemistry.

CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in the malignant progression of cancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and the establishment of the therapy. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterize them. One of the established clones (C44Mab-108; IgG1, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The KD of C44Mab-108 for CHO/CD44 v3-10 was 3.4 × 10-7 M. In western blot analysis, C44Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C44Mab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C44Mab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.

A Novel Anti-CD44 Variant 9 Monoclonal Antibody C44Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers.

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C44Mab-1 (IgG1, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C44Mab-1 recognizes CD44v9. C44Mab-1 could recognize CHO/CD44v3-10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (KD) of C44Mab-1 for CHO/CD44v3-10, COLO201, and COLO205 was 2.5 × 10-8 M, 3.3 × 10-8 M, and 6.5 × 10-8 M, respectively. Furthermore, C44Mab-1 was able to detect the CD44v3-10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C44Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

Variation in adult living donor liver transplantation in the United States: Identifying opportunities for increased utilization.

In the United States, living donor liver transplantation (LDLT) is limited to transplant centers with specific experience. However, the impact of recipient characteristics on procedure selection (LDLT vs. deceased donor liver transplant [DDLT]) within these centers has not been described. Transplant registry data for centers that performed ≥1 LDLT in 2002-2019 were analyzed using hierarchal regression modeling to quantify the impact of patient and center factors on the adjusted odds ratio (aOR) of LDLT (vs DDLT). Among 73,681 adult recipients, only 4% underwent LDLT, varying from <1% to >60% of total liver transplants. After risk adjustment, the likelihood of receiving an LDLT rose by 73% in recent years (aOR 1.73 for 2014-2019 vs. 2002-2007) but remained lower for older adults, men, racial and ethnic minorities, and obese patients. LDLT was less commonly used in patients with hepatocellular carcinoma or alcoholic cirrhosis, and more frequently in those with hepatitis C and with lower severity of illness (Model for End-Stage Liver Disease (MELD) score < 15). Patients with public insurance, lower educational achievement, and residence in the Northwest and Southeast had decreased access. While some differences in access to LDLT reflect clinical factors, further exploration into disparities in LDLT utilization based on center practice and socioeconomic determinants of health is needed.

Design and Synthesis of Poly(2,2'-Bipyridyl) Ligands for Induction of Cell Death in Cancer Cells: Control of Anticancer Activity by Complexation/Decomplexation with Biorelevant Metal Cations.

Chelation therapy is a medical procedure for removing toxic metals from human organs and tissues and for the treatment of diseases by using metal-chelating agents. For example, iron chelation therapy is designed not only for the treatment of metal poisoning but also for some diseases that are induced by iron overload, cancer chemotherapy, and related diseases. However, the use of such metal chelators needs to be generally carried out very carefully, because of the side effects possibly due to the non-specific complexation with intracellular metal cations. Herein, we report on the preparation and characterization of some new poly(bpy) ligands (bpy: 2,2'-bipyridyl) that contain one-three bpy ligand moieties and their anticancer activity against Jurkat, MOLT-4, U937, HeLa S3, and A549 cell lines. The results of MTT assays revealed that the tris(bpy) and bis(bpy) ligands exhibit potent activity for inducing the cell death in cancer cells. Mechanistic studies suggest that the main pathway responsible for the cell death by these poly(bpy) ligands is apoptotic cell death. It was also found that the anticancer activity of the poly(bpy) ligands could be controlled by the complexation (anticancer activity is turned OFF) and decomplexation (anticancer activity is turned ON) with biorelevant metal cations. In this paper, these results will be described.

A nationwide study of patients hospitalized with indeterminate colitis: a comparison with Crohn's disease and ulcerative colitis.

PURPOSE: Indeterminate colitis (IC) is subtype of colonic inflammatory bowel disease (IBD) that has features of both Crohn's disease (CD) and ulcerative colitis (UC). There have also been no studies to date examining patients hospitalized with IC in the United States (US). METHODS: We examined the demographic and clinical characteristics of patients admitted with IC and compared them with patients admitted with CD and UC. We also analyzed trends in cost and length of stay (LOS). RESULTS: Patients admitted with IC tended to be female (58%), Caucasian (72%), and younger [39 (SD: 23) years]. Patients with IC underwent lower endoscopy at higher rates (26%; CD: p < 0.001, UC: p = 0.08) but bowel surgery at lower rates compared to those with CD (11% vs. 16%; p = 0.04). Patients with IC were found to have a higher rate of bowel obstruction (4% vs. 0.7%, p = 0.004) than those with UC, but lower rates of abscess and obstruction compared to patients with CD (p < 0.001). When the analysis was confined to patients who underwent bowel surgery, IC patients still demonstrated higher rates of lower endoscopy (p = 0.03) but lower rates of abscess compared to CD patients (p = 0.049). Costs increased significantly over time for CD- and UC-related hospitalizations, but not for admissions related to IC. CONCLUSION: This is the first nationwide US study illustrating the demographics and clinical characteristics of patients hospitalized with IC. We conclude that IC has notable differences in hospitalization characteristics compared to the main two IBD subtypes.

Sociodemographic and Clinical Factors Associated with Nonattendance at the Hepatology Clinic.

BACKGROUND: Absenteeism from clinic appointments reduces efficiency, wastes resources, and contributes to longer wait times. There are limited data regarding factors associated with nonattendance in hepatology clinics. Identifying factors related to appointment nonattendance may help in the design of interventions for reducing absenteeism. METHODS: We aim to identify sociodemographic, clinical, and appointment-related factors associated with absenteeism following referral to a liver clinic in a tertiary academic center located in the US Midwest. We designed a case-control study using data from electronic medical records of patients scheduled for appointments between January 2016 and December 2021. Cases were defined as patients who canceled appointments on the same day or resulting in no-shows, and controls were those who completed the referral visit. Information about patients' sociodemographic characteristics, appointment details, and etiology of liver disease were recorded. Hierarchical logistic regression was used to analyze factors related to nonattendance. RESULTS: Of 3404 scheduled appointments, 460 (13.5%) missed visits were recorded. In the multivariable logistic regression models, hepatitis C and alcohol-associated liver disease were associated with greater odds of nonattendance [odds ratio (OR) 4.0 (95% CI 3.2-4.9), OR 2.7 (1.7-4.2), respectively] compared to those with other liver disease. Sociodemographic characteristics associated with risk of nonattendance included being Black [OR 2.6, (1.8-3.7)], Medicaid insurance or no insurance [OR 2.3 (1.7-2.9), OR 2.5 (1.6-3.7), respectively], non-English speaking [OR 1.8 (1.1-3.1)], being unmarried [OR 1.8 (1.4-2.2)], and longer wait time (> 30 days) until appointments [OR 1.8 (1.5-2.2)]. CONCLUSION: Several sociodemographic and administrative characteristics, as well as hepatitis C and alcohol-associated liver disease were associated with appointment nonattendance. Targeted future interventions may help to decrease nonattendance.

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

A Cancer-Specific Monoclonal Antibody against Podocalyxin Exerted Antitumor Activities in Pancreatic Cancer Xenografts.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL is considered a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells, such as vascular and lymphatic endothelial cells. Therefore, cancer specificity or selectivity is required to reduce adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG1, kappa), by immunizing mice with a soluble PODXL ectodomain derived from a glioblastoma LN229 cell. PcMab-6 reacted with the PODXL-positive LN229 cells but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H) but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47, showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG2a-type (PcMab-6-mG2a) and a humanized IgG1-type (humPcMab-6) mAb and further produced the core fucose-deficient types (PcMab-6-mG2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.

A Novel Anti-CD44 Variant 3 Monoclonal Antibody C44Mab-6 Was Established for Multiple Applications.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3-10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3-10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3-10, COLO205, and HSC-3 was 1.5 × 10-9 M, 6.3 × 10-9 M, and 1.9 × 10-9 M, respectively. C44Mab-6 could detect the CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.

Case Series of Patients with Coronavirus Disease 2019 Pneumonia Treated with Hydroxychloroquine.

The efficacy of hydroxychloroquine (HCQ) therapy, a previous candidate drug for coronavirus disease 2019 (COVID-19), was denied in the global guideline. The risk of severe cardiac events associated with HCQ was inconsistent in previous reports. In the present case series, we show the tolerability of HCQ therapy in patients treated in our hospital, and discuss the advantages and disadvantages of HCQ therapy for patients with COVID-19. A representative case was a 66-year-old woman who had become infected with severe acute respiratory syndrome coronavirus 2 and was diagnosed as having COVID-19 pneumonia via polymerase chain reaction. She was refractory to treatment with levofloxacin, lopinavir, and ritonavir, while her condition improved after beginning HCQ therapy without severe side effects. We show the tolerability of HCQ therapy for 27 patients treated in our hospital. In total, 21 adverse events occurred in 20 (74%) patients, namely, diarrhea in 11 (41%) patients, and elevated levels of both aspartate aminotransferase and alanine transaminase in 10 (37%) patients. All seven grade ≥ 4 adverse events were associated with the deterioration in COVID-19 status. No patients discontinued HCQ treatment because of HCQ-related adverse events. Two patients (7%) died of COVID-19 pneumonia. In conclusion, HCQ therapy that had been performed for COVID-19 was well-tolerated in our case series.

Protective effect of d-alanine against acute kidney injury.

Recent studies have revealed the connection between amino acid chirality and diseases. We have previously reported that the gut microbiota produces various d-amino acids in a murine acute kidney injury (AKI) model. Here, we further explored the pathophysiological role of d-alanine (d-Ala) in AKI. Levels of d-Ala were evaluated in a murine AKI model. We analyzed transcripts of the N-methyl-d-aspartate (NMDA) receptor, a receptor for d-Ala, in tubular epithelial cells (TECs). The therapeutic effect of d-Ala was then assessed in vivo and in vitro. Finally, the plasma level of d-Ala was evaluated in patients with AKI. The Grin genes encoding NMDA receptor subtypes were expressed in TECs. Hypoxic conditions change the gene expression of Grin1, Grin2A, and Grin2B. d-Ala protected TECs from hypoxia-related cell injury and induced proliferation after hypoxia. These protective effects are associated with the chirality of d-Ala. d-Ala inhibits reactive oxygen species (ROS) production and improves mitochondrial membrane potential, through NMDA receptor signaling. The ratio of d-Ala to l-Ala was increased in feces, plasma, and urine after the induction of ischemia-reperfusion (I/R). Moreover, Enterobacteriaceae, such as Escherichia coli and Klebsiella oxytoca, produce d-Ala. Oral administration of d-Ala ameliorated kidney injury after the induction of I/R in mice. Deficiency of NMDA subunit NR1 in tubular cells worsened kidney damage in AKI. In addition, the plasma level of d-Ala was increased and reflected the level of renal function in patients with AKI. In conclusion, d-Ala has protective effects on I/R-induced kidney injury. Moreover, the plasma level of d-Ala reflects the estimated glomerular filtration rate in patients with AKI. d-Ala could be a promising therapeutic target and potential biomarker for AKI.NEW & NOTEWORTHY d-Alanine has protective effects on I/R-induced kidney injury. d-Ala inhibits ROS production and improves mitochondrial membrane potential, resulting in reduced TEC necrosis by hypoxic stimulation. The administration of d-Ala protects the tubules from I/R injury in mice. Moreover, the plasma level of d-Ala is conversely associated with eGFR in patients with AKI. Our data suggest that d-Ala is an appealing therapeutic target and a potential biomarker for AKI.

A mouse model of weight gain after nicotine withdrawal.

Smoking cessation increases body weight. The underlying mechanisms, however, have not been fully understood. We here report an establishment of a mouse model that exhibits an augmented body weight gain after nicotine withdrawal. High fat diet-fed mice were infused with nicotine for two weeks, and then with vehicle for another two weeks using osmotic minipumps. Body weight increased immediately after nicotine cessation and was significantly higher than that of mice continued on nicotine. Mice switched to vehicle consumed more food than nicotine-continued mice during the first week of cessation, while oxygen consumption was comparable. Elevated expression of orexigenic agouti-related peptide was observed in the hypothalamic appetite center. Pair-feeding experiment revealed that the accelerated weight gain after nicotine withdrawal is explained by enhanced energy intake. As a showcase of an efficacy of pharmacologic intervention, exendin-4 was administered and showed a potent suppression of energy intake and weight gain in mice withdrawn from nicotine. Our current model provides a unique platform for the investigation of the changes of energy regulation after smoking cessation.

The structure of POMGNT2 provides new insights into the mechanism to determine the functional O-mannosylation site on α-dystroglycan.

Defects in the O-mannosyl glycan of α-dystroglycan (α-DG) are associated with α-dystroglycanopathy, a group of congenital muscular dystrophies. While α-DG has many O-mannosylation sites, only the specific positions can be modified with the functional O-mannosyl glycan, namely, core M3-type glycan. POMGNT2 is a glycosyltransferase which adds ß1,4-linked GlcNAc to the O-mannose (Man) residue to acquire core M3-type glycan. Although it is assumed that POMGNT2 extends the specific O-Man residues around particular amino acid sequences, the details are not well understood. Here, we determined a series of crystal structures of POMGNT2 with and without the acceptor O-mannosyl peptides and identified the critical interactions between POMGNT2 and the acceptor peptide. POMGNT2 has an N-terminal catalytic domain and a C-terminal fibronectin type III (FnIII) domain and forms a dimer. The acceptor peptide is sandwiched between the two protomers. The catalytic domain of one protomer recognizes the O-mannosylation site (TPT motif), and the FnIII domain of the other protomer recognizes the C-terminal region of the peptide. Structure-based mutational studies confirmed that amino acid residues of the catalytic domain interacting with mannose or the TPT motif are essential for POMGNT2 enzymatic activity. In addition, the FnIII domain is also essential for the activity and it interacts with the peptide mainly by hydrophobic interaction. Our study provides the first atomic-resolution insights into specific acceptor recognition by the FnIII domain of POMGNT2. The catalytic mechanism of POMGNT2 is proposed based on the structure.