Potential role for arachidonic acid and eicosanoids in modulating progesterone secretion by ovine chorionic cells.

The present study was conducted to investigate whether arachidonic acid and its metabolites can modulate progesterone (P4) secretion in ovine chorionic cells. At concentrations of 7.5 mumol/l and 12.5 mumol/l, arachidonic acid caused an increase of basal P4 secretion (about 1.8-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, over control). Such a stimulatory effect was suppressed when the concentration of arachidonic acid attained 25 mumol/l, and at 50 mumol/l the fatty acid led to a decline of basal P4 synthesis (about 35%, p < 0.01). Phospholipase A2 (PLA2) and melittin had a similar dual effect to that observed when arachidonic acid was added exogenously. In contrast, eicosatrienoic acid (a closely related fatty acid) did not stimulate P4 secretion but inhibited it at a concentration of 50 mumol/l (about 40% inhibition, p < 0.01). The possible involvement of calcium on the effects of arachidonic acid was explored. Interestingly, 3 mmol/l ethylene glycol bis(beta-aminoethyl ether)-N,N,N,N'-tetraacetic acid (EGTA) and 10 mumol/l 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) further enhanced the steroidogenic effect of 12.5 mumol/l arachidonic acid (p < 0.05 and p < 0.01 vs the corresponding value in the absence of EGTA or TMB-8, respectively). In contrast, these agents failed to modify P4 secretion observed in the presence of 50 mumol/l arachidonic acid. We also tested the effect of inhibition of arachidonic acid metabolism via cyclooxygenase and lipoxygenase pathways. Indomethacin (10 mumol/l) failed to block the effects of arachidonic acid, but nordihydroguaiaretic acid (10 mumol/l) prevented the stimulatory action of this fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

20-alpha-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: obtention and characterization of a monoclonal antibody against the enzyme activity.

The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax.

Activation of phospholipase C by different effectors in rat placental cells.

The present communication documents the accumulation of inositol phosphates in rat placental cells by fluoride as well as by vanadate. These findings suggest the existence of the phosphoinositide pathway and its modulation by a G-protein. A concomitant action of fluoride on phosphoinositide breakdown was also observed. As is often the case in intact cells from different organs, protein kinase C exerts a feedback regulatory control on this signalling system. Gonadotropin-releasing hormone (GnRH) also stimulated the accumulation of inositol phosphates in cultured cells but no effect could be detected in freshly isolated cells. Therefore, the phosphoinositide pathway seems to be involved in the mechanism of action of GnRH in rat placental cells.

Nucleolar organizer region enumeration in keratoacanthomas and squamous cell carcinomas of the skin.

Keratoacanthomas (KA) and squamous cell carcinomas (SSC) are epithelial skin tumors exhibiting distinctive clinical and histologic features. However, the differential diagnosis between them in individual cases may be difficult or even impossible. In this article the authors examine the possibility that enumeration of associated proteins of nucleolar organizer regions (AgNOR) could be of help in differentiating KA from SCC. AgNOR counting, performed on unequivocal cases of SCC (n = 20) and KA (n = 16) showed statistically significant higher AgNOR counts in SCC (6.29 +/- 0.91) compared with KA (3.80 +/- 1.62). This result speaks in favor of the different biologic nature of SCC and KA; however, due to significant overlap between the two groups, AgNOR enumeration alone is not sufficiently discriminating so as to be used diagnostically in cases with borderline histologic features.

Evidence for modulation of progesterone secretion by calcium and protein kinase C activators in ovine chorionic cells.

The hypothesis that calcium-dependent mechanisms may be involved in regulating ovine placental steroidogenesis was investigated using chorionic cells isolated by enzymatic digestion. Treatment of the cells with the calmodulin antagonist trifluoperazine (TFP) or pimozide caused a dose-related inhibition of progesterone (P4) production by 80 percent (P less than 0.001) at 40 microM TFP and 56 per cent (P less than 0.001) at 10 microM pimozide. Moreover, the conversion of 25 hydroxycholesterol (25 OH Chol.) to P4 was impaired in the presence of these compounds. These experiments suggest the involvement of a calcium-calmodulin system in the regulation of ovine placental P4 synthesis. Interestingly, calcium ionophore A23187 caused a gradual decline in P4 secretion and completely blocked it at 1 microM (P less than 0.001) and remains absent even in the presence of 25 OH Chol. In contrast, EGTA increased P4 secretion (P less than 0.01). Further, in the presence of 3 mM EGTA the inhibitory effect of 1 microM A23187 was fully reversed. Taken together these results suggest that extracellular calcium could play a role of negative modulation of P4 secretion in these cells. The possible involvement of protein kinase C (PKC) was tested using tumor-promoting phorbol ester (PMA) or permeant diacylglycerols (OAG or DOG). These compounds were unable to modify basal P4 secretion but reduced 25 OH Chol stimulated secretion to basal level. The phorbol ester that was unable to activate PKC had no effect on the metabolism of 25 OH chol. Thus, PMA and diacylglycerol effects are probably mediated by PKC. These data support the hypothesis that PKC activation plays a role in the modulation of cholesterol side-chain cleavage activity in ovine chorionic cells. These results show that calcium-dependent processes are involved in both positive and negative control of P4 secretion by ovine placenta. Our results also suggest a role for calmodulin and PKC pathways in modulating this secretion.

Oral hairy leukoplakia in a HIV-negative renal graft recipient.

Oral hairy leukoplakia (OHL) is seen almost exclusively in patients infected with HIV. A case is reported of OHL occurring in a patient who was seronegative for HIV and who had a renal graft. This occurred following an increase in his treatment with immunosuppressive drugs.

Human papillomavirus type 1-associated squamous cell carcinoma in a heart transplant recipient.

The occurrence of squamous cell carcinomas in organ transplant recipients with warts represents a good model to study viral carcinogenesis. Most of the cases were reported in renal transplant recipients. We present the case of a heart transplant recipient in whom multiple common warts, preepitheliomatous keratoses, and squamous cell carcinomas developed. The warts began 4 years after the transplantation and the first carcinoma occurred 2 years after the warts, all the lesions being on sun-exposed areas. Histologic signs of human papillomavirus infection were seen in all premalignant and malignant lesions. Furthermore, human papillomavirus type 1 DNA was detected by in situ molecular hybridization within one of the carcinomas. Human papillomaviruses, along with other carcinogenic factors, play an important role in the development of carcinomas, and benign types could be implicated. Further studies are required to evaluate the frequency of cutaneous malignant neoplasms in heart transplant recipients as compared with renal transplant recipients.

Neuron-specific enolase is a marker of cutaneous Langerhans' cell histiocytosis ("X")-a comparative study with S100 protein.

The immunohistochemical expression of neuron-specific enolase (gamma/gamma) (NSE) was studied comparatively with S100 protein in a group of Langerhans-cell-type ("X") (n = 8) and non-Langerhans-cell-type ("non X") (n = 24) cutaneous histiocytoses. NSE was expressed by the majority (70-90%) of histiocytic cells in all cases of Langerhans-cell histiocytoses, whereas it was absent from non-Langerhans-cell histiocytoses. S100 protein was expressed by the majority of Langerhans-cell histiocytosis cells but also by a small percentage (1-5%) of cells in non Langerhans-cell histiocytoses. These results show that NSE is almost as sensitive as, but more specific than, S100 protein in discriminating Langerhans-cell from non-Langerhans cell cutaneous histiocytoses, and that it consequently represents a useful adjunct in the immunohistochemical diagnosis of histiocytic skin diseases.

Reactivity of HMB-45 monoclonal antibody with sweat-gland tumours of the skin.

HMB-45, a monoclonal antibody claimed to be specific for malignant melanoma, has been observed to react with normal eccrine sweat glands and occasionally with normal mammary and bronchial epithelium. In this study we show that HMB-45 also decorates cells in approximately 15% of various sweatgland tumours of the skin. This finding, along with the reported reactivity on mammary carcinomas further outlines the lack of absolute specificity of HMB-45 for cells of the melanocytic lineage.

[Oral hairy leukoplakia in AIDS. Histologic and ultrastructural study of 8 cases]. / La leucoplasie orale chevelue du SIDA. Etude histologique et ultrastructurale de 8 cas.

Oral hairy leukoplakia is a disease of the oral mucosa occurring almost exclusively in HIV-infected (mostly AIDS) patients and due to the opportunistic development of Epstein-Barr virus (EBV) within the oral epithelium. Clinically, it shows as whitish patches with a shaggy surface occurring on the lateral margins of the tongue, less frequently the buccal and labial mucosa or the soft palate. Histologically, it comprises parakeratotic hyperkeratosis, acanthosis and numerous koilocytoid cells within the stratum spinosum, i. e. cells with a pycnotic nucleus surrounded by a clear halo and pale-staining cytoplasm. Electronmicroscopy readily shows abundant Herpes-group viral particles within the upper epithelial layers. By immunohistochemistry, in situ molecular hybridization and Southern-blot EBV antigens and DNA have been demonstrated within the lesions whereas HPV and HIV are generally undetectable. In the present work we studied by light- and electronmicroscopy lesions from 8 HIV-seropositive individuals that fulfilled the clinical and histological criteria of OHL. Ultrastructural examination showed the presence in all cases of Herpes-type virions, which, in two of the cases studied by immunohistochemistry, proved to belong to the EBV. It is concluded that electronmicroscopy is a sufficiently sensitive examination to confirm the diagnosis of OHL suggested in the presence of an appropriate clinico-histological setting.

[Warts and epidermoid carcinoma after renal transplantation]. / Verrues et carcinomes épidermoïdes aprés transplantation rénale.

UNLABELLED: Kidney transplant recipients suffer in the long-term from several cutaneous disorders linked to the transplantation. We had the opportunity to observe several patients presenting with pre-epitheliomatous keratoses and cutaneous carcinomas associated with warts. We report herein on five cases that were subjected to a clinical, histological and virological study. Material and methods. Clinical and histological report. The patients were referred to use by the Kidney Transplantation Department of the Ed. Herriot Hospital (Lyon). They were examined clinically by one of us (S.E.). Virological studies. These were performed on warts, keratoses, keratoacanthomas and squamous cell carcinomas. Human papillomavirus (HPV) antigen was detected by indirect immunofluorescence using rabbit antibodies raised against group-specific HPV antigen; viral DNA was detected by in situ molecular hybridization using biotinylated probes of types 1a, 2a, 16, 18 in all cases and type 5 in 14 lesions under stringent conditions. DNA-DNA hybrids were revealed by an alkaline phosphatase enzymatic system. RESULTS: (a) Clinical data are summarized in table I (see fig. 1-5). (b) Histological examination (fig. 6-9) showed either unequivocal squamous-cell carcinoma or keratoacanthoma . The overall architecture of the lesions was reminiscent of keratoacanthoma; however the lower limit was frequently not sharply demarcated; in that area, cells contained large basophilic nuclei exhibiting atypical features and numerous mitoses. The majority of lesions had an histological appearance reminiscent of warts (table III), with upper epidermal keratinocytes being vacuolized and containing basophilic (c) The results of virological studies (fig. 10-13) are summarized in table III. HPV group specific antigen was detected merely in 5 out of 33 lesions; in contrast, in situ molecular hybridization showed that 25 out of 33 lesions contained HPV DNA, with 14 of them containing the potentially oncogenic types 16 and 18. Only 2 lesions were positive with the prove HPV 5. Discussion. The overall incidence of cancers in Kidney transplant recipients (3 p. 100) is about 100 times higher than in control populations (17). Cutaneous carcinomas account for about 50 p. 100 of cancers. This incidence increases with time after transplantation and sun-exposure. The delay on onset of cutaneous malignancies is relatively long (4 to 7 years) (6,7) and becomes longer with a decreasing age of the patients at the time of transplantation, as can be noted in our cases. Apart from Blohme (1), most authors have reported a prevalence of squamous over basal-cell carcinoma. None of our patients presented basal-cell carcinoma.(ABSTRACT TRUNCATED AT 400 WORDS)

Lack of short-term modulation of in vitro placental progesterone secretion in sheep.

Experiments were performed in order to determine whether progesterone secretion in the ovine placenta can be short-term regulated. There was an increase in progesterone content per unit weight in ovine fetal cotyledons as gestation progressed: 17.0 +/- 4.7 ng/100 mg of wet tissue in ewes between 40 and 54 days of pregnancy (n = 7) and 70.7 +/- 18.8 (n = 9) between 100 and 118 days. At all stages of pregnancy, neither progesterone nor 20 alpha-dihydroprogesterone synthesis were significantly affected when fetal cotyledons were incubated for 3 h in the presence of LH, 8-Br-cAMP, GnRH agonist or GnRH antagonist. Addition of pregnenolone to the incubation medium increased progesterone secretion in a dose-dependent manner while addition of 25-hydroxycholesterol did not. These results suggest that the existent (basal) synthesis of progesterone reflects the maximal capacity of steroidogenesis through the cholesterol side-chain-cleavage system. In the presence of these precursors, LH, 8-Br-cAMP, the phorbol ester derivative PMA and calcium ionophore A23187 were not able to modify progesterone or 20 alpha-dihydroprogesterone synthesis. These results also suggest that LH or GnRH and the two signal mechanisms involved in their action, i.e. cAMP and Ca2+ sensitive-inositol phospholipid-dependent mechanisms are not implicated in the short-term regulation of progesterone synthesis in the ovine placenta.

Adenylate cyclase stimulation and luteinizing hormone-receptor interaction in plasma membranes from rat testicular interstitial cells in relation to the chemical structure of the hormone. Role of Mg2+.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.

Concurrent LH and forskolin action on adenylate cyclase activation and progesterone synthesis in corpora lutea from pregnant ewes.

The present communication documents LH- and forskolin-induced activation of adenylate cyclase (AC) system and progesterone synthesis in corpora lutea from pregnant ewes. The activation of AC in plasma membranes by LH or forskolin was amplified by Gpp(NH)p. These results suggest that regulatory nucleotide component (Ns) of the AC complex is required for forskolin. Simultaneous addition of maximal concentrations of forskolin (10(-4) M), Gpp(NH)p (10(-4) M) and LH (10(-7) M) led to greater than additive (i.e. synergistic) responses: the experimental value was 4.71 +/- 0.19 nmoles cAMP/mg of membrane protein, whereas the theoretical additive effect was 3.17 +/- 0.10 nmoles/mg of membrane protein (p less than 0.001). These data reveal that more Ns or C component is being activated in these cells when combined treatments with these agents are applied. In intact cells maximum stimulatory concentrations of forskolin or LH caused similar increase in progesterone production with similar time courses. In striking contrast, the exposure of the luteal cells to LH and forskolin simultaneously led to a decrease in progesterone synthesis as early as 1h30 (40%, p less than 0.001). Thus, the synergism observed between LH and forskolin on the stimulation of plasma membranes AC activity did not occur in steroidogenesis. The AC responses in crude plasma membranes form these cells to different stimulants were enhanced (i.e. 15%, p less than 0.2 for Gpp(NH)p, 33%, p less than 0.01 for LH plus Gpp(NH)p and 52%, p less than 0.01 for forskolin). These findings suggest that an early desensitization of the AC system cannot explain the impaired steroidogenic response observed.

Eosinophilic cellulitis (Wells' syndrome): ultrastructural study of a case with circulating immune complexes.

A 42-year-old woman was observed during 3 bouts of eosinophilic cellulitis over a 6-year-period. Skin biopsies were taken at each relapse and processed for histological, immunofluorescent and ultrastructural studies. Histologically the eosinophilic infiltrate extended to the deep dermis and the subcutaneous fat. High levels of circulating immune complexes, and complement and IgG deposits around the vessels were detected for as long as the cutaneous lesions lasted. Under the electron microscope eosinophils were numerous, half of them degranulated and some granules had a double cristal core. No injury to the vessel walls was observed. The 3 recurrences occurred respectively after lincomycin, nesdonal, acetyl salicylic acid and pholcodin ingestion and responded to sulfone and steroid therapy.