Association between polyclonal B cell activation and the presence of autoantibodies in mice infected with Yersinia enterocolitica O:3

Eight-week old conventional female Swiss mice were inoculated intravenously with Yersinia enterocolitica O:3. A second group of normal mice was used as control. Five mice from each group were bled by heart puncture and their spleens were removed for spleen cell collection on the 3rd, 5th, 7th, 10th, 14th and 21st day after infection. Immunoglobulin-secreting spleen cells were detected by the isotype-specific protein A plaque assay. Total immunoglobulin levels were determined in mouse serum by single radial immunodiffusion and the presence of autoantibodies was determined by ELISA. We observed a marked increase in the total number of cells secreting immunoglobulins of all isotypes as early as on the 3rd day post-infection and the peak of secretion occurred on the 7th day. At the peak of the immunoglobulin response, the total number of secreting cells was 19 times higher than that of control mice and most immunoglobulin-secreting cells were of the IgG2a isotype. On the 10th day post-infection, total serum immunoglobulin values were 2 times higher in infected animals when compared to the control group, and continued at this level up to the 21st day post-infection. Serum absorption with viable Y. enterocolitica cells had little effect on antibody levels detected by single radial immunodiffusion. Analysis of serum autoantibody levels revealed that Y. enterocolitica infection induced an increase of anti-myosin and anti-myelin immunoglobulins. The sera did not react with collagen. The present study demonstrates that Y. enterocolitica O:3 infection induces polyclonal activation of murine B cells which is correlated with the activation of some autoreactive lymphocyte clones

V-region-related and -unrelated immunosupression accompanying infections

This paper discusses current evidence for the relationship between polyclonal lymphocyte activation, specific immunossupression with decreased resistance, and autoimmune pathology, that are all often found associated with infections by a variety of virus, bacteria and parasites . The central question of class determination of immune effector activities is considered in the context of the cellular targets for nonspecific mitogenic activities associated with infection. A model is presented to integrate these findings: mitogenens produced by the microorganism or the infected cells are preferentially active on CD5 B cells, the resulting over-production of IL-10 will tend to bias all immune activities in to a Th-2mode of effector functions, with high titers of polyclonal antibodies and litle or no production of gamma IFN and other "inflamatory"lymphokines that often mediate resistance. In turn these conditions allow for parasite persistence and the corresponding long-term disregulation of self-directed immune reactivities, resulting in autoimmunity in the chronic phase. This model would predict that selective immunization with the mitogenic principles involved in desregulation, could stand better chances than strategies of vaccination based on immunopotentiation against othere, functionally neutral antigenic epitopes. It is argued, however, that the complexity of immune responses and their regulation together with our ignorance on the genetic controls of class-determination, offer poor prospects for a scientifically-based, rational development of vaccines in the near future. It is suggested that empirically-based and technologically developed vaccines might suceed, while basic scientific approaches are reinforced and given the time provide a better understanding of those process.

Immunoregulatory mechanisms and Chagas' disease

During the course of experimental Chagas' disease, several immune disorders occur. In the acute phase, T and B cell plyclonal activation is associated to immunossupression. At the chronic stage. T cells - of the TH2 subset - participate to the pathology characteristic of Chagas'disease. Data obtained after infection of BALB/Xid mice suggest that polyclonal activation may be dependent on B1 (CD5) cell activation. Moreover, these mice fail to develop the pathological features of the chronic infection. Control of lymphokine secretion might play a key role in the clinical status of Chagas'disease.

Restriction in the repertoire of detectable autoantibodies in polyclonal B cell activations and the mimicry of autoantigens by idiotopes

Despite the existence of erythrocyte-autoreactive B cells in normal animals, erythrocyte-autoantibodies could not be detected during polyclonal B-cell activation (PBA) both in patients with visceral leishmaniasis and in bacterial lipopolysacharide (LPS) - injected mice. The failure to detect these autoantibodies in mice with PBA di not seem to be due to suppressor-cell activity, since (1) transfer of spleen cells from LPS-treated mice to naive recipients did not affect the erythrocyte-autoantibody response elicited by subsequent injections of rat erythrocytes and (2) low doses of X-radiation did no lead to erythrocyte-autoantibody detection in LPS-treated mice. The possibility that the detection of erytrocyte-autoantibodies could be affected by autoantibodies with idiotopes mimicring erythrocyte epitopes, the synthesis of which would also be triggerred in PBA, is discussed. Indirect evidence for the existence in normal animal of an expanded lymphocyte population with DNP-binding. Ia-mimicring antigen receptors is presented.

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

Effect of b-Propiolactone treatment on the complement activation mediated by equine antisera

Reduction of complement activation through an alteration of the Fc fragment of immunoglobulins by b-propiolactone treatment was carried out in equine antisera raised against rabies virus, Bothrops venoms and diphtherial toxin. Results were evaluated by means of an anaphylactic test performed on guinea-pigs, and compared to the ones obtained with the same sera purified by saline precipitation (ammonium sulfate), followed or not by enzymatic digestion with pepsin. Protein purity levels for antibothropic serum were 184.5 mg/g and 488.5 mg/g in b-propiolactone treated and pepsin-digested sera, respectively. The recovery of specific activity was 100% and 62.5% when using antibothropic serum treated by b-propiolactone and pepsin digestion, respectively. The antidiphtherial and anti-rabies sera treated with b-propiolactone and pepsin presented protein purity levels of 5,698 and 7,179 Lf/g, 16,233 and 6,784 IU/g, respectively. The recovery of specific activity for these antisera were 88.8%, 77.7%, 100% and 36,5%, respectively. b-propiolactone treatment induced a reduction in complement activation, tested "in vivo", without significant loss of biological activity. This treatment can be used in the preparation of heterologous immunoglobulins for human use.

Human polyclonal anti-hepatitis B surface antigen immunoglobulin reduces the frequency of acute rejection after liver transplantation for chronic hepatitis B

BACKGROUND: Use of polyclonal anti-hepatitis B surface antigen immunoglobulin (HBIg) has been shown to reduce hepatitis B virus (HBV) recurrence after liver transplantation (LT) and to decrease the frequency of acute cellular rejection (ACR). However, the protective role of HBIg against ACR remains controversial, since HBV infection has been also associated with a lower incidence of ACR. AIM: To assess the relationship between HBIg immunoprophylaxis and the incidence of rejection after LT. METHODS: 260 patients (158 males, 43 ± 14 years old) submitted to LT were retrospectively evaluated and divided into three groups, according to the presence of HBsAg and the use of HBIg. Group I was comprised of HBsAg-positive patients (n = 12) that received HBIg for more than 6 months. Group II was comprised of HBsAg-positive patients that historically have not received HBIg or have been treated irregularly for less than 3 months (n = 10). Group III was composed of 238 HBsAg-negative subjects that have not received HBIg. RESULTS: HBIg-treated patients (group I) had significantly less ACR episodes, when compared to group II and III. No differences between groups II and III were observed. CONCLUSIONS: Long-term HBIg administration contributes independently to reduce the number of ACR episodes after LT.

In vitro and in situ activation of the complement system by the fungus Lacazia loboi

Since there are no studies evaluating the participation of the complement system (CS) in Jorge Lobo's disease and its activity on the fungus Lacazia loboi, we carried out the present investigation. Fungal cells with a viability index of 48% were obtained from the footpads of BALB/c mice and incubated with a pool of inactivated serum from patients with the mycosis or with sterile saline for 30 min at 37 ºC. Next, the tubes were incubated for 2 h with a pool of noninactivated AB+ serum, inactivated serum, serum diluted in EGTA-MgCl2, and serum diluted in EDTA. The viability of L. loboi was evaluated and the fungal suspension was cytocentrifuged. The slides were submitted to immunofluorescence staining using human anti-C3 antibody. The results revealed that 98% of the fungi activated the CS by the alternative pathway and no significant difference in L. loboi viability was observed after CS activation. In parallel, frozen histological sections from 11 patients were analyzed regarding the presence of C3 and IgG by immunofluorescence staining. C3 and IgG deposits were observed in the fungal wall of 100% and 91% of the lesions evaluated, respectively. The results suggest that the CS and immunoglobulins may contribute to the defense mechanisms of the host against L. loboi.

POLYCLONAL OUTBREAK OF BLOODSTREAM INFECTIONS CAUSED BY Burkholderia cepacia COMPLEX IN HEMATOLOGY AND BONE MARROW TRANSPLANT OUTPATIENT UNITS

Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc) in bone marrow transplant and hematology outpatients.Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW) hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC) care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units.Findings: 14 patients had B. multivorans(one patient had also B. cenopacia), six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology); in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30%) were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication), no new cases occurred.Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A) of multi-dose vials.

Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.