Early Immune Reactions Research Articles

Dynamic comparison of early immune reactions and immune cell reconstitution after umbilical cord blood transplantation and peripheral blood stem cell transplantation.

Umbilical cord blood transplantation (UCBT) and peripheral blood stem cell transplantation (PBSCT) are effective allogeneic treatments for patients with malignant and non-malignant refractory hematological diseases. However, the differences in the immune cell reconstitution and the immune reactions during initial stages post-transplantation are not well established between UCBT and PBSCT. Therefore, in this study, we analyzed the differences in the immune reactions during the early stages (days 7-100 post-transplantation) such as pre-engraftment syndrome (PES), engraftment syndrome (ES), and acute graft-versus-host disease (aGVHD) and the immune cell reconstitution between the UCBT and the PBSCT group of patients. We enrolled a cohort of patients that underwent UCBT or PBSCT and healthy controls (n=25 each) and evaluated their peripheral blood mononuclear cell (PBMC) samples and plasma cytokine (IL-10 and GM-CSF) levels using flow cytometry and ELISA, respectively. Our results showed that the incidences of early immune reactions such as PES, ES, and aGVHD were significantly higher in the UCBT group compared to the PBSCT group. Furthermore, in comparison with the PBSCT group, the UCBT group showed higher proportion and numbers of naïve CD4+ T cells, lower proportion and numbers of Tregs, higher proportion of CD8+ T cells with increased activity, and higher proportion of mature CD56dim CD16+ NK cells during the early stages post-transplantation. Moreover, the plasma levels of GM-CSF were significantly higher in the UCBT group compared to the PBSCT group in the third week after transplantation. Overall, our findings demonstrated significant differences in the post-transplantation immune cell reconstitution between the UCBT and the PBSCT group of patients. These characteristics were associated with significant differences between the UCBT and the PBSCT groups regarding the incidences of immune reactions during the early stages post transplantation.

Mast Cells Mediate Inflammatory Injury and Aggravate Neurological Impairment in Experimental Subarachnoid Hemorrhage Through Microglial PAR-2 Pathway

Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease with high mortality and disability. Aberrant neuroinflammation has been identified as a critical factor accounting for the poor prognosis of SAH patients. Mast cells (MCs), the sentinel cells of the immune system, play a critical in the early immune reactions and participate in multiple pathophysiological process. However, the exact role of MCs on the pathophysiological process after SAH has not been fully understood. The current study was conducted to determine the role of MCs and MC stabilization in the context of SAH. Mouse SAH model was established by endovascular perforation process. Mice received saline or cromolyn (MC stabilizer) or compound 48/80 (MCs degranulator). Post-SAH evaluation included neurobehavioral test, western blot, immunofluorescence, and toluidine blue staining. We demonstrated that SAH induced MCs activation/degranulation. Administration of MC stabilizer cromolyn conferred a better neurologic outcome and decreased brain edema when compared with SAH+vehicle group. Furthermore, cromolyn significantly inhibited neuroinflammatory response and alleviated neuronal damage after SAH. However, pharmacological activation of MCs with compound 48/80 dramatically aggravated SAH-induced brain injury and exacerbated neurologic outcomes. Notably, pharmacological inhibition of microglial PAR-2 significantly reversed MCs-induced inflammatory response and neurological impairment. Additionally, the effect of MCs-derived tryptase in mediating neuroinflammation was also abolished by the microglial PAR-2 blockage in vitro. Taken together, MCs yielded inflammatory injury through activating microglia-related neuroinflammation after SAH. These data shed light on the notion that MCs might be a novel and promising therapeutic target for SAH.

Canine Angiostrongylus vasorum-Induced Early Innate Immune Reactions Based on NETs Formation and Canine Vascular Endothelial Cell Activation In Vitro.

Simple SummaryAngiostrongylus vasorum is a cardiopulmonary nematode that affects canids, residing in the pulmonary artery and right atrium/ventricle. Due to its location, the parasite will have a close interaction with the different components of the innate immune system, including endothelial cells and polymorphonuclear neutrophils (PMN). Here we evaluated the expression of adhesion molecules of canine aortic endothelial cells (CAEC), and NETs formation by co-culture of freshly isolated canine PMN with A. vasorum L3. Overall, we found distinct inter-donor variations in adhesion molecule expression among CAEC isolates. Additionally, PMN and A. vasorum co-culture induced NETs release without affecting larval viability.Due to its localization in the canine blood stream, Angiostrongylus vasorum is exposed to circulating polymorphonuclear neutrophils (PMN) and the endothelial cells of vessels. NETs release of canine PMN exposed to A. vasorum infective stages (third stage larvae, L3) and early pro-inflammatory immune reactions of primary canine aortic endothelial cells (CAEC) stimulated with A. vasorum L3-derived soluble antigens (AvAg) were analyzed. Expression profiles of the pro-inflammatory adhesion molecules ICAM-1, VCAM-1, P-selectin and E-selectin were analyzed in AvAg-stimulated CAEC. Immunofluorescence analyses demonstrated that motile A. vasorum L3 triggered different NETs phenotypes, with spread NETs (sprNETs) as the most abundant. Scanning electron microscopy confirmed that the co-culture of canine PMN with A. vasorum L3 resulted in significant larval entanglement. Distinct inter-donor variations of P-selectin, E-selectin, ICAM-1 and VCAM-1 gene transcription and protein expression were observed in CAEC isolates which might contribute to the high individual variability of pathological findings in severe canine angiostrongylosis. Even though canine NETs did not result in larval killing, the entanglement of L3 might facilitate further leukocyte attraction to their surface. Since NETs have already been documented as involved in both thrombosis and endothelium damage events, we speculate that A. vasorum-triggered NETs might play a critical role in disease outcome in vivo.

Early immune innate hallmarks and microbiome changes across the gut during Escherichia coli O157: H7 infection in cattle

The zoonotic enterohemorrhagic Escherichia coli (EHEC) O157: H7 bacterium causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. Cattle are primary reservoirs and EHEC O157: H7; the bacteria predominately inhabit the colon and recto-anal junctions (RAJ). The early innate immune reactions in the infected gut are critical in the pathogenesis of EHEC O157: H7. In this study, calves orally inoculated with EHEC O157: H7 showed infiltration of neutrophils in the lamina propria of ileum and RAJ at 7 and 14 days post-infection. Infected calves had altered mucin layer and mast cell populations across small and large intestines. There were differential transcription expressions of key bovine β defensins, tracheal antimicrobial peptide (TAP) in the ileum, and lingual antimicrobial peptide (LAP) in RAJ. The main Gram-negative bacterial/LPS signaling Toll-Like receptor 4 (TLR4) was downregulated in RAJ. Intestinal infection with EHEC O157: H7 impacted the gut bacterial communities and influenced the relative abundance of Negativibacillus and Erysipelotrichaceae in mucosa-associated bacteria in the rectum. Thus, innate immunity in the gut of calves showed unique characteristics during infection with EHEC O157: H7, which occurred in the absence of major clinical manifestations but denoted an active immunological niche.

Rh-relaxin-2 attenuates degranulation of mast cells by inhibiting NF-\u03baB through PI3K-AKT/TNFAIP3 pathway in an experimental germinal matrix hemorrhage rat model

BackgroundMast cells play an important role in early immune reactions in the brain by degranulation and the consequent inflammatory response. Our aim of the study is to investigate the effects of rh-relaxin-2 on mast cells and the underlying mechanisms in a germinal matrix hemorrhage (GMH) rat model.MethodsOne hundred seventy-three P7 rat pups were subjected to GMH by an intraparenchymal injection of bacterial collagenase. Clodronate liposome was administered through intracerebroventricular (i.c.v.) injections 24 h prior to GMH to inhibit microglia. Rh-relaxin-2 was administered intraperitoneally at 1 h and 13 h after GMH. Small interfering RNA of RXFP1 and PI3K inhibitor LY294002 were given by i.c.v. injection. Post-GMH evaluation included neurobehavioral function, Western blot analysis, immunofluorescence, Nissl staining, and toluidine blue staining.ResultsOur results demonstrated that endogenous relaxin-2 was downregulated and that RXFP1 level peaked on the first day after GMH. Administration of rh-relaxin-2 improved neurological functions, attenuated degranulation of mast cells and neuroinflammation, and ameliorated post-hemorrhagic hydrocephalus (PHH) after GMH. These effects were associated with RXFP1 activation, increased expression of PI3K, phosphorylated AKT and TNFAIP3, and decreased levels of phosphorylated NF-κB, tryptase, chymase, IL-6, and TNF-α. However, knockdown of RXFP1 and PI3K inhibition abolished the protective effects of rh-relaxin-2.ConclusionsOur findings showed that rh-relaxin-2 attenuated degranulation of mast cells and neuroinflammation, improved neurological outcomes, and ameliorated hydrocephalus after GMH through RXFP1/PI3K-AKT/TNFAIP3/NF-κB signaling pathway.

Low-Dose Methotrexate Could Reduce Severe Early Immune Reactions but Probably Increase Garft Failure in Umbilical Cord Blood Transplantation

Early immune disorders, including preengraftment syndrome (PES)and acute graft-versus-host disease(aGvHD)are problematic in cord blood transplantation (CBT), and optimal prophylaxis has not been established. Here, we prospectively investigated whether intensive GvHD prophylaxis by adding low-dose methotrexate (MTX)at day 3 after CBThas a prognostic impact on CBT. 16 consecutive single-unit CBT recipients treated for high-risk hematologic malignancies (10 cases) and non-malignancies (6 cases) between February 2019 and March 2019. The patients, 6 female and 10 males, had a median age of 9 years (range 4-40) and a median weight of 26 kg (range 14-68). Myeloablative preparative regimen comprised fludarabine, busulfan and cyclophosphamide for malignancies and reduced-intensity-conditioning comprised fludarabine, cyclophosphamide and total-body irradiation (4 Gy) for non-malignancies. Graft-versus-host disease (GvHD) prophylaxis was cyclosporine and mycophenolate mofetil plus MTX 3 mg/m2 on day+3. 14 patients achieved engraftment at a median of day 19 and had more than 95% (complete) donor chimerism on day+14.Two patients without hematopoietic reconstitution were engrafted after a second CBT. PES was characterized by high-grade fever, rash, pulmonary edema, weight gain, liver and renal dysfunction developed on a median of day 8 in 11 of the 16 evaluable patients, including 2 who did not achieve engraftment. The PES patients received intravenous corticosteroid at a median dose of 1 mg/kg/day, and of these, 2 patients were requiredBasiliximab to improve clinical symptoms. Grade I to IV and aGvHD developed in 4 and only 1 patient developed severe aGvHD with Grade Ⅲ. Follow-up so far, one death occurred at day 103 because of pneumonia. 15 patients are alive well at a median follow-up of 135 days (131-163). Adding low-dose MTX at day +3 may be offer one optimal regimen to reduce severe early immune reactions and improve outcomes in CBT. Disclosures No relevant conflicts of interest to declare.

Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps.

Heartworm disease is a zoonotic vector-borne disease caused by Dirofilaria immitis mainly affecting canids. Infectious third-stage larvae (L3) are transmitted to the definitive hosts via culicid mosquitoes; adult nematodes reside in the pulmonary arteries and in the right heart releasing unsheathed first-stage larvae (microfilariae) into the bloodstream leading to chronic and sometimes fatal disease. So far, early innate immune reactions triggered by these different D. immitis stages in the canine host have scarcely been investigated. Therefore, D. immitis microfilariae and L3 were analyzed for their capacity to induce neutrophil extracellular traps (NETs) in canine polymorphonuclear neutrophils (PMN). Overall, scanning electron microscopy analysis revealed both larval stages as strong inducers of canine NETosis. Co-localization of PMN-derived extracellular DNA with granulocytic histones, neutrophil elastase, or myeloperoxidase in parasite-entrapping structures confirmed the classical characteristics of NETosis. Quantitative analyses showed that both larval stages triggered canine NETs in a time-dependent but dose-independent manner. Moreover, parasite-induced NET formation was not influenced by the parasites viability since heat-inactivated microfilariae and L3 also induced NETs. In addition, parasite/PMN confrontation promoted significant entrapment but not killing of microfilariae and L3. Both, NETosis and larval entrapment was significantly reversed via DNase I treatments while treatments with the NADPH oxidase inhibitor diphenyleneiodonium failed to significantly influence these reactions. Interestingly, different types of NETs were induced by microfilariae and L3 since microfilarial stages merely induced spread and diffuse NETs while the larger L3 additionally triggered aggregated NET formation.

Eimeria ninakohlyakimovae induces NADPH oxidase-dependent monocyte extracellular trap formation and upregulates IL-12 and TNF-α, IL-6 and CCL2 gene transcription

Extracellular trap (ET) formation has been demonstrated as novel effector mechanism against diverse pathogens in polymorphonuclear neutrophils (PMN), eosinophils, mast cells, macrophages and recently also in monocytes. In the current study, we show that E. ninakohlyakimovae triggers the deliverance of monocyte-derived ETs in vitro. Fluorescence illustrations as well as scanning electron microscopy (SEM) analyses showed that monocyte-derived ET formation was rapidly induced upon exposure to viable sporozoites, sporocysts and oocysts of E. ninakohlyakimovae. Classical features of monocyte-released ETs were confirmed by the co-localization of extracellular DNA adorned with myeloperoxidase (MPO) and histones (H3) in parasite-entrapping structures. The treatment of caprine monocyte ET structures with NADPH oxidase inhibitor diphenylene iodondium (DPI) significantly reduced ETosis confirming the essential role of reactive oxygen species (ROS) in monocyte mediated ETs formation. Additionally, co-culture of monocytes with viable sporozoites and soluble oocyst antigen (SOA) induced distinct levels of cytokine and chemokine gene transcription. Thus, the transcription of genes encoding for IL-12 and TNF-α was significantly upregulated after sporozoite encounter. In contrast IL-6 and CCL2 gene transcripts were rather weakly induced by parasites. Conversely, SOA only induced the up-regulation of IL-6 and CCL2 gene transcription, and failed to enhance transcripts of IL-12 and TNF-α in vitro. We here report on monocyte-triggered ETs as novel effector mechanism against E. ninakohlyakimovae. Our results strongly suggest that monocyte-mediated innate immune reactions might play an important role in early host immune reactions against E. ninakohlyakimovae in goats.

Bilateral Descemet Membrane Endothelial Keratoplasty: Analysis of Clinical Outcome in First and Fellow Eye.

To analyze the clinical outcomes, complications, comparability, and predictors of sequential bilateral Descemet membrane endothelial keratoplasty (DMEK). Retrospective analyses were completed on 60 patients (120 eyes) with corneal endothelial disorders who underwent bilateral sequential DMEK. Main outcome measures were best-corrected visual acuity (BCVA), pachymetry, endothelial cell density, rebubbling frequency, and graft or nongraft-associated complications-as measured 6 and 12 months after surgery. BCVA, pachymetry, and endothelial cell loss did not differ significantly 6 and 12 months after surgery in first versus fellow eyes (P > 0.05, respectively). Graft and nongraft-associated complications such as graft detachment, graft failure, graft rejection, anterior chamber inflammation, and intraocular pressure decompensation did not differ significantly between first and fellow eyes (P > 0.05, respectively). The BCVA of first eyes 6 and 12 months after surgery correlates significantly with the 12-month BCVA of fellow eyes (r = 0.454, P = 0.031; r = 0.602, P = 0.001, respectively). Nevertheless, it was not possible to identify BCVA as an influencing variable on fellow eye outcome in regression modeling. Early sequential DMEK did not increase patients' risk of early immune reactions in their fellow eyes. The clinical outcomes of first and fellow eyes were comparable in patients after DMEK. This suggests patients will experience good fellow eye outcomes after successful first eye DMEK. When certain quality characteristics are maintained, donor differences do not significantly affect DMEK results. We found no significant correlation in postoperative complications in first and fellow eye DMEK surgeries.

Besnoitia besnoiti infections activate primary bovine endothelial cells and promote PMN adhesion and NET formation under physiological flow condition.

Besnoitia besnoiti is an obligate intracellular and emerging coccidian parasite of cattle that mainly infects host endothelial cells during acute infection. We here analyzed early innate immune reactions of B. besnoiti-infected primary bovine umbilical vein endothelial cells (BUVEC). B. besnoiti infections significantly activated BUVEC since the gene transcripts of several adhesion molecules (P-selectin, intercellular adhesion molecule 1(ICAM-1)), chemokines (CXCL1, CXCL8, CCL5), and of COX-2 were significantly upregulated during in vitro infection. Overall, the highest upregulation of most transcripts was observed at 24 or 48h post infection (p.i.). Enhanced adhesion molecule expression in infected host cells was confirmed by PMN adhesion assays being performed under physiological flow conditions revealing a significantly increased PMN adhesion on B. besnoiti-infected BUVEC layers at 24h p.i. Furthermore, we were able to illustrate neutrophil extracellular traps (NETs) being released by PMN under physiological flow conditions after adhesion to B. besnoiti-infected BUVEC layers. The present study shows that B. besnoiti infections of primary BUVEC induce a cascade of pro-inflammatory reactions and triggers early innate immune responses.

Graft-versus-host disease (GVHD) prophylaxis by using methotrexate decreases pre-engraftment syndrome and severe acute GVHD, and accelerates engraftment after cord blood transplantation.

GVHD and graft failure are serious problems in CBT. PES after CBT also occurs frequently and is associated with transplantation-related complications such as acute GVHD. We reviewed medical records for 70 consecutive child CBT recipients between December 1997 and April 2015. Forty-nine patients received prophylaxis against GVHD with CsA or Tac in combination with mPSL from day +7 (mPSL group), and 21 patients received CsA or Tac with MTX on day +1 and day +3 (MTX group). Neutrophil engraftment was detected in 59 patients (84.3%). Neutrophil engraftment rate in the MTX group was significantly higher than that in the mPSL group (21/21 (100%) and 38/49 (77.6%), respectively, p = 0.027). PES developed in 35 patients, and the incidence of PES in the mPSL group was significantly higher than that in the MTX group (p = 0.036). The incidence of severe acute GVHD (grade III or IV) in the MTX group was significantly lower than that in the mPSL group (p = 0.049). Although this study was a small-scale study, the results showed that increase in the rate of engraftment and decrease in the incidence of early immune reactions such as PES and severe acute GVHD could be achieved by early commencement of immunosuppression using MTX.

Infliximab Versus Rabbit Anti-Thymocyte Globulin As a Salvage Treatment For Steroid-Refractory Acute Graft-Versus-Host Disease After Reduced-Intensity Cord Blood Transplantation In Adults

Introduction In reduced intensity cord blood transplantation (CBT) post transplant immune disorders, including early immune reactions (PIR) and acute GVHD, are potential complications following CBT in adult patients. Such reactions might increase the risk of organ dysfunction, leading to high rates of transplantation-related mortality, particularly in patients who do not respond to primary therapy, which usually consists of steroids. There is no consensus on the optimal salvage treatment of steroid-refractory acute GVHD. Patients and Methods We retrospectively reviewed 388 patients who underwent single-unit reduced intensity cord blood transplantation consecutively from June 2008 to October 2012 at Toranomon Hospital. Patients who were in poor performance status (ECOG PS >3), had active bacterial or fungal infections at the time of conditioning were excluded. The most frequently used conditioning regimens were fludarabine, alkylating agent (melphalan or busulfan) with total body irradination (TBI), tacrolimus plus mycophenolate mofetil for GVHD prophylaxis. Patients in whom acute GVHD developed received methylpredonisolone 1-2 mg/kg per day. If no partial or complete resolution of symptoms occurred, they were considered steroid-refractory and proceeded to salvage treatment with infliximab or ATG. The dose of infliximab was 5 mg/kg/day once weekly for at least 1 course. ATG was given at 1mg/kg/day once weekly for at least 1 course. An observational study compared response and survival rates in all consecutive patients receiving 1 of these 2 treatments. Results 52 patients among this group developed steroid refractory acute GVHD. Infliximab group (n=31) had a higher proportion of patients with grade III-IV GVHD (69% VS 59%, p=0.18). ATG group (n=21) had a higher proportion of patients with stage 3-4 liver GVHD (28% VS 5%, p<0.01). Infliximab or ATG therapy was initiated after a median of 12 days (range, 2 to 85 days; 10 days for infliximab group VS 22 days for ATG group; p<0.01) of steroids for primary therapy for GVHD. Both overall response (PR+CR) were significantly higher in the infliximab group compared with the ATG group (54.8% VS 33.3%, p=0.015 ). In multivariate analysis, time from first-line therapy (days<10) was an independent predictor of response (HR, 3.91; 95%CI, 1.10-13.8; p=0.034), infliximab therapy was not significant, (HR, 1.30; 95%CI, 0.42-3.99; p=0.18). Median follow-up of surviving patients from the date of initiation of salvage treatment was 21.6 months (range, 5.5-57.9 months).Overall, median survival was 2 months after salvage treatment (95% CI, 1-3), with 25% of patients surviving at 12 months (95%CI, 11-20). Patients who achieved a CR have better survival rates. GVHD was the main cause of death (50%). When comparing survival according to salvage treatment, better OS with infliximab (33.9% VS 22.8, p=0.08) in univariate analysis was not confirmed in multivariate analysis. In multivariate analysis, grade IV GVHD at steroid refractory GVHD onset (HR,7.1; 95% CI, 1.29-38.7; P=0.021), and gut involvement with bleeding (HR, 7.65; 95% CI, 1.35-43.2; P=0.024) were significantly associated with worse survival. Conclusion We conclude that infliximab has more activity than ATG in the treatment of severe steroid-refractory GVHD, and earlier initiation of salvage therapy may give better response. But outcomes remain poor. New methods to prevent and treat GVHD are needed. Disclosures: No relevant conflicts of interest to declare.

Molecular mechanisms of natural killer cell regulation

Natural Killer (NK) cells are important for early immune reactions against viral infections and cancer. They are regulated by a highly redundant system of different activating and inhibitory receptors. Here we summarize our current understanding about the regulation of these cells and describe how mathematical modeling and systems biology approaches can help to shed some light on the complex regulatory network that governs NK cell reactivity.

Infliximab Treatment for Steroid-Refractory Acute Graft-Versus-Host Disease After Reduced-Intensity Cord Blood Transplantation in Adults

Abstract 4553 IntroductionAcute graft versus host disease (GVHD) remains the most frequent complication after allogeneic hematopoietic stem cell transplantation (SCT). In reduced intensity cord blood transplantation (CBT) previous studies have reported a lower incidence of severe acute GVHD compared with conventional allo-SCT. However, in these studies the incidences of grade II-IV acute GVHD varied widely from 26% to 51% (H.Narimatsu Stem cell int. 2011: 607569). In particular, post transplant immune disorders, including early immune reactions (PIR) and acute GVHD, are potential complications following CBT in adult patients. Such reactions might increase the risk of organ dysfunction, leading to high rates of transplantation-related mortality, particularly in patients who do not respond to primary therapy, which usually consists of steroids. Infliximab, a chimeric monoclonal antibody against tumor necrosis factor alpha, has shown activity against steroid refractory acute GVHD. Patients and MethodsWe retrospectively reviewed 275 patients who underwent single-unit reduced intensity cord blood transplantation consecutively from March 2007 to December 2011 at our institute. Patients in whom PIR or acute GVHD developed received methylprednisolone 1–2 mg/kg per day. If no partial or complete resolution of symptoms occurred, they were considered steroid-refractory and proceeded to infliximab treatment. The dose of infliximab was 5 mg/kg/day once weekly for at least 1 course. An antifungal drug, itraconazole or micafungin or voriconazole was used until all immunosuppressive drus were withdrawn. ResultsIn this study we retrospectively evaluated 54 patients who had steroid refractory acute GVHD. Of these, 21 who received infliximab were analyzed. GVHD prophylaxis was with only tacrolimus(n=15) and mycophenolate mofetil+tacrolimus(n=6). All of them developed grade III to IV GVHD. Median follow-up time of survivors was 548 days (range, 222–1152). The overall response rate was 62% (n=13), and 9 patients (43%) experienced complete response (CR). 5 patients (24%) did not respond and 3 (14%) had progressive GVHD. The Kaplan-Meier estimate of overall survival was 31%, with no signs of chronic GVHD (n=2). Four patients who responded subsequently died, one of exacerbation of GVHD, three of infections. All the 8 unresponsive patients died of GVHD or infections. Five patients (21%) had non-Candida invasive fungal infections. Sixteen patients(79%) had bacterial infections. ConclusionInfliximab was well tolerated and active for the treatment of steroid-resistant acute GVHD even following reduced intensity cord blood transplantation. However, it is associated with a high rate of infections. Controlled studies to assess the pharmacokinetics and most effective dosing regimen of infliximab for the treatment of steroid refractory acute GVHD are warranted. Disclosures:No relevant conflicts of interest to declare.

The cell-mediated immune reaction in the cutaneous lesions of the muriue chromoblastomycosis

Objective To evaluate the cell-mediated immune tissue reactions of mice model of chromoblastomycosis. Methods First we developed a murine model of chromoblastomycosis subcutaneous infection with F. pedrosoi inoculated into the footpads using immunocompetent and immunocompromised mice immuned by CTX, and then by immunohistochemistry methods analyzed the expression of cytokines IL-4, IL-10, TNF-α and IFN-γ at the seventh and thirtieth day, the expression of these cytokines at the same time in the healthy mice footpads were used as control. Results In the immunocompetent mice, The expression of IL-4, IL-10 and TNF-α was significantly higher when compared with healthy control at the seventh day,showing an up-regulation pattern of Th2 and Th1 type cellular immune functions with a predominance of the Th2 response. At the 30th day, the expression of IL-10 was significantly lower when compared with the 7th day and no difference with healthy controls, while IFN-γand TNF-α were gradually increased, the T cellmediated immune drives to Th1 response from Th2. In the immunocompromised mice, the expression of IL4 and IL-10 were significantly higher, meanwhile IFN-γwas lower than those in immunocompetent or healty mice, the levels of TNF-α was not significantly different fiom healty control at the 7th day, it showed that Th2 response was more increased with the Th1 responses was significantly inhibited in the early immune reactions. At the 30th day, the Th1 type cytokines IFN-γ and TNF-α were highly significantly higher than early stage but still lower than immunocompetent levels, the level of Th2 type cytokine IL-10 rradually decreased although it was still above the other two rroups. Conclusion In different immune state, there was an immune defence translation from the predominance of Th2 type cellular immunity to Th1 type in the process of murine model of chromoblastomycosis. Thl type cytokines which favors resistance to fungal disease, played a major role at controlling the development of chromoblastomycosis. Key words: Chromoblastomycosis; Murine model; Cytokines; Th2/Th1

Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis

Eimeria bovis infections are under immunological control and recent studies have emphasized the role of early PMN-mediated innate immune responses in infected calves. Neutrophil extracellular traps (NETs) have recently been demonstrated to act as a killing mechanism of PMN against several pathogens. In the present study, the interactions of bovine PMN with sporozoites of E. bovis were investigated in this respect in vitro. For demonstration and quantification of NET formation, extracellular DNA was stained by Sytox Orange. Fluorescence images after Sytox Orange staining as well as scanning electron microscopy (SEM) showed NET formation to occur upon contact with E. bovis sporozoites. Exposure of PMN to viable sporozoites induced stronger NET formation than to dead or homogenized parasites. NET formation was abolished by treatment with DNase and could be reduced by diphenylene iodonium, which is described as a potent inhibitor of NADPH oxidase. After sporozoite and PMN co-culture, extracellular fibres were found attached to sporozoites and seemed to trap them, strongly suggesting that NETs immobilize E. bovis sporozoites and thereby prevent them from infecting host cells. Thus, transfer of sporozoites, previously being confronted with PMN, to adequate host cells resulted in clearly reduced infection rates when compared to PMN-free controls. NET formation by PMN may therefore represent an effector mechanism in early innate immune reactions against E. bovis. This is the first report indicating Eimeria-induced NET formation.

The tumour-Clostridium phenomenon: 50 years of developmental research (Review)

The tumour-Clostridium phenomenon describes the specific affinity of spore forming anaerobes to tumour growth. The discovery of strictly intratumoral tetanus toxic infections in tumour-bearing mice after intravenous spore administration gave the impulse to search for non-toxic clostridial isolates with tumour-selective properties for clostridial biotherapy, i.e. oncolysis, as well as serologic tumour diagnostics without any toxic side-effects. Systematic studies of the oncolytic process and its variables on diverse experimental tumours and laboratory animals revealed that tumour liquefaction, converting necrotic tumour parts to putrid abscesses filled with masses of clostridial forms, stops sharply at the viable rim of the blood-supplied tumour tissue. Similar results were observed in clinical trials, particular of gliomas. Before oncolysis is initiated, the first stage of clostridial multi-plication in the vascularized tissue is inducing a humoral immune response, preceded by phagocytic activity. The majority of tumour-bearing laboratory and domestic animals, so far tested serologically, clinical cancer patients as well, responded with anti-rod antibodies and, independently, anti-spore antibodies. Oncolytic and non-oncolytic Clostridia were equally immunogenic. During the early, immunizing period of clostridial proliferation, analytical tumour-tetanus experiments were focused on potential relations between tumour growth kinetics and rod proliferation. Based on realistic growth models and target principles, computer simulations could reproduce the results, i.e. cumulative curves of tetanus lethality in groups of mice. Thus, crucial assumptions of the mathematical model were ex post confirmed by further experiments. Our working hypothesis concentrates on temporally hypoxic micro-niches close to a pre-mitotic cell with enhanced oxygen demand which can be utilized by anaerobes (randomly) located there. As early immune reactions to clostridial antigens via phagocytosis and humoral immune response will do without invasion in necrobiotic avascular tumour areas, the pacemaker model of tumour-Clostridium interplay extends the scope of genetically engineered Clostridia to early treatment of metastases. Thus, novel concepts, such as 'Clostridia-directed enzyme prodrug-therapy' and 'Combined bacteriolytic therapy', together with immune activation, can come into play.

Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults

Post transplant immune disorders are problematic in cord blood transplantation (CBT) for adult patients, and optimal prophylaxis has not been established. We investigated whether intensive graft-versus-host disease (GVHD) prophylaxis using short-term methotrexate (MTX) has a prognostic impact on CBT. Post-CBT immune reactions were classified according to time course as pre-engraftment immune reaction (PIR), engraftment syndrome (ES) or acute GVHD. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers. Median age was 48 years (range, 18-69 years). Preparative regimens comprised myeloablative (n=31) or reduced-intensity (n=46). Acute GVHD prophylaxis included cyclosporine alone (n=23), tacrolimus alone (n=12), cyclosporine plus MTX (n=17), tacrolimus plus short-term MTX (n=23) or cyclosporine plus methylprednisolone (n=2). Cumulative incidences of PIR, ES and grade II-IV GVHD were 36, 12 and 23%, respectively. Short-term MTX exerted significant favorable effects on post-CBT immune reactions (hazard ratio, 0.55; 95% confidence interval (95% CI), 0.31-0.98; P=0.04) in multivariate analysis. Overall survival rates for patients with and without short-term MTX at day 180 were 59% (95% CI, 42-73%) and 16% (95% CI, 6.6-30%) (P=0.0001), respectively. Short-term MTX could offer one optimal regimen to reduce immune reactions and improve outcomes in CBT.

Favorable Impact of Short Term Methotrexate on Early Immune Reaction and Non-Relapse Mortality in Umbilical Cord Blood Transplantation for Adult Patients.

Cord blood transplantation (CBT) represents an attractive alternative for patients with advanced hematological malignancy who lack matched related or unrelated donors. Adult patients receiving myeloablative or reduced-intensity CBT display a 90% chance of engraftment, but also experience a 50% rate of transplant-related mortality. Post-transplant immune disorders including early immune reactions and acute GVHD are problematic in CBT for adult patients. These reactions and/or additional immunosuppressive therapy might increase the risk of infection and organ dysfunction, leading to high rates of transplantation-related mortality. However, optimal prophylaxis and management for immune reactions have not been established in CBT. We investigated retrospectively whether intensive graft-versus-host disease (GVHD) prophylaxis has a prognostic impact on CBT. Post-CBT immune reactions were classified according to time course as pre-engraftment immune reaction (PIR), engraftment syndrome (ES) or acute GVHD. Between March 2001 and September 2003, a total of 77 patients underwent CBT at 8 centers of the Nagoya Blood and Marrow Transplantation Group (NBMTG). Median age of patients was 48 years (range, 18–69 years). Preparative regimens were myeloablative (n=31) or reduced-intensity (n=46). Acute GVHD prophylaxis included cyclosporine alone (n=23), tacrolimus alone (n=12), cyclosporine plus short-term methotrexate (n=17), tacrolimus plus short-term MTX (n=23) or cyclosporine plus methylprednisolone (n=2). Cumulative incidences of PIR, ES and grade I-IV GVHD were 38%, 12% and 29%, respectively. Short-term MTX exerted significant favorable effects on PIR in univariate analysis (hazard ratio, 0.41; 95% confidence interval, 0.19–0.89) and on non-relapse mortality (NRM) in multivariate analysis (hazard ratio, 0.36; 95% confidence interval, 0.16–0.81). On the other hand, PIR occurred in 35% of patients with cyclosporine and 37% with tacrolimus. Short-term MTX appears to offer optimal GVHD prophylaxis to reduce PIR and improve NRM. [Display omitted]

Eimeria bovis modulates adhesion molecule gene transcription in and PMN adhesion to infected bovine endothelial cells

Eimeria bovis is an important coccidian parasite of cattle causing severe diarrhea in young animals. Its first schizogony takes place in endothelial cells of the ileum resulting in the formation of macroschizonts 14–18 days p.i. This longlasting development suggests a particular immune evasion strategy of the parasite. Here, we analyse early innate immune reactions to E. bovis by determining the adhesion of polymorphonuclear neutrophils (PMN) to infected endothelial cell layers under flow conditions and the transcription of adhesion molecule genes in infected host cells. Bovine umbilical vein endothelial cells (BUVEC) were infected with E. bovis sporozoites. Sporozoites invaded BUVEC within 1 h and the first mature macroschizonts occurred 14 days p.i. PMN adhesion was enhanced in E. bovis-infected BUVEC layers as early as 8 h p.i.; maximum adhesion occurred 48 h p.i. Increased adhesion rates persisted until the end of the observation period at 14 days p.i. PMN adhered to both infected and uninfected cells within monolayers, suggesting paracrine cell activation. E. bovis infection upregulated the transcription of genes encoding for P-selectin, E-selectin, vascular cellular adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Most marked effects concerned E-selectin followed by P-selectin, VCAM-1 and ICAM-1. Increased transcript levels were found beginning 30 min p.i. and maximum values occurred 1–2 h p.i. (P-selectin) and 2–4 h p.i. (E-selectin, VCAM-1, ICAM-1). By 12–24 h p.i. levels had decreased to those of uninfected controls. Tumor necrosis factor α (TNFα)-induced PMN adhesion was significantly reduced in infected vs. uninfected BUVEC. Eimeria bovis also had suppressive effects on TNFα-mediated upregulation of adhesion molecule gene transcription. The data presented here suggest that infection of BUVEC with E. bovis on one hand induces proinflammatory reactions resulting in enhanced PMN adhesion mediated by upregulated adhesion molecule gene transcription but on the other downregulates TNFα-induced cell activation.