Two-dimensional electrophoretic analysis of humoral responses to culture filtrate of Mycobacterium bovis BCG in patients with leprosy and tuberculosis

Various aspects of our communication are well known to have changed over time (1-3). This article describes a cross-sectional study that examined the acoustic characteristics of two groups of Australian women aged 18-25 years from recordings made in 1945 and 1993 and investigated the possible changes in the voice across generations. Archival recordings from 1945 which had been used in a longitudinal study (4) were compared to recordings made in 1993. The results of this study show that women in 1993 have significantly deeper voices than women of the same age recorded in 1945. The possible factors influencing this change are discussed.     

Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins

Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.     

Association between an early humoral response to Mycobacterium tuberculosis antigens and later development of tuberculosis in human immunodeficiency virus-infected individuals

We describe four cases of inflammatory pseudotumor seen at our institution in the past 4 years. Four children were each found to have a large extraperitoneal mass on imaging studies, three of which were in the pelvis. Malignant sarcomatous tumors were suspected. Surgical biopsy of each mass, however, revealed inflammatory pseudotumor.     

Heterogeneous antibody responses in tuberculosis

Antibody responses during tuberculosis were analyzed by an enzyme-linked immunosorbent assay with a panel of 10 protein antigens of Mycobacterium tuberculosis. It was shown that serum immunoglobulin G antibodies were produced against a variety of M. tuberculosis antigens and that the vast majority of sera from tuberculosis patients contained antibodies against one or more M. tuberculosis antigens. The number and the species of serologically reactive antigens varied greatly from individual to individual. In a given serum, the level of specific antibodies also varied with the antigen irrespective of the total number of antigens recognized by that particular serum. These findings indicate that person-to-person heterogeneity of antigen recognition, rather than recognition of particular antigens, is a key attribute of the antibody response in tuberculosis.     

Sperm-immobilizing monoclonal antibody to human seminal plasma antigens

Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma.     

Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.     

Effective, nonsensitizing vaccination with culture filtrate proteins against virulent Mycobacterium bovis infections in mice

Vaccination of mice with Mycobacterium bovis culture filtrate proteins (CFP), prepared in a variety of adjuvants (aluminum hydroxide, Quil-A, and dimethyldioctyldecyl ammonium bromide [DDA]), provided significant protection against an aerosol challenge of virulent M. bovis. Additionally, vaccination with CFP in DDA or Quil-A did not sensitize mice to M. bovis purified protein derivative.     

A comparison of antibody responses to veterinary vaccine antigens potentiated by different adjuvants

Six adjuvant formulations were compared for their ability to potentiate the primary and memory antibody responses in mice to three companion animal vaccine immunogens--feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and a recombinantly-derived heartworm antigen. The combination of a novel bacterial immunostimulator, gliding bacterial adjuvant (GBA), either adsorbed onto an aluminum hydroxide gel (Rehydragel HPA), or emulsified with a vehicle of polyalcohol and detergent, elicited the strongest memory responses to both virus preparations. Both forms of aluminum hydroxide gels administered without GBA gave similar levels of adjuvant effects, on par with or greater than those generated by incomplete Freund's adjuvant (IFA). The Acemannan immunostimulant was not effective in increasing the responses to the virus antigens, but increased the primary response to the heart-worm antigen over tenfold from control levels. All preparations appeared to be well tolerated, with no detectable adverse reactions observed in any of the 250 mice used. The proven safety of aluminum hydroxide adjuvants and the apparent absence of adverse reactions seen with GBA make this vehicle/adjuvant formulation worthy of additional study.     

Lymphoproliferative responses to antigens mediated by human pulmonary alveolar macrophages

We evaluated the ability of human pulmonary alveolar macrophages (PAMs) to mediate (3H)-thymidine incorporation by blood lymphocytes severely depleted of monocytes when stimulated with soluble microbial and allogeneic lymphocyte antigens. Low (less than 2%) concentrations of PAM's from nonsmokers or blood monocytes did not support optimal responses. Over all, at greater than or equal to 10% concentrations, PAM's from nonsmokers supported higher responses than monocytes. At less than or equal to 10% concentrations, PAM's from heavy cigarette smokers mediated significantly less incorporation than did similar concentrations of PAM's from nonsmokers (p less than 0.05). The findings indicate that PAM's from healthy nonsmokers are functionally competent macrophages in terms of mediating lymphoproliferation in cultures stimulated with antigens. This classical macrophage function is impaired with cigarette smoking.     

Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi

Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 +/- 3.8% and 21.2 +/- 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 +/- 5.9% and 2.6 +/- 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 +/- 3.7 and 21.6 +/- 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 +/- 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 +/- 0.5%). Preincubation of SchC/6d with an F(ab)2 fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 +/- 3.0% but only 1.7-fold (maximum cytolysis 37.4 +/- 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.     

Duration of antibody responses to common enterobacterial and O antigens of children with pyogenic peritonitis

Children with pyogenic peritonitis folloiwng appendicits produce antibodies against the common enterobacterial antigen (CA) in unusually high titers when compared to patients with salmonellosis, shigellosis, or urinary tract infection. The duration of this antibody response was determined in 19 children observed over a period of up to 31 months after the acute illness. CA antibody titers decreased significantly in 70% of the patients during the first year and in 91% of those studied between 13 and 31 months after the infection. In the majority (71%) of patients the antibody titers returned to pre- or near pre-infection levels within 31 months after the acute illness. Only in a few patients (19%) were near maximal titers maintained during the observation period. The titers of antibodies against the O antigens of the infecting microorganisms also decreased in the majority of subjects during the follow-up period of observation. These findings may be of importance in connection with studies on the immune response of patients with urinary tract and other infections.     

Spleno-hepatic tuberculosis due to Mycobacterium kansasii

A case of tuberculosis of the spleen and liver is described. The organism involved was Mycobacterium kansasii, one of the atypical mycobacteria. The lack of evidence in the literature of primary splenic or hepatic involvement by this organism suggests that it is rare. In this instance it complicated a case of myeloproliferative disease, megakaryocytic myelosis with extra-medullary haemopoiesis, and was not diagnosed until autopsy.     

An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 10(7) organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.     

Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.     

Activation of human dendritic cells following infection with Mycobacterium tuberculosis

Dendritic cells (DC) play an essential role in the initiation of primary T cell responses to foreign Ag. It is likely that these potent APC are critical in the initiation of immune responses to pathogens, such as bacteria or parasites. However, little is known about the interaction of these important APC with pathogens. To address this issue, the interaction of the bacterium Mycobacterium tuberculosis with human DC was studied. DC generated from human peripheral blood by short term culture in medium containing recombinant human cytokines granulocyte-macrophage-CSF and IL-4 were capable of phagocytosing M. tuberculosis. Infection of DC with live M. tuberculosis bacilli resulted in increased APC surface expression of the costimulatory molecules CD54, CD40, and B7.1, as well as MHC class I molecules. In addition, infected DC secreted elevated levels of inflammatory cytokines, including TNF-alpha, IL-1, and IL-12. M. tuberculosis-infected human monocytes also secreted inflammatory cytokines, but exhibited no enhancement of costimulatory or MHC class I molecule expression. These data indicate that infection with M. tuberculosis results in the direct activation and maturation of these DC. In vivo, such activation may facilitate migration to the lymph nodes, and enhance presentation of Ag to T cells, thereby facilitating the induction of the immune response against this pathogen.     

Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig

Two antigens from Mycobacterium tuberculosis, ESAT-6 and MPT64, elicited delayed-type hypersensitivity (DTH) skin responses in outbred guinea pigs infected with M. tuberculosis by the aerosol and intravenous routes but not those sensitized with M. bovis BCG or M. avium. The DTH epitope of ESAT-6 was mapped to the C terminus. Nonresponders to the individual antigens were found, but all animals responded to a combination of ESAT-6 and MPT64 or their respective minimal target peptides. Correspondingly, these molecules could form the basis of a new skin test for tuberculosis.     

T cell responses to Mycobacterium bovis in red deer, a large animal model for tuberculosis

Uterine morphology assessed by transvaginal ultrasound and the hemodynamics of intratumoral vessels assessed by color Doppler ultrasound were prospectively correlated with the clinical outcome of 25 patients with trophoblastic tumors. Twenty patients were followed without treatment (observation group) and 16 achieved complete local resolution. The four subjects with local persistence were combined with five patients referred from other institutions and received chemotherapy (treatment group). In the observation group both techniques had 100% accuracy in predicting local resolution or local persistence. Persistence was predicted 1-3 weeks before the increase of beta-human chorionic gonadotropin (beta-hCG) levels, whereas resolution was observed up to 8 weeks before the disappearance of beta-hCG. In one patient normal uterine morphology and vascularization in the presence of elevated hCG levels was associated with extrauterine spread. In the treatment group, normal uterine ultrasound morphology and negative color Doppler results had 100% negative predictive value. False-positive results were observed in two cases. We conclude that ultrasound evidence of abnormal uterine morphology or persistent vascularization on color Doppler examination with persistent hCG levels is indicative of local persistence. Normal uterine morphology with negative color Doppler results may be associated with extrauterine spread.     

Mycobacterium avium complex and Mycobacterium tuberculosis in patients infected with the human immunodeficiency virus

Primary care physicians play an important role in identifying and treating bacterial infections in adults infected with the human immunodeficiency virus (HIV). Mycobacterium avium complex and Mycobacterium tuberculosis are pathogens that can cause systemic or local infection in these patients. We review the epidemiology, pathogenesis, clinical presentation, and principles of treatment for these two mycobacterial pathogens. Because M tuberculosis disease is preventable and curable and yet communicable, physicians should maintain a high degree of suspicion for tuberculosis in HIV-infected adults. In comparison, the goal of treating M avium complex in patients with advanced HIV disease is to reduce constitutional symptoms and improve survival.