Citations

Dent disease 1, an X‐linked inherited kidney disease caused by mutations in the CLCN5 gene, is characterized by low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and progressive renal failure. CLCN5 encodes the CLC‐5 protein found at the apical membrane in the kidney proximal tubule as well as endosome membranes of other tubule segments. Approximately 80 human mutations of the CLCN5 gene have been identified consisting of nonsense, missense, splice site, insertion and deletion mutations. However, the genotype and phenotype correlation are poor, and the mechanisms of CLC‐5 dysfunction that cause disease are still unclear. Moreover, there has been a lack of systematic study of CLC‐5 mutational effects on Cl − /H + exchanger activity. To address this need we used ion selective microelectrodes to monitor intracellular pH (pH i ) and intracellular Cl − concentration ([Cl i ]) of Xenopus oocytes expressing WT and mutant CLC‐5 while clamping at +40 mV. Common and novel mutations were identified in Dent patients by collaborators in the Rare Kidney Stone Consortium. The apparent H + :Cl − coupling ratio through the CLC‐5 protein was calculated using the Gibbs‐free energy equation. Cellular localization and protein trafficking of WT and mutant CLC‐5 in renal epithelial cells were also examined by immunofluorescence microscopy using eGFP/HA double‐tagged CLC‐5 and organelle‐specific markers and quantified by flow cytometry. In the presence of 104 mM Cl − (ND96), the pH i and [Cl − ] i of the CLC‐5 WT increased 340 ± 59 ×10 −4 pH unit/min and 2.8 ± 0.5 mM/min, respectively. T657S increased at a similar rate as WT, while there was a significant decrease in the rate of pH i and [Cl − ] i change for S244L, R345W, and Q629X compared to WT. Decreasing extracellular chloride to 5 mM decreased WT ClC‐5 pH i and [Cl − ] i by reversing transport: −7 ± 65 ×10 −4 pH unit/min and −0.2 ± 0.2 mM/min, respectively. Using these measurements, the stoichiometry of WT CLC‐5 was calculated to be 1.80 ± 0.02 (nCl − :H + ) in ND96, similar to the stoichiometry estimated for T657S. However, the stoichiometry was significantly altered for S244L, R345W, and Q629X. WT ClC‐5 acts as Cl − /H + exchanger at +40mV with 104 mM Cl − in the extracellular solution. Lowering extracellular Cl − reverses the transport direction, and the ClC‐5 exchanger functions at a significantly slower rate. Although S244L has normal surface expression, S244L and R345W mutations have significantly decreased pH and Cl − exchange rates in response to solution manipulations, suggesting these mutations have an altered ion exchange coupling ratio (stoichiometry). Interestingly, while R345W was retained in the ER suggesting defective protein maturation, R345W surface localization was rescued by lowering the incubation temperature from 37 to 30°C. Q629X was retained in the ER, not localized in early endosomes, and also has abolished Cl − /H + exchange activity, suggesting that Q629X affects protein maturation and trafficking while T657S is a benign, non‐disease causing mutation. These data suggest studying CLC‐5 mutational effects on both transport function and cellular localization is essential to better understand divergent phenotypes in Dent disease. Support or Funding Information NIH DK101405, U54KD083908, U54DK100227, Mayo O'Brien Urology Research Center, Mayo Clinic Rare Kidney Stone Consortium

Pathogenesis of Irritable Bowel Syndrome (IBS) is multifactorial, and gut microbes are sought to play a role in development and progression of disease. In this study we examined how constipation‐predominant Irritable Bowel Syndrome (IBS‐C) microbiota affect host intestinal transit and signaling pathways. To determine IBS‐C dysbiosis effects on the host, we introduced fecal microbiota from constipated mice to germ‐free recipients. Recipient mice of IBS‐C microbiota developed delayed GI transit time when compared to controls. Bacterial DNA analysis by HiSeq sequencing showed significant increase in Bacteroides uniformis , Bacteroides ovatus and Parabacteroides distasonis species from Bacteroidetes phylum in constipated mice. Fecal microbiota metabolic potential was measured with tetrazolium dye assay and showed significant differences between recipients of control and IBS‐C microbiota. Microbial metabolites such as short chain fatty acids (SCFA) are known to alter intestinal transit. SCFA levels in cecal contents of recipeints were measured by GC‐MS, and showed lower butyrate, propionate and acetate concentration in IBS‐C mice when compared to controls. To determine contractile properties of intestine after introduction of control and IBS‐C microbiota, we stimulated colonic rings with carbachol and KCl, and observed hypercontractility in IBS‐C microbiota recipients. To determine mechanism for delayed GI transit we analyzed colon tissues in fecal microbiota recipients by qRT‐PCR, and determined dysregulated neuromediator pathways. Overall, these results suggest that IBS‐C dysbiosis contributes to development of delayed transit time through microbial metabolite production and changes in colonic contractile properties. Support or Funding Information R01 DK097268 and T32 DK007074

Abstract Aims In long QT syndrome (LQTS), primary prevention improves outcome; thus, early identification is key. The most common LQTS phenotype is a foetal heart rate (FHR) < 3rd percentile for gestational age (GA) but the effects of cohort, genotype, variant, and maternal β-blocker therapy on FHR are unknown. We assessed the influence of these factors on FHR in pregnancies with familial LQTS and developed a FHR/GA threshold for LQTS. Methods and results In an international cohort of pregnancies in which one parent had LQTS, LQTS genotype, familial variant, and maternal β-blocker effects on FHR were assessed. We developed a testing algorithm for LQTS using FHR and GA as continuous predictors. Data included 1966 FHRs at 7–42 weeks’ GA from 267 pregnancies/164 LQTS families [220 LQTS type 1 (LQT1), 35 LQTS type 2 (LQT2), and 12 LQTS type 3 (LQT3)]. The FHRs were significantly lower in LQT1 and LQT2 but not LQT3 or LQTS negative. The LQT1 variants with non-nonsense and severe function loss (current density or β-adrenergic response) had lower FHR. Maternal β-blockers potentiated bradycardia in LQT1 and LQT2 but did not affect FHR in LQTS negative. A FHR/GA threshold predicted LQT1 and LQT2 with 74.9% accuracy, 71% sensitivity, and 81% specificity. Conclusion Genotype, LQT1 variant, and maternal β-blocker therapy affect FHR. A predictive threshold of FHR/GA significantly improves the accuracy, sensitivity, and specificity for LQT1 and LQT2, above the infant’s a priori 50% probability. We speculate this model may be useful in screening for LQTS in perinatal subjects without a known LQTS family history.

INTRODUCTION: There is a paucity of data on the real-world effectiveness of therapies in patients with Crohn's disease of the pouch. METHODS: This was a prospective multicenter study evaluating the primary outcome of remission at 12 months of therapy for Crohn's disease of the pouch. RESULTS: One hundred thirty-four patients were enrolled. Among the 77 patients with symptoms at baseline, 35 (46.7%) achieved remission at 12 months. Of them, 12 (34.3%) changed therapy. There was no significant association between therapy patterns and remission status. DISCUSSION: Approximately 50% with symptoms at enrollment achieved clinical remission at 12 months, most of whom did so without a change in therapy.

Abstract PURPOSE/OBJECTIVE We seek to establish the feasibility of using spine stereotactic radiosurgery (SSRS) allowing for spinal cord dose constraint relaxation as the primary management of metastatic epidural spinal cord compression (MESCC) in inoperable patients monitoring for radiation myelopathy (RM) and radiographic local control (LC). MATERIAL/METHODS Patients with radiation naïve MESCC were enrolled on this prospective Phase 1 trial. Single fraction SSRS was delivered to a dose of 18 or 24 Gy. Spinal cord constraint relaxation was performed in sequential cohorts from an initial allowable Dmax cohort of 10 Gy up to 16 Gy only if tumor progression occurred without RM. RESULTS Thirty two patients enrolled on the trial of which 4, 12, 8 and 8 were in the 10 Gy, 12 Gy, 14 Gy and 16 Gy cord Dmax cohorts, respectively. At baseline, there were 10 sites with MESCC Grade 1B, 10 sites with Grade 1C, 9 sites with Grade 2, 2 sites with Grade 1A, and 1 site with Grade 3 disease. Of the 28 evaluable patients, the median overall survival was 32.3 months (range 5.8-122.6 mo). The 1-year and 5-year LC was 84.5% and 79.5%. The 2-year LC did not statistically differ between the 14-16 Gy cord Dmax combined cohorts and the 10-12 Gy cord Dmax combined cohorts (92.9% vs. 62.9%, p = 0.15). With a median clinical follow-up of 22.7 months (range 3-122.3 mo), there were no cases of RM including in the 13 patients who survived at least 3 years. In the cohort receiving a cord Dmax of 16 Gy, there were no cases of RM with a median follow-up of 18.6 mo (range 9.0-97.1 mo). CONCLUSION With long-term follow-up, these data demonstrate that SSRS is a safe and effective tool in patients with MESCC. Cord constraint relaxation may be considered in inoperable patients with MESCC.

OBJECTIVE The literature on non–small cell lung cancer (NSCLC) brain metastases (BMs) managed using stereotactic radiosurgery (SRS) relies mainly on single-institution studies or randomized controlled trials (RCTs). There is a literature gap on clinical and radiological outcomes of SRS for NSCLC metastases in real-world practice. The objective of this study was to benchmark mortality and progression outcomes in patients undergoing SRS for NSCLC BMs and identify risk factors for these outcomes using a national quality registry. METHODS The SRS Registry of the NeuroPoint Alliance was used for this study. This registry included patients from 16 enrolling sites who underwent SRS from 2017 to 2022. Data are prospectively collected without a prespecified research purpose. The main outcomes of this analysis were overall survival (OS), out-of-field recurrence, local progression, and intracranial progression. All time-to-event investigations included Kaplan-Meier analyses and multivariable Cox regressions. RESULTS Two hundred sixty-four patients were identified, with a mean age of 66.7 years and a female proportion of 48.5%. Most patients (84.5%) had a Karnofsky Performance Status (KPS) score of 80–100, and the mean baseline EQ-5D score was 0.539 quality-adjusted life years. A single lesion was present in 53.4% of the patients, and 29.1% of patients had 3 or more lesions. The median OS was 28.1 months, and independent predictors of mortality included no control of primary tumor (hazard ratio [HR] 2.1), KPS of 80 (HR 2.4) or lower (HR 2.4), coronary artery disease (HR 2.8), and 5 or more lesions present at the time of SRS treatment (HR 2.3). The median out-of-field progression-free survival (PFS) was 24.8 months, and the median local PFS was unreached. Intralesional hemorrhage was an independent risk factor of local progression, with an HR of 6.0. The median intracranial PFS was 14.0 months and was predicted by the number of lesions at the time of SRS (3–4 lesions, HR 2.2; 5–14 lesions, HR 2.5). CONCLUSIONS In this real-world prospective study, the authors used a national quality registry and found favorable OS in patients with NSCLC BMs undergoing SRS compared with results from previously published RCTs. The intracranial PFS was mainly driven by the emergence of new lesions rather than local progression. A greater number of lesions at baseline was associated with out-of-field progression, while intralesional hemorrhage at baseline was associated with local progression.

Novel targeted therapies (small molecule inhibitors, antibody–drug conjugates, and CD19-directed therapies) have changed the treatment landscape of relapsed/refractory B-cell lymphomas. Bruton’s tyrosine kinase (BTK) inhibitors continue to evolve in the management of mantle cell lymphoma (MCL), in both the relapsed/refractory and the frontline setting. Anti-CD19 CAR T-cell therapies are now effective and approved treatment options for relapsed/refractory follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and MCL. Bispecific T-cell engagers represent a novel immunotherapeutic approach for relapsed FL and DLBCL after multiple lines of therapies, including prior CAR T-cell therapy. These NCCN Guideline Insights highlight the significant updates to the NCCN Guidelines for B-Cell Lymphomas for the treatment of FL, DLBCL, and MCL.

Abstract Background Myeloid cells comprise up to 50% of the total tumor mass in glioblastoma (GBM) and have been implicated in promoting tumor progression and immunosuppression. Modulating the response of myeloid cells to the tumor has emerged as a promising new approach for cancer treatment. In this regard, we focus on the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), which has recently emerged as a novel immune modulator in peripheral tumors. Methods We studied the TREM2 expression profile in various patient tumor samples and conducted single-cell transcriptomic analysis in both GBM patients and the GL261 mouse glioma model. We utilized multiple mouse glioma models and employed state-of-the-art techniques such as invivo 2-photon imaging, spectrum flow cytometry, and in vitro co-culture assays to study TREM2 function in myeloid cell-mediated phagocytosis of tumor cells, antigen presentation, and response of CD4+ T cells within the tumor hemispheres. Results Our research revealed significantly elevated levels of TREM2 expression in brain tumors compared to other types of tumors in patients. TREM2 was predominantly localized in tumor-associated myeloid cells and was highly expressed in nearly all microglia, as well as various subtypes of macrophages. Surprisingly, in preclinical glioma models, TREM2 deficiency did not confer a beneficial effect; instead, it accelerated glioma progression. Through detailed investigations, we determined that TREM2 deficiency impaired the ability of tumor-myeloid cells to phagocytose tumor cells and led to reduced expression of MHCII. This deficiency further significantly decreased the presence of CD4+ T cells within the tumor hemispheres. Conclusions Our study unveiled a previously unrecognized protective role of tumor-myeloid TREM2. Specifically, we found that TREM2 enhances the phagocytosis of tumor cells and promotes an immune response by facilitating MHCII-associated CD4+ T-cell responses against gliomas.