Expression of keratin K2e in cutaneous and oral lesions: association with keratinocyte activation,proliferation, and keratinization

The cytoskeleton in keratinocytes is a complex of highly homologous structural proteins derived from two families of type I and type II polypeptides. Keratin K2e is a type II polypeptide that is expressed in epidermis late in differentiation. Here we report the influence of keratinocyte activation, proliferation, and keratinization on K2e expression in samples of cutaneous and oral lesions. The normal expression of K2e in the upper spinous and granular layers of interfollicular epidermis is increased in keloid scars but showed distinct down-regulation in psoriasis and hypertrophic scars where keratinocytes are known to undergo activation. Unlike normal and psoriatic skin, K2e expression in hypertrophic and keloid scars began in the deepest suprabasal layer. In cutaneous basal and squamous cell carcinomas, K2e was absent in most tumor islands but the overlying epidermis showed strong expression. No significant K2e expression in nonkeratinized or keratinized oral epithelia, including buccal mucosa, lateral border of tongue and gingiva was detected. In oral lichen planus K2e expression was undetectable, but in benign keratoses of lingual mucosa induction of K2e along with K1 and K10 was observed. In mild-to-moderate oral dysplasia with orthokeratinization, K2e was highly expressed compared with parakeratinized areas but in severe dysplasia as well as in oral squamous cell carcinoma, K2e expression was undetectable. Taken together, the data suggest that K2e expression in skin is sensitive to keratinocyte activation but its up-regulation in oral lesions is a reflection of the degree of orthokeratinization.     

The nonfunctional allele TCRBV6S1B is strongly associated with Helicobacter pylori infection

To determine genetic susceptibility factors for Helicobacter pylori infection, polymorphic T-cell receptor gene elements were investigated in 203 H. pylori-infected individuals and 180 uninfected individuals (controls). H. pylori infection is highly associated with individuals homozygous for the nonfunctional TCRBV6S1B element (odds ratio = 5.9; chi(2) = 13; P = 0.00032; P value corrected for multiple comparisons [Bonferroni correction] = 0. 00063).     

A duplication/paracentric inversion associated with familial X-linked deafness (DFN3) suggests the presence of a regulatory element more than 400 kb upstream of the POU3F4 gene

X-linked deafness with stapes fixation (DFN3) is caused by mutationsin the POU3F4 gene at Xq21.1. By employing pulsed field gelelectrophoresis (PFGE) we identified a chromosomal aberrationin the DNA of a DFN3 patient who did not show alterations inthe open reading frame (ORF) of POU3F4. Southern blot analysisindicated that a DNA segment of 150 kb, located 170 kb proximalto the POU3F4 gene, was duplicated. Fluorescence in situ hybridization(FISH) analysis, PFGE, and detailed Southern analysis revealedthat this duplication is part of a more complex rearrangementincluding a paracentric inversion involving the Xq21.1 region,and presumably the Xq21.3 region. Since at least two DFN3-associatedminideletions are situated proximal to the duplicated segment,the inversion most likely disconnects the POU3F4 gene from aregulatory element which is located at a distance of at least400 kb upstream of the POU3F4 gene.     

Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.     

Tyrosine kinase-dependent and -independent events induced by interleukin-2 stimulation: interleukin-2-mediated NO production required for the induction of lymphokine-activated killer cell activity in rat splenocytes is tyrosine kinase independent.

Nitric oxide (NO) has recently been shown to be an indispensable co-factor in the generation of lymphokine-activated killer (LAK) cells induced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediated by protein tyrosine kinases (PTK). In this work we utilized a well-characterized PTK inhibitor, genistein (GEN), to address the role of PTK on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferation, generation of cytotoxic activity and production of NO upon IL-2 stimulation of rat splenocytes. We report here that GEN displays profound inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production, measured as NO2-. In contrast, a specific inhibitor of the NO synthetic pathway (NG-monomethyl-L-arginine; NMMA) blocked generation of LAK cells and NO production without affecting cell proliferation. If added directly to the cytotoxicity tests, GEN exerted minor inhibitory effects, not exceeding 25% of control tests, while NMMA was completely ineffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing substance, restored LAK cell generation in cultures performed in the presence of NMMA, but not in those performed in the presence of GEN. These results indicate that IL-2-induced NO production is a PTK-independent event. IL-2-stimulated LAK cell generation obligatorily requires the concurrent activation of PTK dependent and independent signal transduction pathways.     

Feasibility of simultaneous fluorescence immunophenotyping and fluorescence in situ hybridization study for the detection of estrogen receptor expression and deletions of the estrogen receptor gene in breast carcinoma cell lines

For the first time, combined immunophenotyping and fluorescence in situ hybridization (FISH) technique according to the ”fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms” (FICTION) technique have been successfully applied in solid tumors. Thus, we were able to visualize the antigen expression of cells with chromosomal deletions of a tumor suppressor region directly. In six breast carcinoma cell lines, we investigated the correlation between estrogen receptor (ER) expression status and deletions of the estrogen receptor gene (ESR). To screen for deletions of the ESR gene, dual-color FISH was performed with a YAC (yeast artificial chromosome) probe containing the ESR gene and, as internal control, with a centromeric probe of chromosome 6. Deletions of the ESR gene were detected in four of six cell lines. For direct comparison of ER expression with the copy number of the ESR gene at the single cell level, immunophenotyping with mouse anti-human ER antibody was combined with FISH with the YAC probe containing the ESR gene according to the FICTION technique. There was no correlation between lack of or reduced ER expression and deletions of the ESR gene. One cell line with deletions of the ESR gene did express ER on the protein level, while another cell line without a deletion did not. Cells with deletions of the ESR gene were either ER expression positive or negative. The staining intensity of ER expression was not associated with the copy number of the ESR gene. Thus, this FICTION study unequivocally shows that deletions of the ESR gene are not the major cause of absent or reduced ER expression in breast carcinoma cell lines. Received: 6 September 1999 / Accepted: 14 September 1999     

The feasibility,demand, and effect of integrating primary care services with HIV voluntary counseling and testing: evaluation of a 15-year experience in Haiti, 1985-2000

BACKGROUND: HIV voluntary counseling and testing (VCT) may be an effective strategy to prevent transmission of HIV in developing countries. Hypothesizing that primary care services and HIV VCT have synergistic benefits, we examine the feasibility, the demand, and the effect of integrating on-site primary care services into VCT at a stand-alone VCT center in Port au Prince, Haiti. METHODS: Through a retrospective review of patient records, we describe the integration of primary care services at the Groupe Haitien d'Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) VCT center between1985 and 2000. RESULTS: Between 1985 and 1999, services for HIV care, tuberculosis care, treatment of sexually transmitted diseases, and reproductive health were sequentially integrated into HIV VCT at GHESKIO. The number of new people seeking voluntary counseling and testing at GHESKIO increased from 142 in 1985 to 8175 in 1999, with an increasing percentage of women, adolescents, symptom-free clients, and self-referred clients. Of new adults seeking VCT in 1999, the center was able to provide AIDS care to 17%, tuberculosis treatment to 6%, sexually transmitted infection management to 18%, and family planning to 19%. HIV transmission between discordant couples was 0 infections/100 follow-up years (95% CI, 0-3.2); vertical transmission from mother to child was 11 infections/100 live births (95% CI, 4.6-21.9); These rates are significantly lower than expected rates of transmission in Haiti. CONCLUSIONS: This report demonstrates the feasibility, demand, and effective synergy of integrating on-site primary care services into HIV VCT in Haiti. VCT is a good entry point for people in need of services for communicable diseases and reproductive health, and, reciprocally, services attract more people to VCT, including populations that are at high risk for HIV infection. This program is being duplicated elsewhere in Haiti and can serve as a model for other countries.     

Molecular and genetic characterization of human cell lines resistant tol-asparaginase and albizziin

Human cell lines resistant tol-asparaginase or albizziin were isolated by multistep selection of HT1080 fibrosarcoma and MIA PaCa-2 pancreatic carcinoma cells. Mutants were cross-resistant to both drugs, but more resistant to the drug used for selection. The drug-resistant cell lines expressed elevated levels of asparagine synthetase activity and protein, up to 17-fold over that of the parental cells. Enzyme overproduction was due to gene amplification in the albizziin-resistant cells, whereas increased expression without amplification was observed inl-asparaginase-resistant cells.