Obstructive renal failure due to therapy with sulfadiazine in an AIDS patient.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
M T Silva
Publications and source records attributed to M T Silva.
In vitro effects of interleukin-4 on interferon-gamma-induced macrophage activation.
Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) have been shown to be secreted by distinct T-helper cell subsets which have different roles in the determination of host resistance to infection. We studied the activity of these two cytokines on effector mechanisms of mouse macrophages. In vitro cultured bone marrow-derived macrophages from C57BL/6 mice were treated with IFN-gamma, IL-4, or a combination of both cytokines and the ability to secrete superoxide or nitrite or to restrict growth of Mycobacterium avium and Toxoplasma gondii was then evaluated. We found that IL-4 could inhibit the priming of macrophages for enhanced superoxide production induced by IFN-gamma although IL-4 when used alone did have some enhancing effect of its own. This effect of IL-4 on IFN-gamma-primed superoxide production was dose dependent and could be observed even if the treatment by IL-4 was done 24 hr after treatment by IFN-gamma. IL-4 did not, however, influence the enhanced production of nitrogen reactive intermediates, the induction of bacteriostatic activity against M. avium, or the restriction of T. gondii by IFN-gamma-treated macrophages, and did not have any effect of its own regarding these latter functions.
Animals↗
Activation of human neutrophils by phorbol ester decreases the cytoplasm compactness and the lactoferrin content of the granulocytes.
Human neutrophils isolated from the blood were incubated in vitro for 30 min with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to study its effect on the fine structure of granulocytes and on the subcellular distribution of lactoferrin (Lf). Flow cytometry analysis of the human neutrophils showed that PMA induced a decrease in size and granularity of the cells. By electron microscopy, the PMA-treated cells showed numerous empty vesicles, smoothing of the cell surface, and a marked decrease in the compactness of the cytoplasmic ground substance. High-resolution immunogold ultracytochemistry showed that neutrophils lost most of their content in Lf after the PMA incubation. In thin sections, the cytoplasmic compartment of PMA-treated neutrophils contained an average of 1.03 +/- 0.3 Lf-positive vesicles per microns 2, whereas control granulocytes showed an average of 5.72 +/- 1.49 Lf vesicles per microns 2 of cytoplasm. Most of PMA-treated neutrophils kept some Lf-positive vesicles; a significant minority (10-15%) of the granulocytes showed cytological features of cell death. We conclude that the PMA-induced neutrophil activation is associated with microanatomical changes that include i) exocytosis of most, but not all, of the Lf-bearing vesicles; ii) rounding up of the cell outline; and iii) decrease in the compactness of the cytoplasmic ground substance.
Cytoplasm↗
Host and bacterial factors control the Mycobacterium avium-induced chronic peritoneal granulocytosis in mice.
Persistent peritoneal granulocytosis and elevated macrophage counts have been found in nine mouse strains from 8 to 90 days after infection with Mycobacterium avium. Peritoneal granulocytosis was higher in M. avium-resistant BALB/c. Bcgr (C.D2) mice, compared with congenic M. avium-susceptible BALB/c (Bcgs) animals. Although maximal granulocytosis values were not related to virulence of the inocula, the kinetics of the granulocytic response varied with the virulence of M. avium. Following infections by avirulent (rough) strains of M. avium, the peritoneal granulocytosis progressively declined in BALB/c and C3H/He mice. A similar decline in granulocyte number was observed in resistant C3H/He mice infected with virulent M. avium (smooth transparent strain). In both instances the decline in the peritoneal granulocytosis was associated with a progressive elimination of the inoculum. In the susceptible BALB/c mice, virulent M. avium strains induced progressive infection accompanied with a rapid decline in granulocyte number, whereas the infection with attenuated M. avium, which caused a chronic infection, induced persistent granulocytosis. The ability to recruit granulocytes following the intraperitoneal inoculation of a phlogistic substance (casein hydrolysate) was decreased in infected susceptible but not in infected resistant mice at 90 days of infection with virulent M. avium.
Animals↗
IS901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium.
An insertion sequence (IS901), found in pathogenic strains of Mycobacterium avium, but absent in M. avium complex isolates from patients with acquired immune deficiency syndrome (AIDS), has been isolated and sequenced. This insertion element has a nucleotide sequence of 1472 bp, with one open reading frame (ORF1), which codes for a protein of 401 amino acids. The amino acid sequence, terminal ends and target site of IS901 are similar to those of IS900, present in Mycobacterium paratuberculosis. However, the DNA sequences of these two IS elements exhibit only 60% homology, compared to a DNA homology of 98% between their respective hosts. IS901, like IS900, appears to belong to a family of related insertion elements present in actinomycetes and other bacteria. M. avium strains containing IS901 were found to be more virulent in mice than closely related strains lacking IS901. IS901 may be a useful tool for the study of the genetics of virulence in the M. avium complex and for obtaining stable integration of foreign genes into mycobacteria.
Acquired Immunodeficiency Syndrome↗
Intramacrophagic Mycobacterium avium bacilli are coated by a multiple lamellar structure: freeze fracture analysis of infected mouse liver.
We used freeze fracture electron microscopy to study the fine structure of Mycobacterium avium inside phagosomes of murine macrophages. M. avium-susceptible C57BL/6 mice were infected with M. avium by intraperitoneal inoculation of 10(8) viable bacilli. We studied the microanatomy of the mycobacteria in 3-month infections of mice, a situation in which bacillary multiplication is extensive. In these samples, freeze fracture revealed that intraphagosomal bacilli were surrounded by a multilamellar coat that was apposed to the cell wall. In thin sections, in contrast, the area corresponding to the coat showed no substructure and was electron transparent (the so-called electron-transparent zone that has been previously reported by others). The multiple lamellae resembled an onionlike assembly that was inserted in between the mycobacterial wall outer surface and the phagosomal membrane. Each lamella of the M. avium coat was made up of parallel straight fibrils with a width of 5 nm. A variable number of lamellae, sometimes up to 10 or more elements, coated individual bacilli. The multilamellar coat was absent around both extracellular M. avium and intramacrophagic M. avium after short-term (45-min) inoculation of mice. The supramolecular organization of the M. avium lamellar coat as viewed here by freeze fracture is similar to that of purified mycoside C (P. Draper, J. Gen. Microbiol. 83:431-433, 1974; K.-S. Kim, M.R.J. Salton, and L. Barksdale, J. Bacteriol. 125:739-743, 1976), a mycobacterial component currently known as glycopeptidolipid (W.W. Barrow and P.J. Brennan, J. Bacteriol. 150:381-384, 1982). We conclude that M. avium bacilli growing in macrophages are surrounded by multilamellar capsulelike structures that contain glycopeptidolipid molecules.
Animals↗
Mycobacteria and human autoimmune disease: direct evidence of cross-reactivity between human lactoferrin and the 65-kilodalton protein of tubercle and leprosy bacilli.
We document here by Western immunoblotting and immunogold ultracytochemistry that polyclonal antibodies against human lactoferrin (Lf) bind to tubercle and leprosy bacilli. In situ immunogold labeling of Mycobacterium leprae (present in armadillo liver and in human skin) and of Mycobacterium tuberculosis indicated that receptors for anti-Lf antibodies were present both on the cytoplasm and on the envelope of the bacilli. We found by immunoblotting that the 65-kDa heat shock protein is the major component of M. leprae and M. tuberculosis that is responsible for the binding of the anti-Lf probe. Furthermore, we show that anti-Lf immunoglobulin G eluted from the nitrocellulose-transferred mycobacterial 65-kDa protein band did bind back to Lf. Ultracytochemistry of biopsy samples of human lepromas showed that dead or severely damaged M. leprae was strongly marked by the anti-Lf antibodies; a similar pattern of immunogold marking was observed on M. leprae when antibodies against the 65-kDa mycobacterial protein were used. Our results offer direct evidence that the 65-kDa protein of leprosy and tubercle bacilli is recognized with specificity by antibodies against the human protein Lf. The Lf-65-kDa protein antigenic cross-reactivity may contribute to the formation of autoantibodies and immune complexes as well as to other autoimmune events that are frequent in tuberculosis and leprosy. Our immunocytochemical data also suggest that the cross-reactivity may persist for some time after the death of mycobacteria in infected hosts.
Animals↗
Live but not heat-killed mycobacteria cause rapid chemotaxis of large numbers of eosinophils in vivo and are ingested by the attracted granulocytes.
We studied leukocyte chemotaxis triggered by a local injection of mycobacteria (Mycobacterium avium and M. smegmatis) in BALB/c and C57BL/6 mice. Our experimental model consisted of the induction of a subcutaneous air pouch in the dorsal area of mice and inoculation 6 days later of 10(8) CFU of myocobacteria. Inflammatory exudates were harvested from the air pouch cavities 15, 30, and 45 min after the injection of the inocula. Injection of the microorganisms resulted in the migration of an elevated number of eosinophilic granulocytes into the inflammatory cavities. At 30 min after the inoculation of the mycobacteria, the air pouches contained between (3.9 +/- 0.3) x 10(5) (M. avium) and (3.3 +/- 0.3) x 10(5) (M. smegmatis) eosinophils, corresponding to more than one-third (41.4 to 38.3%) of the leukocytes present in the inflammatory cavities. Less than one-half of the eosinophils were attracted to the air pouches when the same number of heat-killed mycobacteria were inoculated [(1.3 +/- 0.2) x 10(5) cells for M. avium and (1.5 +/- 0.2) x 10(5) cells for M. smegmatis]. Injection of gram-negative bacteria (Escherichia coli), of latex beads, or of casein resulted in the attraction of inflammatory eosinophils in numbers that were comparable to those attracted by the heat-killed mycobacteria. Our data document the fact that live mycobacteria exert a rapid chemotactic effect on eosinophils. We therefore postulate that mycobacteria either contain or induce the production of an eosinophilotactic factor. Because this chemotactic effect occurs during the acute inflammatory response to mycobacteria, it cannot be due to the formation of immune complexes (a major infection-associated chemotactic factor for eosinophils). The attracted eosinophils had an important role in the local phagocytosis of mycobacteria, as indicated by our finding, derived from thin-section electron microscopy quantifications, that at 30 min after M. avium inoculation the inflammatory exudates contained (2.2 +/- 0.5) x 10(5) mycobacterium-bearing eosinophils (corresponding to 57% of the total eosinophils), as compared with (2.1 +/- 0.1) x 10(5) neutrophils and (1.5 +/- 0.2) x 10(5) macrophages with ingested bacilli. We conclude that mycobacteria induce the attraction of eosinophils to inflammatory sites and that these granulocytes have the capacity to phagocytize these bacilli in situ.
Animals↗
Fatal infection with a novel, unidentified mycobacterium in a man with the acquired immunodeficiency syndrome.
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Acquired Immunodeficiency Syndrome↗
Cross-reactivity and sequence homology between the 65-kilodalton mycobacterial heat shock protein and human lactoferrin, transferrin, and DR beta subsets of major histocompatibility complex class II molecules.
Immunogold ultracytochemistry and Western immunoblotting showed that polyclonal antibodies against human lactoferrin bind to the highly immunogenic 65-kilodalton (kDa) heat shock protein of mycobacteria. The fast-growing mycobacterial species Mycobacterium smegmatis showed a higher density of these receptors for antilactoferrin sera than the slow-growing M. avium. Polyclonal antibodies against mycobacteria (M. bovis BCG) recognized human lactoferrin. Comparison of the amino acid sequence of lactoferrin with that of the 65-kDa protein of M. tuberculosis revealed seven instances of four amino acid sequence homology between the microbial and the human iron-binding protein. Four of these tetrapeptide sequences were also shared with the human transferrin molecule. The shared amino acid sequence KDLL was also present in the DR1, DR3, and DR4 subsets of the DR beta subregion of major histocompatibility complex (MHC) class II molecules. The molecular mimicry between the 65-kDa mycobacterial protein and the human proteins (lactoferrin, transferrin, and MHC class II molecules) offers a molecular setting for mycobacteria-associated, T-cell-dependent autoimmune disease, namely, for rheumatoid arthritis.
Amino Acid Sequence↗
Pitfalls in the ultrastructural analysis of mycobacteria, including Mycobacterium leprae.
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Animals↗
High-resolution localization of lactoferrin in human neutrophils: labeling of secondary granules and cell heterogeneity.
We studied by electron microscopy the topography of the iron-binding protein lactoferrin in human peripheral blood neutrophils by immunogold labeling of thin-sectioned cells. We show that fixation in 0.3% glutaraldehyde, supplemented by 2% formaldehyde, and combined with embedding in the hydrophilic resin LR White results in excellent preservation of the lactoferrin antigenicity and of the neutrophil microanatomy. Most of the immunogold marking was seen on the specific (secondary) granules of polymorphonuclear leukocytes, thus strengthening prevailing views on the subcellular distribution of lactoferrin in neutrophils. We document that a minority (8-10%) of neutrophils showed significant amounts of lactoferrin associated with a flocculent, protein-like material seen inside large cytoplasmic vacuoles. We show that circulating neutrophils comprise 6-10% of cells that are virtually devoid of lactoferrin labeling. This finding provides specific cytochemical support for the concept that human polymorphonuclear leukocytes are made up of a heterogeneous population of cells.
Adult↗
Patterns of marihuana smoking among Brazilian students.
Changes in frequency of marihuana smoking over time were investigated by interviewing forty-seven Brazilian students from São Paulo between 18 and 24 years old, of both sexes. From their report, it was concluded that the course of smoking frequency follows two predominant patterns. In one of them, the 'Stable Pattern', subjects report increasing consumption up to a peak frequency where they remain for a relatively long and stable period of time. In the other one, the 'Descending Pattern', subjects report a gradual increase up to a peak, followed by a clear decline in rate of use. Two other patterns were also detected in a small number of subjects. The diversity of patterns identified stresses the difficulty in predicting the course of marihuana smoking frequency by young people. The relationship between marihuana smoking and the use of other drugs was also investigated. The only drugs that were reported to be regularly consumed by the majority of subjects were alcohol and tobacco, while 90% of the subjects reported having tried at least one other illegal drug.
Adult↗
Neutrophil-macrophage cooperation in the host defence against mycobacterial infections.
CD-1 mice inoculated intraperitoneally with Mycobacterium avium, M. bovis, M. microti or M. kansasii showed a persistent peritoneal granulocytosis (above 10(6) cells, i.e. more than 15% of total cells) throughout the 3 month period of infection studied. By contrast, in mice inoculated with the non-pathogenic M. aurum or with heat-killed M. avium the number of granulocytes decreased progressively after the first 15 days. No mycobacteria were found in granulocytes except in the first 2 days of infection. The mycobacteria-induced chronic granulocytosis was accompanied by phagocytosis of granulocytes by macrophages. Throughout the 3 months of infection, macrophages were found to contain intracellular lactoferrin. Macrophages with lactoferrin were also found in subcutaneous infection caused by M. marinum and in systemic infection caused by M. avium or M. kansasii. The in vitro activity of mouse peritoneal macrophages against M. avium and M. microti was increased after ingestion of granulocyte material by macrophages. These results lead us to propose that granulocytes participate in the host response to mycobacterial infections, not as phagocytes but rather through an indirect mechanism, as a source for the macrophages of molecules involved in antimicrobial mechanisms (e.g., lactoferrin and myeloperoxidase) lacking in the mature macrophage.
Animals↗
Induction of non-specific immunosuppression in mice by mycobacterial infections and its relationship to macrophage activation.
The development of non-specific immunosuppression during the infection of different strains of mice with three mycobacterial species was evaluated by studying the immune response to a heterologous antigen (sheep red blood cells) and comparing it with the induction of non-specific resistance to a Listeria monocytogenes challenge. It was shown that early (at 15 days) immunosuppression developed in Mycobacterium avium-susceptible mouse strains infected with a high inoculum dose [2.5 x 10(8) colony forming units (CFU)] of virulent M. avium but not in resistant mice infected with a similar inoculum nor in susceptible mice infected by a smaller inoculum dose (2.5 x 10(6) CFU). In the latter case it developed only during the second month of infection and was of smaller magnitude. An inoculum of M. avium of attenuated virulence did not induce immunosuppression. M. lepraemurium induced a late immunosuppression, which occurred when extensive bacterial proliferation had already taken place. The non-pathogenic M. bovis BCG induced immunosuppression in C57BL/6 mice. The results do not establish a correlation between the development of generalized immunosuppression and susceptibility to infection. It could be seen that the early immunosuppression was observed in those situations where there was extensive macrophage activation as shown by the development of non-specific resistance to a listeria challenge. The late immunosuppression was observed when bacterial proliferation was extensive.
Animals↗
New data on the ultrastructure of the membrane of Mycobacterium leprae.
In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown.
Animals↗
T-helper cell subpopulations and the immune spectrum of leprosy.
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Cytotoxicity, Immunologic↗
T cell-dependent chronic neutrophilia during mycobacterial infections.
Euthymic (nu/+) C57BL/6 mice intraperitoneally inoculated with 2.5 x 10(6) colony-forming units (CFU) of Mycobacterium avium developed a chronic peritoneal neutrophilic granulocytosis during the 30 days of infection studied; in contrast, congenitally athymic nude (nu/nu) mice of C57BL/6 background did not show such persistent neutrophil influx. The acute phase of peritoneal infection, characterized by an extensive accumulation of neutrophils peaking at 6 to 12 h post-inoculation, was similar in euthymic and athymic mice. Subcutaneous vaccination of C57BL/6 mice with BCG enhanced the peritoneal influx of granulocytes after the i.p. inoculation of 2.5 x 10(60 CFU of M. avium. Finally, spleen cells from M. avium-infected mice pulsed in vitro with mycobacterial antigen induced a higher neutrophil accumulation after inoculation into the peritoneal cavity of naive recipient mice than unpulsed spleen cells or spleen cells from noninfected mice. These data indicate that the immune system is involved in the regulation of the chronic neutrophil influx during mycobacterial infection.
Animals↗