The Interplay Between Human Leukocyte Antigen Antibody Profile and COVID-19 Vaccination in Waitlisted Renal Transplant Patients.

CONTEXT.­: Mass COVID-19 vaccination is mandated in vulnerable populations in our renal transplant waitlist cohort. However, the anti-human leukocyte antigen (anti-HLA) profile after COVID-19 vaccination is controversial, and the side effects are yet to be discerned. OBJECTIVE.­: To evaluate the status of HLA antibodies in waitlist renal transplant patients before and 3 weeks after each vaccination and if comorbidities are associated with the HLA antibody profile. DESIGN.­: A total of 59 waitlisted kidney transplant patients were included in this study. The anti-HLA antibodies were analyzed before and 6 months after their last COVID-19 vaccination. The mean fluorescence intensity change in the anti-HLA antibody levels was used to classify patients into 3 groups: high inducers, low inducers, and noninducers. RESULTS.­: There were significant HLA antibody profile changes after COVID-19 vaccination, showing 21 antibodies generated against HLA class I antigens and 7 against HLA class II antigens to their baseline. Compared with the noninducers, the high and low inducers showed a higher prevalence of COVID-19 infection, COVID-19 vaccine type, and background hypertension history. CONCLUSIONS.­: Our data suggest that COVID-19 vaccination propagates anti-HLA class I and II antibodies for waitlisted renal transplant patients. The clinical significance of these antibodies needs further study. Furthermore, comorbidities, such as history of COVID-19 infection and hypertension, supplemented this effect. Anti-HLA antibody monitoring may be warranted in vaccinated, waitlisted renal transplant patients with COVID-19 vaccinations, and a history of COVID-19 infection or hypertension.

Reducing Quantitative Uncertainty Caused by Data Processing in Untargeted Metabolomics.

Processing liquid chromatography-mass spectrometry-based metabolomics data using computational programs often introduces additional quantitative uncertainty, termed computational variation in a previous work. This work develops a computational solution to automatically recognize metabolic features with computational variation in a metabolomics data set. This tool, AVIR (short for "Accurate eValuation of alIgnment and integRation"), is a support vector machine-based machine learning strategy (https://github.com/HuanLab/AVIR). The rationale is that metabolic features with computational variation have a poor correlation between chromatographic peak area and peak height-based quantifications across the samples in a study. AVIR was trained on a set of 696 manually curated metabolic features and achieved an accuracy of 94% in a 10-fold cross-validation. When tested on various external data sets from public metabolomics repositories, AVIR demonstrated an accuracy range of 84%-97%. Finally, tested on a large-scale metabolomics study, AVIR clearly indicated features with computational variation and thus guided us to manually correct them. Our results show that 75.3% of the samples with computational variation had a relative intensity difference of over 20% after correction. This demonstrates the critical role of AVIR in reducing computational variation to improve quantitative certainty in untargeted metabolomics analysis.

Basic Research Protocol: Exome Sequencing in Adults With Loin Pain Hematuria Syndrome: A Pilot Study.

Background: Loin pain hematuria syndrome (LPHS) is a poorly understood clinical syndrome characterized by hematuria and either unilateral or bilateral severe kidney pain in the absence of identifiable urological disease. Loin pain hematuria syndrome imposes a significant health and economic impact with a loss of productivity and quality of life in a young population. Owing to an incomplete understanding of its pathophysiology, treatment has been limited to nonspecific pain management. Nearly 60 years after its initial description, we are no further ahead in understanding the molecular pathways involved in LPHS. Objective: To outline the study design for exome sequencing in adults with LPHS and their families. Methods: In this single-center case series, 24 patients with LPHS and 2 additional first-degree family members per participant will be recruited. DNA extracted from venous blood samples will undergo exome sequencing on the Illumina NovaSeq 6000 System at 100× depth and will be assessed for pathogenic variants in genes associated with hematuria (number of genes in: glomerular endothelium [n = 10] and basement membrane [n = 8]), and pain pathways (number of genes in: pain transduction [n = 17], conduction [n = 8], synaptic transmission [n = 37], and modulation [n = 27]). We will further examine identified potentially pathogenic variants that co-segregate with LPHS features among affected families. Conclusions: This pilot study may identify new directions for an investigation into the molecular mechanisms underlying LPHS.

Contexte: Le syndrome de lombalgie-hématurie est un syndrome clinique encore mal compris qui se caractérise par une hématurie et une forte douleur rénale unilatérale ou bilatérale en l'absence d'une maladie urologique identifiable. Le syndrome de lombalgie-hématurie a une incidence importante sur la santé et l'économie en entraînant une perte de productivité et de qualité de vie dans une population jeune. La compréhension de la physiopathologie de ce syndrome étant incomplète, le traitement a été limité à la gestion non spécifique de la douleur. Près de soixante ans après sa description initiale, nous en sommes au même point dans la compréhension des voies moléculaires impliquées dans le syndrome de lombalgie-hématurie. Objectif: Décrire le plan de l'étude pour le séquençage de l'exome chez les adultes atteints du syndrome de lombalgie-hématurie et des membres de leur famille. Méthodologie: Pour cette série de cas menée dans un seul center, nous recruterons 24 patients atteints du syndrome de lombalgie-hématurie et deux membres au premier degré de leur famille. L'ADN extrait d'échantillons de sang veineux sera soumis à un séquençage de l'exome sur le système Ilumina NovaSeq 6000 réglé à 100X de profondeur. Il sera également analysé pour la présence de variants pathogènes dans les gènes associés à l'hématurie (nombre de gènes dans l'endothélium glomérulaire [n = 10] et la membrane basale [n = 8]), et aux voies de transmission de la douleur (nombre de gènes dans la transduction [n = 17], la conduction [n = 8], la transmission synaptique [n = 37] et la modulation [n = 27] de la douleur). Nous poursuivrons l'examen des variants potentiellement pathogènes identifiés qui co-ségrègent avec les caractéristiques du syndrome de lombalgie-hématurie parmi les familles touchées. Conclusion: Cette étude pilote pourrait révéler de nouveaux axes de recherche sur les mécanismes moléculaires qui sous-tendent le syndrome de lombalgie-hématurie.

Kidney Biopsy and Type IV Collagen Gene Sequencing Fail to Explain Hematuria in Loin Pain Hematuria Syndrome.

Introduction: Loin pain hematuria syndrome (LPHS) is a rare clinical syndrome with a reported prevalence of 1 in 10,000. The syndrome is characterized by severe pain localized to the kidney in the absence of identifiable urinary tract disease. Because of an inadequate understanding of the pathophysiology of the disease, the goal of management has been limited to symptomatic pain management. Through detailed phenotype and genotype assessment we sought to identify possible underlying etiologies. Methods: We completed a chart review, ultrasound imaging, kidney biopsy, and type IV collagen (COL4A3, COL4A4, and COL4A5) gene sequencing in 14 patients with loin pain hematuria recruited from a single center. Results: Red blood cells and red cell casts were observed within the tubules in 10 of 14 patients. The glomerular basement membrane (GBM) was normal in 11 patients and thickened in 1 patient. Staining for IgA kappa was present in 1 patient. C3 deposition without any inflammation was present in 7 patients. Arteriolar hyalinosis was present in 4 patients and endothelial cell injury was present in 6 patients. No pathogenic COL4A3, COL4A4, or COL4A5 variants were identified. Conclusion: Conventional histopathology and genetic testing for type IV collagen variants failed to identify the cause of hematuria in 14 patients with LPHS.

The role of the HLA allelic repertoire on the clinical severity of COVID-19 in Canadians, living in the Saskatchewan province.

AIMS: The HLA system has been implicated as an underlying determinant for modulating the immune response to SARS-CoV-2. In this study, we aimed to determine the association of patients' HLA genetic profiles with the disease severity of COVID-19 infection. METHODS: Prospective study was conducted on COVID-19 patients (n = 40) admitted to hospitals in Saskatoon, Canada, between March and December 2020. Next-generation sequencing was performed on the patient samples to obtain high-resolution HLA typing profiles. The statistical association between HLA allelic frequency and disease severity was examined. The disease severity was categorized based on the length of hospital stay and intensive care needs or demise during the hospital stay. RESULTS: HLA allelic frequencies of the high and low-severity cohorts were normalized against corresponding background allelic frequencies. In the high-severity cohort, A*02:06 (11.8-fold), B*51:01 (2.4-fold), B*15:01(3.1-fold), C*01:02 (3.3-fold), DRB1*08:02 (31.2-fold), DQ*06:09 (11-fold), and DPB1*04:02(4-fold) were significantly overrepresented (p < 0.05) making these deleterious alleles. In the low-severity cohort, A*24:02 (2.8-fold), B*35:01 (2.8-fold), DRB1*04:07 (5.3-fold), and DRB1*08:11 (22-fold) were found to be significantly overrepresented (p < 0.05) making these protective alleles. These above alleles interact with NK cell antiviral activity via the killer immunoglobulin-like receptors (KIR). The high-severity cohort had a higher predilection for HLA alleles associated with KIR subgroups; Bw4-80I (1.1-fold), and C1 (1.6-fold) which promotes NK cell inhibition, while the low-severity cohort had a higher predilection for Bw4-80T (1.6-fold), and C2 (1.6-fold) which promote NK cell activation. CONCLUSION: In this study, the HLA allelic repository with the distribution of deleterious and protective alleles was found to correlate with the severity of the clinical course in COVID-19. Moreover, the interaction of specific HLA alleles with the KIR-associated subfamily modulates the NK cell-mediated surveillance of SARS-CoV-2. Both deleterious HLA alleles and inhibitory KIR appear prominently in the severe COVID-19 group focusing on the importance of NK cells in the convalescence of COVID-19.

Histiocytic Glomerulopathy Associated With Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a systemic inflammatory syndrome characterized by heightened activation and proliferation of nonmalignant macrophages and excessive cytokine release. Whereas acute kidney injury is common in this syndrome, direct glomerular involvement by activated histiocytes is very rare. We present the case of a man in his 20s who presented with fevers, malaise, flank pain, anemia, thrombocytopenia, severe acute kidney injury, and proteinuria. A kidney biopsy revealed histiocytic glomerulopathy and subacute thrombotic microangiopathy, and he was diagnosed with HLH. Recovery of kidney function occurred following steroid therapy. A review of kidney involvement by HLH is provided.

Acute kidney injury from immune checkpoint inhibitor use.

Immune checkpoint inhibitors are novel oncological medications, current classes of which include monoclonal antibodies that target inhibitory receptors cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 protein (PD-1) and programmed death-ligand 1. While they are novel in their ability to treat cancer, they also have a unique spectrum of immune-related adverse events. Renal-related immune adverse events, though rare, are an increasingly recognised clinical entity. We present the case of a 67-year-old man with acute kidney injury (AKI) after the second cycle of combination anti-CTLA-4 and anti-PD-1 antibodies for metastatic cutaneous melanoma. He presented with vomiting and diarrhoea, and AKI secondary to dehydration was treated with aggressive rehydration. After failing to recover biochemically, a renal biopsy was performed, which demonstrated severe acute interstitial nephritis. The culprit medications were held and he was treated with steroids. With immunosuppression, creatinine improved to pretreatment values.

Renal tubular acidosis as the initial presentation of Sjögren's syndrome.

We present a 44-year-old female with an initial presentation with distal renal tubular acidosis (RTA) after she presented with hypokalaemia and normal anion gap acidosis. Three years following the diagnosis, she presented with progressive renal impairment. In the absence of any clinical, biochemical and radiological clues, she underwent a renal biopsy which showed severe tubulitis secondary to lymphocytic infiltration. Serological investigations subsequently revealed positive anti-nuclear, anti-Sjögren's syndrome related antigen A (SS-A), and anti-Sjögren's syndrome related antigen B (SS-B) antibodies, supporting the diagnosis of Sjögren's syndrome. This case is unique in that distal RTA was the presenting clinical manifestation of Sjögren's syndrome. We hope that a consideration for Sjögren's syndrome is made in patients with seemingly idiopathic RTA.

Fibrillary Glomerulonephritis with Crescentic and Necrotizing Glomerulonephritis and Concurrent Thrombotic Microangiopathy.

We present a 77-year-old Caucasian woman who presented with nephrotic-range proteinuria, microhematuria, renal impairment, and extremely elevated blood pressure. She had a long history of well-controlled type 2 diabetes. Renal biopsy revealed fibrillary deposits in the mesangium and glomerular basement membrane consistent with fibrillary glomerulopathy (FGN), with crescentic changes and thrombotic microangiopathy (TMA). We could not identify any radiological, clinical, or laboratory evidence of autoimmune disorders, lymphoproliferative disorders, and malignancy. It was decided not to offer her any immunosuppressive therapy, as she was frail with substantial renal damage on the biopsy. Five months after presentation, she gradually progressed to requiring renal replacement therapy and is currently on maintenance hemodialysis. Crescentic changes in FGN, though rare, have been previously described, but the concurrent presence of TMA has never been previously reported.

Recurrent IgG4-related tubulointerstitial nephritis concurrent with chronic active antibody mediated rejection: A case report.

IgG4-related disease is a relatively newly described entity that can affect nearly any organ, including the kidneys, where it usually manifests as tubulointerstitial nephritis (IgG4-TIN). The diagnosis can be suggested by characteristic histological features, including an inflammatory infiltrate with increased IgG4-positive plasma cells associated with "storiform" fibrosis. Serum IgG4 is usually elevated. In the native kidney and other organs, there is typically a brisk response to treatment with immunosuppression. Recurrence of IgG4-TIN after renal transplant has not been described in the literature. Here, we describe the first case of recurrent IgG4-TIN in a young patient concomitant with chronic active antibody mediated rejection five years after kidney transplant. Recurrent IgG4-TIN could be diagnosed by the characteristic histopathologic features and increased IgG4-positive plasma cells. Despite maintenance immunosuppression, this disease may recur in the kidney allograft.

NKG2D regulates production of soluble TRAIL by ex vivo expanded human γδ T cells.

Soluble TRAIL (sTRAIL) can be produced by myeloid-derived cells to kill cancer cells. Whether this mechanism is used by T cells, and if so, how sTRAIL production is regulated, remains unclear. Our previous studies showed that ex vivo expanded human γδ T cells express TRAIL and NK receptor group 2 (R2), member D (NKG2D), and possess potent anticancer activities both in vitro and in vivo. Here, we investigated in greater detail the mechanisms by which γδ T cells utilize TRAIL and NKG2D to kill lung cancer cells. We demonstrate that human lung cancer cells express TRAIL R2 and NKG2D ligands. Blocking TRAIL or NKG2D during γδ T-cell-lung cancer cell co-cultures significantly reduced γδ T-cell-mediated cytotoxicity. Cross-linking NKG2D with anti-NKG2D antibody to mimic ligand binding promoted γδ T cells to produce sTRAIL, which induced apoptosis in lung cancer cells through TRAIL R2. Either neutralizing sTRAIL or blocking lung cancer cell TRAIL R2 significantly reduced γδ T-cell-mediated cytotoxicity to lung cancer cells. This study demonstrates that γδ T cells can mediate anticancer immunity via NKG2D-regulated production of sTRAIL.

Human Vδ1-T cells regulate immune responses by targeting autologous immature dendritic cells.

Recent studies suggest that tissue resident Vδ1-T cells may downregulate immune responses in human beings. However, the function of peripheral blood Vδ1-T cells and their mechanisms of action remain largely unknown because of their limited numbers and the difficulties encountered in expanding these cells. In this study, we provide direct evidence demonstrating that peripheral human Vδ1-T cells can abrogate adaptive immune responses by direct killing of autologous dendritic cells through a perforin-mediated pathway. These findings advance our basic understanding of this unique T-cell subset.

Adoptive immunotherapy of cancer using ex vivo expanded human gammadelta T cells: A new approach.

gammadelta T cells can be an option for adoptive immunotherapy of cancer. The major obstacle to clinical application of gammadelta T cells is their low number and lack of a reliable method to expand them consistently and efficiently. We were able to expand gammadelta T cells with high purity in all donors regardless of their starting repertoire of gammadelta T cells. These ex vivo expanded gammadelta T cells are in early differentiation stage, can efficiently kill various tumors and inhibit growth of human lung cancer xenografts. This new approach for ex vivo expansion of human gammadelta T cells will open new horizons for clinical use of these cells.

Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy.

PROBLEM: In pregnant women with antithyroglobulin antibody, prevalence of abortion is 2-4 fold higher compared to normal controls. Direct effect of such harmful autoantibodies on female reproductive organs may serve a role in pregnancy loss. METHOD OF STUDY: Expression of thyroglobulin in decidua, placenta, and ovary of pregnant Balb/c mice ((Balb/cxBalb/c and Balb/cxC57BL/6) during early, middle, and late stages of pregnancy was evaluated. Expression of thyroglobulin was investigated in these tissues by semi-quantitative RT-PCR. In addition, polyclonal antithyroglobulin antibody was produced, and expression of thyroglobulin protein in aforesaid tissues was evaluated by immunohistochemistry and dot-blot analysis. RESULTS: The results showed that thyroglobulin message is not expressed in placenta, decidua, or ovary in any stages of pregnancy. The same results were obtained at the protein level. CONCLUSION: It is likely that antithyroglobulin antibodies have no direct detrimental effect on such organs in patients with thyroid autoimmunity suffering from recurrent abortion.

Mutual helper effect in copulsing of dendritic cells with 2 antigens: a novel approach for improvement of dendritic-based vaccine efficacy against tumors and infectious diseases simultaneously.

To develop an efficient dendritic cell (DC)-based immunotherapy protocol, we examined whether simultaneous pulsing of DCs with a given antigen and a third-party antigen could enhance their antigen presentation capacity. Purified splenic DCs of Balb/c mice were pulsed separately with immunoglobulin G, ovalbumin, conalbumin, P15 peptide of Mycobacterium tuberculosis, and prostate-specific antigen or double combinations of the aforementioned antigens. In some settings, DCs pulsed with 1 antigen were mixed equally with those pulsed with another antigen. Antigen-pulsed DCs were injected into the footpad of syngeneic mice and proliferation of whole, CD4 and CD8 depleted lymph node cells was measured after restimulation with cognate antigen. Antigen-specific production of interferon-gamma (IFNgamma) was tested in culture supernatants. Frequency of responding lymph node cells was determined by IFNgamma enzyme-linked immunosorbent spot assay. Our results showed that copulsing of DCs with 2 unrelated antigens increased the capacity of DCs to induce antigen-specific T-cell proliferation against both antigens up to 16-fold. Injection of 2 populations of DCs each pulsed with a different antigen, increased proliferation of primed T cells significantly as well. Both CD4 and CD8 depleted populations showed vigorous proliferative response in copulsing system. In addition, copulsing of DCs with 2 antigens resulted in higher frequency of antigen-specific responding cells and significantly more IFNgamma production. Our results clearly showed that unrelated peptides and proteins could be used to enhance efficacy of DC-based vaccines and in this system, each antigen served to help the other one, a condition that we termed as "mutual helper effect."

Assessment of thyroglobulin expression in reproductive organs at different stages of mouse estrous cycle.

Prevalence of abortion is higher in women with autoimmune thyroid disease. In the majority of cases, however, no abnormality of thyroid function is detected despite the high levels of antithyroid antibodies. The direct influence of such harmful autoantibodies in female reproductive organs may serve a role in pregnancy loss. In this study, expression of thyroglobulin in the reproductive tissues of cycling mice has been evaluated. Stages of estrous cycle were determined by cellular morphology and ratio of epithelial cells to leukocytes in vaginal smear of Balb/C mice. At each phase, the mice were sacrificed and their uterus, ovary and fallopian tubes were removed. Expression of thyroglobulin-specific transcript in endometrium was investigated by two sets of primers using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, expression of thyroglobulin in reproductive tissues was assessed by immunohistochemistry and dot blot analysis. The results showed that thyroglobulin mRNA is not expressed in endometrial tissue of Balb/C mice at any stage of estrous cycle. Immunohistochemical analysis also confirmed that thyroglobulin or its cross reactive-antigens are not expressed at the protein level in the female reproductive organs. The results showed that thyroglobulin was not expressed in the reproductive organs of female mice. It is plausible that antithyroglobulin antibodies could interact with newly-generated antigens during placentation and pregnancy.

Repertoire and clonality of T-cell receptor beta variable genes expressed in endometrium and blood T cells of patients with recurrent spontaneous abortion.

PROBLEM: Recurrent spontaneous abortion (RSA) is a relatively common disorder, the underlying causes of which are thought to be immunological in most cases. METHOD OF STUDY: Expression profile and clonality pattern of T-cell receptor beta variable (TCRBV) genes in endometrium and blood of patients with RSA were investigated by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using BV gene-specific primers. Relative expression of each BV family was determined and clonal expansion of the over-expressed genes was assessed by analysis of CDR3 length polymorphism. RESULTS: Compared to blood, relative expression of four TCRBV genes was significantly higher in the endometrium of RSA group. Over-expressed genes, except for TCRBV3, all had restricted and oligoclonal patterns of expression in the endometrium. CONCLUSION: Endometrial T cells have a skewed TCRBV repertoire with restricted transcript heterogeneity, which is shared by both groups and minor variations observed in this pattern in RSA patients may reflect more recent and/or repeated exposure to nominal antigens or superantigens.

Microenvironment of the feto-maternal interface protects the semiallogenic fetus through its immunomodulatory activity on dendritic cells.

OBJECTIVE: To investigate the immunomodulatory activity of decidual culture supernatant on dendritic cell (DC) functions. DESIGN: In vivo and in vitro experimental study using mice. SETTING: Academic research laboratory. ANIMAL(S): C57BL/6-mated female Balb/c mice. INTERVENTION(S): Culture supernatants of decidual cells obtained from the uteri of allogenic pregnant mice (Balb/c x C57BL/6) were collected. Dendritic cells were purified from Balb/c mice spleens and pulsed with antigen during overnight culture. In some cultures, decidual supernatant was added at 5%, 10%, or 20% final concentration. Endometrial culture supernatant-treated DCs served as a control. Antigen-pulsed DCs were injected into the front footpads of syngeneic mice. MAIN OUTCOME MEASURE(S): Lymph nodes of primed mice were removed 5 days after DC injection. Antigen-specific proliferation and interleukin-10 and interferon gamma production by lymphocytes were measured by (3)H-Thymidine incorporation and ELISA, respectively. RESULT(S): The results showed that decidual culture supernatant markedly blocked in vivo antigen presentation by DCs and inhibited their capacity to induce interferon gamma (but not interleukin-10) production by primed lymphocytes. CONCLUSION(S): It seems that soluble factors produced by decidual cells are important mediators of immunoregulation at the feto-maternal interface, which provide the two fundamental requirements for protection of the semiallogenic fetus, namely immunologic tolerance and predominance of T helper 2 immunity, through modulation of DCs function.

The efficient isolation of murine splenic dendritic cells and their cytochemical features.

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.