Nicholas C. Vamvakopoulos

IMPROVED DETECTION OF DIROFILARIA REPENS DNA BY DIRECT POLYMERASE CHAIN REACTION

Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.

Genotypic assignment of infection by Dirofilaria repens.

Dirofilariasis is a parasitic disease, which if treated inappropriately due to misdiagnosis, can cause unwanted complications particularly when the infection is located in the breast. The numerous obstacles that can cause misdiagnosis of dirofilariases by standard morphological procedures prompted the development of a Dirofilaria repens-specific direct polymerase chain reaction (PCR)-based diagnostic approach using freshly infected dog blood. Reliable amplification of nematode DNA from formalin-fixed infected human specimens by this method is only possible from relatively fresh biological material, preserved in the fixative for up to 20 days. We report here our first case of dirofilariasis since the development of PCR genotyping, where the pathogen was morphologically unrecognizable and the diagnosis was based exclusively on DNA amplification. We complete our methodological contribution to the clinical laboratory diagnosis of dirofilariasis by presenting two more cases, where the primary genotypic assignment of infection by D. repens was further confirmed by conventional morphological means.

Clear detection and typing of herpes simplex virus types 1 and 2 by an indirect ELISA assay: comparison with three different combined methods--capture ELISA, restriction enzymes, and polymerase chain reaction.

The severity and recurrences of Herpes Simplex Virus (HSV) infection depend on the type of the infectious agent (HSV‐1 or HSV‐2), which induces the necessity of a nonambiguous detecting typing. The commonly used capture ELISA technique has to be often supported by DNA analysis to confirm the detection and the typing of HSV viruses in exposed patients. In this report, we describe a rapid and cheap indirect ELISA method using anti‐HSV monospecific polyclonal antibodies prepared in the laboratory. The typing of the studied samples was clear, did not need series of dilution, and allowed the immediate classification of viruses without further control examination. We tested 51 specimens, which were typed 25 HSV‐1 and 26 HSV‐2 strains. The comparison with capture ELISA, restriction enzyme and polymerase chain reaction analysis definitely allowed our method to be assessed as a useful tool for a routine diagnostic. J. Clin. Lab. Anal. 11:146–153, 1997.

Mapping the human corticotropin releasing hormone binding protein gene (CRHBP) to the long arm of chromosome 5 (5q11.2–q13.3)

Unexpected stimulation or stress activates the heat shock protein (hsp) system at the cellular level and the hypothalamic-pituitary-adrenal (HPA) axis at the level of the whole organism. At the molecular level, these two systems communicate through the functional interaction between hsp90 and glucocorticoid receptor (GR). The corticotropin releasing hormone (CRH) system regulates the mammalian stress response by coordinating the activity of the HPA axis. It consists of the 41-amino-acid-long principal hypothalamic secretagogue for pituitary adrenocorticotropic hormone (ACTH), CRH, its receptor (CRHR), and its binding protein (CRHBP). Because of its central role in the coordination of stress response and whole body homeostasis, the CRH system has been implicated in the pathogenesis of neuroendocrine and psychiatric disease. 19 refs., 1 fig.

Classification and Structure of Echovirus 5′-UTR Sequences

Enteroviruses are classified into two genetic clusters on the basis of 5′-UTR and all echoviruses (ECV) are classified together with coxsackie B viruses (CBV), coxsackie A viruses (CAV) types 2–10, 12, 14 and 16, and enteroviruses (EV) 68, 69, 71 and 73. During the present study, 5′-UTR-derived sequences constituting the largest part of the Internal Ribosome Entry Site (IRES) of ECVs were studied with respect to their possible secondary structures, which were predicted following the phenomenon of “covariance”, i.e. the existence of evolutionary pressure in favour of structural conservation in the light of nucleotide sequence variability. In this and previous studies, no correlation between overall 5′-UTR identity and the currently recognised Human Enterovirus species was found, implying that notwithstanding their divergent protein-encoding regions, these species are free to exchange 5′-UTRs by recombination. Secondary structure features which are known to be highly conserved amongst enteroviruses and specifically the GNRA tetraloop in secondary structure domain IV, involved in long-term tertiary interactions and loop B in secondary structure domain V with an as yet unknown function were also conserved in ECVs. In contrast, the C(NANCCA)G motif, which is considered to be important in virus transcription and translation, was not conserved in all ECVs and sequence patterns observed in other enterovirus groups and rhinoviruses were recorded.

Molecular and phylogenetic analysis of haemagglutinin and neuraminidase sequences from recent human influenza type A (H3N2) viral isolates in Southern Greece

Summary. Eighteen haemagglutinin (HA1) gene segments and eleven neuraminidase (NA) genes of human influenza type A (H3N2) viruses isolated from non-vaccinated individuals presenting severe influenza-like illness at peak influenza activity in Southern Greece during the surveillance period 1996–1999, were subjected to sequence and phylogenetic analyses following propagation in embryonated hen’s eggs. The HA1 gene segment of the clinical isolates differed from the recent reference influenza type A (H3N2) vaccine strains in an Ile at residue 186, a Val at residue 194 and a Val at residue 226 for one, two and thirteen isolates of the 1996–1997 and 1996–1999 periods, respectively. The analogous differences in the NA gene were confined in an Asp to Asn substitution at residue 198 in one A/Wuhan/359/95 (H3N2)-like isolate of the 1996–1997 period, primarily. In addition, phylogenetic analysis revealed that an isolate of the 1997–1998 period was a recombinant with its HA1 gene segment being closely related to that of A/Wuhan/359/95-like viruses and its NA to viruses of the A/Sydney/5/97 (H3N2) lineage. These findings confirmed the profound genetic instability of influenza type A (H3N2) viruses and underscored the importance for periodic molecular surveys of HA and NA in the effective prevention and management of viral outbreaks. Most importantly, however, they contributed the first complete epidemiological material for influenza in Southern Greece, the archival nature of which constitutes valuable reference for future surveys.

On the mechanism of phenotypic conversion of human cervical adenocarcinoma HeLa cells surviving infection by influenza B virus: Potential implications for biological management of adenocarcinomas

We infected HeLa cells with low (10(-9) units), medium (10(-6) units), and high (10(-2) units) influenza B titers and compared the resulting human papilloma virus (HPV), retinoic acid receptor alpha subunit (RARalpha) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA content of surviving infected hosts with that of their uninfected precursors by semi-quantitative reverse transcriptase/polymerase chain reaction amplification (RT/PCR). This comparison revealed a moderate and drastic dependence of HPV and RARalpha mRNA content, respectively, but a complete independence of GAPDH mRNA expression on viral titer. A mechanism of adoptive replacement of tolerable cellular with viral gene expression was proposed to explain these findings. We conclude that the reported ability of influenza B viruses to specifically target and eliminate the cervical adenocarcinoma HeLa cell line studied may find practical applications in biological cancer management.

Expression of 6 common antigenic markers in invasive ductal breast carcinoma: potential clinical implications.

Expression of estrogen (ER) and progesterone receptors, c-erbB-2 oncogene, mutant p53 antioncogene (mp53), e-cadherin adhesion, and apoptotic caspase-8 antigens in tumor relative to matched normal tissue specimens from 102 unselected patients with primary ductal breast carcinoma of various tumor grades was assessed by immunohistochemistry and correlated with patients biologic and clinical features, such as age, menstrual status, age of menarche, tumor grade and diameter, the presence or absence of metastases, and number of infiltrated lymph nodes. We observed association of e-cadherin adhesion, ER and progesterone antigen marker expression with low histologic grade tumors and limited number of lymph node metastases and of c-erbB-2, mp53, and casp-8 antigen marker expression with high histologic grade tumors and increased number of lymph node metastases. We also observed strong correlation (P<0.05) between 4 of the 6 biomarkers and 4 of the 7 patient/tumor parameters examined. Our findings support the hypothesis of independent expression of these 4 strong biomarkers and reveal that nearly 40% of all breast tumor cases studied express similar proportions of 2 major phenotypic combinations [ER/c-erbB-2/mp53/casp-8: +/+/−/+ (19.6%) & +/−/−/+ (17.8%)]. We conclude that, in agreement with earlier reports, our findings support the diagnostic and potential prognostic value of these markers in the clinical assessment of breast cancer.

Immunophenotypic Evaluation of Dna Mismatch Repair Markers in 2 Cases of Synchronous Histomorphologically Distinct Gastric Adenocarcinomas With Gastrointestinal Stromal Tumors of the Proximal Small Bowel

ObjectivesTo assess the prognostic value of combined mismatch DNA repair (MMR) phenotyping in 2 synchronous histomorphologically distinct gastric adenocarcinomas (GADCs), each accompanied by gastrointestinal stromal tumors (GISTs) of the proximal small bowel. Summary Background DataA 72-year-old female and a 55-year-old male patient were submitted to partial and total gastrectomy, respectively, with synchronous resection of a GIST in the proximal small bowel. The 2 patients attained contrasting survival outcomes. The female survives disease-free 20 months after surgery having received no chemotherapy. The male who received adjuvant chemotherapy developed metastases in liver and lung, and died 18 months after surgery. MethodsWe phenotype MSH2 and MLH1 protein expression in tumor relative to matched normal tissue by immunohistochemistry. ResultsImmunohistochemistry analysis revealed different combined MMR phenotypes for the 2 histomorhologically distinct GADCs and similar for both GISTs studied. ConclusionsGood and bad prognosis for disease-free survival of patients based on reduced and elevated combined MMR phenotypic expression of the 2 histomorphologically distinct GADCs, could be explained by disease-associated emergence of genomic MMR alterations in the tumor. The impact of synchronous GISTs with common intermediate MMR phenotypes on patient survival is rather incidental and secondary to predominating GADCs.