Improved hematopoietic stem cell transplantation upon inhibition of natural killer cell-derived interferon-gamma.

Hematopoietic stem cell transplantation (HSCT) is a frequent therapeutic approach to restore hematopoiesis in patients with hematologic diseases. Patients receive a hematopoietic stem cell (HSC)-enriched donor cell infusion also containing immune cells, which may have a beneficial effect by eliminating residual neoplastic cells. However, the effect that donor innate immune cells may have on the donor HSCs has not been deeply explored. Here, we evaluate the influence of donor natural killer (NK) cells on HSC fate, concluded that NK cells negatively affect HSC frequency and function, and identified interferon-gamma (IFNγ) as a potential mediator. Interestingly, improved HSC fitness was achieved by NK cell depletion from murine and human donor infusions or by blocking IFNγ activity. Thus, our data suggest that suppression of inflammatory signals generated by donor innate immune cells can enhance engraftment and hematopoietic reconstitution during HSCT, which is particularly critical when limited HSC numbers are available and the risk of engraftment failure is high.

Simultaneous assessment of rotavirus-specific memory B cells and serological memory after B cell depletion therapy with rituximab.

The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM(+) and CD27- subpopulations, which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus, maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total, RV- and TT-Abs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV- and TT-specific mBc [IgM(+), switched (IgA(+)/IgG(+)), IgM(+) only, IgD(+) only, and CD27- (IgA(+)/IgG(+)/IgM(+))] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG1, and TT-IgG titers remained stable. In conclusion, in patients with autoimmunity, serological memory against RV and TT seem to be maintained by long-lived plasma cells, unaffected by RTX, and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells, dependent on mBc precursors depleted by RTX.

Induction antibody therapy in renal transplantation using early steroid withdrawal: long-term results comparing anti-IL2 receptor and anti-thymocyte globulin.

BACKGROUND: The present study intends to investigate the effects of anti-IL2 receptors (anti-IL2R) vs. lymphocyte-depleting agents in the early steroid withdrawal (ESW) scheme. METHODS: This is a retrospective cohort of 167 consecutive adult renal transplant recipients. Immunosuppression was based on tacrolimus and mycophenolate mofetil. Antibody induction therapy was carried out with lymphocyte-depleting agent (thymoglobulin) or anti-IL2R (Basiliximab or Daclizumab). ESW protocol was performed by administering intravenous methlyprednisolone as follows: 500 mg on day 0, 250 mg on day 1, 125 mg on day 2, 60 mg on day 3, and then stopped. RESULTS: Among the 167 studied patients, 79 (47.3%) received anti-IL2R and 88 (52.7%) received thymoglobulin induction. Significantly fewer episodes of acute rejection were seen at one year in patients treated with thymoglobulin as compared to anti-IL2R (25.6% vs. 11.4%, p=0.01). At five years, a significant difference in graft survival was observed in anti-IL2R-treated patients compared with thymoglobulin (83.5% vs. 95.5%, p=0.01). Multivariate analysis disclosed that female sex, antibody induction therapy using thymoglobulin and a trough tacrolimus level higher than 10 were protective factors against acute rejection, while there was a trend to increased risk of acute rejection at first year post-transplantation in patients presenting delayed graft function (DGF). Antibody induction was independently associated with patient and graft survival at five years (OR 0.213, 95% CI 0.046-0.991, p=0.04). CONCLUSION: ESW scheme seems to be safe and its use is beneficial since there are fewer adverse effects. Thymoglobulin induction therapy is associated with fewer rejection episodes. Induction therapy with thymoglobulin is associated with higher patient and allograft survival when comparing with anti-IL2R.

Oral tolerance reduces Th17 cells as well as the overall inflammation in the central nervous system of EAE mice.

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory immune response directed against myelin antigens of the central nervous system. In its murine model, EAE, Th17 cells play an important role in disease pathogenesis. These cells can induce blood-brain barrier disruption and CNS immune cells activation, due to the capacity to secrete high levels of IL-17 and IL-22 in an IL-6+TGF-ß dependent manner. Thus, using the oral tolerance model, by which 200 µg of MOG 35-55 is given orally to C57BL/6 mice prior to immunization, we showed that the percentage of Th17 cells as well as IL-17 secretion is reduced both in the periphery and also in the CNS of orally tolerated animals. Altogether, our data corroborates with the pathogenic role of IL-17 and IFN-γ in EAE, as its reduction after oral tolerance, leads to an overall reduction of pro-inflammatory cytokines, such as IL-1α, IL-6, IL-9, IL-12p70 and the chemokines MIP-1ß, RANTES, Eotaxin and KC in the CNS. It is noteworthy that this was associated to an increase in IL-10 levels. Thus, our data clearly show that disease suppression after oral tolerance induction, correlates with reduction in target organ inflammation, that may be caused by a reduced Th1/Th17 response.

A role for regulatory T cells in renal acute kidney injury.

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1(anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10+/-50.04; PC61: 187.23+/-31.38; DTA-1: 64.53+/-25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61(anti-CD25) mAb treatment is deleterious, while DTA-1(anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI.

Presença de aditivos conservantes (nitrito e sulfito) em carnes bovinas moídas, comercializadas em mercados varejistas / Presence of additive conserving (nitrite and sulphite) in grounded beef, commercialized in the retail markets

No presente trabalho, objetivou-se avaliar a prática fraudulenta da adição de nitrito e sulfito, e parâmetros físico-químicos de qualidade em carnes bovinas moídas in natura e resfriadas, comercializadas em mercados varejistas do estado do Rio de Janeiro. Avaliou-se também o grau de depleção do nitrito pós-cocção. Os resultados obtidos demonstraram que de 35 amostras analisadas, 37,14% apresentaram nitrito, 11,42% sulfito, 17,14% amônia e 17,14% odor desagradável na prova da cocção. Todas as amostras apresentaram o valor de pH dentro do padrão oficial. A maior concentração de nitrito detectada foi 1,17mg.Kg-1 e a menor 0,173 mg.Kg-1. Após cozimento de amostras onde se detectou nitrito, houve depleção significativa desta substância. A detecção de nitrito e sulfito em carnes in natura além de caracterizar o descumprimento da legislação vigente por parte dos comerciantes, pode representar um risco à saúde da população, devido ao potencial tóxico destas substâncias quando ingeridas em excesso.(AU)

So, the present work aims to evaluate the quality physiochemical parameters as well as the nitrite and sulphite fraudulent addition to “in natura” grounded beef which was cooled commercialized in the State of Rio retail markets. The nitrite depletion degree after cooking was also evaluated. Our results have demonstrated that in 35 analyzed samples, 37.14% presented sodium nitrite while 11.42% sulphite, 17.14% ammonia and 17.14% unpleasant smell at cooking test. All the samples presented standard pH value. 1.17 mg.Kg-1 and 0.173 mg.Kg-1 were the highest and the lowest nitrite concentration detected, respectively. We found significant depletion of the nitrite after cookihg the samples with this substance. The detention of nitrite and sulfite in meats “in natura” characterizes the disobedience of the current law. The addition of these substances can represent a risk to the health of the population. They are potentially toxic substances when ingested in excess.(AU)

Presença de aditivos conservantes (nitrito e sulfito) em carnes bovinas moídas, comercializadas em mercados varejistas / Presence of additive conserving (nitrite and sulphite) in grounded beef, commercialized in the retail markets

No presente trabalho, objetivou-se avaliar a prática fraudulenta da adição de nitrito e sulfito, e parâmetros físico-químicos de qualidade em carnes bovinas moídas in natura e resfriadas, comercializadas em mercados varejistas do estado do Rio de Janeiro. Avaliou-se também o grau de depleção do nitrito pós-cocção. Os resultados obtidos demonstraram que de 35 amostras analisadas, 37,14% apresentaram nitrito, 11,42% sulfito, 17,14% amônia e 17,14% odor desagradável na prova da cocção. Todas as amostras apresentaram o valor de pH dentro do padrão oficial. A maior concentração de nitrito detectada foi 1,17mg.Kg-1 e a menor 0,173 mg.Kg-1. Após cozimento de amostras onde se detectou nitrito, houve depleção significativa desta substância. A detecção de nitrito e sulfito em carnes in natura além de caracterizar o descumprimento da legislação vigente por parte dos comerciantes, pode representar um risco à saúde da população, devido ao potencial tóxico destas substâncias quando ingeridas em excesso.

So, the present work aims to evaluate the quality physiochemical parameters as well as the nitrite and sulphite fraudulent addition to “in natura” grounded beef which was cooled commercialized in the State of Rio retail markets. The nitrite depletion degree after cooking was also evaluated. Our results have demonstrated that in 35 analyzed samples, 37.14% presented sodium nitrite while 11.42% sulphite, 17.14% ammonia and 17.14% unpleasant smell at cooking test. All the samples presented standard pH value. 1.17 mg.Kg-1 and 0.173 mg.Kg-1 were the highest and the lowest nitrite concentration detected, respectively. We found significant depletion of the nitrite after cookihg the samples with this substance. The detention of nitrite and sulfite in meats “in natura” characterizes the disobedience of the current law. The addition of these substances can represent a risk to the health of the population. They are potentially toxic substances when ingested in excess.

G-CSF-treated granulocytes inhibit acute graft-versus-host disease.

It has been shown that in vivo and in vitro treatment with G-CSF induces the generation of low-density granulocytes (LDGs), which copurify with PBMCs and inhibit IFN-gamma production by human T cells. These results prompted us to postulate an immunomodulatory role for LDGs in acute graft-versus-host disease (aGVHD). Here it is shown that in the mouse experimental model, in vivo and in vitro G-CSF treatment generates LDGs capable of inhibiting 80% of T-cell IFN-gamma production. To assess the role of these LDGs in aGVHD, lethally irradiated (C57BL/6 x BALB/c) F1 hosts were reconstituted with T cell-depleted bone marrow cells plus nylon wool-purified spleen cells from G-CSF-treated (G-NWS) or -nontreated (NWS) C57BL/6 donors. Recipients of G-NWS had a 75% survival rate in contrast to a rate of 25% in the NWS recipients. The protective effect was completely abolished, and the mortality rate was 100% if donor-cell infusion was treated with anti-Gr1. Moreover, if LDGs were infused with NWS, full protection of aGVHD was observed, and no signs of disease were evidenced by mortality rate, weight loss, or histopathology of target organs. These results revealed the unexpected immunosuppressive capacity of G-CSF based on the generation of LDGs, leading to the possibility of using these cells as inhibitors of aGVHD.

Rejection of grafts without histocompatibility antigen disparity by TAP1-/- mice: a role for CD4+ T cells.

We have previously shown that TAP1-/- mice reject heart and skin grafts lacking an H-2 disparity. TAP1-/- mice, which are deficient for MHC-I molecules, probably have a T-cell repertoire with distinct reactivity to these molecules. We speculated that this rejection could be mediated by CD4+ T cells reactive to H-2(b) class I molecules, or to class I-derived peptides presented by self-APC. This hypothesis was tested in the present work. Presensitization of TAP1-/- mice with H-2K(b) peptides accelerated the rejection of C57BL/6 (H-2(b)) skin grafts (MST 13 days, P <.0057), indicating that these peptides were able to mobilize effector T cells that participate in rejection. In addition, CD4 T-cell depletion before transplantation induced a significant delay in rejection (P <.0011), showing that CD4 T cells have a major role in the rejection process, though other cells may also contribute. In conclusion, these results support our hypothesis that H-2(b) molecules may be targeted in graft rejection without an H-2 disparity. The low expression of MHC-I molecules on TAP1-/- mice may determine the selection of a T-cell repertoire that is reactive to self-MHC-I molecules, a phenomenon that is probably beyond the control of peripheral regulatory mechanisms.

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Low cytoreductive regimen of irradiation associated to unmodified bone marrow infusion (UBM) does not prevent the occurrence of graft versus host disease (GVHD) after transplant. PURPOSE: In this study we evaluated the potential advantages of a long-term immunossupression and T-cell depleted bone marrow infusion (TCDBMI) in preventing the occurrence of GVHD after small bowel transplantation (SBTx). METHODS: Heterotopic SBTX was performed with Lewis rats as recipients and DA as donors and distributed into 5 groups according to the irradiation, duration of immunossupression and the use of UBM or TCDBMI: G1 (n=6), without irradiation and G2 (n=9), G3 (n=4), G4 (n=5) and G5 (n=6) was given 250 rd of irradiation. Groups 1,2,4 and G3 and 5 were infused with 100 x 106 UBM and TCDBM respectively. Animals in G1, 2, 3 were immunossupressed with 1mg/ FK506/Kg/IM for 5 days and G4 and G5 for 15 days. Anti CD3 monoclonal antibodies and immunomagnetic beads were used for T-cell depletion.Animals were examined for rejection, GVHD, chimerism characterization and ileal and skin biopsies. RESULTS: Minimal to mild rejection was observed in all groups; however, GVHD were present only in irradiated groups. Long-term immunossupression changed the severity of GVHD in G4 and G5. Rejection was the cause of death in G1 while GVHD in G2, 3, 4 and 5, not avoided by the use of TCDBMI. Total chimerism and T-cell chimerism was statistically higher in irradiated groups when compared to G1. CONCLUSION: Extended immunossupression associated to low dose of irradiation decrease the severity of GVHD, not avoided by the use of TCDBMI.

Regeneration of natural antibody repertoire after massive ablation of lymphoid system: robust selection mechanisms preserve antigen binding specificities.

Natural Abs (NAbs) are Igs present in the serum and body fluids of healthy vertebrate animals, without any previous intentional immunization. NAbs often exhibit autoreactivity but also play an essential role in immunity, being a first line of defense against infectious microorganisms. We have previously analyzed the natural serum IgM Ab repertoire of normal mice, characterizing their reactivity with hundreds/thousands of self Ags; a significant similarity among different individuals was observed, and it was found that many reactivities of NAbs stably kept during adulthood were established early in life, implicating that period as a critical time window in the physiology of NAb repertoire selection. In the work reported here, experiments were conducted to address the role of normal lymphocyte ontogeny to the formation and stability of adult NAb repertoire. The massive destruction of the lymphoid system was promoted in adult mice with gamma-irradiation, and regeneration of hemopoietic tissues was granted by bone marrow or fetal liver inoculum. NAb repertoire regeneration was followed for 60 days after gamma-irradiation in bone marrow or fetal liver chimeric animals. The analysis of serum IgM reactivity with hundreds/thousands of self Ags showed that the NAb repertoire regenerated most of its original format after massive destruction of lymphoid compartments, characterizing autoreactive repertoire selection as a robust biological process. The data also show that regeneration of the NAb repertoire occurred similarly in fetal liver and bone marrow chimeras, although the latter animals poorly reconstituted their CD5(+) B1 cell compartment, suggesting that B1 cells are not essential for natural Ab regeneration.

Treatment results of 23 cases of severe aplastic anemia with lymphocytapheresis

We report the results of 23 patients with aplastic anemia (AA) treated with a program of 14 lymphocytapheresis (LC). Treatments were performed with apheresis machines, models Haemonetics 30-S and Baxter CS3000, using the standard program. This procedure was done because AA in many cases appears as a result of the action of a T cell population that inhibits hematopoiesis. Theorically, removal of this clonal population would produce hematopoietic recovery. Of the total of 23 patients, 9 were excluded for final evaluation of treatment results because 7 died during or shortly after treatment (0.7-3 months); one patient abandoned treatment after three LC and another died 7 months later because of transformation to acute leukemia. The ramaining 14 patients were included in the final evaluation of treatment; seven females and seven males, average age 46.1 years (range 22-69); 13 with severe, and one with moderate AA; 11 with recently diagnosed, and 3 with chronica AA; 12 without previous treatment and two treated before with antilymphocyte globulin + oxymetholone (OXN) + cyclosporine A (CsA) with transiet partial remission (PR). Besides lymphocytapheresis, 13 patients received OXM; 4 of them GM-CSF ad one low dose CsA. Four patients had complete remission lasting >59.5 months (range 42-78); eight PR (average duration of >38.6 months), and two minimal remission (>37 and 29 months). Platelet, reticulocyte and granulocyte counts increased on average at 48.7, 73.3 and 91.4 days, respectively. In cocnlusion, 14 (60.8 perecent) of 23 patients with AA showed an improvement related to LC treatment, with a survival probability of 63 percent from the fourth month, the latter with and added beneficial effect of the other therapies used. Larger numbers of patients have to be treated with LC to determine its real usefulness, mechanism of action and the best conditions for its use

Is there a role for leukocyte depletion filters to remove Trypanosoma cruzi infection from donor blood?

First and third generation leukocyte filters were used to filter blood units inoculated with trypomastigote forms of Trypanosoma cruzi. After blood filtration an aliquot of preor post-filtered blood was infected in isogenic mice. Infection evolution was followed by microscopic parasite count, indirect immunofluorescence and histopathological examination. All the animals that received pre-filtered blood (n = 27) accquired infection, while 26 percent of the mice injected with post-filtered blood (n = 27) were apparently not infected. A trend for late onset and peak of the parasitaemia was also observed among infected animals that received post-filtered blood (p<0.05). Additionally, the apparently non-infected mice also tested negative for both indirect immunofluorescence and histopathological examination. The results of this study, though very preliminary, suggest that that leukocyte filters might help to improve the safety of blood with regard to Trypanosoma, cruzi transmission.