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.

Autonomous vehicle navigation utilizing fuzzy controls concepts for a next generation wheelchair.

Three different positioning techniques were investigated to create an autonomous vehicle that could accurately navigate towards a goal: Global Positioning System (GPS), compass dead reckoning, and Ackerman steering. Each technique utilized a fuzzy logic controller that maneuvered a four-wheel car towards a target. The reliability and the accuracy of the navigation methods were investigated by modeling the algorithms in software and implementing them in hardware. To implement the techniques in hardware, positioning sensors were interfaced to a remote control car and a microprocessor. The microprocessor utilized the sensor measurements to orient the car with respect to the target. Next, a fuzzy logic control algorithm adjusted the front wheel steering angle to minimize the difference between the heading and bearing. After minimizing the heading error, the car maintained a straight steering angle along its path to the final destination. The results of this research can be used to develop applications that require precise navigation. The design techniques can also be implemented on alternate platforms such as a wheelchair to assist with autonomous navigation.

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.

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.

Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication.

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.

Description of an ectothermic TCR coreceptor, CD8 alpha, in rainbow trout.

We have cloned the first CD8 alpha gene from an ectothermic source using a degenerate primer for Ig superfamily V domains. Similar to homologues in higher vertebrates, the rainbow trout CD8 alpha gene encodes a 204-aa mature protein composed of two extracellular domains including an Ig superfamily V domain and hinge region. Differing from mammalian CD8 alpha V domains, lower vertebrate (trout and chicken) sequences do not contain the extra cysteine residue (C strand) involved in the abnormal intrachain disulfide bridging within the CD8 alpha V domain of mice and rats. The trout membrane proximal hinge region contains the two essential cysteine residues involved in CD8 dimerization (alpha alpha or alpha beta) and threonine, serine, and proline residues which may be involved in multiple O-linked glycosylation events. Although the transmembrane region is well conserved in all CD8 alpha sequences analyzed to date, the putative trout cytoplasmic region differs and, in fact, lacks the consensus p56lck motif common to other CD8 alpha sequences. We then determined that the trout CD8 alpha genomic structure is similar to that of humans (six exons) but differs from that of mice (five exons). Additionally, Northern blotting and RT-PCR demonstrate that trout CD8 alpha is expressed at high levels within the thymus and at weaker levels in the spleen, kidney, intestine, and peripheral blood leukocytes. Finally, we show that trout CD8 alpha can be expressed on the surface of cells via transfection. Together, our results demonstrate that the basic structure and expression of CD8 alpha has been maintained for more than 400 million years of evolution.

Analysis of preventable pediatric trauma deaths and inappropriate trauma care in Montana.

OBJECTIVE: To determine the rates of preventable mortality and inappropriate care, as well as the nature of treatment errors associated with pediatric traumatic deaths occurring in a rural state. METHODS: Retrospective multidisciplinary consensus panel review of deaths attributed to mechanical trauma in children aged 18 years or less, occurring in Montana between October 1, 1989, and September 30, 1992. The care rendered in both preventable and nonpreventable cases was evaluated for appropriateness according to nationally accepted guidelines. Rates of pediatric preventable death and inappropriate care, as well as the nature of inappropriate care, were compared with that of the adult population. RESULTS: One hundred thirty-eight cases were reviewed. One death (less than 1%) was judged frankly preventable, 11 deaths (8%) were judged possibly preventable, giving a total preventability rate of 9% for all cases reviewed. Considering only in-hospital deaths (n = 77), the total preventability rate was 16%. The rate of inappropriate care rendered for all deaths, regardless of preventability, was 36%. The rate of inappropriate care in the prehospital phase was 16%; for in-hospital deaths, it was 47%. In the emergency department (ED), the rate was 36%, and in post-ED care, 22%. In comparison to the adult population, the rates of preventable death (9% vs. 14%) and inappropriate care in the hospital phase (64% vs. 66%) were lower. Inappropriate care for the pediatric group was more prevalent in patients less than or equal to 14 years old. The nature of inappropriate care was most frequently associated with the management of respiratory problems, including airway control and management of chest trauma. CONCLUSION: Preventable mortality from traumatic injuries in children in a rural state appears to be low, and lower than that reported for adult trauma victims in the same state. A preponderance of these preventable deaths occur in the subgroup of children less than or equal to 14 years if age. Inappropriate trauma care in children occurs frequently, particularly in the ED phase of care, and is primarily associated with the management of the airway and chest injuries. Education of ED primary care providers in basic principles of stabilization and initial treatment of the injured child 14 years old or younger may be the most effective method of reducing preventable trauma deaths in the rural setting.

Expression, linkage, and polymorphism of MHC-related genes in rainbow trout, Oncorhynchus mykiss.

The architecture of the MHC in teleost fish, which display a lack of linkage between class I and II genes, differs from all other vertebrates. Because rainbow trout have been examined for a variety of immunologically relevant genes, they present a good teleost model for examining both the expression and organization of MHC-related genes. Full-length cDNA and partial gDNA clones for proteasome delta, low molecular mass polypeptide (LMP) 2, TAP1, TAP2A, TAP2B, class Ia, and class IIB were isolated for this study. Aside from the expected polymorphisms associated with class I genes, LMP2 and TAP2 are polygenic. More specifically, we found a unique lineage of LMP2 (LMP2/delta) that shares identity to both LMP2 and delta but is expressed like the standard LMP2. Additionally, two very different TAP2 loci were found, one of which encodes polymorphic alleles. In general, the class I pathway genes are expressed in most tissues, with highest levels in lymphoid tissue. We then analyzed the basic genomic organization of the trout MHC in an isogenic backcross. The main class Ia region does not cosegregate with the class IIB locus, but LMP2, LMP2/delta, TAP1A, and TAP2B are linked to the class Ia locus. Interestingly, TAP2A (second TAP2 locus) is a unique lineage in sequence composition that appears not to be linked to this cluster or to class IIB. These results support and extend the recent findings of nonlinkage between class I and II in a different teleost order (cyprinids), suggesting that this unique arrangement is common to all teleosts.

Cloning, structure, and function of two rainbow trout Bf molecules.

The factor B (Bf) and C2 complement genes are closely linked within the MHC class III region and are thought to have arisen by gene duplication from a single gene encoding an ancestral molecule; the animal phyla in which this duplication event took place is unknown. Two teleost fish, (zebrafish and medaka fish) have each been shown to possess only a single molecule that shows an equivalent degree of similarity to mammalian Bf and C2. In contrast, here we present the characterization of two factor B molecules (Bf-1 and Bf-2) in another teleost fish (the rainbow trout) that are about 9% more similar to mammalian factor B than C2, yet play a role in both alternative and classical pathways of complement activation. The full lengths of Bf-1 and Bf-2 cDNAs are 2509 and 2560 bp, respectively, and their deduced amino acid sequences are 75% identical. Both trout Bf genes are mainly expressed in liver and appear to be single-copy genes. The isolated Bf-1 and Bf-2 proteins are able to form the alternative pathway C3 convertase and are cleaved (in the presence of purified trout C3, trout factor D, and Mg2+ EGTA) into Ba- and Bb-like fragments in a manner similar to that seen for mammalian factor B. The most remarkable feature of trout Bf-2 is its ability to restore the hemolytic activity of trout Bf-depleted serum through both the alternative and classical pathways; whether Bf-1 possess similar activity is unclear at present.

Lymphocyte development in fish and amphibians.

Recently, molecular markers such as recombination activating genes (RAG), terminal deoxynucleotidyl transferase (TdT), stem cell leukemia hematopoietic transcription factor (SCL), Ikaros and gata-binding protein (Gata)-family members have been isolated and characterized from key lower vertebrates, adding to our growing knowledge of lymphopoiesis in ectotherms. In all gnathostomes there appear to be two main embryonic locations derived from the early mesoderm, both intra- and extraembryonic, which contribute to primitive and definitive hematopoiesis based upon their differential expression of SCL, Gata-1, Gata-2 and myeloblastosis oncogene (c-myb). In teleosts, a unique intraembryonic location for hematopoietic stem cells termed the intermediate cell mass (ICM) of Oellacher appears to be responsible for primitive or definitive hematopoiesis depending upon the species being investigated. In Xenopus, elegant grafting studies in combination with specific molecular markers has led to a better definition of the roles that ventral blood islands and dorsal lateral plate play in amphibian hematopoiesis, that of primitive and definitive lymphopoiesis. After the early embryonic contribution to hematopoiesis, specialized tissues must assume the role of providing the proper microenvironment for T and B-lymphocyte development from progenitor stem cells. In all gnathostomes, the thymus is the major site for T-cell maturation as evidenced by strong expression of developmental markers such as Ikaros, Rag and TdT plus expression of T-cell specific markers such as T-cell receptor beta and lck. In this respect, several zebrafish mutants have provided new insights on the development of the thymopoietic environment. On the other hand, the sites for B-cell lymphopoiesis are less clear among the lower vertebrates. In elasmobranchs, the spleen, Leydig's organ and the spiral valve may all contribute to B-cell development, although pre-B cells have yet to be fully addressed in fish. In teleosts, the kidney is undeniably the major source of B-cell development based upon functional, cellular and molecular indices. Amphibians appear to use several different sites (spleen, bone marrow and/or kidney) depending upon the species in question.

Conservation of a master hematopoietic switch gene during vertebrate evolution: isolation and characterization of Ikaros from teleost and amphibian species.

The generation of T, B and NK lymphocyte lineages from pluripotent hematopoietic stem cells is dependent upon the early expression of the Ikaros locus which by means of alternative splicing produces a variety of zinc finger DNA binding transcription factors. We assessed the general biological importance of Ikaros by studying its conservation and expression in teleost fish and amphibians. Portions of Ikaros cDNA from rainbow trout and Xenopus were amplified by reverse transcription-polymerase chain reaction (RT-PCR). They show roughly 75% conservation of the amino acid sequence with mammalian Ikaros. The trout fragment was then used to isolate full-length Ikaros clones from a trout thymocyte cDNA library. In mice and humans, Ikaros produces six alternatively spliced isoforms, but in trout two additional novel splice variants designated Ik-7 and Ik-8 were also found. Ik-7 is expressed in a similar fashion to Ik-1 and Ik-2, the predominant isoforms expressed in mammalian lymphocytes. In trout and zebrafish, as in mammals, Ikaros is a single-copy gene, but in Xenopus segregation analysis demonstrates that Ikaros has been duplicated, most likely a result of polyploidization. We then examined the expression of Ikaros in trout and Xenopus tumor T cell lines via Northern blot, RT-PCR, immunofluorescence and Western blot analysis. Overall, Ikaros is expressed in a lymphoid-specific fashion similar to that found in mice and humans. In addition Ikaros is expressed early in trout ontogeny, beginning roughly at days 3-4 in the yolk sac and at day 5-6 in the embryo proper. The conservation of Ikaros structure and expression confirms it as a master switch of hematopoiesis.

Characterization of rainbow trout terminal deoxynucleotidyl transferase structure and expression. TdT and RAG1 co-expression define the trout primary lymphoid tissues.

One component of antigen receptor diversity shared by all gnathostomes characterized to date is mediated by a unique DNA polymerase, terminal deoxynucleotidyl transferase (TdT), which generates significant functional diversity during immunoglobulin and T-cell receptor rearrangement. To gain further insight into the evolutionary origin(s) of this unique enzyme and the immune system, a thymic cDNA clone encoding TdT was isolated from rainbow trout. The 2.3 kilobase (kb) full-length clone contained an open reading frame of 1 506 base pairs with a deduced protein product of Mr 57 000. Sequence comparisons demonstrate that TdT has been highly conserved in both sequence (>70% aa similarity, >50 aa identity) and overall structure during the course of vertebrate evolution. An amino acid alignment of all known TdT sequences (chicken, Xenopus, mouse, human, cattle, and trout) reveals that some, but not all, structural motifs believed to be critical for TdT activity have been conserved. The TdT alignment, in conjuction with the recently solved crystal structure for rat beta-polymerase, supports the hypothesis that both may have evolved from a common ancestral repair gene. In addition, four PKC phosphorylation sites are conserved, and hence may be involved in TdT regulation. Because TdT contributes to the generation of junctional diversity in antigen receptors of immature lymphocytes, its expression serves as a developmental marker for the sites of teleost lymphopoiesis. Northern blot (2.3 kb message) analysis shows that TdT mRNA is highly expressed within the thymus and to a lesser extent in the pronephros. In addition, reverse transcriptase-polymerase chain reaction analysis detected transcipts of both RAG1 and TdT in the thymus, pronephros, mesonephros, spleen, and intestine, but not within muscle, liver, or brain. Finally, TdT cDNA was amplified from embryos at 20 days post-fertilization (pf), which correlates with the appearence of the thymus and pronephros anlage during trout ontogeny.

The glossy1 locus of maize and an epidermis-specific cDNA from Kleinia odora define a class of receptor-like proteins required for the normal accumulation of cuticular waxes.

Mutations at the glossy1 (gl1) locus of maize (Zea mays L.) quantitatively and qualitatively affect the deposition of cuticular waxes on the surface of seedling leaves. The gl1 locus has been molecularly cloned by transposon tagging with the Mutator transposon system. The epi23 cDNA was isolated by subtractive hybridization as an epidermis-specific mRNA from Senecio odora (Kleinia odora). The deduced amino acid sequence of the GL1 and EPI23 proteins are very similar to each other and to two other plant proteins in which the sequences were deduced from their respective mRNAs. These are the Arabidopsis CER1 protein, which is involved in cuticular wax deposition on siliques, stems, and leaves of that plant, and the protein coded by the rice expressed sequence tag RICS2751A. All four proteins are predicted to be localized in a membrane via a common NH2-terminal domain, which consists of either five or seven membrane-spanning helices. The COOH-terminal portion of each of these proteins, although less conserved, is predicted to be a water-soluble, globular domain. These sequence similarities indicate that these plant orthologs may belong to a superfamily of membrane-bound receptors that have been extensively characterized from animals, including the HIV co-receptor fusin (also termed CXCR4).

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.

The recombination activating gene 2 (RAG2) of the rainbow trout Oncorhynchus mykiss.

We have previously described the isolation and expression of RAG1 in trout to provide an initial understanding regarding the tissues involved in V(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating gene 2 (RAG2) of rainbow trout has now been cloned and characterized. The rainbow trout genomic RAG2 gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical of RAG2 sequences. The proximity of RAG1 and -2 within this teleost is similar to that found in other vertebrates. The genes are convergently transcribed and share a 3' untranslated (UT) region [2. 8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6 - 8 kb). The entire 3' UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for both RAG genes. Northern blot analysis of one-year-old trout demonstrated strong expression of RAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of both RAG1 and -2 in the thymus followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both genes are expressed in embryos beginning at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based upon RAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of the RAG genes as well as that of the immune system itself.

Analysis of preventable trauma deaths and inappropriate trauma care in a rural state.

OBJECTIVE: The goal of this study was to determine the rate of preventable mortality and inappropriate care in cases of traumatic death occurring in a rural state. DESIGN: This is a retrospective case review. MATERIALS AND METHODS: Deaths attributed to mechanical trauma throughout the state and occurring between October 1, 1990 and September 30, 1991 were examined. All cases meeting inclusion criteria were reviewed by a multidisciplinary panel of physicians and nonphysicians representing the prehospital as well as hospital phases of care. Deaths were judged frankly preventable, possibly preventable, or nonpreventable. The care rendered in both preventable and nonpreventable cases was evaluated for appropriateness according to nationally accepted guidelines. MEASUREMENTS AND MAIN RESULTS: The overall preventable death rate was 13%. Among those patients treated at a hospital, the preventable death rate was 27%. The rate of inappropriate care was 33% overall and 60% in-hospital. The majority of inappropriate care occurred in the emergency department phase and was rendered by one or more members of the resuscitation team, including primary contact physicians and surgeons. Deficiencies were predominantly related to the management of the airway and chest injuries. CONCLUSIONS: The rural preventable death rate from trauma is not dissimilar to that found in urban areas before the implementation of a trauma care system. Inappropriate care rendered in the emergency department related to airway and chest injury management occurs at a high rate. This seems to be the major contributor to preventable trauma deaths in rural locations. Education of emergency department primary care providers in basic principles of stabilization and initial treatment may be the most cost-effective method of reducing preventable deaths in the rural setting.

The recombination activation gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phylogenetic analysis.

The characterization of genes involved in the generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) genes, leading to the generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and -T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and examined its tissue- and lymphocyte-specific expression. The polymerase chain reaction, coupled with degenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone. Sequence analysis of this genomic clone confirmed the RAG1 nature of this gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAG1 sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAG1 to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.