Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting ß-cell regeneration

Type 1 diabetes mellitus results from a cell-mediated autoimmune attack against pancreatic ß-cells. Traditional treatments involve numerous daily insulin dosages/injections and rigorous glucose control. Many efforts toward the identification of ß-cell precursors have been made not only with the aim of understanding the physiology of islet regeneration, but also as an alternative way to produce ß-cells to be used in protocols of islet transplantation. In this review, we summarize the most recent studies related to precursor cells implicated in the regeneration process. These include embryonic stem cells, pancreas-derived multipotent precursors, pancreatic ductal cells, hematopoietic stem cells, mesenchymal stem cells, hepatic oval cells, and mature ß-cells. There is controversial evidence of the potential of these cell sources to regenerate ß-cell mass in diabetic patients. However, clinical trials using embryonic stem cells, umbilical cord blood or adult bone marrow stem cells are under way. The results of various immunosuppressive regimens aiming at blocking autoimmunity against pancreatic ß-cells and promoting ß-cell preservation are also analyzed. Most of these regimens provide transient and partial effect on insulin requirements, but new regimens are beginning to be tested. Our own clinical trial combines a high dose immunosuppression with mobilized peripheral blood hematopoietic stem cell transplantation in early-onset type 1 diabetes mellitus.

Immune factors and immunoregulation in tuberculosis

Pathogens causing tuberculosis and other chronic infectious diseases of major public health importance commonly have complex mechanisms involved in their persistence in the host despite specific and sometimes strong immune responses. These diseases are also associated with the lack of efficient vaccines, difficult therapeutics and a high mortality rate among susceptible individuals. Here, we will review features of the host immune response that contribute to the occurrence of disease. In addition, we propose that the immune responses observed in tuberculosis cannot be interpreted solely on the basis of a Th1-Th2 counter-regulatory paradigm since there is growing evidence that natural regulatory T cells may play an important role in the regulation of host immune responses against Mycobacterium tuberculosis. Thus, the development of more effective vaccines against this bacterial disease should take into account the role of natural regulatory T cells in the progression to severe disease and persistence of infection. Finally, new treatments based on manipulation of regulatory T cells should be investigated.

Aging reduces the primary humoral response and the in vitro cytokine production in mice

Aging is accompanied by a decrease in several physiological functions that make older individuals less responsive to environmental challenges. In the present study, we analyzed the immune response of female BALB/c mice (N = 6) of different ages (from 2 to 96 weeks) and identified significant age-related alterations. Immunization with hapten-protein (trinitrophenyl-bovine serum albumin) conjugates resulted in lower antibody levels in the primary and secondary responses of old mice (72 weeks old). Moreover, young mice (2, 16, and 32 weeks old) maintained specific antibodies in their sera for longer periods after primary immunization than did old mice. However, a secondary challenge efficiently induced memory in old mice, as shown by the increased antibody levels in their sera. The number of CD4+ and CD8+ T cells in the spleen increased until 8 weeks of age but there was no change in the CD4+/CD8+ ratio with aging. Splenic T cells from old mice that had or had not been immunized were less responsive to concanavalin-A and showed reduced cytokine production compared to young mice (IL-2: 57-127 vs 367-1104 pg/mL, IFN-g: 2344-12,836 vs 752-23,106 pg/mL and IL-10: 393-2172 vs 105-2869 pg/mL in old and young mice, respectively). These data suggest that there are significant changes in the organization of the immune system throughout life. However, the relevance of these alterations for the functioning of the immune system is unknown.

The use of stem cells for the treatment of autoimmune diseases

Autoimmune diseases constitute a heterogeneous group of conditions commonly treated with anti-inflammatory, immunosuppressant and immunomodulating drugs, with satisfactory results in most cases. Nevertheless, some patients become resistant to conventional therapy. The use of high doses of drugs in such cases results in the need for bone marrow reconstitution, a situation which has stimulated research into the use of hematopoietic stem cells in autoimmune disease therapy. Stem cell transplantation in such diseases aims to destroy the self-reacting immune cells and produce a new functional immune system, as well as substitute cells for tissue damaged in the course of the disease. Significant results, such as the reestablishment of tolerance and a decrease in the recurrence of autoimmune disease, have been reported following stem cell transplantation in patients with autoimmune disease in Brazil and throughout the world. These results suggest that stem cell transplantation has the potential to become an important therapeutic approach to the treatment of various autoimmune diseases including rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, Crohn's disease, autoimmune blood cytopenias, and type I diabetes mellitus.

Impairment of cytomegalovirus-specific cellular immune response as a risk factor for cytomegalovirus disease in transplant recipients

Human cytomegalovirus (CMV) infection is common in most people but nearly asymptomatic in immunocompetent individuals. After primary infection the virus persists throughout life in a latent form in a variety of tissues, particularly in precursor cells of the monocytic lineage. CMV reinfection and occurrence of disease are associated with immunosuppressive conditions. Solid organ and bone marrow transplant patients are at high risk for CMV disease as they undergo immunosuppression. Antiviral treatment is effective in controlling viremia, but 10-15% of infected patients can experience CMV disease by the time the drug is withdrawn. In addition, long-term antiviral treatment leads to bone marrow ablation and renal toxicity. Furthermore, control of chronic CMV infection in transplant recipients appears to be dependent on the proper recovery of cellular immunity. Recent advances in the characterization of T-cell functions and identification of distinct functional signatures of T-cell viral responses have opened new perspectives for monitoring transplant individuals at risk of developing CMV disease.

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

Intrinsic denervation of the colon is associated with a decrease of some colonic preneoplastic markers in rats treated with a chemical carcinogen

Denervation of the colon is protective against the colon cancer; however, the mechanisms involved are unknown. We tested the hypothesis that the denervated colonic mucosa could be less responsive to the action of the chemical carcinogen dimethylhydrazine (DMH). Three groups of 32 male Wistar rats were treated as follows: group 1 (G1) had the colon denervated with 0.3 mL 1.5 mM benzyldimethyltetradecylammonium (benzalkonium chloride, BAC); G2 received a single ip injection of 125 mg/kg DMH; G3 was treated with BAC + the same dose and route of DMH. A control group (Sham, N = 32) did not receive any treatment. Each group was subdivided into four groups according to the sacrifice time (1, 2, 6, and 12 weeks after DMH). Crypt fission index, ß-catenin accumulated crypts, aberrant crypt foci, and cell proliferation were evaluated and analyzed by ANOVA and the Student t-test. G3 animals presented a small number of aberrant crypt foci and low crypt fission index compared to G2 animals after 2 and 12 weeks, respectively. From the second week on, the index of ß-catenin crypt in G3 animals increased slower than in G2 animals. From the 12th week on, G2 animals presented a significant increase in cell proliferation when compared to the other groups. Colonic denervation plays an anticarcinogenic role from early stages of colon cancer development. This finding can be of importance for the study of the role of the enteric nervous system in the carcinogenic process.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Attenuation of phosphamidon-induced oxidative stress and immune dysfunction in rats treated with N-acetylcysteine

The effect of N-acetylcysteine, a thiolic antioxidant, on attenuation of phosphamidon-induced oxidative stress and immune dysfunction was evaluated in adult male Wistar rats weighing 200-250 g. Rats were divided into four groups, 8 animals/group, and treated with phosphamidon, N-acetylcysteine or the combination of both for 28 days. Oral administration of phosphamidon (1.74 mg/kg), an organophosphate insecticide, increased serum malondialdehyde (3.83 ± 0.18 vs 2.91 ± 0.24 nmol/mL; P < 0.05) and decreased erythrocyte superoxide dismutase (567.8 ± 24.36 vs 749.16 ± 102.61 U/gHb; P < 0.05), catalase activity (1.86 ± 0.18 vs 2.43 ± 0.08 U/gHb; P < 0.05) and whole blood glutathione levels (1.25 ± 0.21 vs 2.28 ± 0.08 mg/gHb; P < 0.05) showing phosphamidon-induced oxidative stress. Phosphamidon exposure markedly suppressed humoral immune response as assessed by antibody titer to ovalbumin (4.71 ± 0.51 vs 8.00 ± 0.12 -log2; P < 0.05), and cell-mediated immune response as assessed by leukocyte migration inhibition (25.24 ± 1.04 vs 70.8 ± 1.09%; P < 0.05) and macrophage migration inhibition (20.38 ± 0.99 vs 67.16 ± 5.30%; P < 0.05) response. Phosphamidon exposure decreased IFN-у levels (40.7 ± 3.21 vs 55.84 ± 3.02 pg/mL; P < 0.05) suggesting a profound effect of phosphamidon on cell-mediated immune response. A phosphamidon-induced increase in TNF-α level (64.19 ± 6.0 vs 23.16 ± 4.0 pg/mL; P < 0.05) suggests a contributory role of immunocytes in oxidative stress. Co-administration of N-acetylcysteine (3.5 mmol/kg, orally) with phosphamidon attenuated the adverse effects of phosphamidon. These findings suggest that oral N-acetylcysteine treatment exerts protective effect and attenuates free radical injury and immune dysfunction caused by subchronic phosphamidon exposure.

Humoral and cellular immune responses to Blomia tropicalis and concanavalin A-binding fractions in atopic patients

Blomia tropicalis, Dermatophagoides pteronyssinus and D. farinae are prevalent house dust mites. Concanavalin A-binding components derived from B. tropicalis (Bt-ConA extract) are highly immunogenic in allergic diseases. The aim of the present study was to evaluate the humoral and cellular immune responses to B. tropicalis in mite-sensitized patients. A total of 137 patients with allergic rhinitis with/without asthma and 109 non-atopic subjects were selected and analyzed by the skin prick test, and for total serum IgE and specific IgE levels to both Bt-total and Bt-ConA extracts, their proliferative response and cytokine (IFN-γ and IL-5) production by peripheral blood mononuclear cells (PBMC) stimulated with both extracts. Skin prick test showed that 70% of the patients were sensitized to Bt (Bt+) and similar levels of specific IgE to Bt-total and Bt-ConA extracts were demonstrable in Bt+ patients. Significant PBMC proliferation was observed in response to Bt-total extract in Bt+, but not in Bt- patients and non-atopic subjects (P < 0.001). Bt-ConA extract induced increased proliferative responses in all patient groups compared to medium alone (P < 0.05), but these responses were significantly decreased in the presence of the mannopyranoside ConA inhibitor (P < 0.05). Significant IFN-γ production was observed after Bt-ConA stimulation of Bt+ patients (P < 0.05), while Bt-total extract had no effect. IL-5 production was consistently detected in Bt+ patients after allergen-specific stimulation or with no stimulus, indicating that PBMC from allergic patients are prone to produce Th2 profile cytokines, spontaneously or inductively by allergen restimulation. These data showed that ConA-binding components isolated from B. tropicalis may contain relevant antigens that are involved in both humoral and cellular immune responses. However, without an additional purification procedure to eliminate the residual contamination with ConA, its use in immunotherapeutic procedures cannot be recommended.

Effects on prolactin secretion and binding to dopaminergic receptors in sleep-deprived lupus-prone mice

Sleep disturbances have far-reaching effects on the neuroendocrine and immune systems and may be linked to disease manifestation. Sleep deprivation can accelerate the onset of lupus in NZB/NZWF1 mice, an animal model of severe systemic lupus erythematosus. High prolactin (PRL) concentrations are involved in the pathogenesis of systemic lupus erythematosus in human beings, as well as in NZB/NZWF1 mice. We hypothesized that PRL could be involved in the earlier onset of the disease in sleep-deprived NZB/NZWF1 mice. We also investigated its binding to dopaminergic receptors, since PRL secretion is mainly controlled by dopamine. Female NZB/NZWF1 mice aged 9 weeks were deprived of sleep using the multiple platform method. Blood samples were taken for the determination of PRL concentrations and quantitative receptor autoradiography was used to map binding of the tritiated dopaminergic receptor ligands [³H]-SCH23390, [³H]-raclopride and [³H]-WIN35,428 to D1 and D2 dopaminergic receptors and dopamine transporter sites throughout the brain, respectively. Sleep deprivation induced a significant decrease in plasma PRL secretion (2.58 ± 0.95 ng/mL) compared with the control group (25.25 ± 9.18 ng/mL). The binding to D1 and D2 binding sites was not significantly affected by sleep deprivation; however, dopamine transporter binding was significantly increased in subdivisions of the caudate-putamen - posterior (16.52 ± 0.5 vs 14.44 ± 0.6), dorsolateral (18.84 ± 0.7 vs 15.97 ± 0.7) and ventrolateral (24.99 ± 0.5 vs 22.54 ± 0.7 µCi/g), in the sleep-deprived mice when compared to the control group. These results suggest that PRL is not the main mechanism involved in the earlier onset of the disease observed in sleep-deprived NZB/NZWF1 mice and the reduction of PRL concentrations after sleep deprivation may be mediated by modifications in the dopamine transporter sites of the caudate-putamen.

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.

Advances in biliary atresia: from patient care to research

Biliary atresia, the most common cause of liver transplantation in children, remains a challenge for clinicians and investigators. The development of new therapeutic options, besides the typical hepatoportoenterostomy, depends on a greater understanding of its pathogenesis and how it relates to the clinical phenotypes at diagnosis and the rate of disease progression. In this review, we present a perspective of how recent research has advanced the understanding of the disease and has improved clinical care protocols. Molecular and morphological analyses at diagnosis point to the potential contributions of polymorphism in the CFC1 and VEGF genes to the pathogenesis of the disease, and to an association between the degree of bile duct proliferation and long-term outcome. In experimental models, cholangiocytes do not appear to have antigen-presenting properties despite a substantial innate and adaptive immune response that targets the biliary epithelium and produces duct obstruction. Initial clinical trials assessing the efficacy of corticosteroids in decreasing the inflammation and improving outcome do not show a superior effect of corticosteroids as an adjuvant treatment following hepatoportoenterostomy. The best outcome still remains linked to an early diagnosis and surgical treatment. In this regard, the Yellow Alert campaign by the Sociedade Brasileira de Pediatria and the inclusion of the Stool Color Card in the health booklet given to every neonate in Brazil have the potential to decrease the age of diagnosis, shorten the time between diagnosis and surgical treatment, and improve the long-term outcome of children with this devastating disease.

Evaluation of the immune humoral response of Brazilian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, mental and growth deficiency, and recurrent respiratory infections. RTS has been associated with CREBBP gene mutations, but EP300 gene mutations have recently been reported in 6 individuals. In the present study, the humoral immune response in 16 RTS patients with recurrent respiratory infections of possible bacterial etiology was evaluated. No significant differences between patients and 16 healthy controls were detected to explain the high susceptibility to respiratory infections: normal or elevated serum immunoglobulin levels, normal salivary IgA levels, and a good antibody response to both polysaccharide and protein antigens were observed. However, most patients presented high serum IgM levels, a high number of total B cell and B subsets, and also high percentiles of apoptosis, suggesting that they could present B dysregulation. The CREBBP/p300 family gene is extremely important for B-cell regulation, and RTS may represent an interesting human model for studying the molecular mechanisms involved in B-cell development.

Evasion of immune responses by Trypanosoma cruzi, the etiological agent of Chagas disease

Infection with the protozoan parasite Trypanosoma cruzi leads to Chagas disease, which affects millions of people in Latin America. Infection with T. cruzi cannot be eliminated by the immune system. A better understanding of immune evasion mechanisms is required in order to develop more effective vaccines. During the acute phase, parasites replicate extensively and release immunomodulatory molecules that delay parasite-specific responses mediated by T cells. This immune evasion allows the parasite to spread in the host. In the chronic phase, parasite evasion relies on its replication strategy of hijacking the TGF-β signaling pathway involved in inflammation and tissue regeneration. In this article, the mechanisms of immune evasion described for T. cruzi are reviewed.