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1.
J Clin Microbiol ; 52(8): 3078-81, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24899020

RESUMO

Matched vaginal and cervical specimens from 96 subjects were analyzed by quantitative PCR for the presence and concentration of bacterial vaginosis-associated microbes and commensal Lactobacillus spp. Detection of these microbes was 92% concordant, indicating that microbial floras at these body sites are generally similar.


Assuntos
Carga Bacteriana , Biota , Colo do Útero/microbiologia , Lactobacillus/isolamento & purificação , Vagina/microbiologia , Vaginose Bacteriana/microbiologia , Adolescente , Adulto , Estudos Transversais , Feminino , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
2.
Diagn Microbiol Infect Dis ; 110(1): 116381, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38901330

RESUMO

Bartonella henselae is a gram-negative rod-shaped bacterium and is the primary causative agent of Cat Scratch Disease (CSD). Although the prevalence of CSD is low in the human population, the possibility of developing multi-organ complications, especially in vulnerable individuals, remains a serious cause for concern. The immunofluorescent assay (IFA) is currently one of the most common laboratory tests for the detection of antibodies to B. henselae in serum, however, it has several disadvantages. The enzyme-linked immunosorbent assay (ELISA) technique offers a more quantitative, sensitive, and cost-effective alternative to conventional IFAs. Here, we report the purification of a novel bioidentical polyclonal antibody from discarded human serum for use as a standard in ELISAs against B. henselae. This novel method of antibody production overcomes the many limitations of animal-derived antibodies while also offering a more robust, reproducible, and scalable antibody production alternative for the diagnosis of CSD.


Assuntos
Anticorpos Antibacterianos , Bartonella henselae , Doença da Arranhadura de Gato , Ensaio de Imunoadsorção Enzimática , Bartonella henselae/imunologia , Bartonella henselae/isolamento & purificação , Humanos , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Doença da Arranhadura de Gato/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Sensibilidade e Especificidade
3.
Front Cell Infect Microbiol ; 14: 1409774, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39006741

RESUMO

Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.


Assuntos
Gardnerella vaginalis , Lactobacillus , Microbiota , Reação em Cadeia da Polimerase em Tempo Real , Vagina , Vaginose Bacteriana , Feminino , Vaginose Bacteriana/diagnóstico , Vaginose Bacteriana/microbiologia , Humanos , Vagina/microbiologia , Microbiota/genética , Lactobacillus/isolamento & purificação , Lactobacillus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Gardnerella vaginalis/isolamento & purificação , Gardnerella vaginalis/genética , Adulto Jovem , Sensibilidade e Especificidade , Prevotella/isolamento & purificação , Prevotella/genética , Megasphaera/isolamento & purificação , Megasphaera/genética , Actinobacteria/isolamento & purificação , Actinobacteria/genética , Actinobacteria/classificação , Pessoa de Meia-Idade , Lactobacillus crispatus/isolamento & purificação , Lactobacillus crispatus/genética , Adolescente , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/classificação , Gravidez , RNA Ribossômico 16S/genética
4.
Microbiol Spectr ; 11(4): e0467622, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37347202

RESUMO

Vaginal dysbiosis in women reduces the abundance of Lactobacillus species and increases that of anaerobic fastidious bacteria. This dysbiotic condition in the vagina, called bacterial vaginosis (BV), can be symptomatic with odorous vaginal discharges or asymptomatic and affects a third of women of reproductive age. Three unclassified bacterial species designated BV-associated bacteria 1, 2, and 3 (BVAB-1, -2, and -3) in 2005 were found to be highly preponderant in the vagina of females with BV. Here, we used sequence homology and phylogenetics analyses to identify the actual species of BVAB-1, -2, and -3 and found BVAB-1 to be Clostridiales genomosp. BVAB-1, BVAB-2 to be Oscillospiraceae bacterium strain CHIC02, and BVAB-3 to be Mageeibacillus indolicus, respectively. These are anaerobic and uncultured species that can be identified only through metagenomics. Long-read sequencing of BV specimens can also enable a genomic reassembly of these species' genomes from metagenomes. Species-specific identification of these pathogens and the availability of their genomes from assembled metagenomes will advance our understanding of their biology, facilitate the design of sensitive diagnostics and drugs, and enhance the treatment of BV. IMPORTANCE For many years since 2005, BVAB, an important pathogen of the female vaginal tract that is associated with BV, has been identified using PCR without knowing its actual species. Without a full genome of these pathogens, a better understanding of their pathogenicity, treatment, resistance, and diagnostics cannot be reached. In this analysis, we use the DNA of BVAB-1, -2, and -3 to determine their actual species to enhance further research into their pathogenicity, resistance, diagnosis, and treatment.


Assuntos
Vaginose Bacteriana , Feminino , Humanos , Vaginose Bacteriana/diagnóstico , Vaginose Bacteriana/genética , Vaginose Bacteriana/microbiologia , Bactérias/genética , Vagina/microbiologia , Metagenoma , Reação em Cadeia da Polimerase
5.
J Urol ; 188(6): 2101-7, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23083854

RESUMO

PURPOSE: Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. MATERIALS AND METHODS: We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. RESULTS: In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. CONCLUSIONS: We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.


Assuntos
Biomarcadores Tumorais/urina , Moléculas de Adesão Celular/genética , Metilação de DNA , Genes bcl-2 , Genes p16 , Reação em Cadeia da Polimerase , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Reação em Cadeia da Polimerase/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/urina
6.
J Low Genit Tract Dis ; 16(4): 352-7, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22968052

RESUMO

OBJECTIVE: The study aimed to compare the overall detection rate of Trichomonas vaginalis to Chlamydia trachomatis and Neiserria gonorrhea and report detection rates by age groups. MATERIALS AND METHODS: Real-time polymerase chain reaction was used to detect the presence of T. vaginalis, C. trachomatis, and N. gonorrhea in cervical samples obtained from patients during gynecological examinations. A total of 78,428, 119,451, and 117,494 samples from women age 12 to 75 years were retrospectively analyzed for the presence of T. vaginalis, C. trachomatis, and N. gonorrhea, respectively. T. vaginalis and C. trachomatis detection rates in Florida, New Jersey, and Texas were calculated in different age groups. RESULTS: The overall detection rate was 4.3% for T. vaginalis, 3.8% for C. trachomatis, and 0.6% for N. gonorrhea. The overall detection rate of T. vaginalis in Florida was 4.7% (n = 22,504), in New Jersey was 3.6% (n = 22,249), and in Texas was 4.5% (n = 33,675). Calculation of infection rates with T. vaginalis revealed differences between selected age groups with the highest detection rates in all 3 states found in age group 46 to 55 years (6.2%), which was higher than the overall detection rates in other age groups (p < .05 for all states). For C. trachomatis, the highest detection rate was found in age group 12 to 25 years (7.3%). CONCLUSIONS: The overall infection rates of T. vaginalis were higher compared with those of C. trachomatis and N. gonorrhea. Detection rates of T. vaginalis were found to be highest among women age 46 to 55 years and may be due to T. vaginalis infiltrating the subepithelial glands and being detected only during hormone-induced or antibiotic-induced changes in the vaginal flora.


Assuntos
Gonorreia/epidemiologia , Linfogranuloma Venéreo/epidemiologia , Neisseria gonorrhoeae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tricomoníase/epidemiologia , Trichomonas vaginalis/isolamento & purificação , Adolescente , Adulto , Idoso , Criança , Chlamydia trachomatis/isolamento & purificação , Feminino , Florida/epidemiologia , Gonorreia/microbiologia , Humanos , Linfogranuloma Venéreo/microbiologia , Pessoa de Meia-Idade , New Jersey/epidemiologia , Texas/epidemiologia , Tricomoníase/parasitologia , Adulto Jovem
7.
Open Forum Infect Dis ; 9(11): ofac595, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36438621

RESUMO

Background: A retrospective study of a single laboratory's results from patients in the United States to investigate high-risk human papillomavirus (HPV) genotype distribution according to cervical cytology and age was performed. Methods: Anonymous results of 23 580 patients' cervical specimens sent to Medical Diagnostic Laboratories, LLC, for cervical cytology and HPV testing between August 2020 and August 2021 were analyzed. Results: Overall, any of the 14 high-risk HPV genotypes identified were detected in 2302 of the 23 580 patients (9.8%), with HPV 52 (1.4%), HPV 39 (1.3%), HPV 51 (1.3%), and HPV 16 (1.2%) being the most frequent in all patients. Multiple high-risk HPV infection was observed in 1.3% of all patients. HPV 16 was most likely to be a single high-risk genotype detected as compared with detection with other high-risk HPV genotypes, in contrast to HPV 33, which is least likely to be a single high-risk genotype detected as compared with detection with other high-risk HPV genotypes. High-risk HPV detection was greatest in patients under 25 years old (<21-year-olds, 24.6%, and 21-25-year-olds, 25.4%). In patients with low-grade squamous intraepithelial lesions, the most frequently detected high-risk HPV genotypes were HPV 51 (10.5%) and HPV 39 (9.1%), and in patients with high-grade squamous intraepithelial lesions, the most frequently detected high-risk HPV genotypes were HPV 16 (25.6%) and HPV 52 (17.1%). Conclusions: HPV genotyping and cervical cytology data analysis may contribute to and inform cervical cancer screening and HPV vaccination programs.

8.
BMC Cancer ; 11: 234, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21663673

RESUMO

BACKGROUND: Bladder cancer is a significant healthcare problem in the United States of America with a high recurrence rate. Early detection of bladder cancer is essential for removing the tumor with preservation of the bladder, avoiding metastasis and hence improving prognosis and long-term survival. The objective of this study was to analyze the presence of DEK protein in voided urine of bladder cancer patients as a urine-based bladder cancer diagnostic test. METHODS: We examined the expression of DEK protein by western blot in 38 paired transitional cell carcinoma (TCC) bladder tumor tissues and adjacent normal tissue. The presence of DEK protein in voided urine was analyzed by western blot in 42 urine samples collected from patients with active TCC, other malignant urogenital disease and healthy individuals. RESULTS: The DEK protein is expressed in 33 of 38 bladder tumor tissues with no expression in adjacent normal tissue. Based on our sample size, DEK protein is expressed in 100% of tumors of low malignant potential, 92% of tumors of low grade and in 71% of tumors of high grade. Next, we analyzed 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals for DEK protein expression by western blot analysis. We are the first to show that the DEK protein is present in the urine of bladder cancer patients. Approximately 84% of TCC patient urine specimens were positive for urine DEK. CONCLUSION: Based on our pilot study of 38 bladder tumor tissue and 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals; DEK protein is expressed in bladder tumor tissue and voided urine of bladder cancer patients. The presence of DEK protein in voided urine is potentially a suitable biomarker for bladder cancer and that the screening for the presence of DEK protein in urine can be explored as a noninvasive diagnostic test for bladder cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/urina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/urina , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/urina , Neoplasias da Bexiga Urinária/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Ligação a Poli-ADP-Ribose
9.
Microbes Infect ; 10(4): 439-46, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18403235

RESUMO

Bacterial vaginosis is the most common vaginal disorder among women of reproductive age. The pathogenesis of bacterial vaginosis is poorly understood, but is defined by a transition in the vaginal flora from the predominant Lactobacillus species to other bacterial species such as Atopobium vaginae and Gardnerella vaginalis. This change is associated with an increase in vaginal cytokine secretion. We hypothesize that vaginal epithelial cells respond to bacterial vaginosis-associated bacteria by triggering an innate immune response. We observed that vaginal epithelial cells secreted interleukin-6 and interleukin-8 in response to Atopobium vaginae and Gardnerella vaginalis, but not to Lactobacillus crispatus. Atopobium vaginae induced increased levels of interleukin-6 and interleukin-8 transcripts, as well as increased transcripts for the antimicrobial peptide beta-defensin 4. This innate immune response required live bacteria capable of protein synthesis in direct contact with vaginal epithelial cells. The response of vaginal epithelial cells was mediated by Toll-like receptor 2, required the adaptor protein MyD88, and involved activation of the NFkappaB signaling pathway. These results suggest that Atopobium vaginae stimulates an innate immune response from vaginal epithelial cells, leading to localized cytokine and defensin production, and possibly contributes to the pathogenesis of bacterial vaginosis.


Assuntos
Actinobacteria/imunologia , Imunidade Inata , Vaginose Bacteriana/imunologia , Vaginose Bacteriana/microbiologia , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Gardnerella vaginalis/imunologia , Perfilação da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lactobacillus/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/imunologia , RNA Mensageiro/biossíntese , Receptor 2 Toll-Like/imunologia , beta-Defensinas/biossíntese , beta-Defensinas/genética
10.
Microbes Infect ; 10(2): 114-21, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18248759

RESUMO

Urinary tract infections are a major source of morbidity among women, with the majority caused by uropathogenic Escherichia coli. Our objective was to test if uropathogenic E. coli suppress the innate immune response of bladder epithelial cells. We found that bladder epithelial cells secrete interleukin-6 and interleukin-8 in response to non-pathogenic E. coli, whereas they failed to do so in response to uropathogenic E. coli. Uropathogenic E. coli prevented interleukin-6 secretion in response to non-pathogenic E. coli and a panel of Toll-like receptor agonists, as well as to interleukin-1beta, but not to tumor necrosis factor alpha. These results indicate that receptors with a Toll/interleukin-1 receptor domain are specifically targeted, and that suppression is not a consequence of toxicity. One candidate for mediating immune suppression is bacterial lipopolysaccharide. However, lipopolysaccharide isolated from either uropathogenic or non-pathogenic E. coli stimulated interleukin-6 secretion to similar levels. In addition, uropathogenic E. coli did not stimulate interleukin-6 secretion from cells expressing a dominant negative Toll-like receptor 4, and prevented cells lacking Toll-like receptor 4 from secreting interleukin-6 in response to synthetic lipoprotein. We conclude that uropathogenic E. coli suppress the innate immune response through a pathway partially independent of lipopolysaccharide and Toll-like receptor 4.


Assuntos
Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Escherichia coli/imunologia , Tolerância Imunológica , Lipopolissacarídeos/imunologia , Receptor 4 Toll-Like/imunologia , Bexiga Urinária/imunologia , Linhagem Celular , Humanos , Interleucina-1beta/imunologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Receptor 4 Toll-Like/agonistas , Fator de Necrose Tumoral alfa/imunologia
12.
J Clin Microbiol ; 46(4): 1501-3, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18305136

RESUMO

A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests performed on cervicovaginal swabs over a 4-year period demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%). However, the species distributions among different age groups revealed increases in the percentages of non-albicans species with increases in age.


Assuntos
Candida/classificação , Candida/isolamento & purificação , Candidíase Vulvovaginal/epidemiologia , Candidíase Vulvovaginal/microbiologia , Vagina/microbiologia , Adulto , Fatores Etários , Idoso , Candida/genética , Candida albicans/classificação , Candida albicans/genética , Candida albicans/isolamento & purificação , Colo do Útero/microbiologia , DNA Fúngico , Feminino , Humanos , Pessoa de Meia-Idade , Técnicas de Tipagem Micológica , Reação em Cadeia da Polimerase/métodos , Especificidade da Espécie , Estados Unidos/epidemiologia
13.
J Clin Virol ; 40(4): 325-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17997134

RESUMO

BACKGROUND: Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. OBJECTIVES: To develop a rapid method for identifying patients infected with MCV via swab sampling. STUDY DESIGN: Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. RESULTS: Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. CONCLUSIONS: These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.


Assuntos
Genitália Feminina/virologia , Molusco Contagioso/virologia , Vírus do Molusco Contagioso/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Primers do DNA , Sondas de DNA , DNA Viral/análise , DNA Viral/genética , Feminino , Genótipo , Humanos , Dados de Sequência Molecular , Molusco Contagioso/diagnóstico , Vírus do Molusco Contagioso/classificação , Vírus do Molusco Contagioso/genética , Sensibilidade e Especificidade
14.
Microb Drug Resist ; 13(2): 119-23, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17650964

RESUMO

A multiplex PCR assay was used to detect the erythromycin (EM) and clindamycin (CM) antibiotic resistance genes, ermB, ermTR, and mefA/E, in Group B Streptococcal (GBS) clinical isolates and in DNA extracted from the corresponding cervicovaginal-rectal (CVR) swabs. We compared these results to the standard EM/CM double disk diffusion assay of 46 isolates. Given that these genes are present in other CVR flora and are found on mobile genetic elements, the PCR assay was unable to predict GBS resistance directly from the swabs. Therefore, PCR can only accurately detect resistance genes and predict the resistance phenotype from purified GBS isolates.


Assuntos
Antibacterianos/farmacologia , Clindamicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Eritromicina/farmacologia , Streptococcus agalactiae/efeitos dos fármacos , Colo do Útero/microbiologia , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Feminino , Genes Bacterianos , Humanos , Reação em Cadeia da Polimerase , Reto/microbiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/isolamento & purificação , Vagina/microbiologia
15.
J Clin Virol ; 33(1): 25-34, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15797362

RESUMO

BACKGROUND: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions. OBJECTIVE: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype. STUDY DESIGN: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5' biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced. RESULTS: The quantitative range of the assay extended from 10(8) through 10(0) copies of each virus (r(2) > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2. CONCLUSIONS: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.


Assuntos
Herpes Genital/epidemiologia , Herpes Simples/epidemiologia , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Adulto , Colo do Útero/virologia , Primers do DNA , DNA Viral/análise , Feminino , Herpes Genital/diagnóstico , Herpes Genital/virologia , Herpes Simples/diagnóstico , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Humanos , Oligonucleotídeos/análise , Prevalência , Sensibilidade e Especificidade , Software , Manejo de Espécimes/métodos
16.
Mol Immunol ; 41(4): 435-47, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15163540

RESUMO

Quiescent T lymphocytes reorganize the actin cytoskeleton subsequent to interaction with antigen presenting cells bearing the appropriate peptide antigen. Although both biochemical and genetic evidence indicate that T cell receptor-dependent cytoskeletal reorganization is critical to T cell activation, the mechanisms that mediate this process remain poorly defined. In this study, the cytoskeletal regulatory protein alpha-adducin was identified as a novel target of TCR signaling in primary T lymphocytes through the biochemical purification of an unknown 120 kDa protein (p120) defined by a fortuitously cross-reactive phospho-sensitive antiserum. The epitope identified by this antiserum defines a previously unrecognized site of phosphorylation localized to amino acids 590-620. Both TCR cross-linking and exposure to phorbol ester resulted in the phosphorylation-dependent elimination of this epitope. However, while phorbol ester induced rapid phosphorylation of both the phospho-sensitive epitope and the functionally defined major protein kinase C site present near the carboxy-terminus (serine 724) of alpha-adducin, only the phospho-sensitive epitope was modified upon activation through the TCR. Moreover, inhibition of actin polymerization by cytochalasin D blocked this modification. Of particular importance, alpha-adducin was not expressed in T cell lines, was completely down-regulated in primary T cells within 24h of activation and was reduced in quiescent memory T cells. These results suggest a model in which reorganization of the unique cytoskeletal network that defines a primary quiescent T lymphocyte is mediated in part through the TCR-dependent modification and subsequent down-regulation of alpha-adducin, thereby resulting in a cytoskeletal architecture compatible with T cell effector and memory functions.


Assuntos
Proteínas de Ligação a Calmodulina/fisiologia , Citoesqueleto/ultraestrutura , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/fisiologia , Linfócitos T/imunologia , Animais , Proteínas de Ligação a Calmodulina/biossíntese , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Epitopos/imunologia , Regulação da Expressão Gênica , Memória Imunológica , Ativação Linfocitária , Camundongos , Fosforilação , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T/ultraestrutura
17.
Cancer Med ; 1(1): 76-81, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23342256

RESUMO

Bladder cancer is one of the most common cancers in the United States. Numerous markers have been evaluated for suitability of bladder cancer detection and surveillance. However, few of them are acceptable as a routine tool. Therefore, there exists a continuing need for an assay that detects the presence of bladder cancer in humans. It would be advantageous to develop an assay with a protein that is associated with the development of bladder cancer. We have identified the cancerous inhibitor of PP2A (CIP2A) protein as a novel bladder cancer biomarker. In this study, Western blot analysis was used to assess the expression level of CIP2A protein in bladder cancer cell lines and bladder cancer patient tissues (n = 43). Our studies indicated CIP2A protein was abundantly expressed in bladder cancer cell lines but not in nontumor epithelial cell lines. Furthermore, CIP2A was specifically expressed in transitional cell carcinoma (TCC) of the bladder tumor tissues but not in adjacent nontumor bladder tissue. Our data showed that CIP2A protein detection in high-grade TCC tissues had a sensitivity of 65%, which is 3.4-fold higher than that seen in low-grade TCC tissues (19%). The level of CIP2A protein expression increased with the stage of disease (12%, 27%, 67%, and 100% for pTa, pT1, pT2, and pT3 tumor, respectively). In conclusion, our studies suggest that CIP2A protein is specifically expressed in human bladder tumors. CIP2A is preferentially expressed in high-grade and high-stage TCC tumors, which are high-risk and invasive tumors. Our studies reported here support the role of CIP2A in bladder cancer progression and its usefulness for the surveillance of recurrence or progression of human bladder cancer.


Assuntos
Autoantígenos/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Idoso , Idoso de 80 Anos ou mais , Autoantígenos/genética , Linhagem Celular , Feminino , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias da Bexiga Urinária/genética
18.
Microbes Infect ; 14(7-8): 628-38, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22326301

RESUMO

Uropathogenic Escherichia coli is the primary cause of urinary tract infections, which affects over 60% of women during their lifetime. UPEC exhibits a number of virulence traits that facilitate colonization of the bladder, including inhibition of cytokine production by bladder epithelial cells. The goal of this study was to identify the mechanism of this inhibition. We observed that cytokine suppression was associated with rapid cytotoxicity toward epithelial cells. We found that cytotoxicity, cytokine suppression and alpha-hemolysin production were all tightly linked in clinical isolates. We screened a UPEC fosmid library and identified clones that gained the cytotoxicity and cytokine-suppression phenotypes. Both clones contained fosmids encoding a PAI II(J96)-like domain and expressed the alpha-hemolysin (hlyA) encoded therein. Mutation of the fosmid-encoded hly operon abolished cytotoxicity and cytokine suppression. Similarly, mutation of the chromosomal hlyCABD operon of UPEC isolate F11 also abolished these phenotypes, and they could be restored by introducing the PAI II(J96)-like domain-encoding fosmid. We also examined the role of alpha-hemolysin in cytokine production both in the murine UTI model as well as patient specimens. We conclude that E. coli utilizes alpha-hemolysin to inhibit epithelial cytokine production in vitro. Its contribution to inflammation during infection requires further study.


Assuntos
Citocinas/biossíntese , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/patogenicidade , Animais , Células Clonais , Feminino , Variação Genética , Humanos , Camundongos , Virulência/genética
19.
Cancer Biomark ; 8(6): 309-17, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-22072119

RESUMO

Early detection of cervical cancer is critical for a favorable prognosis. Standard cytological detection methods, such as Pap smear, are highly subjective and HPV detection is not a reliable marker for predicting the malignancy potential of cervical lesions. As a result, there is a demand for a diagnostic assay capable of sensitive and specific detection of cervical cancer. In this preclinical exploratory study, qRT-PCR and western blotting were used to assess expression levels of CIP2A and p16INK4a in cervical tissue samples (n(normal adjacent) = 23, n(tumor) = 29). CIP2A was abundantly expressed in cervical cancer cell lines and was not expressed in normal epithelial cells. CIP2A mRNA levels were higher in cervical tumor tissues in comparison to the level of CIP2A mRNA in normal adjacent tissue from cervical cancer patients. CIP2A protein was specifically expressed in cervical tumor tissues at different cancer grades and stages, and was not observed in normal adjacent tissue. Elevated CIP2A mRNA levels in cervical tissues had a sensitivity of 80% and specificity of 91% and CIP2A protein expression detection had a sensitivity of 83% and specificity of 100%, similar to that of p16INK4a, with no correlation of CIP2A expression with HPV infection, age, race, or other patient characteristics. However the number of samples analyzed in this preliminary study is limited and a large prospective cohort study is necessary to further evaluate CIP2A as a biomarker for cervical cancer.


Assuntos
Autoantígenos/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias do Colo do Útero/metabolismo , Adulto , Idoso , Autoantígenos/genética , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , RNA Mensageiro/genética , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética
20.
Aging (Albany NY) ; 1(3): 335-49, 2009 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-20195489

RESUMO

Although the transcription factor NF-kappaB is most clearly linked to the inhibition of extrinsic apoptotic signals such as TNFalpha by upregulating known anti-apoptotic genes, NF-kappaB has also been proposed to be required for p53-induced apoptosis in transformed cells. However, the involvement of NF-kappaB in this process is poorly understood. Here we investigate this mechanism and show that in transformed MEFs lacking NF-kappaB (p65-null cells) genotoxin-induced cytochrome c release is compromised. To further address how NF-kappaB contributes to apoptosis, gene profiling by microarray analysis of MEFs was performed, revealing that NF-kappaB is required for expression of Noxa, a pro-apoptotic BH3-only protein that is induced by genotoxins and that triggers cytochrome c release. Moreover, we find that in the absence of NF-kappaB, genotoxin treatment cannot induce Noxa mRNA expression. Noxa expression had been shown to be regulated directly by genes of the p53 family, like p73 and p63, following genotoxin treatment. Here we show that p73 is activated after genotoxin treatment only in the presence of NF-kappaB and that p73 induces Noxa gene expression through the p53 element in the promoter. Together our data provides an explanation for how loss of NF-kappaB abrogates genotoxin-induced apoptosis.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Fator de Transcrição RelA/fisiologia , Ativação Transcricional , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Linhagem Celular , Citocromos c/metabolismo , Etoposídeo/farmacologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Tumoral p73
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