Cross-sectional analysis reveals autoantibody signatures associated with COVID-19 severity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with increased levels of autoantibodies targeting immunological proteins such as cytokines and chemokines. Reports further indicate that COVID-19 patients may develop a broad spectrum of autoimmune diseases due to reasons not fully understood. Even so, the landscape of autoantibodies induced by SARS-CoV-2 infection remains uncharted territory. To gain more insight, we carried out a comprehensive assessment of autoantibodies known to be linked to diverse autoimmune diseases observed in COVID-19 patients in a cohort of 231 individuals, of which 161 were COVID-19 patients (72 with mild, 61 moderate, and 28 with severe disease) and 70 were healthy controls. Dysregulated IgG and IgA autoantibody signatures, characterized mainly by elevated concentrations, occurred predominantly in patients with moderate or severe COVID-19 infection. Autoantibody levels often accompanied anti-SARS-CoV-2 antibody concentrations while stratifying COVID-19 severity as indicated by random forest and principal component analyses. Furthermore, while young versus elderly COVID-19 patients showed only slight differences in autoantibody levels, elderly patients with severe disease presented higher IgG autoantibody concentrations than young individuals with severe COVID-19. This work maps the intersection of COVID-19 and autoimmunity by demonstrating the dysregulation of multiple autoantibodies triggered during SARS-CoV-2 infection. Thus, this cross-sectional study suggests that SARS-CoV-2 infection induces autoantibody signatures associated with COVID-19 severity and several autoantibodies that can be used as biomarkers of COVID-19 severity, indicating autoantibodies as potential therapeutical targets for these patients.

MS-Driven Metabolic Alterations Are Recapitulated in iPSC-Derived Astrocytes.

OBJECTIVE: Astrocytes play a significant role in the pathology of multiple sclerosis (MS). Nevertheless, for ethical reasons, most studies in these cells were performed using the Experimental Autoimmune Encephalomyelitis model. As there are significant differences between human and mouse cells, we aimed here to better characterize astrocytes from patients with MS (PwMS), focusing mainly on mitochondrial function and cell metabolism. METHODS: We obtained and characterized induced pluripotent stem cell (iPSC)-derived astrocytes from three PwMS and three unaffected controls, and performed electron microscopy, flow cytometry, cytokine and glutamate measurements, gene expression, in situ respiration, and metabolomics. We validated our findings using a single-nuclei RNA sequencing dataset. RESULTS: We detected several differences in MS astrocytes including: (i) enrichment of genes associated with neurodegeneration, (ii) increased mitochondrial fission, (iii) increased production of superoxide and MS-related proinflammatory chemokines, (iv) impaired uptake and enhanced release of glutamate, (v) increased electron transport capacity and proton leak, in line with the increased oxidative stress, and (vi) a distinct metabolic profile, with a deficiency in amino acid catabolism and increased sphingolipid metabolism, which have already been linked to MS. INTERPRETATION: Here we describe the metabolic profile of iPSC-derived astrocytes from PwMS and validate this model as a very powerful tool to study disease mechanisms and to perform non-invasive drug targeting assays in vitro. Our findings recapitulate several disease features described in patients and provide new mechanistic insights into the metabolic rewiring of astrocytes in MS, which could be targeted in future therapeutic studies. ANN NEUROL 2022;91:652-669.

Photobiomodulation Reduces the Cytokine Storm Syndrome Associated with COVID-19 in the Zebrafish Model.

Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a and coa1) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.

Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

Butyrate is a short-chain fatty acid derived from the metabolism of indigestible carbohydrates by the gut microbiota. Butyrate contributes to gut homeostasis, but it may also control inflammatory responses and host physiology in other tissues. Butyrate inhibits histone deacetylases, thereby affecting gene transcription, and also signals through the metabolite-sensing G protein receptor (GPR)109a. We produced an mAb to mouse GPR109a and found high expression on podocytes in the kidney. Wild-type and Gpr109a-/- mice were induced to develop nephropathy by a single injection of Adriamycin and treated with sodium butyrate or high butyrate-releasing high-amylose maize starch diet. Butyrate improved proteinuria by preserving podocyte at glomerular basement membrane and attenuated glomerulosclerosis and tissue inflammation. This protective phenotype was associated with increased podocyte-related proteins and a normalized pattern of acetylation and methylation at promoter sites of genes essential for podocyte function. We found that GPR109a is expressed by podocytes, and the use of Gpr109a-/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Mariño, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

A high level of urinary retinol-binding protein is associated with cytomegalovirus infection in kidney transplantation.

The indirect effects of cytomegalovirus (CMV) viremia can be related to chronic changes in renal allograft structure, but its real impact in early and late graft function remains speculative. A total of 159 patients undergoing renal transplantation using a preemptive therapeutic strategy to prevent CMV disease were included in the present study. The patients were prospectively followed, with serial measurements of urinary retinol-binding protein (uRBP), a marker of proximal tubule injury. uRBP levels and their dynamic performance were compared according to CMV viremia and the 5-year estimated glomerular filtration rate (eGFR), as measured with the modification of diet in renal disease (MDRD) equation. CMV viremia was detected in 79.9% of the patients, with high uRBP levels being detected in 76.0% of these patients (compared with 40.7% in CMV-, P=0.005). High uRBP was associated with male recipients (P=0.02), the number of mismatches (P=0.02) and CMV infection (P=0.001). Five-year eGFR was worse in patients with high uRBP levels (50.3 ± 25.8 compared with 59.8 ± 26.4 ml/min, P=0.04). In a multivariate model, eGFR <60 ml/min was associated with donor age (P<0.001), the number of mismatches (P=0.04), thymoglobulin dose (P=0.02), the presence of and time with delayed graft function (DGF) (P=0.005 and P=0.04), 1-month tacrolimus levels (P=0.03), and uRBP levels after CMV treatment (P=0.01). Patients with CMV viremia in whom uRBP levels were normalized up to 3 months after treatment showed significantly better 5-year eGFR than those in whom uRBP remained high: 61.0 ± 24.2 compared with 42.3 ± 23.9 ml/min, P<0.001. CMV viremia was associated with high uRBP levels, which represent a profile of proximal tubule injury, and the dynamic performance of uRBP after treatment was associated with long-term kidney graft function.

Acute lung injury is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile.

Nicotinic α-7 acetylcholine receptor (nAChRα7) is a critical regulator of cholinergic anti-inflammatory actions in several diseases, including acute respiratory distress syndrome (ARDS). Given the potential importance of α7nAChR as a therapeutic target, we evaluated whether PNU-282987, an α7nAChR agonist, is effective in protecting the lung against inflammation. We performed intratracheal instillation of LPS to generate acute lung injury (ALI) in C57BL/6 mice. PNU-282987 treatment, either before or after ALI induction, reduced neutrophil recruitment and IL-1ß, TNF-α, IL-6, keratinocyte chemoattractant (KC), and IL-10 cytokine levels in the bronchoalveolar lavage fluid (P < 0.05). In addition, lung NF-κB phosphorylation decreased, along with collagen fiber deposition and the number of matrix metalloproteinase-9+ and -2+ cells, whereas the number of tissue inhibitor of metalloproteinase-1+ cells increased (P < 0.05). PNU-282987 treatment also reduced lung mRNA levels and the frequency of M1 macrophages, whereas cells expressing the M2-related markers CD206 and IL-10 increased, suggesting changes in the macrophage profile. Finally, PNU-282987 improved lung function in LPS-treated animals. The collective results suggest that PNU-282987, an agonist of α7nAChR, reduces LPS-induced experimental ALI, thus supporting the notion that drugs that act on α7nAChRs should be explored for ARDS treatment in humans.-Pinheiro, N. M., Santana, F. P. R., Almeida, R. R., Guerreiro, M., Martins, M. A., Caperuto, L. C., Câmara, N. O. S., Wensing, L. A., Prado, V. F., Tibério, I. F. L. C., Prado, M. A. M., Prado, C. M. Acute lung injury is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile.

Minocycline alters expression of inflammatory markers in autonomic brain areas and ventilatory responses induced by acute hypoxia.

NEW FINDINGS: What is the central question of this study? Microglia are presumed to be the source of inflammatory mediators that contribute to hypoxia-induced neuroinflammation. However, the relationship between microglial activity during hypoxia and inflammatory responses in specific autonomic brain regions is not well understood. Therefore, we hypothesized that acute hypoxia initiates an immune response in the central nervous system elicited by an increased expression of inflammatory mediators in specific brain areas related to autonomic control. What is the main finding and its importance? Acute hypoxia initiated neuroinflammatory mechanisms specifically in brain autonomic nuclei responsible for cardiorespiratory control, i.e. the rostral ventrolateral medulla and paraventricular nucleus of the hypothalamus. Our findings emphasize the importance of microglia for the maintenance of autonomic adjustments during physiological challenges, such as hypoxia, or during cardiorespiratory reflex activation elicited by the arterial chemoreceptors. ABSTRACT: Prolonged and continuous exposure of mammals to a low oxygen environment (chronic hypoxia) elicits remarkable morphological and physiological adjustments. These include altered gene expression, increased peripheral chemosensitivity, enhanced respiratory drive and sympathoexcitation. The current study examines the hypothesis that acute hypoxia (AH) initiates an immune response in the central nervous system elicited by an increased expression of inflammatory mediators in specific brain areas related to autonomic control. Male Wistar rats pretreated with vehicle or minocycline (30 mg kg-1  day-1 for 5 days) were subjected to AH (8% O2 , balance N2 ) or normoxia (21% O2 ) for 3 h. AH increased interleukin (IL)-6, IL-1ß and matrix metalloprotease 9 (MMP9) mRNA expression in the paraventricular nucleus of the hypothalamus (PVH) and rostral ventrolateral medulla (RVLM) and tumour necrosis factor α (TNFα) in the RVLM. Treatment with minocycline, an inhibitor of microglial activation, decreased IL-1ß, TNFα and MMP9 mRNA expression in the RVLM, and increased IL-6 mRNA expression in the RVLM and PVH of rats exposed to AH. Minocycline treatment also elicited a decrease in the number of activated neurons in the RVLM/C1 neurons (expressed as Fos+ /tyrosine hydroxylase+ ), the number of Fos-activated neurons in the PVH and the increase in ventilation elicited by AH. When viewed together, these results suggest that AH modulates the expression of inflammatory mediators in autonomic brain nuclei that may be involved in the responses to chemoreceptor activation.

Serum Iron Protects from Renal Postischemic Injury.

Renal transplants remain a medical challenge, because the parameters governing allograft outcome are incompletely identified. Here, we investigated the role of serum iron in the sterile inflammation that follows kidney ischemia-reperfusion injury. In a retrospective cohort study of renal allograft recipients (n=169), increased baseline levels of serum ferritin reliably predicted a positive outcome for allografts, particularly in elderly patients. In mice, systemic iron overload protected against renal ischemia-reperfusion injury-associated sterile inflammation. Furthermore, chronic iron injection in mice prevented macrophage recruitment after inflammatory stimuli. Macrophages cultured in high-iron conditions had reduced responses to Toll-like receptor-2, -3, and -4 agonists, which associated with decreased reactive oxygen species production, increased nuclear localization of the NRF2 transcription factor, increased expression of the NRF2-related antioxidant response genes, and limited NF-κB and proinflammatory signaling. In macrophage-depleted animals, the infusion of macrophages cultured in high-iron conditions did not reconstitute AKI after ischemia-reperfusion, whereas macrophages cultured in physiologic iron conditions did. These findings identify serum iron as a critical protective factor in renal allograft outcome. Increasing serum iron levels in patients may thus improve prognosis of renal transplants.

Adipocyte mTORC1 deficiency promotes adipose tissue inflammation and NLRP3 inflammasome activation via oxidative stress and de novo ceramide synthesis.

Mechanistic target of rapamycin complex (mTORC)1 activity is increased in adipose tissue of obese insulin-resistant mice, but its role in the regulation of tissue inflammation is unknown. Herein, we investigated the effects of adipocyte mTORC1 deficiency on adipose tissue inflammation and glucose homeostasis. For this, mice with adipocyte raptor deletion and controls fed a chow or a high-fat diet were evaluated for body mass, adiposity, glucose homeostasis, and adipose tissue inflammation. Despite reducing adiposity, adipocyte mTORC1 deficiency promoted hepatic steatosis, insulin resistance, and adipose tissue inflammation (increased infiltration of macrophages, neutrophils, and B lymphocytes; crown-like structure density; TNF-α, interleukin (IL)-6, and monocyte chemoattractant protein 1 expression; IL-1ß protein content; lipid peroxidation; and de novo ceramide synthesis). The anti-oxidant, N-acetylcysteine, partially attenuated, whereas treatment with de novo ceramide synthesis inhibitor, myriocin, completely blocked adipose tissue inflammation and nucleotide oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3)-inflammasome activation, but not hepatic steatosis and insulin resistance induced by adipocyte raptor deletion. Rosiglitazone treatment, however, completely abrogated insulin resistance induced by adipocyte raptor deletion. In conclusion, adipocyte mTORC1 deficiency induces adipose tissue inflammation and NLRP3-inflammasome activation by promoting oxidative stress and de novo ceramide synthesis. Such adipose tissue inflammation, however, is not an underlying cause of the insulin resistance displayed by these mice.

NLRP3 Inflammasome Mediates Aldosterone-Induced Vascular Damage.

BACKGROUND: Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. METHODS: We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout (NLRP3-/-), caspase-1 knockout (Casp-1-/-), and interleukin-1 receptor knockout (IL-1R-/-) mice treated with vehicle or aldosterone (600 µg·kg-1·d-1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. RESULTS: Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1ß levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1ß secretion by bone marrow-derived macrophages by activating nuclear factor-κB signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1ß in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. CONCLUSIONS: Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels.

Prophylactic and therapeutic treatment with the flavonone sakuranetin ameliorates LPS-induced acute lung injury.

Sakuranetin is the main isolate flavonoid from Baccharis retusa (Asteraceae) leaves and exhibits anti-inflammatory and antioxidative activities. Acute respiratory distress syndrome is an acute failure of the respiratory system for which effective treatment is urgently necessary. This study investigated the preventive and therapeutic effects of sakuranetin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Animals were treated with intranasal sakuranetin 30 min before or 6 h after instillation of LPS. Twenty-four hours after ALI was induced, lung function, inflammation, macrophages population markers, collagen fiber deposition, the extent of oxidative stress, and the expression of matrix metalloprotease-9 (MMP-9), tissue inhibitor of MMP-9 (TIMP-1) and NF-κB were evaluated. The animals began to show lung alterations 6 h after LPS instillation, and these changes persisted until 24 h after LPS administration. Preventive and therapeutic treatment with sakuranetin reduced the neutrophils in the peripheral blood and in the bronchial alveolar lavage. Sakuranetin treatment also reduced macrophage populations, particularly that of M1-like macrophages. In addition, sakurnaetin treatment reduced keratinocyte-derived chemokines (IL-8 homolog) and NF-κB levels, collagen fiber formation, MMM-9 and TIMP-1-positive cells, and oxidative stress in lung tissues compared with LPS animals treated with vehicle. Finally, sakuranetin treatment also reduced total protein, and the levels of TNF-α and IL-1ß in the lung. This study shows that sakuranetin prevented and reduced pulmonary inflammation induced by LPS. Because sakuranetin modulates oxidative stress, the NF-κB pathway, and lung function, it may constitute a novel therapeutic candidate to prevent and treat ALI.

MATE-1 modulation by kinin B1 receptor enhances cisplatin efflux from renal cells.

Cisplatin is a drug widely used in chemotherapy that frequently causes severe renal dysfunction. Organic transporters have an important role to control the absorption and excretion of cisplatin in renal cells. Deletion and blockage of kinin B1 receptor has already been show to protect against cisplatin-induced acute kidney injury. To test whether it exerts its protective function by modulating the organic transporters in kidney, we studied kinin B1 receptor knockout mice and treatment with a receptor antagonist at basal state and in presence of cisplatin. Cisplatin administration caused downregulation of renal organic transporters; in B1 receptor knockout mice, this downregulation of organic transporters in kidney was absent; and treatment by a B1 receptor antagonist attenuated the downregulation of the transporter MATE-1. Moreover, kinin B1 receptor deletion and blockage at basal state resulted in higher renal expression of MATE-1. Moreover we observed that kinin B1 receptor deletion and blockage result in less accumulation of platinum in renal tissue. Thus, we propose that B1 receptor deletion and blockage protect the kidney from cisplatin-induced acute kidney injury by upregulating the expression of MATE-1, thereby increasing the efflux of cisplatin from renal cells.

Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis.

Stem cells from human exfoliated deciduous teeth (SHED) have great therapeutic potential and here, by the first time, we evaluated their immunomodulatory effect on experimental model of autoimmune encephalomyelitis (EAE). Specifically, we investigated the effect of SHED administration on clinical signs and cellular patterns in EAE model using Foxp3 GFP + transgenic mice (C57Bl/6-Foxp3GFP). The results showed that SHED infusion ameliorated EAE clinical score with reduced number of infiltrating IFN-γ+CD8+, IL-4+CD8+, IFN-γ+CD4+ and IL-4+CD4+ T cells into the central nervous system (CNS). In addition, we observed that SHED promoted a significant increase in CD4+FOXP3+ T cells population in the spleen of EAE-affected animals. Taken together, our results provide strong evidence that SHED can modulate peripherally the CD4+ T cell responses suggesting that SHED would be explored as part of cellular therapy in autoimmune diseases associated with CNS.

Mast cells control insulitis and increase Treg cells to confer protection against STZ-induced type 1 diabetes in mice.

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-ß, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.

Hyperglycemia reduces integrin subunits alpha v and alpha 5 on the surface of dermal fibroblasts contributing to deficient migration.

Deficient wound healing is a common multifactorial complication in diabetic patients, but the cellular and molecular mechanisms involved are poorly defined. In the present study, we analyzed the effects of hyperglycemia on integrins expression in rat dermal fibroblasts and addressed its role in cell adhesion and migration. Diabetes Mellitus was induced in rats by streptozotocin injection and maintained for 30 days. Primary cultures of dermal fibroblasts from control and diabetic rats were maintained under low glucose (5 mM D-glucose) or high glucose (30 mM D-glucose) for 7 days. Cell adhesion and migration were studied by kymography, transwell, and time-lapse assays, and the expressions of integrin subunits αv and α5 were studied by immunocytochemistry and western blotting. Fibroblasts derived from diabetic rats confirmed a reduced migration speed and delayed spreading compared to fibroblasts derived from control rats. The membrane fraction of diabetic-derived fibroblasts showed a decrease of integrin subunits α5 and αv, which was confirmed by immunocytochemistry assays. A reduction in the pericellular fibronectin matrix was also observed. The exposure of diabetic-derived cells to a higher concentration of exogenous fibronectin improved migration velocity and the expression of αv but did not completely restore their migration capacity. In conclusion, the mechanisms involved in the deleterious effects of Diabetes Mellitus on wound healing include the ability of fibroblasts to secrete and to adhere to fibronectin.

Presence of arteriolar hyalinosis in post-reperfusion biopsies represents an additional risk to ischaemic injury in renal transplant.

AIM: The role of post-reperfusion biopsy findings as a predictor of early and long-term graft function and survival is still a target of research. METHODS: We analyzed data from 136 post-reperfusion biopsies performed in deceased donor renal transplanted patients from November 2008 to May 2012. We analyzed the presence of acute tubular necrosis (ATN), arteriolar hyalinosis (AH), intimal thickness (IT), interstitial fibrosis (IF) and glomerulosclerosis (GS). We also analyzed the impact of donor features on the following outcomes: delayed graft function (DGF) and chronic allograft dysfunction defined as eGFR < 60 mL/min at 1 year. RESULTS: The mean donor age was 41 years, 26% of whom were extended criteria donors (ECD), 33% had hypertension and 50% had cerebral vascular accident (CVA) as the cause of death. ATN was present in 87% of these biopsies, AH in 31%, IF in 21%, IT in 27% and GS in 32%. DGF occurred in 80% and chronic allograft dysfunction was present in 53%. AH was the only histological finding associated with DGF and chronic allograft dysfunction at 1 year. Patients with AH had a lower eGFR at 1 year than patients without it (49.8 mL/min × 64.5 mL/min, P = 0.02). In the multivariate analysis, risk variables for development of chronic graft dysfunction were male sex (odds ratio [OR] = 3.159 [CI: 1.22-8.16]; P = 0.018), acute rejection (OR = 8.91 [CI: 2.21-35.92]; P = 0.002), donor hypertension (OR = 2.94 [CI: 1.10-7.84]; P = 0.031), AH (OR = 3.96 [CI: 1.46-10.70]; P = 0.007) and eGFR at discharge (OR = 0.96 [CI: 0.93-0.98]; P = 0.005). In multivariate analysis, risk factors for AH were donor age ≥ 50 years (OR = 2.46 [CI: 1.10-5.44]; P = 0.027) and CVA as the cause of donor death (OR = 2.33 [CI: 1.05-5.15]; P = 0.007). CONCLUSION: The presence of AH in post-reperfusion biopsies is a marker of ageing and vascular disease and was associated with DGF and a one year poorer renal function. AH in donor biopsies superimposed to long ischaemic time is a predictor of renal function. The management of immunosuppression based on the presence of AH in post-reperfusion biopsy could be useful to improve long term graft function.

Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury.

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

Leptin deficiency impairs maturation of dendritic cells and enhances induction of regulatory T and Th17 cells.

Leptin is an adipose-secreted hormone that plays an important role in both metabolism and immunity. Leptin has been shown to induce Th1-cell polarization and inhibit Th2-cell responses. Additionally, leptin induces Th17-cell responses, inhibits regulatory T (Treg) cells and modulates autoimmune diseases. Here, we investigated whether leptin mediates its activity on T cells by influencing dendritic cells (DCs) to promote Th17 and Treg-cell immune responses in mice. We observed that leptin deficiency (i) reduced the expression of DC maturation markers, (ii) decreased DC production of IL-12, TNF-α, and IL-6, (iii) increased DC production of TGF-ß, and (iv) limited the capacity of DCs to induce syngeneic CD4(+) T-cell proliferation. As a consequence of this unique phenotype, DCs generated under leptin-free conditions induced Treg or TH 17 cells more efficiently than DCs generated in the presence of leptin. These data indicate important roles for leptin in DC homeostasis and the initiation and maintenance of inflammatory and regulatory immune responses by DCs.

Anti-metastatic immunotherapy based on mucosal administration of flagellin and immunomodulatory P10.

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.