Mycobacterium tuberculosis promotes genomic instability in macrophages.

BACKGROUND: Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. OBJECTIVES: To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. METHODS: We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. FINDINGS: Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. MAIN CONCLUSIONS: Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection.

Human papillomavirus genotyping by Linear Array and Next-Generation Sequencing in cervical samples from Western Mexico.

BACKGROUND: The Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. However, the assay is limited to a restricted number of HPV, and sequence variations in the detection region of the HPV probes could give false negatives results. Recently, 454 Next-Generation sequencing (NGS) technology has been efficiently used also for HPV genotyping; this methodology is based on massive sequencing of HPV fragments and is expected to be highly specific and sensitive. In this work, we studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS. METHODS: Two hundred thirty three cervical samples from women Without cervical lesions (WCL, n = 48), with Cervical intraepithelial neoplasia grade 1 (CIN I, n = 98), or with Cervical cancer (CC, n = 87) were recruited, DNA was extracted, and HPV positivity was determined by PCR amplification using PGMY09/11 primers. All HPV- positive samples were genotyped individually by LA. Additionally, pools of amplicons from the PGMY-PCR products were sequenced using 454 NGS technology. Results obtained by NGS were compared with those of LA for each group of samples. RESULTS: We identified 35 HPV genotypes, among which 30 were identified by both technologies; in addition, the HPV genotypes 32, 44, 74, 102 and 114 were detected by NGS. These latter genotypes, to our knowledge, have not been previously reported in Mexican population. Furthermore, we found that LA did not detect, in some diagnosis groups, certain HPV genotypes included in the test, such as 6, 11, 16, 26, 35, 51, 58, 68, 73, and 89, which indicates possible variations at the species level. CONCLUSIONS: There are HPV genotypes in Mexican population that cannot be detected by LA, which is, at present, the most complete commercial genotyping test. More studies are necessary to determine the impact of HPV-44, 74, 102 and 114 on the risk of developing CC. A greater number of samples must be analyzed by NGS for the most accurate determination of Mexican HPV variants.

Human papillomavirus as a single infection in pregnant women from Northeastern Mexico: Cross-sectional study.

BACKGROUND: The role of human papillomavirus (HPV) as single or multiple infections in pregnant women would be relevant to determine the time to progression and/or the time to regression of cervical lesions. OBJECTIVE: In this preliminary study, we determined the prevalence of HPV as single or multiple infections in pregnant women from Northeastern Mexico. MATERIALS AND METHODS: Samples from 31 pregnant and 62 nonpregnant women were examined between January 2015 and November 2015 at UMAE-23 of the Instituto Mexicano del Seguro Social (IMSS). The samples of cervicovaginal exudate were obtained for HPV DNA detection using the INNO-LiPA test, and HPV infections were analyzed as single or multiple infections. Participants completed a questionnaire on sociodemographic, gynecological, obstetric, and sexual behavior characteristics. RESULTS: The mean age of the pregnant women was 25.7 ± 4.8 yr, with an average time of pregnancy of 6 ± 1 months at the time of the study. With respect to age, parity, smoking history, or oral contraceptive use no statistically significant differences between the two studied groups was observed. The HPV infection was 2.7 times higher in pregnant women (35%) than in the control group (13%). In total, 78% of the pregnant women who were HPV-positive presented with single infections compared with 28% of the nonpregnant women. CONCLUSION: A higher prevalence of HPV as a single infection was found in this sample of pregnant Mexican women. Follow-up is necessary to evaluate the persistence or regression of the infection.

Chromosome segregation and chromatin integrity in spermatozoa from a t(2;8)(p24;p21)mat carrier: A case-report.

BACKGROUND: Chromosome rearrangements can produce genomic imbalance in gametes which causes a drastic decrease in fertility. Several studies have described the relationship between high levels of DNA damage and chromosomal alterations in the spermatozoa of infertile or subfertile males. However, the nature of this relation is poorly understood. In this study, the meiotic segregation pattern and chromatin integrity were analyzed in the ejaculated spermatozoa of a 46, XY, t(2;8)(p24;p21)mat carrier with normozoospermia and a lack of conception. CASE: A 39-year-old infertile man with a 46, XY, t(2;8)(p24;p21)mat inherited from his mother, was studied. The wife of the proband (30 yrs old) had a normal karyotype and no reproductive problems. The meiotic segregation pattern and aneuploidy of chromosome-8 and chromosome-2 were analyzed by FISH. Sperm DNA damage was evaluated by the Sperm Dispersion Chromatin, alkaline comet assay and DNA breaking detection. Five healthy male donors were included as controls. The frequency of genetically unbalanced spermatozoa was 61.6%. Analysis of the aneuploidy of chromosome-8 and chromosome-Y revealed approximately three and 24 fold increased level respectively in comparison with that of the control group. CONCLUSION: We suggest that the accumulation of genetically unbalanced spermatozoa, and increased sperm aneuploidy level is related to male infertility. Interestingly, the case described here has a high level of sperm chromosomal imbalance appears to be linked to sperm DNA fragmentation status. This information could be useful in assisted reproductive techniques.

[Chromosomal abnormalities in patients from Obstetrics and Gynaecology hospital]. / Anormalidades cromosómicas en pacientes de un hospital de ginecoobstetricia.

BACKGROUND: Chromosomal abnormalities are present in 2-4 % of all newborns, and they cause 20 % of deaths in the first year of life. The estimated prevalence of chromosomal abnormalities is one for each 500-1000 newborns. These abnormalities can be numerical or structural, and they can affect autosomal or sexual chromosomes. They affect from 1 to 3 % of general population, and from 6 to 7 % of individuals with congenital anomalies. METHODS: Descriptive study, which included all the registries of cytogenetic analysis (of adults and newborns) made in a genetic laboratory in a period of 14 years. The prevalence of polymorphisms and chromosomal abnormalities in the patients from the Hospital de Ginecoobstetricia 23, Instituto Mexicano del Seguro Social (Monterrey, Nuevo León) was assessed. RESULTS: Of 4006 cytogenetic studies, 253 (6.3 %) did not show in vitro growth, 2667 (66.5 %) were normal, and 1175 (29.3 %) were abnormal. Of these, 614 (52.2 %) had polymorphisms, and 561 (47.7 %) structural or numerical chromosomal abnormalities. In regards to these chromosomopathies (561), trisomy 21 was observed in 429 (36.5 %); Turner's syndrome, in 84 (7.1 %); trisomy 18, in 57 (4.8 %); and trisomy 13, in 32 (2.7 %). With G-band technique, we found 93 % of in vitro cell growth. CONCLUSIONS: Of these studies, 55 % was performed due to non-numerical abnormalities; 14.4 %, due to structural abnormalities; and the rest, due to polymorphisms.

INTRODUCCIÓN: las anormalidades cromosómicas se presentan en 2 a 4 % de los recién nacidos y causan 20 % de las muertes en el primer año de vida. Su prevalencia es de uno por cada 500 a 1000 recién nacidos vivos. Pueden ser numéricas o estructurales y afectar a los cromosomas autosómicos o sexuales. Se presentan en 1 a 3 % de la población general y en 6 a 7 % de los individuos con anomalías congénitas. MÉTODOS: estudio descriptivo en el que se incluyeron todos los resultados citogenéticos de cariotipos tomados de sangre periférica de adultos y neonatos. Se evaluó la prevalencia de polimorfismos y alteraciones cromosómicas en derechohabientes del Hospital de Ginecoobstetricia 23 del Instituto Mexicano del Seguro Social, en Monterrey, Nuevo León. RESULTADOS: de 4006 estudios citogenéticos, en 253 no se obtuvo crecimiento de linfocitos (6.3 %), 2667 fueron normales (66.5 %) y 1175, anormales (29.3 %); de estos últimos, en 614 (52.2 %) se identificaron polimorfismos cromosómicos y en 561 (47.7 %), aberraciones cromosómicas estructurales o numéricas. De las cromosomopatías (561, 47.7 %), la trisomía 21 se observó en 429 (36.5 %), el síndrome de Turner en 84 (7.1 %), la trisomía 18 en 57 (4.8 %) y la trisomía 13 en 32 (2.7 %). Con la técnica de bandeo G se obtuvo un crecimiento celular in vitro de 93 %. CONCLUSIONES: 55 % de los estudios se realizó por anormalidades diferentes a las numéricas, 14.4 % por alteraciones estructurales y el resto se debió a polimorfismos.

Mycobacterium tuberculosis promotes genomic instability in macrophages

BACKGROUND Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. OBJECTIVES To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. METHODS We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. FINDINGS Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. MAIN CONCLUSIONS Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection.

Immunohistochemical analysis of prostate apoptosis response-4 (Par-4) in Mexican women with breast cancer: a preliminary study.

BACKGROUND AND AIMS: We undertook this study to compare the expression level of prostate apoptosis response-4 (Par-4) among patient outcome in two groups of women with breast cancer (short and long survival) and two groups without breast cancer (benign lesion and control). METHODS: We included breast specimens with nonhistological abnormalities (eight samples) as a control group. Semiquantitative and quantitative analysis of immunohistochemical staining by image analysis software were used to study the intensity of Par-4 expression. Both methods produced similar results (p>0.05). RESULTS: No significant expression of Par-4 was observed in normal breast tissue. Benign lesions and breast cancer tissue showed strong nuclear expression of Par-4, predominantly on epithelial cells and specifically in ductal cells. Par-4 expression was lower in myoepithelial cells and there was no appreciable stromal staining. Significantly less Par-4 reactivity was detected in tissue from patients with a short survival compared with patients with benign lesions and those with a long survival. CONCLUSIONS: Our findings suggest that a lower expression level of Par-4 is related to an unfavorable prognosis. A larger prospective study of samples of all patient groups with a longer follow-up is needed to validate this finding.