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An acute kidney injury (AKI) is the most common complication following cardiac surgery, and can lead to the initiation of continuous renal replacement therapy (CRRT). However, there is still insufficient evidence for when patients should be liberated from CRRT. Proenkephalin A 119-159 (PENK) is a novel biomarker that reflects kidney function independently of other factors. This study investigated whether PENK could guide successful liberation from CRRT. Therefore, we performed a prospective, observational, single-center study at the Medical University of Vienna between July 2022 and May 2023, which included adult patients who underwent cardiac surgery for a cardiopulmonary bypass; patients on preoperative RRT were excluded. The PENK levels were measured at the time of AKI diagnosis and at the initiation of and liberation from CRRT, and were subsequently compared to determine whether the patients were successfully liberated from CRRT. We screened 61 patients with postoperative AKI; 20 patients experienced a progression of AKI requiring CRRT. The patients who were successfully liberated from CRRT had mean PENK levels of 113 ± 95.4 pmol/L, while the patients who were unsuccessfully liberated from CRRT had mean PENK levels of 290 ± 175 pmol/L (p = 0.018). For the prediction of the successful liberation from CRRT, we found an area under the curve of 0.798 (95% CI, 0.599-0.997) with an optimal threshold value of 126.7 pmol/L for PENK (Youden Index = 0.53, 95% CI, 0.10-0.76) at the time of CRRT liberation (sensitivity = 0.64, specificity = 0.89). In conclusion, PENK is a novel biomarker that has the potential to predict the successful liberation from CRRT for patients with AKI after cardiac surgery.
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Injúria Renal Aguda , Biomarcadores , Procedimentos Cirúrgicos Cardíacos , Terapia de Substituição Renal Contínua , Humanos , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/terapia , Injúria Renal Aguda/diagnóstico , Masculino , Feminino , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Idoso , Pessoa de Meia-Idade , Estudos Prospectivos , Terapia de Substituição Renal Contínua/métodos , Encefalinas/metabolismo , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/terapia , Precursores de Proteínas/metabolismo , Precursores de Proteínas/sangue , Terapia de Substituição Renal/métodosRESUMO
Primary focal segmental glomerulosclerosis (FSGS) is a disease of the podocytes and glomerulus, leading to nephrotic syndrome and progressive loss of renal function. One of the most serious aspects is its recurrence of disease in over 30% of patients following allogeneic kidney transplantation, leading to early graft loss. This research investigates the individual genetic predispositions and differences in the immune responses leading to recurrence of FSGS after transplantation. We performed exome sequencing on six patients with recurrent FSGS to identify variants in fifty-one genes and found significant variations in the alpha-2-macroglobulin (A2M). Immunoblotting was used to investigate effects of specific gene variants at the protein level. Further expression analysis identified A2M, exophilin 5 (EXPH5) and plectin (PLEC) as specific proteins linked to podocytes, endothelial cells, and the glomerulus. Subsequent protein array screening revealed the presence of non-HLA-specific antibodies, including TRIM21, after transplantation. Using Metascape for pathway and process enrichment analysis, we focused on the IL-17 signaling and chemotaxis pathways. ELISA measurements showed significantly elevated IL-17 levels in patients with recurrent FSGS (32.30 ± 9.12 pg/mL) compared to individuals with other glomerular diseases (23.16 ± 2.49 pg/mL; p < 0.01) and healthy subjects (22.28 ± 0.94 pg/mL; p < 0.01), with no significant difference in plasma CCL2/MCP-1 levels between groups. This study explores the molecular dynamics underlying recurrence of FSGS after transplantation, offering insights into potential biomarkers and therapeutic targets for the future development of individualized treatments for transplant patients.
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Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.
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Vírus BK , Vesículas Extracelulares , Infecções por Polyomavirus , Eliminação de Partículas Virais , Vírus BK/fisiologia , Vírus BK/metabolismo , Vírus BK/genética , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Infecções por Polyomavirus/virologia , Infecções por Polyomavirus/metabolismo , Replicação Viral , Transplante de Rim , Vírion/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Núcleo Celular/metabolismo , Montagem de Vírus , Infecções Tumorais por Vírus/virologia , Infecções Tumorais por Vírus/metabolismo , Transformação Celular Viral , Masculino , AnimaisRESUMO
Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca2+-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca2+-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. Kd differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and ß-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.
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Fusão de Membrana , Secretagoginas , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Secretagoginas/metabolismo , Membrana Celular/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Exocitose , Comunicação Celular , Sintaxina 1/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Early detection of acute kidney injury (AKI) is crucial for timely intervention and improved patient outcomes after cardiac surgery. This study aimed to evaluate the potential of urinary collectrin as a novel biomarker for AKI in this patient population. METHODS: In this prospective, observational cohort study, 63 patients undergoing elective cardiac surgery with cardiopulmonary bypass (CPB) were studied at the Medical University of Vienna between 2016 and 2018. We collected urine samples prospectively at four perioperative time points, and urinary collectrin was measured using an enzyme-linked immunosorbent assay. Patients were divided into two groups, AKI and non-AKI, defined by Kidney Disease: Improving Global Outcomes Guidelines, and differences between groups were analyzed. RESULTS: Postoperative AKI was found in 19 (30%) patients. Urine sample analysis revealed an inverse correlation between urinary collectrin and creatinine and AKI stages, as well as significant changes in collectrin levels during the perioperative course. Baseline collectrin levels were 5050 ± 3294 pg/mL, decreased after the start of CPB, reached their nadir at the end of surgery, and began to recover slightly on postoperative day (POD) 1. The most effective timepoint for distinguishing between AKI and non-AKI patients based on collectrin levels was POD 1, with collectrin levels of 2190 ± 3728 pg/mL in AKI patients and 3768 ± 3435 pg/mL in non-AKI patients (p = 0.01). CONCLUSIONS: Urinary collectrin shows promise as a novel biomarker for the early detection of AKI in patients undergoing cardiac surgery on CPB. Its dynamic changes throughout the perioperative period, especially on POD 1, provide valuable insights for timely diagnosis and intervention. Further research and validation studies are needed to confirm its clinical usefulness and potential impact on patient outcomes.
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Polyomaviruses are widespread, with BK viruses being most common in humans who require immunosuppression due to allotransplantation. Infection with BK polyomavirus (BKV) may manifest as BK virus-associated nephropathy and hemorrhagic cystitis. Established diagnostic methods include the detection of polyomavirus in urine and blood by PCR and in tissue biopsies via immunohistochemistry. In this study, 79 patients with pathological renal retention parameters and acute kidney injury (AKI) were screened for BK polyomavirus replication by RNA extraction, reverse transcription, and virus-specific qPCR in urine sediment cells. A short fragment of the VP2 coding region was the target of qPCR amplification; patients with (n = 31) and without (n = 48) a history of renal transplantation were included. Urine sediment cell immunofluorescence staining for VP1 BK polyomavirus protein was performed using confocal microscopy. In 22 patients with acute renal injury, urinary sediment cells from 11 participants with kidney transplantation (KTX) and from 11 non-kidney transplanted patients (nonKTX) were positive for BK virus replication. BK virus copies were found more frequently in patients with AKI stage III (n = 14). Higher copy numbers were detected in KTX patients having experienced BK polyoma-nephropathy (BKPyVAN) in the past or diagnosed recently by histology (5.6 × 109-3.1 × 1010). One patient developed BK viremia following delayed graft function (DGF) with BK virus-positive urine sediment. In nonKTX patients with BK copies, decoy cells were absent; however, positive staining of cells was found with epithelial morphology. Decoy cells were only found in KTX patients with BKPyVAN. In AKI, damage to the tubular epithelium itself may render the epithelial cells more permissive for polyoma replication. This non-invasive diagnostic approach to assess BK polyomavirus replication in urine sediment cells has the potential to identify KTX patients at risk for viremia and BKPyVAN during AKI. This method might serve as a valuable screening tool for close monitoring and tailored immunosuppression decisions.
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Injúria Renal Aguda , Vírus BK , Transplante de Rim , Infecções por Polyomavirus , Polyomavirus , Humanos , Vírus BK/genética , Viremia/diagnóstico , Viremia/etiologia , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Rim/patologia , Injúria Renal Aguda/etiologiaRESUMO
Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6-32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68-94% of them losing parts of their DNA and exhibiting membrane damage, appearing as "ghost cells" but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.
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Mycophenolic acid (MPA) is a widely used immunosuppressive agent and exerts its effect by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH), the main regulating enzyme of purine metabolism. However, significant unexplained differences in the efficacy and tolerability of MPA therapy pose a clinical challenge. Therefore, broad pharmacogenetic, pharmacokinetic, and pharmacodynamic approaches are needed to individualize MPA therapy. In this prospective cohort study including 277 renal transplant recipients, IMPDH2 rs11706052 SNP status was assessed by genetic sequencing, and plasma MPA trough levels were determined by HPLC and IMPDH enzyme activity in peripheral blood mononuclear cells (PBMCs) by liquid chromatography-mass spectrometry. Among the 277 patients, 84 were identified with episodes of biopsy-proven rejection (BPR). No association was found between rs11706052 SNP status and graft rejection (OR 1.808, and 95% CI, 0.939 to 3.479; p = 0.076). Furthermore, there was no association between MPA plasma levels and BPR (p = 0.69). However, the patients with graft rejection had a significantly higher predose IMPDH activity in PBMCs compared to the controls without rejection at the time of biopsy (110.1 ± 50.2 vs. 95.2 ± 45.4 pmol/h; p = 0.001), and relative to the baseline IMPDH activity before transplantation (p = 0.042). Our results suggest that individualization of MPA therapy, particularly through pharmacodynamic monitoring of IMPDH activity in PBMCs, has the potential to improve the clinical outcomes of transplant patients.
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Background: The SARS-CoV-2 pandemic increased mortality and morbidity among immunocompromised populations. Vaccination is the most important preventive measure, however, its effectiveness among patients depending on maintenance immunoglobulin G (IgG) apheresis to control autoimmune disease activity is unknown. We aimed to examine the humoral immune response after mRNA-1273 Moderna® vaccination in immunoapheresis patients. Methods: We prospectively monitored SARS-CoV-2 IgG spike (S) protein antibody levels before and after each IgG (exposure) or lipid (LDL) apheresis (controls) over 12 weeks and once after 24 weeks. Primary outcome was the difference of change of SARS-CoV-2 IgG S antibody levels from vaccination until week 12, secondary outcome was the difference of change of SARS-CoV-2 IgG S antibody levels by apheresis treatments across groups. Results: We included 6 IgG and 18 LDL apheresis patients. After 12 weeks the median SARS-CoV-2 IgG S antibody level was 115 (IQR: 0.74, 258) in the IgG and 1216 (IQR: 788, 2178) in the LDL group (p=0.03). Median SARS-CoV-2 IgG S antibody reduction by apheresis was 76.4 vs. 23.7% in the IgG and LDL group (p=0.04). The average post- vs. pre-treatment SARS-CoV-2 IgG S antibody rebound in the IgG group vs. the LDL group was 46.1 and 6.44%/week from prior until week 12 visit. Conclusions: IgG apheresis patients had lower SARS-CoV-2 IgG S antibody levels compared to LDL apheresis patients, but recovered appropriately between treatment sessions. We believe that IgG apheresis itself probably has less effect on maintaining the immune response compared to concomitant immunosuppressive drugs. Immunization is recommended independent of apheresis treatment.
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COVID-19 , Imunoglobulina G , Vacina de mRNA-1273 contra 2019-nCoV , Anticorpos Antivirais , Formação de Anticorpos , COVID-19/prevenção & controle , Estudos de Coortes , Humanos , Lipídeos , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
Background: Acute kidney injury (AKI) is a serious condition associated with chronic kidney disease, dialysis requirement and a high risk of death. However, there are specialized repair mechanisms for the nephron, and migrated committed progenitor cells are the key players. Previous work has described a positive association between renal recovery and the excretion of tubular progenitor cells in the urine of kidney transplant recipients. The aim of this work was to describe such structures in non-transplanted AKI patients and to focus on their differentiation. Methods: Morning urine was obtained from four patients with AKI stage 3 and need for RRT on a consecutive basis. Urine sediment gene expression was performed to assess which part of the tubular or glomerular segment was affected by injury, along with measurement of neprilysin. Urine output and sediment morphology were monitored, viable hyperplastic tubular epithelial clusters were isolated and characterized by antibody or cultured in vitro. These cells were monitored by phase contrast microscopy, gene, and protein expression over 9 days by qPCR and confocal immunofluorescence. Furthermore, UMOD secretion into the supernatant was quantitatively measured. Results: Urinary neprilysin decreased rapidly with increasing urinary volume in ischemic, toxic, nephritic, and infection-associated AKI, whereas the decrease in sCr required at least 2 weeks. While urine output increased, dead cells were present in the sediment along with debris followed by hyperplastic agglomerates. Monitoring of urine sediment for tubular cell-specific gene transcript levels NPHS2 (podocyte), AQP1 and AQP6 (proximal tubule), and SLC12A1 (distal tubule) by qPCR revealed different components depending on the cause of AKI. Confocal immunofluorescence staining confirmed the presence of intact nephron-specific epithelial cells, some of which appeared in clusters expressing AQP1 and PAX8 and were 53% positive for the stem cell marker PROM1. Isolated tubule epithelial progenitor cells were grown in vitro, expanded, and reached confluence within 5-7 days, while the expression of AQP1 and UMOD increased, whereas PROM1 and Ki67 decreased. This was accompanied by a change in cell morphology from a disproportionately high nuclear/cytoplasmic ratio at day 2-7 with mitotic figures. In contrast, an apoptotic morphology of approximately 30% was found at day 9 with the appearance of multinucleated cells that were associable with different regions of the nephron tubule by marker proteins. At the same time, UMOD was detected in the culture supernatant. Conclusion: During renal recovery, a high replicatory potential of tubular epithelial progenitor cells is found in urine. In vitro expansion and gene expression show differentiation into tubular cells with marker proteins specific for different nephron regions.
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Injúria Renal Aguda , Neprilisina , Humanos , Neprilisina/metabolismo , Injúria Renal Aguda/metabolismo , Rim , Túbulos Renais Proximais/metabolismo , Isquemia/metabolismoRESUMO
Acute kidney injury (AKI) is a leading complication in hospitalized patients of different disciplines due to various aetiologies and is associated with the risk of chronic kidney disease, the need for dialysis and death. Since nephrons are not supplied with pain signals, kidney injury is mostly diagnosed by serum creatinine with a time delay. Recent work has shown that certain urinary biomarkers are available for early detection of AKI. In total, 155 subjects, including 102 patients with AKI at various stages and 53 subjects without AKI, were enrolled, and their course and laboratory data were recorded. Urinary collectrin (TMEM27) was measured by a commercially available ELISA assay. Changes in serum creatinine were used to determine AKI stage. Patients with AKI presented with significantly lower levels of urinary collectrin compared to patients without AKI (1597 ± 1827 pg/mL vs. 2855 ± 2073; p = 0.001). Collectrin was found to inversely correlate with serum creatinine and stages of AKI. Collectrin levels were lowest in AKI stage III (1576 ± 1686 pg/mL; p = 0.001) and also significantly lower in stage II (1616 ± 2148 pg/mL; p = 0.021) and stage I (1630 ± 1956 pg/mL; p = 0.019) compared to subjects without AKI. An optimal minimum collectrin cut-off value of 1606 [95% CI 1258 to 1954] pg/mL was determined to detect AKI. In conclusion, urinary collectrin represents an indicator of AKI that, unlike all other established AKI biomarkers, decreases with stage of AKI and thus may be associated with a novel pathogenic pathway.
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SARS-CoV-2 variants of concern (VOCs) have caused a significant increase in infections worldwide. Despite high vaccination rates in industrialized countries, the fourth VOC, Omicron, has outpaced the Delta variant and is causing breakthrough infections in individuals with two booster vaccinations. While the magnitude of morbidity and lethality is lower in Omicron, the infection rate and global spread are rapid. Using a specific IgG multipanel-ELISA with the spike protein's receptor-binding domain (RBD) from recombinant Alpha, Gamma, Delta, and Omicron variants, sera from health-care workers from the Medical University of Vienna were tested pre-pandemic and post-vaccination (BNT162b2; ChAdOx1 nCoV-19). The cohort was continuously monitored by SARS-CoV-2 testing and commercial nucleocapsid IgG ELISA. RBD IgG ELISA showed significantly lower reactivity against the Omicron-RBD compared to the Alpha variant in all individuals (p < 0.001). IgG levels were independent of sex, but were significantly higher in BNT162b2 recipients <45 years of age for Alpha, Gamma, and Delta (p < 0.001; p = 0.040; p = 0.004, respectively). Pre-pandemic cross-reactive anti-Omicron IgG was detected in 31 individuals and was increased 8.78-fold after vaccination, regardless of vaccine type. The low anti-RBD Omicron IgG level could explain the breakthrough infections and their presence could also contribute to a milder COVID-19 course by cross-reactivity and broadening the adaptive immunity.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Teste para COVID-19 , ChAdOx1 nCoV-19 , Humanos , Imunoglobulina G , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , VacinaçãoRESUMO
First-generation vaccines against SARS-CoV-2 do not provide adequate immune protection. Therefore, we engineered a divalent gene construct combining the receptor-binding domain (RBD) of the spike protein and the immunodominant region of the viral nucleocapsid. This fusion protein was produced in either E. coli or a recombinant baculovirus system. Subsequently, the fusion protein was mixed with adjuvant and administered to mice in a prime-booster mode. Mice (72%) produced an IgG response against both proteins (titer: 10-4-10-5) 14 days after the first booster injection, which was increased to 100% by a second booster. Comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed IgGs beyond 90 days. In addition, cytolytic effector cell molecules were increased in lymphocytes isolated from peripheral blood. Ex vivo stimulation of T cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 among the CD4+ and CD8+ T cells of vaccinated mice. No side effect was documented in the central nervous system. Cumulatively, these data represent a proof-of-principle approach alternative to existing mRNA vaccination strategies.
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Acute kidney injury (AKI) is an abrupt deterioration of renal function often caused by severe clinical disease such as sepsis, and patients require intensive care. Acute-phase parameters for systemic inflammation are well established and used in routine clinical diagnosis, but no such parameters are known for AKI and inflammation at the local site of tissue damage, namely the nephron. Therefore, we sought to investigate complement factors C3a/C3 in urine and urinary sediment cells. After the development of a C3a/C3-specific mouse monoclonal antibody (3F7E2), urine excretion from ICU sepsis patients was examined by dot blot and immunoblotting. This C3a/C3 ELISA and a C3a ELISA were used to obtain quantitative data over 24 hours for 6 consecutive days. Urine sediment cells were analyzed for topology of expression. Patients with severe infections (n = 85) showed peak levels of C3a/C3 on the second day of ICU treatment. The majority (n = 59) showed C3a/C3 levels above 20 µg/ml at least once in the first 6 days after admission. C3a was detectable on all 6 days. Peak C3a/C3 levels correlated negatively with peak C-reactive protein (CRP) levels. No relationship was found between peak C3a/C3 with peak leukocyte count, age, or AKI stage. Analysis of urine sediment cells identified C3a/C3-producing epithelial cells with reticular staining patterns and cells with large-granular staining. Opsonized bacteria were detected in patients with urinary tract infections. In critically ill sepsis patients with AKI, urinary C3a/C3 inversely correlated with serum CRP. Whether urinary C3a/C3 has a protective function through autophagy, as previously shown for cisplatin exposure, or is a by-product of sepsis caused by pathogenic stimuli to the kidney must remain open in this study. However, our data suggest that C3a/C3 may function as an inverse acute-phase parameter that originates in the kidney and is detectable in urine.
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Injúria Renal Aguda/urina , Complemento C3/urina , Sepse/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto JovemRESUMO
BACKGROUND: Immunosuppression in solid organ transplantation is associated with frequent infections. Renal allograft recipients are susceptible to opportunistic infections and can acquire human cytomegalovirus (HCMV) infections even within the allograft. There, HCMV can be found in both the glomerulus and tubular cells, but is mostly restricted to specific and circumscribed sites. Therefore, not all organ infections are identifiable by immunohistology for HCMV proteins in fine needle core biopsies. Thus, we performed a urinalysis study to search for HCMV-specific RNA transcripts in the urine sediment of patients with acute kidney injury. METHODS: Urinary sediment of 90 patients with acute kidney injury (AKI), including 48 renal transplant recipients (RTX) and 42 non-transplant recipients (nRTX), was collected from morning urine for RNA extraction and reverse transcription. The copy number of HCMV transcripts was evaluated using a UL132 HCMV-specific probe set and by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Of the 48 RTX patients, ten showed HCMV copies in their urine sediment cells. Within this group, three recipients had negative HCMV serology and received an allograft from an HCMV-seropositive donor. In addition, all three RTX patients on a belatacept-based immunosuppressive regimen had HCMV transcripts in their urine. Of the 42 nRTX patients, only two had detectable HCMV transcripts in urine sediment cells and both were under immunosuppression. CONCLUSIONS: Ten immunosuppressed renal allograft recipients and two immunosuppressed non-transplant patients with AKI showed HCMV copies in urine sediment. Thus, HCMV positivity in urinary sediment appears to be associated with immunosuppression. This study describes a novel noninvasive method for detection of HCMV in urinary sediment. Whether all HCMV infections can be detected or only those with viral replication warrants further investigation.
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Injúria Renal Aguda/microbiologia , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/urina , Citomegalovirus/isolamento & purificação , Hospedeiro Imunocomprometido , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/urina , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/urina , Adulto , Idoso , Infecções por Citomegalovirus/imunologia , Feminino , Humanos , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas/imunologia , RNA Viral/urina , Reação em Cadeia da Polimerase em Tempo Real , Transplante Homólogo , Urina/microbiologiaRESUMO
PURPOSE: In primary brain tumors, the efficacy of immune-modulating therapies is still under investigation as inflammatory responses are restricted by tight immunoregulatory mechanisms in the central nervous system. Here, we measured soluble PD-L1 (sPD-L1) in the plasma of patients with recurrent glioblastoma (GBM) and recurrent WHO grade II-III glioma treated with bevacizumab-based salvage therapy. METHODS: Thirty patients with recurrent GBM and 10 patients with recurrent WHO grade II-III glioma were treated with bevacizumab-based salvage therapy at the Medical University of Vienna. Prior to each treatment cycle, EDTA plasma was drawn and sPD-L1 was measured applying a sandwich ELISA with a lower detection limit of 0.050 ng/ml. Leukocyte counts and C-reactive protein (CRP) levels were measured according to institutional practice. RESULTS: Median number of sPD-L1 measurements was 6 per patient (range: 2-24). At baseline, no significant difference in sPD-L1 concentrations was observed between WHO grade II-III glioma and GBM. Intra-patient variability of sPD-L1 concentrations was significantly higher in WHO grade II-III glioma than in GBM (p = 0.014) and tendentially higher in IDH-mutant than in IDH-wildtype glioma (p = 0.149) In WHO grade II-III glioma, sPD-L1 levels were significantly lower after one administration of bevacizumab than at baseline (median: 0.039 ng/ml vs. 0.4855 ng/ml, p = 0.036). In contrast, no significant change could be observed in patients with GBM. CONCLUSIONS: Changes in systemic inflammation markers including sPD-L1 are observable in patients with recurrent glioma under bevacizumab-based treatment and differ between WHO grade II-III glioma and GBM.
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Antígeno B7-H1/sangue , Bevacizumab/uso terapêutico , Glioma/sangue , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Idoso , Biomarcadores Tumorais/sangue , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/tratamento farmacológico , Feminino , Glioblastoma/sangue , Glioblastoma/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Chronic kidney disease patients show a high mortality in cases of a severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) infection. Thus, information on the sero-status of nephrology personnel might be crucial for patient protection; however, limited information exists about the presence of SARS-CoV2 antibodies in asymptomatic individuals. METHODS: We examined the seroprevalence of SARS-CoV2 IgG and IgM antibodies among healthcare workers of a tertiary care kidney center during the the first peak phase of the corona virus disease 2019 (COVID-19) crisis in Austria using an orthogonal test strategy and a total of 12 commercial nucleocapsid protein or spike glycoprotein-based assays as well as Western blotting and a neutralization assay. RESULTS: At baseline 60 of 235 study participants (25.5%, 95% confidence interval, CI 20.4-31.5%) were judged to be borderline positive or positive for IgM or IgG using a high sensitivity/low specificity threshold in one test system. Follow-up analysis after about 2 weeks revealed IgG positivity in 12 (5.1%, 95% CI: 2.9-8.8%) and IgM positivity in 6 (2.6%, 95% CI: 1.1-5.6) in at least one assay. Of the healthcare workers 2.1% (95% CI: 0.8-5.0%) showed IgG nucleocapsid antibodies in at least 2 assays. By contrast, positive controls with proven COVID-19 showed antibody positivity among almost all test systems. Moreover, serum samples obtained from healthcare workers did not show SARS-CoV2 neutralizing capacity, in contrast to positive controls. CONCLUSION: Using a broad spectrum of antibody tests the present study revealed inconsistent results for SARS-CoV2 seroprevalence among asymptomatic individuals, while this was not the case among COVID-19 patients. TRIAL REGISTRATION NUMBER: CONEC, ClinicalTrials.gov number NCT04347694.
Assuntos
COVID-19 , Nefrologia , Anticorpos Antivirais , Pessoal de Saúde , Humanos , SARS-CoV-2 , Estudos SoroepidemiológicosRESUMO
BACKGROUND: Acute kidney injury (AKI) predicts adverse outcomes after cardiac surgery. The accuracy of using changes in serum creatinine for diagnosis and grading of AKI is limited in the peri-operative cardiac surgical setting and AKI may be underdiagnosed due to haemodilution from cardiopulmonary bypass priming and the need for intra-operative and postoperative volume resuscitation. OBJECTIVES: To determine whether the urinary biomarker neprilysin can be used as a marker for the early detection of AKI after cardiac surgery. DESIGN: Prospective, observational cohort study. SETTING: Austrian tertiary referral centre. PATIENTS: 96 Patients undergoing elective cardiac surgery with cardiopulmonary bypass. MAIN OUTCOME MEASURES: Differences and discriminatory power of neprilysin levels early after cardiac surgery and on postoperative day 1 between patients with or without AKI, as defined by the Kidney Disease Improving Global Outcomes Group. RESULTS: AKI was found in 27% (n=26). The median neprilysin levels on postoperative day 1 were significantly higher in the AKI than in the non-AKI group, 4.0 [interquartile range (IQR): 2 to 6.25] vs. 2.0ângâml [IQR: 1.0 to 4.5], Pâ=â0.0246, respectively. In addition, the median neprilysin levels at the end of surgery were significantly different between both groups, 5.0 [IQR: 2.0 to 9.0] vs. 2.0ângâml [IQR: 1.0 to 4.0], Pâ=â0.0055, respectively. The discriminatory power of neprilysin for detecting early AKI corresponded to an area under the curve of 0.77 (95% confidence interval, 0.65 to 0.90). CONCLUSION: Urinary neprilysin has potential as a biomarker for the early detection of AKI after cardiac surgery and has comparable discriminatory power to recently studied AKI biomarkers. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov (NCT03854825, https://clinicaltrials.gov/ct2/show/NCT03854825).
Assuntos
Injúria Renal Aguda , Procedimentos Cirúrgicos Cardíacos , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/epidemiologia , Injúria Renal Aguda/etiologia , Áustria , Biomarcadores , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Creatinina , Humanos , Neprilisina , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/epidemiologia , Estudos ProspectivosRESUMO
BACKGROUND: Immune-modulatory treatments have so far shown limited clinical activity in primary brain tumours. We aimed to investigate soluble programmed death receptor ligand 1 (sPD-L1) as systemic inflammation parameter in patients with brain tumour. METHODS: EDTA plasma was collected from 81 glioma (55 glioblastoma (GBM), 26 lower-grade glioma (LGG)), 17 meningioma and 44 brain metastasis (BM) patients and 24 controls. sPD-L1 concentrations were determined by ELISA. Correlations with the local tumour microenvironment were assessed by immunohistochemical analysis for PD-L1, CD3 and CD8. RESULTS: sPD-L1 was detected in 62 out of 166 (37.7%) patients (glioma: 41/81, 50.6%; meningioma: 5/17, 29.4%; BM: 7/44, 15.9%; controls: 9/24, 37.5%; p=0.002). sPD-L1 concentrations were lower in BM than in LGG (p=0.003) or GBM (p<0.001). Membranous PD-L1 expression on tumour cells was not associated with sPD-L1 concentrations (p=0.953). sPD-L1 concentration was inversely correlated with the density of CD8+ (r=-0.713, p=0.001) and CD3+ (r=-0.484, p=0.042) tumour-infiltrating lymphocytes in LGG. sPD-L1 is correlated with neutrophil counts (r=-0.318, p=0.045) and C reactive protein levels (r=-0.363, p=0.008) in GBM. sPD-L1+ patients had longer overall survival in GBM (p=0.006) and worse OS in LGG (p=0.028). CONCLUSIONS: sPD-L1 is detectable in a fraction of patients with brain tumour. Although it is not correlated with tissue PD-L1 expression, correlations with other local and systemic inflammation parameters could be detected in LGG and GBM.
Assuntos
Antígeno B7-H1 , Neoplasias Encefálicas , Biomarcadores Tumorais , Humanos , Inflamação , Receptor de Morte Celular Programada 1 , Microambiente TumoralRESUMO
BACKGROUND: Sepsis-related acute kidney injury (AKI) is associated with high morbidity and mortality among patients. Underlying pathomechanisms include capillary leakage and fluid loss into the interstitial tissue and constant exposure to pathogens results in activation of inflammatory cascades, organ dysfunction and subsequently organ damage. METHODS: To identify novel factors that trigger sepsis-related acute kidney injury, plasma levels of Granzyme A, as representative of a lymphocyte-derived protease, and heparin-binding protein as indicator for neutrophil-derived mediators, were investigated retrospectively in 60 sepsis patients. RESULTS: While no association was found between plasma levels of lymphocyte-derived Granzyme A and the incidence of sepsis-related AKI, sepsis patients with AKI had significantly higher plasma levels of heparin-binding protein compared to those without AKI. This applies both to heparin-binding protein peak values (43.30 ± 23.34 vs. 30.25 ± 15.63 pg/mL; p = 0.005) as well as mean values (27.93 ± 14.39 vs. 22.02 ± 7.65 pg/mL; p = 0.021). Furthermore, a heparin-binding protein cut-off value of 23.89 pg/mL was established for AKI diagnosis. CONCLUSION: This study identifies the neutrophil-derived heparin-binding protein as a valuable new biomarker for AKI in sepsis. Beyond the diagnostic perspective, this offers prospect for further research on pathogenesis of AKI and novel therapeutic approaches.