Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

The role of human T cell lymphotrophic virus type 1, hepatitis B virus and hepatitis C virus coinfections in leprosy

Leprosy spectrum and outcome is associated with the host immune response against Mycobacterium leprae. The role of coinfections in leprosy patients may be related to a depression of cellular immunity or amplification of inflammatory responses. Leprosy remains endemic in several regions where human T cell lymphotrophic virus type 1 (HTLV-1), hepatitis B virus (HBV) or hepatitis C virus (HCV) are also endemic. We have evaluated the evidence for the possible role of these viruses in the clinical manifestations and outcomes of leprosy. HTLV-1, HBV and HCV are associated with leprosy in some regions and institutionalization is an important risk factor for these viral coinfections. Some studies show a higher prevalence of viral coinfection in lepromatous cases. Although HBV and HCV coinfection were associated with reversal reaction in one study, there is a lack of information about the consequences of viral coinfections in leprosy. It is not known whether clinical outcomes associated with leprosy, such as development of reactions or relapses could be attributed to a specific viral coinfection. Furthermore, whether the leprosy subtype may influence the progression of the viral coinfection is unknown. All of these important and intriguing questions await prospective studies to definitively establish the actual relationship between these entities.

Sympathetic glial cells and macrophages develop different responses to Trypanosoma cruzi infection or lipopolysaccharide stimulation

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagas disease and the proximity of parasitised glial cells and neurons in damaged myenteric ganglia is a frequent finding. Glial cells have crucial roles in many neuropathological situations and are potential sources of NO. Here, we investigate peripheral glial cell response to Trypanosoma cruzi infection to clarify the role of these cells in the neuronal lesion pathogenesis of Chagas disease. We used primary glial cell cultures from superior cervical ganglion to investigate cell activation and NO production after T. cruzi infection or lipopolysaccharide (LPS) exposure in comparison to peritoneal macrophages. T. cruzi infection was greater in glial cells, despite similar levels of NO production in both cell types. Glial cells responded similarly to T. cruzi and LPS, but were less responsive to LPS than macrophages were. Our observations contribute to the understanding of Chagas disease pathogenesis, as based on the high susceptibility of autonomic glial cells to T. cruzi infection with subsequent NO production. Moreover, our findings will facilitate future research into the immune responses and activation mechanisms of peripheral glial cells, which are important for understanding the paradoxical responses of this cell type in neuronal lesions and neuroprotection.

Selenium and vitamin E enriched diet increases NK cell cytotoxicity in cattle

A number of studies has shown that antioxidants, fatty acids and trace minerals may modulate different immune cell activities, and that their deficiency may be associated with diseases and impaired immune responses. In innate immunity, natural killer (NK) cells have a central role, killing virally infected and cancerous cells, and also secreting cytokines that shape adaptive immune responses. Thus, the aim of this study was to evaluate the effect of enriched diets in selenium plus vitamin E and/or canola oil on complete blood count and on NK cell cytotoxicity from blood lymphocytes of Nellore bulls. Bulls that received selenium plus vitamin E had (P=0.0091) higher NK cell cytotoxicity than control bulls. This result positively correlated with serum selenium levels. To the best of our knowledge, this is the first study that showed immunostimulatory effects of selenium plus vitamin E on NK cell cytotoxicity of Nellore bulls.

Minimal doses of hydroxyurea for sickle cell disease

The use of hydroxyurea (HU) can improve the clinical course of sickle cell disease. However, several features of HU treatment remain unclear, including the predictability of drug response and determination of adequate doses, considering positive responses and minimal side effects. In order to identify adequate doses of HU for treatment of sickle cell disease, 10 patients, 8 with sickle cell anemia and 2 with Sß thalassemia (8SS, 2Sß), were studied for a period of 6 to 19 months in an open label dose escalation trial (10 to 20 mg kg-1 day-1). Hemoglobin (Hb), fetal hemoglobin (Hb F) and mean corpuscular volume (MCV) values and reticulocyte, neutrophil and platelet counts were performed every two weeks during the increase of the HU dose and every 4 weeks when the maximum HU dose was established. Reduction in the number of vasoocclusive episodes was also considered in order to evaluate the efficiency of the treatment. The final Hb and Hb F concentrations, and MCV values were significantly higher than the initial values, while the final reticulocyte and neutrophil counts were significantly lower. There was an improvement in the concentration of Hb (range: 0.7-2.0 g/dl) at 15 mg HU kg-1 day-1, but this concentration did not increase significantly when the HU dose was raised to 20 mg kg-1 day-1. The concentration of Hb F increased significantly (range: 1.0-18.1%) when 15 mg HU was used, and continued to increase when the dose was raised to 20 mg kg-1 day-1. The final MCV values increased 11-28 fl (femtoliters). However, reticulocyte (range: 51-205 x 109/l) and neutrophil counts (range: 9.5-1.3 x 109/l) obtained at this dose were significantly lower than those obtained with 15 mg kg-1 day-1. All patients reported a decrease in frequency or severity of vasoocclusive episodes. These results suggest that a hydroxyurea dose of 15 mg kg-1 day-1 seems to be adequate for treatment of sickle cell disease in view of the minimal side effects observed and the improvement in laboratory and clinical parameters.

Molecular mimicry between cardiac myosin and Trypanosoma cruzi antigen B13: identification of a B13-driven human T cell clone that recognizes cardiac myosin

Previous reports from our group have demonstrated the association of molecular mimicry between cardiac myosin and the immunodominant Trypanosoma cruzi protein B13 with chronic Chagas' disease cardiomyopathy at both the antibody and heart-infiltrating T cell level. At the peripheral blood level, we observed no difference in primary proliferative responses to T. cruzi B13 protein between chronic Chagas' cardiopathy patients, asymptomatic chagasics and normal individuals. In the present study, we investigated whether T cells sensitized by T. cruzi B13 protein respond to cardiac myosin. T cell clones generated from a B13-stimulated T cell line obtained from peripheral blood of a B13-responsive normal donor were tested for proliferation against B13 protein and human cardiac myosin. The results showed that one clone responded to B13 protein alone and the clone FA46, displaying the highest stimulation index to B13 protein (SI = 25.7), also recognized cardiac myosin. These data show that B13 and cardiac myosin share epitopes at the T cell level and that sensitization of a T cell with B13 protein results in response to cardiac myosin. It can be hypothesized that this also occurs in vivo during T. cruzi infection which results in heart tissue damage in chronic Chagas' disease cardiomyopathy

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

Feeding manipulation elicits different proliferative responses in the gastrointestinal tract of suckling and weanling rats

Food deprivation has been found to stimulate cell proliferation in the gastric mucosa of suckling rats, whereas the weanling period has been reported to be unresponsive in terms of proliferative activity. In the present study we analyze regional differences in the effect of milk or food deprivation on cell proliferation of the epithelia of the esophagus and of five segments of small intestine in suckling, weanling and newly weaned Wistar rats of both sexes. DNA synthesis was determined using tritiated thymidine to obtain labeling indices (LI); crypt depth and villus height were also determined. Milk deprivation decreased LI by 50% in the esophagus (from 15 to 8.35%) and small intestine (from 40 to 20%) of 14-day-old rats. In 18-day-old rats, milk and food deprivation decreased LI in the esophagus (from 13 to 5%) and in the distal segments of the small intestine (from 36-40 to 24-32%). In contrast, the LI of the epithelia of the esophagus (5%) and of all small intestine segments (around 30%) of 22-day-old rats were not modified by food deprivation. Crypt depth did not change after treatment (80 to 120 µm in 14- and 22-day-old rats, respectively). Villus height decreased in some small intestine segments of unfed 14- (from 400 to 300 µm) and 18-day-old rats (from 480 to 360 µm). The results show that, contrary to the stomach response, milk deprivation inhibited cell proliferation in the esophagus and small intestine of suckling rats, demonstrating the regional variability of each segment of the gastrointestinal tract in suckling rats. In newly weaned rats, food deprivation did not alter the proliferation of these epithelia, similarly to the stomach, indicating that weanling is a period marked by the insensitivity of gastrointestinal epithelia to dietary alterations

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.

Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation

The purpose of the present study was to evaluate the mixed lymphocyte culture as a predictive assay of acute and chronic graft-versus-host disease (GVHD). We studied 153 patients who received a first bone marrow transplantation from human leukocyte antigen-identical siblings. Acute GVHD was observed in 26 of 128 (20.3%) patients evaluated and chronic GVHD occurred in 60 of 114 (52.6%). One-way mixed lymphocyte culture (MLC) assays were performed by the standard method. MLC results are reported as the relative response (RR) from donor against patient cells. The responses ranged from -47.0 to 40.7%, with a median of 0.5%. The Kaplan-Meier probability of developing GVHD was determined for patients with positive and negative MLC. There was no significant difference in incidence of acute GVHD between the groups studied. However, the incidence of chronic GVHD was higher in recipients with RR >4.5% than in those with RR <=4.5%. The Cox Proportional Hazards model was used to examine the effect of MLC levels on incidence of chronic GVHD, while adjusting for the potential confounding effect of others suspected or observed risk factors. The relative risk of chronic GVHD was 2.5 for patients with positive MLC (RR >4.5%), 2.9 for those who received peripheral blood progenitor cells as a graft, and 2.2 for patients who developed previous acute GVHD. MLC was not useful for predicting acute GVHD, but MLC with RR >4.5% associated with other risk factors could predict the development of chronic GVHD, being of help for the prevention and/or treatment of this late complication.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Long-term ethanol intoxication reduces inflammatory responses in rats

The anti-inflammatory effects of long-term ethanol intoxication were determined during ethanol treatment and withdrawal on the basis of neutrophil and eosinophil migration, hind paw edema and mast cell degranulation. Male Wistar rats (180-200 g, around 2 months of age) were exposed to increasing concentrations of ethanol vapor over a 10-day period. One group was evaluated immediately after exposure (treated group - intoxicated), and another was studied 7 h later (withdrawal group). Ethanol inhalation treatment significantly inhibited carrageenan- (62% for the intoxicated group, N = 5, and 35% for the withdrawal group, N = 6) and dextran-induced paw edema (32% for intoxicated rats and 26% for withdrawal rats, N = 5 per group). Ethanol inhalation significantly reduced carrageenan-induced neutrophil migration (95% for intoxicated rats and 41% for withdrawn rats, N = 6 per group) into a subcutaneous 6-day-old air pouch, and Sephadex-induced eosinophil migration to the rat peritoneal cavity (100% for intoxicated rats and 64% for withdrawn rats, N = 6 per group). A significant decrease of mast cell degranulation was also demonstrated (control, 82%; intoxicated, 49%; withdrawn, 51%, N = 6, 6 and 8, respectively). Total leukocyte and neutrophil counts in venous blood increased significantly during the 10 days of ethanol inhalation (leukocytes, 13, 27 and 40%; neutrophils, 42, 238 and 252%, respectively, on days 5, 9 and 10, N = 7, 6 and 6). The cell counts decreased during withdrawal, but were still significantly elevated (leukocytes, 10%; neutrophils, 246%, N = 6). These findings indicate that both the cellular and vascular components of the inflammatory response are compromised by long-term ethanol intoxication and remain reduced during the withdrawal period.

Prostanoids modulate inflammation and alloimmune responses during graft rejection

Acute rejection of a transplanted organ is characterized by intense inflammation within the graft. Yet, for many years transplant researchers have overlooked the role of classic mediators of inflammation such as prostaglandins and thromboxane (prostanoids) in alloimmune responses. It has been demonstrated that local production of prostanoids within the allograft is increased during an episode of acute rejection and that these molecules are able to interfere with graft function by modulating vascular tone, capillary permeability, and platelet aggregation. Experimental data also suggest that prostanoids may participate in alloimmune responses by directly modulating T lymphocyte and antigen-presenting cell function. In the present paper, we provide a brief overview of the alloimmune response, of prostanoid biology, and discuss the available evidence for the role of prostaglandin E2 and thromboxane A2 in graft rejection.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

A low-protein diet leads to functional and structural pancreatic islet alterations, including islet hypotrophy. Insulin-signaling pathways are involved in several adaptive responses by pancreatic islets. We determined the levels of some insulin-signaling proteins related to pancreatic islet function and growth in malnourished rats. Adult male Wistar rats (N = 20 per group) were fed a 17% protein (normal-protein diet; NP) or 6% protein (low-protein diet; LP), for 8 weeks. At the end of this period, blood glucose and serum insulin and albumin levels were measured. The morphometric parameters of the endocrine pancreas and the content of some proteins in islet lysates were determined. The β-cell mass was significantly reduced (≅65%) in normoglycemic but hypoinsulinemic LP rats compared to NP rats. Associated with these alterations, a significant 30% reduction in insulin receptor substrate-1 and a 70% increase in insulin receptor substrate-2 protein content were observed in LP islets compared to NP islets. The phosphorylated serine-threonine protein kinase (pAkt)/Akt protein ratio was similar in LP and NP islets. The phosphorylated forkhead-O1 (pFoxO1)/FoxO1 protein ratio was decreased by 43% in LP islets compared to NP islets (P < 0.05). Finally, the ratio of phosphorylated-extracellular signal-related kinase 1/2 (pErk1/2) to total Erk1/2 protein levels was decreased by 71% in LP islets compared to NP islets (P < 0.05). Therefore, the reduced β-cell mass observed in LP rats is associated with the reduction of phosphorylation in mitogenic-related signals, FoxO1 and Erk proteins. The cause/effect basis of this association remains to be determined.

The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70S6K1 and AMPK signaling pathway responses in rat skeletal muscle

The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70S6K1 pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr172AMPK (p-AMPK), Akt, phospho-Ser473Akt (p-Akt), p70S6K1, phospho-Thr389-p70S6K1(p-p70S6K1), mTOR, phospho-Ser2448mTOR (p-mTOR), and phospho-Thr1462-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70S6K1 ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle.