Bi-specific autoantigen-T cell engagers as targeted immunotherapy for autoreactive B cell depletion in autoimmune diseases.

Introduction: In autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need. Methods: Here, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A2 receptor (PLA2R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA2R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker. Results: BiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig+ and T cells. Ex vivo, the BiAATE successfully induced T cell-dependent depletion of PLA2R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA2R antibody levels following active immunization with PLA2R. Discussion: Should this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases.

Sirt3 deficiency promotes endothelial dysfunction and aggravates renal injury.

Sirtuin 3 (SIRT3), the main deacetylase of mitochondria, modulates the acetylation levels of substrates governing metabolism and oxidative stress. In the kidney, we showed that SIRT3 affects the proper functioning of high energy-demanding cells, such as tubular cells and podocytes. Less is known about the role of SIRT3 in regulating endothelial cell function and its impact on the progression of kidney disease. Here, we found that whole body Sirt3-deficient mice exhibited reduced renal capillary density, reflecting endothelial dysfunction, and VEGFA expression compared to wild-type mice. This was paralleled by activation of hypoxia signaling, upregulation of HIF-1α and Angiopietin-2, and oxidative stress increase. These alterations did not result in kidney disease. However, when Sirt3-deficient mice were exposed to the nephrotoxic stimulus Adriamycin (ADR) they developed aggravated endothelial rarefaction, altered VEGFA signaling, and higher oxidative stress compared to wild-type mice receiving ADR. As a result, ADR-treated Sirt3-deficient mice experienced a more severe injury with exacerbated albuminuria, podocyte loss and fibrotic lesions. These data suggest that SIRT3 is a crucial regulator of renal vascular homeostasis and its dysregulation is a predisposing factor for kidney disease. By extension, our findings indicate SIRT3 as a pharmacologic target in progressive renal disease whose treatments are still imperfect.

SARS-CoV-2 spike protein induces lung endothelial cell dysfunction and thrombo-inflammation depending on the C3a/C3a receptor signalling.

The spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can interact with endothelial cells. However, no studies demonstrated the direct effect of the spike protein subunit 1 (S1) in inducing lung vascular damage and the potential mechanisms contributing to lung injury. Here, we found that S1 injection in mice transgenic for human angiotensin converting enzyme 2 (ACE2) induced early loss of lung endothelial thromboresistance at 3 days, as revealed by thrombomodulin loss and von Willebrand factor (vWF) increase. In parallel, vascular and epithelial C3 deposits and enhanced C3a receptor (C3aR) expression were observed. These changes preceded diffuse alveolar damage and lung vascular fibrin(ogen)/platelets aggregates at 7 days, as well as inflammatory cell recruitment and fibrosis. Treatment with C3aR antagonist (C3aRa) inhibited lung C3 accumulation and C3a/C3aR activation, limiting vascular thrombo-inflammation and fibrosis. Our study demonstrates that S1 triggers vascular dysfunction and activates complement system, instrumental to lung thrombo-inflammatory injury. By extension, our data indicate C3aRa as a valuable therapeutic strategy to limit S1-dependent lung pathology.

Long-term adaptive response in COVID-19 vaccine recipients and the effect of a booster dose.

We examined the immune response in subjects previously infected with SARS-CoV2 and infection-naïve 9 months after primary 2-dose COVID-19 mRNA vaccination and 3 months after the booster dose in a longitudinal cohort of healthcare workers. Nine months after primary vaccination, previously infected subjects exhibited higher residual antibody levels, with significant neutralizing activity against distinct variants compared to infection-naïve subjects. The higher humoral response was associated with higher levels of receptor binding domain (RBD)-specific IgG+ and IgA+ memory B cells. The booster dose increased neither neutralizing activity, nor the B and T cell frequencies. Conversely, infection-naïve subjects needed the booster to achieve comparable levels of neutralizing antibodies as those found in previously infected subjects after primary vaccination. The neutralizing titer correlated with anti-RBD IFNγ producing T cells, in the face of sustained B cell response. Notably, pre-pandemic samples showed high Omicron cross-reactivity. These data show the importance of the booster dose in reinforcing immunological memory and increasing circulating antibodies in infection-naïve subjects.

Low Nephron Number Induced by Maternal Protein Restriction Is Prevented by Nicotinamide Riboside Supplementation Depending on Sirtuin 3 Activation.

A reduced nephron number at birth, due to critical gestational conditions, including maternal malnutrition, is associated with the risk of developing hypertension and chronic kidney disease in adulthood. No interventions are currently available to augment nephron number. We have recently shown that sirtuin 3 (SIRT3) has an important role in dictating proper nephron endowment. The present study explored whether SIRT3 stimulation, by means of supplementation with nicotinamide riboside (NR), a precursor of the SIRT3 co-substrate nicotinamide adenine dinucleotide (NAD+), was able to improve nephron number in a murine model of a low protein (LP) diet. Our findings show that reduced nephron number in newborn mice (day 1) born to mothers fed a LP diet was associated with impaired renal SIRT3 expression, which was restored through supplementation with NR. Glomerular podocyte density, as well as the rarefaction of renal capillaries, also improved through NR administration. In mechanistic terms, the restoration of SIRT3 expression through NR was mediated by the induction of proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α). Moreover, NR restored SIRT3 activity, as shown by the reduction of the acetylation of optic atrophy 1 (OPA1) and superoxide dismutase 2 (SOD2), which resulted in improved mitochondrial morphology and protection against oxidative damage in mice born to mothers fed the LP diet. Our results provide evidence that it is feasible to prevent nephron mass shortage at birth through SIRT3 boosting during nephrogenesis, thus providing a therapeutic option to possibly limit the long-term sequelae of reduced nephron number in adulthood.

SARS-CoV-2 Spike Protein 1 Activates Microvascular Endothelial Cells and Complement System Leading to Platelet Aggregation.

Microvascular thrombosis is associated with multiorgan failure and mortality in coronavirus disease 2019 (COVID-19). Although thrombotic complications may be ascribed to the ability of SARS-CoV-2 to infect and replicate in endothelial cells, it has been poorly investigated whether, in the complexity of viral infection in the human host, specific viral elements alone can induce endothelial damage. Detection of circulating spike protein in the sera of severe COVID-19 patients was evaluated by ELISA. In vitro experiments were performed on human microvascular endothelial cells from the derma and lung exposed to SARS-CoV-2-derived spike protein 1 (S1). The expression of adhesive molecules was studied by immunofluorescence and leukocyte adhesion and platelet aggregation were assessed under flow conditions. Angiotensin converting enzyme 2 (ACE2) and AMPK expression were investigated by Western Blot analysis. In addition, S1-treated endothelial cells were incubated with anti-ACE2 blocking antibody, AMPK agonist, or complement inhibitors. Our results show that significant levels of spike protein were found in the 30.4% of severe COVID-19 patients. In vitro, the activation of endothelial cells with S1 protein, via ACE2, impaired AMPK signalling, leading to robust leukocyte recruitment due to increased adhesive molecule expression and thrombomodulin loss. This S1-induced pro-inflammatory phenotype led to exuberant C3 and C5b-9 deposition on endothelial cells, along with C3a and C5a generation that further amplified S1-induced complement activation. Functional blockade of ACE2 or complement inhibition halted S1-induced platelet aggregates by limiting von Willebrand factor and P-selectin exocytosis and expression on endothelial cells. Overall, we demonstrate that SARS-CoV-2-derived S1 is sufficient in itself to propagate inflammatory and thrombogenic processes in the microvasculature, amplified by the complement system, recapitulating the thromboembolic complications of COVID-19.

Post-translational modifications by SIRT3 de-2-hydroxyisobutyrylase activity regulate glycolysis and enable nephrogenesis.

Abnormal kidney development leads to lower nephron number, predisposing to renal diseases in adulthood. In embryonic kidneys, nephron endowment is dictated by the availability of nephron progenitors, whose self-renewal and differentiation require a relatively repressed chromatin state. More recently, NAD+-dependent deacetylase sirtuins (SIRTs) have emerged as possible regulators that link epigenetic processes to the metabolism. Here, we discovered a novel role for the NAD+-dependent deacylase SIRT3 in kidney development. In the embryonic kidney, SIRT3 was highly expressed only as a short isoform, with nuclear and extra-nuclear localisation. The nuclear SIRT3 did not act as deacetylase but exerted de-2-hydroxyisobutyrylase activity on lysine residues of histone proteins. Extra-nuclear SIRT3 regulated lysine 2-hydroxyisobutyrylation (Khib) levels of phosphofructokinase (PFK) and Sirt3 deficiency increased PFK Khib levels, inducing a glycolysis boost. This altered Khib landscape in Sirt3-/- metanephroi was associated with decreased nephron progenitors, impaired nephrogenesis and a reduced number of nephrons. These data describe an unprecedented role of SIRT3 in controlling early renal development through the regulation of epigenetics and metabolic processes.

COVID-19 and lombardy: TESTing the impact of the first wave of the pandemic.

BACKGROUND: Italy was the first western country to experience a large Coronavirus Disease 2019 (COVID-19) outbreak and the province of Bergamo experienced one of the deadliest COVID-19 outbreaks in the world. Following the peak of the epidemic in mid-March, the curve has slowly fallen thanks to the strict lockdown imposed by the Italian government on 9th March 2020. METHODS: We performed a cross-sectional study to assess the prevalence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in 423 workers in Bergamo province who returned to the workplace after the end of the Italian lockdown on 5th May 2020. To this end, we performed an enzyme-linked immunosorbent assay (ELISA) to detect the humoral response against SARS-CoV-2 and a nasopharyngeal swab to assess the presence of SARS-CoV-2 RNA by real-time reverse transcription polymerase chain reaction (rRT-PCR). As a secondary aim of the study, we validated a lateral flow immunochromatography assay (LFIA) for the detection of anti-SARS-CoV-2 antibodies. FINDINGS: ELISA identified 38.5% positive subjects, of whom 51.5% were positive for both IgG and IgM, 47.3% were positive only for IgG, but only 1.2% were positive for IgM alone. Only 23 (5.4%) participants tested positive for SARS-CoV-2 by rRT-PCR, although with high cycle thresholds (between 34 and 39), indicating a very low residual viral load that was not able to infect cultured cells. All these rRT-PCR positive subjects had already experienced seroconversion. When the ELISA was used as the comparator, the estimated specificity and sensitivity of the rapid LFIA for IgG were 98% and 92%, respectively. INTERPRETATION: the prevalence of SARS-CoV-2 infection in the province of Bergamo reached 38.5%, significantly higher than has been reported for most other regions worldwide. Few nasopharyngeal swabs tested positive in fully recovered subjects, though with a very low SARS-CoV-2 viral load, with implications for infectivity and discharge policies for positive individuals in the post-pandemic period. The rapid LFIA used in this study is a valuable tool for rapid serologic surveillance of COVID-19 for population studies. FUNDING: The study was supported by Regione Lombardia, Milano Serravalle - Milano Tangenziali S.p.A., Brembo S.p.A, and by MEI System.

Protective Effects of Human Nonrenal and Renal Stromal Cells and Their Conditioned Media in a Rat Model of Chronic Kidney Disease.

Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic option for limiting chronic kidney disease progression. Conditioned medium (CM) containing bioactive compounds could convey similar benefits, avoiding the potential risks of cell therapy. This study compared the efficacy of nonrenal and renal cell-based therapy with the corresponding CM in rats with renal mass reduction (RMR). Infusions of human kidney stromal cells (kPSCs) and CM-kPSCs, but not umbilical cord (uc) MSCs or CM-ucMSCs, reduced proteinuria and preserved podocyte number and nephrin expression in RMR rats. Glomerular fibrosis, microvascular rarefaction, and apoptosis were reduced by all treatments, while the peritubular microvascular loss was reduced by kPSCs and CM-kPSCs treatment only. Importantly, kPSCs and CM-kPSCs reduced NG2-positive pericytes, and all therapies reduced α-smooth muscle actin expression, indicating reduced myofibroblast expansion. Treatment with kPSCs also significantly inhibited the accumulation of ED1-positive macrophages in the renal interstitium of RMR rats. These findings demonstrate that the CM of ucMSCs and kPSCs confers similar renoprotection as the cells. kPSCs and CM-kPSCs may be superior in attenuating chronic renal injury as a cell source.

A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood.

Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.

Renal progenitors derived from human iPSCs engraft and restore function in a mouse model of acute kidney injury.

Acute kidney injury (AKI) is one of the most relevant health issues, leading to millions of deaths. The magnitude of the phenomenon remarks the urgent need for innovative and effective therapeutic approaches. Cell-based therapy with renal progenitor cells (RPCs) has been proposed as a possible strategy. Studies have shown the feasibility of directing embryonic stem cells or induced Pluripotent Stem Cells (iPSCs) towards nephrogenic intermediate mesoderm and metanephric mesenchyme (MM). However, the functional activity of iPSC-derived RPCs has not been tested in animal models of kidney disease. Here, through an efficient inductive protocol, we directed human iPSCs towards RPCs that robustly engrafted into damaged tubuli and restored renal function and structure in cisplatin-mice with AKI. These results demonstrate that iPSCs are a valuable source of engraftable cells with regenerative activity for kidney disease and create the basis for future applications in stem cell-based therapy.

Effects of MCP-1 inhibition by bindarit therapy in a rat model of polycystic kidney disease.

BACKGROUND/AIMS: Experimental and clinical evidence suggested that monocyte chemoattractant protein-1 (MCP-1/CCL2) has a role in the development of interstitial inflammation and renal failure in polycystic kidney disease (PKD). We investigated whether bindarit, an inhibitor of MCP-1/CCL2 synthesis, could influence the evolution of PKD in PCK rats. METHODS: PCK rats were treated from 5 to 15 weeks of age with vehicle or bindarit. Sprague-Dawley rats served as control. For in vitro studies, murine podocytes were exposed to albumin with or without bindarit. RESULTS: MCP-1 mRNA was upregulated in the kidney of PCK rats and reduced by bindarit. Treatment limited overexpression of MCP-1 protein by epithelial cells of dilated tubules and cysts, and interstitial inflammatory cells. Excessive renal accumulation of monocytes/macrophages was lowered by bindarit by 41%. Serum creatinine slightly increased in PCK rats on vehicle and was similar to controls after bindarit. Kidney and liver cysts were not affected by treatment. Bindarit significantly reduced progressive proteinuria of PCK rats. The antiproteinuric effect was associated with the restoration of the defective nephrin expression in podocytes of PCK rats. Bindarit limited podocyte foot process effacement and ameliorated slit diaphragm frequency. In cultured podocytes, bindarit reduced MCP-1 production in response to albumin and inhibited albumin-induced cytoskeletal remodeling and cell migration. CONCLUSION: This study showed that although bindarit did not prevent renal cyst growth, it limited interstitial inflammation and renal dysfunction and reduced proteinuria in PKD. Thus, bindarit could be considered a therapeutic intervention complementary to therapies specifically acting to block renal cyst growth.

Shiga toxin promotes podocyte injury in experimental hemolytic uremic syndrome via activation of the alternative pathway of complement.

Shiga toxin (Stx)-producing Escherichia coli is the offending agent of postdiarrhea-associated hemolytic uremic syndrome (HUS), a disorder of glomerular ischemic damage and widespread microvascular thrombosis. We previously documented that Stx induces glomerular complement activation, generating C3a responsible for microvascular thrombosis in experimental HUS. Here, we show that the presence of C3 deposits on podocytes is associated with podocyte damage and loss in HUS mice generated by the coinjection of Stx2 and LPS. Because podocyte adhesion to the glomerular basement membrane is mediated by integrins, the relevance of integrin-linked kinase (ILK) signals in podocyte dysfunction was evaluated. Podocyte expression of ILK increased after the injection of Stx2/LPS and preceded the upregulation of Snail and downregulation of nephrin and α-actinin-4. Factor B deficiency or pretreatment with an inhibitory antibody to factor B protected mice against Stx2/LPS-induced podocyte dysregulation. Similarly, pretreatment with a C3a receptor antagonist limited podocyte loss and changes in ILK, Snail, and α-actinin-4 expression. In cultured podocytes, treatment with C3a reduced α-actinin-4 expression and promoted ILK-dependent nuclear expression of Snail and cell motility. These results suggest that Stx-induced activation of the alternative pathway of complement and generation of C3a promotes ILK signaling, leading to podocyte dysfunction and loss in Stx-HUS.

ß-arrestin-1 drives endothelin-1-mediated podocyte activation and sustains renal injury.

Activation of endothelin-A receptor (ET(A)R) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through ß-arrestin signaling. Here, we investigated whether this pathogenetic pathway could affect podocyte phenotype in proliferative glomerular disorders. In cultured mouse podocytes, ET-1 caused loss of the podocyte differentiation marker synaptopodin and acquisition of the mesenchymal marker α-smooth muscle actin. ET-1 promoted podocyte migration via ET(A)R activation and increased ß-arrestin-1 expression. Activated ET(A)R recruited ß-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and ß-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1-induced migration as confirmed by Snail knockdown experiments. Silencing of ß-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ET(A)R-driven signaling. In vitro findings were confirmed in doxorubicin (Adriamycin)-induced nephropathy. Mice receiving Adriamycin developed renal injury with loss of podocytes and hyperplastic lesion formation; ß-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ET(A)R antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular ß-arrestin-1 and Snail. Increased ß-arrestin-1 levels in podocytes retrieved from crescents of patients with proliferative glomerulopathies confirmed the translational relevance of these findings and suggest the therapeutic potential of ET(A)R antagonism for a group of diseases still needing a specific treatment.

Erythropoietin, but not the correction of anemia alone, protects from chronic kidney allograft injury.

Anemia can contribute to chronic allograft injury by limiting oxygen delivery to tissues, particularly in the tubulointerstitium. To determine mechanisms by which erythropoietin (EPO) prevents chronic allograft injury we utilized a rat model of full MHC-mismatched kidney transplantation (Wistar Furth donor and Lewis recipients) with removal of the native kidneys. EPO treatment entirely corrected post-transplant anemia. Control rats developed progressive proteinuria and graft dysfunction, tubulointerstitial damage, inflammatory cell infiltration, and glomerulosclerosis, all prevented by EPO. Normalization of post-transplant hemoglobin levels by blood transfusions, however, had no impact on chronic allograft injury, indicating that EPO-mediated graft protection went beyond the correction of anemia. Compared to syngeneic grafts, control allografts had loss of peritubular capillaries, higher tubular apoptosis, tubular and glomerular oxidative injury, and reduced expression of podocyte nephrin; all prevented by EPO treatment. The effects of EPO were associated with preservation of intragraft expression of angiogenic factors, upregulation of the anti-apoptotic factor p-Akt in tubuli, and increased expression of Bcl-2. Inhibition of p-Akt by Wortmannin partially antagonized the effect of EPO on allograft injury and tubular apoptosis, and prevented EPO-induced Bcl-2 upregulation. Thus non-erythropoietic derivatives of EPO may be useful to prevent chronic renal allograft injury.

Alternative pathway activation of complement by Shiga toxin promotes exuberant C3a formation that triggers microvascular thrombosis.

Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti-P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.

Propionyl-L-carnitine prevents early graft dysfunction in allogeneic rat kidney transplantation.

Ischemia-reperfusion injury is an important cause of graft failure. Because carnitine regulates substrate flux and energy balance across membranes which may be deranged in ischemia we determined whether its use was effective in preventing kidney injury in an allogeneic transplant model. Brown Norway rats received a Lewis rat kidney transplant and were then treated with cyclosporine A to avoid rejection. The grafts were stored in Belzer solution supplemented with propionyl-L-carnitine during the cold ischemia period. Compared to rats receiving untreated kidneys but with equal cold ischemia times, the post-transplant serum creatinine values of the carnitine-treated transplants were significantly lower. Histological evaluation 16 h after transplant showed that propionyl-L-carnitine significantly inhibited tubular necrosis and neutrophil infiltration of the allografts and improved the 3 month graft survival. Treated transplants also had decreased lipid peroxidation, inducible nitric oxide synthase expression and protein nitration compared to the untreated grafts. Post-transplant serum creatinine levels were significantly reduced and graft survival was slightly prolonged in rats not receiving cyclosporine A treatment and transplanted with a kidney treated with propionyl-L-carnitine. The efficacy of propionyl-L-carnitine to modulate ischemia-reperfusion injury during transplantation suggests that its use in human transplantation is worth testing.

Fractalkine and CX3CR1 mediate leukocyte capture by endothelium in response to Shiga toxin.

Shiga toxins (Stx) are the virulence factors of enterohemorrhagic Escherichia coli O157:H7, a worldwide emerging diarrheal pathogen, which precipitates postdiarrheal hemolytic uremic syndrome, the leading cause of acute renal failure in children. In this study, we show that Stx2 triggered expression of fractalkine (FKN), a CX3C transmembrane chemokine, acting as both adhesion counterreceptor on endothelial cells and soluble chemoattractant. Stx2 caused in HUVEC expression of FKN mRNA and protein, which promoted leukocyte capture, ablated by Abs to either endothelial FKN or leukocyte CX3CR1 receptor. Exposure of human glomerular endothelial cells to Stx2 recapitulated its FKN-inducing activity and FKN-mediated leukocyte adhesion. Both processes required phosphorylation of Src-family protein tyrosine kinase and p38 MAPK in endothelial cells. Furthermore, they depended on nuclear import of NF-kappaB and other stress-responsive transcription factors. Inhibition of their nuclear import with the cell-penetrating SN50 peptide reduced FKN mRNA levels and FKN-mediated leukocyte capture by endothelial cells. Adenoviral overexpression of IkappaBalpha inhibited FKN mRNA up-regulation. The FKN-mediated responses to Stx2 were also dependent on AP-1. In mice, both virulence factors of Stx-producing E. coli, Stx and LPS, are required to elicit hemolytic uremic syndrome. In this study, FKN was detected within glomeruli of C57BL/6 mice injected with Stx2, and further increased after Stx2 plus LPS coadministration. This was associated with recruitment of CX3CR1-positive cells. Thus, in response to Stx2, FKN is induced playing an essential role in the promotion of leukocyte-endothelial cell interaction thereby potentially contributing to the renal microvascular dysfunction and thrombotic microangiopathy that underlie hemolytic uremic syndrome due to enterohemorrhagic E. coli O157:H7 infection.

Effect of seliciclib (CYC202, R-roscovitine) on lymphocyte alloreactivity and acute kidney allograft rejection in rat.

BACKGROUND: T cell stimulation by alloantigens is followed by cell cycle progression, an event that is critically dependent on cyclin-dependent kinases. METHODS: We conducted a study to evaluate whether the cyclin-dependent kinase inhibitor seliciclib affected rat lymph node cells (LNc) activation and proliferation induced by either concanavalin A or allogeneic splenocytes in vitro and studied the mechanisms underlying the suppressive effect. We also investigated the immunosuppressive properties of seliciclib in vivo. RESULTS: Seliciclib completely inhibited in vitro proliferation of LNc and CD8 T cells, in response to either concanavalin A or allogeneic splenocytes. The percentage of activated LNc was lower in mixed leukocyte reactions (MLR) added with seliciclib than in MLR added with vehicle. The percentages of viable and apoptotic cells at the end of MLR with seliciclib were comparable to those of MLR with vehicle. LNc pre-exposed in MLR to seliciclib did not respond to further stimulation with alloantigens, and neither IL-2 nor IL-15 restored proliferation. These data indicate that the inhibitory effect of seliciclib on T cell alloreactivity is not because of cytotoxic effect but is associated with induction of profound T cell anergy. LNc harvested at the end of the primary MLR with seliciclib did not suppress the proliferation of syngeneic LNc cells toward allogeneic splenocytes, thus excluding that seliciclib induced the formation of regulatory cells. Finally, seliciclib partially prolonged grafted animal survival in a rat model of fully major histocompatibility complex-mismatched kidney transplantation. CONCLUSIONS: Altogether these results document that seliciclib regulates lymphocyte reactivity and may exert an immunosuppressive effect in vivo in the setting of transplantation.

DnIKK2-transfected dendritic cells induce a novel population of inducible nitric oxide synthase-expressing CD4+CD25- cells with tolerogenic properties.

BACKGROUND: We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4 T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. METHODS: Brown Norway (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4 T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4 T cells pre-exposed to dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. RESULTS: CD4 T cells pre-exposed to dnIKK2-DCs were CD4CD25 and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:10 ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). CONCLUSIONS: DnIKK2-Treg is a unique population of CD4CD25 T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.