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.

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.

Macrophage esterase: identification, purification and properties of a chymotrypsin-like esterase from lung that hydrolyses and transfers nonpolar amino acid esters.

A chymotrypsin-like esterase was purified from beef lung. This lysosomal enzyme, not previously characterized, seemed to be composed of two or more forms with molecular weights of about 52 000. It hydrolysed N-benzoyl-DL-phenylalanine beta-naphthol ester at acid and neutral pH; it polymerized L-phenylalanine methyl ester(Phe-OMe) at neutral pH; and it transferred the Phe-residue from Phe-OMe to hydroxylamine at neutral pH. Phenylmethanesulfonyl fluoride, an inhibitor of hydrolytic enzymes with serine in their catalytic site, inhibited this enzyme, but pepstatin, the cathepsin D (EC 3.4.4.23) inhibitor, did not. Sulfhydryl reagents were not required for activity. Macrophages, especially pulmonary alveolar macrophages, were a rich source of this esterase, so it is likely that the enzyme purified from lung came from its macrophages. The esterase hydrolysed and transferred monoamino acid esters, especially those of the aromatic type. Cathepsin C, the dipeptidyl peptide hydrolase (EC 3.4.14.1), acted only on dipeptide esters and amides. Pancreatic chymotrypsin acted on both monoamino acid and dipeptide esters. The chymotrypsin-like esterase did not hydrolyse hemoglobin, casein, or plasma albumin. Thus its proteolytic activity, if present, must be limited to specific substrates, as yet unknown.

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.

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.

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.

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.

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.

Phagocytosis in leprosy. 2. Production of superoxide by circulating blood leukocytes from lepromatous patients.

The ability of polymorphonuclear leukocytes from lepromatous patients to produce superoxide (O-2) anion while phagocytizing latex particles was studied. The results were compared with those obtained in a group composed of normal individuals. No differences were found between lepromatous and normal groups. No differences were found either when the comparison was made between patients showing any form of leprosy reaction and patients without leprosy complications at the time of the study. Together with other results previously reported, our findings suggest that PMN from lepromatous patients are not different from PMN from healthy individuals with respect to their ability to generate and show the metabolic oxidative changes (as measured by the NBT- and the O-2-tests) induced by in vitro phagocytosis of latex particles. These metabolic activities remain normal even when PMN may show alterations at other levels of the phagocytic process.

Serum lactate dehydrogenase in murine leprosy: source and isozymes.

The bulk of the lactate dehydrogenase (LDH) that increases in the serum of mice infected with Mycobacterium lepraemurium (MLM) derives from the liver and corresponds to the isozyme V. MLM-induced granulomas continuously arise in the liver and steadily increase in size until the animal's death. Growing granulomas push the adjacent hepatocytes away and cause them to disrupt and to release their cytoplasmic contents, including LDH. The LDH is then picked up by the infiltrating phagocytes and/or admixed with the circulating blood. Other LDH-containing organs (including the testis with its additional isozyme LDH-X) in the infected or normal animals do not seem to significantly contribute to the serum levels of LDH. The study of the liver-associated histochemical and biochemical changes in this controlled model of murine leprosy allows us to gain insight into the overall pathology of this mycobacteriosis. In some respects this sheds light on the liver involvement in human leprosy; a subject on which results of all sorts have been published.

[Sequential activation of cellular and humoral immunity in leprosy: considerations based on recent findings]. / Activación secuencial de la inmunidad celular y la inmunidad humoral en la lepra: consideraciones con base en hallazgos recientes.

Lepromatous leprosy in the human being evolves showing a progressive loss of cell mediated immunity (CMI) to the antigens of Mycobacterium leprae (ML). This does not prevent the host to respond with antibodies to the same microorganism. On the other hand, the production of antibodies to the great majority of exogenous antigens results from cell-to-cell interactions that involve the participation of helper T cells. On this ground, a satisfactory explanation for the loss of CMI to M. leprae (which indicates either the loss or inactivation of specific helper T cells), with no effect on the humoral response to the same microorganism (this implying the participation of functional specific helper T cells), was difficult to found. It was not until Mosmann established, in the mouse, the existence of two subpopulations of helper T cells, that a feasible explanation for the apparent immunological paradox observed in leprosy was possible to offer. The work described here, based to a great extent in our experience on murine leprosy, refers to recent concepts concerning this issue.

Inhibition of complement activity in murine leprosy.

NIH mice infected with Mycobacterium lepraemurium (MLM) show a marked depression in their levels of hemolytic complement that is proportional to the degree of infection. The defect affects more the activation of complement through the classical pathway (CPW) than the activation of complement through the alternative pathway. Although this low activity of CPW-complement may be due to different causes (complement consumption by the infecting microorganism, lack of biosynthesis of complement components, or the presence of complement inhibitory factors), our results seem to support the last possibility. The generation of factors in the infected animals that inhibit the autologous activity of complement as the infection goes on reduces the risk of complement-mediated tissue damage and prolongs the survival time of the host, a wise strategy on the part of the MLM to assure its own survival as a parasite.