Biomedical subjects

Henrik Leffers

Publications and source records attributed to Henrik Leffers.

At least 19 recordsLinked to original sources

PubMed2006

Estrogenic effect of soy isoflavones on mammary gland morphogenesis and gene expression profile.

We examined the effect of 17beta-estradiol (E2) and soy isoflavones' exposure on morphogenesis and global gene expression in the murine mammary gland. Three exposure regimens were applied: isoflavones added to the diet throughout either the lactational period (via the dams) or the postweaning period and E2 administered orally during the lactational period. Whole mounts of mammary glands were evaluated both in juvenile and adult animals with respect to branching morphogenesis and terminal end bud (TEB) formation. At postnatal day (PND) 28, we observed a significant increase in branching morphogenesis in all treated groups with the most pronounced effect after E2 exposure. For the E2-treated animals there was also a significant increase in TEB formation. At PNDs 42-43 the postweaning isoflavone and the E2 groups showed a transient reduction in the number of TEBs. A similar response after isoflavone and E2 exposure was further substantiated by changes in gene expression, since the same groups of genes were up- and downregulated, particularly in the E2 and postweaning isoflavone regimen. All changes in gene expression correlated with changes in the cellular composition of the gland, i.e., more and larger TEBs and ducts. The results suggest an estrogenic response of physiological doses of isoflavones on mammary gland development at both the morphological and molecular level, which resembled that induced by puberty.

Animals↗

PubMed2006

Identity of M2A (D2-40) antigen and gp36 (Aggrus, T1A-2, podoplanin) in human developing testis, testicular carcinoma in situ and germ-cell tumours.

Testicular germ-cell tumours of young adults are derived from a pre-invasive intratubular lesion, carcinoma in situ (CIS). In a recent genome-wide gene expression screening using cDNA microarrays, we found PDPN over-expressed in CIS compared to normal adult testis. PDPN encodes podoplanin (Aggrus, human gp36, T1A-2), a transmembrane glycoprotein expressed in lymphatic endothelium and various solid tumours. To examine a potential role for PDPN in testicular neoplasms and during testicular development, we investigated its expression pattern during the development of human testis and in a series of testicular CIS, gonadoblastoma and overt germ-cell tumours. We established by RT-PCR and by immunohistochemistry with a gp36 antibody that PDPN mRNA and the protein product were expressed in testes with germ-cell neoplasms but not in the normal adult testis. We also found gp36 expression in early foetal gonocytes and immature Sertoli cells, similar to the expression pattern of M2A antigen, a previously identified marker for CIS and seminoma. This reinforced our previous proposal that M2A (D2-40) antigen was identical to gp36 (podoplanin, Aggrus, T1A-2). Our findings also suggest that podoplanin has a function in developing testis, most likely at the level of cell-cell interactions among pre-meiotic germ cells and immature Sertoli cells.

Adolescent↗

PubMed2006

The sensitivity of the child to sex steroids: possible impact of exogenous estrogens.

The current trends of increasing incidences of testis, breast and prostate cancers are poorly understood, although it is assumed that sex hormones play a role. Disrupted sex hormone action is also believed to be involved in the increased occurrence of genital abnormalities among newborn boys and precocious puberty in girls. In this article, recent literature on sex steroid levels and their physiological roles during childhood is reviewed. It is concluded that (i) circulating levels of estradiol in prepubertal children are lower than originally claimed; (ii) children are extremely sensitive to estradiol and may respond with increased growth and/or breast development even at serum levels below the current detection limits; (iii) no threshold has been established, below which no hormonal effects can be seen in children exposed to exogenous steroids or endocrine disruptors; (iv) changes in hormone levels during fetal and prepubertal development may have severe effects in adult life and (v) the daily production rates of sex steroids in children estimated by the Food and Drug Administration in 1999 and still used in risk assessments are highly overestimated and should be revised. Because no lower threshold for estrogenic action has been established, caution should be taken to avoid unnecessary exposure of fetuses and children to exogenous sex steroids and endocrine disruptors, even at very low levels.

Adolescent↗

PubMed2006

CLLU1 expression levels predict time to initiation of therapy and overall survival in chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. IgV(H) mutational status, chromosomal aberrations, CD38 expression and ZAP-70 expression are prognostic markers in CLL, however, they are not exclusively confined to this disease. We recently identified a novel CLL-specific gene (CLL upregulated gene1, CLLU1) that is exclusively upregulated in CLL cells. Here we describe our evaluation of the prognostic significance of CLLU1 in CLL. METHODS: A cohort of 59 previously untreated CLL patients was studied. We determined the expression levels of two CLLU1 transcripts, cDNA1 and CDS, by quantitative RT-PCR. The relation between CLLU1 expression and time to therapy, overall survival and presence or absence of ZAP-70, CD38, chromosomal aberrations or IgV(H) mutations in the 59 patients was analyzed. RESULTS: Analyzed as a continuous, quantitative parameter CLLU1 levels significantly predicted time from diagnosis to initiation of therapy (P < or = 0.0003) Analyzed as a categorical parameter, by segregation of the patients into groups with cDNA1 or CDS expression above or below the median, the CLLU1 levels significantly predicted time from diagnosis to initiation of therapy (P = 0.001) and predicted overall survival with borderline significance (P < or = 0.05). Patient stratification according to clinical stage, cytogenetics, IgV(H) mutational status, ZAP-70 and CD38, demonstrated significantly increased CLLU1 expression in all investigated CLL poor risk groups. CLLU1 expression levels contributed additional prognostic information to ZAP-70-positive patients. CONCLUSIONS: CLLU1 is the first identified CLL specific gene. The CLLU1 mRNA expression level can predict time to initiation of treatment and survival in CLL patients.

ADP-ribosyl Cyclase 1↗

PubMed2006

Identification and expression profiling of 10 novel spermatid expressed CYPT genes.

To identify candidate genes for poor sperm morphology, we have screened for genes expressed during spermiogenesis. We identified 10 new members of the cysteine-rich perinuclear theca (CYPT) family showing that this family contains at least 15 members, which also includes the casein kinase II target genes. Based on similarity the CYPT sequences could be divided into two groups, Cypt1-10 and the novel members Cypt12-15. The 5'-end of the CYPT family is highly similar to exon1A and part of the first intron of Zfy2. Seven CYPT genes mapped to the X chromosome; six contained an intron and one was intron-less. One CYPT gene mapped to chromosome 3 and one mapped to chromosome 9 which were both intron-less. The upstream region of the CYPT family and Zfy2 genes is conserved. For some the conservation extended over a large region, however, only about 150 nucleotides is conserved among all CYPT members and Zfy2. Nevertheless, the short conserved promoter leads to essentially identical expression profiles for the CYPT family members and Zfy2, which was clearly different from the profile of Zfy1. Expression of the CYPT family and Zfy2 preceded the expression of other spermatid-specific genes such as the transition proteins and the protamines. In situ hybridization revealed a low expression in pachytene spermatocytes from stages IX-X followed by a strong upregulation in spermatids from stage VI with maximum expression in spermatids in stages VII-VIII. The CYPT family may function in the remodeling of the spermatid nucleus before condensation of the DNA.

Animals↗

PubMed2006

Is human fecundity declining?

Summary The decreasing trends in fertility rates in many industrialized countries are now so dramatic that they deserve much more scientific attention. Although social and behavioural factors undoubtedly play a major role for these trends, it seems premature, and not based on solid information, to conclude that these trends can be ascribed to social and behavioural changes alone. There is evidence to suspect that changing lifestyle and increasing environmental exposures, e.g. to endocrine disrupters, are behind the trends in occurrence of male reproductive health problems, including testis cancer, undescended testis and poor semen quality. These biological factors may also contribute to the extremely low fertility rates. However, the necessary research is complex and requires non-traditional collaboration between demographers, epidemiologists, clinicians, biologists, wild life researchers, geneticists and molecular biologists. This research effort can hardly be carried out without major support from governments and granting agencies making it possible to fund collaborative projects within novel research networks of scientists.

Environmental Pollutants↗

PubMed2006

From embryonic stem cells to testicular germ cell cancer-- should we be concerned?

Since the discovery of testicular carcinoma in situ (CIS) -- the precursor cell for the vast majority of germ cell tumours -- it has been proposed that CIS cells could be derived from transformed primordial germ cells or gonocytes. Here, we review recent discoveries not only substantiating that initial hypothesis but also indicating that CIS cells have a striking phenotypic similarity to embryonic stem cells (ESC). Many cancers have been proposed to originate from tissue-specific stem cells [so-called 'cancer stem cells' (CSC)] and we argue that CIS may be a very good example of a CSC, but with exceptional features due to the retention of embryonic pluripotency. In addition, considering the fact that pre-invasive CIS cells are transformed from early fetal cells, possibly due to environmentally induced alterations of the niche, we discuss potential risks linked to the uncontrolled therapeutic use of ESC.

Carcinoma in Situ↗

PubMed2005

Identification of a gene on chromosome 12q22 uniquely overexpressed in chronic lymphocytic leukemia.

The pathogenesis of chronic lymphocytic leukemia (CLL) is unknown but may involve aberrant activation of signaling pathways. Somatic hypermutations in rearranged immunoglobulin heavy-chain (IgVH) genes allow a division of CLL patients into 2 categories: mutated IgVH genes are associated with an indolent disease, whereas unmutated IgVH genes define an aggressive form. Using differential display to compare gene expression in CLL cells with and without IgVH hypermutations, we identified a novel gene, CLL up-regulated gene 1 (CLLU1), that was highly up-regulated in CLL cells without IgVH hypermutations. CLLU1 mapped to chromosome 12q22, within a cluster of genes that are active in germinal center B cells. However, appreciable levels of CLLU1 were detectable only in CLL cells and not in a panel of normal tissue extracts or in any other tested hematologic malignancy. High expression of CLLU1 in CLL samples occurred irrespective of trisomy 12 or large chromosomal rearrangements. CLLU1 encodes 6 mRNAs with no sequence homology to any known gene, and most transcripts appear to be noncoding. Two transcripts, however, potentially encode a peptide with remarkable structural similarity to human interleukin 4. These data, in particular the unique and restricted expression pattern, suggest that CLLU1 is the first disease-specific gene identified in CLL.

Chromosome Mapping↗

PubMed2005

Genomic and gene expression signature of the pre-invasive testicular carcinoma in situ.

Testicular cancer is the most common malignancy among men in the reproductive age and the incidence is increasing, probably caused by environmental factors. Most testicular cancers are testicular germ cell tumours and all originate from a carcinoma in situ (CIS) pattern. In this review, we focus on the pre-invasive CIS and its possible fetal origin by reviewing recent data originating from DNA microarrays and comparative genomic hybridisations. A comparison of gene expression and genomic aberrations reveal chromosomal "hot spots" with mutual clustering of gene expression and genomic amplification. Some of the genes found in the hot spots may be involved in creating the CIS phenotype. On the other hand, many genes that are highly expressed in CIS are not present in the hot-spot areas. The gene expression profile of CIS thus most likely reflects the combined result of genomic amplification and increased transcriptional activation and/or deficiency in the epigenetic silencing of specific loci. Amplification of chromosome 12p, appears to be a good genomic marker of the transition from the pre-malignant to malignant CIS cell; this is consistent with recent findings of propagation advantages in cultured undifferentiated embryonic stem cells after spontaneous amplification in similar regions. The gene expression profile of CIS cells has remarkable similarity to that of embryonic stem cells and supports our long-standing hypothesis of an early developmental origin of CIS and testicular germ cell cancer.

Carcinoma in Situ↗

PubMed2005

Determination of phthalate monoesters in human milk, consumer milk, and infant formula by tandem mass spectrometry (LC-MS-MS).

Daily exposure of humans to phthalates may be a health risk because animal experiments have shown these compounds can affect the differentiation and function of the reproductive system. Because milk is the main source of nutrition for infants, knowledge of phthalate levels is important for exposure and risk assessment. Here we describe the development and validation of a quantitative analytical procedure for determination of phthalate metabolites in human milk. The phthalate monoesters investigated were: monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mono-(2-ethylhexyl) phthalate (mEHP), and monoisononyl phthalate (mNP). The method is based on liquid extraction with a mixture of ethyl acetate and cyclohexane (95:5) followed by two-step solid-phase extraction (SPE). Detection and quantification of the phthalate monoesters were accomplished by high-pressure liquid chromatography using a Betasil phenyl column (100 mmx2.1 mmx3 microm) and triple tandem mass spectrometry (LC-MS-MS). Detection limits were in the range 0.01 to 0.5 microg L(-1) and method variation was from 5 to 15%. Analysis of 36 milk samples showed that all these phthalates were present, albeit at different concentrations. Median values (microg L(-1)) obtained were 0.11 (mMP), 0.95 (mEP), 3.5 (mBP), 0.8 (mBzP), 9.5 (mEHP), and 101 (mNP). We also analysed seven samples of consumer milk and ten samples of infant formula. Only mBP and mEHP were detected in these samples, in the ranges 0.6-3.9 microg L(-1) (mBP) and 5.6-9.9 microg L(-1) (mEHP).

Chromatography, Liquid↗

PubMed2004

Transcription factor AP-2gamma is a developmentally regulated marker of testicular carcinoma in situ and germ cell tumors.

PURPOSE: Transcription factor activator protein-2gamma (TFAP2C, AP-2gamma) was reported previously in extraembryonic ectoderm and breast carcinomas but not in the testis. In our recent gene expression study we detected AP-2gamma in carcinoma in situ testis (CIS, or intratubular germ cell neoplasia), precursor of testicular germ cell tumors. In this study we aimed to investigate the expression pattern of AP-2gamma and to shed light on this factor in germ cell differentiation and the pathogenesis of germ cell neoplasia. EXPERIMENTAL DESIGN: We analyzed expression pattern of AP-2gamma at the RNA and protein level in normal human tissues and a panel of tumors and tumor-derived cell lines. In the gonads, we established the ontogeny of expression of AP-2gamma in normal and dysgenetic samples. We also investigated the regulation of AP-2gamma by steroids and retinoic acid. RESULTS: We detected abundant AP-2gamma in testicular CIS and in testicular germ cell tumors of young adults and confirmed differential expression of AP-2gamma in somatic tumors. We found that AP-2gamma expression was regulated by retinoic acid in an embryonal carcinoma cell line (NT2). The investigation of ontogeny of AP-2gamma protein expression in fetal gonads revealed that it was confined to oogonia/gonocytes and was down-regulated with germ cell differentiation. In some prepubertal intersex cases, AP-2gamma was detected outside of the normal window of expression, probably marking neoplastic transformation of germ cells. CONCLUSIONS: AP-2gamma is developmentally regulated and associated with the undifferentiated phenotype in germ cells. This transcription factor may be involved in self-renewal and survival of immature germ cells and tissue-specific stem cells. AP-2gamma is a novel marker of testicular CIS and CIS-derived tumors.

Adolescent↗

PubMed2004

Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling.

Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly expressed in testicular CIS, including many never reported in testicular neoplasms. Expression was further verified by semiquantitative reverse transcription-PCR and in situ hybridization. Among the highest expressed genes were NANOG and POU5F1, and reverse transcription-PCR revealed possible changes in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported as unstable in cultured ESCs. The close similarity between CIS and ESCs explains the pluripotency of CIS. Moreover, the findings are consistent with an early prenatal origin of TGCTs and thus suggest that etiologic factors operating in utero are of primary importance for the incidence trends of TGCTs. Finally, some of the highly expressed genes identified in this study are promising candidates for new diagnostic markers for CIS and/or TGCTs.

Carcinoma in Situ↗

PubMed2004

Analysis of cell-type-specific gene expression during mouse spermatogenesis.

In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume of specific cells, or changes in the cell-type composition. This reflects the cellularity of the tissue. Here we have combined techniques that assess the expression profiles of genes at the whole-tissue level, differential display and DNA array, and, at the level of cellularity, in situ hybridization. Combining results from these techniques allows determination of the cell-type-specific gene-expression patterns of many genes during spermatogenesis. Differential display was used to determine expression profiles with high sensitivity and independent of prior knowledge of the sequence, whereas DNA arrays quickly assess the expression profiles of all the genes. This identified three groups of gene-expression profiles. The major group corresponds to genes that are upregulated in spermatocytes during either the mid- or late- pachytene phase of spermatogenesis (stages VII-XI). This pachytene cluster was gradually extinguished in the later spermatid stages but was followed by another cluster of genes expressed in spermatids. Finally, a group of genes was downregulated during spermatogenesis and probably expressed in nongerm cells. We believe that expression of most genes can be described by a combination of these cell-type-specific expression patterns.

Animals↗

PubMed2004

Frequent polymorphism of the mitochondrial DNA polymerase gamma gene (POLG) in patients with normal spermiograms and unexplained subfertility.

BACKGROUND: Male fertility largely depends on the quality of sperm production, which may be affected by environmental and genetic factors. In this study, we explored a possible role of the polymerase gamma (POLG) gene polymorphism, recently reported to be associated with male infertility in some populations. METHODS: The polymorphic CAG repeat (usually 10 codons long) in the POLG gene was studied in 1298 male subjects: 429 patients with infertility/subfertility, and 869 controls (495 men from the general population with unknown fertility and 374 recent fathers). In all subjects, the POLG polymorphism was assessed in relation to their semen quality, and--in the fertile controls--with biological fecundity measured as waiting time-to-pregnancy (TTP) for the couples. In the patients lacking the common POLG allele, the outcome of the assisted reproductive techniques (ART) for the couples was evaluated. RESULTS: The absence of one (10/ not equal to 10) or both common POLG alleles (not equal to 10/not equal to 10) was more frequent among the subfertile patients than among fertile controls (P=0.021 and P=0.04 respectively). The estimated predictive value for infertility in a man homozygous for the POLG polymorphism was 15.5% (95% CI: 4.8-51%). There was a positive association with sperm concentration: 14.3% of the normospermic subfertile patients were homozygous for the absence of the common POLG allele (not equal to 10/not equal to 10), in comparison with 2.3% of unselected controls (P=0.001) and 0.9% of the fertile men (P=0.0001). No association with sperm motility, morphology and TTP was found. Spermatozoa of the three not equal to 10/not equal to 10 patients treated with IVF retained the ability to penetrate the egg, but the fertilization rate was low. Nine homozygous not equal to 10/ not equal to 10 patients were treated with ICSI, resulting in pregnancy in seven couples. CONCLUSIONS: The POLG gene polymorphism should be considered as a possible contributing factor in patients with unexplained subfertility and normal spermiograms. The oocyte penetration ability of sperm may be partially impaired in the not equal to 10/not equal to 10 patients but most of them can be successfully treated with ICSI.

Adult↗

PubMed2003

High-resolution comparative genomic hybridization detects extra chromosome arm 12p material in most cases of carcinoma in situ adjacent to overt germ cell tumors, but not before the invasive tumor development.

High-resolution comparative genomic hybridization (HR-CGH) analysis was performed on DNA purified from laser-capture microdissected carcinoma in situ (CIS) cells from nine cases of CIS, either from tissue without any invasive tumor or from testicular parenchyma adjacent to seminoma, nonseminoma, or a combined germ cell tumor. Before CGH analysis, DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and directly labeled with a mixture of FITC-dUTP and FITC-dCTP. CGH analysis revealed extra chromosome arm 12p material in six out of seven cases with CIS adjacent to overt tumors, but only a diminutive gain of 12q was noted in one of the two cases of CIS without invasive elements. In addition, gains of parts of chromosome 8 (3/7) and losses of chromosome 5 (2/7) were demonstrated in CIS adjacent to invasive tumors. Gains of parts of chromosome 7 were found in CIS adjacent to seminoma (4/4), whereas relative gains of chromosome 15 were identified in some cases of CIS adjacent to seminoma and in isolated CIS in comparison to CIS adjacent to nonseminoma. Our data seem to indicate that extra 12p material is not present in the "dormant" CIS cell before development of an invasive tumor. The gain of extra chromosome 12 material may not be an early event in the neoplastic transformation, but is most likely associated with a more malignant progression of the CIS cell.

Adolescent↗

PubMed2003

No AZF deletion in 160 patients with testicular germ cell neoplasia.

Testicular germ cell cancer is aetiologically linked to genital malformations and male infertility and is most probably caused by a disruption of embryonic programming and gonadal development during fetal life. In some cases, germ cell neoplasia is associated with a relative reduction of Y chromosomal material (e.g. 45,X/46,XY) or other abnormalities of the Y chromosome. The euchromatic long arm of the human Y chromosome (Yq11) contains three azoospermia factors (AZFa, AZFb, AZFc) functionally important in human spermatogenesis. Microdeletions encompassing one of these three AZF loci result in the deletion of multiple genes normally expressed in testis tissue and are associated with spermatogenic failure. The aim of our study was to investigate whether AZF microdeletions, in addition to causing infertility, predispose also to germ cell neoplasia, since subjects with poor spermatogenesis have an increased risk of testicular cancer. We screened for putative deletions of AZF loci on the Y chromosome in DNA isolated from white blood cells of 160 Danish patients with testicular germ cell neoplasia. Interestingly, although AZF microdeletions are found frequently in patients with idiopathic infertility, in all cases studied of testicular germ cell cancer the Yq region was found to be intact. We conclude that the molecular aetiology of testicular germ cell neoplasia of the young adult type most likely does not involve the same pathways as male infertility caused by AZF deletions. Malignant transformation of germ cells is thus caused by the dysfunction of some other genes that still need to be identified.

Adult↗

PubMed2002

Analysis of the polymorphic CAG repeat length in the androgen receptor gene in patients with testicular germ cell cancer.

Changes in the length of a polymorphic trinucleotide (CAG) repeat in the androgen receptor (AR) gene, which may lead to altered transactivation of the AR gene, have been implicated to play a role in the pathogenesis of several forms of endocrine cancer and certain reproductive disorders. Subjects with reproductive disorders that are associated with a relative deficiency of androgen function carry an increased risk for testicular cancer, therefore we have examined the (CAG)n in the AR gene in DNA isolated from peripheral blood cells of 102 patients diagnosed with testicular germ cell neoplasia and compared them with a control group of 110 healthy men with proven fertility. All patients and control subjects underwent comprehensive andrological examination that included reproductive hormone profiles and the analysis of the (CAG)n in the AR gene that was done by means of PCR and DNA sequencing. There was no difference in the distribution of (CAG)n between the subjects and controls, no association of (CAG)n and the tumor type and no association with severity of the disease. We conclude that the high risk of testicular germ cell cancer in the Danish population is not associated with the (CAG)n polymorphism in the AR gene.

Adult↗

PubMed2002

CAG repeat length in androgen-receptor gene and reproductive variables in fertile and infertile men.

Several reports implicated a relation between the trinucleotide (CAG) repeat length in the androgen-receptor gene and male infertility, whereas others failed to find an association. We investigated the CAG repeat length in relation to sperm production and reproductive hormones in 119 infertile men and 110 men with proven fertility. We found no difference in the distribution of CAG repeat lengths between the groups and no association with reproductive parameters. This finding suggests that, within the normal range of 14-33 repeats, there is no biological link between the CAG repeat length and fertility. This lack of association was comfirmed by an analysis of the data from all previously published European studies (in total 674 infertile men and 660 controls).

Adolescent↗