Subcellular localization of the Iitracellular survival-enhancing Eis protein of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designated eis, was found to enhance intracellular survival of Mycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377-384, 2000). When eis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eis gene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product of eis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

HIV phenotype correlates with the relative amounts of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II in the virion envelope.

OBJECTIVE: The biological phenotype of HIV-1 has been associated with various aspects of its infectivity, including syncytium formation and coreceptor usage. Adhesion molecules, present on both the target cell and the virus, have also been shown to play a role in the infectious process. A possible correlation between the presence of adhesion molecules in the envelope of HIV-1 with the biological phenotype of the virus is examined. DESIGN: The envelopes of 56 isolates of HIV-1 of known biological phenotype were analyzed for the presence of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II molecules. METHODS: The coreceptor usage of each isolate was determined in a GHOST cell or a U87.CD4 infectivity assay. The presence of LFA-1 and MHC class II in each virus envelope was then determined using a virus-binding enzyme-linked immunosorbent assay (ELISA). RESULTS: Viruses using the chemokine receptor CCR5 have relatively higher levels of MHC class II than LFA-1 in their envelopes compared with those using CXCR4. CONCLUSIONS: The finding that there is a differential incorporation of MHC class II and LFA-1 molecules by CXCR4- and CCR5-using viruses augments the list of properties contributing to the biological phenotype of HIV-1. This may explain, in part, how CXCR4-using viruses are able to bind to and infect a broader range of cell types than CCR5-using viruses, and why CXCR4-using viruses are associated with a more aggressive disease course.

Isolation and serological characterization of a Plasmodium vivax recombinant antigen.

A genomic library for Plasmodium vivax was constructed in lambda gt11 and immunologically screened with pooled serum samples from vivax patients. Six seroreactive clones were isolated, and one clone, denoted PV9, was studied further. This clone has an unusual base composition (65% G + C), does not share any homology with P. falciparum, and codes for an entirely new antigenic determinant. Antibodies (immunoglobulin G type) against the PV9-encoded polypeptide were produced in all vivax patients older than 15 years. This seroreactivity was lower among patients younger than 15 years (53%). The antigenic epitope(s) of the PV9-encoded polypeptide was recognized at a similar rate by serum samples from P. vivax patients who were living 350 to 973 km apart. Fifty percent of uninfected Indian adults were also seropositive, whereas all European and American (United States) sera tested were negative, suggesting that anti-PV9 antibodies persist after infection. The seroreactivity pattern of this antigen is similar to that of the immunity developed in malaria after repeated infections.

Recombinant fusion protein identified by lepromatous sera mimics native Mycobacterium leprae in T-cell responses across the leprosy spectrum.

Pooled polyvalent sera from lepromatous leprosy patients were used to screen a lambda gt11 recombinant DNA expression library of Mycobacterium leprae in order to identify the relevant antigens recognized by the human immune response. Of the 300,000 phages screened, 4 clones were identified that coded for fusion proteins of the same molecular mass. The fusion protein from clone LSR2 was tested for immunoreactivity in assays using peripheral blood cells and sera from 11 laboratory personnel and 105 patients across the leprosy spectrum. LSR2 protein appears to be predominantly a T-cell antigen. It evokes similar lymphoproliferative responses as the native bacillus both at the individual level and in the leprosy spectrum as a whole. Though only 50% of patient sera with anti-M. leprae antibodies reacted with the fusion protein, the pattern of reactivity in the antibody responses was also similar for the various clinical types. The coding regions of clones LSR1 and LSR2 are identical. They show no homology with sequences stored in data banks and encode a protein of 89 amino acids with a calculated molecular mass of approximately 10 kDa.

The nature and kinetics of a delayed immune response to purified protein derivative of tuberculin in the skin of lepromatous leprosy patients.

We have analyzed the nature and kinetics of a delayed, cell-mediated immune response to a purified protein derivative of tuberculin (PPD) in the skin of 154 naturally sensitized patients with lepromatous leprosy. After the intradermal injection of 5 U of PPD, biopsies were taken at 1-21 d and studied for the composition, extent, persistence, and organization of the emigratory cell response by light and electron microscopy. Induration of positive sites occurred promptly, reached a maximum diameter at 4 d, displayed a major extravasatory element, and was evident for as long as 21 d. The cellularity of the site exhibited a biphasic course, reached a maximum at 7 d, involved as much as 70% of the dermis and millions of new cells, and was elevated threefold above preinjection levels at 21 d. The emigratory cells were limited to T cells and circulating monocytes. T cells were more evident as they entered a preexisting lepromatous lesion containing parasitized macrophages and only occasional T cells many of the CD8+ phenotype. The predominant emigratory T cell was CD4+ although CD8+ cells were in evidence. The CD4/CD8 ratio of the lesions started at less than unity and in two distinct steps reached levels as high as 5:1. In most sites CD4+ cells were in the majority at 21 d. A well-defined granulomatous response with epithelioid and giant cells was apparent at 4 d, reached a maximum at 7 d, and involved all PPD sites at this time point. The generation of these differentiated mononuclear phagocytes from newly emigrated monocytes was never observed in the underlying lepromatous lesion but is a constant feature of the tuberculoid leprosy response. Epidermal thickening and keratinocyte proliferation, sequellae of the dermal reaction, reached a maximum at 7 d and gradually resolved by 3 wk. A constant feature of the PPD response was the extensive destruction of preexisting macrophages containing Mycobacterium leprae bacilli or their products. This was associated with the presence of and intimate contact with highly polarized lymphoid cells of unknown phenotype. Cell destruction did not involve other elements of the dermis and spared parasitized Schwann cells. Newly emigrated T cells and monocytes were never seen within the perineural sheath in contact with neural elements. It appears that a single antigenic stimulus leads to a very long-term, defined series of events with distinct temporal patterns. It includes waves of emigratory T cells, the maturation and organization of monocytes, the generation of killer cells, and the extensive destruction of parasitized macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Type 1 reactions in leprosy--heterogeneity in T-cell functions related to the background leprosy type.

Nineteen each of paucibacillary borderline tuberculoid (BT) and multibacillary borderline borderline (BB)/borderline lepromatous (BL) leprosy patients undergoing type 1 reactions were compared with nonreactional stable patients of the appropriate leprosy type. In the BT reactional group, both phytohemagglutinin-induced and, more importantly, antigen-induced lymphoproliferation was reduced in 80%-90% of the patients. On the other hand, leukocyte migration inhibition was reduced in 40% and remained unchanged in the others. Suppressor-cell activity as evaluated by a costimulant assay was also reduced in a majority of the reactional BT individuals. In contrast, the bacilliferous BB and BL patients in reaction showed significant general improvement in leukocyte migration inhibition (p less than 0.001) and antigen-induced lymphoproliferation (p less than 0.05) as compared to the expected hyporesponsive/anergic uncomplicated BB-BL patients. Suppressor-cell activity also recovered during the reactional phase. However, no significant differences were observed in either of the reactional or stable leprosy types in the numbers of total T cells (OKT3+) and their subsets as defined by OKT4+ (helper/inducer) and OKT8+ (suppressor/cytotoxic) functional phenotypes. Moreover, during type 1 reactions the 48-hr delayed-type hypersensitivity (DTH) responses after intradermal injection of Mycobacterium leprae antigens continued to reflect the background leprosy type rather than the functional perturbations in the circulating T cells. Only a marginal increase in DTH was observed in some BT reactional individuals. No consistent pattern in the above in vitro T-cell-related responses was discernable in the same individuals 4-6 months after subsidence of reactions. The clinical entity of type 1 reactions encompassing paucibacillary and multibacillary leprosy shows a heterogeneity/dichotomy in T-cell responses which may reflect different immunological mechanisms underlying the reactional state.

Natural emergence of antigen-reactive T cells in lepromatous leprosy patients during erythema nodosum leprosum.

Fifteen lepromatous leprosy (LL) patients undergoing erythema nodosum leprosum (ENL) reactions were compared with 13 stable, uncomplicated, anergic individuals of the same leprosy background. ENL patients showed significant antigen-induced leukocyte migration inhibition (migration index = 0.058 +/- 0.01), paralleling the values obtained with a responder tuberculoid leprosy population (migration index = 0.04 +/- 0.004). Both phytohemagglutinin-induced general T-cell proliferation and, more significantly, antigen-induced lymphoproliferation were enhanced during the acute phase of the reaction. Suppressor cell activity, monitored by a costimulant assay, showed enhanced antigen-stimulated suppression of mitogen responses. Interestingly, the improvement in in vitro T-cell responses was not reflected in dermal reactivity, since 48-h delayed-type hypersensitivity responses after intradermal injection of soluble Mycobacterium leprae antigens continued to be poor. After subsidence of reactional lesions, leukocyte migration inhibition, lymphoproliferation, and suppressor cell activity were reduced to the unresponsive state seen in stable LL patients. Significantly, perturbations of T-cell reactivity are detectable in ENL reactions, indicating the natural but transient emergence of antigen-induced T cells in LL.

Differences in predominant T cell phenotypes and distribution pattern in reactional lesions of tuberculoid and lepromatous leprosy.

The nature and histological pattern of the cutaneous infiltrates of 17 leprosy patients in reversal reactions (Type I) and erythema nodosum leprosum (Type II, ENL) were compared with tissues from 18 non-reactional borderline leprosy (BT, BL) and lepromatous leprosy (LL) patients using monoclonal antibodies and immunofluorescence. Reactional BT lesions showed a mild increase in OKT11+ pan T cells as compared to non-reactional tissues and a significant influx of OKT8+ (suppressor/cytotoxic) cells which were peripherally localized in the lymphocyte mantle surrounding the epithelioid cells. The Leu 3a+ (helper/inducer) cells were scattered amongst the lymphocytes and macrophages. The mean ratio (+/- s.d.) of Leu 3a+/OKT8+ cells was 1.88 +/- 0.64 in Type I BT reactions as compared to 2.95 +/- 0.95 in BT lesions. In contrast, lesions of BL reversal reactions and ENL showed a more marked increase in pan T cells with a preponderance of the helper/inducer subset, Leu 3a+/OKT8+ ratio being 2.26 +/- 0.61 and 0.93 +/- 0.57 in BL reactional and non-reactional lesions, respectively. Interestingly, this increase in the numbers of the T cells reached levels observed in BT lesions. The distribution pattern of OKT8+ cells was similar to Leu 3a+, both being diffusely scattered amongst the bacilli laden macrophages. Ia like antigens were present in all granulomas and were abundant on lymphocytes and macrophages and less conspicuous on epithelioid cells. T6+ Langerhans cells were uniformly increased in all reactional lesions. It would appear that the changes observed in both Type I and Type II reactions are similar in the lepromatous group of patients. They differ significantly from the BT reversal reaction in terms of the dominant T cell subset and the microanatomical distribution of the OKT8+ cells in the lesions.