Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines.

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections.

Single cell gene expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

The CD8 T cell response generatedby gene-based vaccines is importantfor protective immunity againstmany infectious diseases but its complexityis incompletely understood.Here, we report that different vaccinesencoding HIV Env elicit qualitativelydistinct CD8 T cells that wereidentified by patterns of gene expressionin individual cells. Three alternativeprime-boost vector combinationsstimulated antigen-specific CD8 Tcell populations of similar magnitudeand function by intracellular cytokinestaining; however, single cell geneexpression profiling enabled the discriminationof distinct CM and EMCD8 cells elicited by the three vaccines.Two previously unrecognizedCD8 T cell subsets have been definedby their coexpression of Eomes,Cxcr3 and Ccr7; or Klrk1, Klrg1 andCcr5 in CM and EM cells respectively.

Diagnosis of tuberculosis infection using interferon-γ-based assays

Interferon-γ-based assays, collectively known as IFN-γ release assays (IGRAs), have emerged as a reliable alternative to the old tuberculin skin test (TST) for the immunodiagnosis of tuberculosis (TB) infection. The 2 commercially available tests, the enzyme-linked immunosorbent assay (ELISA), QuantiFERON-TB Gold Intube (QFT-IT), and the enzyme-linked immunospot assay (ELISPOT), T-SPOT.TB, are more accurate than TST for the diagnosis of TB, since they are highly specific and correlate better with the existence of risk factors for the infection. According to the available data, T-SPOT.TB obtains a higher number of positive results than QFT-IT, while its specificity seems to be lower. Although the sensitivity of the IFN-γ -based assays may be impaired to some extent by cellular immunosuppression and extreme ages of life, they perform better than TST in these situations. Data from longitudinal studies suggest that IFN-γ-based tests are better predictors of subsequent development of active TB than TST; however this prognostic value has not been consistently demonstrated. This review focuses on the clinical use of the IFN-γ -based tests in different risk TB groups, and notes the main limitations and areas for future development.

Estimation of the diagnostic accuracy of the glyco-C and US9 gene-based polymerase chain reaction technique for the detection of bovine Herpesvirus type 5 DNA in decomposed brain suspension from a slaughter house using Bayesian analysis, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Consequences of DNA damage for gene transcription : direct effects of modified nucleobases and the role of base excision repair = Folgen von DNA-Schäden für die Gentranskription

The presence of damaged nucleobases in DNA can negatively influence transcription of genes. One of the mechanisms by which DNA damage interferes with reading of genetic information is a direct blockage of the elongating RNA polymerase complexes – an effect well described for bulky adducts induced by several chemical substances and UV-irradiation. However, other mechanisms must exist as well because many of the endogenously occurring non-bulky DNA base modifications have transcription-inhibitory properties in cells, whilstrnnot constituting a roadblock for RNA polymerases under cell free conditions. The inhibition of transcription by non-blocking DNA damage was investigated in this work by employing the reporter gene-based assays. Comparison between various types of DNA damage (UV-induced pyrimidine photoproducts, oxidative purine modifications induced by photosensitisation, defined synthetic modified bases such as 8-oxoguanine and uracil, and sequence-specific single-strand breaks) showed that distinct mechanisms of inhibition of transcription can be engaged, and that DNA repair can influence transcription of the affectedrngenes in several different ways.rnQuantitative expression analyses of reporter genes damaged either by the exposure of cells to UV or delivered into cells by transient transfection supported the earlier evidence that transcription arrest at the damage sites is the major mechanism for the inhibition of transcription by this kind of DNA lesions and that recovery of transcription requires a functional nucleotide excision repair gene Csb (ERCC6) in mouse cells. In contrast, oxidisedrnpurines generated by photosensitisation do not cause transcriptional blockage by a direct mechanism, but rather lead to transcriptional repression of the damaged gene which is associated with altered histone acetylation in the promoter region. The whole chain of events leading to transcriptional silencing in response to DNA damage remains to be uncovered. Yet, the data presented here identify repair-induced single-strand breaks – which arise from excision of damaged bases by the DNA repair glycosylases or endonucleases – as arnputative initiatory factor in this process. Such an indirect mechanism was supported by requirement of the 8-oxoguanine DNA glycosylase (OGG1) for the inhibition of transcription by synthetic 8-oxodG incorporated into a reporter gene and by the delays observed for the inhibition of transcription caused by structurally unrelated base modifications (8-oxoguanine and uracil). It is thereby hypothesized that excision of the modified bases could be a generalrnmechanism for inhibition of transcription by DNA damage which is processed by the base excision repair (BER) pathway. Further gene expression analyses of plasmids containing single-strand breaks or abasic sites in the transcribed sequences revealed strong transcription inhibitory potentials of these lesions, in agreement with the presumption that BER intermediates are largely responsible for the observed effects. Experiments with synthetic base modifications positioned within the defined DNA sequences showed thatrninhibition of transcription did not require the localisation of the lesion in the transcribed DNA strand; therefore the damage sensing mechanism has to be different from the direct encounters of transcribing RNA polymerase complexes with DNA damage.rnAltogether, this work provides new evidence that processing of various DNA basernmodifications by BER can perturb transcription of damaged genes by triggering a gene silencing mechanism. As gene expression can be influenced even by a single DNA damage event, this mechanism could have relevance for the endogenous DNA damage induced in cells under normal physiological conditions, with a possible link to gene silencing in general.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Gene-based approach to human gene-phenotype correlations

Elucidating the genetic basis of human phenotypes is a major goal of contemporary geneticists. Logically, two fundamental and contrasting approaches are available, one that begins with a phenotype and concludes with the identification of a responsible gene or genes; the other that begins with a gene and works toward identifying one or more phenotypes resulting from allelic variation of it. This paper provides a conceptual overview of phenotype-based vs. gene-based procedures with emphasis on gene-based methods. A key feature of a gene-based approach is that laboratory effort first is devoted to developing an assay for mutations in the gene under regard; the assay then is applied to the evaluation of large numbers of unrelated individuals with a variety of phenotypes that are deemed potentially resulting from alleles at the gene. No effort is directed toward chromosomally mapping the loci responsible for the phenotypes scanned. Example is made of my laboratory’s successful use of a gene-based approach to identify genes causing hereditary diseases of the retina such as retinitis pigmentosa. Reductions in the cost and improvements in the speed of scanning individuals for DNA sequence anomalies may make a gene-based approach an efficient alternative to phenotype-based approaches to correlating genes with phenotypes.

Assessing Methods for Assigning SNPs to Genes in Gene-Based Tests of Association Using Common Variants

Gene-based tests of association are frequently applied to common SNPs (MAF>5%) as an alternative to single-marker tests. In this analysis we conduct a variety of simulation studies applied to five popular gene-based tests investigating general trends related to their performance in realistic situations. In particular, we focus on the impact of non-causal SNPs and a variety of LD structures on the behavior of these tests. Ultimately, we find that non-causal SNPs can significantly impact the power of all gene-based tests. On average, we find that the “noise” from 6–12 non-causal SNPs will cancel out the “signal” of one causal SNP across five popular gene-based tests. Furthermore, we find complex and differing behavior of the methods in the presence of LD within and between non-causal and causal SNPs. Ultimately, better approaches for a priori prioritization of potentially causal SNPs (e.g., predicting functionality of non-synonymous SNPs), application of these methods to sequenced or fully imputed datasets, and limited use of window-based methods for assigning inter-genic SNPs to genes will improve power. However, significant power loss from non-causal SNPs may remain unless alternative statistical approaches robust to the inclusion of non-causal SNPs are developed.

Novel inactivating mutations in the GH secretagogue receptor gene in patients with constitutional delay of growth and puberty

Background: A limited number of mutations in the GH secretagogue receptor gene (GHSR) have been described in patients with short stature. Objective: To analyze GHSR in idiopathic short stature (ISS) children including a subgroup of constitutional delay of growth and puberty (CDGP) patients. Subjects and methods: The GHSR coding region was directly sequenced in 96 independent patients with ISS, 31 of them with CDGP, in 150 adults, and in 197 children with normal stature. The pharmacological consequences of GHSR non-synonymous variations were established using in vitro cell-based assays. Results: Five different heterozygous point variations in GHSR were identified (c.-6 G>C, c.251G>T (p.Ser84Ile), c.505G>A (p.Ala169Thr), c.545 T>C (p.Val182Ala), and c.1072G>A (p.Ala358Thr)), all in patients with CDGP. Neither these allelic variants nor any other mutations were found in 694 alleles from controls. Functional studies revealed that two of these variations (p.Ser84Ile and p. Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. These mutations were identified in two female patients with CDGP (at the age of 13 years, their height SDS were -2.4 and -2.3). Both patients had normal progression of puberty and reached normal adult height (height SDS of -0.7 and -1.4) without treatment. Conclusion: This is the first report of GHSR mutations in patients with CDGP. Our data raise the intriguing possibility that abnormalities in ghrelin receptor function may influence the phenotype of individuals with CDGP.