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

The effect of bacterial lipopolysaccharide on gastric emptying in rats suffering from moderate renal insufficiency

The objective of the present study was to evaluate the response of rats suffering from moderate renal insufficiency to bacterial lipopolysaccharide (LPS, or endotoxin). The study involved 48 eight-week-old male SPF Wistar rats (175-220 g) divided into two groups of 24 animals each. One group underwent 5/6 nephrectomy while the other was sham-operated. Two weeks after surgery, the animals were further divided into two subgroups of 12 animals each and were fasted for 20 h but with access to water ad libitum. One nephrectomized and one sham-treated subgroup received E. coli LPS (25 µg/kg, iv) while the other received a sterile, pyrogen-free saline solution. Gastric retention (GR) was determined 10 min after the orogastric infusion of a standard saline test meal labeled with phenol red (6 mg/dl). The gastric emptying of the saline test meal was studied after 2 h. Renal function was evaluated by measuring the plasma levels of urea and creatinine. The levels of urea and creatinine in 5/6 nephrectomized animals were two-fold higher than those observed in the sham-operated rats. Although renal insufficiency did not change gastric emptying (median %GR = 26.6 for the nephrectomized subgroup and 29.3 for the sham subgroup), LPS significantly retarded the gastric emptying of the sham and nephretomized groups (median %GR = 42.0 and 61.0, respectively), and was significantly greater (P<0.01) in the nephrectomized rats. We conclude that gastric emptying in animals suffering from moderate renal insufficiency is more sensitive to the action of LPS than in sham animals

Cardiorespiratory adaptations induced by aerobic training in middle-aged men: the importance of a decrease in sympathetic stimulation for the contribution of dynamic exercise tachycardia

We investigated the effects of aerobic training on the efferent autonomic control of heart rate (HR) during dynamic exercise in middle-aged men, eight of whom underwent exercise training (T) while the other seven continued their sedentary (S) life style. The training was conducted over 10 months (three 1-h sessions/week on a field track at 70-85% of the peak HR). The contribution of sympathetic and parasympathetic exercise tachycardia was determined in terms of differences in the time constant effects on the HR response obtained using a discontinuous protocol (4-min tests at 25, 50, 100 and 125 watts on a cycle ergometer), and a continuous protocol (25 watts/min until exhaustion) allowed the quantification of the parameters (anaerobic threshold, VO2 AT; peak O2 uptake, VO2 peak; power peak) that reflect oxygen transport. The results obtained for the S and the T groups were: 1) a smaller resting HR in T (66 beats/min) when compared to S (84 beats/min); 2) during exercise, a small increase in the fast tachycardia (D0-10 s) related to vagal withdrawal (P<0.05, only at 25 watts) was observed in T at all powers; at middle and higher powers a significant decrease (P<0.05 at 50, 100 and 125 watts) in the slow tachycardia (D1-4 min) related to a sympathetic-dependent mechanism was observed in T; 3) the VO2 AT (S = 1.06 and T = 1.33 l/min) and VO2 peak (S = 1.97 and T = 2.47 l/min) were higher in T (P<0.05). These results demonstrate that aerobic training can induce significant physiological adaptations in middle-aged men, mainly expressed as a decrease in the sympathetic effects on heart rate associated with an increase in oxygen transport during dynamic exercise.

Inhibitory effect of high [Mg2+] on the vasopressin-stimulated hydroosmotic permeability of the isolated perfused cortical collecting duct

High magnesium concentration inhibits the effect of arginine vasopressin (AVP) on smooth muscle contraction and platelet aggregation and also influences hepatocyte AVP receptor binding. The aim of this study was to determine the role of magnesium concentration [Mg2+] in AVP-stimulated water transport in the kidney collecting duct. The effect of low and high peritubular [Mg2+] on the AVP-stimulated osmotic water permeability coefficient (Pf) was evaluated in the isolated perfused rabbit cortical collecting duct (CCD). Control tubules bathed and perfused with standard Ringer bicarbonate solution containing 1 mM Mg2+ presented a Pf of 223.9 ± 27.2 µm/s. When Mg2+ was not added to the bathing solution, an increase in the AVP-stimulated Pf to 363.1 ± 57.2 µm/s (P<0.05) was observed. An elevation of Mg2+ to 5 mM resulted in a decrease in Pf to 202.9 ± 12.6 µm/s (P<0.05). This decrease in the AVP-stimulated Pf at 5 mM Mg2+ persisted when the CCDs were returned to 1 mM Mg2+, Pf = 130.2 ± 20.3 µm/s, and was not normalized by the addition of 8-[4-chlorophenylthio]-adenosine 3',5'-cyclic monophosphate, a cAMP analogue, to the preparation. These data indicate that magnesium may play a modulatory role in the action of AVP on CCD osmotic water permeability, as observed in other tissues.

Structural, functional and immunological studies on a polymeric bacterial protein

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-Å resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

A moderate decrease in temperature inhibits the calcium signaling mechanism(s) of the regulatory volume decrease in chick embryo cardiomyocytes

Chick cardiomyocytes, when submitted to hyposmotic swelling, exhibit a partial regulatory volume decrease (RVD). A Ca2+ influx by stretch-activated channels signals a taurine efflux and the RVD at 37°C. We evaluated the cell's performance at room temperature. Cardiomyocytes isolated and cultured from 11-day-old chick embryos were submitted to a hyposmotic solution (180 mOsm/kg H2O) at 37°C and at room temperature (26°C). Under these conditions we measured the changes in cell volume as well as the intracellular free Ca2+ (using fura-2). During hyposmotic swelling, cells at 37°C displayed a peak relative volume of 1.61 ± 0.03 and recovery to 1.22 ± 0.04 (N = 14), while cells at 26°C presented a peak swell relative volume of 1.74 ± 0.06 and did not recover (1.59 ± 0.09, N = 9). Transient increases in intracellular Ca2+, which are characteristic of the normal RVD, were observed at both temperatures (29.1 ± 4.5% (N = 8) and 115.2 ± 42.8% (N = 5) increase at 37° and 26°C (P<0.05), respectively). A delay in the Ca2+ transient increase was also observed when the cells were at 26°C (109 ± 34 s compared to 38 ± 9 s at 37°C, P<0.05). At room temperature the RVD does not occur because the calcium transient increase, which is an early event in the signaling of the RVD, is delayed. Also, free calcium is not cleared as in the 37°C RVD. In the normal RVD the free calcium returns to baseline levels. The very high and persistent free calcium levels seen at room temperature can lead to unregulated enzyme activities and may promote irreversible injury and cell death.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 µmol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 µmol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

Bone mineral density of the lumbar spine of Brazilian children and adolescents aged 6 to 14 years

The authors performed a study of bone mass in eutrophic Brazilian children and adolescents using dual-energy X-ray absorptiometry (DXA) in order to obtain curves for bone mineral content (BMC) and bone mineral density (BMD) by chronological age and correlate these values with weight and height. Healthy Caucasian children and adolescents, 120 boys and 135 girls, 6 to 14 years of age, residents of São Paulo, Brazil, were selected from the Pediatric Department outpatient clinic of Hospital São Paulo (Universidade Federal de São Paulo). BMC, BMD and the area of the vertebral body of the L2-L4 segment were obtained by DXA. BMC and BMD for the lumbar spine (L2-L4) presented a progressive increase between 6 and 14 years of age in both sexes, with a distribution that fitted an exponential curve. We identified an increase of mineral content in female patients older than 11 years which was maintained until 13 years of age, when a new decrease in the velocity of bone mineralization occurred. Male patients presented a period of accelerated bone mass gain after 11 years of age that was maintained until 14 years of age. At 14 years of age the mean BMD values for boys and girls were 0.984 and 1.017 g/cm², respectively. A stepwise multiple regression analysis of paired variables showed that the "vertebral area-age" pair was the most significant in the determination of BMD values and the introduction of a third variable (weight or height) did not significantly increase the correlation coefficient.

Uniform decrease of alpha-global field power induced by intermittent photic stimulation of healthy subjects

Nineteen-channel EEGs were recorded from the scalp surface of 30 healthy subjects (16 males and 14 females, mean age: 34 years, SD: 11.7 years) at rest and under trains of intermittent photic stimulation (IPS) at rates of 5, 10 and 20 Hz. Digitalized data were submitted to spectral analysis with fast fourier transformation providing the basis for the computation of global field power (GFP). For quantification, GFP values in the frequency ranges of 5, 10 and 20 Hz at rest were divided by the corresponding data obtained under IPS. All subjects showed a photic driving effect at each rate of stimulation. GFP data were normally distributed, whereas ratios from photic driving effect data showed no uniform behavior due to high interindividual variability. Suppression of alpha-power after IPS with 10 Hz was observed in about 70% of the volunteers. In contrast, ratios of alpha-power were unequivocal in all subjects: IPS at 20 Hz always led to a suppression of alpha-power. Dividing alpha-GFP with 20-Hz IPS by alpha-GFP at rest (R = alpha-GFP IPS/alpha-GFPrest) thus resulted in ratios lower than 1. We conclude that ratios from GFP data with 20-Hz IPS may provide a suitable paradigm for further investigations.

The decrease in uroporphyrinogen decarboxylase activity induced by ethanol predisposes rats to the development of porphyria and accelerates xenobiotic-triggered porphyria, regardless of hepatic damage

We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58% reduction of tyrosine hydroxylase in the substantia nigra, and a 35% reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 ± 32.5 to 58 ± 16.5 ng/mg protein (47.2% reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 ± 44 protein in control and 175 ± 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Occurrence of Haemophilus influenzae strains in three Brazilian states since the introduction of a conjugate Haemophilus influenzae type b vaccine

Few vaccines in history have induced such a dramatic decline in incidence over such a short period of time as the Haemophilus influenzae type b (Hib) conjugate. This vaccine was introduced in 1988 in the United States, but only in 1999 was Hib immunization introduced by the Brazilian Ministry of Health as part of the routine infant National Immunization Program. The authors analyzed 229 H. influenzae (Hi) isolates from Public Health Laboratories in three Brazilian states: Pernambuco (Northeast, N = 54), Santa Catarina (South, N = 19), and Rio de Janeiro (Southeast, N = 156). The isolates were collected from Brazilian children 0-10 years of age with meningitis and other infections from 1990 to 2003 and were part of the research collection of the National Institute of Quality Control in Health, FIOCRUZ. Bacterial strains were characterized by serotyping and biotyping. During the pre-vaccination period the prevalence infection due to Hib was of 165 isolates and only 2 non-b Hi among all the notified meningitis infections caused by Hi. Our results showed a significant decrease in the prevalence of Hib meningitis from 165 to 33 isolates after 1999. However, during the post-vaccination period of 2001-2003 we observed an increase in the number of non-b Hi isolates: only 2 non-b strains isolated from 1990 to 1999 and 29 from 1999 to 2003. Based on the present data, the authors emphasize the need for more sensitive epidemiological and bacteriological studies aiming the improvement of the available Hib vaccine, in order to protect the susceptible population to infections due to other serological types of Hi and the reevaluation of immunization schedules used by the National Immunization Program.

Effect of high hydrostatic pressure on the barrier properties of polyamide-6 films

Little is known about the barrier properties of polymer films during high pressure processing of prepackaged foods. In order to learn more about this, we examined the influence of high hydrostatic pressure on the permeation of raspberry ketone (dissolved in ethanol/water) through polyamide-6 films at temperatures between 20 and 60ºC. Permeation was lowered by increasing pressure at all temperatures. At 23°C, the increasing pressure sequence 0.1, 50, 100, 150, and 200 MPa correlated with the decreasing permeation coefficients P/(10(9) cm² s-1) of 6.2, 3.8, 3.0, 2.2, and 1.6. Analysis of the permeation kinetics indicated that this effect was due to a reduced diffusion coefficient. Pressure and temperature acted antagonistically to each other. The decrease in permeation at 200 MPa was compensated for by a temperature increase of 20ºC. After release of pressure, the former permeation coefficients were recovered, which suggests that this `pressure effect' is reversible. Taken together, our data revealed no detrimental effects of high hydrostatic pressure on the barrier properties of polymer films.

Early Life Intestinal Microbiota in Health and in Atopic Eczema

Decrease in microbial contacts in affluent societies is considered to lie behind the rise in allergic and other chronic inflammatory diseases during the last decades. Indeed, deviations in the intestinal microbiota composition and diversity have been associated with several diseases, such as atopic eczema. However, there is no consensus yet on what would constitute a beneficial or harmful microbiota. The aim of this thesis was to study the microbiota development in healthy infants and to characterize intestinal microbiota signatures associated with disease status and severity in infants with atopic eczema. The methodological aim was to compare and optimize methods for DNA extraction from fecal samples to be used in high-throughput microbiota analyses. It was confirmed that the most critical step in successful microbial DNA extraction from fecal samples is the mechanical cell lysis procedure. Based on this finding, an efficient semi-automated extraction process was developed that can be scaled for use in high-throughput platforms such as phylogenetic microarray used in this series of studies. By analyzing a longitudinal motherchild cohort for 3 years it was observed that the microbiota development is a gradual process, where some bacterial groups reach the degree of adult-type pattern earlier than others. During the breast-feeding period, the microbiota appeared to be relatively simple, while major diversification was found to start during the weaning process. By the age of 3 years, the child’s microbiota composition started to resemble that of an adult, but the bacterial diversity has still not reached the full diversity, indicating that the microbiota maturation extends beyond this age. In addition, at three years of age, the child’s microbiota was more similar to mother’s microbiota than to microbiota of nonrelated women.In infants with atopic eczema, a high total microbiota diversity and abundance of butyrate-producing bacteria was found to correlate with mild symptoms at 6 months. At 18 months, infants with mild eczema had significantly higher microbiota diversity and aberrant microbiota composition when compared to healthy controls at the same age. In conclusion, the comprehensive phylogenetic microarray analysis of early life microbiota shows the synergetic effect of vertical transmission and shared environment on the intestinal microbiota development. By the age of three years, the compositional development of intestinal microbiota is close to adult level, but the microbiota diversification continues beyond this age. In addition, specific microbiota signatures are associated with the existence and severity of atopic eczema and intestinal microbiota seems to have a role in alleviating the symptoms of this disease.

Bacterial community structure and petroleum hydrocarbon degradation in the Baltic Sea

The Baltic Sea is unique by its biological, geochemical and physical features. The number of species of larger organisms is small and the species composition is distinctive. On the contrary microbial communities are diverse. Because of the low salinity levels, bacterial communities differ from the ones in the oceans. Knowing the structure of these communities better and how they response to different environmental conditions helps us to estimate how different factors affect the balance and function of the Baltic Sea ecosystem. Bacteria are the key players when it comes to natural biogeochemical processes and human-induced phenomena like eutrophication, oil spills or disposal of other harmful substances to the sea ecosystem. In this thesis, bacterial community structure in the sea surface microlayer and subsurface water of the Archipelago Sea were compared. In addition, the effect of diatom derived polyunsaturated aldehydes on bacterial community structure was studied by a mesocosm experiment. Diesel, crude oil and polycyclic aromatic hydrocarbon degradation capacity of the Baltic Sea bacteria was studied in smaller scale microcosm experiments. In diesel oil experiments bacteria from water phase of the Archipelago Sea was studied. Sediment and iron manganese concretions collected from the Gulf of Finland were used in the crude oil and polycyclic aromatic hydrocarbon experiments. The amount of polycyclic aromatic hydrocarbon degradation genes was measured in all of the oil degradation experiments. The results show how differences in bacterial community structure can be seen in the sea surface when compared to the subsurface waters. The mesocosm experiment demonstrated how diatom-bacteria interactions depend on other factors than diatom derived polyunsaturated aldehydes, which do not seem to have an effect on the bacterial community structure as has been suggested in earlier studies. The dominant bacterial groups in the diesel microcosms differed in samples taken from a pristine site when compared to a site with previous oil exposure in the Archipelago Sea area. Results of the study with sediment and iron-manganese concretions indicate that there are diverse bacterial communities, typical to each bottom type, inhabiting the bottoms of the Gulf of Finland capable to degrade oil and polycyclic aromatic hydrocarbon compounds.