Identification of a 2244 base pair interstitial deletion within the human ESR1 gene in the Spanish population.

BACKGROUND: The ESR1 AGATA haplotype is composed of five markers located within introns 5 and 6 of the human oestrogen receptor 1 (ESR1) gene. This haplotype has been studied in several male urogenital tract anomalies and male infertility. In one of these studies, a deviation from Hardy-Weinberg equilibrium (DHW) was found for the ESR1 AGATA marker rs3020375 in two groups of healthy controls. In the present study, we investigated whether the observed DHW is caused by structural variants present within the ESR1 gene. PARTICIPANTS: 229 family units achieving pregnancy through assisted reproductive technologies (n = 129) or by natural means (n = 100), 2465 general Spanish population controls and 162 men with idiopathic infertility. MAIN OUTCOME MEASUREMENTS: Segregation analyses of genetic markers in family units and case-control genetic association studies. RESULTS: We identified a new interstitial deletion of 2244 base pairs within intron 6 of the human ESR1 gene as the cause for the observed DHW. This new variant presents a 10% allelic frequency in the general Spanish population and it is associated with idiopathic male infertility (OR = 1.51; p = 0.03). The percentage of infertile couples in which both members carried the ESR1 deletion (10.08%) was also a higher than expected value of 6% (p = 0.03). CONCLUSIONS: We have characterised a novel structural variation in human ESR1 gene. The available data indicate a deleterious action of the ESR1 deletion in both male and couple fertility. The potential effects of this deletion on other oestrogen-related diseases need to be determined.

Cryptic chromosomal rearrangement screening in 30 patients with mental retardation and dysmorphic features.

Mental retardation affects 1-3% of the general population, and the genetic causes in many cases are unknown. Cytogenetically undetected chromosomal imbalances have been indicated as an explanation. Nowadays, due to the development of molecular cytogenetic techniques, it is possible to identify cryptic rearrangements involving the ends of chromosomes. We report a screening using chromosome-specific telomere fluorescence in-situ hybridization (FISH) probes, in a group of 30 patients with a well-characterized phenotype including mental retardation, dysmorphic features, and a normal karyotype. Among them, two subtelomeric rearrangements have been detected and characterized. One of them is a de novo deletion of 1p36, which has been previously described as a new contiguous gene syndrome. The second is an unbalanced product of a cryptic translocation involving chromosomes 1 and 13, which results in a partial 1q trisomy and partial 13q monosomy. These findings highlight, the importance of searching for cryptic subtelomeric rearrangements in non-syndromic mentally retarded patients.

Protein-bound polysaccharide K and interleukin-2 regulate different nuclear transcription factors in the NKL human natural killer cell line.

The activation of natural killer cells and induction of cytotoxicity are complex processes whose molecular mechanisms have not been clearly elucidated. Stimulation of the NKL human NK cell line with interleukin-2 (IL-2) or protein-bound polysaccharide K (PSK) leads to sustained growth and cytolytic activity in comparison to unstimulated NKL cells. However, it is not known whether both agents give rise to the same or different intracellular signals. To determine the molecular basis for the action of IL-2 and PSK, the binding activity of AP-1, CRE, NF-kappaB, PU.1, SP-1, NFAT, STAT1, STAT5/6, GAS/ISRE and IRF-1 transcription factors was compared in IL-2- and PSK-stimulated NKL cells. Here we report that PSK enhanced AP-1 and CRE binding activities, whereas IL-2 increased AP-1 and SP-1 and modified GAS/ISRE, IRF-1 and STAT5. Our results indicate that IL-2 and PSK regulate different nuclear transcription factors in NKL cells, and that the signal transduction pathway used by these inducers is different.

Protein-bound polysaccharide (PSK) induces cytotoxic activity in the NKL human natural killer cell line.

We studied the effect of protein-bound polysaccharide PSK on the activation of the human natural killer cell line NKL. We observed an increased natural killer cytotoxic activity against different tumor cells (K562, Daudi, and U937) when a standard 2- to 3-h 51chromium release assay was performed. The results parallel those obtained after treatment of the NKL cell line with interleukin-2. The highest cytotoxic activity was reached at a concentration of 100 microg/ml of PSK. This natural killer activation was inhibited when the PSK dose was 1,000 microg/ml. None of the cell surface markers that were analyzed by fluorescence-activated cell sorting showed variations after PSK or interleukin-2 treatment of NKL cells. These markers included CD2, CD11b, CD11c, CD18, CD16, CD54, CD56, CD98, CD25, CD122, HLA class I, HLA class II, CD94, ILT2, p58.1, p70, and NKp46. One of these markers (NKp46) is a major triggering receptor reported to be involved in the natural cytotoxicity of fresh or cultured human natural killer cells. In our study, another triggering receptor must be implicated in PSK-induced natural killer lysis. Our data suggest that PSK is an important biological response modifier of natural killer cells in vitro and may prove to be useful for the study of human natural killer cell biology.

Selective upregulation of MHC class I expression in metastatic colonies derived from tumor clones of a murine fibrosarcoma.

Eighteen metastatic nodes derived from the wild-type (GR9) and from 4 different clones (G2, D8, B10, and B9) obtained from a fibrosarcoma were analyzed for H-2 class I and II expression, as well as for adhesion molecules (CD44, CDIIb, CD18, CD49, and CD54). When metastatic nodes were cultured, typed for H-2 antigens, and compared with the H-2 expression of the inducing tumor cell, H-2 Kd and Dd class I expression was greater in most nodes analyzed. In contrast, the Ld molecule remained negative, or showed a minor increase. Class II expression was negative in the wild-type and the tumor clones, and remained so in the metastatic colonies. Analysis of the adhesion molecules revealed no differences between the inducing tumor cells and the metastatic nodes. The only molecule expressed was CD44, which was present in all cells studied and was also inducible by interferon-gamma. The increase in H-2K and H-2D expression was associated with resistance to natural killer cytotoxicity, as observed in the G2 tumor clone and some autologous metastases, such as B9MP2, G2MK2, and G2MLI. In three independent clones of this tumor system (D8, BIOMP6, and B9MP6) we found that tumor cells treated with interferon-gamma had the same altered phenotype, i.e., a selective lack of response of the Ld molecule to induction. These findings add a cautionary note to the well-established idea that tumor cells may lose all class I antigens during tumor progression, and suggest that sometimes this may not be the case. The selective downregulation of Ld and upregulation of Kd and Dd class I expression may give some tumor cells means of escaping both cytotoxic lymphocyte and natural killer immune surveillance.

Hierarchy in the assembly of HLA-B27 and HLA-Cw3 molecules in transgenic mice.

Cell surface expression of human class I molecules in transgenic mice is dependent upon the available pool of beta 2-microglobulin (beta 2m) and the affinity between mouse beta 2m and human class I molecules. HLA-B27 and HLA-Cw3 transgenes can be expressed in mouse strains of the H-2 haplotypes b, f, k, and s which encode two endogenous class I genes mapping to H-2K and H-2D. The human class I genes cannot be expressed on H-2d and H-2q haplotypes which encode three endogenous class I molecules (K,D,L). This suggests that there may be only enough mouse beta 2m molecules to support three class I molecules. When both the HLA-B27 and HLA-Cw3 genes are introduced into H-2b mice, only HLA-Cw3 reaches the cell surface. This suggests that HLA-Cw3 has a higher affinity than HLA-B27 for mouse beta 2m. The possible implications of our findings regarding the assembly, transport, and expression of class I MHC molecules in vivo are discussed.

Human class I major histocompatibility complex transgene prevents virus-induced demyelination in susceptible mutant B10.D2dml mice.

Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelination in susceptible strains of mice, providing an excellent model for multiple sclerosis. Class I genes within the major histocompatibility complex locus (H-2D region) play a major role in determining whether strains of mice develop chronic demyelination and TMEV persistence. B10.D2dml mice with deletion in the 3' end of Dd and the 5' end of Ld genes develop the most prominent demyelination in comparison with resistant B10.D2 mice with normal complementation of H-2D region genes. We tested whether expression of a class I human transgene (HLA-B27) would modulate virus-induced demyelination in mutant B10.D2dml mice. Transgenic B10.D2dml (HLA-B27+) mice infected with virus showed dramatic decrease in the extent of demyelination (p < 0.0001) and virus antigen expression in spinal cord compared with littermate controls without the human class I transgene. These experiments demonstrate that transgenic expression of a human class I major histocompatibility complex locus molecule can prevent demyelination induced by a virus in mutant mice.

Characterization of CD44 antigen during lymphoid ontogeny.

We studied immunohistological and biochemical aspects of the CD44 molecule with a mAb produced in our lab: GRHL-1. The characteristic expression of this antigen in cells of B lineage was analyzed. This mAb showed identical immunohistological patterns of reactivity to other mAbs included in CD44 cluster, on a variety lymphoid and nonlymphoid human tissues, and demonstrated similar bands on SDS-PAGE of 125I labeled lymphocyte lysates. This antigen is limited to cells of mature phenotype, and disappears in proliferating B cells in the germinal centers of the lymphoid follicles. CD44 is absent in pre-B and Burkitt cell lines. PKC activation mediate in vitro differentiation of pre-B cell lines. However, it is not involved in up-regulation of CD44 antigen expression.

Protein kinase C-mediated regulation of the expression of CD14 and CD11/CD18 in U937 cells.

We used the U937 cell line to analyze CD14, CD11/CD18, HLA class-I and DR antigen expression during PMA-induced differentiation. Treatment of U937 cells with PMA markedly increased CD14, CD11a, CD11b and CD18 antigen expression, and slightly increased CD11c expression. Protein kinase C may play a major role in regulating the expression of these antigens. The protein kinase inhibitor H7 abrogated the inductive effect of PMA. Calcium ionophore, when added alone or in the presence of PMA, had no effect. The inhibitory effect of the calcium antagonist verapamil, EGTA, and of chlorpromazine, an antagonist of calcium-binding proteins, supports a role for calcium-dependent protein kinase C in the up-regulation of CD14 and CD11/CD18 surface expression. The specific calmodulin inhibitors R24571 and W7 had no effect on antigen expression. Our findings suggest that protein kinase C activation is an important step in the PMA-induced differentiation of U937 cells.

Histocompatibility antigens in primary and metastatic squamous cell carcinoma of the larynx.

A series of 38 primary laryngeal and hypopharyngeal tumours, 15 lymph-node metastases and normal tissue were evaluated in frozen sections for the expression of MHC class I and II antigens, using monomorphic monoclonal antibodies (MAbs) to HLA-ABC, beta 2-microglobulin, DR, DP, DQ, HLA-B and polymorphic HLA-ABC antigens. Normal distant mucosa of larynx reacted to anti-class I antibodies but not to anti-class II. In 9 primary tumours (23.7%) HLA class I antigens were not observed. The remaining 29 showed a strong reaction to not observed. The remaining 29 showed a strong reaction to anti-HLA-ABC (heavy chain) and anti-beta 2-microglobulin, although in 3 cases out of 29 no staining was observed with anti-HLA-B locus-specific MAbs. These selective losses were confirmed using the corresponding anti-HLA polymorphic MAbs. For HLA class II molecules, only DR was observed in 3 of 38 cases. Defective HLA class I expression statistically correlates with high scores according to Jakobsson's criteria for histopathological tumour grading. Loss of HLA-ABC antigens was most frequent among the cases with poor differentiation (6/8 cases). On the contrary, class II antigen expression was correlated with a well differentiated pattern and a more favourable prognosis (p less than 0.001). We have found differences in HLA class I expression when comparing primary tumours and autologous metastases (3/9 cases). Immunoprecipitation and SDS-PAGE of class I antigens, Northern and Southern blot analyses of MHC class I genes were performed. We have not detected class I gene rearrangement using HLA coding and locus-specific non-coding probes. However, we have found a class I transcription defect that corresponds with a class-I-negative phenotype.

Different CD45 and CD45R epitopes involved in T cell proliferation and NK cytotoxicity.

Different monoclonal antibodies detecting the leucocyte common antigen have been obtained, for CD45 (GRT2, GRT3 and GRT4) and for CD45R (GRT22). Several epitopes have been defined with these monoclonal antibodies (MAbs). We have performed a comparative study with CD45 and CD45R MAbs on NK and T cell proliferation. Common epitopes of CD45 antigen were found to be involved in blocking of NK activity but not CD45R restricted determinants. In the cell proliferation assays, fresh human peripheral blood mononuclear cells were stimulated with PHA and soluble CD3 MAbs. CD45 and CD45R MAbs failed to demonstrate the capacity to modify the mitogenic response when optimal and suboptimal doses of PHA were used. In contrast, both (CD45 and CD45R) MAbs led to a significant rise in anti CD3 response. A CD18 (GRF1) was used as control and inhibited both PHA and CD3 T cell proliferation as well as NK activity. We think these results can be explained on the basis of different activation pathways (PHA versus anti CD3) and the accessory signals induced by these MAbs as recorded only in anti CD3 induced mitogenesis.

Studies on CD11a and CD18 molecules with two new monoclonal antibodies: differential myelomonocytic antigen expression of PMA treated HL60 and U937 cell lines.

Biochemical and functional aspects as well as features of cellular distribution of the differentiation groups CD11a and CD18 were studied in the course of a detailed characterization of two new monoclonal antibodies which recognize the alpha (GRS3) and beta (GRF1) chains of the LFA-1 antigen. Both MAbs inhibited homotypic aggregation of an EBV cell line. In contrast, only GRF1 (anti-beta chain) was able to inhibit granulocyte aggregation as well. Different myeloid-monocyte antigen modulation was noted in PMA induced macrophage differentiation of the U937 and HL60 cell lines. PMA treated HL60 cells showed increased expression of alpha M (CD11b) and alpha X (CD11c) antigens but no change in HLA-DR or CD14 antigen expression. No variation in the expression of LFA complex antigen (CD11a, b and c, or CD18) was observed on U937 cells, which on the other hand presented de novo expression of HLA-DR and CD14 antigens.

Distribution of the CD45R antigen in the maturation of lymphoid and myeloid series: the CD45R negative phenotype is a constant finding in T CD4 positive lymphoproliferative disorders.

We have compared the expression of the common leucocyte antigen (CD45) and the restricted leucocyte antigen (CD45R) on normal haematopoietic cells, cell lines, and a total number of 136 cases of myeloid and lymphoid proliferative syndromes. CD45, the conventional leucocyte antigen, presents a generalized distribution along the lymphoid and myeloid maturation pathway with the exception of some myelomas and pre-B leukaemias. In contrast, the expression of the CD45R determinant is more limited. Although it is found in the majority of the differentiation stages of B cells and monocytes, it is present only in the early stages of myeloid differentiation. On T cells it is expressed on mature thymocytes and in the majority of CD8+ lymphocytes and a subset of CD4+ cells on peripheral blood. Finally, our results also indicated that CD4+ T lymphoproliferative syndromes are derived from the CD4+ CD45R- subset (20/20 cases).