Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a serious health-threatening complication of diabetes mellitus characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are a potential therapeutic tool for DCM and myocardial fibrosis via mechanisms such as the regulation of microRNA (miRNA) expression and inflammation. It remains unclear, however, whether hUC-MSC therapy has beneficial effects on cardiac function following different durations of diabetes and which mechanistic aspects of DCM are modulated by hUC-MSC administration at different stages of its development. This study aimed to investigate the therapeutic effects of intravenous administration of hUC-MSCs on DCM following different durations of hyperglycemia in an experimental male model of diabetes and to determine the effects on expression of candidate miRNAs, target mRNA and inflammatory mediators. METHODS: A male mouse model of diabetes was induced by multiple low-dose streptozotocin injections. The effects on severity of DCM of intravenous injections of hUC-MSCs and saline two weeks previously were compared at 10 and 18 weeks after diabetes induction. At both time-points, biochemical assays, echocardiography, histopathology, polymerase chain reaction (PCR), immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis and expression of fibrosis-related mRNA, miRNA and inflammatory mediators. RESULTS: Saline-treated diabetic male mice had impaired cardiac function and increased cardiac fibrosis after 10 and 18 weeks of diabetes. At both time-points, cardiac dysfunction and fibrosis were improved in hUC-MSC-treated mice. Pro-fibrotic indicators (α-SMA, collagen I, collagen III, Smad3, Smad4) were reduced and anti-fibrotic mediators (FGF-1, miRNA-133a) were increased in hearts of diabetic animals receiving hUC-MSCs compared to saline. Increased blood levels of pro-inflammatory cytokines (IL-6, TNF, IL-1ß) and increased cardiac expression of IL-6 were also observed in saline-treated mice and were reduced by hUC-MSCs at both time-points, but to a lesser degree at 18 weeks. CONCLUSION: Intravenous injection of hUC-MSCs ameliorated key functional and structural features of DCM in male mice with diabetes of shorter and longer duration. Mechanistically, these effects were associated with restoration of intra-myocardial expression of miRNA-133a and its target mRNA COL1AI as well as suppression of systemic and localized inflammatory mediators.

Amelioration of diabetic nephropathy in mice by a single intravenous injection of human mesenchymal stromal cells at early and later disease stages is associated with restoration of autophagy.

BACKGROUND AND AIMS: Mesenchymal stromal cells (MSCs) a potentially effective disease-modulating therapy for diabetic nephropathy (DN) but their clinical translation has been hampered by incomplete understanding of the optimal timing of administration and in vivo mechanisms of action. This study aimed to elucidate the reno-protective potency and associated mechanisms of single intravenous injections of human umbilical cord-derived MSCs (hUC-MSCs) following shorter and longer durations of diabetes. METHODS: A streptozotocin (STZ)-induced model of diabetes and DN was established in C57BL/6 mice. In groups of diabetic animals, human (h)UC-MSCs or vehicle were injected intravenously at 8 or 16 weeks after STZ along with vehicle-injected non-diabetic animals. Diabetes-related kidney abnormalities was analyzed 2 weeks later by urine and serum biochemical assays, histology, transmission electron microscopy and immunohistochemistry. Serum concentrations of pro-inflammatory and pro-fibrotic cytokines were quantified by ELISA. The expression of autophagy-related proteins within the renal cortices was investigated by immunoblotting. Bio-distribution of hUC-MSCs in kidney and other organs was evaluated in diabetic mice by injection of fluorescent-labelled cells. RESULTS: Compared to non-diabetic controls, diabetic mice had increases in urine albumin creatinine ratio (uACR), mesangial matrix deposition, podocyte foot process effacement, glomerular basement membrane thickening and interstitial fibrosis as well as reduced podocyte numbers at both 10 and 18 weeks after STZ. Early (8 weeks) hUC-MSC injection was associated with reduced uACR and improvements in multiple glomerular and renal interstitial abnormalities as well as reduced serum IL-6, TNF-α, and TGF-ß1 compared to vehicle-injected animals. Later (16 weeks) hUC-MSC injection also resulted in reduction of diabetes-associated renal abnormalities and serum TGF-ß1 but not of serum IL-6 and TNF-α. At both time-points, the kidneys of vehicle-injected diabetic mice had higher ratio of p-mTOR to mTOR, increased abundance of p62, lower abundance of ULK1 and Atg12, and reduced ratio of LC3B to LC3A compared to non-diabetic animals, consistent with diabetes-associated suppression of autophagy. These changes were largely reversed in the kidneys of hUC-MSC-injected mice. In contrast, neither early nor later hUC-MSC injection had effects on blood glucose and body weight of diabetic animals. Small numbers of CM-Dil-labeled hUC-MSCs remained detectable in kidneys, lungs and liver of diabetic mice at 14 days after intravenous injection. CONCLUSIONS: Single intravenous injections of hUC-MSCs ameliorated glomerular abnormalities and interstitial fibrosis in a mouse model of STZ-induced diabetes without affecting hyperglycemia, whether administered at relatively short or longer duration of diabetes. At both time-points, the reno-protective effects of hUC-MSCs were associated with reduced circulating TGF-ß1 and restoration of intra-renal autophagy.

Quantitative evaluation of trastuzumab deruxtecan pharmacokinetics and pharmacodynamics in mouse models of varying degrees of HER2 expression.

Trastuzumab deruxtecan (T-DXd; DS-8201; ENHERTU®) is a human epithelial growth factor receptor 2 (HER2)-directed antibody drug conjugate (ADC) with demonstrated antitumor activity against a range of tumor types. Aiming to understand the relationship between antigen expression and downstream efficacy outcomes, T-DXd was administered in tumor-bearing mice carrying NCI-N87, Capan-1, JIMT-1, and MDA-MB-468 xenografts, characterized by varying HER2 levels. Plasma pharmacokinetics (PK) of total antibody, T-DXd, and released DXd and tumor concentrations of released DXd were evaluated, in addition to monitoring γΗ2AX and pRAD50 pharmacodynamic (PD) response. A positive relationship was observed between released DXd concentrations in tumor and HER2 expression, with NCI-N87 xenografts characterized by the highest exposures compared to the remaining cell lines. γΗ2AX and pRAD50 demonstrated a sustained increase over several days occurring with a time delay relative to tumoral-released DXd concentrations. In vitro investigations of cell-based DXd disposition facilitated the characterization of DXd kinetics across tumor cells. These outputs were incorporated into a mechanistic mathematical model, utilized to describe PK/PD trends. The model captured plasma PK across dosing arms as well as tumor PK in NCI-N87, Capan-1, and MDA-MB-468 models; tumor concentrations in JIMT-1 xenografts required additional parameter adjustments reflective of complex receptor dynamics. γΗ2AX longitudinal trends were well characterized via a unified PD model implemented across xenografts demonstrating the robustness of measured PD trends. This work supports the application of a mechanistic model as a quantitative tool, reliably projecting tumor payload concentrations upon T-DXd administration, as the first step towards preclinical-to-clinical translation.

Social deprivation and diabetic kidney disease: A European view.

There is a large body of literature demonstrating a social gradient in health and increasing evidence of an association between social deprivation and diabetes complications. Diabetic kidney disease (DKD) increases mortality in people with diabetes. Socioeconomic deprivation is increasingly recognized as a modifier of risk factors for kidney disease but also an independent risk factor itself for kidney disease. This may not be truly appreciated by clinicians and warrants further attention and exploration. In this review we explore the literature to date from Europe on the relationship between social deprivation and DKD. The majority of the studies showed at least an association with microalbuminuria, an early marker of DKD, while many showed an association with overt nephropathy. This was seen across many countries in Europe using a variety of different measures of deprivation. We reviewed and considered the mechanisms by which deprivation may lead to DKD. Health related behaviors such as smoking and suboptimal control of risk factors such as hypertension, hyperglycemia and elevated body mass index (BMI) accounts for some but not all of the association. Poorer access to healthcare, health literacy, and stress are also discussed as potential mediators of the association. Addressing deprivation is difficult but starting points include targeted interventions for people living in deprived circumstances, equitable roll out of diabetes technology, and flexible outpatient clinic arrangements including virtual and community-based care.

Safety and Preliminary Efficacy of Mesenchymal Stromal Cell (ORBCEL-M) Therapy in Diabetic Kidney Disease: A Randomized Clinical Trial (NEPHSTROM).

SIGNIFICANCE STATEMENT: Mesenchymal stromal cells (MSCs) may offer a novel therapy for diabetic kidney disease (DKD), although clinical translation of this approach has been limited. The authors present findings from the first, lowest dose cohort of 16 adults with type 2 diabetes and progressive DKD participating in a randomized, placebo-controlled, dose-escalation phase 1b/2a trial of next-generation bone marrow-derived, anti-CD362 antibody-selected allogeneic MSCs (ORBCEL-M). A single intravenous (iv) infusion of 80×10 6 cells was safe and well-tolerated, with one quickly resolved infusion reaction in the placebo group and no subsequent treatment-related serious adverse events (SAEs). Compared with placebo, the median annual rate of decline in eGFR was significantly lower with ORBCEL-M, although mGFR did not differ. The results support further investigation of ORBCEL-M in this patient population in an appropriately sized phase 2b study. BACKGROUND: Systemic therapy with mesenchymal stromal cells may target maladaptive processes involved in diabetic kidney disease progression. However, clinical translation of this approach has been limited. METHODS: The Novel Stromal Cell Therapy for Diabetic Kidney Disease (NEPHSTROM) study, a randomized, placebo-controlled phase 1b/2a trial, assesses safety, tolerability, and preliminary efficacy of next-generation bone marrow-derived, anti-CD362-selected, allogeneic mesenchymal stromal cells (ORBCEL-M) in adults with type 2 diabetes and progressive diabetic kidney disease. This first, lowest dose cohort of 16 participants at three European sites was randomized (3:1) to receive intravenous infusion of ORBCEL-M (80×10 6 cells, n =12) or placebo ( n =4) and was followed for 18 months. RESULTS: At baseline, all participants were negative for anti-HLA antibodies and the measured GFR (mGFR) and estimated GFR were comparable between groups. The intervention was safe and well-tolerated. One placebo-treated participant had a quickly resolved infusion reaction (bronchospasm), with no subsequent treatment-related serious adverse events. Two ORBCEL-M recipients died during follow-up of causes deemed unrelated to the trial intervention; one recipient developed low-level anti-HLA antibodies. The median annual rate of kidney function decline after ORBCEL-M therapy compared with placebo did not differ by mGFR, but was significantly lower by eGFR estimated by the Chronic Kidney Disease Epidemiology Collaboration and Modification of Diet in Renal Disease equations. Immunologic profiling provided evidence of preservation of circulating regulatory T cells, lower natural killer T cells, and stabilization of inflammatory monocyte subsets in those receiving the cell therapy compared with placebo. CONCLUSIONS: Findings indicate safety and tolerability of intravenous ORBCEL-M cell therapy in the trial's lowest dose cohort. The rate of decline in eGFR (but not mGFR) over 18 months was significantly lower among those receiving cell therapy compared with placebo. Further studies will be needed to determine the therapy's effect on CKD progression. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrial.gov NCT02585622 .

N-Arylpyrazole NOD2 Agonists Promote Immune Checkpoint Inhibitor Therapy.

The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N-arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N-arylpyrazole NOD2 agonist is enantiomer-specific and effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo. Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.

Novel Arginase Inhibitor, AZD0011, Demonstrates Immune Cell Stimulation and Antitumor Efficacy with Diverse Combination Partners.

Antitumor immunity can be hampered by immunosuppressive mechanisms in the tumor microenvironment, including recruitment of arginase (ARG) expressing myeloid cells that deplete l-arginine essential for optimal T-cell and natural killer cell function. Hence, ARG inhibition can reverse immunosuppression enhancing antitumor immunity. We describe AZD0011, a novel peptidic boronic acid prodrug to deliver an orally available, highly potent, ARG inhibitor payload (AZD0011-PL). We demonstrate that AZD0011-PL is unable to permeate cells, suggesting that this compound will only inhibit extracellular ARG. In vivo, AZD0011 monotherapy leads to arginine increases, immune cell activation, and tumor growth inhibition in various syngeneic models. Antitumor responses increase when AZD0011 is combined with anti-PD-L1 treatment, correlating with increases in multiple tumor immune cell populations. We demonstrate a novel triple combination of AZD0011, anti-PD-L1, and anti-NKG2A, and combination benefits with type I IFN inducers, including polyI:C and radiotherapy. Our preclinical data demonstrate AZD0011's ability to reverse tumor immunosuppression and enhance immune stimulation and antitumor responses with diverse combination partners providing potential strategies to increase immuno-oncology therapies clinically.

Chronic Kidney Disease Is Characterized by Expansion of a Distinct Proinflammatory Intermediate Monocyte Subtype and by Increased Monocyte Adhesion to Endothelial Cells.

SIGNIFICANCE STATEMENT: CKD is accompanied by abnormal inflammation, which contributes to progressive loss of functional renal tissue and accelerated cardiovascular disease. Although studies have documented that dysregulation of monocyte maturation and function is associated with CKD and its complications, it is not well characterized. This study reveals that a distinctive human monocyte subtype with high propensity for releasing proinflammatory mediators and activating endothelial cells is increased in adults with CKD compared with adults with high cardiovascular risk and normal kidney function. It also demonstrates that human monocyte adhesion to endothelial layers and responses to specific inflammatory migration signals are enhanced in CKD. These findings offer insights into the mechanisms of CKD-associated intravascular and localized inflammation and may suggest potential targets for therapeutic interventions. BACKGROUND: Cardiovascular disease (CVD) in patients with CKD is associated with increased circulating intermediate monocytes (IMs). Dysregulation of monocyte maturation and function is associated with CKD and its complications, but it is incompletely characterized. METHODS: To explore monocyte repertoire abnormalities in CKD, we studied properties of monocyte subpopulations, including IM subpopulations distinguished by HLA-DR expression level, in individuals with or without CKD. Using flow cytometry, we profiled monocyte populations in blood samples from adults with CKD, healthy volunteers (HVs), and patient controls (PCs) with high CVD risk. Monocyte subpopulations were also derived from single-cell RNA-sequencing profiles of paired blood and biopsy samples from kidney transplant recipients. We quantified intracellular cytokine production, migration, and endothelial adhesion in ex vivo assays of PBMCs. RESULTS: Of four predefined blood monocyte subpopulations, only HLA-DR hi IMs were increased in individuals with CKD compared with HVs and PCs. In HVs and patients with CKD, LPS-stimulated HLA-DR hi IMs isolated from blood produced higher amounts of TNF and IL-1 ß than other monocyte populations. Single-cell analysis revealed four monocyte clusters common to blood and kidneys, including an HLA-DR hi IM-like cluster that was enriched in kidneys versus blood. Migration toward CCL5 and CX3CL1 and adhesion to primary endothelial cell layers were increased in monocyte subpopulations in individuals with CKD compared with HVs. Monocyte adhesion to endothelial cells was partly dependent on CX3CR1/CX3CL1 interaction. CONCLUSIONS: CKD is associated with an increased number of a distinctive proinflammatory IM subpopulation and abnormalities of monocyte migration and endothelial adhesion. Dysregulated monocyte maturation and function may represent targetable factors contributing to accelerated CVD in CKD.

Peptidoglycan NlpC/P60 peptidases in bacterial physiology and host interactions.

The bacterial cell wall is composed of a highly crosslinked matrix of glycopeptide polymers known as peptidoglycan that dictates bacterial cell morphology and protects against environmental stresses. Regulation of peptidoglycan turnover is therefore crucial for bacterial survival and growth and is mediated by key protein complexes and enzyme families. Here, we review the prevalence, structure, and activity of NlpC/P60 peptidases, a family of peptidoglycan hydrolases that are crucial for cell wall turnover and division as well as interactions with antibiotics and different hosts. Understanding the molecular functions of NlpC/P60 peptidases should provide important insight into bacterial physiology, their interactions with different kingdoms of life, and the development of new therapeutic approaches.

The synergistic effect of physicochemical in vitro microenvironment modulators in human bone marrow stem cell cultures.

Modern bioengineering utilises biomimetic cell culture approaches to control cell fate during in vitro expansion. In this spirit, herein we assessed the influence of bidirectional surface topography, substrate rigidity, collagen type I coating and macromolecular crowding (MMC) in human bone marrow stem cell cultures. In the absence of MMC, surface topography was a strong modulator of cell morphology. MMC significantly increased extracellular matrix deposition, albeit in a globular manner, independently of the surface topography, substrate rigidity and collagen type I coating. Collagen type I coating significantly increased cell metabolic activity and none of the assessed parameters affected cell viability. At day 14, in the absence of MMC, none of the assessed genes was affected by surface topography, substrate rigidity and collagen type I coating, whilst in the presence of MMC, in general, collagen type I α1 chain, tenascin C, osteonectin, bone sialoprotein, aggrecan, cartilage oligomeric protein and runt-related transcription factor were downregulated. Interestingly, in the presence of the MMC, the 1000 kPa grooved substrate without collagen type I coating upregulated aggrecan, cartilage oligomeric protein, scleraxis homolog A, tenomodulin and thrombospondin 4, indicative of tenogenic differentiation. This study further supports the notion for multifactorial bioengineering to control cell fate in culture.

Risk stratification of patients listed for heart transplantation while supported with extracorporeal membrane oxygenation.

OBJECTIVES: Extracorporeal membrane oxygenation (ECMO) is used to support patients in severe cardiogenic shock. In the absence of recovery, these patients may need to be listed for heart transplant (HT), which offers the best long-term prognosis. However, posttransplantation mortality is significantly elevated in patients who receive ECMO. The objective of the present study was to describe and risk-stratify different profiles of patients listed for HT supported by ECMO. METHODS: Patients listed for HT in the United Network for Organ Sharing database were analyzed. The primary outcome was 1-year survival and was assessed in patients bridged to transplant with ECMO (ECMOBTT) and patients who were previously supported on ECMO but had it removed before HT (ECMOREMOVED). RESULTS: Among 65,636 adult candidates listed for HT (between 2001 and 2017), 712 were supported on ECMO, 292 of whom (41%) underwent HT (ECMOBTT, n = 202; ECMOREMOVED, n = 90). Most of the patients with ECMOREMOVED were transplanted with a ventricular assist device. In ECMOBTT, recipient age (each 10-year increase), time on the waitlist (both defined as minor risk factors), need for dialysis, and need for mechanical ventilation (both defined as major risk factors) were independent predictors of mortality. ECMOREMOVED and ECMOBTT with no risk factors showed 1-year survival comparable to that in patients who were never supported on ECMO. Compared with patients who were never on ECMO, patients in ECMOBTT group with minor risk factors, 1 major risk factor, and 2 major risk factors had ~2-, ~5-, and >10-fold greater 1-year mortality, respectively (P < .05). CONCLUSIONS: The HT recipients in the ECMOREMOVED and ECMOBTT groups with no risk factors showed similar survival as the HT recipients who were never supported on ECMO. In the ECMOBTT group, posttransplantation mortality increased significantly with increasing risk factors.

Mesenchymal stromal cell therapy compared to SGLT2-inhibitors and usual care in treating diabetic kidney disease: A cost-effectiveness analysis.

BACKGROUND AND OBJECTIVES: To simulate the cost-effectiveness of Mesenchymal Stromal Cell (MSC) therapy compared to sodium/glucose co-transporter 2 inhibitors (SGLT2i) or usual care (UC) in treating patients with Diabetic Kidney Disease (DKD). DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: This Markov-chain Monte Carlo model adopted a societal perspective and simulated 10,000 patients with DKD eligible for MSC therapy alongside UC using a lifetime horizon. This cohort was compared with an SGLT2i alongside UC arm and a UC only arm. Model input data were extracted from the literature. A threshold of $47,000 per quality-adjusted life year and a discount rate of 3% were used. The primary outcome measure was incremental net monetary benefit (INMB). Sensitivity analysis was conducted to examine: parameter uncertainty; threshold effects regarding MSC effectiveness and cost; and INMB according to patient age (71 vs 40 years), sex, and jurisdiction (UK, Italy and Ireland). RESULTS: While MSC was more cost-effective than UC, both the UC and MSC arms were dominated by SLGT2i. Relative to SGLT2i, the INMB's for MSC and UC were -$4,158 and -$10,085 respectively indicating that SGLT2i, MSC and UC had a 64%, 34% and 1% probability of being cost-effective at the given threshold, respectively. This pattern was consistent across most scenarios; driven by the relatively low cost of SGLT2i and demonstrated class-effect in delaying kidney failure and all-cause mortality. When examining younger patients at baseline, SGLT2i was still the most cost-effective but MSC performed better against UC given the increased lifetime benefit from delaying progression to ESRD. CONCLUSIONS: The evidence base regarding the effectiveness of MSC therapy continues to evolve. The potential for these therapies to reverse kidney damage would see large improvements in their cost-effectiveness as would targeting such therapies at younger patients and/or those for whom SGLT2i is contra-indicated.

Microbial mechanisms to improve immune checkpoint blockade responsiveness.

The human microbiota acts as a diverse source of molecular cues that influence the development and homeostasis of the immune system. Beyond endogenous roles in the human holobiont, host-microbial interactions also alter outcomes for immune-related diseases and treatment regimens. Over the past decade, sequencing analyses of cancer patients have revealed correlations between microbiota composition and the efficacy of cancer immunotherapies such as checkpoint inhibitors. However, very little is known about the exact mechanisms that link specific microbiota with patient responses, limiting our ability to exploit these microbial agents for improved oncology care. Here, we summarize current progress towards a molecular understanding of host-microbial interactions in the context of checkpoint inhibitor immunotherapies. By highlighting the successes of a limited number of studies focused on identifying specific, causal molecules, we underscore how the exploration of specific microbial features such as proteins, enzymes, and metabolites may translate into precise and actionable therapies for personalized patient care in the clinic.

Temozolomide and Radiotherapy versus Radiotherapy Alone in Patients with Glioblastoma, IDH-wildtype: Post Hoc Analysis of the EORTC Randomized Phase III CATNON Trial.

PURPOSE: In a post hoc analysis of the CATNON trial (NCT00626990), we explored whether adding temozolomide to radiotherapy improves outcome in patients with IDH1/2 wildtype (wt) anaplastic astrocytomas with molecular features of glioblastoma [redesignated as glioblastoma, isocitrate dehydrogenase-wildtype (IDH-wt) in the 2021 World Health Organization (WHO) classification of central nervous system tumors]. PATIENTS AND METHODS: From the randomized phase III CATNON study examining the addition of adjuvant and concurrent temozolomide to radiotherapy in anaplastic astrocytomas, we selected a subgroup of IDH1/2wt and H3F3Awt tumors with presence of TERT promoter mutations and/or EGFR amplifications and/or combined gain of chromosome 7 and loss of chromosome 10. Molecular abnormalities including MGMT promoter methylation status were determined by next-generation sequencing, DNA methylation profiling, and SNaPshot analysis. RESULTS: Of the 751 patients entered in the CATNON study, 670 had fully molecularly characterized tumors. A total of 159 of these tumors met the WHO 2021 molecular criteria for glioblastoma, IDH-wt. Of these patients, 47 received radiotherapy only and 112 received a combination of radiotherapy and temozolomide. There was no added effect of temozolomide on either overall survival [HR, 1.19; 95% confidence interval (CI), 0.82-1.71] or progression-free survival (HR, 0.87; 95% CI, 0.61-1.24). MGMT promoter methylation was prognostic for overall survival, but was not predictive for outcome to temozolomide treatment either with respect to overall survival or progression-free survival. CONCLUSIONS: In this cohort of patients with glioblastoma, IDH-wt temozolomide treatment did not add benefit beyond that observed from radiotherapy, regardless of MGMT promoter status. These findings require a new well-powered prospective clinical study to explore the efficacy of temozolomide treatment in this patient population.

The utility of a genetic kidney disease clinic employing a broad range of genomic testing platforms: experience of the Irish Kidney Gene Project.

BACKGROUND AND AIMS: Genetic testing presents a unique opportunity for diagnosis and management of genetic kidney diseases (GKD). Here, we describe the clinical utility and valuable impact of a specialized GKD clinic, which uses a variety of genomic sequencing strategies. METHODS: In this prospective cohort study, we undertook genetic testing in adults with suspected GKD according to prespecified criteria. Over 7 years, patients were referred from tertiary centres across Ireland to an academic medical centre as part of the Irish Kidney Gene Project. RESULTS: Among 677 patients, the mean age was of 37.2 ± 13 years, and 73.9% of the patients had family history of chronic kidney disease (CKD). We achieved a molecular diagnostic rate of 50.9%. Four genes accounted for more than 70% of identified pathogenic variants: PKD1 and PKD2 (n = 186, 53.4%), MUC1 (8.9%), and COL4A5 (8.3%). In 162 patients with a genetic diagnosis, excluding PKD1/PKD2, the a priori diagnosis was confirmed in 58% and in 13% the diagnosis was reclassified. A genetic diagnosis was established in 22 (29.7%) patients with CKD of uncertain aetiology. Based on genetic testing, a diagnostic kidney biopsy was unnecessary in 13 (8%) patients. Presence of family history of CKD and the underlying a priori diagnosis were independent predictors (P < 0.001) of a positive genetic diagnosis. CONCLUSIONS: A dedicated GKD clinic is a valuable resource, and its implementation of various genomic strategies has resulted in a direct, demonstrable clinical and therapeutic benefits to affected patients.

Comparison of Single and Repeated Dosing of Anti-Inflammatory Human Umbilical Cord Mesenchymal Stromal Cells in a Mouse Model of Polymicrobial Sepsis.

Mesenchymal stromal cells (MSCs) ameliorate pre-clinical sepsis and sepsis-associated acute kidney injury (SA-AKI) but clinical trials of single-dose MSCs have not indicated robust efficacy. This study investigated immunomodulatory effects of a novel MSC product (CD362-selected human umbilical cord-derived MSCs [hUC-MSCs]) in mouse endotoxemia and polymicrobial sepsis models. Initially, mice received intra-peritoneal (i.p.) lipopolysaccharide (LPS) followed by single i.p. doses of hUC-MSCs or vehicle. Next, mice underwent cecal ligation and puncture (CLP) followed by intravenous (i.v.) doses of hUC-MSCs at 4 h or 4 and 28 h. Analyses included serum/plasma assays of biochemical indices, inflammatory mediators and the AKI biomarker NGAL; multi-color flow cytometry of peritoneal macrophages (LPS) and intra-renal immune cell subpopulations (CLP) and histology/immunohistochemistry of kidney (CLP). At 72 h post-LPS injections, hUC-MSCs reduced serum inflammatory mediators and peritoneal macrophage M1/M2 ratio. Repeated, but not single, hUC-MSC doses administered at 48 h post-CLP resulted in lower serum concentrations of inflammatory mediators, lower plasma NGAL and reversal of sepsis-associated depletion of intra-renal T cell and myeloid cell subpopulations. Hierarchical clustering analysis of all 48-h serum/plasma analytes demonstrated partial co-clustering of repeated-dose hUC-MSC CLP animals with a Sham group but did not reveal a distinct signature of response to therapy. It was concluded that repeated doses of CD362-selected hUC-MSCs are required to modulate systemic and local immune/inflammatory events in polymicrobial sepsis and SA-AKI. Inter-individual variability and lack of effect of single dose MSC administration in the CLP model are consistent with observations to date from early-phase clinical trials.

Improving immunotherapy response through the use of designer bacteria.

Reprogramming the tumor microenvironment may be a key strategy to broaden the efficacy of current cancer immunotherapies. In a recent Nature paper, Canale et al. use synthetic biology to alter intratumoral arginine levels via engineered bacteria, leading to improved responsiveness to anti-PD-L1 checkpoint blockade in a murine model of cancer.

Enterococcus peptidoglycan remodeling promotes checkpoint inhibitor cancer immunotherapy.

The antitumor efficacy of cancer immunotherapy can correlate with the presence of certain bacterial species within the gut microbiome. However, many of the molecular mechanisms that influence host response to immunotherapy remain elusive. In this study, we show that members of the bacterial genus Enterococcus improve checkpoint inhibitor immunotherapy in mouse tumor models. Active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in nonprotective E. faecalis was sufficient to promote immunotherapy response, and its activity required the peptidoglycan sensor NOD2. Notably, SagA-engineered probiotics or synthetic muropeptides also augmented anti-PD-L1 antitumor efficacy. Taken together, our data suggest that microbiota species with specialized peptidoglycan remodeling activity and muropeptide-based therapeutics may enhance cancer immunotherapy and could be leveraged as next-generation adjuvants.