Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specifics probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Autosomal-Recessive Posterior Microphthalmos Is Caused by Mutations in PRSS56, a Gene Encoding a Trypsin-Like Serine Protease.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

Monoamine oxidase A down-regulation contributes to high metanephrine concentration in pheochromocytoma.

CONTEXT: The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. OBJECTIVE: The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. DESIGN: A multicenter comparative study was conducted. STUDY PARTICIPANTS: The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. MAIN OUTCOME MEASURES: CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. RESULTS: At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. CONCLUSION: High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

Pattern and clinical significance of cancer-testis gene expression in head and neck squamous cell carcinoma.

Cancer-testis (CT) antigens comprise families of tumor-associated antigens that are immunogenic in patients with various cancers. Their restricted expression makes them attractive targets for immunotherapy. The aim of this study was to determine the expression of several CT genes and evaluate their prognostic value in head and neck squamous cell carcinoma (HNSCC). The pattern and level of expression of 12 CT genes (MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, MAGE-C2, NY-ESO-1, LAGE-1, SSX-2, SSX-4, BAGE, GAGE-1/2, GAGE-3/4) and the tumor-associated antigen encoding genes PRAME, HERV-K-MEL, and NA-17A were evaluated by RT-PCR in a panel of 57 primary HNSCC. Over 80% of the tumors expressed at least 1 CT gene. Coexpression of three or more genes was detected in 59% of the patients. MAGE-A4 (60%), MAGE-A3 (51%), PRAME (49%) and HERV-K-MEL (42%) were the most frequently expressed genes. Overall, the pattern of expression of CT genes indicated a coordinate regulation; however there was no correlation between expression of MAGE-A3/A4 and BORIS, a gene whose product has been implicated in CT gene activation. The presence of MAGE-A and NY-ESO-1 proteins was verified by immunohistochemistry. Analysis of the correlation between mRNA expression of CT genes with clinico-pathological characteristics and clinical outcome revealed that patients with tumors positive for MAGE-A4 or multiple CT gene expression had a poorer overall survival. Furthermore, MAGE-A4 mRNA positivity was prognostic of poor outcome independent of clinical parameters. These findings indicate that expression of CT genes is associated with a more malignant phenotype and suggest their usefulness as prognostic markers in HNSCC.

DIFFERENTIAL EXPRESSION OF GLUTARYL-CoA DEHYDROGENASE IN ADULT RAT CNS, PERIPHERAL TISSUES AND DURING EMBRYONIC DEVELOPMENT

Glutaryl-CoA dehydrogenase (GCDH, EC 1.3.99.7) deficiency, known as glutaric acidemia type I, is one of the more common organic acidurias. To investigate the role of this pathway in different organs we studied the tissue-specific expression pattern of rat Gcdh. The open reading frame cDNA of the rat Gcdh gene was cloned from rat brain mRNA by RT-PCR, allowing the synthesis of digoxigenin-labeled in situ hybridization (ISH) riboprobes. Gcdh mRNA expression was analyzed by ISH on cryosections of adult rat brain, kidney, liver, spleen and heart muscle, as well as on E15 and E18 rat embryos. Gcdh was found expressed in the whole rat brain, almost exclusively in neurons. Gcdh was absent from astrocytes but expressed in rare oligodendrocytes. Strong Gcdh expression was found in liver and spleen, where expression appears predominant to lymphatic nodules. In kidney, the highest Gcdh expression is found in the juxtamedullar cortex (but not in glomerula), and at lower levels in medulla. Heart muscle was negative. During embryonic development, Gcdh was found well expressed in liver, intestinal mucosa and skin, as well as at lower levels in CNS. Further studies are ongoing to provide evidence on the presence of the entire pathway in CNS in order to understand the mechanisms leading to neurotoxicity in glutaric aciduria. The high expression of Gcdh in kidney may explain why certain patients with residual enzyme activity are low excretors at the urine metabolite level.

Variation in novel exons (RACEfrags) of the MECP2 gene in Rett syndrome patients and controls.

The study of transcription using genomic tiling arrays has lead to the identification of numerous additional exons. One example is the MECP2 gene on the X chromosome; using 5'RACE and RT-PCR in human tissues and cell lines, we have found more than 70 novel exons (RACEfrags) connecting to at least one annotated exon.. We sequenced all MECP2-connected exons and flanking sequences in 3 groups: 46 patients with the Rett syndrome and without mutations in the currently annotated exons of the MECP2 and CDKL5 genes; 32 patients with the Rett syndrome and identified mutations in the MECP2 gene; 100 control individuals from the same geoethnic group. Approximately 13 kb were sequenced per sample, (2.4 Mb of DNA resequencing). A total of 75 individuals had novel rare variants (mostly private variants) but no statistically significant difference was found among the 3 groups. These results suggest that variants in the newly discovered exons may not contribute to Rett syndrome. Interestingly however, there are about twice more variants in the novel exons than in the flanking sequences (44 vs. 21 for approximately 1.3 Mb sequenced for each class of sequences, p=0.0025). Thus the evolutionary forces that shape these novel exons may be different than those of neighboring sequences.

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.

Eokines: synthesis, storage and release from human eosinophils

Eosinophils are prominent inflammatory cells in asthma and other allergic disorders, as well as in helminthic parasite infections. Recently, eosinophils have been reported to synthesize and store a range of regulatory proteins within their secretory granules (eokines). Eokines comprise a group of cytokines, chemokines, and growth factors which are elaborated by eosinophils. These proteins, and the messages which encode them, appear to be identical to those produced by lymphocytes and other tissues. Interestingly, immunoreactivity to many of these eokines has been found to co-localize to the eosinophil´s secretory granules. In this review, we have discussed the repertoire of 18 eokines so far identified in eosinophils, and focused on four of these, namely, interleukin-2 (IL-2), IL-4, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES. These four eokines co-localize to the crystalloid granules in eosinophils, as shown in studies using subcellular fractionation and immunogold labeling in electron microscopy. During stimulation by physiological triggers, for example, with serum-coated particles, eosinophils release these mediators into the surrounding supernatant. In addition, eokines are likely to be synthesized within eosinophils rather than taken up by endocytosis, as show in detection of mRNA for each of these proteins using in situ hybridization, RT-PCR, and in the case of RANTES, in situ RT-PCR. Eokines synthesis and release from eosinophils challenges the commonly held notion that these cells act downstream of key elements in immune system, and indicate that they may instead belong to the afferent arm of immunity.

Rapid detection of enterovirus in cerebrospinal fluid by a fully-automated PCR assay is associated with improved management of aseptic meningitis in adult patients.

BACKGROUND: Enterovirus (EV) is the most frequent cause of aseptic meningitis (AM). Lack of microbiological documentation results in unnecessary antimicrobial therapy and hospitalization. OBJECTIVES: To assess the impact of rapid EV detection in cerebrospinal fluid (CSF) by a fully-automated PCR (GeneXpert EV assay, GXEA) on the management of AM. STUDY DESIGN: Observational study in adult patients with AM. Three groups were analyzed according to EV documentation in CSF: group A=no PCR or negative PCR (n=17), group B=positive real-time PCR (n=20), and group C=positive GXEA (n=22). Clinical, laboratory and health-care costs data were compared. RESULTS: Clinical characteristics were similar in the 3 groups. Median turn-around time of EV PCR decreased from 60h (IQR (interquartile range) 44-87) in group B to 5h (IQR 4-11) in group C (p<0.0001). Median duration of antibiotics was 1 (IQR 0-6), 1 (0-1.9), and 0.5 days (single dose) in groups A, B, and C, respectively (p<0.001). Median length of hospitalization was 4 days (2.5-7.5), 2 (1-3.7), and 0.5 (0.3-0.7), respectively (p<0.001). Median hospitalization costs were $5458 (2676-6274) in group A, $2796 (2062-5726) in group B, and $921 (765-1230) in group C (p<0.0001). CONCLUSIONS: Rapid EV detection in CSF by a fully-automated PCR improves management of AM by significantly reducing antibiotic use, hospitalization length and costs.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.