An adverse immune-endocrine profile in patients with tuberculosis and type 2 diabetes.

Diabetes is a risk factor for the development of pulmonary tuberculosis (TB) and both diseases present endocrine alterations likely to play a role in certain immuno-endocrine-metabolic associated disorders. Patients with TB, or with TB and type 2 diabetes (TB + T2DM) and healthy controls (HCo) were assessed for plasma levels of cortisol, dehydroepiandrosterone (DHEA), estradiol, testosterone, growth hormone (GH), prolactin, insulin-like growth factor-1 (IGF-1), cytokines (IL-6, IL-10, IFN-γ) and the specific lymphoproliferative capacity of peripheral blood mononuclear cells. All patients had higher levels of cortisol with a reduction in DHEA, thus resulting in an increased cortisol/DHEA ratio (Cort/DHEA). Increased prolactin and particularly GH levels were found in both groups of TB patients. This was not paralleled by increased concentrations of IGF, which remained within the levels of HCo. Estradiol levels were significantly augmented in patients TB, and significantly more in TB + T2DM, whereas testosterone levels were decreased in both groups of patients. IFN- γ and IL-6 concentrations were significantly increased in all TB, even further in TB + T2DM; while IL-10 was equally increased in both groups of TB patients. The in vitro specific proliferative capacity was decreased in both groups of patients as compared to that of HCo. The adverse immune-endocrine profile of TB seems to be slightly more pronounced in patients who also have T2DM.

Immunoneuroendocrine alterations in patients with progressive forms of chronic Chagas disease.

We studied the features of parallel immunoneuroendocrine responses in patients with different degrees of chronic Chagas myocarditis (indeterminate, mild/moderate or severe). A systemic inflammatory scenario was evident in patients with severe myocarditis compared to healthy subjects. This was paralleled by a disrupted activation of the hypothalamus-pituitary-adrenal axis, characterized by decreased concentrations of dehydroepiandrosterone-sulfate (DHEA-s) and an unbalanced cortisol/DHEA-s ratio, reinforcing the view that severe Chagas disease is devoid of an adequate anti-inflammatory milieu, likely involved in pathology. Our study constitutes the first demonstration of neuroendocrine disturbances, in parallel to a systemic inflammatory profile, during progressive human Chagas disease.

Short treatment with the tumour necrosis factor-alpha blocker infliximab diminishes chronic chagasic myocarditis in rats without evidence of Trypanosoma cruzi reactivation.

Tumour necrosis factor (TNF)-alpha is crucial for resistance to Trypanosoma cruzi acute infection, but there is scant information on its role during the chronic phase. To address this issue, we analysed whether a short treatment with a TNF-alpha blocker affected the course and characteristics of chronic disease in a rat experimental model of T. cruzi infection. An anti-TNF-alpha agent (infliximab) was administered during the chronic phase for a period of 4 weeks (3 mg/kg/week), while control infected rats were inoculated with saline physiological solution. Search for parasites yielded non-successful results in all infected groups, irrespective of treatment. Nevertheless, the presence of T. cruzi kDNA in heart tissue was detected in infected and infected plus treated animals. Because infliximab might induce changes in the anti-parasite cytokine response, circulating levels of interleukin (IL)-10, interferon-gamma and nitric oxide were evaluated. An increase in IL-10 levels was observed only in the infected group treated with the anti-TNF-alpha blocker compared to the remaining groups (P < 0.05). A clear attenuation of histological damage associated with a diminution of cardiac TNF-alpha mRNA expression was observed in the infected and treated animals compared to the infected and non-treated group. Blocking of TNF-alpha during a relatively short period in chronically infected rats did not lead to evident parasite reactivation but reduced myocarditis severity significantly, indicating a role of this cytokine in the pathogenesis of chronic myocardial damage.

Chronic inflammation as a manifestation of defects in immunoregulatory networks: implications for novel therapies based on microbial products.

Based on a unifying theory presented here, it is predicted that the immune defects resulting in chronic inflammation rather than effective immune responses could be rectified by the therapeutic use of agents prepared from micro-organisms. With appropriate molecular patterns, these should be able to induce protective immunoregulatory networks or to reprogramme defective ones. In contrast to acute inflammation, chronic inflammation appears to have no beneficial role, but is a state of sustained immune reactivity in the presence or progression of a disease process. This results in an escalating cycle of tissue damage followed by unproductive tissue repair, breaks in self-tolerance, malignant transformation or deleterious changes in tissue morphology and function. Such inappropriate immune reactivity is an underlying characteristic, either in initiation or maintenance, of a diverse range of disease states including chronic infection, autoimmunity, allergy, cancer, vascular disease and metabolic alterations. Evidence is presented that the inappropriate immune reactivity is due, at least to some extent, to failures in the establishment of immunoregulatory networks as a result of hygiene-related factors. Such networks are the result of activation of antigen-presenting cells, principally dendritic cells, by molecular patterns of micro-organisms encountered sequentially during life and establishing the 'biography' of the immune system.

The use of mycobacterial adjuvant-based agents for immunotherapy of cancer.

A heat-killed preparation of Mycobacterium vaccae (SRL172) has been shown, in recent studies, to be effective in the treatment of adenocarcinoma of the lung and renal cell cancer. It is postulated that the mechanisms of this form of immunotherapy is, at least in part, due to immune regulation, reflected in the selective enhancement of Th1 and down-regulation of Th2 T cell activity. These beneficial effects are attributed to the ability of adjuvants in the bacterial cell walls to modify and optimise the response to antigens shared by the bacteria and stressed host tissues, resulting in the destruction of cancer cells by programmed cell death or apoptosis. The M. vaccae-induced apoptosis appears to be most effective against carcinomas, perhaps especially those of glandular tissue, in contrast to pyrexia-induced necrosis which is most effective against tumours of mesodermal origin. In view of the great range of adjuvants, especially in the genus Mycobacterium and related genera, it may prove possible to develop a range of immunotherapeutic agents with useful activity against a wide range of cancers.

Altered cortisol/DHEA ratio in tuberculosis patients and its relationship with abnormalities in the mycobacterial-driven cytokine production by peripheral blood mononuclear cells.

We have investigated the relationship between cortisol and dehydroepiandrosterone (DHEA) levels and the immune response to mycobacterial antigens in peripheral venous blood, from a male population of active tuberculosis patients and age-matched healthy controls of the same sex (HCo). Peripheral blood mononuclear cells were cultured for 36 or 96 h with whole sonicated Mycobacterium tuberculosis (WSA) for measurement of proliferation, interferon gamma (IFN-gamma) and interleukin-10 (IL-10) in culture supernatants. Comparisons on the in vitro mycobacterial-driven immune responses demonstrated that TB patients had a higher IL-10 production, a decreased lymphoproliferation and a trend to reduced IFN-gamma synthesis, in relation to HCo. Active disease was also characterized by increases in the plasma levels of glucocorticoids (GC) and reduced concentrations of DHEA which resulted in a higher cortisol/DHEA ratio respect the HCo group. Plasma DHEA levels were positively correlated with IFN-gamma values. An inverse correlation was found between the cortisol/DHEA ratio and IFN-gamma levels. Novel evidence is provided showing that the balance between cortisol and DHEA is partly responsible for the immune perturbations seen in TB patients.

Altered production of IFN-gamma? And other inflammation-related cytokines by mycobacteria- stimulated peripheral blood mononuclear cells from rheumatoid arthritis patients.

Peripheral blood mononuclear cells taken from 32 patients with Rheumatoid Arthritis (RA) receiving neither steroids nor methotrexate and 34 healthy controls were examined for lymphoproliferation in the presence of ultrasonic extracts of 14 different mycobacterial species or serotypes, of an extract of Candida albicans and of 2 mitogens. Additionally, cells were incubated for 96 hours alone, or with Mycobacterium tuberculosis (M.tb) sonicate or Concanavalin-A (Con-A), and supernatants were tested for a range of cytokines. Lymphocytes of rheumatoid patients were less reactive than controls to all the mycobacterial preparations, but no different in their responses to mitogens. Stimulation of patients' cells with M.tb sonicate induced significantly less interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) but more transforming growth factor- beta (TGF-beta) than controls. Even stimulation with Con-A induced much less IFN-gamma in patient's cells than in those of controls. The combination of reduced responses to the mycobacterial reagents and reduced stimulation of type 1 cytokines by the sonicate of M.tb, suggests reduced responsiveness to group i, common mycobacterial antigens. Such findings need not indicate involvement of mycobacteria specifically in the disease aetiology, but provide novel information on the immunopathological abnormalities, which may explain the reported increased susceptibility to mycobacteria of RA patients.

Deficient control of Trypanosoma cruzi infection in C57BL/6 mice is related to a delayed specific IgG response and increased macrophage production of pro-inflammatory cytokines.

Earlier work in Trypanosoma cruzi-infected C57BL/6 and BALB/c mice revealed an acute disease, of lethal outcome in the former group and lesser severity in BALB/c mice. Fatal course was not accompanied by an increased parasite load, but by a substantial imbalance between pro- and anti-inflammatory cytokine serum levels. To better characterise the mechanisms allowing the host to restrain the infection, we have now studied the specific IgG production and in vitro behaviour of peritoneal macrophages (PMs) when exposed to T. cruzi. BALC/c mice displayed higher serum levels of specific immunoglobulins in the first weeks of acute infection. In vitro infected PMs showed no between-group differences in the number of intracellular parasites, although TNFalpha levels were significantly higher in culture supernatants from C57BL/6 mice. Because an LPS-based pretreatment (desensitisation protocol followed by a sublethal LPS dose) reduced disease severity of C57BL/6 mice, we next explored the features of the in vitro infection in PMs from mice subjected to such protocol. PMs from LPS-pretreated mice had a decreased production of TNFalpha and IL-1beta, becoming more permissive to parasite replication. It is concluded that deficient control of T. cruzi infection in C57BL/6 mice may also involve a less satisfactory specific IgG response and increased TNFalpha production by PMs. Improved disease outcome in LPS-pretreated mice may be associated with the reduced inflammatory cytokine production by PMs, but the impaired ability of these cells to control parasite growth suggests that compensatory mechanisms are operating in the in vivo situation.

Age-related increase in resistance to acute Trypanosoma cruzi infection in rats is associated with an appropriate antibody response.

Inoculation at weaning with Trypanosoma cruzi in inbred "l" rats resulted in a self-resolving acute infection characterized by marked parasitaemias, whereas challenge to adult rats revealed a mild disease with extremely low parasitaemias. To explore the mechanisms underlying such age-associated differences in disease outcome, we analysed the in vitro replication of T. cruzi, nitric oxide and tumour necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages (PMs), the serum concentrations of the specific immunoglobulins (Igs) IgM and IgG, antibodies exhibiting lytic activity against bloodstream forms of T. cruzi and circulating levels of nitrate, TNF-alpha and interferon-gamma (IFN-gamma). Macrophages from young rats were as effective as their adult counterparts for restraining intracellular parasite replication. When stimulated with IFN-gamma, culture supernatants from young PMs contained higher amounts of nitrite and TNF-alpha. Serum samples from 4 and 7 days post infection revealed easily detectable amounts of nitrate, with values being further augmented by day 7 post infection and significantly higher in the young group. TNF-alpha levels were only detected in the young group by day 7 post infection. Both groups had increased amounts of IFN-gamma in their sera, although in adult rats, this trend was followed by a significant drop at day 7, with young rats showing values still higher by the same time point evaluation. In contrast, young rats presented significantly lower levels of IgM and IgG antibodies during the first week of infection. Increased resistance in adult rats seems to be the result of a more appropriate antibody production.

Trypanocidal drug benznidazole impairs lipopolysaccharide induction of macrophage nitric oxide synthase gene transcription through inhibition of NF-kappaB activation.

In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-gamma with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-kappaB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-kappaB in stimulated macrophages. NF-kappaB is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IkappaB. Within this setting, Western blot was also performed to show that BZL blocked IkappaBalpha degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-kappaB activation after LPS plus IFN-gamma stimulation.

Influence of disease severity on nitrite and cytokine production by peripheral blood mononuclear cells (PBMC) from patients with pulmonary tuberculosis (TB).

Earlier studies in patients with pulmonary TB have revealed a higher production of Th1 cell type cytokines in moderate TB, with predominant Th2-like responses in advanced disease. Given the influence of IL-12 in T cell differentiation, as well as the roles of transforming growth factor-beta (TGF-beta), nitric oxide and tumour necrosis factor-alpha (TNF-alpha) in the immune response against intracellular pathogens, we decided to analyse the interferon-gamma (IFN-gamma), IL-4, IL-12, TGF-beta, TNF-alpha and nitrite concentrations in culture supernatants of PBMC from TB patients showing different degrees of lung involvement. The sample population comprised 18 untreated TB patients with either moderate (n = 9) or advanced (n = 9) disease and 12 age- and sex-matched healthy controls (total population (patients and controls) 12 women, 18 men, aged 37 +/- 13 years (mean +/- s.d.)). PBMC were stimulated with whole sonicate from Mycobacterium tuberculosis and the supernatants were collected on day 4 for measurement of cytokine and nitrite levels. Antigen-stimulated IFN-gamma, TGF-beta and TNF-alpha production was found to be significantly increased in TB patients, both moderate and advanced, compared with the controls. Levels of IFN-gamma were significantly higher in moderate disease than advanced cases, whereas advanced cases showed significantly higher IL-12, TGF-beta and TNF-alpha concentrations when compared with cases of moderate TB. Nitrite levels were also increased in TB patients and the increase was statistically significant when advanced cases were compared with controls. These findings may contribute to a clearer picture of the net effect of cytokine interactions in TB, essential for a better understanding of the immunopathological mechanisms underlying the distinct clinical forms of the disease.

Protected Trypanosoma cruzi infection in rats born to mothers receiving interferon-gamma during gestation is associated with a decreased intramacrophage parasite growth and preferential synthesis of specific IgG2b antibodies.

We demonstrated that administration of interferon gamma (IFN-gamma) to pregnant rats conferred partial resistance in their offspring to further challenge with Trypanosoma cruzi. Because of the effects of IFN-gamma on macrophage activation and immunoglobulin isotype selection, offspring were now studied to ascertain whether this intervention modifies the in vitro replication of T. cruzi and nitric oxide (NO) production by peritoneal macrophages (PE), together with the anti-T. cruzi IgG isotypes. To evaluate the possibility of a detrimental effect of IFN-gamma, serum levels of anti-sulphatide autoantibodies were also investigated. Offspring were born to mothers undergoing one of the following procedures during gestation: treatment with recombinant rat IFN-gamma, 50,000 IU/rat, five times/week for 3 weeks, which was started on the day of mating; infection with 10(6) trypomastigotes of T. cruzi at 7, 14, and 21 days after mating plus IFN-gamma treatment as given to the former group; the same protocol except that physiological saline was injected instead of IFN-gamma; injection of physiological saline only. Offspring were challenged at weaning with a similar dose of T. cruzi, to constitute four groups of infected young, plus an additional group of age-matched uninfected rats born to control mothers. PE were harvested at day 7 postinfection (pi), exposed to parasites and further investigated for the replication of T. cruzi and NO production, whereas ELISA studies for measuring serum anti-T. cruzi IgG subclasses and anti-sulphatide autoantibodies were performed at day 30 pi. The number of intracellular parasites in PE was markedly decreased in young born to IFN-gamma-treated mothers, this not being accompanied by higher nitrite levels in culture supernatants. Offspring delivered by IFN-gamma-treated mothers showed no higher serum concentrations of anti-sulphatide autoantibodies, but exhibited a preferential synthesis of anti-T. cruzi IgG2b antibodies. This rat isotype is known to fix complement and constitutes the rat counterpart of IgG2a mouse immunoglobulins whose synthesis is favoured by IFN-gamma.

Benznidazole, a drug employed in the treatment of Chagas' disease, down-regulates the synthesis of nitrite and cytokines by murine stimulated macrophages.

Benznidazole (BZL) is a nitroheterocyclic drug employed in the chemotherapy of Chagas' disease, a protozoan disease caused by Trypanosoma cruzi. Because this parasite mostly replicates in macrophages, we investigated whether BZL was likely to modify the synthesis of macrophage mediators such as nitrite, tumour necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and IL-10. Control and stimulated murine macrophages (lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma)) were treated with BZL and measurements were carried out in culture supernatants collected 24 h later. Synthesis of nitrite, IL-6 and IL-10 was maximal upon combined stimulation with LPS + IFN-gamma, whereas lower amounts of the three mediators were detected when both stimuli were given alone. BZL treatment significantly reduced nitrite, IL-6 and IL-10 production, to undetectable levels in some cases, particularly IL-6 and IL-10. LPS was the most potent stimulus of IL-1beta and TNF-alpha production, followed by LPS + IFN-gamma and IFN-gamma in decreasing order. BZL partly inhibited TNF-alpha synthesis, but this effect was smaller than that observed for nitrite, IL-6 and IL-10. LPS-induced production of IL-1beta was also affected by BZL. Semiquantification of gene expression for inducible nitric oxide synthase (iNOS) showed that BZL completely inhibited iNOS gene induction by IFN-gamma, and resulted in respective inhibitions of 30% and 50% with LPS- and LPS + IFN-gamma-stimulated cells. BZL was not cytotoxic on macrophage cultures, as shown by the lactate dehydrogenase activity. Besides its trypanocidal activity, BZL may also alter the balance between pro- and anti-inflammatory mediators with important consequences for the course of T. cruzi infection.

Clinical and serological studies of tuberculosis patients in Argentina receiving immunotherapy with Mycobacterium vaccae (SRL 172).

Two small, placebo-controlled studies of immunotherapy with heat killed Mycobacterium vaccae added to routine chemotherapy for pulmonary tuberculosis, together involving 40 HIV seronegative patients, were carried out in Argentina. The immunotherapy was associated with reduced sputum smear positivity of AFB at 1 month and a greater reduction in ESR at 2 months. In the first study radiological improvement was better (P < 0.05) among immunotherapy recipients. In the second study, weight regain and time to become apyrexial were measured and were found to be improved amongst immunotherapy recipients (P < 0.05). In the first month of treatment the levels of IgG to the 65 kDa and 70 kDa heat-shock proteins showed greater falls following immunotherapy (P < 0.05 and P < 0.001, respectively). On admission serum cytokine levels of interleukins 4 and 10 (IL-4, IL-10), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) were grossly raised in comparison with a matched control group (P < 0.001). After 1 month. Levels of IL-4, IL-10 and TNF-alpha fell (P < 0.001, P < 0.01 and P < 0.01, respectively) and levels of IFN-gamma rose more (P = 0.005) in immunotherapy recipients than in those receiving chemotherapy alone. The results are in accord with a switch towards a TH1 immunological status and clinical benefit for immunotherapy recipients.

Administration of interferon-gama to pregnant rats reverses the depressed adjuvant-induced arthritis of their chronically Trypanosoma cruzi-infected offspring

We demonstrated that administration of interferon gamma (IFN-gama) to the inbred "l" strain of pregnant rats conferred partial resistance on their offspring to challenge with Trypanosoma cruzi. We now examine if this intervention also modifies the reportedly immunodepressed cellular responses which occur during chronic infection. Offspring were born to mothers undergoing one of the following procedures during gestation: subcutaneous injections of recombinant rat IFN-gama, 50,000 IU/rat, five times/week for 3 weeks, which was started on the day of mating (IFN-Mo); infection with 106 trypomastigotes of T. cruzi at 7, 14, and 21 days after mating plus IFN-gama treatment as given to the former group (TcIFN-Mo); the same protocol except that physiological saline was injected instead of IFN-gama (Tc-Mo); injection of physiological saline only (control-Mo). All offspring groups (N = 8-10/group) were infected at weaning and were assessed 90 days later for their adjuvant-induced arthritic response or levels of major T cell subsets in spleen and lymph nodes. TcIFN-Mo and IFN-Mo offspring showed a reestablished arthritic response, which remained within the range seen in controls. Immunolabeling studies on parallel groups of 90-day-infected offspring showed that the inverse CD4/CD8 cell ratio that is usually seen in lymphoid organs from these chronically infected rats (median 0.61) appeared to have recovered in the TcIFN-Mo and IFN-Mo groups (median 1.66 and 1.78, respectively) and was not different from uninfected controls (1.96). These studies indicate that early stimulation with IFN-gama is able to reverse the immunosuppressive state that is usually present during the chronic period of the experimental infection.

Levels of tumor necrosis factor alpha, gamma interferon, and interleukins 4,6, and 10 as determined in mice infected with virulent or attenuated strains of Trypanosoma cruzi.

Inoculation of BALB/c mice with the virulent Tulahuen (TUL) strain of T. cruzi was shown to lead to progressive and eventually lethal infections, whereas infection with an attenuated strain (TCC) resulted in a hardly noticeable experimental disease producing no tissue damage. To determine whether differences in such infection outcome are associated with a particular pattern of cytokine response, a study was undertaken to investigate the serum levels of TNF-alpha, IFN-gamma, IL-4, IL-6, and IL-10 by using an enzyme-linked immunosorbent assay. Mice from both infected groups were bled at 5, 9, 15, 22, 30 and 48 days post-infection (p.i.), with the same interval being applied for obtention of serum samples in age-matched uninfected mice, a group that yielded negative results in all cases. Infection with the TUL strain of T. cruzi was accompanied by a significant increase of TNF-alpha serum concentrations at day 5 p.i., and detectable amounts of IFN-gamma by day 15 p.i., which were exclusively recorded in this group. Serum IL-4 was mostly present in TCC mice with values at day 15 pi being statistically significant in relation to TUL-infected mice. IL-10 was firstly detected at 3 weeks after infection, and showed higher levels in the TCC group, although comparisons with TUL-infected group were not significant. At our limit of detection, no samples were found to contain IL-6 serum concentrations. Infection with virulent parasites seems to be associated with presence of Th1-type cytokines, whereas challenge with the attenuated TCC strain appears as being related to a Th2-type profile.

Macrophage activity, IL-6 levels, antibody response and heart histology in rats undergoing an attenuated Trypanosoma cruzi acute infection upon treatment with recombinant interferon gamma.

Earlier experiments in Trypanosoma cruzi-infected rats showed that recombinant rat (Rr) interferon (IFN)-gamma given shortly after infection ameliorated acute disease without modifying the serum titers of endogenously synthesized IFN-gamma and tumor necrosis factor. To gain some insight into the processes underlying this protective effect, 21-day old 'I' rats that were infected with T. cruzi and the following day started with a 20-day cycle of RrIFN-gamma injections (20000 IU/rat/day) were investigated for the in vitro replication of T. cruzi and nitric oxide (NO) production by peritoneal macrophages (day 7 post-infection, pi), antibodies with lytic activity against T. cruzi (days 7, 20, and 28 pi), and serum levels of biologically active interleukin (IL)-6 (days 15 and 30 pi). Therapy with RrIFN-gamma rendered cultured peritoneal macrophages less permissive to infection with T. cruzi. Such an effect was not accompanied by higher amounts of NO in macrophage cultured supernatants, compared with those from T. cruzi-infected rats receiving no RrIFN-gamma, which appeared not to be protected from in vitro infection. Acutely T. cruzi-infected rats had significant amounts of IL-6 in their sera - this not being the case in infected rats given RrIFN-gamma, whose levels appeared decreased as in control rats. The presence of complement-mediated anti-T. cruzi lytic antibodies was not modified by RrIFN-gamma. Likewise, heart histology at day 7 pi revealed that treatment with RrIFN-gamma made no differences as to the amount of acute inflammation, but tended to reduce the myocardial parasite load.