Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody.

Detection of immunoglobulin M (IgM) precipitin antibody to coccidioidin, the autolysate of mycelial-phase cells of Coccidioides immitis, is an important serologic aid in establishing a diagnosis of primary coccidioidomycosis. In the present study, the component of coccidioidin that reacts with IgM precipitin antibody was isolated by a combination of immunoaffinity and anion-exchange chromatography. Antigenic analysis of the purified antigen in two-dimensional immunoelectrophoresis against goat anti-coccidioidin revealed a precipitinogen characterized by a complete cathodal leg and a partial anodal leg. The reactivity of this incomplete precipitating antigen with anti-C. immitis IgM was established by serologic assays and by the adsorption of reference IgM precipitin antibody on solid-phase immunosorbents containing the purified precipitinogen. The isolation of the coccidioidin component that reacts with IgM precipitin antibody and the production of monospecific antibody will provide the necessary reagents for the development of a sensitive immunoassay for detecting this serodiagnostic response.     

T-cell recognition of the HspX protein of Mycobacterium tuberculosis correlates with latent M. tuberculosis infection but not with M. bovis BCG vaccination

During stationary growth or in vitro conditions mimicking relevant aspects of latency, the HspX protein (Rv2031c) is specifically upregulated by Mycobacterium tuberculosis. In this study we compared T-cell responses against HspX and the secreted M. tuberculosis protein Ag85B (Rv1886c) in tuberculosis (TB) patients, tuberculin skin test-positive individuals, M. bovis BCG-vaccinated individuals, and healthy negative controls. Gamma interferon responses to HspX were significantly higher in M. tuberculosis-exposed individuals than in M. tuberculosis-unexposed BCG vaccinees. In contrast, no such differences were found with respect to T-cell responses against Ag85B. Therefore, BCG-based vaccines containing relevant fragments of HspX may induce improved responses against this TB latency antigen. To identify relevant major histocompatibility complex class I- and class II-restricted HspX-specific T-cell epitopes, we immunized HLA-A2/K(b) and HLA-DR3.Ab(0) transgenic (tg) mice with HspX. Two new T-cell epitopes were identified, p91-105 and p31-50, restricted via HLA-A*0201 and HLA-DRB1*0301, respectively. These epitopes were recognized by human T cells as well, underlining the relevance of HspX T-cell recognition both in vivo and in vitro. In line with the data in humans, BCG immunization of both tg strains did not lead to T-cell responses against HspX-derived epitopes, whereas nonlatency antigens were efficiently recognized. These data support the notion that BCG vaccination per se does not induce T-cell responses against the latency antigen, HspX. Thus, we suggest that subunit vaccines incorporating HspX and/or other latency antigens, as well as recombinant BCG strains expressing latency antigens need to be considered as new vaccines against TB.     

Sequence-specific binding of normal serum immunoglobulin M to exposed protein C-termini

Both the timely clearance of degraded endogenous structures and the presence of secreted natural immunoglobulin M (IgM) are needed to avoid autoimmunity. These requirements may be causally related provided that natural IgM preferentially reacts with degraded antigens and, by activating complement, mediates their non-inflammatory clearance through complement receptors. We have previously shown that normal serum IgM reacts in vivo and in vitro with virtually all randomly generated C-terminal peptides displayed on T7 phage. The resultant multivalent IgM-peptide complexes activate complement and are detected by a loss of phage infectivity. A striking feature of these reactions is that different C-terminal peptides ( approximately 3-4 amino acids) specifically react with different 'C-terminal' IgM (C-IgM) antibodies. This suggests that degraded supramolecular structures, expressing elevated levels of identical C-termini as a result of proteolysis, denaturation and abnormal exposure of repetitive protein constituents, may be preferential targets of C-IgM-mediated complement activation in the physiological environment. The specificity of C-IgM-peptide reactions is much higher than one would expect, assuming that normal serum IgM mostly comprises polyspecific natural antibodies. However, it is possible that polyspecific IgM is not adequately registered by our 'functional' phage-inactivation assays. In this study, we resolve the issue of C-IgM specificity by directly characterizing the binding reactivity of normal serum IgM with phage-displayed C-terminal peptides.     

Cold urticaria associated with immunoglobulin M serum factor

A case of cold urticaria, thought to be due to IgM, is presented. Passive transfer (PT) activity was manifested in cryoglobulin-free serum and in up to 20-fold dilutions of fraction I, the IgM-containing fraction obtained by Sephadex G-200 column chromatography. PT test results were positive with serum absorbed with cold agglutinins, and with heat-treated (56 °C, 4 hr) serum. These results were negative with cryoglobulin and after absorbing the supernatant of fraction I with anti-IgM. Skin adhesion time was within 24 hr. The in vivo histamine release during cold stimulation was recorded and in this patient, antihistamine administration proved the best method to alleviate the symptoms.     

Elimination of false-positive cytomegalovirus immunoglobulin M-fluorescent-antibody reactions with immunoglobulin M serum fractions.

The cytomegalovirus fluorescent-antibody test for immunoglobline M (IgM) antibody was found positive in seven of nine infants with congenital rubella infection, in addition to eight of eight infants with confirmed cytomegalovirus infection. When the test was repeated on IgM fractions of the same sera freed from IgG by ultracentrifugation, only negative reactions were observed in those from the rubella-infected infants, whereas IgM fractions from the cytomegalovirus-infected infants remained positive.     

The separation of immunoglobulin M from human serum by fast protein liquid chromatography

A fast protein liquid chromatography (FPLC) system was evaluated as a method for rapid separation of serum immunoglobulin M (IgM) from immunoglobulin G (IgG) and immunoglobulin A (IgA). The system incorporates the use of a strong anion exchanger. Evaluation was carried out in 3 ways. The effect of increasing the serum percentage in the 500 microliters volumes loaded on to the column was tested. Samples containing up to 60% serum resulted in only small concentrations of contaminating IgG and IgA in the IgM fraction. Reproducibility was tested by fractionating the same serum sample several times; the coefficient of variation (CV) of the IgM concentration in the IgM fraction was 6%. A number of sera which varied considerably in immunoglobulin concentration were fractionated without any significant adverse effects on the immunoglobulin ratios in the IgM fraction. One serum sample containing a high concentration of IgG and IgA was included. In contrast to gel filtration chromatography, FPLC can separate IgM from IgG and IgA within 6 min. On loading 500 microliters samples containing from 20 to 60% serum, less than 0.01 g/l IgG was detected in the IgM fractions when tested by the radial immunodiffusion method.     

Enhancing the protective efficacy of Mycobacterium bovis BCG vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein

Tuberculosis continues to ravage humanity, killing 2 million people yearly. Most cases occur in areas of the world to which the disease is endemic, where almost everyone is vaccinated early in life with Mycobacterium bovis BCG, the currently available vaccine against tuberculosis. Thus, while more-potent vaccines are needed to replace BCG, new vaccines are also needed to boost the immune protection of the 4 billion people already vaccinated with BCG. Until now, no booster vaccine has been shown capable of significantly enhancing the level of protective immunity induced by BCG in the stringent guinea pig model of pulmonary tuberculosis, the "gold standard" for testing tuberculosis vaccines. In this paper, we describe a booster vaccine for BCG comprising the purified recombinant Mycobacterium tuberculosis 30-kDa protein, the major secreted protein of this pathogen. In the guinea pig model of pulmonary tuberculosis, boosting BCG-immunized animals once with the 30-kDa protein greatly increased cell-mediated and humoral immune responses to the protein in three consecutive experiments. Most importantly, boosting BCG-immunized animals once with the 30-kDa protein significantly enhanced protective immunity against aerosol challenge with highly virulent M. tuberculosis, as evidenced by a significantly reduced lung and spleen burden of M. tuberculosis compared with those for nonboosted BCG-immunized animals (mean additional reduction in CFU of 0.4 +/- 0.1 log in the lung [P = 0.03] and 0.6 +/- 0.1 log in the spleen [P = 0.002]). This study suggests that administering BCG-immunized people a booster vaccine comprising the 30-kDa protein may enhance their level of immunoprotection against tuberculosis.     

Clearance and organ distribution of Mycobacterium tuberculosis lipoarabinomannan (LAM) in the presence and absence of LAM-binding immunoglobulin M

Lipoarabinomannan (LAM) is a component of the mycobacterial surface which has been associated with a variety of deleterious effects on immune system function. Despite the importance of LAM to the pathogenesis of mycobacterial infection, there is no information available on its fate in vivo. In this study, we determined the pharmacokinetics and tissue distribution of exogenously administered LAM in mice. For measurements of serum and tissue LAM concentrations, we developed an enzyme-linked immunosorbent assay which used monoclonal antibodies of different isotypes to capture and detect LAM at concentrations of >/=0.4 microg/ml. Intravenous administration of LAM to mice resulted in transient serum levels with organ deposition in the spleen and in the liver. Immunohistochemical studies localized LAM to the spleen marginal zone macrophages and, to a lesser degree, to liver macrophages. When LAM was administered to mice previously given a LAM-binding immunoglobulin M (IgM), LAM was very rapidly cleared from circulation. In those mice, deposition of LAM in the spleen was significantly reduced while LAM deposition in the liver increased. Administration of LAM-binding IgM resulted in significant levels of IgM to LAM in bile consistent with an increased hepatobiliary excretion of LAM in the presence of specific antibody. Bile, liver extracts, and bile salts were found to rapidly inactivate the immunoreactivity of LAM. The results indicate that serum clearance and organ deposition of LAM in mice are affected by the presence of LAM-binding antibody and suggest a mechanism by which antibody could modify the course of mycobacterial infection.     

The human X-chromosome and the levels of serum immunoglobulin M

The serum concentrations of immunoglobulins G, A and M were measured in a sample of 93 pairs of monozygotic twins, their spouses, and their offspring. The hypothesis that the human X-chromosome carries genes which control the levels of immunoglobulin M was tested with three different approaches. Our results indicate that environmental factors are primarily responsible for the observed variation in the levels of IgG and IgA. The variance of IgM seems to be mostly the result of X-linked gene effects, with women having higher IgM levels than men.     

Changes in avidity and level of immunoglobulin G antibodies to Mycobacterium tuberculosis in sera of patients undergoing treatment for pulmonary tuberculosis

Much is known about specific antibodies and their titers in patients with tuberculosis. However, little is known about the avidity of these antibodies or whether changes in avidity occur during the progression of the disease or during treatment. The aims of this study were to determine the avidity of antibodies to Mycobacterium tuberculosis in patients with pulmonary tuberculosis, to explore the value of avidity determination for the diagnosis of tuberculosis, and to study changes in levels of antibodies and their avidity during treatment. Antibody avidity was measured by an enzyme-linked immunosorbent assay with thiocyanate elution. Avidity indices and serum levels of immunoglobulin G to M. tuberculosis were determined for 22 patients with pulmonary tuberculosis before and during treatment and for 24 patients with other pulmonary diseases. Antibody levels and avidity were both significantly higher in untreated tuberculosis patients than in the controls. Avidity determination had more diagnostic potential than determination of the antibody levels. Tuberculosis patients with a long duration of symptoms had higher antibody avidity than those with a recent onset of symptoms, indicating affinity maturation of specific antibodies during active disease. In the early phase of treatment, a decrease in antibody avidity was observed for 73% of all tuberculosis patients, accompanied by an initial increase in antibody levels in 36% of these patients. These phenomena could be explained by an intense stimulation of the humoral response by antigens released from killed bacteria, reflecting early bactericidal activity of antituberculous drugs leading to the production of low-affinity antibodies against these released antigens.     

Use of the hupB gene encoding a histone-like protein of Mycobacterium tuberculosis as a target for detection and differentiation of M. tuberculosis and M. bovis

The gene for histone-like protein (hupB [Rv2986c]) of Mycobacterium tuberculosis has been identified as a singular target which allows differentiation of two closely related mycobacterial species, namely, M. tuberculosis and M. bovis of the MTB complex, by a PCR assay. The N and S primer-generated PCR amplicons differed in M. tuberculosis and M. bovis; these amplicons were determined to be 645 and 618 bp, respectively. This difference was localized to the C-terminal part of the gene by using primers M and S. The C-terminal PCR amplicons of M. tuberculosis and M. bovis were determined to be 318 and 291 bp, respectively. The differences in the C-terminal portion of the gene were confirmed by restriction fragment length polymorphism analysis and sequencing. Sequence analysis indicated that in M. bovis there was a deletion of 27 bp (9 amino acids) in frame after codon 128 in the C-terminal part of the hupB gene. In the present study 104 mycobacterial strains and 11 nonmycobacterial species were analyzed for hupB gene sequences. Of the 104 mycobacterial strains included, 62 belonged to the MTB complex and 42 were non-MTB complex strains and species. Neither the hupB gene-specific primers (N and S) nor the C-terminal primers (M and S) amplify DNA from any other mycobacteria, making the assay suitable for distinguishing members of the MTB complex from other mycobacterial species, as well as for differentiating between members of the MTB complex, namely, M. tuberculosis and M. bovis.     

Impact of protein malnutrition on exogenous reinfection with Mycobacterium tuberculosis.

Malnutrition may be a predisposing host factor in the development of exogenous-reinfection tuberculosis. Outbred Hartley guinea pigs were given isocaloric diets containing either 30% ovalbumin (control animals) or 10% ovalbumin (low-protein-fed [LP] animals). Equal numbers of control and LP animals were assigned to one of three infection groups: (i) primary pulmonary infection with a low-virulence, streptomycin-resistant (LVsr) isolate of Mycobacterium tuberculosis and then reinfection 6 weeks later by the same route with a high-virulence (HV) isolate; (ii) only the primary infection (LVsr isolate); and (iii) only the secondary infection (HV isolate). Each infection resulted in the development of 4 to 12 pulmonary tubercles. Guinea pigs were skin tested with purified protein derivative and killed 6 weeks after the second infection. Protein deprivation suppressed the dermal responses to purified protein derivative in all infection groups. Primary infection of well-nourished animals with the LVsr isolate induced significant protection against infection with the HV isolate in the reinfected group, based upon the numbers of viable mycobacteria in the lung and spleen. Protein malnutrition did not exacerbate disease in the reinfected group beyond that observed in malnourished animals infected with the HV isolate only, but neither did the infection with the LVsr isolate protect the LP animals against reinfection with the HV isolate. We conclude that malnutrition interferes with the protection normally afforded by primary infection but does not result in more severe disease in reinfected individuals than would be observed in singly infected subjects.     

Therapeutic efficacy of high-dose intravenous immunoglobulin in Mycobacterium tuberculosis infection in mice

Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies and, at high doses, to treat a range of autoimmune and inflammatory disorders. With high-dose IVIg (hdIVIg), immunomodulatory mechanisms act on a range of cells, including T cells, B cells, and dendritic cells. Here, we demonstrate that the treatment of M. tuberculosis-infected mice with a single cycle of hdIVIg resulted in substantially reduced bacterial loads in the spleen and lungs when administered at either an early or late stage of infection. Titration of the IVIg showed a clear dose-response effect. There was no reduction in bacterial load when mice were given equimolar doses of another human protein, human serum albumin, or maltose, the stabilizing agent in the IVIg preparation. HdIVIg in vitro had no inhibitory effect on the growth of M. tuberculosis in murine bone marrow-derived macrophages. In addition, the effect of hdIVIg on bacterial loads was not observed in nude mice, suggesting the involvement of conventional T cells. Analysis of T cells infiltrating the lungs revealed only small increases in CD8(+) but not CD4(+) T-cell numbers in hdIVIg-treated mice. The mechanism of action of hdIVIg against tuberculosis in mice remains to be determined. Nevertheless, since hdIVIg is already widely used clinically, the magnitude and long duration of the therapeutic effect seen here suggest that IVIg, or components of it, may find ready application as an adjunct to therapy of human tuberculosis.     

Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle

The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.     

Genetic biodiversity of Mycobacterium tuberculosis complex strains from patients with pulmonary tuberculosis in Cameroon

We analyzed DNA polymorphisms in 455 Mycobacterium tuberculosis complex isolates from 455 patients to evaluate the biodiversity of tubercle bacilli in Ouest province, Cameroon. The phenotypic and genotypic identification methods gave concordant results for 99.5% of M. tuberculosis isolates (413 strains) and for 90% of Mycobacterium africanum isolates (41 strains). Mycobacterium bovis was isolated from only one patient. Analysis of regions of difference (RD4, RD9, and RD10) proved to be an accurate and rapid method of distinguishing between unusual members of the M. tuberculosis complex. Whereas M. africanum strains were the etiologic agent of tuberculosis in 56% of cases 3 decades ago, our results showed that these strains now account for just 9% of cases of tuberculosis. We identified a group of closely genetically related M. tuberculosis strains that are currently responsible for >40% of smear-positive pulmonary tuberculosis cases in this region of Cameroon. These strains shared a spoligotype lacking spacers 23, 24, and 25 and had highly related IS6110 ligation-mediated (LM) PCR patterns. They were designated the "Cameroon family." We did not find any significant association between tuberculosis-causing species or strain families and patient characteristics (sex, age, and human immunodeficiency virus status). A comparison of the spoligotypes of the Cameroon strains with an international spoligotype database (SpolDB3) containing 11,708 patterns from >90 countries, showed that the predominant spoligotype in Cameroon was limited to West African countries (Benin, Senegal, and Ivory Coast) and to the Caribbean area.     

Persistence of rubellavirus-specific immunoglobulin M and immunoglobulin A antibodies: investigation of successive serum samples with lowered immunoglobulin G concentration.

The persistence of rubellavirus-specific immunoglobulin (Ig) M and IgA antibodies has been studied in seven patients with primary postnatal rubella infections. Successive blood samples obtained over a period of several years after the onset of disease have been investigated, employing the fluorescent antibody technique and the sucrose gradient centrifugation method. IgM antibodies were found to persist for 4 to 5 weeks after the onset of disease, with only moderate variation being observed with respect to the different patients and the method of investigation being studied. The persistence of IgA antibodies (as examined by the fluorescent antibody technique) varied from a few weeks to several years after the onset of the disease. The detection of IgA antibodies cannot be considered as conclusive for the diagnosis of recent rubella infections.     

Interpreting the results of the amplified Mycobacterium tuberculosis direct test for detection of M. tuberculosis rRNA

The Amplified Mycobacterium tuberculosis Direct (AMTD) test detects M. tuberculosis rRNA. By using culture of M. tuberculosis as a gold standard, a number of different diagnostic indices were examined in an attempt to determine the diagnostic performance of the AMTD test and demonstrate how it might usefully be interpreted during the early management of disease.     

Spoligotyping of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mumbai, India

Tuberculosis remains a major health problem in India, with 2 million new cases and 421,000 deaths each year. In this paper, we describe the spoligotyping results of 216 Mycobacterium tuberculosis culture isolates from patients with pulmonary tuberculosis in Mumbai, India. As spoligotyping data from India have rarely been described until now, and as there is limited information on the major circulating clades of M. tuberculosis, the data obtained were also compared to an international spoligotype database (SpolDB4) that contained patterns from 22,546 isolates from more than 100 countries. Eighty-four (39%) of the isolates were definitively marked as orphan strains, indicating the paucity of such data from India. The remaining 132 isolates clustered among 59 shared types; among these, 42 shared types were already present in the database, 17 were newly created, and 5 of them were specifically reported from Mumbai. A total of 9 major types in this study clustered 32% of the isolates. At the phylogenetic level, 30% of the isolates belonged to the Central Asian families CAS1 and CAS2, of the major genetic group (MGG) 1, 29% to MGG 2 and 3 families (spacers 33-36 missing) and 17% to the ancestral East African Indian (EAI) family. Finally, nearly 10% of the isolates belonged to the W-Beijing family in a broad sense, also in the MGG 1 group. In conclusion, historic clones of the MGG 1 group of M. tuberculosis are responsible for roughly 60% of all tuberculosis cases in Mumbai. Together with the fact that organisms presumably of European descent (such as the Haarlem family) were only rarely found, our observations suggest that tuberculosis in Mumbai, India is essentially caused by historical clones of tubercle bacilli undergoing active circulation due to uncontrolled demography, high prevalence of the disease, and a paucity of resources.