Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study.

BACKGROUND: Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach. METHODS: We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls. FINDINGS: The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68+ and CD15+ innate immune cells, as well as NKp46+ cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models. INTERPRETATION: Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results. FUNDING: OrganX and MSD Avenir.

Impact of the COVID-19 pandemic on publication dynamics and non-COVID-19 research production.

BACKGROUND: The COVID-19 pandemic has severely affected health systems and medical research worldwide but its impact on the global publication dynamics and non-COVID-19 research has not been measured. We hypothesized that the COVID-19 pandemic may have impacted the scientific production of non-COVID-19 research. METHODS: We conducted a comprehensive meta-research on studies (original articles, research letters and case reports) published between 01/01/2019 and 01/01/2021 in 10 high-impact medical and infectious disease journals (New England Journal of Medicine, Lancet, Journal of the American Medical Association, Nature Medicine, British Medical Journal, Annals of Internal Medicine, Lancet Global Health, Lancet Public Health, Lancet Infectious Disease and Clinical Infectious Disease). For each publication, we recorded publication date, publication type, number of authors, whether the publication was related to COVID-19, whether the publication was based on a case series, and the number of patients included in the study if the publication was based on a case report or a case series. We estimated the publication dynamics with a locally estimated scatterplot smoothing method. A Natural Language Processing algorithm was designed to calculate the number of authors for each publication. We simulated the number of non-COVID-19 studies that could have been published during the pandemic by extrapolating the publication dynamics of 2019 to 2020, and comparing the expected number to the observed number of studies. RESULTS: Among the 22,525 studies assessed, 6319 met the inclusion criteria, of which 1022 (16.2%) were related to COVID-19 research. A dramatic increase in the number of publications in general journals was observed from February to April 2020 from a weekly median number of publications of 4.0 (IQR: 2.8-5.5) to 19.5 (IQR: 15.8-24.8) (p < 0.001), followed afterwards by a pattern of stability with a weekly median number of publications of 10.0 (IQR: 6.0-14.0) until December 2020 (p = 0.045 in comparison with April). Two prototypical editorial strategies were found: 1) journals that maintained the volume of non-COVID-19 publications while integrating COVID-19 research and thus increased their overall scientific production, and 2) journals that decreased the volume of non-COVID-19 publications while integrating COVID-19 publications. We estimated using simulation models that the COVID pandemic was associated with a 18% decrease in the production of non-COVID-19 research. We also found a significant change of the publication type in COVID-19 research as compared with non-COVID-19 research illustrated by a decrease in the number of original articles, (47.9% in COVID-19 publications vs 71.3% in non-COVID-19 publications, p < 0.001). Last, COVID-19 publications showed a higher number of authors, especially for case reports with a median of 9.0 authors (IQR: 6.0-13.0) in COVID-19 publications, compared to a median of 4.0 authors (IQR: 3.0-6.0) in non-COVID-19 publications (p < 0.001). CONCLUSION: In this meta-research gathering publications from high-impact medical journals, we have shown that the dramatic rise in COVID-19 publications was accompanied by a substantial decrease of non-COVID-19 research. META-RESEARCH REGISTRATION: https://osf.io/9vtzp/ .

[New technologies for renal pathology: Transcriptomics on paraffin-embedded fixed tissue]. / Nouvelles technologies au service de la pathologie rénale : transcriptomique sur tissu fixé et inclus en paraffine.

The development of new high-throughput technologies in genomics and then in transcriptomics has modified clinical approach in nephrology. At the interface between high-throughput technologies (microarray, new generation sequencing «NGS¼) and few mRNA analysis (reverse transcriptase quantitative PCR [RT-qPCR]), the nCounter® of NanoString® offers a new and complementary approach. Capable of analyzing formalin-fixed paraffin-embedded samples, this technology is a credible candidate for implanting transcriptomics in clinical routine.

Sunitinib-induced cardiac hypertrophy and the endothelin axis.

Anti-angiogenics drugs in clinical use for cancer treatment induce cardiotoxic side effects. The endothelin axis is involved in hypertension and cardiac remodelling, and addition of an endothelin receptor antagonist to the anti-angiogenic sunitinib was shown to reduce cardiotoxicity of sunitinib in mice. Here, we explored further the antidote effect of the endothelin receptor antagonist macitentan in sunitinib-treated animals on cardiac remodeling. Methods: Tumor-bearing mice treated per os daily by sunitinib or vehicle were imaged before and after 1, 3 and 6 weeks of treatment by positron emission tomography using [18F]fluorodeoxyglucose and by echocardiography. Non-tumor-bearing animals were randomly assigned to be treated per os daily by vehicle or sunitinib or macitentan or sunitinib+macitentan, and imaged by echocardiography after 5 weeks. Hearts were harvested for histology and molecular analysis at the end of in vivo exploration. Results: Sunitinib treatment increases left ventricular mass and ejection fraction and induces cardiac fibrosis. Sunitinib also induces an early increase in cardiac uptake of [18F]fluorodeoxyglucose, which is significantly correlated with increased left ventricular mass at the end of treatment. Co-administration of macitentan prevents sunitinib-induced hypertension, increase in ejection fraction and cardiac fibrosis, but fails to prevent increase of the left ventricular mass. Conclusion: Early metabolic changes predict sunitinib-induced cardiac remodeling. Endothelin blockade can prevent some but not all cardiotoxic side-effects of sunitinib, in particular left ventricle hypertrophy that appears to be induced by sunitinib through an endothelin-independent mechanism.

Calpastatin prevents Angiotensin II-mediated podocyte injury through maintenance of autophagy.

The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin II promoting progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy) specifically in the podocyte resulted in accelerated angiotensin II-induced podocytopathy, accentuated albuminuria and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.

Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation.

This meeting report from the XV Banff conference describes the creation of a multiorgan transplant gene panel by the Banff Molecular Diagnostics Working Group (MDWG). This Banff Human Organ Transplant (B-HOT) panel is the culmination of previous work by the MDWG to identify a broadly useful gene panel based on whole transcriptome technology. A data-driven process distilled a gene list from peer-reviewed comprehensive microarray studies that discovered and validated their use in kidney, liver, heart, and lung transplant biopsies. These were supplemented by genes that define relevant cellular pathways and cell types plus 12 reference genes used for normalization. The 770 gene B-HOT panel includes the most pertinent genes related to rejection, tolerance, viral infections, and innate and adaptive immune responses. This commercially available panel uses the NanoString platform, which can quantitate transcripts from formalin-fixed paraffin-embedded samples. The B-HOT panel will facilitate multicenter collaborative clinical research using archival samples and permit the development of an open source large database of standardized analyses, thereby expediting clinical validation studies. The MDWG believes that a pathogenesis and pathway based molecular approach will be valuable for investigators and promote therapeutic decision-making and clinical trials.

The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell- and antibody-mediated rejection.

The XV. Banff conference for allograft pathology was held in conjunction with the annual meeting of the American Society for Histocompatibility and Immunogenetics in Pittsburgh, PA (USA) and focused on refining recent updates to the classification, advances from the Banff working groups, and standardization of molecular diagnostics. This report on kidney transplant pathology details clarifications and refinements to the criteria for chronic active (CA) T cell-mediated rejection (TCMR), borderline, and antibody-mediated rejection (ABMR). The main focus of kidney sessions was on how to address biopsies meeting criteria for CA TCMR plus borderline or acute TCMR. Recent studies on the clinical impact of borderline infiltrates were also presented to clarify whether the threshold for interstitial inflammation in diagnosis of borderline should be i0 or i1. Sessions on ABMR focused on biopsies showing microvascular inflammation in the absence of C4d staining or detectable donor-specific antibodies; the potential value of molecular diagnostics in such cases and recommendations for use of the latter in the setting of solid organ transplantation are presented in the accompanying meeting report. Finally, several speakers discussed the capabilities of artificial intelligence and the potential for use of machine learning algorithms in diagnosis and personalized therapeutics in solid organ transplantation.

The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression.

The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and ß1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.

Vasa nervorum angiogenesis in prostate cancer with perineural invasion.

BACKGROUND: Perineural invasion (PNI) is generally accepted as a major route of cancer dissemination in malignancies associated with highly enervated organs. However, the effect of cancer cells on vasa nervorum remains unknown. We studied this effect in locally advanced prostate cancer, a high-risk feature associated with approximately 20% of prostate cancer specific mortality. METHODS: We used immunohistochemistry for CD34, fibroblast growth factor-2 (FGF-2), FSHR, podoplanin, vascular endothelial growth factor (VEGF), and VEGFR-2 as well as histochemical methods to examine the vasa nervorum of nerves invaded by cancer cells in tissue samples from 85 patients. RESULTS: The percentage of the nerve area occupied by CD34-positive vasa nervorum endothelial cells in nerves with PNI was much higher than in nerves without PNI (7.3 ± 1.2 vs 1.9 ± 0.4; P < 0.001 and 5.8 ± 0.6 vs 1.23 ± 0.8; P < 0.001 in pT3a and pT3b prostate cancer specimens, respectively). In 19/85 of the patients the CD34-positive vasa nervorum microvessels have a thick basement membrane, similar to the vessels in diabetic microangiopathy. This subendothelial layer contains collagen fibers. Vasa nervorum endothelia and Schwann cells express FGF-2 (nuclear localization) and FSHR (plasma membrane and cytoplasmic staining). Prostate cancer cells invading nerves express VEGF, a critical cytokine in tumor angiogenesis. The vasa nervorum of prostatic nerves with PNI did not express detectable levels of VEGFR-2. No podoplanin-positive lymphatic vessels were seen in nerves. CONCLUSION: In locally advanced prostate cancer, PNI of cancer cells is associated with formation of new endoneurial capillaries and changes of vasa nervorum morphology.

Endothelial Epas1 Deficiency Is Sufficient To Promote Parietal Epithelial Cell Activation and FSGS in Experimental Hypertension.

FSGS, the most common primary glomerular disorder causing ESRD, is a complex disease that is only partially understood. Progressive sclerosis is a hallmark of FSGS, and genetic tracing studies have shown that parietal epithelial cells participate in the formation of sclerotic lesions. The loss of podocytes triggers a focal activation of parietal epithelial cells, which subsequently form cellular adhesions with the capillary tuft. However, in the absence of intrinsic podocyte alterations, the origin of the pathogenic signal that triggers parietal epithelial cell recruitment remains elusive. In this study, investigation of the role of the endothelial PAS domain-containing protein 1 (EPAS1), a regulatory α subunit of the hypoxia-inducible factor complex, during angiotensin II-induced hypertensive nephropathy provided novel insights into FSGS pathogenesis in the absence of a primary podocyte abnormality. We infused angiotensin II into endothelial-selective Epas1 knockout mice and their littermate controls. Although the groups presented with identical high BP, endothelial-specific Epas1 gene deletion accentuated albuminuria with severe podocyte lesions and recruitment of pathogenic parietal glomerular epithelial cells. These lesions and dysfunction of the glomerular filtration barrier were associated with FSGS in endothelial Epas1-deficient mice only. These results indicate that endothelial EPAS1 has a global protective role during glomerular hypertensive injuries without influencing the hypertensive effect of angiotensin II. Furthermore, these findings provide proof of principle that endothelial-derived signaling can trigger FSGS and illustrate the potential importance of the EPAS1 endothelial transcription factor in secondary FSGS.

Hmox1 Deficiency Sensitizes Mice to Peroxynitrite Formation and Diabetic Glomerular Microvascular Injuries.

OBJECTIVE: Indirect evidence suggests a role for heme oxygenase-1 (HO-1) in limiting diabetic vasculopathy. The goal of this study was to assess the role of HO-1 in the development of microvascular lesions within glomeruli during diabetes mellitus using a mouse model with specific alteration of the Hmox1 gene. APPROACH AND RESULTS: The effects of Hmox1 haploinsufficiency were studied as a means of assessing the intrinsic contribution of HO-1 in the development of renal microvascular lesions during diabetes. Renal function and histology were analyzed 10 weeks after diabetes induction with streptozotocin. Diabetic Hmox1+/- mice showed higher levels of albuminuria and blood urea compared to their wild-type diabetic littermates. More severe glomerular microvascular lesions were also observed in the diabetic Hmox1+/- mice. This was associated with a renal increase in the expression of the oxidative stress marker, nitrotyrosine. CONCLUSIONS: Genetic Hmox1 partial deficiency is sufficient to sensitize mice to the development of diabetic glomerular microvascular lesions. HO-1 exerts antioxidant effects in the kidney during diabetes mellitus. These have protective effects on the development of glomerular endothelial injury.

Follicle-Stimulating Hormone Receptor Expression in Endometriotic Lesions and the Associated Vasculature: An Immunohistochemical Study.

Follicle-stimulating hormone receptor (FSHR) is present on endothelial cells of blood vessels and endometrial glands of the proliferative and secretory endometrium. So far, the expression of FSHR in endometriosis has not been studied. We evaluated FSHR expression in 194 tissue specimens representing 3 relevant types of endometriosis: rectovaginal endometriotic nodules, ovarian endometriotic cysts, and peritoneal endometriotic implants. Specimens of normal endometrium were used as controls. Archival formalin-fixed and paraffin-embedded material was analyzed immunohistochemically with a highly specific monoclonal antihuman FSHR antibody using the peroxidase method. A robust vascular FSHR expression was found in all 194 patients, irrespective of the endometriosis lesion location. Follicle-stimulating hormone receptor was not detected in normal host tissues located more than 5 mm from the lesions. The endometriotic lymphatic vessels do not express FSHR. The density of FSHR-positive vessels in patients with rectovaginal endometriotic nodules was 46.0 ± 5.7 vessels/mm(2) Similar values were obtained for ovarian endometriotic cysts and peritoneal endometriosis. The density of FSHR-positive vessels associated with the core of rectovaginal endometriotic nodules was 2-fold higher than that of the perilesional, adjacent normal host tissue (64.2 ± 8.2 vs 27.2 ± 3.2 vessels/mm(2), respectively). Expression of FSHR was also detected either in endometriotic glandular epithelial cells, endometriotic stromal cells, or in both cell types (23%, 25%, and 21% of patients, respectively). Normal endometrium expressed FSHR predominately in basalis, in a cellular distribution dependent on hormonal environment. In conclusion, our data suggest novel FSHR expression in endometriotic lesions, qualitatively and quantitatively different from that of normal endometrium.

Endothelial follicle-stimulating hormone receptor expression in invasive breast cancer and vascular remodeling at tumor periphery.

BACKGROUND: Follicle-stimulating hormone receptor (FSHR) is expressed on the endothelial surface of blood vessels associated with solid tumor periphery, where angiogenesis is known to occur. The correlation between FSHR expression and formation of new peritumoral vessels has not been previously investigated. METHODS: We used immunohistochemical techniques involving specific antibodies to detect FSHR and the endothelial markers (CD34, VEGFR2, and D2-40) in tissue samples from 83 patients with lymph node-negative, invasive breast cancer representing four main clinical treatment groups: HR+/HER2-, HR+/HER2+, HR-/HER2+ and triple-negative. RESULTS: The FSHR+ vessels were exclusively located at breast cancer periphery, in a layer that extended 2 mm into and 5 mm outside of the tumor. The percentage of blood vessels expressing FSHR reached a maximum of 100% at the demarcation line between the tumor and the normal tissue. Common among FSHR+ vessels, regardless of breast cancer type, were the high densities of arterioles and venules (6.4 ± 1.4 and 13.9 ± 2.1 vessels/mm(2), respectively). These values were 3-fold higher that those noticed for CD34+ arterioles and venules associated with normal breast tissue located at a distance greater than 10 mm outside the tumors. The average density of FSHR+ and CD34+ blood vessels as well as of D2-40+ lymphatic vessels did not differ significantly among breast cancer subgroups. FSHR+ vessels did not express VEGFR2. The endothelial FSHR expression correlated significantly with the peritumoral CD34+ vessels' density (p < 0.001) and tumor size (p = 0.01). CONCLUSION: Endothelial FSHR expression in breast cancer is associated with vascular remodeling at tumor periphery.

The PI3K/AKT pathway promotes gefitinib resistance in mutant KRAS lung adenocarcinoma by a deacetylase-dependent mechanism.

To select the appropriate patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), it is important to gain a better understanding of the intracellular pathways leading to EGFR-TKI resistance, which is a common problem in patients with lung cancer. We recently reported that mutant KRAS adenocarcinoma is resistant to gefitinib as a result of amphiregulin and insulin-like growth factor-1 receptor overexpression. This resistance leads to inhibition of Ku70 acetylation, thus enhancing the BAX/Ku70 interaction and preventing apoptosis. Here, we determined the intracellular pathways involved in gefitinib resistance in lung cancers and explored the impact of their inhibition. We analyzed the activation of the phosphatidyl inositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) pathway in lung tumors. The activation of AKT was associated with disease progression in tumors with wild-type EGFR from patients treated with gefitinib (phase II clinical trial IFCT0401). The administration of IGF1R-TKI or amphiregulin-directed shRNA decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. The combination of PI3K/AKT inhibition with gefitinib restored apoptosis via Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which decreased the BAX/Ku70 interaction, inhibited AKT signaling and induced gefitinib-dependent apoptosis. The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in lung tumors with KRAS mutation, through the regulation of the BAX/Ku70 interaction. This finding suggests that combined treatments could improve the outcomes for this subset of lung cancer patients, who have a poor prognosis.