Development of Anxiolytic and Depression-like Behavior in Mice Infected with Mycobacterium lepraemurium.

Murine leprosy is a systemic infectious disease of mice caused by Mycobacterium lepraemurium (MLM) in which the central nervous system (CNS) is not infected; nevertheless, diseased animals show measurable cognitive alterations. For this reason, in this study, we explored the neurobehavioral changes in mice chronically infected with MLM. BALB/c mice were infected with MLM, and 120 days later, the alterations in mice were evaluated based on immunologic, histologic, endocrine, neurochemical, and behavioral traits. We found increases in the levels of IL-4 and IL-10 associated with high bacillary loads. We also found increase in the serum levels of corticosterone, epinephrine, and norepinephrine in the adrenal gland, suggesting neuroendocrine deregulation. Mice exhibited depression-like behavior in the tail suspension and forced swimming tests and anxiolytic behavior in the open field and elevated plus maze tests. The neurobehavioral alterations of mice were correlated with the histologic damage in the prefrontal cortex, ventral hippocampus, and amygdala, as well as with a blood-brain barrier disruption in the hippocampus. These results reveal an interrelated response of the neuroimmune--endocrinological axis in unresolved chronic infections that result in neurocognitive deterioration.

Successive Intramuscular Boosting with IFN-Alpha Protects Mycobacterium bovis BCG-Vaccinated Mice against M. lepraemurium Infection.

Leprosy caused by Mycobacterium leprae primarily affects the skin and peripheral nerves. As a human infectious disease, it is still a significant health and economic burden on developing countries. Although multidrug therapy is reducing the number of active cases to approximately 0.5 million, the number of cases per year is not declining. Therefore, alternative host-directed strategies should be addressed to improve treatment efficacy and outcome. In this work, using murine leprosy as a model, a very similar granulomatous skin lesion to human leprosy, we have found that successive IFN-alpha boosting protects BCG-vaccinated mice against M. lepraemurium infection. No difference in the seric isotype and all IgG subclasses measured, neither in the TH1 nor in the TH2 type cytokine production, was seen. However, an enhanced iNOS/NO production in BCG-vaccinated/i.m. IFN-alpha boosted mice was observed. The data provided in this study suggest a promising use for IFN-alpha boosting as a new prophylactic alternative to be explored in human leprosy by targeting host innate cell response.

Adjuvant treatment with a dialyzable leukocytes extract contributes to maintain HPV-infected women free of low-grade cervical lesions.

PURPOSE OF INVESTIGATION: To investigate if adjuvant treatment with a dialyzable extract of leukocytes (DLE), may help HPV-infected patients with low-grade intraepithelial squamous cervical lesions (LIS) to get free of HPV infection and cervical lesions. MATERIALS AND METHODS: Patients with untreated, low-grade cervical lesions were treated either with surgery (Group A) or with DLE (Group B). Pa- tients with low-grade but recurrent cervical lesions were newly treated with surgery plus DLE (Group C). RESULTS: A decreased or ab- sent cervical lesion correlated with a diminished or absent HPV viral load at one year of treatment (r = 0.6,p <0.05). Seventy-nine percent of Group B but only 50 % of Group C and 38 % of Group A patients were free of cervical lesion after 24 months of treatment (p < 0.05). CONCLUSION: The present data support the benefit of adding DLE as adjuvant for treating HPV-infected women with LIS.

Dengue virus serotype-2 impairs proliferation of healthy donors' T lymphocytes.

OBJECTIVES: T lymphocytes are not infected by dengue virus (DENV), nevertheless it is possible that exposure to DENV may affect their function. T lymphocytes from DENV-infected individuals are impaired in their proliferative capacity, although this effect has been attributed to altered function of antigen-presenting cells rather than to an intrinsic defect on T lymphocytes. Here we analyzed whether T lymphocytes from healthy donors became impaired in their proliferative capacity following in vitro exposure to DENV serotype-2 (DENV-2), as well as the possible mechanisms for this. METHODS: Isolated CD4+ and CD8+ T lymphocytes from healthy donors were in vitro exposed to DENV-2, before polyclonal activation, cell proliferation, IL-2 synthesis. IL-2Rα expression, nuclear translocation of NF-AT and NF-κB, and intracellular calcium flux were assessed. RESULTS: In vitro exposure of both CD4+ and CD8+ T lymphocytes from healthy donors to DENV-2 impairs cell proliferation, IL-2 synthesis, and IL-2Rα (CD25) cell membrane expression. Signalling wise, exposure to DENV-2 impairs the nuclear translocation of NF-AT, downstream of intracellular calcium mobilization, as well as that of NF-κB. CONCLUSION: In the course of a dengue infection, direct exposure of T lymphocytes to DENV could affect cell-mediated immune responses.

The line blot assay: problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi-antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call "line blot" is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm-long segment line (see Results), depending on the strength of the secondary, enzyme-labelled antibody.

Palsy of the rear limbs in Mycobacterium lepraemurium-infected mice results from bone damage and not from nerve involvement.

A small but relatively constant proportion (3-5%) of mice chronically infected with Mycobacterium lepraemurium (MLM) develops bilateral paralysis of the rear limbs. The aim of the study was to investigate whether or not the bilateral leg palsy results from nerve involvement. Direct bacterial nerve infection or acute/delayed inflammation might possibly affect the nerves. Therefore, palsied animals were investigated for the presence of: (a) histopathological changes in the leg tissues including nerves, bones and annexes, and (b) serum antibodies to M. lepraemurium and M. leprae lipids, including phenolic glycolipid I from M. leprae. Histopathological study of the palsied legs revealed that the paralysis was not the result of direct involvement of the limb nerves, as neither bacilli nor inflammatory cells were observed in the nerve branches studied. Antibodies to brain lipids and cardiolipin were not detected in the serum of the palsied animals, thus ruling out an immune response to self-lipids as the basis for the paralysis. Although high levels of antibodies to MLM lipids were detected in the serum of palsied animals they were not related to limb paralysis, as the nerves of the palsied legs showed no evidence of inflammatory damage. In fact, nerves showed no evidence of damage. Paralysis resulted from severe damage of the leg bones. Within the bones the bone marrow became replaced by extended bacilli-laden granulomas that frequently eroded the bone wall, altering the normal architecture of the bone and its annexes, namely muscle, tendons and connective tissue. Although this study rules out definitively the infectious or inflammatory damage of nerves in murine leprosy, it opens a new avenue of research into the factors that participate in the involvement or the sparing of nerves in human and murine leprosy, respectively.

Antibodies to diverse lipids in the serum of patients with clinically cured leprosy and tuberculosis.

In this study we looked for the presence of antibodies to cardiolipin, cerebrosides, and whole lipids extracted from M. leprae, M. tuberculosis and M. habana, in the serum of patients with clinically cured lepromatous leprosy (sixteen) or tuberculosis (sixteen), 8 to 12 months after arresting the corresponding multi-drug therapy (MDT). Compared to healthy controls (sixteen), both leprosy and tuberculosis ex-patients had still significant levels of antibodies to the three mycobacterial lipids but no detectable levels of antibodies to cardiolipin or cerebroside lipids. Although leprosy and tuberculosis sera recognized the homologous mycobacterial lipids in a preferential fashion, all of them, on the average, reacted more strongly with the lipids of M. habana. This observation backs up, in a certain way, the proposition of using M. habana as a prospective vaccine for leprosy and tuberculosis.

Mycobacterium leprae and Mycobacterium lepraemurium infections in domestic and wild animals.

Mycobacterium leprae, the aetiological agent of leprosy in humans, gives rise to a chronic granulomatous disease that affects primarily the skin and peripheral nerves, and secondarily some internal organs such as the testis and the eye; viscera are seldom involved. Depending on host resistance, leprosy may present as a benign disease (tuberculoid leprosy) or as a malignant disease (lepromatous leprosy), with a spectrum of intermediate stages appearing between the two. Immunity against leprosy depends on the cell-mediated immunity of the host, and this is severely compromised in the malignant (lepromatous) form of leprosy. Although culture of M. leprae has never been achieved in artificial media, the bacterium may be grown in several experimental animals, including the armadillo, non-human primates, and to a certain extent, rodents. Naturally acquired leprosy has been reported in wild nine-banded armadillos (Dasypus novemcinctus) and in three species of non-human primates (chimpanzees [Pan troglodytes], sooty mangabey monkeys [Cercocebus atys] and cynomolgus macaques [Macaca fascicularis]), thus qualifying leprosy as a zoonosis. Murine leprosy is a leprosy-like disease of rats and mice, caused by Mycobacterium lepraemurium. The disease affects primarily viscera and the skin, and very rarely peripheral nerves. Depending on the host strain, rodent leprosy may also evolve as 'lepromatous' or 'tuberculoid' leprosy, and strains of mouse that develop intermediate forms of the disease may exist. Growth of M. lepraemurium on conventional media for mycobacteria is not successful, but the bacterium has been cultured on an egg yolk-based medium. Naturally acquired murine leprosy has been observed in rats, mice and cats, but not in humans or any other species. Thus, in contrast to human leprosy, murine leprosy is not a zoonosis.

Recognition of phenolic glycolipid-I (Mycobacterium leprae) and sulfolipid-I (M. tuberculosis) by serum from Mexican patients with leprosy or tuberculosis.

SETTING: Differential diagnosis of leprosy and tuberculosis in regions where both illnesses are endemic is a prerequisite for proper identification and treatment. OBJECTIVE: To evaluate the recognition of phenolic glycolipid-I (PGL-I) of Mycobacterium leprae and sulfolipid-I (SL-I) of M. tuberculosis by serum from patients with leprosy (LL) or pulmonary tuberculosis (PTB). DESIGN: Purified PGL-I and SL-I were used as antigens in an ELISA test set up to assess recognition of these lipids by serum from 43 LL patients, 44 PTB patients and 38 healthy individuals. RESULTS: Leprosy patients gave higher IgM than IgG responses to PGL-I and had comparable IgM and IgG responses to SL-I. A similar situation was observed with PTB serum. Some healthy individuals were found to contain significant levels of antibodies to both lipids. CONCLUSION: There is no specific recognition of either of the two lipid antigens tested by serum from both leprosy and tuberculosis patients; this rules out the possibility of using PGL-I and SL-I as tools for the differential diagnosis of these two mycobacterial diseases.

Susceptibility of "et," the spontaneously mutating CD1-derived nude mouse, to infection of M. lepraemurium.

We have studied the susceptibility to infection by Mycobacterium lepraemurium (MLM) of a nude, hypothymic, CD1-derived, spontaneous mouse mutant called "et" because of its extraterrestrial appearance. We found that despite their hypothymia, et/et mice were not more susceptible to infection by MLM than their euthymic et/+ counterparts. Infection of both et/et and et/+ mice with 50 x 10(6) bacilli by the intraperitoneal route led only to a mild infection with low levels of antimycobacterial antibodies and a small number of lesions. These lesions were indicative of reactive hepatitis and hyaline perisplenitis with lymphoid hyperplasia. Some small bacilliferous granulomas were also observed at the end of the experiment (5 months of infection). CD1 mice behave in a rather "resistant" manner to the infection by MLM. It is clear that the nu gene is not necessarily linked to the thymus defect, and it is also clear that the hypothymia of et/et mice does not obviously affect their general cell-mediated immune competence.

Secretion antigens of Mycobacterium tuberculosis: a comparison between a reference strain and seven wild isolates.

BACKGROUND: This study was carried out with the aim of detecting possible differences between proteins secreted by fresh wild isolates of Mycobacterium tuberculosis and from a reference strain of this microorganism, H37Rv TMCC 102. MATERIALS AND METHODS: This reference strain of M. tuberculosis has been in our laboratory for over 10 years, where it has been maintained by serial subcultures in PBY and Lowenstein-Jensen media. Patterns of protein secretion and recognition by sera derived from both tuberculosis patients and normal individuals were analyzed by electrophoresis and Western blotting. RESULTS: No major qualitative differences were observed among the several strains studied with respect to protein patterns or recognition of these proteins by test sera. Normal sera were found to react with almost all antigens recognized by tuberculosis sera, but with less intensity. However, a small protein of 14.5 kDa, secreted by both the wild and reference strains of M. tuberculosis, was recognized by 32 of the 40 tuberculous patient sera tested (80%), and was not recognized by any of the 40 serum samples derived from healthy individuals. CONCLUSIONS: This small protein seems to be a potentially important antigen for the serological diagnosis of tuberculosis and/or for use in the follow-up of patients who received treatment.

Altered pattern of connectivity in serum immunoglobulins from pemphigus vulgaris patients.

Pemphigus vulgaris is a cutaneous autoimmune disease in which the occurrence of autoantibodies directed against desmoglein-3 and other self-antigens has been well established in patient sera. However, V-region interactions (connectivity) of serum IgG and IgM have not been analysed to date. In this report, it has been demonstrated that IgG and IgM in the sera of pemphigus vulgaris patients bind a preparation of F(ab')2 fragments fractionated according to their isoelectric points, and that a pattern of connectivity distinguishable from that of healthy donor sera arises when the sera are tested against 20 individual isoelectric-focusing-separated F(ab')2-containing fractions. This suggests that there are alterations in regulatory networks. In spite of the fact that prednisolone-based treatment of pemphigus patients has proved to be effective in controlling the disease, some undesirable effects associated with this form of treatment have prompted investigation into other therapeutic approaches. One possible approach to the treatment of this autoimmune disease is the use of high doses of normal polyclonal immunoglobulins. In fact there are a few reports of the empirical intravenous administration of immunoglobulins to pemphigus vulgaris patients. The results presented here provide the rational basis for using such a treatment, since it is demonstrated that a deviation from healthy V-region interactions can be attributed to pemphigus patients and that such a condition is considered to be modified by this type of immunotherapy.

Do antibodies to phospholipid antigens play any role in murine leprosy?

In order to know whether antibodies to phospholipids and other host lipids play a role in the pathology of murine leprosy, we looked for the presence of antibodies to cardiolipin, cerebroside sulfatide, and to lipids extracted from normal murine spleen, liver and brain in the sera of mice bearing a 6-month infection with Mycobacterium lepraemurium. We also looked for the presence of antibodies to lipids isolated from M. lepraemurium. We found that all of the 16 animals examined contained high levels of antibodies to the mycobacterial lipids of intermediate polarity (mostly glycolipids) but none of them had antibodies to the other lipids tested, including those isolated from mouse liver, spleen and brain, bovine cardiolipin and sulfatide, nor any significant levels of antibodies to mycobacterial lipids of high or low polarity. The infected animals also had high levels of antibodies to antigens sonically extracted from the microorganism. Antibodies to the socially extracted antigens (mostly proteins) were mainly IgG, while antibodies to the lipid antigens were predominantly IgM. Despite the low but significant percentage (1%-3%) of infected animals developing bilateral paralysis of the rear limbs, autoimmunity (due to antibodies to phospholipids and other host lipids) does not seem to be a feature of murine leprosy.

Mycobacterium lepraemurium, a well-adapted parasite of macrophages: I. Oxygen metabolites.

We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide anion)] by murine peritoneal macrophages challenged in vitro with Mycobacterium lepraemurium (MLM), complement-opsonized yeast, M. bovis BCG, M. phlei, or phorbol myristate acetate (PMA). We found that except for MLM, all of the other materials provoked the release of significant amounts of hydrogen peroxide and superoxide. MLM entered the macrophages without triggering their oxidative metabolism. Pre-infection of macrophages with MLM did not alter these cells' capacity to release the normal amounts of ROI in response to other microorganisms or PMA. Killing of MLM did not revert the macrophages' failure to release ROI upon ingestion of the microorganism, nor were macrophages able to produce these toxic metabolites when pre-incubated in the presence of murine gamma interferon (IFN-gamma). MLM has several attributes that allow it to survive within macrophages: a) it is a nontoxigenic microorganism (it does not harm its host), b) it resists the harsh conditions of the intraphagolysosomal milieu (a property perhaps dependent on its thick lipidic envelope), and c) it penetrates the macrophages without triggering their oxidative response (thus avoiding the generation of the toxic intermediaries of oxygen). For these attributes (and others discussed in this paper), we recognize MLM as a highly evolved, well-adapted parasite of macrophages. In addition, the results of the present study prompted the analysis of the biochemical pathways used by MLM and M. bovis BCG to penetrate into their cellular hosts, a subject now under investigation in our laboratory.

Recognition of lipid antigens by sera of mice infected with Mycobacterium lepraemurium.

Lipids extracted from mouse tissues infected with Mycobacterium lepraemurium (MLM) were analyzed by thin-layer chromatography. Although the extracted lipids were heterogeneous in polarity, the lipids of intermediate polarity were the ones that predominated. All of the lipids of intermediate polarity were glycosylated species. There were also lipids of low and high polarity, the latter being glycolipids. Compared to lipids extracted from normal tissue (mostly to lipids of high and low polarity), all of the additional lipids extracted from the infected tissue corresponded to lipids present in the purified bacteria. Enzyme-linked immunoassays (ELISAs) were then performed with the whole lipids extracted from purified bacilli, the lipids of high, intermediate and low polarity, and the sera from 20 normal and 20 MLM-infected mice. Lipids of intermediate polarity were specifically recognized by MLM-infected mice. Neither sera (diluted 1:500) from normal mice nor infected mice reacted with the lipids of high or low polarity, but a higher concentration (sera diluted 1:100) of some sera from mice in both groups reacted significantly with these lipids. In the ELISAs the whole-lipid extract and the lipids of intermediate polarity were similarly recognized by the sera of the infected mice. Thus, as observed in human leprosy, the mycobacterial disease in the mouse (murine leprosy) is also accompanied by the development of antibodies to the glycolipids of the infecting microorganism.