bluuebunny/crossref_metadata_2025 huggingface dataset:
Neural differentiation of rhesus embryonic stem cells
Authors: THOMSON JAMES A. , MARSHALL VIVIENNE S. , TROJANOWSKI JOHN Q.
Published: August 2009

Primate embryonic stem (ES) cells are capable of indefinite, undifferentiated proliferation and maintain the potential to differentiate to trophoblast and derivatives of embryonic endoderm, mesoderm, and ectoderm. We previously reported that neural differentiation by rhesus ES cells in teratomas includes tissue with a remarkable resemblance to neural tube (Thomson et al. 1995). Here we examine a series of markers including a cell proliferation marker, neurofilament proteins, and glial fibrillary acidic protein (GFAP) in teratomas at 5, 6, 7, 9, and 12 weeks after rhesus ES cell transplantation into muscles of immunodeficient mice. All teratomas examined contained derivatives of all three embryonic germ layers. Neural differentiation included tissues resembling neural tube and embryonic ganglia, as well as individual dispersed neurons, and brain‐like gray matter. Tumours of all ages contained neurons and proliferating cells, indicated by staining for neurofilament subunits and Ki67 antigens. Younger tumours contained no or few astrocytes indicated by the absence of GFAP staining, but as these tumours developed, there was an increase in astrocyte differentiation. The results indicate that normal neural differentiation is recapitulated, in part, by the differentiation of rhesus ES cells in teratomas. The differentiation of rhesus ES cells provides an important new model for understanding human neural differentiation.
CD30 and its ligand: Possible role in regulation of teratoma stem cells
Authors: PERA MARTIN F. , BENNETT WILLIAM , CERRETTI DOUGLAS P.
Published: August 2009

Like the oocyte, the cells of the early embryo, and primordial germ cells, human teratocarcinoma stem cells are pluripotent, capable of giving rise to a wide range of somatic and extraembryonic tissues. Growth factors which regulate the growth of multipotent stem cells in the mouse have been identified, but none of these have been shown conclusively to have similar effects on human or primate multipotent stem cells. CD30 is a member of the tumour necrosis factor receptor superfamily with a restricted pattern of tissue distribution, limited to immune cells, decidual tissue, and human embryonal carcinoma: in common with other embryonal carcinoma markers, CD30 is found in foci of cells in a sub‐population of seminomas. CD30 ligand is a transmembrane protein, structurally related to tumour necrosis superfamily members TNFα, TNFβ, and CD40. CD30 ligand is expressed by T and B lymphocytes, macrophages, and a variety of normal haematopoietic cells and tumours derived from them, and exerts pleiotropic effects on normal and malignant lymphoid cells, including death, differentiation, or cell division. Studies on cultured cell lines derived from human embryonal carcinomas and yolk sac carcinomas confirm CD30 expression in the former but not the latter, and show that CD30 expression is down‐regulated during stem cell differentiation in vitro. Transcripts for CD30 ligand are found at highest levels in yolk sac carcinoma cell lines, but are also found in embryonal carcinoma. CD30 ligand protein is detected in yolk sac carcinoma and nullipotent embryonal carcinoma cell lines. Exogenous CD30 ligand has no effect on multipotent human stem cell growth in vitro. However, the receptor‐ligand pair may function in autocrine regulation of embryonal carcinoma stem cells. CD30 and its ligand are candidate stem cell identity factors, juxtacrine regulators whose sole function is to identify a cell's position in a developmental hierarchy.
Testicular teratocarcinogenesis in mice — a review
Authors: MATIN ANGABIN , COLLIN GAYLE B. , VARNUM DON S. , NADEAU JOSEPH H.
Published: August 2009

Spontaneous testicular germ cell tumours in humans and mice are remarkable for their diverse composition. These tumours are usually composed of an extraordinary variety of cell and tissue types including muscle, skin, bone, cartilage, and neuroepithelia. Their diverse composition reflects their origin from totipotent primordial germ cells at about Day 12 of fetal development. Although much is known about the development of these tumours, remarkably little is known about the genetics of the mammalian primordial germ cell lineage or about the genes that control susceptibility to spontaneous testicular germ cell tumours in humans or mice. Conventional genetic analysis of susceptible 129/Sv mice is difficult because of the large number of susceptibility genes and their low penetrance. We are taking advantage of the Ter mutation to simplify the genetic analysis. Various evidence suggests that Ter is neither necessary nor sufficient for tumourigenesis. Instead, Ter acts as a modifier, dramatically increasing tumour incidence from ˜1% in +/+ males, to ˜17% in Ter/+ males and ˜94% in Ter/Ter males. Segregation analysis suggests that Ter increases tumour incidence by requiring some, but perhaps not all, of the 129/Sv‐derived susceptibility genes. With standard crosses that segregate for the Ter mutation, identification not only of Ter but also of these 129/Sv‐derived susceptibility genes should be possible. In this paper, we review the genetics and development of germ cell tumours in 129/Sv mice, summarize the status of Ter mapping, and provide evidence that different genetic pathways lead to unilateral and bilateral tumours.
Carcinoma in situ and seminoma in equine testis
Authors: VEERAMACHANENI D. N. RAO , SAWYER HEYWOOD R.
Published: August 2009

The presence of atypical germ cells resembling carcinoma in situ of human testis is reported for the first time in an unilaterally cryptorchid stallion. These cells were found in association with developing intratubular seminoma indicating they represented carcinoma in situ.
Genomic imprinting in testicular germ cell tumours
Authors: LOOIJENGA LEENDERT H. J. , VERKERK ANNEMIEKE J. M. H. , DEKKER MARJOLEIN C. , VAN GURP RUUD J. H. L. M. , GILLIS AD J. M. , OOSTERHUIS J. WOLTER
Published: August 2009

Genomic imprinting refers to the parental origin‐specific functional difference between the paternally and maternally‐derived mammalian haploid genome. Normal embryogenesis depends on the presence of both a paternal and a maternal copy of particular chromosomal regions, containing the so‐called imprinted genes. Genomic imprinting is established somewhere in the maturation from a primordial germ cell to a mature gamete, either spermatid or oocyte. We discuss the value of testicular cancers, especially those derived from the germ cell lineage, as a model to study erasement of the biparental pattern of genomic imprinting as present in the zygote and establishment of the paternal pattern during spermatogenesis. In addition, we will present data on the presence of X‐inactivation in these cancers.
Developmental arrest of germ cells in the pathogenesis of germ cell neoplasia
Authors: MEYTS EWA RAJPERT‐DE , JØRGENSEN NIELS , BRØNDUM‐NIELSEN KAREN , MÜLLER JØRN , SKAKKEBÆK NIELS E.
Published: August 2009

Clinical observations and epidemiological evidence suggest that important aetiopathological events that cause neoplastic transformation of the male germ cell may occur in fetal life or early infancy. The incidence of germ cell neoplasia is high in individuals with various disorders of gonadal development and sexual differentiation, such as gonadal dysgenesis or androgen insensitivity syndrome. Increased risk has also been noted in individuals with trisomy 21, idiopathic infertility and low birth weight. Infertility is sometimes associated with small aberrations of sex chromosomes (e.g. low frequency mosaicism XY/XO) which can also be found in patients with testicular cancer. The variety of conditions that predispose to testicular neoplasia and the rise in its incidence in many countries speaks for the influence of environmental factors which may affect genetically predisposed individuals. We hypothesise that if the development of the testis is disturbed or delayed, primordial germ cells or gonocytes undergo maturation delay or differentiation arrest which may render them susceptible to neoplastic transformation. Morphologically homogenous premalignant carcinoma in situ (CIS) cells have the potential to differentiate into a variety of histological forms of overt testicular tumours. Analysis of cell surface antigens expressed by CIS cells found in the vicinity of pure and mixed tumours demonstrates that CIS cells are phenotypically heterogeneous. Comparison of the phenotypes of CIS cells, primordial germ cells, human embryonal carcinoma cells and closely related primate embryonal stem cells reveals various similarities but also differences. We speculate that phenotypical heterogeneity of CIS cells may be associated with their potential to give rise to different tumour types, and may be related to the developmental stage of the early germ cell which has undergone malignant transformation.
The platelet‐derived growth factor α‐receptor 1.5 kb transcript: Target for molecular detection of testicular germ cell tumours of adolescents and adults
Authors: OOSTERHUIS J. WOLTER , GILLIS AD J. M. , VAN ROOZENDAAL CORNELIUS E. P. , VAN ZOELEN EVERARDUS J. J. , LOOIJENGA LEENDERT H. J.
Published: August 2009

An accurate and relatively simple method to detect testicular germ cell tumours of adolescents and adults (TGCTs) could be useful for early detection and may help to avoid unnecessary orchidectomies. We report a highly sensitive reverse‐transcription polymerase chain reaction assay for the 1.5 kb transcript of the platelet‐derived growth factor‐α receptor for molecular diagnosis of TGCTs. As a simulation of the clinical application of the assay the approach was applied on through‐cut‐biopsies from orchidectomy specimens. In a series of 31 specimens, the 1.5 kb transcript was detected in all samples containing either carcinoma in situ, or an invasive TGCT, with mature teratoma as only exception. No expression was detected in normal parenchyma. On the basis of the through‐cut‐biopsies the assay shows a sensitivity of 0.87 and a specificity of 1.00. The positive and negative predictive values of the test are 1.00 and 1.00. For carcinoma in situ alone these values are 0.86, 1.00, 1.00, and 0.80, respectively. The figures at least equal the results obtained with the most sensitive morphological/enzyme‐histochemical study of duplicate biopsies. We conclude that the 1.5 kb transcript of the platelet‐derived growth factor‐α receptor is a useful molecular marker for TGCTs, and therefore of interest in a clinical setting.
Human endogenous retrovirus (HERV)‐K transcripts in germ cell and trophoblastic tumours
Authors: HERBST H. , SAUTER M. , KÜHLER‐OBBARIUS C. , LÖNING T. , MUELLER‐LANTZSCH N.
Published: August 2009

Prompted by the observation of retroviral particle formation in teratocarcinoma cell lines and the consistent finding of antibodies against Gag and Env proteins encoded by human endogenous retrovirus (HERV)‐K genomes in the sera of patients with classical seminoma, we studied ovarian and testicular germ cell tumours, their precursor lesions, dysgenetic gonads, and trophoblast lesions for expression of HERV‐K sequences by in situ hybridization using radioactive and non‐radioactive probes. HERV‐K transcripts were detected in all testicular and ovarian germ cell tumours with the exception of teratomas and spermatocytic seminomas. HERV‐K expression was also common to testicular carcinoma in situ as well as gonocytes of dysgenetic gonads. Among gestational trophoblastic lesions, HERV‐K expression was regularly found in choriocarcinomas, but not in molar lesions. The patterns of HERV‐K expression suggest a common molecular pathogenesis of most germ cell tumour entities and malignant gestational trophoblastic disease. They furthermore support the concept of carcinoma in situ as a precursor lesion common to most testicular germ cell neoplasms. The detection of HERV‐K gene products in body fluids and tissues may aid diagnosis and monitoring of germ cell tumours and related lesions.
The origin and biology of CIS cells: General discussion
Authors: GRIGOR KENNETH M. , WYLIE CHRISTOPHER C.
Published: August 2009

Participants at the 4th Copenhagen Workshop on Carcinoma in situ and Cancer of the Testis, representing cell biologists and tumour biologists, met together to discuss the similarities and differences between primordial germ cells (PGCs) of the embryo, and the carcinoma in situ (CIS) stem cell of human testicular germ cell tumours (GCTs). Much has been discovered about PGCs in the last 10 years and we still do not know the exact nature of CIS cells. Knowledge of PGCs comes mainly from mouse experiments and knowledge of CIS comes from the study of human tumours. A mouse model of human GCT would help to investigate the nature of CIS cells. Grafting mouse male genital ridges into mouse fetal testes results in the development of testicular tissue and the formation of teratomatous tumour components. Amplification of PGCs in culture is possible but this results in their transformation into embryonic germ (EG) cells. CIS cells die by apoptosis if they are isolated, and short‐term culture is only possible if the CIS cells are cultured in their normal environment within seminiferous tubules. It may be possible for CIS cells to differentiate in culture although they cannot be maintained in culture as isolated cells. Human CIS cells are likely to be formed as a result of in utero factors rather than agents acting on normal adult testicular germ cells. EG cells stimulate feeder cells by paracrine factors but it is not known if these cells produce autocrine factors.
Trends in sex‐ratio, testicular cancer and male reproductive hazards: Are they connected?
Authors: MØLLER HENRIK
Published: August 2009

In the last few decades, the male proportion of newborn babies has been decreasing in several populations. The changes are very small and without practical importance per se, but the underlying biological mechanisms are not known. In the same period, testicular cancer incidence has increased, and there has been indications of decreasing sperm counts in men in several populations. The available knowledge on factors that influence the sex‐ratio in humans supports the idea that an excess of girls in the offspring of a man may be an indicator of reproductive hazards. Data from a Danish case‐control study show strong associations between testicular cancer, low fertility and a low M:F sex‐ratio in the offspring. It is proposed as a hypothesis that there may exist common aetiological factors for testicular cancer, low fertility and low offspring sex‐ratio, and that a search for the causal factors involved may focus on agents that can act prenatally to disrupt the normal development and differentiation of the male reproductive organs.
Are estrogens carcinogenic during development of the testes?
Authors: McLACHLAN JOHN A. , NEWBOLD RETHA R. , LI SHAUNFANG , NEGISHI MASAHIKO
Published: August 2009

Many chemicals in the environment mimic the female sex hormone, estrogen. Exposure to environmental estrogens during early fetal development was proposed by Sharpe & Skakkebœk as a potential risk factor for subsequent testicular disease, including neoplasia and poor semen quality. To understand the mechanisms of action of estrogenic chemicals during differentiation of the male genital tract, we have studied developmental exposure to the synthetic estrogen, diethylstilboestrol (DES). While DES is a much more potent estrogen than most environmental chemicals examined, several of these compounds share some of the same properties as DES, such as a relative lack of binding to serum estrogen carrying proteins. Prenatal exposure to DES is associated with poor semen quality, prostatic disease, cryptorchidism and testicular neoplasia in mice. A rare form of testicular cancer, rete testis carcinoma, was observed in five percent of male mice treated in utero with DES. We also demonstrated altered regulation of an estrogen responsive gene, lactotransferrin (LTF) in the seminal vesicles of treated mice, but not the controls. Likewise, LTF was irreversibly altered in the uteri of developmentally treated females; at the molecular level altered methylation of the gene appears to be involved, thus, providing a potential marker for hormonal effects during development. The induction of permanent or “imprinted” responses during the development of a relatively estrogen‐free reproductive tract cell suggests that undifferentiated targets for estrogen action may be sites for subsequent growth and differentiation defects associated with neoplasia.
Detection of oestrogenic chemicals by assaying the expression level of oestrogen regulated genes
Authors: JØRGENSEN MARIANNE , HUMMEL RENÉ , BÉVORT MAJA , ANDERSSON ANNA‐MARIA , SKAKKEBÆK NIELS E. , LEFFERS HENRIK
Published: August 2009

The oestrogen receptor belongs to the superfamily of nuclear receptors. Classically, nuclear receptors are thought to reside either in the nucleus or in the cytoplasm where they interact with their ligand which induces a conformational change that exposes the DNA binding domain. This is followed by dimerisation and binding of their corresponding response elements. By interacting with the transcriptional apparatus they then either activate or repress the transcription of target genes. However, this is a highly simplified view, since the activated oestrogen receptor interacts with other signal transduction pathways and its intrinsic transcriptional activity is highly influenced by phosphorylation and by its interaction with other proteins. This is clearly observed when the oestrogenicity of antioestrogens is tested since some compounds activate the receptor in yeast, but not in mammalian cells. However, when specific kinases are activated antioestrogens can also function as oestrogens in mammalian cells. Moreover, components of the MAP kinase and perhaps the cAMP and other pathways are activated before the receptor even enters the nucleus. Thus, when analysing the effects of oestrogenic compounds, it is important to assay both their potency as activators of transcription as the effects caused by interactions with other signal transduction pathways. This may be possible by combining assay methods, such as direct in vitro measurement of interaction between a potential oestrogenic chemical and the receptor or the yeast E‐screen, with methods that are based on mammalian cells or whole animals. An alternative is to assay gene expression directly by methods such as differential display, where the expression of both genes known to be regulated directly by the receptor and genes regulated by other pathways can be monitored. Thereby it may be possible to assign different responses to the activation of distinct pathways.
The 4th Copenhagen Workshop on Carcinoma in situ and Cancer of the Testis: Concluding remarks
Authors: de KRETSER DAVID M. , DAMJANOV IVAN
Published: August 2009

The 4th Copenhagen Workshop “Carcinoma in situ Germ Cell and Testicular Cancer: Molecular and Endocrine Aspects” was held in Copenhagen, Denmark, May 18–21, 1997. This paper discusses the major themes that emerged during the workshop and summarises the most important contributions.
Lipofuscin: Mechanisms of formation and increase with age
Authors: TERMAN ALEXEI , BRUNK ULF T.
Published: August 2009

Lipofuscin (age pigment) is a brown‐yellow, electron‐dense, autofluorescent material that accumulates progressively over time in lysosomes of postmitotic cells, such as neurons and cardiac myocytes. The exact mechanisms behind this accumulation are still unclear. This review outlines the present knowledge of age pigment formation, and considers possible mechanisms responsible for the increase of lipofuscin with age. Numerous studies indicate that the formation of lipofuscin is due to the oxidative alteration of macromolecules by oxygen‐derived free radicals generated in reactions catalyzed by redox‐active iron of low molecular weight. Two principal explanations for the increase of lipofuscin with age have been suggested. The first one is based on the notion that lipofuscin is not totally eliminated (either by degradation or exocytosis) even at young age, and, thus, accumulates in postmitotic cells as a function of time. Since oxidative reactions are obligatory for life, they would act as age‐independent enhancers of lipofuscin accumulation, as well as of many other manifestations of senescence. The second explanation is that the increase of lipofuscin is an effect of aging, caused by an age‐related enhancement of autophagocytosis, a decline in intralysosomal degradation, and/or a decrease in exocytosis.
Triggering of renal tissue damage in the rabbit by IgG Fc‐receptor‐positive group A streptococci
Authors: BUROVA LARISSA A. , NAGORNEV VLADIMIR A. , PIGAREVSKY PETER V. , GLADILINA MARIA M. , SELIVERSTOVA VALENTINA G. , SCHALEN CLAES , TOTOLIAN ARTEM A.
Published: August 2009

Our previous studies have shown that streptococcal IgG Fc receptors (FcR) act to elicit circulating anti‐IgG as well as renal glomerular deposition of IgG in rabbits immunized with group A streptococci (GAS). In order to study if other FcR‐positive bacteria might have similar effects, rabbits were immunized with either group G streptococci (GGS; strain G148) or Staphylococcus aureus (strain Cowan I) for two periods of 8 and 6 weeks, respectively. At the end of immunization, circulating anti‐IgG was found in 6 of 20 (30%) and 4 of 19 (21%) animals receiving G148 and Cowan I, respectively, compared to all 28 receiving FcR‐positive GAS strains of types M1, M4, M15 or M22 (p < 0.05 for both comparisons); furthermore, anti‐IgG appeared earlier and at higher levels in the GAS groups. Weak glomerular IgG deposits occurred in 5 out of 10 (50%) and 2 out of 8 (25%) animals immunized with G148 and Cowan I, respectively. In contrast, all 11 rabbits examined, given GAS of types M1 or M15, displayed heavy deposits. None of four control animals immunized with either of two FcR‐negative strains, GAS type T27 or group B streptococci (GBS) type la, exhibited any renal IgG deposits or circulating anti‐IgG. Renal tissue materials from rabbits immunized with any of the four FcR‐positive GAS strains showed strong inflammatory and degenerative glomerular changes, compatible with the picture seen in acute poststreptococcal glomerulonephritis (APSGN). Only transient renal changes were found in those rabbits immunized with G148 or Cowan I, or the controls injected with the FcR‐negative strains, GAS type T27 or GBS. Thus, only the FcR‐positive GAS strains showed capacity to induce high levels of anti‐IgG, pronounced tissue deposition of IgG as well as irreversible glomerular changes. Our experimental data suggest that streptococcal IgG FcR activity might play an important role in triggering APSGN.
Estimation of type‐specific anti‐streptococcal M‐protein antibodies with biotinylated peptides in human sera
Authors: KÜHNEMUND O. , SCHUMACHER F. , CASARETTO M. , HOLM S. E. , LÜTTICKEN R.
Published: August 2009

The design of a novel enzyme‐linked immunosorbent assay for the estimation of antibodies directed against the N‐terminus of the M‐protein of Streptococcus pyogenes is described. The ELISA employs biotinylated peptide antigens of the types 1, 4, 12 and 19 immobilized by (strept‐)avidin on the surface of polystyrene microtiter wells. In rabbit hyperimmune sera and in human serum samples, antibodies against the corresponding serotype could be detected with high sensitivity and specificity.
No signs of activity markers in peripheral blood despite increased bronchial reactivity after repeated low‐dose allergen exposure
Authors: ROQUET ANNIKA , LAGGING EVA , IHRE ELISABETH , VAN HAGE‐HAMSTEN MARIANNE , HALLDÉN GUNILLA , HÄRFAST BENGT , ZETTERSTRÖM O.
Published: August 2009

The allergen inhalation test can be used as an experimental model to study pathophysiological events in allergic asthma. Repeated low‐dose inhalations of allergen induce increased bronchial hyperresponsiveness (BHR) and resemble natural allergen exposure. The objective of the present study was to investigate whether eosinophil recruitment and activation in peripheral blood, differences in expression of lymphocyte surface antigens and increased bronchial responsiveness to histamine occur during and after repeated low‐dose bronchial allergen challenge. Fourteen atopic asthmatic patients were challenged in a randomized double‐blind manner for 7 days with either allergen in very low doses or placebo. We measured the concentration of eosinophils, eosinophil cationic protein (ECP) and the expression of the EG2‐epitope on intracellular ECP in eosinophils and the expression of lymphocyte surface antigen markers in peripheral blood. The challenge period started and ended with a histamine provocation. The repeated low‐dose allergen exposure resulted in a significant increase in BHR. No changes were seen in the placebo group. Concerning the inflammatory parameters in peripheral blood, no significant changes were seen during or after the week of low‐dose allergen inhalations. Our results show that very low, repeated doses of allergen induce increased airway reactivity despite lack of evident clinical symptoms or signs of activation of inflammatory cells in peripheral blood.
Lung hypoplasia – a possible teratogenic effect of valproate
Authors: JANAS METTE STAGAARD , ARRØE METTE , HANSEN STEEN HOLGER , GRÆM NIELS
Published: August 2009

Three cases of lung hypoplasia occurring in two siblings and in an unrelated child are reported. All three cases had been exposed to valproate in utero. The two female siblings had no other malformations, whereas the third female had a number of defects consistent with the fetal valproate syndrome. Thus, none of the known major causes of lung hypoplasia was present in any of the three cases. It is discussed whether pulmonary hypoplasia may be a separate disease entity, or caused by prenatal exposure to valproate.
Induction of cvtokines and expression of surface receptors in Mono Mac 6 cells after infection with different Legionella species
Authors: NEUMEISTER B. , KLEIHAUER A. , ROSSMANN V. , FEHRENBACH E. , FAIGLE M. , BAUMBACH S. , NORTHOFF H.
Published: August 2009

The ability of Legionella species to multiply within human mononuclear phagocytes is usually regarded as being associated with their pathogenicity. Activation of host cells results in inhibition of intracellular Legionella multiplication. The most effective substance to induce macrophage activation, both in vivo and in vitro, is interferon‐γ. In addition, some evidence exists that macrophage‐derived cytokines may contribute to the host defense against L. pneumophila, but the production of pro‐ and antiinflammatory cytokines by monocytes after infection with different Legionella species has not been reported with regard to their ability to multiply within the host cells. We therefore examined the production of TNF‐α, IL‐1, IL‐6, IL‐8, IL‐10 and TGF‐β by Mono Mac 6 cells after infection with Legionella species of different human prevalence that differ in their ability to replicate within this macrophage‐like cell line. After infection, Mono Mac 6 cells showed a cytokine response with time kinetics characteristic for the cytokine. Maximum cytokine levels produced differed with Legionella species, but were not related to intracellular multiplication rates. Moreover, LPS‐tolerant Mono Mac 6 cells, which failed to produce cytokines, showed intracellular increase or decrease of bacterial numbers identical to that of untreated Mono Mac 6 cells. By FACS analysis, an up‐regulation of CD14 (LPS receptor) and CD54 (ICAM‐1) could be demonstrated. We conclude that, in the Mono Mac 6 cell line, induction of macrophage‐derived cytokines after infection with members of the genus Legionella mimics an inflammatory reaction without association with intracellular multiplication rate.
Viremia in chronic hepatitis C patients evaluated by the Amplicor RT‐PCR, a nested RT‐PCR, and transaminase levels
Authors: LAURSEN ALEX L. , INGERSLEV JØRGEN , ANDERSEN PAUL L. , ØTERGAARD LARS
Published: August 2009

A commercially available kit, Amplicor®, was compared with a locally developed nested reverse‐transcriptase (RT) PCR for qualitative detection of HCV‐RNA. Sixty‐one serum samples from sixty‐one patients with liver disease, and 60 samples from 60 hemophiliacs without symptoms, but known to have been heavily exposed to hepatitis C virus, were investigated. There was a high degree of concordance between the two diagnostic tests (97%), the Amplicor® kit being slightly more sensitive than the in‐house PCR, when evaluated using serial dilutions of samples showing discrepant results. The relationship between viremia and abnormal ALT levels was studied in the two groups of patients. Among those with chronic liver disease, 8.3% of patients with viremia had normal ALT levels, whereas transaminases were normal in 20% of hemophiliacs with viremia. This points to ALT as being a poor marker of ongoing viral replication.
Veto cell suppression mechanisms in the prevention of allograft rejection
Authors: JACOBSEN IDA MARIE , CLAESSON MOGENS H.
Published: August 2009

Substantial evidence has accumulated to suggest that in the near future implementation of the veto‐cell‐suppressor concept in the treatment of kidney allograft recipients might lead to the establishment of life‐long specific allograft tolerance in the absence of further immunosuppressive therapy. Veto suppression prevents the generation of antigen‐specific T‐helper and cytotoxic T lymphocytesin vitroprovided that the T‐lymphocyte precursors specifically recognize antigenic peptides associated with the major histocompatibility complex molecules class II and class I, respectively, expressed on the surface of the veto‐active cell. Data from a large number of experimental and clinical studies strongly indicate that veto‐active cells functionin vivoand are capable of preventing allograft rejection. Thus, donor‐cell‐mediated veto activity is the most likely explanation for the well‐known graft tolerizing effect of pretransplant donor blood transfusions in kidney graft recipients. A prerequisite for a veto‐active environmentin vivois the establishment of lymphoid microchimerism, in which veto‐active donor and recipient cells mutually downregulate potential alloaggression.
T‐cell subsets in adenoids and peripheral blood related to age, otitis media with effusion and allergy
Authors: LAGGING EVA , PAPATZIAMOS GEORGIOS , HALLDÉN GUNILLA , HEMLIN CLAES , HÄRFAST BENGT , VAN HAGE‐HAMSTEN MARIANNE
Published: August 2009

Adenoids and peripheral blood samples from 29 children (20–120 months of age) undergoing adenoidectomy for long‐standing otitis media with effusion (OME) (n=16) or obstructive adenoid hyperplasia (n=13) were investigated by flow cytometry for their T‐lymphocyte profile. Eleven of the enrolled children were allergic to inhalant and/or food allergens. For the whole group, the percentage of helper T cells belonging to the memory phenotype (CD4+/CD45RO+ cells) was significantly higher in adenoids than in blood (p<0.0001), while the same cell category increased with age in peripheral blood (p<0.01). A highly significant negative regression (p<0.001) was found between age and the percentage ratio of CD4+ cells that were CD45RO+ in adenoids and blood. Allergic children had a higher CD4+/CD8+ ratio for cells expressing CD45RO+ (p<0.05) in adenoids. The results of this study indicate that adenoids participate in the development of an immunological memory. Our findings support a relationship between allergy and memory cells in adenoids.
Molecular genetic analysis of easily accessible breast tumour DNA, purified from tissue left over from hormone receptor measurement
Authors: HANSEN LISE LOTTE , ANDERSEN JØRN , OVERGAARD JENS , KRUSE TORBEN A.
Published: August 2009

In order to establish a large panel of normal and tumour DNA from primary breast cancer patients, we looked for a source of easily accessible, good quality breast tumour DNA. Following routine hormone receptor analysis at the hospital the leftover pellets contained the nuclei from the tumour tissue. We collected 670 pellets over a period of 2 1/4 years and isolated a large amount of DNA (on average 400 μg per pellet). To control the quality of this tumour DNA, we analysed 41 pellets and matching normal DNA for loss of heterozygosity (LOH), with 11 microsatellite markers along chromosome 17. This chromosome is well described for breast cancer. LOH is a sensitive method, requiring good quality and pure tumour DNA. Contamination with normal DNA will blur the results. We found a high rate of LOH, ranging from 33 to 74%, which is in agreement with other reports, and therefore recommend this rich source of breast tumour DNA for molecular biological analysis.
Autopsy‐verified venous thromboembolism within a defined urban population – the city of Malmö, Sweden
Authors: NORDSTRÖM MONICA , LINDBLAD BENGT
Published: August 2009

Objective. To analyse the frequency of autopsy‐proven venous thromboembolism within a defined region and evaluate if certain risk groups not earlier recognized could be found. The incidence of objectively diagnosed venous thromboembolism was also calculated.

Setting. The city of Malmö, Sweden, with 230,000 inhabitants.

Main outcome measure. Analysis of 2,356 autopsies for the year 1987 of deceased inhabitants from the city of Malmö (2,981 deceased, autopsy frequency 79.0%) regarding venous thromboembolism.

Results. 25% of autopsies revealed venous thromboembolism. At the acute‐care hospital 31%, at the chronic hospital 37%, but in forensic autopsies of non‐hospital deaths only 5% (p<0.001) revealed venous thromboembolism. Major pulmonary embolism was seen in 13% and was more frequent in in‐hospital deaths (p<0.001). Two in‐hospital and two non‐hospital deaths due to pulmonary embolism and fractures were found: two patients with knee fractures, one hip fracture and one ankle fracture patient. The incidence of objectively diagnosed venous thromboembolism (autopsy, phlebography, perfusion scintigraphy) was calculated and an incidence of 42.5/10,000 inhabitants/year was found, (strongly age‐dependent).

Conclusion. Venous thromboembolism is a common finding in autopsies of hospitalized patients. Patients with fractures other than hip fractures are less well studied as regards venous thromboembolic complications. Further studies on these fracture patients are warranted.
Association between in vivo iododeoxyuridine labeling, MIB‐1 expression, malignancy grade and clinical stage in human prostate cancer
Authors: BORRE MICHAEL , HØYER MORTEN , SØRENSEN FLEMMING BRANDT , NERSTRØM BENNI , OVERGAARD JENS
Published: August 2009

Large variability in the biological behavior of prostate cancer makes prognostic markers important. The extent of tumor cell proliferation has been suggested as an important predictor of clinical outcome. Fifty‐five patients suspected of having or with previously diagnosed prostate cancer were labeled in vivo with IdUrd (a thymidine analogue incorporated into DNA in S‐phase cells) by intravenous infusion before transurethral resection. IdUrd‐labeled cells and MIB‐1‐positive cells were detected by immunohistochemistry. We found statistically significant associations between the tumor cell proliferation rates measured by in vivo IdUrd labeling and MIB‐1 expression in formalin‐fixed paraffin‐embedded tumors. Good correlations were also found between S‐phase fraction, MIB‐1 expression, clinical stage and malignancy grade. These results make larger retrospective studies on archival tissue meaningful.
Pyogenic hepatic abscess. A 10‐year population‐based retrospective study
Authors: HANSEN PER SYRAK , SCHØNHEYDER HENRIK CARL
Published: August 2009

A 10‐year retrospective survey was undertaken of patients with pyogenic hepatic abscesses (PHA). Fifty‐two patients fulfilled the criteria of PHA, equivalent to a mean annual incidence of 11/1,000,000. The main symptom was fever. Laboratory tests were compatible with infection, slightly elevated alkaline phosphatase being the only test pointing towards the liver as the focus of infection. Forty‐one patients (79%, 95% CL, 68–90%) had positive cultures from aspirated pus, with a total of 79 isolates. Enteric Gram‐negative rods accounted for 45% and anaerobic bacteria for 31% of PHA isolates. Gram‐positive cocci, predominantly non‐haemolytic streptococci, were the third largest group (19%), but were rare among blood isolates. Positive blood cultures were found in 21 patients (40%, 95% CL, 27–54%), with a total of 28 isolates. Percutaneous drainage was performed in 26, percutaneous needle aspiration in 10, combinations thereof in 5, and abdominal surgery in 5. Forty‐nine patients received systemic antibiotic therapy, four of whom were treated with antibiotics only. Seven recurrences occurred and the overall case fatality rate was 6% (95%, CL 0–12%), which might reflect a low rate of underlying malignant diseases in our study material.
Coagulase‐negative staphylococci in a major Danish university hospital: diversity in antibiotic susceptibility between wards
Authors: JARLØV JENS OTTO , HØIBY NIELS
Published: August 2009

Over a one‐year period, all coagulase‐negative staphylococci (CoNS) from blood cultures, cerebrospinal fluids and peritoneal effluents from patients in a major Danish university hospital were investigated for susceptibility to penicillin G; methicillin; gentamicin; netilmicin; amikacin; erythromycin; clindamycin; fusidic acid; rifampicin; tetracycline; chloramphenicol; ciprofloxacin; teicoplanin; and vancomycin. Among the CoNS‐isolates, 56% were resistant to methicillin, 51% to gentamicin, 28% to ciprofloxacin, and 5% to teicoplanin. Blood culture CoNS‐isolates from patients with a central venous catheter (CVC) were more often resistant to various antibiotics compared to CoNS‐isolates from patients without a CVC, e.g. methicillin (72%vs 21%), gentamicin (65%vs 22%) (p<0.00000001). Great diversity in antibiotic resistance between the wards was found; methicillin resistance (in most cases multiple antibiotic resistance) was in particular associated with consumption of broad‐spectrum (β‐lactams, quinolones, and total antibiotic consumption in a ward. Thus, the antibiotic policy of a ward is an important factor for antibiotic resistance among CoNS.
Epstein‐Barr virus strain characterization
Authors: MUNCH METTE
Published: August 2009

The Epstein‐Barr virus (EBV), a ubiquitous human herpesvirus originally described in cultured lymphoblasts from African Burkitt's lymphoma, is the causative agent of infectious mononucleosis, but appears to be involved in the pathogenesis of a variety of diseases. EBV has the ability to establish lifelong persistent infection, where only a restricted pattern of the viral proteins is expressed. Periodic reactivation of EBV occurs, where mature EBV particles are produced. Strain variation is potentially important in the biology and epidemiology of EBV, and in attempts to relate EBV to associated diseases. Analysis of the EBV genomes isolated from patients with EBV‐associated diseases and from various parts of the world has so far failed to identify conclusive disease‐specific viral subtypes. This review focuses on the different strategies which have been used for strain characterization or subtyping of EBV.
Clinical microbiology laboratories do not always detect resistance of Haemophilus influenzae with disk or tablet diffusion methods
Authors: MANNINEN RAIJA , HUOVINEN PENTTI , NISSINEN ANTTI ,   AND THE FINNISH STUDY GROUP FOR ANTIMICROBIAL RESISTANCE (FiRe)
Published: August 2009

The performance of disk diffusion testing of Haemophilus influenzae was evaluated in 20 laboratories. Thirteen disk‐medium‐breakpoint‐inoculum modifications were used in Finnish clinical microbiology laboratories. The performance of various methods was evaluated by testing a susceptible control strain and one with non‐β‐lactamase‐mediated ampicillin resistance 10 times in 16 laboratories. Gaps in millimeters were measured between these two groups of results. The strains were separated by a gap of at least 5 mm in 8/16 laboratories testing ampicillin, in 7/15 laboratories testing cefaclor, in 5/16 laboratories testing cefuroxime, and in 15/16 laboratories testing trimethoprim‐sulfa. Detection of ampicillin resistance was better with 2.5 μg tablets than with 10 μg disks or 33 μg tablets. For MIC‐determinations, 785 isolates and their disk diffusion results were collected. None of the 12 clinical isolates with non‐β‐lactamase‐mediated ampicillin resistance was detected as resistant in the participating laboratories. The ampicillin and cefaclor results of the isolates were no better even when a laboratory was able to separate the control strains. Cefaclor results were unreliable because of poor disk diffusion‐MIC correspondence and incoherent breakpoint references. Interlaboratory variation of the zone diameters caused false intermediate results of cefuroxime‐susceptible strains. When ampicillin, cefaclor and cefuroxime were tested, the discrimination of laboratories using disks and tablets was equal, whereas the laboratories using paper disks were better able to detect trimethoprim‐sulfa resistance.
Adherence of Staphylococcus aureus, Klebsiella pneumoniae from healthy persons and HIV carriers, under the influence of Broncho Vaxom in vitro and ascorbic acid in vivo
Authors: Fortis A. A. , Lianou P. E. , Papavassilliou J. T.
Published: August 2009

We examined the in vitro effect of Broncho Vaxom (BV) (an immunobiotherapeutic preparation containing a lysate made from bacteria often involved in respiratory tract infections) on adherence of Staphylococcus aureus, Klebsiella pneumoniae and Candida albicans to human buccal epithelial cells (BEC) of healthy volunteers and HIV carriers. We also examined the ex vivo effect of ascorbic acid on the adherence of the same microorganisms to BEC of HIV carriers. The study reached the following conclusions: The presence of BV in vitro significantly reduces the adherence of the tested strains to BEC from healthy persons and HIV carriers. No significant difference was observed between healthy persons and HIV carriers regarding the adherence of the tested strains to BEC. Significant difference in the adherence of the tested strains to BEC was observed between HIV carriers who had been taking ascorbic acid over a 3‐month period and those who had not. There was no further reduction in the adherence of the tested strains to BEC from HIV carriers who had been taking ascorbic acid in the presence of BV in vitro.
Ribotyping of Pseudomonas aeruginosa from infected patients: evidence of common strain types
Authors: FERRUS MARÍA A. , HERNANDEZ MANUEL , HABA JAVIER HERNANDEZ
Published: August 2009

Sixty‐nine strains of Pseudomonas aeruginosa isolated from infected patients at three hospitals in Valencia were serotyped and analyzed by comparison of restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping). Genomic Southern blots of EcoRI restriction digests were hybridized with a universal rRNA gene probe from Escherichia coli 16S and 23S rRNA. Strains were genetically diverse and 12 different ribotypes of 2 to 7 bands between 5 and 21.5 kb were defined. All strains shared a common band of 6.0 kb. The predominant ribotypes were R1 and R2, representing 25% and 41% of all isolates, respectively. Ribotypes were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype. This study demonstrated the prevalence of certain strain types associated with infected patients at Valencia hospitals, confirming the high typ‐ability and reproducibility of a single enzyme ribotyping for epidemiological studies of Pseudomonas aeruginosa. Ribotyping could be particularly useful if used in conjunction with serotyping.
Immunohistochemical determination of tumor angiogenesis measured by the maximal microvessel density in human prostate cancer
Authors: OFFERSEN BIRGITTE V. , BORRE MICHAEL , OVERGAARD JENS
Published: August 2009

Tumor growth beyond a certain size requires angiogenesis. Experimental evidence shows that once tumors leave the pre‐angiogenic phenotype to become angiogenic, metastases often start to evolve. The aim of this study was to develop a reproducible immunohistochemical technique and method to characterize the neovascularization in archival prostate cancer tissue by quantifying the microvessel density (MVD). Archival tumor specimens from 64 consecutively diagnosed prostate cancer patients were immunostained for von Willebrand Factor (vWF), endothelial antigen and for CD31 combined with the use of different digestive enzymes (trypsin and pronase) and heating in a microwave oven. Both the mean and the maximal MVD, and the reproducibility of the method were estimated. Finally, the mean MVD, the maximal MVD, and clinical characteristics were correlated with the crude survival of the patient population. The immunohistochemical staining for vWF to measure the maximal MVD was found to be a reproducible method of characterizing the individual tumor. Both a univariate and a multivariate analysis demonstrated that the maximal MVD, in contrast to the mean MVD, was significantly associated with survival in prostate cancer patients. We conclude that evaluation of angiogenesis by immunostaining the endothelial cells for vWF measured by the MVD in the most vascularized areas of the tumor is a reproducible method of characterizing the individual prostate tumor. Maximal MVD proved to be an independent prognostic parameter useful in conjunction with other known prognostic markers in human prostate cancer.
Experimental porcine neosporosis
Authors: JENSEN LENE , JENSEN TIM K. , LIND PETER , HENRIKSEN SVEND AAGE , UGGLA ARVID , BILLE‐HANSEN VIVI
Published: August 2009

Six gilts were inoculated intramuscularly with 2.5X106tachyzoites ofNeospora caninumon three different days of gestation to study the pathogenic effect ofNeosporainfection in pigs, including possible transplacental transmission. The gilts were euthanized 59, 30, and 9/10 days postinoculation (p.i.), corresponding to days 107, 102/106 and 110/111 of pregnancy. With the exception of one animal (euthanized day 9 p.i.) all gilts seroconverted as measured by the indirect, fluorescent antibody test (IFAT). Neosporosis with multifocal intralobular necrotizing hepatitis was seen in the two gilts inoculated 9/10 days before euthanasia. The uterus of one gilt inoculated 59 days before euthanasia revealed granulomatous and focal necrotizing endometritis with a corresponding multifocal necrosis of the trophoblasts of two fetuses. Transplacental neosporosis was indicated in the two fetuses by strongly elevatedNeosporaIFAT titres in pleural fluid and by the presence of multifocal necrotizing encephalitis and hepatitis together with non‐suppurative myocarditis, pneumonitis, nephritis and hepatitis. Furthermore,N. caninumwas re‐isolated in cell culture from one of these fetuses. A third fetus from the same gilt revealed only disseminated, pinpoint necroses in the liver. Immunohistochemically,N. caninumtachyzoites were detected in association with histopathological changes in the liver and the endometrium of the gilts, and in the brain, liver, and allantochorion of the three fetuses.
Identification ofProtothecafrom bovine mastitis in Denmark
Authors: AALBÆK BENT , JENSEN HENRIK ELVANG , HUDA ANNA
Published: August 2009

In the present study, algal isolates recovered during the period 1991 through 1996 from Danish cases of bovine mastitis were characterized and identified. Sixteen isolates ofProtothecawere obtained, all of which were identified asPrototheca zopfiiaccording to assimilation pattern, absence of capsules and resistance to clotrimazole.