Objective: Evaluate the Monocyte Locomotion Inhibitory Factor (MLIF) effect upon the expression o... more Objective: Evaluate the Monocyte Locomotion Inhibitory Factor (MLIF) effect upon the expression of genes encoding human cytokines, receptors and related factors in the human cell line U-937. MLIF (Met-Gln-Cys-Asn-Ser) is an anti-infl ammatory pentapeptide produced by Entamoeba histolytica that inhibits many human monocyte functions. Material and Methods: U-937 cell line cultured (24 hrs/ RPMI). RNA extracted by Trizol method. 385 genes were analyzed on microarray membranes, complement by real-time RT-PCR and protein expression of some affected genes. Results: MLIF had a preferentially inhibitory effect on gene expression; four genes were over-expressed and 13 underexpressed in MILF vs. simple medium -constitutive expression. Three genes are over-expressed and 19 under-expressed in MLIF/PMA vs. PMA -induced expression. Conclusions: Many modifi ed genes are products regulated by the Nuclear Factor-κB and Mitogen Activated Protein Kinase pathways, suggesting MLIF involvement with these two major pathways for the modulation of the infl ammation and immune responses.
I. Estrada-García). Tuberculosis (2008) 88, 212-220 modified cytokine, TLR and MHC class II expre... more I. Estrada-García). Tuberculosis (2008) 88, 212-220 modified cytokine, TLR and MHC class II expression. These findings may help to unravel the complex mechanisms used by virulent mycobacteria to evade and subvert the immune response.
Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often char... more Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often characterized by immunological dysfunction of peripheral blood mononuclear cells, especially at chronic stages. Lipids from the Mycobacterium tuberculosis cell wall have been shown to produce various suppressive effects on cell-mediated immunity. The cell-surface lipid di-O-acyl-trehalose (DAT) is able to inhibit T-cell proliferation and cytokine secretion in cells from naïve mice. In the present study, we addressed the mechanisms involved in the suppressive effect caused by DAT. We found that DAT decreased the proliferation of spleen cells induced with PMA-ionomycin, suggesting that the suppressive mechanisms target intracellular functions just after phospholipase C-g activation. Addressing this possibility, the effect of DAT was found to involve down-modulation of the di-acyl glycerol-dependent activation of the MAPK-ERK1/2 pathway, one of the crucial signaling pathways leading to adaptive cell immune response against TB. Moreover, the inhibitory effect of DAT on proliferation was reproduced in antigen-stimulated T cells from M. tuberculosis-infected mice, involving the lowering of Th1-type cytokine transcription levels. The present findings thus reveal a new kind of bioactivity for a long-known M. tuberculosis cell wall lipid, DAT.
Background: Methylotrophs are a diverse group of bacteria that can utilize single-carbon compound... more Background: Methylotrophs are a diverse group of bacteria that can utilize single-carbon compounds as a sole energy source, and are often catalase-positive. Known as environmental symbionts, they are emerging as disease-causing organisms in patients with CGD. Methods: We present a case of lymphadenitis due to Granulibacter bethesdensis, a facultative methylotroph, and review 8 other infections caused by methylotrophs in patients with CGD. Results: There have been 9 reported cases of infections due to methylotrophs in patients with CGD. Seven cases were due to G. bethesdensis, one was due to Acidomonas methanolica and one was due to a Methylobacter. In all cases, 16s rRNA gene sequencing was required for diagnosis. Conclusions: Methylotrophs are fastidious and difficult to identify. Although the mechanisms underlying susceptibility to infection with methylotrophs in CGD remain to be elucidated, these bacteria should be included in the spectrum of pathogens associated with infections ...
The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned ... more The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned from a lambda gtll clone into the high-copy-number plasmid pUC8. Escherichia coli containing these recombinants produced large amounts of the antigen, which was purified by polyacrylamide gel electrophoresis in the presence of urea. The ability of E. coli to recognize the mycobacterial promoter was confirmed by constructing additional clones in which the gene is flanked by transcriptional terminators from phage fd. A similar approach was used to demonstrate the expression of this gene in Streptomyces lividans. Mice immunized with killed M. leprae showed cell-mediated immune reactivity to the purified 65-kilodalton protein which stimulated both in vitro lymphoproliferative and in vivo delayed-type hypersensitivity responses.
International Journal of Leprosy and Other Mycobacterial Diseases
We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide ... more We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide anion)] by murine peritoneal macrophages challenged in vitro with Mycobacterium lepraemurium (MLM), complement-opsonized yeast, M. bovis BCG, M. phlei, or phorbol myristate acetate (PMA). We found that except for MLM, all of the other materials provoked the release of significant amounts of hydrogen peroxide and superoxide. MLM entered the macrophages without triggering their oxidative metabolism. Pre-infection of macrophages with MLM did not alter these cells' capacity to release the normal amounts of ROI in response to other microorganisms or PMA. Killing of MLM did not revert the macrophages' failure to release ROI upon ingestion of the microorganism, nor were macrophages able to produce these toxic metabolites when pre-incubated in the presence of murine gamma interferon (IFN-gamma). MLM has several attributes that allow it to survive within macrophages: a) it is a nontoxigenic microorganism (it does not harm its host), b) it resists the harsh conditions of the intraphagolysosomal milieu (a property perhaps dependent on its thick lipidic envelope), and c) it penetrates the macrophages without triggering their oxidative response (thus avoiding the generation of the toxic intermediaries of oxygen). For these attributes (and others discussed in this paper), we recognize MLM as a highly evolved, well-adapted parasite of macrophages. In addition, the results of the present study prompted the analysis of the biochemical pathways used by MLM and M. bovis BCG to penetrate into their cellular hosts, a subject now under investigation in our laboratory.
This review focuses on the principal strategies for conviviality in mice parasite relationships, ... more This review focuses on the principal strategies for conviviality in mice parasite relationships, specially in those genetically determined, and discussed as: a) Innate susceptibility of the host to the parasite; b) Specificity of the parasite for the host, and c) Susceptibility derived from genetic interactions between host and parasite. It is concluded that susceptibility is regulated by multiple genetic facts from both host and parasite, which can interact and modify each other, making each instance of host-parasite relationship unique.
... ALBERT0 A. PALACIOS-BOIX,* IRIS ESTRADA-G,' M. JOSEPH COLSTON,&a... more ... ALBERT0 A. PALACIOS-BOIX,* IRIS ESTRADA-G,' M. JOSEPH COLSTON,' AND GABRIEL S. PANAY12* From the *Rheumatology Unit, Dfuision of Medicine. Guy's Hospftal, and the tLaboratory for Leprosy and Mycobacterial Research, National Instftute for Medical Research. ...
International Journal of Leprosy and Other Mycobacterial Diseases
In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segm... more In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segments of the amino acid sequence of proteins from Mycobacterium leprae: 65 kDa, 28 kDa, 18 kDa, and 28 kDa superoxide dismutase, recently renamed antigens 2L, 9L, 12L, and 14L, respectively. They were assayed in the guinea pig model for the induction of a delayed-type hypersensitivity response in M. leprae and BCG-sensitized animals. To sensitize the animals two schemes were used: either a single dose of 5 x 10(9) irradiated or autoclaved whole bacilli, or four weekly intramuscular injections each containing 500 micrograms of soluble extract of M. leprae (MLSE) in incomplete Freund's adjuvant. Because the second scheme used far too much antigen, we decided to use the first scheme for the experiments we report here. DTH reactions of sensitized animals were induced after 30 days with intradermal injections of 5 micrograms of MLSE and with each of the 10 peptides at three different concentrations: 250 micrograms, 100 micrograms, and 0.05 micrograms. All M. leprae-sensitized guinea pigs gave indurations of 10 mm or more with MLSE, which indicates that the animals were sensitized. None of them gave DTH indurations with 250 micrograms or 100 micrograms, but some of them had positive DTH reactions with the 0.05 micrograms doses of the synthetic peptides. This is most likely due to the fact that we have used an outbred strain of guinea pigs. The peptides were also tested at 0.05 micrograms in animals sensitized with BCG. P7 and P10 seem to be nonspecific peptides; the remaining peptides only induced DTH in the M. leprae-sensitized guinea pigs. P3 (segments 65-85 of the 65-kDa protein) induced a positive DTH in 58% of the tested animals. In other experiments, guinea pigs were sensitized with a single injection (500 micrograms) of each of the synthetic peptides. All animals, except those sensitized with P4 and P8, had positive DTH responses when the homologous peptide was used. Those sensitized with P2, P4, P5, P7, and P8 were able to produce indurations when MLSE was used for the induction of the DTH reaction.
We have investigated changes in gene expression in mouse peritoneal macrophages following infecti... more We have investigated changes in gene expression in mouse peritoneal macrophages following infection with virulent Mycobacterium tuberculosis. Using differential-display reverse transcription-PCR (RT-PCR), we have identified a gene that was markedly down-regulated within 6 h of infection and remained so for the duration of the experiment (5 days). On sequencing, this gene was found to encode the murine cytochrome c oxidase subunit VIIc (COX VIIc). Down-regulation of COX VIIc during M. tuberculosis infection was confirmed by three independent techniques: limiting-dilution RT-PCR, RNase protection assay, and Northern analysis. Limitingdilution RT-PCR and Northern analysis were also used to analyze the specificity of this regulation; heat-killed M. tuberculosis, Mycobacterium bovis BCG, and latex beads had no effect on expression of COX VIIc. Downregulation of this enzyme was also confirmed by using adherent cells isolated from spleens of M. tuberculosisinfected mice. These ex vivo macrophages showed apoptotic features, suggesting a possible involvement of cytochrome c oxidase in the programmed cell death of the host cells.
To classify Mycobacterium leprae isolates from multiple areas in Mexico based on variable number ... more To classify Mycobacterium leprae isolates from multiple areas in Mexico based on variable number of tandem repeats of 6 base within the rpoT gene and three single nucleotide polymorphism (SNP), and to analyse their geographic distribution in the context of the origin of leprosy in Mexico. Analysis for rpoT genotyping of 64 samples collected in the west and southwestern areas revealed that 46 isolates were of the 4 copy type and 18 isolates were of the 3 copy type. All samples from the eastern coastal area (n = 24) and from the Yucatan peninsula (n = 12) were of the 3 copy type. Six isolates from the west and southwestern area were SNP-type 1, 13 isolates were SNP-type 2 and 45 isolates were SNP-type 3. Nineteen of 24 isolates from the eastern coastal area were SNP-type 3 and one was SNP-type 4. Seven isolates from the Yucatan peninsula were SNP-type 3 and one was SNP-type 4. The difference of the proportion of each genotype between the western areas and the eastern areas indicated the expansion of leprosy through different paths in Mexico.
Introducción: Los linfocitos T juegan un papel importante en la inmunopatogénesis de la queratiti... more Introducción: Los linfocitos T juegan un papel importante en la inmunopatogénesis de la queratitis estromal herpética (QEH). Se ha demostrado, en modelos animales, que los linfocitos T infiltran la córnea y producen grandes cantidades de IFN-γ En contraste, la respuesta inmune en humanos no ha sido estudiada ampliamente. Objetivo: Evaluar el perfil de citocinas en células mononucleadas de sangre periférica (CMN) de pacientes con QEH. Métodos: Se determinó en CMN de pacientes (p) con QEH e individuos sanos (IS) la expresión de CD4, CD8, citocinas intracelulares (IFN-γ/IL-4) y citocinas proinflamatorias (TNF-α/IL-6/IL-1β) en sobrenadantes de cultivo (SN) antes/después del estímulo policlonal. Los resultados fueron obtenidos por citometría de flujo y ELISA. Resultados: Porcentaje (%) de linfocitos T(LT) CD4 + IFN-γ + en p-QEH 16 vs 8 en IS (p=0.024); %LT CD4 + IL-4 + en p-QEH 7 vs 4 en IS; %LT CD8 + IFN-γ + en p-QEH 33 vs 31 en IS; %LT CD8 + IL-4 + en p-QEH 5 vs 6 en IS; %LT CD4 + IL-4 + 7 vs 16 en CD4 + IFN-γ + de p-QEH (p=0.006); Citocinas en SN (pg/ml): TNF-± en células no estimuladas (CNE) de p-QEH 285 vs 3740 de células estimuladas (CE) en p-QEH (p<0.001); TNF-α en CNE de IS 172 vs 3364 en CE de IS (p<0.001); IL-1β en CNE de p-QEH 53 vs 107 en CE de p-QEH (p=0.038) ; IL-1β en CNE de IS 37 vs 54 en CE de IS; IL-1β en CE de p-QEH 107 vs 54 en CE de IS (p=0.029); IL-6 en CNE de p-QEH 328 vs 481 en CE de p-QEH (p=0.026). Conclusiones: Los linfocitos T estimulados de p-QEH son predominantemente IFN-γ + . En nuestro estudio encontramos que IL-1β es la citocina que más importantemente se eleva posterior al estímulo in vitro en pacientes con queratitis herpética. Estos resultados sugieren que los linfocitos T IFN-γ + circulantes pueden migrar a la córnea humana y colaborar con células residentes o infiltrantes para producir citocinas proinflamatorias, incrementando el daño inflamatorio como ha sido demostrado en modelos animales.
In this work the biological properties and the capability of the outer membrane proteins (OMPs) f... more In this work the biological properties and the capability of the outer membrane proteins (OMPs) from different strains of Bordetella to induce protection against challenge with B. pertussis 18323 were examined. The OMPs from each strain were isolated using Schnaitmann's method. Two OMPs (30 and 32 kDa) were found to be specific for the vaccine strains of B. pertussis and were absent in the OMPs preparation from both B. parapertussis and B. bronchiseptica. When the OMPs from the vaccine strains of B. pertussis were assayed in the mouse intracerebral protection test, they were found to be highly protective (75%-88%) against a challenge with 250 50% lethal doses (LD50) of B. pertussis 18323. However, no correlation was observed between the protective activity and the lymphocytosis-promoting factor (LPF) content of different preparations. Moreover, neither LPF activity or histamine-sensitizing activity (HSA) were found in any of the OMPs assayed. Our results show that OMPs from B. p...
Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned a... more Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region. The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli. The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of precursor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 16S rRNA were also investigated. Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tubercul...
Objective: Evaluate the Monocyte Locomotion Inhibitory Factor (MLIF) effect upon the expression o... more Objective: Evaluate the Monocyte Locomotion Inhibitory Factor (MLIF) effect upon the expression of genes encoding human cytokines, receptors and related factors in the human cell line U-937. MLIF (Met-Gln-Cys-Asn-Ser) is an anti-infl ammatory pentapeptide produced by Entamoeba histolytica that inhibits many human monocyte functions. Material and Methods: U-937 cell line cultured (24 hrs/ RPMI). RNA extracted by Trizol method. 385 genes were analyzed on microarray membranes, complement by real-time RT-PCR and protein expression of some affected genes. Results: MLIF had a preferentially inhibitory effect on gene expression; four genes were over-expressed and 13 underexpressed in MILF vs. simple medium -constitutive expression. Three genes are over-expressed and 19 under-expressed in MLIF/PMA vs. PMA -induced expression. Conclusions: Many modifi ed genes are products regulated by the Nuclear Factor-κB and Mitogen Activated Protein Kinase pathways, suggesting MLIF involvement with these two major pathways for the modulation of the infl ammation and immune responses.
I. Estrada-García). Tuberculosis (2008) 88, 212-220 modified cytokine, TLR and MHC class II expre... more I. Estrada-García). Tuberculosis (2008) 88, 212-220 modified cytokine, TLR and MHC class II expression. These findings may help to unravel the complex mechanisms used by virulent mycobacteria to evade and subvert the immune response.
Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often char... more Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often characterized by immunological dysfunction of peripheral blood mononuclear cells, especially at chronic stages. Lipids from the Mycobacterium tuberculosis cell wall have been shown to produce various suppressive effects on cell-mediated immunity. The cell-surface lipid di-O-acyl-trehalose (DAT) is able to inhibit T-cell proliferation and cytokine secretion in cells from naïve mice. In the present study, we addressed the mechanisms involved in the suppressive effect caused by DAT. We found that DAT decreased the proliferation of spleen cells induced with PMA-ionomycin, suggesting that the suppressive mechanisms target intracellular functions just after phospholipase C-g activation. Addressing this possibility, the effect of DAT was found to involve down-modulation of the di-acyl glycerol-dependent activation of the MAPK-ERK1/2 pathway, one of the crucial signaling pathways leading to adaptive cell immune response against TB. Moreover, the inhibitory effect of DAT on proliferation was reproduced in antigen-stimulated T cells from M. tuberculosis-infected mice, involving the lowering of Th1-type cytokine transcription levels. The present findings thus reveal a new kind of bioactivity for a long-known M. tuberculosis cell wall lipid, DAT.
Background: Methylotrophs are a diverse group of bacteria that can utilize single-carbon compound... more Background: Methylotrophs are a diverse group of bacteria that can utilize single-carbon compounds as a sole energy source, and are often catalase-positive. Known as environmental symbionts, they are emerging as disease-causing organisms in patients with CGD. Methods: We present a case of lymphadenitis due to Granulibacter bethesdensis, a facultative methylotroph, and review 8 other infections caused by methylotrophs in patients with CGD. Results: There have been 9 reported cases of infections due to methylotrophs in patients with CGD. Seven cases were due to G. bethesdensis, one was due to Acidomonas methanolica and one was due to a Methylobacter. In all cases, 16s rRNA gene sequencing was required for diagnosis. Conclusions: Methylotrophs are fastidious and difficult to identify. Although the mechanisms underlying susceptibility to infection with methylotrophs in CGD remain to be elucidated, these bacteria should be included in the spectrum of pathogens associated with infections ...
The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned ... more The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned from a lambda gtll clone into the high-copy-number plasmid pUC8. Escherichia coli containing these recombinants produced large amounts of the antigen, which was purified by polyacrylamide gel electrophoresis in the presence of urea. The ability of E. coli to recognize the mycobacterial promoter was confirmed by constructing additional clones in which the gene is flanked by transcriptional terminators from phage fd. A similar approach was used to demonstrate the expression of this gene in Streptomyces lividans. Mice immunized with killed M. leprae showed cell-mediated immune reactivity to the purified 65-kilodalton protein which stimulated both in vitro lymphoproliferative and in vivo delayed-type hypersensitivity responses.
International Journal of Leprosy and Other Mycobacterial Diseases
We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide ... more We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide anion)] by murine peritoneal macrophages challenged in vitro with Mycobacterium lepraemurium (MLM), complement-opsonized yeast, M. bovis BCG, M. phlei, or phorbol myristate acetate (PMA). We found that except for MLM, all of the other materials provoked the release of significant amounts of hydrogen peroxide and superoxide. MLM entered the macrophages without triggering their oxidative metabolism. Pre-infection of macrophages with MLM did not alter these cells&#39; capacity to release the normal amounts of ROI in response to other microorganisms or PMA. Killing of MLM did not revert the macrophages&#39; failure to release ROI upon ingestion of the microorganism, nor were macrophages able to produce these toxic metabolites when pre-incubated in the presence of murine gamma interferon (IFN-gamma). MLM has several attributes that allow it to survive within macrophages: a) it is a nontoxigenic microorganism (it does not harm its host), b) it resists the harsh conditions of the intraphagolysosomal milieu (a property perhaps dependent on its thick lipidic envelope), and c) it penetrates the macrophages without triggering their oxidative response (thus avoiding the generation of the toxic intermediaries of oxygen). For these attributes (and others discussed in this paper), we recognize MLM as a highly evolved, well-adapted parasite of macrophages. In addition, the results of the present study prompted the analysis of the biochemical pathways used by MLM and M. bovis BCG to penetrate into their cellular hosts, a subject now under investigation in our laboratory.
This review focuses on the principal strategies for conviviality in mice parasite relationships, ... more This review focuses on the principal strategies for conviviality in mice parasite relationships, specially in those genetically determined, and discussed as: a) Innate susceptibility of the host to the parasite; b) Specificity of the parasite for the host, and c) Susceptibility derived from genetic interactions between host and parasite. It is concluded that susceptibility is regulated by multiple genetic facts from both host and parasite, which can interact and modify each other, making each instance of host-parasite relationship unique.
... ALBERT0 A. PALACIOS-BOIX,* IRIS ESTRADA-G,&amp;amp;amp;#x27; M. JOSEPH COLSTON,&amp;a... more ... ALBERT0 A. PALACIOS-BOIX,* IRIS ESTRADA-G,&amp;amp;amp;#x27; M. JOSEPH COLSTON,&amp;amp;amp;#x27; AND GABRIEL S. PANAY12* From the *Rheumatology Unit, Dfuision of Medicine. Guy&amp;amp;amp;#x27;s Hospftal, and the tLaboratory for Leprosy and Mycobacterial Research, National Instftute for Medical Research. ...
International Journal of Leprosy and Other Mycobacterial Diseases
In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segm... more In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segments of the amino acid sequence of proteins from Mycobacterium leprae: 65 kDa, 28 kDa, 18 kDa, and 28 kDa superoxide dismutase, recently renamed antigens 2L, 9L, 12L, and 14L, respectively. They were assayed in the guinea pig model for the induction of a delayed-type hypersensitivity response in M. leprae and BCG-sensitized animals. To sensitize the animals two schemes were used: either a single dose of 5 x 10(9) irradiated or autoclaved whole bacilli, or four weekly intramuscular injections each containing 500 micrograms of soluble extract of M. leprae (MLSE) in incomplete Freund&amp;amp;#39;s adjuvant. Because the second scheme used far too much antigen, we decided to use the first scheme for the experiments we report here. DTH reactions of sensitized animals were induced after 30 days with intradermal injections of 5 micrograms of MLSE and with each of the 10 peptides at three different concentrations: 250 micrograms, 100 micrograms, and 0.05 micrograms. All M. leprae-sensitized guinea pigs gave indurations of 10 mm or more with MLSE, which indicates that the animals were sensitized. None of them gave DTH indurations with 250 micrograms or 100 micrograms, but some of them had positive DTH reactions with the 0.05 micrograms doses of the synthetic peptides. This is most likely due to the fact that we have used an outbred strain of guinea pigs. The peptides were also tested at 0.05 micrograms in animals sensitized with BCG. P7 and P10 seem to be nonspecific peptides; the remaining peptides only induced DTH in the M. leprae-sensitized guinea pigs. P3 (segments 65-85 of the 65-kDa protein) induced a positive DTH in 58% of the tested animals. In other experiments, guinea pigs were sensitized with a single injection (500 micrograms) of each of the synthetic peptides. All animals, except those sensitized with P4 and P8, had positive DTH responses when the homologous peptide was used. Those sensitized with P2, P4, P5, P7, and P8 were able to produce indurations when MLSE was used for the induction of the DTH reaction.
We have investigated changes in gene expression in mouse peritoneal macrophages following infecti... more We have investigated changes in gene expression in mouse peritoneal macrophages following infection with virulent Mycobacterium tuberculosis. Using differential-display reverse transcription-PCR (RT-PCR), we have identified a gene that was markedly down-regulated within 6 h of infection and remained so for the duration of the experiment (5 days). On sequencing, this gene was found to encode the murine cytochrome c oxidase subunit VIIc (COX VIIc). Down-regulation of COX VIIc during M. tuberculosis infection was confirmed by three independent techniques: limiting-dilution RT-PCR, RNase protection assay, and Northern analysis. Limitingdilution RT-PCR and Northern analysis were also used to analyze the specificity of this regulation; heat-killed M. tuberculosis, Mycobacterium bovis BCG, and latex beads had no effect on expression of COX VIIc. Downregulation of this enzyme was also confirmed by using adherent cells isolated from spleens of M. tuberculosisinfected mice. These ex vivo macrophages showed apoptotic features, suggesting a possible involvement of cytochrome c oxidase in the programmed cell death of the host cells.
To classify Mycobacterium leprae isolates from multiple areas in Mexico based on variable number ... more To classify Mycobacterium leprae isolates from multiple areas in Mexico based on variable number of tandem repeats of 6 base within the rpoT gene and three single nucleotide polymorphism (SNP), and to analyse their geographic distribution in the context of the origin of leprosy in Mexico. Analysis for rpoT genotyping of 64 samples collected in the west and southwestern areas revealed that 46 isolates were of the 4 copy type and 18 isolates were of the 3 copy type. All samples from the eastern coastal area (n = 24) and from the Yucatan peninsula (n = 12) were of the 3 copy type. Six isolates from the west and southwestern area were SNP-type 1, 13 isolates were SNP-type 2 and 45 isolates were SNP-type 3. Nineteen of 24 isolates from the eastern coastal area were SNP-type 3 and one was SNP-type 4. Seven isolates from the Yucatan peninsula were SNP-type 3 and one was SNP-type 4. The difference of the proportion of each genotype between the western areas and the eastern areas indicated the expansion of leprosy through different paths in Mexico.
Introducción: Los linfocitos T juegan un papel importante en la inmunopatogénesis de la queratiti... more Introducción: Los linfocitos T juegan un papel importante en la inmunopatogénesis de la queratitis estromal herpética (QEH). Se ha demostrado, en modelos animales, que los linfocitos T infiltran la córnea y producen grandes cantidades de IFN-γ En contraste, la respuesta inmune en humanos no ha sido estudiada ampliamente. Objetivo: Evaluar el perfil de citocinas en células mononucleadas de sangre periférica (CMN) de pacientes con QEH. Métodos: Se determinó en CMN de pacientes (p) con QEH e individuos sanos (IS) la expresión de CD4, CD8, citocinas intracelulares (IFN-γ/IL-4) y citocinas proinflamatorias (TNF-α/IL-6/IL-1β) en sobrenadantes de cultivo (SN) antes/después del estímulo policlonal. Los resultados fueron obtenidos por citometría de flujo y ELISA. Resultados: Porcentaje (%) de linfocitos T(LT) CD4 + IFN-γ + en p-QEH 16 vs 8 en IS (p=0.024); %LT CD4 + IL-4 + en p-QEH 7 vs 4 en IS; %LT CD8 + IFN-γ + en p-QEH 33 vs 31 en IS; %LT CD8 + IL-4 + en p-QEH 5 vs 6 en IS; %LT CD4 + IL-4 + 7 vs 16 en CD4 + IFN-γ + de p-QEH (p=0.006); Citocinas en SN (pg/ml): TNF-± en células no estimuladas (CNE) de p-QEH 285 vs 3740 de células estimuladas (CE) en p-QEH (p<0.001); TNF-α en CNE de IS 172 vs 3364 en CE de IS (p<0.001); IL-1β en CNE de p-QEH 53 vs 107 en CE de p-QEH (p=0.038) ; IL-1β en CNE de IS 37 vs 54 en CE de IS; IL-1β en CE de p-QEH 107 vs 54 en CE de IS (p=0.029); IL-6 en CNE de p-QEH 328 vs 481 en CE de p-QEH (p=0.026). Conclusiones: Los linfocitos T estimulados de p-QEH son predominantemente IFN-γ + . En nuestro estudio encontramos que IL-1β es la citocina que más importantemente se eleva posterior al estímulo in vitro en pacientes con queratitis herpética. Estos resultados sugieren que los linfocitos T IFN-γ + circulantes pueden migrar a la córnea humana y colaborar con células residentes o infiltrantes para producir citocinas proinflamatorias, incrementando el daño inflamatorio como ha sido demostrado en modelos animales.
In this work the biological properties and the capability of the outer membrane proteins (OMPs) f... more In this work the biological properties and the capability of the outer membrane proteins (OMPs) from different strains of Bordetella to induce protection against challenge with B. pertussis 18323 were examined. The OMPs from each strain were isolated using Schnaitmann's method. Two OMPs (30 and 32 kDa) were found to be specific for the vaccine strains of B. pertussis and were absent in the OMPs preparation from both B. parapertussis and B. bronchiseptica. When the OMPs from the vaccine strains of B. pertussis were assayed in the mouse intracerebral protection test, they were found to be highly protective (75%-88%) against a challenge with 250 50% lethal doses (LD50) of B. pertussis 18323. However, no correlation was observed between the protective activity and the lymphocytosis-promoting factor (LPF) content of different preparations. Moreover, neither LPF activity or histamine-sensitizing activity (HSA) were found in any of the OMPs assayed. Our results show that OMPs from B. p...
Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned a... more Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region. The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli. The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of precursor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 16S rRNA were also investigated. Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tubercul...
Uploads
Papers by Iris Estrada