IFNG +874 T>A single nucleotide polymorphism is associated with leprosy among Brazilians.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, a low virulence mycobacterium, and the outcome of disease is dependent on the host genetics for either susceptibility per se or severity. The IFNG gene codes for interferon-γ (IFN-γ), a cytokine that plays a key role in host defense against intracellular pathogens. Indeed, single nucleotide polymorphisms (SNPs) in IFNG have been evaluated in several genetic epidemiological studies, and the SNP +874T>A, the +874T allele, more specifically, has been associated with protection against infectious diseases, especially tuberculosis. Here, we evaluated the association of the IFNG locus with leprosy enrolling 2,125 Brazilian subjects. First, we conducted a case-control study with subjects recruited from the state of São Paulo, using the +874 T>A (rs2430561), +2109 A>G (rs1861494) and rs2069727 SNPs. Then, a second study including 1,370 individuals from Rio de Janeiro was conducted. Results of the case-control studies have shown a protective effect for +874T carriers (OR(adjusted) = 0.75; p = 0.005 for both studies combined), which was corroborated when these studies were compared with literature data. No association was found between the SNP +874T>A and the quantitative Mitsuda response. Nevertheless, the spontaneous IFN-γ release by peripheral blood mononuclear cells was higher among +874T carriers. The results shown here along with a previously reported meta-analysis of tuberculosis studies indicate that the SNP +874T>A plays a role in resistance to mycobacterial diseases.

Genetic, epidemiological and biological analysis of interleukin-10 promoter single-nucleotide polymorphisms suggests a definitive role for -819C/T in leprosy susceptibility.

Leprosy is a complex infectious disease influenced by genetic and environmental factors. The genetic contributing factors are considered heterogeneous and several genes have been consistently associated with susceptibility like PARK2, tumor necrosis factor (TNF), lymphotoxin-alpha (LTA) and vitamin-D receptor (VDR). Here, we combined a case-control study (374 patients and 380 controls), with meta-analysis (5 studies; 2702 individuals) and biological study to test the epidemiological and physiological relevance of the interleukin-10 (IL-10) genetic markers in leprosy. We observed that the -819T allele is associated with leprosy susceptibility either in the case-control or in the meta-analysis studies. Haplotypes combining promoter single-nucleotide polymorphisms also implicated a haplotype carrying the -819T allele in leprosy susceptibility (odds ratio (OR)=1.40; P=0.01). Finally, we tested IL-10 production in peripheral blood mononuclear cells stimulated with Mycobacterium leprae antigens and found that -819T carriers produced lower levels of IL-10 when compared with non-carriers. Taken together, these data suggest that low levels of IL-10 during the disease outcome can drive patients to a chronic and unprotective response that culminates with leprosy.

Influence of tumour condition on the macrophage activity in Candida albicans infection.

To better understand the interactions between opportunistic fungi and their hosts, we investigated hydrogen peroxide (H2O2), nitric oxide and TNF-alpha production by peritoneal macrophages from Ehrlich tumour-bearing mice (TBM) during microbial infections. For this purpose, TBM at days 7, 14 and 21 of tumour progression were inoculated intraperitoneally with C. albicans and evaluated after 24 and 72 h. We observed that TBM showed significant increases in H2O2, TNF-alpha levels and fungal clearance at day 7 after C. albicans infection. However, as the tumour advanced, there was a progressive decline in the release of H2O2 and TNF-alpha that was paired with the dissemination of C. albicans. These results demonstrate that protective macrophage activities against Candida albicans are limited to the initial stages of tumour growth; continued solid tumour growth weakened the macrophage response and as a consequence, weakened the host's susceptibility to opportunistic infections.

Variations in DNA synthesis and mitotic indices in hepatocytes and sinusoid litoral cells of adult intact male mouse along a circadian time span.

Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.

Experimental murine mycobacteriosis: evaluation of the functional activity of alveolar macrophages in thalidomide- treated mice.

Thalidomide is a selective inhibitor of tumor necrosis factor-alpha (TNF-alpha), a cytokine involved in mycobacterial death mechanisms. We investigated the role of this drug in the functional activity of alveolar macrophages in the presence of infection induced by intranasal inoculation of Mycobacterium avium in thalidomide-treated and untreated adult Swiss mice. Sixty animals were inoculated with 5 x 10(6) M. avium by the respiratory route. Thirty animals received daily thalidomide (30 mg/kg mouse) and 30 received water by gavage up to sacrifice. Ten non-inoculated mice were used as a control group. Lots of animals from each group were evaluated until 6 weeks after inoculation. Infection resulted in an increased total number of inflammatory cells as well as increased activity of pulmonary macrophages. Histologically, intranasal inoculation of bacilli resulted in small mononuclear infiltrates located at the periphery of the organ. Culture of lung fragments revealed the presence of bacilli only at the beginning and at the end of the experimental period. Thalidomide administration did not affect the microbiological or histological features of the infection. Thalidomide-treated and untreated animals showed the same amount of M. avium colonies 3 weeks after infection. Although it did not affect bacillary clearance, thalidomide administration resulted in a decreased percent of spread cells and release of hydrogen peroxide, suggesting that factors other than TNF-alpha play a role in the killing of mycobacteria by alveolar macrophages. Thalidomide administration also reduced the number of spread cells among resident macrophages, suggesting a direct effect of the drug on this phenomenon.

[The reaction to trichophytin in dermatophytoses]. / Contribuição ao estudo da reação à tricofitina nas dermatofitoses.

The authors investigated the specific immunological competence of 31 patients with dermatophytosis using tricophytin antigen. Among them, 54.8% showed reaction to the delay phase (48 h) in the following proportions: tinea inguinale, 75%; tinea pedis, 61.5%; tinea unguium, 50% and tinea corporis, 20%. Other 62.5% showed positive result to the early phase (30 m). The association between these reactions revealed that, although the majority of cases with early positive reaction showed negativity to the delayed reaction, 20.8% presented positively to both phases of the reaction. Out of the non-reactive patients to the delayed phase, 8 were submitted to the other cutaneous tests such as PPD, streptokinase, candidin, vaccinia and DNCB and showed preserved cellular immunity in 75%. These results suggest that, while using this reaction for immunological evaluation of patients with dermatophytosis, one should consider the overall immune status of the patient, the presence of early hypersensibility and the localization of the infection.

Experimental dermatophytosis in hamsters inoculated with Trichophyton mentagrophytes in the cheek pouch.

This study presents the results of T. mentagrophytes inoculation in the cheek pouch of the hamster, an immunologically privileged site. Forty two animals were used: 21 inoculated with 10(6) fungi in the cheek pouch (group 1) and 21 inoculated initially with 10(6) fungi in the foot pad and 15 days later in the cheek pouch, with the same amount of fungi (group 2). Animals were sacrificed at 20 hours, 3, 7, 14, 30, 60, and 120 days; samples from inoculated cheek pouch, and foot pads submitted to the foot pad test (FPT), were collected. Independent of group and time of evolution of infection, animals did not develop delayed hypersensitivity evaluated through the FPT. The pre-inoculation of fungi in the foot pad did not change the morphology of lesions induced in the cheek pouch. Therefore, in animals of group 1 and 2, the introduction of the fungus in the cheek pouch resulted in focal lesion composed of a sterile acute inflammatory infiltrate, with abscess formation that evolved to a macrophagic reaction, and later to resolution even in the absence of immune response detectable by FPT. Our results indicate that in spite of the important role of the immune response in the spontaneous regression of dermatophytosis, other factors are also an integral part in the defense against this fungal infection.

Dermatophytosis: association between ABO blood groups and reactivity to the trichophytin.

The authors investigated the relationship between dermatophytosis and ABO blood groups through blood typing, identification of isolated dermatophytes and specific cellular immune response of 40 individuals carriers of this mycosis. They verified that the fungus Trichophyton rubrum, isolated from 54.5% of the patients, was more frequent in individuals belonging to blood group A. The cellular immune response, evaluated through the trichophytin antigen, was positive in 25% of the studied patients; the presence of immediate reactions (30 minutes) was verified in 35%. The blood group distribution among patients with dermatophytosis and control groups was, respectively: 47.5% X 36% in group A, 40% X 50% in group O, 12. 5% X 11% in group B. Even though the authors have found a higher number of patients belonging to blood group A infected by T. rubrum, these results suggest that there is no statistical evidence that these individuals are more susceptible to dermatophytosis.

Jorge Lobo's disease: experimental inoculation in Swiss mice.

Sixty-four isogenic Swiss mice were intradermically inoculated in both hind foot pads. The inocula, consisting of fungal suspensions from biopsies obtained from Jorge Lobo's Disease patients, had the total number of fungi and the viability index determined using a Neubauer chamber and the fluorescein diacetate-ethidium bromide technique (FD-EB), respectively. The animals were sacrificed at times ranging from ten days to eighteen months after inoculation. The cellular infiltrate, mainly consisting of macrophages containing fungi, increased progressively up to end of the study; however, no macroscopic alterations were observed in the inoculated feet. After nine months, small numbers of Langhans' giant cells started to appear in the infiltrate. A considerable number of fungi was observed at the end of the experimental period, but only a few were viable when stained by the FD-EB technique. This fact suggests that there is a multiplication of fungal cells, which are destroyed by the macrophages but remain in the tissue for a long time due perhaps to the difficulties in their elimination. These findings led us to conclude that in spite of the maintenance of the infection in these animals, Swiss mice cannot be considered an ideal model to study Jorge Lobo's Disease. However, the authors call attention to the possibility of other mouse strains being more susceptible to Paracoccidioides loboi.

[Mitotic activity of duodenal-crypt enterocytes in mice with hepatocarcinoma]. / Actividad mitótica de los enterocitos de las criptas duodenales en ratones portadores de un hepatocarcinoma.

Tumors grafted into mice may modify the proliferation of normal cell populations. In this paper, we have studied the evolution of mitotic activity (MA) in duodenal-crypt enterocytes of ES12a hepatocarcinoma-bearing mice; a total of 87 28-day-old female animals of the C3H/S strain were used after standardization for circadian-periodicity analysis. The mice were distributed into two groups: those remaining intact and those receiving tumor grafts. Each group was then divided into six batches (n = 6-10), one of which was sacrificed every 4 h over a period of one day. A dose of colchicine (2 micrograms/g) was administered to each animal 4 h before killing. Samples of duodenum were fixed in 10% (v/v) buffered formalin and processed for assessment of mitotic activity. The number and topographic localization of the colchicine-arrested metaphases were recorded among the entero-cytes within 20 longitudinal sections of the duodenal crypts in each animal. From these data the mitotic indices over the total crypt-cell population as well as within each previously-established zone were determined along with mean +/- SEM for each experimental group. The statistical significance of the differences among the data were analyzed by Student t test. The results show that the presence of ES12a tumor inhibits the mitotic activity of the duodenal-crypt enterocytes and produces an apparent temporal shift in the peak and trough within the circadian curve for this growth parameter.

Effect of gonadal activity on the cranial dimorphism of the rat.

Weanling rats of both sexes were submitted to either castration, or castration together with periodic injections of testicular extract to males or of ovarian extract to females. Control and experimental animals were sampled at 63 days of age. Cranial differentiation between sexes was estimated by Mahalanobis D2 distances. The controls showed a significant sexual cranial difference. Orchidectomy decreased cranial differences and this effect was compensated by injections of testicular extract. On the other hand, oophorectomy increased cranial differences, which were diminished by injections of ovarian extract. Sexual cranial dimorphism in the normal rat seems to be the result of a counteracting effect between testicular and ovarian hormonal secretions.

Ultrastructural study of somatotroph cells from mice bearing a fast growing transplanted hepatoma, in different periods of the tumor development.

The presence of a transplanted, fast-growing hepatoma (SS1-K) produces conspicuous ultrastructural changes in pituitary STH cells of C3H-S male mice. These changes are suggestive of an increased secretion of growth hormone only during the first stages of the tumor development. The hepatoma influence does not seem to be clearly related to the illumination regimen or time of killing.

Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver.

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibiting effect of a hepatoma extract on the mitotic rate of regenerating liver.

Aqueous tumor extracts were prepared by the homogenization of a fast-growing, undifferentiated, transplantable malignant murine hepatoma in distilled water. After centrifugation, an aliquot of 0.01 ml of the supernatant g body weight was injected intraperitoneally into partially hepatectomized mice. Control animals were injected with saline. Groups of mice were killed at various times in relation to the hepatectomy. Four h before killing the animals were given Colcemid (1 microgram/g body weight). The number of Colcemid-arrested mitoses in the hepatocytes and in the littoral cells, respectively, were counted in 140 microscopic fields. The extract significantly inhibited the mitotic rate in hepatocytes when the injection was given between 22 h before, and up to 26 h after hepatectomy. In the littoral cells, a slight initial stimulation was followed by a slight but significant inhibition which occurred when the injection was given at hepatectomy or until 18 h after hepatectomy. The effect was not modified by exposing the extracts to temperatures of 47 degrees C for 30 min or 22 degrees C for 24 h, but 10 min of boiling destroyed their inhibitory effect. Lyophilization and storing at -18 degrees C for up to 4 weeks did not modify the effect. The mitosis-inhibiting effect was also measurable when the extract was injected subcutaneously. There was an almost linear dose-response curve. The results are discussed in relation to circadian rhythms, the pattern of liver cell proliferation after hepatectomy, and recent similar reports from the literature. The conclusion is drawn that extracts of a hepatoma contain one or more growth-inhibitory factors significantly active on regenerating liver cells, and less significantly on littoral cells.

Circadian rhythm in the mitotic activity of the pars intermedia endocrine cell population of the mouse.

A mitotic circadian rhythm is reported for the endocrine cells of the pars intermedia of mouse pituitary. The peak is observed at 0000 and the trough at 1600. This curve is about 12h out of phase with the curves of mitotic activity of most of the cell populations of the organism of the mouse which show peaks about noon (1200). A similar inverted mitotic rhythm has been reported for corticoadrenal since a long time ago. The results are discussed in relation with the contradiction existing between proliferation and function in cell populations. The conclusion is drawn that the endocrine cells of the pars intermedia could represent another link in the circadian system controlling periodicity.

Prolactin secretion in normal male mice during a circadian period. An ultrastructural and biochemical study.

A quantitative ultrastructural study on the prolactin (PRL) cells of normal male mice was correlated with serum PRL immunoassay determination at different times in a circadian period. Significant time fluctuations in the percentages of cytoplasmic volume occupied by most of the organelles, as well as cyclic variations in serum PRL concentrations were detected. The endoplasmic reticulum appeared more developed at the beginning of the light period, suggesting a higher synthesis of PRL at this time. The evaluation of the Golgi complex and the secretory granules would indicate that packaging of secretory material and storing of PRL granules occurred mainly during the second half of the light period. This coincided with the lowest values in serum PRL concentration and an increment in mitochondrial mass. The highest emiocytotic activity together with a diminution of total secretory granules were found during the dark period. The peak of PRL serum concentration was also present in this phase, but some hours later. Evaluation of microtubules and lysosomes gave some additional information. The results are in keeping with those from some biochemical investigations performed in mice and rats, but are not in agreement with others. The importance of a morphometric approach to evaluate and corroborate the immunoassay methods in the study of cyclic hormone secretion is emphasized.

Liver regeneration in mice bearing a transplanted hepatoma.

The hepatocyte mitotic index curve in hepatectomized hepatoma-bearing mice, rises earlier, has a greater amplitude and is less synchronized than that of normal hepatectomized mice. This indicates a stimulation (more mitosis in a shorter time period) produced by the presence of the tumors. The sinusoid litoral cells mitotic index curve in hepatectomized hepatoma-bearing mice appears earlier and is much less synchronized than that of normal hepatectomized mice. Nevertheless both curves have the same amplitude for the whole sampling period and the early stimulation is quickly compensated by lower values (apparent inhibition) appearing in the resting (light) period.

Evaluation of the vital staining method for Lacazia loboi through the experimental inoculation of BALB/c mice.

The viability of the currently unculturable fungal pathogen Lacazia loboi can be determined by means of fluorescein diacetate-ethidium bromide (FD-EB) staining. This technique can be used in experimental study of the mycosis, in attempts to cultivate the fungus and in attempts to gauge the success of therapies. In the present study, the potential applications of FD-EB vital staining were studied using a proposed murine experimental model of lobomycosis. BALB/c mice were inoculated in the footpads with an L. loboi suspension that appeared in FD-EB staining to have lost viability after being held for 15 days at room temperature, whereas a control group of mice was inoculated with apparently viable fungi. The animals were killed after 1, 2, 4, 6, 8, 11 and 13 months. Both inoculated footpads were excised, one for determination of viability and the other for histological examination. In the group injected with nonviable material, no active infection was noted; inoculation sites showed small quantities of macrophage-laden infiltrate and no viable fungal cells. In the control group, the infection progressed with exuberant infiltrates surrounding copious fungal growth, most of which consisted of cells staining as viable in FD-EB. These results suggest that the FD-EB vital staining is a sensitive and specific method that can reliably be used for viability determination in L. loboi.