A1 and A2A adenosine receptors play a protective role to reduce prevalence of autoimmunity following tissue damage.

Adenosine is a potent modulator that has a tremendous effect on the immune system. Adenosine affects T cell activity, and is necessary in maintaining the T helper/regulatory T cell (Treg ) ratio. Adenosine signalling is also involved in activating neutrophils and the formation of neutrophil extracellular traps (NETs), which has been linked to autoimmune disorders. Therefore, adenosine, through its receptors, is extremely important in maintaining homeostasis and involved in the development of autoimmune diseases. In this study, we aim to evaluate the role of adenosine A1 and A2A receptors in involvement of autoimmune diseases. We studied adenosine regulation by NETosis in vitro, and used two murine models of autoimmune diseases: type I diabetes mellitus (T1DM) induced by low-dose streptozotocin and pristane-induced systemic lupus erythematosus (SLE). We have found that A1 R enhances and A2A R suppresses NETosis. In addition, in both models, A1 R-knock-out (KO) mice were predisposed to the development of autoimmunity. In the SLE model in wild-type (WT) mice we observed a decline of A1 R mRNA levels 6 h after pristane injection that was parallel to lymphocyte reduction. Following pristane, 43% of A1 R-KO mice suffered from lupus-like disease while WT mice remained without any sign of disease at 36 weeks. In WT mice, at 10 days A2A R mRNA levels were significantly higher compared to A1R-KO mice. Similar to SLE, in the T1DM model the presence of A1 R and A2A R was protective. Our data suggest that, in autoimmune diseases, the acute elimination of lymphocytes and reduction of DNA release due to NETosis depends upon A1 R desensitization and long-term suppression of A2A R.

Comparison of the etiologic, microbiologic, clinical and outcome characteristics of febrile vs. non-febrile neutropenia in hospitalized immunocompetent children.

We compared the etiologic, microbiologic, clinical, and outcome picture among febrile and non-febrile immunocompetent children hospitalized during 2013-2015 with acute neutropenia (absolute neutrophil count < 1.5 × 109/L). Serious bacterial infections (SBI) were defined as culture-positive blood, urine, cerebrospinal fluid, articular fluid or stool infections, pneumonia, brucellosis, and rickettsiosis. Overall, 664 children < 18 years of age were enrolled; 407 (62.2%) had fever > 38.0 °C and 247 (37.8%) were non-febrile at admission. There were 425 (64.0%), 125 (18.8%), 48 (7.2%), and 66 (9.9%) patients aged 0-24 months, 2-6, 7-12, and > 12 years, respectively. No differences were recorded in the distribution of febrile vs. non-febrile patients among the age groups nor among the 3 neutropenia severity groups (< 0.5, 0.5-1.0 and 1.0-1.5 × 109/L). SBI infections were diagnosed in 98 (14.8%) patients, with higher rates among febrile patients vs. non-febrile patients (16.8% vs. 11.5%, P = 0.06). Brucellosis and rickettsiosis were diagnosed in 15.4% and 23.1% tests performed, respectively. 295/688 (42.9%) virologic examinations returned positive. Among patients < 24 months, more febrile ones had viral infectious compared with afebrile patients (P = 0.025). Acute leukemia was diagnosed in 6 patients. Neutropenia resolved in 163/323 (50.5%) patients during a 1-month follow-up. No differences were recorded in neutropenia resolution between febrile and non-febrile children among all 3 severity groups. Severe neutropenia was rare and occurred mainly in very young patients. SBIs were more common among febrile patients compared with non-febrile patients, but there was no association between severity of neutropenia or its resolution and the presence or absence of fever at diagnosis.

Clinical and Laboratory Findings in Jewish and Bedouin Patients in Southern Israel Who Were Diagnosed with Factor VII Deficiency.

BACKGROUND: Congenital factor VII deficiency is a rare recessive autosomal bleeding disorder with a wide spectrum of clinical manifestations. OBJECTIVES: To compare the clinical and laboratory findings in Jewish and Bedouin patients with factor VII deficiency. METHODS: The clinical and laboratory findings of patients with factor VII deficiency treated at Soroka Medical Center, a tertiary hospital in Israel, from 2005 to 2015 were analyzed regarding blood factor levels, illness severity, treatment administration, and disease outcome. RESULTS: Seventy-eight patients were enrolled (1:13,000 of the population in southern Israel) of whom 26 were diagnosed with severe factor VII deficiency (1:40,000). Sixty (76.9%) patients were Jewish and 18 (23.1%) were Bedouin. In univariable analysis, Bedouin patients exhibited a more severe illness, with significantly higher complication and fatality rates, and required more preventive treatment than the Jewish patients. CONCLUSIONS: The prevalence of congenital factor VII deficiency (including severe deficiency) in the Jewish and Bedouin populations of southern Israel is higher than previously reported. The clinical spectrum of the disease was found to be more severe in the Bedouin population.

The etiologic, microbiologic, clinical and outcome characteristics of immunocompetent young children <2 years of age hospitalized with acute neutropenia.

BACKGROUND: To describe the etiologic, microbiologic, clinical and outcome characteristics of acute neutropenia (absolute neutrophil count, ANC, <1.5 × 109/L) in hospitalized immunocompetent children. METHODS: Serious bacterial infections (SBI) were defined as culture-positive blood, urine, cerebrospinal fluid, articular fluid or stool infections, alveolar pneumonia, Brucellosis and Rickettsiosis. RESULTS: 431/671 (64.2%) healthy infants and children hospitalized with acute neutropenia were <2 years of age; 176 (40.8%), 167 (38.8%) and 88 (20.4%) patients were aged 0-3, 4-12 and 13-24 months, respectively. There were 19 (4.4%), 53 (12.3%), 140 (32.5%) and 209 (50.8%) patients with ANC count <200, 200-500, 501-1000 and 1001-1500 × 109 cells/L, respectively. Severe neutropenia (<500 × 109/L) was recorded in 72 (16.7%) patients. Fever >38 °C was present in 208/431 (48.3%) patients. Blood cultures were positive in 10 (2.3%), with Brucella melitensis, Staphylococcus aureus and Enterobacter spp. identified in 4, 3 and 2 patients, respectively; 5/10 patients with positive blood cultures were <3 months of age. Overall, 55/431 (12.7%) and 65/431 (15.1%) patients were diagnosed with SBIs and bacterial infections, respectively. Nasal washings-PCR for respiratory viruses was positive in 139/293 (47.4%) patients tested. An infectious etiology (bacterial and/or viral) was diagnosed in 190/431 (44.1%) patients. Three patients were diagnosed with acute lymphocytic leukemia. Resolution of neutropenia was achieved in 111/208 (53.4%) evaluable patients (63%, 50.6% and 48% of patients aged 0-3, 4-12 and >12 months, respectively and 56.8%, 53.5% and 52% of patients with severe, moderate and mild neutropenia, respectively). CONCLUSION: Acute neutropenia is common in immunocompetent children <2 years of age and is frequently associated with viral infections. We showed a substantial involvement of bacterial infections and particularly SBIs in the etiology of acute neutropenia. After a 1-month follow-up, resolution of neutropenia occurred in half of the patients, without association with age subgroups and with neutropenia severity.

Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes.

By studying the inactivation of malaria parasite culture by cysteine protease inhibition using confocal microscopy of living cells and electron microscopy of high-pressure frozen and freeze-substituted cells, we report the precise step in the release of malaria parasites from erythrocytes that is likely regulated by cysteine proteases: the opening of the erythrocyte membrane, liberating parasites for the next round of infection. Inhibition of cysteine proteases within the last few minutes of cycle does not affect rupture of the parasitophorus vacuole but irreversibly blocks the subsequent rupture of the host cell membrane, locking in resident parasites, which die within a few hours of captivity. This irreversible inactivation of mature parasites inside host cells makes plasmodial cysteine proteases attractive targets for antimalarials, as parasite-specific cysteine protease inhibitors may significantly augment multi-target drug cocktails.

Pharmacological preconditioning with adenosine A1 receptor agonist induces immunosuppression and improves graft survival in novel allogeneic transplantation models.

Adenosine is widely known as a potent modulator of innate and acquired immunity. It is released during transplants, and acts on four subtype receptors. In previous studies, we demonstrated that pharmacological preconditioning (PPC), pre-administration of the selective A1 receptor (A1R) agonist led to A1R desensitization, is followed by upregulation of the adenosine A2A receptor. This immunosuppressive effect resulted in lymphopenia, and it reduced T-cell reactivity. The aim of the current study was to challenge the immunosuppressive effects of A1R-PPC in models of allogeneic grafts. PPC mice were treated by intraperitoneal injection using specific adenosine A1R agonist 24 h and 12 h before starting any procedure. We challenged our method in novel allogeneic muscle and skin grafts models. Mice and grafts were assessed by complete blood counts, MLR from PPC splenocytes, and pathological evaluation. We found a significant reduction in WBC and lymphocyte counts in PPC-treated mice. Two-way MLR with splenocytes from PPC grafted mice showed decreased proliferation and anergy. Histology of PPC allogeneic grafts revealed profoundly less infiltration and even less muscle necrosis compared to vehicle treated allografts. Similar results observed in PPC skin transplantation. To conclude, PPC moderated graft rejection in separate allogeneic challenges, and reduced lymphocytes infiltration and ischemic damage.

Systemic inflammatory response syndrome-related lymphopenia is associated with adenosine A1 receptor dysfunction.

SIRS is associated with lymphopenia, and prolonged lymphopenia of septic patients has been associated with increased mortality risk. We hypothesize that elevated adenosine during SIRS down-regulates Gi-coupled A1R, which signals an effect that sensitizes a cAMP-dependent lymphotoxic response. In this study, we evaluate the role of adenosine in SIRS-mediated lymphopenia and impaired IL-15 production. Cecal ligation and puncture was used to induce sepsis-associated SIRS in mice. BMDCs were cultured and used to measure the effect of adenosine on IL-15. We found that A1R mRNA levels were significantly down-regulated and A1R-dependent Gi activity was abolished in T cells of septic mice. In accordance, cAMP was elevated in isolated T cells from cecal ligation and puncture compared with sham-treated mice. Similar to septic mice, leukopenia was evident in sham A1R-KO mice, after treatment with the A1R antagonist (8-cyclopentyl-1,3-dipropylxanthine), or after A1R desensitization. In contrast, A2AR-KO mice were protected from leukopenia. In addition, we observed that septic A1R-KO mice exhibited low IL-15 levels. Cultured BMDC agonists of A2AR and A2BR inhibited IL-15 production and adenosine blocked IL-15-dependent proliferation of cytotoxic T cells that were cocultured with stimulated BMDCs. To conclude, we suggest that SIRS-associated lymphopenia is initiated by A1R desensitization and adenosine-mediated inhibition of IL-15 production is part of the mechanism that accounts for the delay in leukopenia recovery in patients with severe sepsis. Interference with adenosine signaling may thus be potentially beneficial for septic patients with leukopenia.

Ketamine improves survival in burn injury followed by sepsis in rats.

Ketamine was reported to decrease cytokine production and improve survival after Escherichia coli-induced sepsis. We examined whether ketamine decreased interleukin (IL)-6 production and improved survival after 1) burn injury or 2) burn injury combined with sepsis (E. coli) at 24 h. Ketamine (10 mg/kg) or saline was given at 1 h after burn injury (G 1, 2, 5, 6), 24 h after burn injury (G 3, 4), or at E. coli inoculation (G 7, 8). Mortality was recorded for 7 days and IL-6 was measured in serum at 6 h after burn (G 1-2), 30 h after burn (G 3-4), or 6 h after sepsis (30 h after burn) (G 5-8). Burn injury only: Ketamine given immediately (1 h) after burn injury but not 24 h after, decreased the burn-induced increase of IL-6 but did not improve survival. Burn injury + sepsis: Ketamine given immediately after burn injury did not significantly decrease the sepsis-induced increase of IL-6 or improve survival. In contrast, ketamine given immediately after sepsis significantly improved survival (46.1% versus 13.3%, P = 0.008) and decreased IL-6 production (72,640 +/- 40,990 vs 332,300 +/- 32,300 pg/mL, P = 0.008). We conclude that ketamine therapy improves survival in burn injury followed by sepsis. This beneficial effect is probably achieved through interference with the inflammatory cascade, as evidenced by attenuation of the proinflammatory marker IL-6.

CD40 ligand expression correlates with resolution of peritonitis and mononuclear cell recruitment.

BACKGROUND: CD40 belongs to the tumor necrosis factor receptor family and its ligation is a central event in major inflammatory and immune reactions. We have previously demonstrated that CD40 ligation upregulates the secretion of mononuclear chemokines from peritoneal mesothelial cells (PMC), and that blocking the CD40 ligand (CD154) reduced the mononuclear infiltrate in a model of peritonitis. OBJECTIVE: To characterize the kinetics of CD154 expression on peritoneal Leukocytes and examine the correlation of this occurrence with the mononuclear transition at the resolution phase of peritonitis. METHODS: Leukocytes were collected from the effluent of 11 patients during episodes of peritonitis while undergoing peritoneal dialysis (PD). The effluent was then analyzed by flow cytometry to characterize CD154 expression. RESULTS: CD154 expression on peritoneal mononuclear cells gradually increased during the resolution phase of peritonitis, peaking first on T cells (CD4+ and CD8* cells at 20-45 hours) and then on macrophages (CD14' at 20-50 hours). The maximal expression of CD154 on macrophages, CD4* cells, and CD8* cells during peak hours reached values of 33% * 23%, 4%-3%, and 24%-17%, respectively. The increase in CD154 expression was in-negative correlation (r= -0.44, p = 0.032) with total Leukocyte numbers and in positive correlation (r = 0.52, p = 0.009) with the increase of mononuclear cells. Deterioration of peritonitis was associated with a decrease in CD154 levels, while recurrence of peritonitis was related to high CD154 Levels. CONCLUSION: Our data, which show a positive correlation between CD154 Levels and mononuclear dominance, suggest that CD40-CD154 Ligation plays an important role in the transition to mononuclear predominance in the late phase of peritonitis.

Isolation and ultrastructural characterization of squid synaptic vesicles.

Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.

Cytotoxicity mediated by the Fas ligand (FasL)-activated apoptotic pathway in stem cells.

Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(-/-) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues.

A molecular mechanism for mimosine-induced apoptosis involving oxidative stress and mitochondrial activation.

Mimosine, a non-protein amino acid, is mainly known for its action as a reversible inhibitor of DNA replication and, therefore, has been widely used as a cell cycle synchronizing agent. Recently, it has been shown that mimosine also induces apoptosis, as mainly reflected in its ability to elicit characteristic nuclear changes. The present study elucidates the mechanism underlying mimosine's apoptotic effects, using the U-937 leukemia cell line. We now demonstrate that in isolated rat liver mitochondria, mimosine induces mitochondrial swelling that can be inhibited by cyclosporine A, indicative of permeability transition (PT) mega-channel opening. Mimosine-induced apoptosis was accompanied by formation of hydrogen peroxide and a decrease in reduced glutathione levels. The apoptotic process was partially inhibited by cyclosporine A and substantially blocked by the antioxidant N-acetylcysteine, suggesting an essential role for reactive oxygen species formation during the apoptotic processes. The apoptosis induced by mimosine was also accompanied by a decrease in mitochondrial membrane potential, cytochrome c release and caspase 3 and 9 activation. Our results thus imply that mimosine activates apoptosis through mitochondrial activation and formation of H2O2, both of which play functional roles in the induction of cell death.

Involvement of graft-derived interleukin-15 in islet allograft rejection in mice.

The possibility that islets play a role in graft rejection during islet transplantation for type-1 diabetes patients holds promise for ex vivo islet manipulation and for specific anti-rejection therapy. Interleukin (IL)-15 is a T cell growth factor and chemoattractant that is expressed by non-T cells. Intragraft expression of IL-15 is elevated during acute rejection in patients and in mice, and systemic blockade of IL-15 in mice prolongs allograft survival. However, the source of IL-15 in these conditions is undetermined. Since epithelial cell-derived IL-15 promotes lymphocyte proliferation in culture, we sought to determine whether islet-derived IL-15 promotes rejection in mice. We designed antisense oligodeoxyribonucleotide molecules that target mouse IL-15. Uptake of FITC-labeled antisense molecules and efficacy of IL-15 inhibition in IFNgamma-stimulated islets were evaluated. Islets exhibited typical cytoplasmatic distribution of antisense molecules and produced IL-15 levels that were comparable to non-stimulated cells. Antisense-treated islet allografts, that were transplanted across multiple minor-histocompatibility-antigen mismatched strains of mice, were accepted at a higher rate than control-antisense treated islets or untreated islets (88.9% vs. 37.5% and 20%, respectively). Our results suggest that islet-derived IL-15 may be involved in acute islet allograft rejection.

CD40 ligand (CD154) takes part in regulation of the transition to mononuclear cell dominance during peritonitis.

BACKGROUND: CD40 is a member of the tumor necrosis factor (TNF) family of receptors whose ligand (CD154) is found mainly on membranes of activated mononuclear cells. CD154-CD40 cross-linking is a central event in antigen presentation, B-cell activation, and regulation of cytokine and chemokine secretion from various types of cells. We have previously demonstrated in vitro the presence of CD40 on human peritoneal mesothelial cells (PMC) and have also shown that CD40 ligation synergizes with interferon-gamma (IFN-gamma) to up-regulate CC chemokine secretion from these cells. The aim of the present study was to investigate the role of CD40 ligation in leukocyte recruitment during peritonitis. METHODS: Peritonitis was induced in mice by bacterial inoculation, CD40 levels were analyzed on PMC by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. CD154 levels on leukocytes were analyzed by flow cytometry and RT-PCR. Chemokines mRNA levels were analyzed by RT-PCR. CD154 was blocked in vivo using monoclonal antibodies. Results. In mice inoculated by Staphylococcus epidermidis or Escherichia coli, CD40 in PMC increased twofold at 24 hours and CD154 was induced and reached a peak at 48 hours. In both Gram-positive and Gram-negative-peritonitis, peritoneal macrophages were the main peritoneal leukocyte population to express CD154. Similar results were observed in human subjects during peritonitis. Injection of CD154 blocking monoclonal antibody (MR1) reduced the mononuclear infiltrate by 50% and had no effect on granulocyte recruitment 48 hours after inoculation of S. epidermidis. CONCLUSION: Our data suggest that CD40 plays a significant role in the process of the mononuclear infiltration during peritonitis.

Involvement of adenosine in the antiinflammatory action of ketamine.

BACKGROUND: Ketamine is an anesthetic drug. Subanesthetic doses of ketamine have been shown to reduce interleukin-6 concentrations after surgery and to reduce mortality and the production of tumor necrosis factor alpha and interleukin 6 in septic animals. Similarly, adenosine was shown to reduce tumor necrosis factor alpha and mortality of septic animals. The aim of this study was to determine whether adenosine mediates the antiinflammatory effects of ketamine. METHODS: Sepsis was induced in mice by lipopolysaccharide or Escherichia coli inoculation. Leukocyte recruitment and cytokine concentrations were used as inflammation markers. Adenosine concentrations were assayed by high-performance liquid chromatography, and the involvement of adenosine in the effects of ketamine was demonstrated by adenosine receptor agonists and antagonists. RESULTS: Ketamine markedly reduced mortality from sepsis, leukocyte recruitment, and tumor necrosis factor-alpha and interleukin-6 concentrations. Ketamine administration in mice and rats was associated with a surge at 20-35 min of adenosine in serum (up to 5 microm) and peritoneal fluid. The adenosine A2A receptor agonist CGS-21680 mimicked the effect of ketamine in peritonitis, whereas the A2A receptor antagonists DMPX and ZM 241385 blocked its antiinflammatory effects. In contrast, A1 and A3 receptor antagonists had no effect. ZM 241385 reversed the beneficial effect of ketamine on survival from bacterial sepsis. CONCLUSIONS: The current data suggest that the sepsis-protective antiinflammatory effects of ketamine are mediated by the release of adenosine acting through the A2A receptor.