The genetic basis of early-onset hereditary ataxia in Iran: results of a national registry of a heterogeneous population.

BACKGROUND: To investigate the genetics of early-onset progressive cerebellar ataxia in Iran, we conducted a study at the Children's Medical Center (CMC), the primary referral center for pediatric disorders in the country, over a three-year period from 2019 to 2022. In this report, we provide the initial findings from the national registry. METHODS: We selected all early-onset patients with an autosomal recessive mode of inheritance to assess their phenotype, paraclinical tests, and genotypes. The clinical data encompassed clinical features, the Scale for the Assessment and Rating of Ataxia (SARA) scores, Magnetic Resonance Imaging (MRI) results, Electrodiagnostic exams (EDX), and biomarker features. Our genetic investigations included single-gene testing, Whole Exome Sequencing (WES), and Whole Genome Sequencing (WGS). RESULTS: Our study enrolled 162 patients from various geographic regions of our country. Among our subpopulations, we identified known and novel pathogenic variants in 42 genes in 97 families. The overall genetic diagnostic rate was 59.9%. Notably, we observed PLA2G6, ATM, SACS, and SCA variants in 19, 14, 12, and 10 families, respectively. Remarkably, more than 59% of the cases were attributed to pathogenic variants in these genes. CONCLUSIONS: Iran, being at the crossroad of the Middle East, exhibits a highly diverse genetic etiology for autosomal recessive hereditary ataxia. In light of this heterogeneity, the development of preventive strategies and targeted molecular therapeutics becomes crucial. A national guideline for the diagnosis and management of patients with these conditions could significantly aid in advancing healthcare approaches and improving patient outcomes.

Effect of ultrasound geometry on the production efficiency of damaged starch: Determining rheology parameters, and non-isothermal reaction kinetics.

Present study investigates the effects of probe size geometry on thermodynamic kinetics, rheology, and microstructure of wheat and tapioca starch. Ultrasound treatment using different probe diameters (20 mm and 100 mm) significantly influenced the gelatinization process. Results showed reduced enthalpy (ΔH) and Gibbs energy (ΔG), indicating enhanced gelatinization efficiency. According to the results, using a 20 mm and 100 mm probe leads to a reduction of 52.7 % and 68.6 % in reaction enthalpy for wheat starch compared to native starch, respectively. Microstructure analysis revealed structural changes, with ultrasound treatment leading to granular fractures and a sheet-like structure with air bubbles. The rheological behavior of the starches is found to exhibit shear thinning behavior, with the Casson model providing the best fit for the experimental data. Moreover, rheology modeling using Herschel-Bulkley and power law models showed increased viscosity and shear stress in larger probes. Numerical simulation data demonstrated that probe size influenced ultrasonic pressure, sound pressure level, and thermal power dissipation density, affecting fluid motion and velocity field components. Moreover, the maximum dissipated power decreases from 8.43 to 0.655 mW/m3 with an increase in probe diameter from 20 to 100 mm. The average yield shear stress values are calculated as 3.36 and 3.14 for wheat and tapioca starches, respectively. The larger probe diameter leads to greater entropy increases, with tapioca starch showing a 4.72 % increase and wheat starch a 4.97 % increase, compared to 2.56 % and 3.11 %, respectively, with the smaller probe. Additionally, the Keller-Miksis model provided insights into bubble dynamics, revealing increased pressure and temperature with higher pressure amplitudes.

Exploring the degradation of silver nanowire networks under thermal stress by coupling in situ X-ray diffraction and electrical resistance measurements.

The thermal instability of silver nanowires (AgNWs) leads to a significant increase of the electrical resistance of AgNW networks. A better understanding of the relationship between the structural and electrical properties of AgNW networks is primordial for their efficient integration as transparent electrodes (TEs) for next-generation flexible optoelectronics. Herein, we investigate the in situ evolution of the main crystallographic parameters (i.e. integrated intensity, interplanar spacing and peak broadening) of two Ag-specific Bragg peaks, (111) and (200), during a thermal ramp up to 400 °C through in situ X-ray diffraction (XRD) measurements, coupled with in situ electrical resistance measurements on the same AgNW network. First, we assign the (111) and (200) peaks of χ-scans to each five crystallites within AgNWs using a rotation matrix model. Then, we show that the thermal transition of bare AgNW networks occurs within a temperature range of about 25 °C for the electrical properties, while the structural transition spans over 200 °C. The effect of a protective tin oxide coating (SnO2) on AgNW networks is also investigated through this original in situ coupling approach. For SnO2-coated AgNW networks, the key XRD signatures from AgNWs remain constant, since the SnO2 coating prevents Ag atomic surface diffusion, and thus morphological instability (i.e. spheroidization). Moreover, the SnO2 coating does not affect the strain of both (111) and (200) planes. The thermal expansion for bare and SnO2-coated AgNW networks appears very similar to the thermal expansion of bulk Ag. Our findings provide insights into the underlying failure mechanisms of AgNW networks subjected to thermal stress, helping researchers to develop more robust and durable TEs based on metallic nanowire networks.

Inactivation of Foodborne Pathogens by Lactiplantibacillus Strains during Meat Fermentation: Kinetics and Mathematical Modelling.

This study examined the effect of beef fermentation with Lactiplantibacillus paraplantarum (L) PTCC 1965, Lactiplantibacillus (L) plantarum subsp. plantarum PTCC 1745, and Lactiplantibacillus (L) pentosus PTCC 1872 bacteria on the growth of pathogenic bacteria, including Salmonella (S) Typhi PTCC 1609 and Staphylococcus (S) aureus PTCC 1826. The growth of lactic acid bacteria (LAB) and the effect of fermentation on pathogenic bacteria were studied using Weibull: biphasic linear and competitive models. The results showed that the rate of pH reduction was lower in the early stages and increased as the microbial population grew. The α parameter was lower for L. plantarum subsp. plantarum compared to L. paraplantarum and L. pentosus. The comparison of the α parameter for bacterial growth and pH data showed that the time interval required to initiate the rapid growth phase of the bacteria was much shorter than that for the rapid pH reduction phase. The pH value had a 50% greater effect on the inactivation of S. Typhi when compared to the samples containing L. plantarum subsp. plantarum and L. pentosus. The same parameter was reported to be 72% for the inactivation of St. aureus. In general, during the fermentation process, LAB strains caused a decrease in pH, and as a result, reduced the growth of pathogens, which improves consumer health and increases the food safety of fermented meat.

Activin A-Expressing Polymorphonuclear Myeloid-Derived Suppressor Cells Infiltrate Skeletal and Cardiac Muscle and Promote Cancer Cachexia.

Cachexia is a major cause of death in cancer and leads to wasting of cardiac and skeletal muscle, as well as adipose tissue. Various cellular and soluble mediators have been postulated in driving cachexia; however, the specific mechanisms behind this muscle wasting remain poorly understood. In this study, we found polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) to be critical for the development of cancer-associated cachexia. Significant expansion of PMN-MDSCs was observed in the cardiac and skeletal muscles of cachectic murine models. Importantly, the depletion of this cell subset, using depleting anti-Ly6G Abs, attenuated this cachectic phenotype. To elucidate the mechanistic involvement of PMN-MDSCs in cachexia, we examined major mediators, that is, IL-6, TNF-α, and arginase 1. By employing a PMN-MDSC-specific Cre-recombinase mouse model, we showed that PMN-MDSCs were not maintained by IL-6 signaling. In addition, PMN-MDSC-mediated cardiac and skeletal muscle loss was not abrogated by deficiency in TNF-α or arginase 1. Alternatively, we found PMN-MDSCs to be critical producers of activin A in cachexia, which was noticeably elevated in cachectic murine serum. Moreover, inhibition of the activin A signaling pathway completely protected against cardiac and skeletal muscle loss. Collectively, we demonstrate that PMN-MDSCs are active producers of activin A, which in turn induces cachectic muscle loss. Targeting this immune/hormonal axis will allow the development of novel therapeutic interventions for patients afflicted with this debilitating syndrome.

Expanding the genetic spectrum of giant axonal neuropathy: Two novel variants in Iranian families.

BACKGROUND: Giant axonal neuropathy (GAN) is a progressive childhood hereditary polyneuropathy that affects both the peripheral and central nervous systems. Disease-causing variants in the gigaxonin gene (GAN) cause autosomal recessive giant axonal neuropathy. Facial weakness, nystagmus, scoliosis, kinky or curly hair, pyramidal and cerebellar signs, and sensory and motor axonal neuropathy are the main symptoms of this disorder. Here, we report two novel variants in the GAN gene from two unrelated Iranian families. METHODS: Clinical and imaging data of patients were recorded and evaluated, retrospectively. Whole-exome sequencing (WES) was undertaken in order to detect disease-causing variants in participants. Confirmation of a causative variant in all three patients and their parents was carried out using Sanger sequencing and segregation analysis. In addition, for comparing to our cases, we reviewed all relevant clinical data of previously published cases of GAN between the years 2013-2020. RESULTS: Three patients from two unrelated families were included. Using WES, we identified a novel nonsense variant [NM_022041.3:c.1162del (p.Leu388Ter)], in a 7-year-old boy of family 1, and a likely pathogenic missense variant [NM_022041.3:c.370T>A (p.Phe124Ile)], in two affected siblings of the family 2. Clinical examination revealed typical features of GAN-1 in all three patients, including walking difficulties, ataxic gait, kinky hair, sensory-motor polyneuropathy, and nonspecific neuroimaging abnormalities. Review of 63 previously reported cases of GAN indicated unique kinky hair, gait problem, hyporeflexia/areflexia, and sensory impairment were the most commonly reported clinical features. CONCLUSIONS: One homozygous nonsense variant and one homozygous missense variant in the GAN gene were discovered for the first time in two unrelated Iranian families that expand the mutation spectrum of GAN. Imaging findings are nonspecific, but the electrophysiological study in addition to history is helpful to achieve the diagnosis. The molecular test confirms the diagnosis.

Clinical and Molecular Findings of Autosomal Recessive Spastic Ataxia of Charlevoix Saguenay: an Iranian Case Series Expanding the Genetic and Neuroimaging Spectra.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is now increasingly identified from all countries over the world, possibly rendering it one of the most common autosomal recessive ataxias. Here, we selected patients harboring SACS variants, the causative gene for ARSACS, in a large cohort of 137 patients with early-onset ataxia recruited from May 2019 to May 2021 and were referred to the ataxia clinic. Genetic studies were performed for 111 out of 137 patients (81%) which led to a diagnostic rate of 72.9% (81 out of 111 cases). Ten patients with the molecular diagnosis of ARSACS were identified. We investigated the phenotypic and imaging spectra of all confirmed patients with ARSACS. We also estimated the frequency of ARSACS in this cohort and described their clinical and genetic findings including seven novel variants as well as novel neuroimaging findings. While the classic clinical triad of ARSACS is progressive cerebellar ataxia, spasticity, and sensorimotor polyneuropathy, it is not a constant feature in all patients. Sensorimotor axonal-demyelinating neuropathy was detected in all of our patients, but spasticity and extensor plantar reflex were absent in 50% (5/10). In all patients, brain magnetic resonance imaging (MRI) showed symmetric linear hypointensities in the pons (pontine stripes) and anterior superior cerebellar atrophy as well as a hyperintense rim around the thalami (thalamic rim). Although infratentorial arachnoid cyst has been reported in ARSACS earlier, we report anterior temporal arachnoid cyst in two patients for the first time, indicating that arachnoid cyst may be an associated imaging feature of ARSACS. We also extended molecular spectrum of ARSACS by presenting 8 pathogenic and one variant of unknown significance (VUS) sequence variants, which 7 of them have not been reported previously. MetaDome server confirmed that the identified VUS variant was in the intolerant regions of sacsin protein encoded by SACS.

Atmospheric atomic layer deposition of SnO2 thin films with tin(II) acetylacetonate and water.

Due to its unique optical, electrical, and chemical properties, tin dioxide (SnO2) thin films attract enormous attention as a potential material for gas sensors, catalysis, low-emissivity coatings for smart windows, transparent electrodes for low-cost solar cells, etc. However, the low-cost and high-throughput fabrication of SnO2 thin films without producing corrosive or toxic by-products remains challenging. One appealing deposition technique, particularly well-adapted to films presenting nanometric thickness is atomic layer deposition (ALD). In this work, several metalorganic tin-based complexes, namely, tin(IV) tert-butoxide, bis[bis(trimethylsilyl)amino] tin(II), dibutyltin diacetate, tin(II) acetylacetonate, tetrakis(dimethylamino) tin(IV), and dibutyltin bis(acetylacetonate), were explored thanks to DFT calculations. Our theoretical calculations suggest that the three last precursors are very appealing for ALD of SnO2 thin films. The potential use of these precursors for atmospheric-pressure spatial atomic layer deposition (AP-SALD) is also discussed. For the first time, we experimentally demonstrate the AP-SALD growth of SnO2 thin films using tin(II) acetylacetonate (Sn(acac)2) and water. We observe that Sn(acac)2 exhibits efficient ALD activity with a relatively large ALD temperature window (140-200 °C), resulting in a growth rate of 0.85 ± 0.03 Å per cyc. XPS analyses show a single Sn 3d5/2 characteristic peak for Sn4+ at 486.8 ± 0.3 eV, indicating that a pure SnO2 phase is obtained within the ALD temperature window. The as-deposited SnO2 thin films are in all cases amorphous, and film conductivity increases with the deposition temperature. Hall effect measurements confirm the n-type nature of SnO2 with a free electron density of about 8 × 1019 cm-3, electron mobility up to 11.2 cm2 V-1 s-1, and resistivity of 7 × 10-3 Ω cm for samples deposited at 270 °C.

Supplemental hydrogen sulfide in models of renal transplantation after cardiac death.

BACKGROUND: The increasing use of kidneys from donations after cardiac death (DCD) for renal transplantation is hindered by negative outcomes owing to organ injury after prolonged warm and cold ischemia-reperfusion. Recently, hydrogen sulfide (H2S) has shown cytoprotective effects against ischemia-reperfusion injury; however, its effectiveness in the context of DCD renal transplantation is unknown. METHODS: We tested a novel 30-day in vivo syngeneic murine model of DCD renal transplantation, in which the donor kidney was clamped for 30 minutes and stored for 18 hours in cold University of Wisconsin (UW) solution or UW with 150 µM sodium hydrogen sulfide (UW + NaHS) before transplantation. We also tested a 7-day in vivo porcine model of DCD renal autotransplantation, in which the left kidney was clamped for 60 minutes and preserved for 24 hours using hypothermic perfusion with UW or UW + 150 µM NaHS before autotransplantation. We collected blood and urine samples periodically, and collected kidney samples at the end point for histopathology and quantitative reverse transcription polymerase chain reaction. RESULTS: Rats that received H2S-treated kidneys showed significantly higher survival, faster recovery of graft function and significantly lower acute tubular necrosis than controls. Pig kidneys perfused with UW + NaHS showed significantly higher renal blood flow and lower renal resistance than control kidneys after 24 hours of perfusion. After autotransplantation, pigs that received H2S-treated kidneys showed significantly lower serum creatinine on days 1 and 7 after transplantation. Rat and pig kidneys treated with H2S also showed more protective gene expression profiles than controls. CONCLUSION: Our findings support the potential use of H2S-supplemented UW solution during cold storage as a novel and practical means to improve DCD graft survival and function.

PDMS hydrogel-coated tissue culture plates for studying the impact of substrate stiffness on dendritic cell function.

The mechanical properties of polydimethylsiloxane hydrogels can be tuned to mimic physiological tensions, an underappreciated environmental parameter in immunology studies. We describe a workflow to prepare PDMS-coated tissue culture plates with biologically relevant substrate stiffness, and the use of these hydrogel plates to condition isolated primary splenic CD11c+ dendritic cells (DC). Finally, we suggest downstream applications to study the impact of substrate stiffness on DC function and metabolism. The protocol could be adapted to study other mechanosensitive immune cell subsets. For complete details on the use and execution of this protocol, please refer to Chakraborty et al. (2021).

Sodium thiosulfate-supplemented UW solution protects renal grafts against prolonged cold ischemia-reperfusion injury in a murine model of syngeneic kidney transplantation.

INTRODUCTION: Cold ischemia-reperfusion injury (IRI) is an inevitable event that increases post-transplant complications. We have previously demonstrated that supplementation of University of Wisconsin (UW) solution with non-FDA-approved hydrogen sulfide (H2S) donor molecules minimizes cold IRI and improves renal graft function after transplantation. The present study investigates whether an FDA-approved H2S donor molecule, sodium thiosulfate (STS), will have the same or superior effect in a clinically relevant rat model of syngeneic orthotopic kidney transplantation. METHOD: Thirty Lewis rats underwent bilateral nephrectomy followed by syngeneic orthotopic transplantation of the left kidney after 24-hour preservation in either UW or UW+STS solution at 4 °C. Rats were monitored to post-transplant day 14 and sacrificed to assess renal function (urine output, serum creatinine and blood urea nitrogen). Kidney sections were stained with H&E, TUNEL, CD68, and myeloperoxidase (MPO) to detect acute tubular necrosis (ATN), apoptosis, macrophage infiltration, and neutrophil infiltration. RESULT: UW+STS grafts showed significantly improved graft function immediately after transplantation, with improved recipient survival compared to UW grafts (p < 0.05). Histopathological examination revealed significantly reduced ATN, apoptosis, macrophage and neutrophil infiltration and downregulation of pro-inflammatory and pro-apoptotic genes in UW+STS grafts compared to UW grafts (p < 0.05). CONCLUSION: We show for the first time that preservation of renal grafts in STS-supplemented UW solution protects against prolonged cold IRI by suppressing apoptotic and inflammatory pathways, and thereby improving graft function and prolonging recipient survival. This could represent a novel clinically applicable therapeutic strategy to minimize the detrimental clinical outcome of prolonged cold IRI in kidney transplantation.

Silver Nanowire Networks: Ways to Enhance Their Physical Properties and Stability.

Silver nanowire (AgNW) networks have been intensively investigated in recent years. Thanks to their attractive physical properties in terms of optical transparency and electrical conductivity, as well as their mechanical performance, AgNW networks are promising transparent electrodes (TE) for several devices, such as solar cells, transparent heaters, touch screens or light-emitting devices. However, morphological instabilities, low adhesion to the substrate, surface roughness and ageing issues may limit their broader use and need to be tackled for a successful performance and long working lifetime. The aim of the present work is to highlight efficient strategies to optimize the physical properties of AgNW networks. In order to situate our work in relation to existing literature, we briefly reported recent studies which investigated physical properties of AgNW networks. First, we investigated the optimization of optical transparency and electrical conductivity by comparing two types of AgNWs with different morphologies, including PVP layer and AgNW dimensions. In addition, their response to thermal treatment was deeply investigated. Then, zinc oxide (ZnO) and tin oxide (SnO2) protective films deposited by Atmospheric Pressure Spatial Atomic Layer Deposition (AP-SALD) were compared for one type of AgNW. We clearly demonstrated that coating AgNW networks with these thin oxide layers is an efficient approach to enhance the morphological stability of AgNWs when subjected to thermal stress. Finally, we discussed the main future challenges linked with AgNW networks optimization processes.

Subnormothermic Perfusion with H2S Donor AP39 Improves DCD Porcine Renal Graft Outcomes in an Ex Vivo Model of Kidney Preservation and Reperfusion.

Cold preservation is the standard of care for renal grafts. However, research on alternatives like perfusion at higher temperatures and supplementing preservation solutions with hydrogen sulfide (H2S) has gained momentum. In this study, we investigated whether adding H2S donor AP39 to porcine blood during subnormothermic perfusion at 21 °C improves renal graft outcomes. Porcine kidneys were nephrectomized after 30 min of clamping the renal pedicles and treated to 4 h of static cold storage (SCS) on ice or ex vivo subnormothermic perfusion at 21 °C with autologous blood alone (SNT) or with AP39 (SNTAP). All kidneys were reperfused ex vivo with autologous blood at 37 °C for 4 h. Urine output, histopathology and RNAseq were used to evaluate the renal graft function, injury and gene expression profiles, respectively. The SNTAP group exhibited significantly higher urine output than other groups during preservation and reperfusion, along with significantly lower apoptotic injury compared to the SCS group. The SNTAP group also exhibited differential pro-survival gene expression patterns compared to the SCS (downregulation of pro-apoptotic genes) and SNT (downregulation of hypoxia response genes) groups. Subnormothermic perfusion at 21 °C with H2S-supplemented blood improves renal graft outcomes. Further research is needed to facilitate the clinical translation of this approach.

Mechanical Stiffness Controls Dendritic Cell Metabolism and Function.

Stiffness in the tissue microenvironment changes in most diseases and immunological conditions, but its direct influence on the immune system is poorly understood. Here, we show that static tension impacts immune cell function, maturation, and metabolism. Bone-marrow-derived and/or splenic dendritic cells (DCs) grown in vitro at physiological resting stiffness have reduced proliferation, activation, and cytokine production compared with cells grown under higher stiffness, mimicking fibro-inflammatory disease. Consistently, DCs grown under higher stiffness show increased activation and flux of major glucose metabolic pathways. In DC models of autoimmune diabetes and tumor immunotherapy, tension primes DCs to elicit an adaptive immune response. Mechanistic workup identifies the Hippo-signaling molecule, TAZ, as well as Ca2+-related ion channels, including potentially PIEZO1, as important effectors impacting DC metabolism and function under tension. Tension also directs the phenotypes of monocyte-derived DCs in humans. Thus, mechanical stiffness is a critical environmental cue of DCs and innate immunity.

Patterns of Expression of H2S-Producing Enzyme in Human Renal Cell Carcinoma Specimens: Potential Avenue for Future Therapeutics.

BACKGROUND: Renal cell carcinoma (RCC) is the most common cancer of the kidney. The most common histotype is clear-cell (cc) RCC. Hydrogen sulfide (H2S) is an angiogenic and anti-apoptotic gasotransmitter that is elevated under pseudohypoxic conditions. H2S is endogenously produced by three enzymes: Cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST). Seeing as increased expression of these enzymes has been observed in other human cancer types, this study aimed to quantify H2S-producing enzyme expression in human RCC samples and evaluate whether it correlated with clinical outcomes. PATIENTS AND METHODS: Eighty-eight human kidney tissue specimens, with healthy and cancerous tissue components, were immunohistochemically stained for CSE, CBS, and MPST. The mean pixel intensity of positively stained areas was quantified. A retrospective analysis was conducted to obtain patient demographics, rates of metastasis/recurrence, and prognostic characteristics. Statistical correlations between enzyme expressions and subsequent patient outcomes were evaluated. RESULTS: There was significantly greater expression of CSE, CBS, and MPST in cc-RCC compared to paired healthy tissue (p<0.0001). The difference in expression of CSE in cancerous versus normal tissue was significantly greater than that for CBS and MPST (p<0.0001 and p<0.01, respectively). Enzyme expression patterns in cancerous versus normal tissue did not correlate with nuclear grade, stage, histological type or cancer recurrence/metastasis. CONCLUSION: To our knowledge, this is the first report of the differential increase in expression of CSE, CBS, and MPST in human RCC. Although these patterns do not appear to correlate with cancer recurrence, metastasis, size or nuclear grade, their differential increase suggests a potential therapeutic target.

Hydrogen Sulfide: Emerging Role in Bladder, Kidney, and Prostate Malignancies.

Hydrogen sulfide (H2S) is the latest member of the gasotransmitter family and known to play essential roles in cancer pathophysiology. H2S is produced endogenously and can be administered exogenously. Recent studies showed that H2S in cancers has both pro- and antitumor roles. Understanding the difference in the expression and localization of tissue-specific H2S-producing enzymes in healthy and cancer tissues allows us to develop tools for cancer diagnosis and treatment. Urological malignancies are some of the most common cancers in both men and women, and their early detection is vital since advanced cancers are recurrent, metastatic, and often resistant to treatment. This review summarizes the roles of H2S in cancer and looks at current studies investigating H2S activity and expression of H2S-producing enzymes in urinary cancers. We specifically focused on urothelial carcinoma, renal cell carcinoma, and prostate cancer, as they form the majority of newly diagnosed urinary cancers. Recent studies show that besides the physiological activity of H2S in cancer cells, there are patterns between the development and prognosis of urinary cancers and the expression of H2S-producing enzymes and indirectly the H2S levels. Though controversial and not completely understood, studying the expression of H2S-producing enzymes in cancer tissue may represent an avenue for novel diagnostic and therapeutic strategies for addressing urological malignancies.

Reconstitution of T-Cell Subsets Following Thymoglobulin-Induced Depletion in High Immunologic Risk and Donation After Cardiac Death Renal Transplant Recipients.

INTRODUCTION: Depletion therapy in high immunologic risk (HR) patients by antithymocyte globulin (rATG) induces lymphopenia and subsequent compartmental repopulation of T-cell subsets. rATG is also given to patients receiving kidneys from donations after cardiac death (DCDs) to mitigate innate immune activation associated with the DCD process. METHODS: We compared the T-cell response with rATG in both HR and DCD kidney recipients. We examined the reconstitution of T-cell subsets after rATG treatment in HR and DCD recipients (n = 19 per group) by multicolor flow cytometry. RESULTS: Following treatment, there was a rapid drop in the frequency of T cells in both groups, which persisted over 28 days. HR patients had an early surge in the frequency of CD4+ naïve, effector-memory, and regulatory T cells. Although we found a significant proliferation of the T cells in both groups, the DCD cohort had a blunted response as well as reduced CD4+ T-cell immune-reactivity compare with the HR group. CONCLUSIONS: Our data suggest that there is a lack of significant homeostatic proliferative response in DCD recipients following rATG, and CD4+ T cells may be less reactive in the DCD group than previously thought, indicating that rATG treatment may not have to be considered a first-line induction therapy in DCD recipients.

Novel therapeutic strategies for renal graft preservation and their potential impact on the future of clinical transplantation.

PURPOSE OF REVIEW: The current review aims to examine recent evidence about improvements, therapeutics and novel approaches for renal graft preservation along with presenting a pragmatic outlook on their potential for clinical translation. RECENT FINDINGS: Modifying established cold preservation methods (4 °C) with oxygenation, gene therapies and gasotransmitters such as hydrogen sulfide has been shown to improve renal graft outcomes with minimum modifications to current protocols. These strategies have also shown promise in the context of normothermic preservation (34-37 °C), which circumvents the damage caused by cold preservation. Although normothermic machine perfusion (NMP) is being evaluated in clinical trials, it is limited by high cost, the use of blood and the lack of standardized protocols. Recent studies confirmed that preservation at subnormothermic temperatures (∼20 °C) is effective with approved preservation solutions and, in conjunction with exogenous hydrogen sulfide therapy, this approach may expedite a static preservation alternative to NMP. SUMMARY: Progress has been made in investigating improvements and alternatives to cold preservation. Promising therapeutic strategies have also been studied in the context of cold, subnormothermic and normothermic preservation. Further research is needed to optimize clinical renal graft preservation.

Endogenous H2S production deficiencies lead to impaired renal erythropoietin production.

INTRODUCTION: Patients suffering from chronic kidney disease (CKD) experience a number of associated comorbidities, including anemia. Relative deficiency in renal erythropoietin (EPO) production is thought to be a primary cause of anemia. Interestingly, CKD patients display low levels of hydrogen sulfide (H2S), an endogenously derived renal oxygen sensor. Previous in vitro experiments have revealed that H2S-deficient renal cell lines produce less EPO than wild-type renal cell lines during hypoxia. METHODS: We postulated that H2S might be a primary mediator of EPO synthesis during hypoxia, which was tested using an in vivo murine model of whole-body hypoxia and in clinical samples obtained from CKD patients. RESULTS: Following a 72-hour period of hypoxia (11% O2), partial H2S knockout mice (lacking the H2S biosynthetic enzyme cystathionine γ-lyase [CSE]) displayed lower levels of hemoglobin, EPO and cystathionine-ß-synthase (CBS) (another H2S biosynthetic enzyme) compared to wild-type mice, all of which was rescued by exogenous H2S supplementation. We also found that anemic CKD patients requiring exogenous EPO exhibited lower urinary thiosulfate levels compared to non-anemic CKD patients of similar CKD classification. CONCLUSIONS: Together, our results confirm an interplay between the actions of H2S during hypoxia and EPO production.

H2S supplementation: A novel method for successful organ preservation at subnormothermic temperatures.

Renal transplantation is the preferred treatment for end-stage renal disease. Currently, there is a large gap between the supply and demand for transplantable kidneys. The use of sub-optimal grafts obtained via donation after cardiac death (DCD) is on the rise. While static cold storage (SCS) in University of Wisconsin (UW) solution on ice (4°C) is the clinical standard of care for renal graft preservation, cold storage has been associated with negative graft outcomes. The alternative, normothermic machine perfusion, involves mechanical perfusion of the organ at physiological or normothermic temperature (37°C) and this technique is expensive, complicated and globally inaccessible. As such, simpler alternatives are of interest. Preliminary results revealed that UW solution is more protective at 21°C than 37°C and subnormothermic preservation is of interest because it may facilitate the use of existing solutions while preventing cold injury. We have previously shown that SCS in UW solution supplemented with mitochondria-targeted H2S donor AP39 improves renal graft outcomes. As such, it was hypothesized subnormothermic preservation at 21°C with AP39 will also improve renal outcomes. Using an in vitro model of hypoxia and reoxygenation, we found that treating porcine tubular epithelial cells with UW+5 µM AP39 during 18 h hypoxia at 21°C significantly increased renal tubular epithelial cell viability after 24 h of reoxygenation at 37°C compared to UW alone. Also, AP39-supplemented UW solution was significantly more cytoprotective during hypoxia at 21°C than hypoxia at 37°C, regardless of AP39 concentration. Using an ex vivo DCD organ preservation model, we found that DCD porcine kidneys stored for 24 h in UW+200 nM AP39 at 21°C showed significantly lower tissue necrosis than DCD porcine kidneys preserved using SCS in UW solution, the clinical standard of care. Overall, our findings suggest that exogenous H2S supplementation improves the viability of the gold standard organ preservation solution, UW solution, for subnormothermic preservation at 21°C.