Involvement of suppressor cells induced with membrane fractions of trypanosomes in immunosuppression of trypanosomiasis

We showed that infection with Trypanosoma congolense in mice led to suppression of listeria-induced delayed type hypersensitivity (DTH). Mice were pre-treated with irradiated T. evansi, which caused rapid and effective suppression of DTH. A membrane fraction obtained by homogenizing T. evansi variant in a hypotonic buffer solution and centrifuging it at 150,000g produced suppression of listeria-induced DTH when injected i.p. into mice as early as 1 day before listeria immunization. Furthermore, we demonstrated by an adoptive transfer system that the suppressor cells involved in this suppression had developed in the spleen and that the activity of the splenic suppressor cells was due to the presence of a macrophage population.     

Genetic manipulation of African trypanosomes as a tool to dissect the immunobiology of infection

The variant surface glycoprotein (VSG) coat of African trypanosomes exhibits immunobiological functions distinct from its prominent role as a variant surface antigen. In order to address questions regarding immune stealth effects of VSG switch-variant coats, and the innate immune system activating effects of shed VSG substituents, several groups have genetically modified the ability of trypanosomes to express or release VSG during infection of the mammalian host. The role of mosaic surface coats expressed by VSG switch-variants (VSG double-expressors) in escaping early immune detection, and the role of VSG glycosylphosphatidylinositol (GPI) anchor substituents in regulating host immunity have been revealed, respectively, by stable co-expression of an exogenous VSG gene in trypanosomes expressing an endogenous VSG gene, and by knocking out the genetic locus for GPI-phospholipase C (PLC) that releases VSG from the membrane. Both approaches to genetic modification of African trypanosomes have suggested interesting and unexpected immunobiological effects associated with surface coat molecules.     

Polyamine oxidase-mediated killing of African trypanosomes

African trypanosomes, Trypanosoma brucei brucei, T. vivax and T. congolense , were killed when incubated in vitro with ruminant sera in the presence of exogeneous spermidine, and were non-infective for mice. Purified polyamine oxidase in the presence of spermidine-mediated similar killing of trypanosomes. The abundance of polyamine oxidase activity in ruminant sera which can react with polyamines to produce products with cytotoxic properties may explain the trypanosome killing. This system may contribute to non-specific parasite killing in vivo.     

Species-specific anticoagulant and mitogenic activities of murine protein S

Background

The protein C pathway down-regulates thrombin generation and promotes cytoprotection during inflammation and stress. In preclinical studies using models of murine injury (e.g., sepsis and ischemic stroke), murine protein S may be required because of restrictive species specificity.

Design and Methods

We prepared and characterized recombinant murine protein S using novel coagulation assays, immunoassays, and cell proliferation assays.

Results

Purified murine protein S had good anticoagulant co-factor activity for murine activated protein C, but not for human activated protein C, in mouse or rat plasma. In human plasma, murine protein S was a poor co-factor for murine activated protein C and had no anticoagulant effect with human activated protein C, suggesting protein S species specificity for factor V in addition to activated protein C. We estimated that mouse plasma contains 22±1 μg/mL protein S and developed assays to measure activated protein C co-factor activity of the protein S in murine plasma. Activated protein C-independent anticoagulant activity of murine protein S was demonstrable and quantifiable in mouse plasma, and this activity was enhanced by exogenous murine protein S. Murine protein S promoted the proliferation of mouse and human smooth muscle cells. The potency of murine protein S was higher for mouse cells than for human cells and similarly, human protein S was more potent for human cells than for mouse cells.

Conclusions

The spectrum of bioactivities of recombinant murine protein S with mouse plasma and smooth muscle cells is similar to that of human protein S. However, in vitro and in vivo studies of the protein C pathway in murine disease models are more appropriately performed using murine protein S. This study extends previous observations regarding the remarkable species specificity of protein S to the mouse.     

Regulation of innate and acquired immunity in African trypanosomiasis

African trypanosomes are well known for their ability to avoid immune elimination by switching the immunodominant variant surface glycoprotein (VSG) coat during infection. However, antigenic variation is only one of several means by which trypanosomes manipulate the immune system of their hosts. In this article, the role of parasite factors such as GPI anchor residues of the shed VSG molecule and the release of CpG DNA, in addition to host factors such as IFN-gamma, in regulating key aspects of innate and acquired immunity during infection is examined. The biological relevance of these immunoregulatory events is discussed in the context of host and parasite survival.     

New immunosuppressive strategies and the risk of infection

Abstract: Newer immunosuppressive strategies have resulted in a marked reduction in graft rejection after transplantation, with the price being an increase of infectious complications, such as BK‐related nephropathy. The targeting of new immunosuppressive pathways, such as interleukin‐2–mammalian target of rapamycin inhibition, may have unexpected consequences for the immune response. Cell‐depleting agents have long‐lasting effects on cellular recovery and function, with the activation of latent viral infections and late viral and fungal infections. The multitude of different induction and maintenance protocols renders the detection of small increases of often rare infections very difficult. At the same time, preemptive and prophylactic strategies have gained widespread acceptance and may further offset small changes in infection rates. Other factors related to an increase or shift of infections may be of equal importance, such as increased use of marginal donors, older age at transplantation, or more patients receiving a second transplant. Not all the changes observed result in an increased immunosuppression. Steroid‐ and calcineurin inhibitor‐sparing protocols may have a beneficial impact on infectious complications. Antimycotic or antiviral activity has been described for specific immunosuppressive agents, although the in vivo effect of these activities is uncertain. The possible role of specific drugs in the occurrence of infections is discussed, with emphasis on the antibodies and fusion proteins. The unequivocal attribution of a given infection to a specific drug is often impossible, as the risk of infection is dependent on the entirety of immunosuppression and the epidemiological pressure (‘net immunosuppression’). It is important to remain vigilant for unexpected infections, not only in the context of clinical studies with selected patients, but also in the routine follow‐up of our transplant patients.     

The effects of nucleoside analogue prophylactic treatment on HBV activation in HBcAb+ patients undergoing immunosuppressive therapy

We investigated the effects of prophylactic nucleoside analogue treatment on HBV activation in patients with antibodies against core antigen (HBcAb+) patients undergoing immunosuppressive therapy. Patients (113), who were HBcAb+, with various autoimmune diseases, undergoing immunosuppressive therapy, were divided into two groups. The control group, not treated with antivirals, and the prophylactic group, treated with antiviral drugs. The two groups were evaluated for changes in serum biochemical marker (alanine aminotransferase ALT), virological marker (HBV DNA) and for seroconversion. In the control group, the number of patients with an increase in ALT in patients with isolated HBcAb and HBcAb and antibodies against HBsAg (HBsAb +) were five (20.0%) and one (2.8%), respectively (P < 0.05). There were six cases (24.0%) with an increase in HBV DNA in the isolated HBcAb+ subgroup and one case (2.8%) in HBsAb+/HBcAb+ subgroup (P < 0.05). In the HBcAb+ only population, six patients (24.0%) in the control group had an increase in HBV DNA compared with none in the antiviral prophylactic group (P < 0.05). One patient (4.0%) with HBcAb+ in the control group underwent an HBsAg seroconversion when receiving immunosuppressive therapy for 18 months, while none in the antiviral prophylactic group underwent reversion to HBsAg positivity (P = 0.4949). Under immunosuppressive condition, the risk of HBV activation was much higher in patients with HBcAb than in patients with both HBcAb and antibodies to HBsAb group. Antiviral prophylactic therapy could significantly reduce the risk of HBV reactivation.     

Immune responses and serum levels of cytokines in adult T-cell leukemia patients and human T-cell leukemia virus type-I carriers.

To find predictive parameters for development and progression of adult T-cell leukemia (ATL) in human T-cell leukemia virus type-I (HTLV-I) carriers, we investigated cellular immune responses such as mitogenic responses and natural killer activity of the peripheral blood mononuclear cells (PBMC). And serum or plasma levels of cytokines, including tumor-necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and immunosuppressive acidic protein (IAP), were also measured in patients with ATL, healthy HTLV-I carriers and healthy HTLV-I non-carriers as controls. Results are as follows: (1) increased spontaneous proliferation and decreased mitogenic responses of PBMC already existed in HTLV-I carriers; (2) IAP was significantly higher in patients with acute/lymphoma type ATL than in those with chronic/smoldering type, HTLV-I carriers and HTLV-I non-carriers. These results suggest that spontaneous proliferation or mitogenic responses and IAP may be useful parameters for the development and progression of ATL from the carriers. Since HTLV-I carriers already have various grades of immunosuppression, we should seriously try to prevent further HTLV-I transmission.     

Evidence of cytogenetic and molecular remission by allogeneic cells after immunosuppressive therapy alone

An immunosuppressive but not myeloablative regimen followed by HLA-matched donor mobilized haemopoietic stem cell transplantation was employed in two high-risk patients. The first patient had refractory anaemia with excess blasts (RAEB) and cytogenetic evidence of translocation 1;3(p36;q21). The second patient had Philadelphia-negative but p190 BCR-ABL chimaeric gene positive chronic myelogenous leukaemia in accelerated phase (AP-CML). The conditioning regimen consisted of fludarabine (30 mg/m²/d, days 1–3) with cyclophosphamide (300 mg/m²/d, days 1–3). Cyclosporine and methotrexate were employed for acute graft-versus-host disease (aGVHD) prophylaxis. In both cases the engraftment of donor cells was demonstrated by cytogenetics and short tandem repeat polymorphisms via PCR. Both patients are alive with normal cytogenetic (RAEB) and molecular (AP-CML) remissions, 100 and 150 d after allografting, respectively. In particular, in the AP-CML patient, the BCR-ABL became undetectable and the BCR-ABL/ABL ratio was <0.0001.     

Chronic non-A, non-B hepatitis: lack of correlation between biochemical and morphological activity, and effects of immunosuppressive therapy on disease progression

A study was made of 52 patients considered to probably have chronic non-A, non-B hepatitis who were seen during an eight-year period at Westmead Hospital, Sydney. The patients were followed for a median of 28 months to assess the natural history of the disease and, in a small number of patients, the effect of immunosuppressive therapy on disease progression was examined. In 94% of cases, infection appeared to have been acquired by a parenteral route; the remainder were sporadic infections. Fifty-six per cent of the patients had mild constitutional symptoms and the remainder were asymptomatic. Similarly, 54% of patients had no signs of chronic liver disease and none exhibited signs of hepatic decompensation. Liver biopsies were performed in 42 patients; chronic active hepatitis with or without cirrhosis was present in 90%. However, neither the presence of symptoms nor the degree of biochemical abnormality were predictive of disease severity as determined histologically. Among eight patients treated with corticosteroids (with or without azathioprine), six underwent follow-up liver biopsy. Quantitative analysis of inflammatory and fibrotic changes indicated significant (p < 0.01) progression of histological severity during a median 33 months (range 7–98 months) between biopsies with cirrhosis developing in four instances. In contrast, among the seven untreated patients rebiopsied after a median of 16.0 months (range 11–37 months) there was no overall change in histological severity and only one patient developed cirrhosis. It is concluded that histological assessment is required in all patients suspected of having chronic non-A, non-B hepatitis as other means of assessment are unreliable. At present, immunosuppressive therapy cannot be recommended for patients with this form of chronic viral hepatitis; as well as being unhelpful, such treatment may accelerate disease progression.     

Efficacy and safety of immunosuppressive therapies in the treatment of high-risk IgA nephropathy: A network meta-analysis

Background:IgA nephropathy (IgAN) is one of the significant contributing factors of end-stage renal disease (ESRD). It is reported that over half of patients with IgAN accompany multiple high-risk factors, which increase the risk of ESRD progression. Studies have shown that immunosuppressive agents were beneficial in high-risk IgAN, but the efficacy and safety have not been fully demonstrated yet. The present study aims to elucidate the efficacy of commonly used immunosuppressants in high-risk IgAN and their relative safety profiles via a network meta-analysis strategy.Methods:Randomized controlled trials (RCTs) eligible for this network meta-analysis were included to evaluate the efficacy and safety of different immunosuppressants for high-risk IgAN. Main outcomes and measures include incidence of renal composite end point, the rate of total remission, adverse events, and proteinuria. Besides, subgroup analysis and cluster analysis were carried out.Results:This network meta-analysis of 37 RCTs involving 3012 participants found that Mycophenolate mofetil (MMF) combined with corticosteroids (CS) was superior to other interventions in end point events and proteinuria. Cyclosporine A (CsA) plus CS was the best option for clinical remission rate, and supportive care (SC) was the safest treatment. Cluster analysis showed that MMF+CS and Leflunomide (LEF)+CS were best protocols in efficacy and safety. Subgroup analysis indicated the best benefits of MMF were presented among the Asian population, and the benefits increased with the increase of follow-up duration. The effect of Cyclophosphamide (CTX) +CS on crescent IgAN was better than that of other risk factors. Moreover, the increasing follow-up duration was negatively associated with the effect.Conclusions:MMF+CS and LEF+CS appear to serve as the best choice for treating high-risk IgAN than other immunosuppressive therapies.     

Efficacy of combination therapy of antiviral and immunosuppressive drugs for the treatment of severe acute exacerbation of chronic hepatitis B

BACKGROUND: Patients with severe exacerbation of chronic hepatitis B, sometimes developing into fulminant liver failure, are at high risk for mortality even with antiviral therapy. The efficacy of immunosuppressive therapy in clinically severe exacerbation of chronic hepatitis B has not been well demonstrated. In this study, we evaluated the efficacy of the early introduction of immunosuppressive therapy in combination with antiviral therapy in such patients. METHODS: Forty-two patients, 29 men and 13 women, were defined as having severe exacerbation of chronic hepatitis B based on our uniform criteria, and were enrolled in this study. Sixteen patients between 1982 and 1996 were analyzed retrospectively. We defined the criteria of severe disease in 1997, and then began to introduce sufficient doses of corticosteroids prospectively. Nucleoside analogs were administered in combination with corticosteroids after 1999. Twenty-six patients between 1997 and 2007 were analyzed prospectively. RESULTS: In the retrospective study between 1982 and 1996, four of 16 (25%) patients recovered. In the prospective study between 1997 and 2007, 17 of 26 (65%) patients recovered; 15 of 17 patients treated with corticosteroids with or without antiviral drugs within 10 days after the diagnosis of severe disease recovered, none of five treated similarly but later than 10 days after the diagnosis recovered, and two of three treated with antiviral drugs recovered. CONCLUSIONS: The early introduction of sufficient doses of corticosteroids and nucleoside analogs could be one option for reversing the potential deterioration of patients with clinically severe, life-threatening exacerbation of chronic hepatitis B.     

Coexistence of normal and clonal haemopoiesis in aplastic anaemia patients treated with immunosuppressive therapy

Cytogenetic abnormalities and paroxysmal nocturnal haemoglobinuria (PNH) phenotype are frequent findings in aplastic anaemia patients treated with immunosuppressive therapy (IST). In this study we investigated whether the appearance of clonal haemopoiesis influences patient outcome and survival. 97 patients entered this study and were followed from the onset of the disease for a median follow-up (FU) of 53 months. 93% are alive, 56% achieved complete remission, 30% partial remission, both transfusion independent, and 14% did not respond. Three groups were identified: (A) patients without evidence of emerging clones (71/97); (B) patients who acquired chromosomal abnormalities (13/97); (C) patients who showed low expression of glycosyl phosphatidylinositol anchored proteins (GPI-AP) (PNH phenotype) at presentation or later (16/97). Three patients showed both PIG-AP deficiency and chromosomal abnormalities. The actuarial survival of patients without clonal haemopoiesis (n = 71) at 6 years was 95%, for patients with chromosomal abnormalities (n = 13), 88%, and for patients with PIG-AP deficiency (n = 16), 89%. There was no difference in the probability of becoming transfusion independent in the three groups (93%, 92% and 88% respectively). This study confirmed that a proportion of severe aplastic anaemia (SAA) patients exhibit clonal markers during the time after IST, often coexisting with cytogenetically or phenotypically normal haemopoiesis. There was no significant clinical impact of these abnormalities on transfusion independence and survival at the median follow-up of 4 years.     

The effectiveness of active population screening and treatment for sleeping sickness control in the Democratic Republic of Congo

BACKGROUND: The human African trypanosomiasis (HAT) control programme of the Democratic Republic of Congo (DRC) uses mass screening with the card agglutination test for trypanosomes (CATT). We looked at the contribution of CATT and improved parasitological confirmation to the effectiveness of screening and treatment. METHOD: The effectiveness of the screening and treatment process is measured by the percentage of HAT cases that is effectively cured after a single round of screening. The process is analysed in five steps: (i) the attendance at the screening, (ii) the sensitivity of the screening procedure, (iii) the sensitivity of the parasitological confirmation, (iv) the proportion of the confirmed cases that effectively receive treatment and (v) the cure rate of the treatment. We used a simplified model that multiplies proportions of infected persons that go through each step. We estimated these parameters using a combination of routine data collected by the national control programme over the period January 1997 to December 1998 and published data. For varying attendance rates we compared the effectiveness of screening strategies based on CATT or on CATT combined with improved parasitological confirmation by mini anion exchange column technique (mAECT) with the previously used strategy based on palpation of neck glands and microscopy alone. RESULTS: The model shows that overall effectiveness of the active case detection and treatment strategy is <50% under most scenarios. Attendance rates averaged 74% but showed considerable regional variability and are a major problem in some areas of DRC. The CATT and replacing traditional parasitology by mAECT increases the sensitivity of the screening but a substantial part of the gains are lost at other stages of the screening process. CONCLUSION: Improvements of the HAT screening process such as introduction of CATT or mAECT only make sense if other parameters and attendance rate in particular are optimized at the same time.     

Complement (C3) levels and the effect of C3 depletion in infections of Trypanosoma brucei in mice

Summary Circulating C3 levels and parasitaemias have been measured in four groups of mice chronically infected with Trypanosoma brucei : group A (normal); group B (irradiated, reconstituted); Group C (T cell-deprived); and group D (T cell-deprived, C3-depleted by treatment with cobra venom factor). In groups A-C, C3 levels first rose two to three times normal, C3 thus behaving as an acute phase reactant. Three weeks after infection C3 returned to normal levels for the remainder of the infection. It is thus unlikely that the severe generalized immunodepression seen in mice infected with this trypanosome, is in any way dependent upon a reduction of circulating C3. The curves of parasitaemia in all four groups of mice were essentially similar, even though in group D mice, C3 levels were reduced to about 10% of normal for the first 3 weeks of infection. From this finding, it is argued that complement has no essential role in the mechanisms whereby mice control successive variant populations of T. brucei in the blood. Variant-specific IgM antibodies, acting as trypagglutinins, are probably all that are required to control blood stream infections.     

Graft-versus-host disease (GVHD)-specific survival and duration of systemic immunosuppressive treatment in patients who developed chronic GVHD following allogeneic haematopoietic cell transplantation

We investigated graft-versus-host disease (GVHD)-specific survival (GSS) and the duration of systemic immunosuppressive treatment (IST) in 82 patients who developed chronic GVHD following allogeneic haematopoietic cell transplantation (HCT). These two major study endpoints were calculated using the Kaplan-Meier method. Deaths solely due to the relapse of underlying disease or accidental deaths were censored at the time of occurrence for the analysis of GSS. The probability of GSS at 5 years was 74.2%. The median duration of systemic IST for chronic GVHD was 272 d (range: 7-1450), and the probability of withdrawal of systemic IST at 1, 2 and 3 years was 67.3%, 82.4% and 89.0% respectively. Analysis based on a multivariate model showed that a diagnosis other than leukaemia or myelodysplastic syndrome (P = 0.049), prior occurrence of grade III-IV acute GVHD (P = 0.021), onset of chronic GVHD before d 120 (P = 0.013), serum alkaline phosphatase over 120 IU/l (P = 0.034), and serum bilirubin over 34.2 micromol/l (P = 0.015) were independent adverse prognostic factors for GSS. Prior occurrence of grade III-IV acute GVHD significantly influenced the duration of systemic IST (P = 0.048). In conclusion, analyses of GSS and the duration of systemic IST will allow patients with different outcomes to be stratified for appropriate treatment application and will provide important parameters in prospective trials for the treatment of chronic GVHD.     

Immune depression in bovine trypanosomiasis: effects of acute and chronic Trypanosoma congolense and chronic Trypanosoma vivax infections on antibody response to Brucella abortus vaccine

Summary Cattle were vaccinated with Brucella abortus (S19) vaccine during acute (25 days) and chronic (25 weeks) Trypanosoma congolense and chronic Trypanosoma vivax (25 weeks) infections in order to determine the effect of such infections on the antibody response to the vaccine. It was found that the specific antibody responses of IgG₁ and IgG₂ sub-classes were profoundly depressed (80%) in both the acute and chronic infections with T. congolense. Whereas IgM antibody response was also profoundly depressed (90%) in cattle with the acute infection, it was only 50% depressed in those with chronic infection. There was no depression of IgG₁, IgG₂, or IgM in cattle infected with T. vivax. These animals, however, had no detectable parasitaemia at the time of vaccination and thereafter. These results suggest that during acute infection with T. congolense depressive mechanisms could be acting on the afferent arm of the immune response, namely, antigen recognition and/or processing.     

Predictors of response to immunosuppressive therapy with antithymocyte globulin and cyclosporine and prognostic factors for survival in patients with severe aplastic anemia

Background: Immunosuppressive therapy (IST) with antithymocyte globulin (ATG) plus cyclosporine (CSA) is standard therapy in patients with severe aplastic anemia (SAA) who do not have an available HLA‐matched sibling donor. Methods and patients: The current study aimed to determine the predictive factors for response to IST in patients with SAA and to identify prognostic factors following IST. A total of 62 patients diagnosed with SAA who received IST with either rabbit ATG (n = 33) or horse ATG (n = 29) plus CSA between October 1994 and December 2007 were included. Results: With a median follow‐up duration of 60.5 months, complete response and overall response were estimated to be 31% and 53%, respectively. The 4 yr overall survival rate was 75 ± 6%. In terms of predicting the response to IST, neutrophil counts above 0.3 × 10⁹/L prior to IST were the only significant predictive factor (P = 0.02). Survival following IST was significantly different in favor of both the group showing high absolute reticulocyte counts (ARC) above 10.9 × 10⁹/L prior to IST (P = 0.004) and the group achieving any response following IST (P = 0.002). Conclusions: Pre‐IST neutrophil counts might predict the response to IST, while absolute ARCs prior to IST and response status after IST could be prognostic factors following IST.     

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cell''s sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.