Detection of human cytomegalovirus myocardial involvement by polymerase chain reaction during systemic infection and correlation with pp65 antigenemia and DNAemia in infected heart recipients.

The presence of human cytomegalovirus DNA was investigated in 103 unfixed endomyocardial biopsies, performed during the first 4 months in 17 heart transplant recipients by polymerase chain reaction. Results were correlated with human cytomegalovirus systemic infection, as detected by the test for the viral lower matrix phosphoprotein pp65 (antigenemia) and by polymerase chain reaction for viral DNA in blood leukocytes (DNAemia). Three patients out of 17 did not develop cytomegalovirus infection and 14 did: 5 had symptomatic disease treated with ganciclovir and 9 developed asymptomatic infection and were not treated. Viral DNA was detected in 24 out of 103 biopsies (23%) from 13 patients: 5 with symptomatic infection during the acute phase of disease (mean levels of pp65: 125+/-232 pp65 positive leukocytes/200,000 examined cells) and 8 patients with asymptomatic infection when the mean antigenemia was 5+/-15/200,000 (4 patients) or when DNAnemia was present in the blood (4 patients). No histological evidence of myocarditis was shown in viral DNA-positive biopsies. No difference in acute rejection was found in viral DNA-positive and DNA-negative biopsy specimens in symptomatic and asymptomatic infected patients. Our experience suggests that during systemic symptomatic and asymptomatic cytomegalovirus infection, polymerase chain reaction can detect a relatively frequent myocardial involvement, but this involvement is not associated with myocarditis or with a higher incidence of acute rejection. THe presence of viral DNA in myocardial biopsies can be a result of high viremia, but it also can be due to low level of viral DNA in circulating infected leukocytes. Polymerase chain reaction is the most sensitive method for cytomegalovirus DNA detection in biopsies, but its results need to be evaluated together with morphology-preserving methods and systemic markers of infection in order to make a correct diagnosis.

Glucocorticoid receptors immunoreactivity in tissue of human embryos.

The exact period when glucocorticoid receptors (GR) appear in human embryos is unknown, however their presence is acknowledged in target tissues before the fetal adrenal cortex secretes cortisol. Determining when GR develop could serve as an index of the importance of glucocorticoids in the morphological and functional development of tissues. The aim of this study was to determine time of onset of GR in human tissues using an immunohistochemical method. Results indicate GR are present in tissues of 8-10-week-old human embryos: in most tissues, the immune reaction was only or predominantly nuclear.

Intermediate biomarkers in the colorectal tumor progression.

Intermediate biomarkers are biological alterations in tissue which signal a stage of carcinogenesis between initiation and the development of a malignant tumor. Proliferation biomarkers are those that most satisfy the requisites for premorphological intermediate markers in the colorectal tumor progression. Cell proliferation changes in histologically normal intestinal mucosa are early events directly and closely associated with the morphogenesis of colorectal neoplasia. The transition from the morphological stage and the latter's further progression can be reliably monitored through the use of differentiation and genomic markers. In particular the incidence and the degree of DNA aneuploidy are indicators of the risk of malignant transformation in colorectal adenomas. New "regression-related" biomarkers should be investigated for the planning of measures designed to bring about the regression of premalignant lesions.

An alternative approach to the assessment of gamma delta T-cell clonality in celiac disease intestinal lesions through cDNA heteroduplex analysis of T-cell receptor VJ junctions.

We have investigated the clonality of the gamma delta T lymphocytes infiltrating the intestinal mucosa of CD patients and control subjects by means of a simple and powerful method based on the heteroduplex analysis of the TCR VJ junctions. Each V-specific TCR chain, amplified either from fresh biopsy material or intestinal T-cell-line cDNA, is denatured and renatured to allow the random reshuffling of the various strands carrying different junctional sequences, coamplified in the same reaction. The mismatched chains (heteroduplexes) are separated from the matched ones (homoduplexes) through polyacrylamide gel electrophoresis, and whenever one or more T-cell clones are emerging over the polyclonal background, discrete bands are visible by ethidium-bromide staining. Through this method, we have estimated the diversity of the V delta 1-3 chains and a newly described V gene (V delta 8) whose homologue in mice is abundantly expressed in gamma delta iLs. We demonstrate that the well-documented expansion of V gamma 1+ gamma delta lymphocytes in the jejunum of CD patients is polyclonal. Overall, the heteroduplex analysis on fresh intestinal and peripheral blood lymphocytes from both healthy and affected subjects shows a polyclonal pattern of all the V delta+ subsets. In contrast, most intestinal T-cell lines produce oligoclonal patterns, suggesting a dramatic in vitro selection effect. The cell expansion in culture is generally not required for the TCR heteroduplex analysis, which can therefore be applied to rapidly monitor the T-cell response in a variety of physiologic and autoimmune reactions, substituting the standard approach of TCR cloning and multiple VJ sequencing.

T cell receptor V delta 2-C alpha transcripts are present in the thymus but virtually absent in the periphery.

To investigate whether the V delta 2-(D)-J alpha gene configuration, characteristically associated with the major subset of acute lymphoblastic leukemias in humans, might have a physiologic role in T cell ontogeny, we have looked for V delta 2-C alpha transcripts in the thymus and peripheral blood of normal donors. Here we show by PCR analysis that these transcripts are virtually absent in the PBMC, whereas they are present in fetal and postnatal thymus. Interestingly, over 80% of 43 V delta 2-C alpha cDNAs randomly isolated from one postnatal thymus appeared to maintain an open reading frame. This suggests that in the thymus the V delta 2-C alpha products might be exposed to selective pressure. Furthermore, in two of three thymuses tested for J alpha usage, it was found overrepresented in a J alpha element (J alpha 58) located 2 kb downstream to a pseudo-J (J alpha 61), known to be a hot spot of recombination in alpha beta committed cells. A possible alternative pathway to alpha beta T cell differentiation via a V delta 2-J alpha intermediate is discussed.

T cell receptor heterogeneity in gamma delta T cell clones from intestinal biopsies of patients with celiac disease.

In celiac disease large numbers of gamma delta T lymphocytes infiltrate the intestinal epithelia. We have isolated intestinal gamma delta T cell clones from patients with celiac disease and have analyzed their T cell receptor repertoire. T cell lines and clones were obtained from jejunal biopsies of 14 celiac patients and 12 individuals without celiac disease. These were analyzed by staining with monoclonal antibodies against CD3, alpha beta and gamma delta T cell receptor, by Southern blot with gamma- and delta-specific probes and by polymerase chain reaction using V delta-specific oligonucleotides. Intestinal gamma delta cells from patients with celiac disease differed from those of controls with normal jejunal histology in that V delta 1+ cells and V delta 1-V delta 2- cells were significantly increased. There was no evidence of the expansion of one or more clones expressing particular types of gamma delta T cell receptor.