Screening of subfertile men for testicular carcinoma in situ by an automated image analysis-based cytological test of the ejaculate.

Testicular cancer (TC) is usually diagnosed after manifestation of an overt tumour. Tumour formation is preceded by a pre-invasive and asymptomatic stage, carcinoma in situ (CIS) testis, except for very rare subtypes. The CIS cells are located within seminiferous tubules but can be exfoliated and detected in ejaculates with specific CIS markers. We have built a high throughput framework involving automated immunocytochemical staining, scanning microscopy and in silico image analysis allowing automated detection and grading of CIS-like stained objects in semen samples. In this study, 1175 ejaculates from 765 subfertile men were tested using this framework. In 5/765 (0.65%) cases, CIS-like cells were identified in the ejaculate. Three of these had bilateral testicular biopsies performed and CIS was histologically confirmed in two. In total, 63 bilateral testicular biopsy were performed in conjunction with analysis of the ejaculates because of infertility work-up. Histological analysis of the biopsies for the presence of CIS yielded a test sensitivity of 0.67 and a specificity of 0.98. In addition, ejaculates from 45 patients with clinical signs of an overt TC were investigated and yielded a slightly lower sensitivity (0.51), possibly because of obstruction. We conclude that this novel non-invasive test combining automated immunocytochemistry and advanced image analysis allows identification of TC at the CIS stage with a high specificity, but a negative test does not completely exclude CIS. On the basis of the results, we propose that the assay could be offered to subfertile men and other patients who are at increased risk of TC.

Evolutionary history of the ABCB2 genomic region in teleosts.

Gene duplication, silencing and translocation have all been implicated in shaping the unique genomic architecture of the teleost MH regions. Previously, we demonstrated that trout possess five unlinked regions encoding MH genes. One of these regions harbors ABCB2 which in all other vertebrate classes is found in the MHC class II region. In this study, we sequenced a BAC contig for the trout ABCB2 region. Analysis of this region revealed the presence of genes homologous to those located in the human class II (ABCB2, BRD2, psiDAA), extended class II (RGL2, PHF1, SYGP1) and class III (PBX2, Notch-L) regions. The organization and syntenic relationships of this region were then compared to similar regions in humans, Tetraodon and zebrafish to learn more about the evolutionary history of this region. Our analysis indicates that this region was generated during the teleost-specific duplication event while also providing insight about potential MH paralogous regions in teleosts.

Lowering of plasma cholesterol levels in free-living adolescent males; use of natural and synthetic polyunsaturated foods to provide balanced fat diets.

Two hundred and twenty-nine adolescent male pupils, attending two boarding schools, participated in a study, under free-living dietary conditions, designed to assess the effects on plasma lipids of altering only the type and not the amount of dietary fat. The students were monitored for 6 weeks on three different diets. During the first study period, dietary changes comprised substituting a polyunsaturated dried "filled" milk and products derived therefrom for conventional dairy products (diet A). The second dietary phase involved replacing all meat and dairy products with equivalent polyunsaturated ruminant fat products (diet B). The third period consisted of a control diet of conventional dairy and meat products. During both polyunsaturated diets a 14% reduction in plasma cholesterol was achieved compared with control levels. Low-density lipoprotein cholesterol was similarly suppressed. A greater decrease in plasma cholesterol of 19-21% was recorded in those pupils with initial cholesterol levels greater than 230 mg/dl. There was no significant difference in plasma triglycerides between diet A and the control diet whereas the triglyceride levels were 16.5% lower during diet B. The fall in plasma cholesterol coincided with an increase in plasma triglyceride linoleate from 8% to 19% of total triglyceride fatty acids. Dietary analysis of the pupils' diet indicated an increase in the ratio of polyunsaturates to saturates from 0.27 during the control period to 1.08 on the dried filled milk and 0.90 on the polyunsaturated ruminant fat products. A palatability survey showed that both the dried filled milk and the polyunsaturated ruminant fat products were as well accepted by the pupils as the conventional products. The results suggest that such products could, if introduced to the general population, play an important part in plasma cholesterol suppression in the hope that this would significantly reduce the incidence of coronary artery disease.

Conservation of an alpha 2 domain within the teleostean world, MHC class I from the rainbow trout Oncorhynchus mykiss.

A full-length cDNA clone (Onmy-UA-C32) encoding a major histocompatibility complex (MHC) class I heavy chain was isolated from a rainbow trout thymus cDNA library. Onmy-UA-C32 alpha I and III extracellular domains were most similar to other salmonids (92 and 86% at the nucleotide and amino acid level) but interestingly the alpha II domain is closer to that of the carp (74 and 73%) and zebrafish (75 and 70%). In addition, Onmy-UA-C32 displays conservation of residues known to be essential for the function and structure of MHC class Ia molecules. Northern blot hybridization with alpha 2 or 2-3 domain probes of Onmy-UA-C32 detected high expression (2.6 kb) of this gene in the spleen, thymus, kidney, heart and intestine with lower levels being observed in the brain and liver. No tissues were found to be negative indicating a ubiquitous pattern of expression for Onmy-UA-C32. Onmy-UA-C32 may therefore represent a MHC class Ia gene in trout as well as providing new insights regarding the evolution of the MHC within teleost species.

Potential involvement of rainbow trout thrombocytes in immune functions: a study using a panel of monoclonal antibodies and RT-PCR.

The functional relationship between fish and mammalian thrombocytes is relatively unknown. In this study, a panel of monoclonal antibodies (mAbs) was used to investigate the functional properties of rainbow trout thrombocytes. The mAbs recognize cell-surface molecules on thrombocytes with molecular weights ranging from 17 to 160 kDa. Flow cytometric and immuno-electron microscopic analyses demonstrate that these molecules are expressed at different levels and that surface expression increased upon activation with bovine collagen. Two of these cell-surface molecules (17 and 21 kDa) were directly involved in collagen-induced aggregation of thrombocytes since aggregation was blocked upon pre-treatment with mAbs that recognize the two surface markers. Interestingly, the percentage of thrombocytes in blood increased after stimulation using different antigens. The transcriptional profile of trout thrombocytes was then examined after immuno-magnetic enrichment using the described mAbs to assess potential roles of trout thrombocytes in immune functions. Trout thrombocytes express components of the MHC class Ia pathway, IL1beta, TNFalpha, TGFbeta, the interleukin receptor common gamma chain as well as CXC and CC chemokines. MHC class IIB and TNFalpha were expressed at low levels in resting thrombocytes. No evidence was found for the expression of TCRalphabeta, Ig heavy chain, CD8alpha or CK1 mRNA. Taken together, these results suggest that rainbow trout thrombocytes express molecules involved in activation, aggregation and genes encoding proteins, that are involved in antigen presentation and immune regulation.