The immune responses in tuberculosis: role for pathogenesis, diagnosis and prevention.
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BCG Vaccine↗
Biomedical subjects
P H Lagrange
Publications and source records attributed to P H Lagrange.
Characterisation of CMY-4, an AmpC-type plasmid-mediated beta-lactamase in a Tunisian clinical isolate of Proteus mirabilis.
A strain of Proteus mirabilis resistant to beta-lactams, including cefoxitin, was isolated from the urine of a woman from Tunisia. Its antibiotic susceptibility pattern and that of the Escherichia coli transconjugant suggested the presence of an AmpC-type beta-lactamase. Two bands of beta-lactamase activity (pI 5.4 and 9.2) were detected by isoelectric focusing. The nucleotide sequence of the gene encoding the AmpC-type enzyme was determined. The deduced amino acid sequence was 98-99% identical to CMY-3 and to those of the plasmid-mediated AmpC-type beta-lactamases originated from Citrobacter freundii and 97% identical to the chromosome-encoded beta-lactamase of a Tunisian clinical isolate of C. freundii. This enzyme differs from CMY-2 by one substitution (Arg for Trp at position 221) and from CMY-3 by two substitutions (Glu for Gly at position 42 and Ser for Asn at position 363) and we propose the denomination CMY-4.
Aged↗
IgG subclass distribution of antibody responses to protein and polysaccharide mycobacterial antigens in leprosy and tuberculosis patients.
Immunoenzymatic assays were developed for the measurement of antibodies against mycobacterial lipoarabinomannan (LAM), a cell-free proteic extract (CFX) of Mycobacterium leprae, and the 38-kD protein antigen of M. tuberculosis. Sera from 108 leprosy patients, belonging to all clinical-immunological forms of the spectrum, and 81 patients with localized or disseminated tuberculosis (TB) were tested for antibodies of the four IgG subclasses. Standard calibration curves were used to allow comparisons between results of different isotypes and specificities. Mean concentrations of total IgG antibodies were higher in the overall leprosy population than in TB patients. In leprosy, levels of anti-CFX increased from tuberculoid toward lepromatous forms, with a clear switch from IgG1 to IgG2 subclass predominance. A similar IgG1 to IgG2 conversion was observed in anti-LAM antibodies, although total levels of anti-LAM were similar in patients with tuberculoid and lepromatous forms. In TB, antibodies against polysaccharide and protein antigens were both predominantly of IgG1 subclass, whatever the patient's clinical status, although lower in disseminated forms, probably due to concomitant HIV infection. A hypergammaglobulinaemia was also found in most leprosy and TB patients. In TB this was due to increased IgG1 and IgG3, especially in HIV co-infected patients. Based on the current knowledge of the influence of T cell-secreted cytokines on human immunoglobulin isotype expression, these results do not fit with a putative role of Th1 (such as found in TB and tuberculoid leprosy (TT)) and Th2 (such as found in leprosy lepromatous (LL) leprosy) environment in the isotypy of antibody responses in mycobacterial infections. Nor do variations of isotypy according to pathological conditions seem to be related to the biochemical nature of antigens, since antibodies to LAM and protein antigens had comparable evolutions of their subclass distribution. Other factors are to be investigated in order to understand better the significance and possible roles of antibodies in mycobacterial diseases.
Adolescent↗
Susceptibility to leprosy is linked to the human NRAMP1 gene.
Leprosy is a debilitating infectious disease of human skin and nerves. Genetic factors of the host play an important role in the manifestation of disease susceptibility. The human NRAMP1 gene is a leprosy susceptibility candidate locus since its murine homologue Nramp1 (formerly Lsh/Ity/Bcg) controls innate resistance to Mycobacterium lepraemurium. In this study, 168 members of 20 multiplex leprosy families were genotyped for NRAMP1 alleles and 4 closely linked polymorphic markers. Highly informative haplotypes overlapping the NRAMP1 gene were constructed, and the haplotype segregation into leprosy-affected offspring was analyzed. It was observed that the segregation of NRAMP1 haplotypes into affected siblings was significantly nonrandom. This finding is consistent with the hypothesis that NRAMP1 itself is a leprosy susceptibility locus.
Alleles↗
Human immunodeficiency virus infection alters antigen-induced cytokine responses in patients with active mycobacterial diseases.
Peripheral blood cells from 29 patients with active Mycobacterium avium (MAC) or Mycobacterium tuberculosis diseases were tested for mycobacterial antigen-induced interferon (IFN)-gamma and interleukin (IL)-4 production. Among MAC patients, human immunodeficiency virus (HIV) infection was associated with an 80% decrease in those who produced IFN-gamma, resulting in a predominantly type 2 cytokine profile. HIV infection in patients with tuberculosis correlates with a 37% increase in those producing IL-4 and a type 1 to type 0 profile shift. These qualitative changes were independent of CD4+ or CD8+ cell numbers. The amounts of both IFN-gamma and IL-4 were decreased in the HIV-infected population. Quantitative reduction of IFN-gamma was the result of fewer secreting cells rather than a down-regulation at the single-cell level. Disseminated disease was restricted to 2 of 5 HIV-infected MAC patients with a type 2 cytokine profile and 4 of 5 HIV-infected tuberculosis patients with a type 0 profile. These results demonstrated a shift in mycobacterial antigen-specific cytokine profiles from type 1 to type 0 and to type 2, in parallel with AIDS progression.
AIDS-Related Opportunistic Infections↗
Characterization of FOX-3, an AmpC-type plasmid-mediated beta-lactamase from an Italian isolate of Klebsiella oxytoca.
Klebsiella oxytoca 1731, which showed a wide spectrum of resistance to beta-lactams, including cefoxitin, was isolated in 1994 from a patient in Genoa, Italy. This strain contained a plasmid-mediated AmpC beta-lactamase with a pI of 7.25. Sequencing of the corresponding DNA of K. oxytoca 1731 revealed 96 and 97% identities of the deduced amino acid sequence with FOX-1 and FOX-2, respectively.
Amino Acid Sequence↗
Salmonella enteritidis: AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii.
DHA-1, a plasmid-mediated cephalosporinase from a single clinical Salmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable to Escherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coli JM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98. 7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC and ampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampG gene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type beta-lactamase, DHA-1, probably originated from M. morganii.
Amino Acid Sequence↗
An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon.
A survey of an emerging tuberculosis epidemic among the Yanomami Indians of the Amazonian rain forest provided a unique opportunity to study the impact of tuberculosis on a population isolated from contact with the tubercle bacillus for millennia until the mid-1960s. Within the Yanomami population, an extraordinary high prevalence of active tuberculosis (6.4% of 625 individuals clinically examined) was observed, indicating a high susceptibility to disease, even among bacille Calmette-Guérin-vaccinated individuals. Observational studies on cell-mediated and humoral immune responses of the Yanomami Indians compared with contemporary residents of the region suggest profound differences in immunological responsiveness to Mycobacterium tuberculosis infection. Among the Yanomami, a very high prevalence of tuberculin skin test anergy was found. Of patients with active tuberculosis, 46% had purified protein derivative of tuberculosis reactions <10 mm; similarly 58% of recent bacillus Calmette-Guérin vaccines exhibited skin test reactions <5 mm. The Yanomami also had higher titers of antibodies against M. tuberculosis glycolipid antigens (>70%) than the control subjects comprised of Brazilians of European descent (14%). The antibodies were mostly of the IgM isotype. Among the tuberculosis patients who also produced IgG antibodies, the titers of IgG4 were significantly higher among the Yanomami than in the control population. Although it was not possible to analyze T-cell responses or patterns of lymphokine production in vitro because of the remoteness of the villages from laboratory facilities, the results suggest that the first encounter of the Yanomami Indian population with tuberculosis engenders a diminished cell-mediated immune response and an increased production antibody responses, relative to other populations with extensive previous contact with the pathogen. These findings suggest that tuberculosis may represent a powerful selective pressure on human evolution that over centuries has shaped the nature of human immune responses to infection.
Antibodies, Bacterial↗
Circulating immune complexes in human tuberculosis sera: demonstration of specific antibodies against Mycobacterium tuberculosis glycolipid (DAT, PGLTb1, LOS) antigens in isolated circulating immune complexes.
An enzyme-linked immunosorbent assay (ELISA) for IgG using three glycolipid antigens from Mycobacterium tuberculosis in 65 tuberculosis (TB) patients and 50 healthy control subjects was performed. The circulating immune complexes (CICs) were isolated by precipitation with polyethylene glycol 6000 (PEG). This method associated to ELISA measured the specific antibodies present in these CICs. PEG [optical density (OD) 280] was shown to be significantly elevated (P < 0.001) in tuberculous samples. The concentrations of IgG antibodies complexed to the three glycolipid antigens were shown to be higher in patient with tuberculosis than in normal control subjects (P < 0.001). No correlation was observed between levels of free and CIC-bound antibodies. These antibodies isolated from CICs were responsible for almost all of the false-negative serological results. However, great heterogeneity was noticed depending on the antigen used, showing a more positive ELISAs against DAT (77%) than against LOS (71%) or PGLTb1 (18.5%). No correlation was established between the presence of specific CIC-complexed IgG and the bacteriological load or the tuberculosis localization (pulmonary vs. extrapulmonary). The sensitivity of ELISA for CIC-complexed IgG to DAT and LOS was lower in HIV-infected TB patients. From these results, we conclude that detection of complexed IgG DAT and LOS glycolipid antigen will be useful as a complementary technique for the serodiagnosis of tuberculosis.
Adult↗
Recycling of ELISA plates for the serological diagnosis of tuberculosis using a Mycobacterium tuberculosis-specific glycolipid antigen.
A Mycobacterium tuberculosis-specific glycolipid antigen (diacyl-trehalose-DAT) and a battery of defined sera from tuberculous patients were used to evaluate the reliability of recycling ELISA microplates for serodiagnosis of tuberculosis. The reuse of plates was evaluated by the same ELISA technique before and after recycling with an acid buffer to dissociate and release antibodies from antigen-antibody complexes fixed on used plates. The correlation coefficients for comparisons between several new plates used on different days were always higher than r = 0.90, and the variation between tests was always lower than 12%. For comparisons performed systematically between new plates and recycled plates, the values were variable. Up to the fifth recycling, the correlation coefficient was higher than r = 0.60. From the sixth recycling, correlation coefficient values fell to r = 0.381. The results obtained with recycled plates washed manually were much poorer than those obtained after automatic washing. The results described in this report demonstrate that an ELISA test can be performed on recycled plates under the conditions described.
Enzyme-Linked Immunosorbent Assay↗
Detection of SHV-5 extended-spectrum beta-lactamase in Klebsiella pneumoniae strains isolated in Italy.
Thirty-five Klebsiella pneumoniae strains isolated during 1993-1994 in intensive care units of a large Italian hospital were examined for the presence of extended-spectrum beta-lactamases. Five strains showed a high level of simultaneous resistance to beta-lactam agents, including ceftazidime and aztreonam, conferred by a large (130 kb) self-transferable plasmid (in 4 of 5 strains). Isoelectrofocusing and hybridisation studies suggest that these enzymes can be identified as SHV-5 extended-spectrum beta-lactamases. Pulsed-field get electrophoresis analysis showed three different genomic fingerprinting profiles, while plasmid restriction enzyme digestion revealed three different patterns, demonstrating that the diffusion of SHV-5 beta-lactamase is not the result of a single strain or plasmid dissemination.
Conjugation, Genetic↗
In-vitro susceptibility of Klebsiella oxytoca strains to 13 beta-lactams in the presence and absence of beta-lactamase inhibitors.
The susceptibility of Klebsiella oxytoca isolates was tested by an agar diffusion method (167 strains collected in six countries) and an agar dilution method (38 strains). Multivariate analysis of inhibition zone diameters by principal component analysis clearly individualised four susceptibility patterns, including the phenotype of strains overproducing beta-lactamase and resistant to penicillins, first-generation cephalosporins, cefuroxime and aztreonam, but susceptible to ceftazidime. This phenotype was different from that conferred by plasmid-mediated extended-spectrum beta-lactamases; strains expressing these enzymes were also resistant to ceftazidime and cefotaxime. The bla(oxy) gene from K. oxytoca was introduced into Escherichia coli and K. oxytoca recipients and conferred increased resistance to beta-lactams in the recipient cells. Clavulanic acid was effective in association with piperacillin (MIC decreased 36-fold), ceftriaxone (35-fold) and aztreonam (19-fold) against overproducing strains, in spite of a relatively high IC50 (0.3 microM). Sulbactam (IC50, 400 microM) was ineffective in this context when combined with piperacillin (MIC decreased 1.5-fold), ceftriaxone (1.6-fold) and aztreonam (1.6-fold). The inhibitory activity of tazobactam (IC50, 8.2 microM) was heterogeneous depending on the strain and the beta-lactam with which it was combined. When combined with piperacillin or ceftriaxone little potentiation in antibiotic activity occurred (MIC decreased 3.9-fold and 4.5-fold, respectively); however, tazobactam plus aztreonam resulted in a 50-fold decrease in MIC of antibiotic.
Enzyme Inhibitors↗
Interaction of macrolides with alpha dornase during DNA hydrolysis.
Since patients with cystic fibrosis are often treated with alpha dornase to reduce sputum viscosity, and because of preliminary reports of efficacy of long-term low-dose erythromycin therapy in chronic airway diseases, it is likely that alpha dornase and macrolides might be given together in such patients. A possible interaction between these drugs was therefore investigated. Using hyperchromic effect to quantify alpha dornase activity, a time- and dose-dependent inhibitory effect on human DNA hydrolysis has been observed for erythromycin, roxithromycin and azithromycin. Inhibitory doses 50% for alpha dornase were graphically determined. Azithromycin exhibited the strongest inhibitory effect.
Azithromycin↗
Chromosomal beta-lactamase genes of Klebsiella oxytoca are divided into two main groups, blaOXY-1 and blaOXY-2.
The chromosomally encoded beta-lactamase gene (blaOXY-2) of the wild-type Klebsiella oxytoca SL911 was cloned and sequenced. Its nucleotide sequence similarity with the previously sequenced K. oxytoca beta-lactamase gene (blaOXY-1) (Y. Arakawa, M. Ohta, N. Kido, M. Mori, H. Ito, T. Komatsu, Y. Fujii, and N. Kato, Antimicrob. Agents Chemother. 33:63-70, 1989) is 87.3%, and its amino acid similarity is 89.7%. This group of K. oxytoca beta-lactamases is related to chromosomal beta-lactamases of Citrobacter diversus, Proteus vulgaris, and Yersinia enterocolitica and to the plasmid-mediated extended-spectrum beta-lactamases MEN-1 and Toho-1. By colony hybridization with 86 strains susceptible and resistant to aztreonam, isolated in six countries, K. oxytoca beta-lactamase genes hybridized with either a specific blaOXY-1 DNA probe (668 bp) or a blaOXY-2 DNA probe (723 bp). Thus, beta-lactamase genes could be divided into two groups: blaOXY-1 (47% of the strains) and blaOXY-2 (53% of the strains). A study of isoelectric points confirmed the great variability reported in the literature. However, the two beta-lactamase groups were each represented by four different pIs: for OXY-2, 5.2, 5.7, 6.4, and 6.8, with the 5.2 form representing 59% of all OXY-2 enzymes, and for OXY-1, 7.1, 7.5, 8.2, and 8.8, with the 7.5 form representing 88% of all OXY-1 enzymes.
Amino Acid Sequence↗
beta-lactamase gene promoters of 71 clinical strains of Klebsiella oxytoca.
beta-Lactamase gene promoters of 45 clinical Klebsiella oxytoca isolates resistant to beta-lactams and exhibiting beta-lactamase hyperproduction differed from those in 26 susceptible strains. Direct sequencing revealed one mutation in either the -10 or -35 conserved sequences: a G-to-A transition of the fifth base (67%) or a G-to-T transversion of the first base of the -10 sequence (27%) or a T-to-A transversion in the fourth base in the -35 sequence (4%). One strain carried both the -10 transition and the -35 transversion.
Base Sequence↗
[The genetic susceptibility to leprosy in humans].
The capacity of certain individuals to resist certain diseases, including leprosy, has for a long time been considered as being influenced by genetic factors. The clinical and pathological spectrum of leprosy, epidemiological heterogeneity, both geographic and ethnic, in the prevalence of polar forms, may be explained by genetic differences in host resistance. While the specific genes in question have not been identified, recent studies suggest a genetic basis for differences in the capacity of macrophages in the host to reduce bacterial multiplication. Experimental models analyzing the reactions of antimycobacterial defence have underscored at existing differences in resistance or vulnerability to infection (M. bovis, BCG, M. lepraemurium, M. tuberculosis) were guided by a dominant gene which exists in two allelic forms, bcgr and bcg5. The bcgr allele confers resistance and is more dominant than the bcgs allele which represents greater vulnerability to infection. The murine candidate gene for the bcg gene has been named NRAMP (Natural Resistance-associated Macrophage Protein). Even though the exact function of NRAMP is not currently known, it has been demonstrated that this gene is expressed mainly in macrophages, and that it brings about increased bacteriostatic capacity in these cells. NRAMP is structurally homologous to the family of membranous proteins having a transport function linking ATP. NRAMP is similar to the membranous bacterial system transporting nitrites. The NRAMP protein is also involved as a signal of transduction during the activation of macrophages. It is therefore possible to conceive of genetic polymorphism at this locus intervening in specific and non-specific immune responses to infection. Apart from such potential polymorphism during the initial phase of infection, immunogenetic studies suggest that the polymorphism of class II HLA molecules could intervene in the evolution of secondary immune response to M. leprae. Knowing that HLA molecules are expressed in a co-dominant form, and attributing extraordinary allelic polymorphism to this locus, there may be a rather wide range of immune responses to the M. leprae antigens in subjects with discordant HLA and in populations which have varied genetic profiles. In general it has been acknowledged that HLA-DR isotypes are associated with protective response, while HLA-DQ isotypes are said to be associated with multibacillary lepromatous forms. The chief role of the HLA systems controlling cell-mediated immunity leads to the probability that differences in HLA haplotypes could contribute to the wide spectrum of immune responses observed in leprosy. Genetic determinants of resistance to leprosy cannot be described in a straightforward manner using a classic approach because the complex mechanisms of resistance, yet to be clarified and for which at least two loci are believed to be contributory, may be re-assessed like a multifactorial, multigenetic complex in which environmental events linked to the transmission of M. leprae, its duration, intensity and host factors, varying as a function of time, intervene. A close study of each element and better understanding of the physiological and pathological mechanisms of infection and disease are necessary in order to state the influence of genetic factors on each of them with greater precision.
Adenosine Triphosphate↗
Molecular characterisation by PCR-restriction fragment length polymorphism of TEM beta-lactamases.
To rapidly characterise TEM-derived extended-spectrum beta-lactamases a fast and easy method using polymerase chain reaction-restriction fragment length polymorphism was developed. This method was validated with ten reference TEM-type extended-spectrum beta-lactamases. The mutations involved in TEM-20 and TEM-21, which were previously reported only with biochemical analysis, were then characterised. TEM-20 differed from TEM-19 by a silent mutation at position 925 (A for G), and TEM-21 differed from TEM-3 and TEM-14 by a single mutation (G for A) in an unreported position 660. beta-lactamase conferring low resistance to ceftazidime (TEM-29), was described. TEM-29 derived from TEM-1, with an amino acid substitution, his-164. Finally, the combination of polymerase chain reaction-restriction fragment length polymorphism and plasmid analysis allowed us to investigate nosocomial outbreaks due to clinical isolates of multi-resistant Klebsiella pneumoniae in three hospitals.
Cross Infection↗
Complex segregation analysis of leprosy in southern Vietnam.
To investigate the nature of the genetic component controlling susceptibility to leprosy and its subtypes, 402 nuclear families were ascertained through a leprosy patient followed at the Dermatology Hospital in Ho Chi Minh City, Vietnam; 285 families were of Vietnamese origin and 117 were of Chinese origin with a higher proportion of lepromatous forms among Chinese patients. Segregation analyses were conducted using the model developed by Abel and Bonney [(1990) Genet Epidemiol 7:391-407], which accounted for variable age of onset and time-dependent covariates. Three phenotypes were considered: leprosy per se (all forms of leprosy together), nonlepromatous leprosy, and lepromatous leprosy. For each of this phenotype, analyses were performed on the whole sample and separately on the Vietnamese and the Chinese families. The results showed that a single Mendelian gene could not account for the familial distributions of leprosy per se and its two subtypes in the whole sample. However, these results were different according to the ethnic origin of the families. In the Vietnamese subsample, there was evidence for a codominant major gene with residual familial dependences for the leprosy per se phenotype, and borderline rejection of the Mendelian transmission hypothesis for the nonlepromatous phenotype. In Chinese families, strong rejection of Mendelian transmission was obtained in the analysis of leprosy per se, and no evidence for a familial component in the distribution of the nonlepromatous phenotype was observed. For the lepromatous phenotype, the discrimination between models was poor, and no definitive conclusion could be reached. Referring to immunological data, we suggest that these results could be explained by a heterogeneity in the definition of the lepromatous phenotype. It is likely that progress in the understanding of the genetic components involved in the expression of leprosy will come from a better definition of the phenotype under study, and immunological studies are ongoing in this population to investigate this hypothesis.
Adolescent↗