Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

Development of a Novel Anti-CD44 Variant 8 Monoclonal Antibody C₄₄Mab-94 against Gastric Carcinomas

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells. These results indicate that C44Mab-94 is useful for detecting CD44v8 in a variety of experimental methods and is expected to become usefully applied to GC diagnosis and therapy

Development of a Novel Anti−CD44 Monoclonal Antibody for Multiple Applications against Esophageal Squamous Cell Carcinomas

CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo−resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti−CD44 monoclonal antibodies (mAbs) by immunizing mice with a CD44 variant (CD44v3−10) ectodomain and screening using enzyme−linked immunosorbent assay. We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established clones (C44Mab−46; IgG1, kappa) reacted with CD44 standard isoform (CD44s)−overexpressed Chinese hamster ovary−K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The apparent KD of C44Mab−46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1 × 10−8 M, 4.9 × 10−8 M, and 4.1 × 10−8 M, respectively. C44Mab−46 detected CD44s of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore, C44Mab−46 strongly stained the formalin−fixed paraffin−embedded ESCC tissues in immunohistochemistry. Collectively, C44Mab−46 is very useful for detecting CD44 in various applications

EMab-300 Detects Mouse Epidermal Growth Factor Receptor-Expressing Cancer Cell Lines in Flow Cytometry

Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the KD of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10−8 M and 1.9 × 10−8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies

Predictors and impact of time to clinical stability in community-acquired pneumococcal pneumonia

SummaryBackgroundA clinical stability (CS) evaluation is thought to be important in community-acquired pneumonia (CAP) treatment, but evidence concerning the time to CS (TCS) remains lacking.MethodsAmong consecutive patients hospitalized with pneumococcal pneumonia, relationships between TCS and other clinical outcomes were examined, and predictors and a predictive TCS score were derived from patient characteristics on admission.ResultsA total of 144 patients were enrolled, including 46% and 27% with moderate and severe pneumonia, respectively, defined by the pneumonia severity index (PSI). The median TCS was 2 days, and was significantly correlated with the length of hospital stay (r = 0.595); a longer TCS was significantly associated with the more presence of poor clinical outcomes and ICU stays (adjusted odds ratios: 1.359 and 1.366, respectively). A multivariate Cox proportional hazard model revealed an absence of bilateral pneumonia (hazard rate (HR): 2.107) or bacteremia (HR: 2.520), and mild or moderate pneumonia (HR: 2.798 and 2.515, respectively, versus severe) as predictors of CS. A predictive score had moderate discriminating power for the prolonged TCS (area under the curve: 0.76), and provided similar predictive values for poor clinical outcomes and ICU stays. A score of 3 or more points indicated the prolonged TCS, with a sensitivity and specificity of 73.3% and 70.9%, respectively.ConclusionsBecause TCS has a significant relationship with other clinical outcomes of pneumococcal CAP, the prediction of TCS might lead to the prevention of complications or an earlier transition to oral therapy. Future studies are warranted to validate these results

Linagliptin inhibits lipopolysaccharide-induced inflammation in human U937 monocytes

Abstract Background Atherosclerosis and inflammation are more common in patients with diabetes than in patients without diabetes, and atherosclerosis progression contributes to inflammation. Therefore, anti-inflammatory therapy is important for the prognosis of patients with diabetes. Linagliptin is the only bile-excreted, anti-diabetic oral dipeptidyl peptidase-4 (DPP-4) inhibitor. Although the anti-inflammatory effects of DPP-4 inhibitors in vivo and in vitro have been reported, few in vitro studies have examined the effects of linagliptin using monocytes, which play a central role in arteriosclerosis-related inflammation. Herein, we assessed the anti-inflammatory effects of linagliptin in human U937 monocytes. Methods U937 cells at densities of 1 × 106 cells/mL were cultured in Roswell Park Memorial Institute medium supplied with 10% fetal bovine serum and treated with 100 nM phorbol myristate acetate for 48 h for differentiation into macrophages. The media were replaced, and the cells were pretreated with 1, 5, 10, 50, and 100 nM linagliptin for 1 h or were left untreated. The media were then replaced again, and the cells were treated with 1 μg/mL lipopolysaccharide (LPS) or 10 nM interleukin (IL)-1β only, in combination with 1, 5, 10, 50, and 100 nM linagliptin or were left untreated. The extracted media were used to measure IL-6 and tumor necrosis factor (TNF)-α levels using enzyme-linked immunosorbent assay kits. Results LPS alone significantly increased IL-6 and TNF-α production compared with the control treatment. The treatment of cells with linagliptin at all concentrations significantly inhibited the LPS-stimulated IL-6 and TNF-α production. Meanwhile, IL-1β alone significantly increased IL-6 production compared with the control treatment. No significant difference in IL-6 production was noted between the cells treated with IL-1β and simultaneous treatment with IL-1β and linagliptin. Conclusions Linagliptin inhibited LPS-induced inflammation in human monocytic U937 cells

Detection of Mycoplasma wenyonii and “Candidatus Mycoplasma haemobos” from Japanese Black breeding cows in Kyushu and Okinawa region, southern part of Japan

This study aimed to elucidate the epidemiological status of hemoplasma infection and investigate the interaction between Theileria orientalis and hemoplasmas in Japanese Black breeding cows raised in the Kyushu and Okinawa regions. Blood samples were collected from 400 cows from 80 different farms in eight prefectures (five samples per farm and 10 farms per prefecture). Mycoplasma wenyonii (Mw), “Candidatus Mycoplasma haemobos” (CMh), and T. orientalis were examined by polymerase chain reaction (PCR) assay using whole blood samples. PCR results showed that 91.5% (366/400) of cows were positive for bovine hemoplasma: 40.3% were infected with Mw only, 9.5% with CMh only, and 41.8% with both species. T. orientalis was detected in 36% (144/400) of cows. The infection rate of T. orientalis was higher in the grazing group (P<0.001) than in the housed group, while the rate of CMh infection was higher (P<0.05) in the housed group than in the grazing group, suggesting that not only the tick but also other arthropod vectors may contribute to hemoplasma transmission. Although the cows with hemoplasma dual infection showed higher (P<0.05) white blood cell counts compared with hemoplasma-negative cows, there was no difference in hematologic parameters related to the anemia between the hemoplasma-positive and -negative animals. This may indicate that Japanese Black cattle could have resistance to the anemia caused by infection with hemoplasma.Citation: Tatsukawa, F., Nohara, R., Taniguchi, T., Goto, A., Misawa, N., & Katamoto, H. (2021). Detection of Mycoplasma wenyonii and "Candidatus Mycoplasma haemobos" from Japanese Black breeding cows in Kyushu and Okinawa region, southern part of Japan. The Journal of veterinary medical science, 83(1), 9–16. https://doi.org/10.1292/jvms.20-050