Cross-reactivities between date palm (Phoenix dactylifera L.) polypeptides and foods implicated in the oral allergy syndrome.

BACKGROUND: Date fruit and pollen antigens share a number of cross-reactive epitopes. Date pollen has been shown to cross-react with antigens from Artemisia, cultivated rye (Secale cereale), Timothy grass (Phleum pratense), Sydney golden wattle (Acacia longifolia) and Bermuda grass (Cynodon dactylon) pollen. The present study was carried out to examine any cross-reactivities between date palm polypeptides and antigens of some common foods and vegetables that have been implicated in the oral allergy syndrome (OAS). Because most of such cross-reactivities in other allergens are attributable to the presence of carbohydrate chains and profilin, their role was also investigated. METHODS: Fresh extracts of 20 common fruits and vegetables were prepared. Putative date profilins were isolated by affinity chromatography using a poly L-proline column. Date fruit extracts were digested by various endoglycosidases and the immunoglobulin (Ig)E binding of the postdigest products was assessed in immunoblots. Rabbit antisera to whole date fruit extracts, Timothy grass profilin and putative date profilins, as well as human sera from date sensitive individuals were used in immunoblotting, ELISA and in inhibition experiments. RESULTS: IgG, ELISA and immunoblot results with the different rabbit antisera and date-sensitive atopic sera showed several antigenic cross-reactivities and similar cross-reactivities were seen with birch, date and timothy grass profilins. IgE, ELISA and immunoblot experiments with pooled date sensitive human sera showed a range of cross-reactivities with some food extracts. A number of the IgE cross-reactivities could be inhibited after preabsorption of pooled sera with date extracts. Sixty-six percent of individual date hypersensitive human sera bound IgE in putative date fruit profilin and their pooled sera bound IgE in birch pollen profilin. IgE-binding of the endoglycosidase digested date fruit extracts to atopic serum pool was restricted to only a very low molecular weight band of 6.5-8 kDa. CONCLUSION: These results indicate that date palm polypeptides share cross-reactive IgG and IgE epitopes with a number of foods implicated in the oral allergy syndrome, bind to birch and Timothy grass profilins and bind IgE through glycosyl residues. The clinical relevance of these cross-reactivities needs to be further elucidated.

Cultivar-specific IgE-epitopes in date (Phoenix dactylifera L.) fruit allergy. Correlation of skin test reactivity and ige-binding properties in selecting date cultivars for allergen standardization.

BACKGROUND: Date fruits are allergenic and standardized extracts are required for diagnosis and therapy of this allergy. Since there are several cultivars of dates, this study was carried out to assess the allergenicity of different cultivars in order to select suitable source material for standardization. METHODS: The protein profiles of 18 of the most commonly sold varieties were compared by SDS-PAGE and their relative allergenicity assessed by SPT and IgE-based ELISA and immunoblotting. Thirty-two date fruit-sensitive patients were skin tested with a pooled extract from all the cultivars. Six of the patients with high SPT results (> or =3+) who volunteered were further tested with the 18 cultivars and their sera used in ELISA and immunoblotting. RESULTS: Six of the cultivars gave high SPT-positive reactions in > or =4 of patients. Five of these high SPT-reactive cultivars gave high IgE ELISA scores (> or =0.58) but individual cultivars varied in their number of IgE immunoblot bands. Cultivar-specific IgE-binding patterns indicated that only certain cultivars bound IgE at molecular weights of < or =14.3 and 27-33 kDa whilst all cultivars bound to a 54-58 kDa doublet. Cultivars that bind to the < or =14.3 and 27-33 kDa bands appeared to form the majority of the high SPT-reactive cultivars. When individual sera of 24 of the 32 SPT-positive patients were used in IgE immunoblots with the pooled cultivar extract, all sera bound IgE at < or =14.3 and 27-33 kDa and about 60% of sera bound to a 54-58 kDa doublet bands. CONCLUSIONS: These results indicate that allergenicity of date fruits is a cultivar-specific phenomenon. Sixty to 100% of sera from date fruit-allergic patients bind IgE to three major allergens of < or =14.3, 27-33 and 54-58 kDa. Five of the cultivars that evoke high SPT reactions, high IgE ELISA scores and bind IgE to the major allergens, can be selected for the preparation of 'in-house' allergen extracts and for allergen standardization.

Serum and synovial fluid concentrations of endothelin-1 in patients with rheumatoid arthritis.

To determine the endothelin-1 (ET-1) concentrations in synovial fluid and serum of rheumatoid arthritis (RA) patients, this study was designed to examine if serum ET-1 concentration of control subjects has any correlation either with the ET-1 concentration of synovial fluid or ET-1 concentration of serum from RA patients. Twenty-eight patients were studied of whom eight males and twenty females with confirmed rheumatoid arthritis. Twenty-eight healthy volunteers were also included as controls. The immunoreactive concentration of ET-1 was measured using commercially available radioimmunoassay (RIA) kits (Peninsula Laboratories, Belmont CA) specific for ET-1. All the samples were performed in duplicate and after plotting % B/Bo for each standard directly on Y axis and endothelin concentrations on the X axis, the "best fit" curve was drawn and the amount of ET-1 was calculated. Mean ET-1 level in synovial fluid was 15.53 +/- 2.82 pg/me. In serum samples from RA patients, the mean ET-1 level was detected as 16.42 +/- 3.07 pg/ml (n = 28). Sera from twenty-eight healthy volunteers were analyzed as controls and mean ET-1 concentration was 8.68 +/- 1.96 pg/ml. A significant difference (P < 0.001) was found between ET-1 level of sera from RA patients and ET-1 levels from control sera. Highly significant difference (P < 0.001) was also detected between synovial fluid ET-1 and control ET-1 levels. However, no significant difference was found between ET-1 levels of synovial fluid and serum ET-1 levels of RA patients. Results of this study confirmed the presence of elevated levels of ET-1 concentration in synovial fluid and serum samples of patients with RA. The clinical significance and physiological role of endothelin in synovial fluids and sera of patients suffering from a variety of pathophysiological conditions of arthritis deserves further studies.

FHIT gene abnormalities in both benign and malignant thyroid tumours.

FHIT, a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancers. Loss of heterozygosity (LOH) at this region has also been found frequently in follicular thyroid carcinoma (FTC). To investigate the potential role of FHIT in thyroid tumorigenesis, we examined 57 thyroid tumour specimens (eight benign adenomas, 40 papillary, four follicular and five anaplastic carcinomas), and two thyroid carcinoma cell lines (NPA, SW579) for genetic alterations by using reverse transcription-polymerase chain reaction (RT-PCR), PCR product sequencing, single-strand conformation polymorphism (SSCP) and Southern blot analysis. Two cervical carcinoma cell lines (C-33A, HeLa) were included as positive controls. We detected truncated FHIT transcripts in three of eight (38%) benign adenomas, nine of 40 (23%) papillary, and two of five (40%) anaplastic carcinomas, and in three cell lines (SW579, C-33A, HeLa). Most of the truncated transcripts lacked exons 4 or 5 to 7 or 8 of the gene and were presumably non-functional as the translation start site is located in exon 5. SSCP analysis of the coding exons failed to detect any point mutations among the samples without abnormal FHIT transcripts. Southern blot analysis demonstrated either loss or reduced intensity of major Bam HI restriction fragments in the three cell lines found to have abnormal FHIT transcripts, indicating, respectively, either intragenic homozygous or heterozygous deletions of the FHIT gene. Intragenic homozygous deletions were also found in two papillary thyroid carcinoma specimens: one was missing a 13 kb Bam HI fragment which contains exon 4, the other had deletions of 15.5, 13 and 4.2 kb fragments which contain exons 2 and 9, 4, and 5, respectively. The absence of a defective FHIT gene in FTC indicates that an additional tumour suppressor gene may reside in this region and be involved in the development of FTC. Given that defective FHIT genes were found in both benign and malignant thyroid tumours, the inactivation of this putative tumour suppressor gene is likely to be an early event in the pathogenesis of some forms of thyroid neoplasms.

Immunomodulatory effect of Nigella sativa proteins fractionated by ion exchange chromatography.

Whole Nigella sativa (N. sativa) proteins were purified on a DEAE Sephadex A50 ion exchange column. Complete fractionation was achieved in four peaks. Analysis of the purified peaks was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Whole N. sativa showed a number of protein bands ranging from 94-10 kDa molecular mass. In mixed lymphocyte cultures (MLC), whole N. sativa and its purified proteins were found stimulatory as well as suppressive and this effect varied from one donor to another. Maximum stimulation (mean + S.E. of % relative index was 63.73 + 20.78) was observed with fractionated N. sativa proteins (P1) (10 microg/ml) in MLC. In MLC, also N. sativa peaks (P1 and P2) were stimulatory at all concentrations (10 microg/ml, 1 microg/ml or 0.1 microg/ml) used. However, a uniformly suppressive effect of N. sativa and its all four peaks at a concentration of 10 microg/ml was noticed when lymphocytes were activated with pokeweed mitogen (PWM). The effect of N. sativa proteins was further evaluated on the production of cytokines which were measured by using specific enzyme-linked immunosorbent assay. Large quantities of IL-1beta were secreted by whole N. sativa in culture medium with non-activated peripheral blood mononuclear cells (PBMC) (450 pg/ml) and with allogeneic cells (410 pg/ml). Fractionated N. sativa was less effective when compared with whole N. sativa proteins. No effect on IL-4 secretion was seen either by using non-activated, PWM-activated or allogeneic-cells. Whole N. sativa suppressed as well as stimulated the production of IL-8 in non-activated and PWM-activated PBMC respectively. All N. sativa peaks with protein concentration of 2 microg/ml were stimulatory for the induction of IL-8 by PWM-activated cells. However, no effect on IL-8 was seen either with whole N. sativa or its peaks when allogeneic PBMC were used. Stimulatory effect of whole N. sativa and fractionated proteins was also noticed on the production of TNF-alpha either using non-activated or mitogen activated cells.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA is elevated in advanced stages of thyroid carcinoma.

Tumour cell invasion and metastasis is a multistep process that involves the degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs). Tissue inhibitors of metalloproteinases (TIMPs) act as negative regulators of MMPs and thus prevent tumour cell invasion and metastasis by preserving extracellular matrix (ECM) integrity. In the present study we examined the expression of one member of TIMPs, TIMP-1, in 39 thyroid tumour specimens and two thyroid carcinoma cell lines (NPA and SW579). We also investigated the effect of high TIMP-1 expression on the invasive potential of NPA cells. Northern blot analysis showed that TIMP-1 mRNA levels correlated directly with tumour aggressiveness: the highest number of TIMP-1 transcripts was found in stages III and IV vs benign goitre (P < 0.0001). However, TIMP-1 expression was not increased in NPA and SW579 cells, both of which are derived from poorly differentiated thyroid tumours. Immunohistochemical study showed strong TIMP-1 staining in the stroma cells of advanced stages of carcinomas. Overexpression of TIMP-1 by gene transfer resulted in a significant suppression of the malignant phenotype of NPA cells as judged by an in vitro tumour invasion assay. These results suggest that high levels of TIMP-1 transcripts in advanced stages of thyroid carcinoma likely come from stroma rather than thyroid cancer cells, and TIMP-1 may function as a thyroid tumour invasion/metastasis suppressor.

Allergy to date fruits: characterization of antigens and allergens of fruits of the date palm (Phoenix dactylifera L.).

BACKGROUND: Date-palm (Phoenix dactylifera L.) fruits are eaten daily by most inhabitants of the Middle East and the neighboring countries. Recent reports have indicated that dates are allergenic. This study aimed to investigate the antigenic and allergenic potential of date fruits. METHODS: Date-fruit extracts from eight cultivars were evaluated in skin prick tests (SPT) in an atopic population, used to produce antisera, analyzed by SDS-PAGE, and fractionated by gel-filtration chromatography. Sera from SPT-positive individuals were evaluated by ELISA and RAST, and in anti-igE immunoblot experiments. RESULTS: About 13% of patients were SPT-positive for at least two extracts. SDS-PAGE of whole extracts revealed 15-18 protein bands of 6.5->100 kDa, and Sephacryl S-200 fractions gave distinct peptide bands. RAST and anti-IgE ELISA gave a range of positive results, which could be abrogated when sera were preabsorbed with fruit extracts. IgE immunoblots of different extracts with pooled positive sera revealed different anti-IgE-binding immunoprints. All the positive sera from fruit-allergic and pollen-allergic individuals bound strongly to two anti-IgE reactive bands of 6.5 to 12-14 kDa and 28-33 kDa, respectively, and about 50% of sera bound to a 54-58-kDa band. CONCLUSIONS: These results strongly indicate that 1) date-palm fruit is a potent allergen 2) sera from fruit-allergic as well as pollen-allergic patients recognize common fruit-specific epitopes 3) there is heterogeneity in patient responses to the different extracts.

Human sensitization to Prosopis juliflora antigen in Saudi Arabia.

BACKGROUND: Allergenicity to Prosopis juliflora pollen antigen has been reported from only a few countries, including the US, South Africa, India and Kuwait. In some parts of Saudi Arabia, species of Prosopis have been introduced by the millions as roadside ornamentation. There appear to be four flowering seasons during which pollen grains float in all directions. However, the role of Prosopis pollen as the sensitizing and/or triggering agent of allergic asthma and/or rhinitis in the Kingdom has never been evaluated. PATIENTS AND METHODS: A total of 473 allergic patients suffering from bronchial asthma in four different geographical regions (Abha, Qassim, Hofuf and Gizan), and attending allergy clinics and chest disease centers of university and Ministry of Health hospitals in the region were tested for immediate hypersensitivity reaction to Prosopis juliflora allergens. Airborne pollen grains at one center were also studied for one full year, using volumetric sampling techniques. RESULTS: A total of 76.1% patients in Qassim, 37.5% in Gizan, 29% in Abha and 11% in Hofuf reacted positively to Prosopis antigen. Multiple sensitivities to other pollen antigens were detected in all patients. The level of airborne Prosopis pollen detected in Gizan exceeded 90 grains m -3 of air. CONCLUSION: In view of the documented evidence of Prosopis-involved allergenicity, the role of Prosopis pollen as a sensitizing factor in Saudi Arabia has been confirmed. However, the cause of elicitation of symptoms in many multiple sensitive patients, together with the question of cross-reactivities, needs thorough and detailed investigation. In vitro confirmation of all positive results is also required to incriminate Prosopis as one of the major allergens in parts of Saudi Arabia.

A carbocyclic nucleoside analogue is a TNF-alpha inhibitor with immunosuppressive action: role of prostaglandin E2 and protein kinase C and comparison with pentoxifylline.

Tumor necrosis factor-alpha (TNF-alpha) is associated with several acute and chronic inflammatory conditions. New therapies directed at inhibiting TNF-alpha will be important in treating pathological processes mediated by TNF-alpha. In this study, we studied and compared the effect of the carbocyclic nucleoside analogue (9-[(1R, 3R)-trans-cyclopentan-3-ol] adenine) with pentoxifylline on modulating TNF-alpha production. The carbocyclic nucleoside analogue inhibited TNF-alpha production in a dose-dependent manner (1 microM-1 mM) by stimulated peripheral blood mononuclear cells and cell lines of both monocyte (THP-1) and T-lymphocyte phenotypes (CEM x 174). The drug potently inhibited TNF production in cells stimulated by endotoxin, the superantigen (staphylococci enterotoxin A), the mitogen (phytohemagglutinin), and the protein kinase C activator (phorbol myristate acetate) with ED50 ranging from 5 to 30 microM. At moderate concentrations, the carbocyclic nucleoside analogue inhibited superantigen (ED50 = 300 microM) and alloantigen (mixed lymphocyte reaction) T cell proliferative responses (ED50 = 150 microM). The involvement of protein kinase C and prostaglandin E2 (PGE2), mediators that regulate TNF-alpha production, was also investigated. Unlike PTX, the nucleoside analogue did not upregulate PGE2 production. The inhibition of TNF-alpha production appeared to be mediated at least partly by PKC, since the nucleoside analogue caused suppression of PKC activity in stimulated cells. The results show that the carbocyclic nucleoside analogue is a TNF-alpha inhibitor that may be appropriate in the therapy of TNF-alpha-associated complications. The suppressive properties of the carbocyclic nucleoside analogue on antigen and alloantigen (mixed lymphocyte reaction) responses may be appropriate in disease conditions in which inhibiting both TNF-alpha and T-cell reactivity is desirable.

Presence of leukemia inhibitory factor and interleukin-12 in human follicular fluid during follicular growth.

PROBLEM: Cytokines have been shown to be present in human follicular fluid and have regulatory functions on follicular maturation. The presence of leukemia inhibitory factor (LIF) and interleukin (IL)-12 in human follicular fluid obtained at different stages of maturation was investigated. METHOD OF STUDY: Follicular fluids and granulosa cells were obtained from preovulatory and immature follicles. Follicular fluids from both groups were assayed for IL-12 and LIF by enzyme-linked immunosorbent assay. Granulosa cells from preovulatory and immature follicles were treated with human chorionic gonadotropin (hCG) in vitro and subsequent LIF and IL-12 production were measured. RESULTS: The average concentration of LIF was significantly higher in preovulatory follicles (7.6 +/- 1.3 pg/ml, n = 24) than in immature follicles (2.0 +/- 1.3 pg/ml, n = 6). The concentration of IL-12 was significantly higher in follicular fluid obtained from immature follicles (10.9 +/- 5.0 pg/ml) than in preovulatory follicles (1.3 +/- 0.4 pg/ml). hCG only stimulated LIF production from mature granulosa cells; it had no effect on IL-12 production. CONCLUSIONS: IL-12 and LIF are present in follicular fluid and their levels are regulated differently during follicular maturation. hCG stimulates LIF production from granulosa cells in vitro.

Inverse association between cyclin D1 overexpression and retinoblastoma gene mutation in thyroid carcinomas.

Cyclin D1 plays a key role in the regulation of the G1/S transition through the cell cycle. Deregulation of cyclin D1, most often leading to overexpression of the gene, has been reported in many tumor types. It has been suggested that cyclin D1 overexpression could be an alternative mechanism for pRb inactivation. We have previously found Rb gene mutations in 55% of malignant thyroid tumors. In the present study, we examined the cyclin D1 gene expression and amplification in 24 tumor samples (two of them are benign goiters) randomly selected from the same series of thyroid tumors, to see whether cyclin D1 overexpression is present in those specimens without Rb gene mutations. We found a four- to fivefold increase in cyclin D1 expression in 7 of 22 thyroid carcinomas as compared with that in benign nodular goiters. Six of them were found in carcinomas without Rb gene mutations. Among the remaining 15 thyroid carcinoma samples, 11 were found previously to have Rb gene mutations. The association between increased cyclin D1 expression and absence of Rb mutation is statistically significant (p < 0.05). We found no evidence of the cyclin D1 gene amplification or rearrangement to account for such an increase in cyclin D1 expression. We conclude that cyclin D1 overexpression may be relevant to thyroid carcinogenesis. Two mechanisms may be involved in the inactivation of pRb: one is through Rb gene mutations, and the other is by cyclin D1 overexpression.

Aeroallergens and viable microbes in sandstorm dust. Potential triggers of allergic and nonallergic respiratory ailments.

Aeroallergens and antigens in sandstorm dust, extracts of which were skin prick test (SPT) positive in allergic patients, were detected by rocket immunoelectrophoresis and ELISA. Fungi and bacteria isolated by agar settle plates and soil dilution and soil washing methods were enumerated and identified. Cat dander, Acacia, Alternaria, Aspergillus, Chenopodium, Cladosporium, Bermuda grass, Pithecellobium, Prosopis, Rumex, cultivated rye, and Washingtonia palm allergens were detected by both methods. Viable microbes including 1892 +/- 325 colony-forming units (cfu) of bacteria, and 869 +/- 75 cfu of fungi were isolated per gram of dust by the soil dilution method. Randomly selected microbial colonies on streaking and subculture were found to consist of between two and seven mixed colonies. Fungi including Alternaria, Aspergillus, Botrytis, Cladosporium, Mortierella, Mucor, Mycelia sterilia, Penicillium, Pythium, Ulocladium, Verticillium, and some yeasts were isolated. Actinomyces, Bacillus, Pseudomonas, and mostly coagulase-negative Staphylococcus species were identified, but the bulk of unidentified bacterial isolates were mainly mixed colonies of rods, cocci, coccobacilli, and some filamentous types. Six-hour agar settle-plate counts during sandstorms were 100 and 40% higher for bacteria and fungi, respectively, than without sandstorms. The most abundant aeroallergens were those of Acacia, Alternaria, Aspergillus, Bermuda grass, Cladosporium, cultivated rye, Prosopis, and cat dander. Pithecellobium dulce, Rumex crispus, and Washingtonia palm allergens were detectable for the first time in Riyadh. IgE reactivities of the dust in man were demonstrated by ELISA using sera from atopic, exposed, and normal subjects. These results indicate that sandstorm dust is a prolific source of potential triggers of allergic and nonallergic respiratory ailments, and the methods mentioned here should be routinely used for quick sampling of the environment.

The alpha chemokine, interleukin 8, inhibits the antiviral action of interferon alpha.

Interferon (IFN) exhibits a potent antiviral activity in vitro and plays a major role in the early defense against viruses. Like IFN, the proinflammatory chemokine, interleukin (IL)-8, is induced by viruses and appears in circulation during viral infections. In an in vitro cytopathic effect assay for IFN, we found that IL-8 can inhibit IFN-alpha activity in a dose-dependent manner. This action was reversed by specific monoclonal antibodies to IL-8. The chemokine was able to attenuate the IFN-mediated inhibition of viral replication as determined by measuring infectious virus yield. IL-8 also diminished the ability of IFN to inhibit an early stage of viral replication since IL-8 attenuated the inhibition of the formation of viral proteins. It appeared that IL-8 interfered with a late rather than an early step of IFN-mediated pathway such as early gene expression. The IL-8 inhibitory action on IFN-alpha antiviral activity was associated with reduced 2',5'-A oligoadenylate synthetase activity, a pathway well correlative with the anti- encephalomyocarditis virus action of IFN-alpha. Understanding pathways that antagonize IFN action may lead to novel approaches to potentiate endogenous and therapeutic IFN.

The effect of antibodies against cell-surface adhesion molecules (LFA-1 alpha and ICAM-1) on the migration and localization of 99mTc-labeled leukocytes in acute infection.

The deficiency of adhesion molecules on leukocytes could severely impair their ability to migrate and perform effective immunological functions leading to clinical situation such as LAD (leukocyte adhesion deficiency) syndrome. We investigated the effects of blocking anti-LFA-1 alpha and ICAM-1 antibody-treated 99mTc-labeled leukocytes on the migration and localization to the site of E. coli-induced acute infection in CBA/J mice. A significant inhibition of migration and localization of antibody-treated leukocytes to the site of infection was observed, reaffirming the vital role of these adhesion molecules, especially during scintigraphic examination of patients for deep infections or abscess using labeled leukocytes.

House dust mite allergens in Saudi Arabia: Regional variations and immune response.

In order to assess the causative extrinsic allergic factor(s) in school-age children diagnosed as having bronchial asthma and allergic rhinitis, and to qualitatively and quantitatively evaluate the presence of house dust mites (HDMs) in the homes of these children in Saudi Arabia, a study analyzing mite contents in 165 samples collected from patientsâ indoor environment was conducted. The dust samples were collected from four regions of Saudi Arabia, showing variation in their geography and climate. Immunochemical assays were performed using ALK reagents by ELISA technique. A total of 462 children were also tested using skin prick test (SPT) method for IgE-mediated reactions to HDMs. The samples from the Central dry region revealed a very low amount of the potent house dust mites (Dermatophagoides pteronyssinus and D. farinae, the two dominant species in various parts of the world). The samples from the Southern mountainous region contained a very high concentration of Der p I (84,000 ng/g of dust), while the Western coastal region showed a high concentration of Der f I (up to 22,000 ng/g). The mid-Western agricultural region did not exhibit any significant level of either Der p I or Der f I. The maximum level of D. pteronyssinus detected in the Central dry region was 106 ng/g of dust. The data exhibit both qualitative and quantitative variations of HDMs in the three regions and may be attributed to variation in geography and climate, particularly humidity of the regions, which vary significantly. Riyadh in the Central region is considered to have low humidity (<40%), while humidity in the Western coastal region, Jeddah, and the Southern region of Abha is comparatively higher, which helps house dust mites thrive. SPT results in these regions with house dust mite allergens (in addition to other common inhalant allergens) also revealed a considerable number of IgE-mediated reactions, consistent with the frequency of house dust mites in the region. Though more data are being accumulated on the subject to conduct a statistical comparison and more skin tests are underway in the Southern region, the study suggests the presence of at least two HDMs as well as qualitative diversity and quantitative variation of house dust mites in Saudi Arabia. The study also indicates, with a considerable number of IgE-mediated reactions, the possible influence of mites in the allergic manifestations of many patients, which is not only common, but increasing in parts of the country.

Evidence of gene deletion of p21 (WAF1/CIP1), a cyclin-dependent protein kinase inhibitor, in thyroid carcinomas.

Eukaryotic cell cycle progression is controlled by a host of cyclin/cyclin-dependent kinases (Cdks), that are themselves regulated by multiple factors, including a group of small cyclin-Cdk inhibitor proteins (p15, p16, p21 and p27). The involvement of Cdk inhibitors in carcinogenesis has been demonstrated by the studies of p16. p53 is frequently mutated in thyroid carcinomas and p21/Waf1 is a downstream effector of p53. It is conceivable that genetic defects of genes downstream in the p53 pathway could also be oncogenic. We, therefore, examined a series of 57 thyroid tumour specimens (eight follicular adenomas and 49 carcinomas) for deletion and point mutation of the p21/Waf1 gene. Three different kinds of deletions ranging from 349 to 450 bp were detected in five papillary carcinoma specimens by reverse transcription-polymerase chain reaction (RT-PCR). All the deletions were involved in the second exon of the p21/Waf1 gene. RT-PCR single strand conformational polymorphism (SSCP) analysis of remaining samples failed to reveal any point mutations in the coding region of the gene, except for a polymorphism at codon 31 (Ser to Arg). Genomic Southern blot analysis did not demonstrate any gene deletion or rearrangement in these samples, indicating abnormal RNA splicing may be involved. Analysis of intron-exon boundary and the coding region of the second exon did not reveal any mutation except for a point mutation (C to G) located 16 bp downstream from the splice donor site of the second intron in three out of five samples with p21/Waf1 deletions. Whether the mutation plays any role in aberrant RNA splicing remains to be determined. Among the five samples with p21/Waf1 gene deletions, none of them simultaneously carried a p53 or retinoblastoma (Rb) gene mutation. No p21/Waf1 abnormality was found in the benign adenomas. Thus, 12.5% (5/40) of thyroid papillary carcinoma specimens harboured p21/Waf1 gene deletions. Our data suggest that p21/Waf1 gene deletion is involved in thyroid carcinogenesis and may play an important role in thyroid cell transformation.

Immunoreactive endothelin-1, endothelin-2 and big endothelin-1 in follicular fluids of women undergoing ovulation induction for in-vitro fertilization.

Endothelin-like immunoreactivity specific for endothelin-1 (ET-1), endothelin-2 (ET-2) or big endothelin-1 (big ET-1) was measured, using commercially available radioimmunoassay kits, in follicular fluid collected at the time of oocyte aspiration from 36 women undergoing ovulation induction by human menopausal gonadotrophin (HMG). The relationship of ET concentrations to HMG dose, peak serum oestradiol concentration, the number and size of follicles (by ultrasound), the number of retrieved oocytes and the fertilization rate per retrieved oocyte were studied. Overall, 94% of follicular fluid samples were positive for ET-1, 92% were positive for ET-2, and 100% were positive for big ET-1. Mean ET-1, ET-2 and big ET-1 concentrations were 17.23 +/- 12.20, 32.42 +/- 14.32 and 34.55 +/- 16.34 pg/ml respectively. Endothelin-like immunoreactivity in follicular fluid samples was found in an order of ET-1 < ET-2 < big ET-1. There was a highly significant positive correlation (r = 0.8711,P = 0.001, n = 32) between follicular ET-1 and ET-2 concentrations. No significant correlation of follicular big ET-1 was established either with ET-1 or ET-2. However, big ET-1 was found to be negatively correlated with number of oocytes (P = 0.03) and number of follicles (P = 0.04). Control plasma ET-1 and follicular ET-1 were not significantly different. There was no significant correlation between ET concentrations and any of the other studied parameters. The results demonstrated that immunoreactive ET-1, ET-2 and big ET-1 exist in human follicular fluid collected at the time of oocytes retrieval for in-vitro fertilization and may be involved in the regulation of reproductive function. The clinical significance and physiological role of follicular fluid ET deserve further studies.

Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody.

The study was designed in order to investigate the action of progesterone on the spontaneous and ionophore-induced human spermatozoa acrosome reaction in vitro. The principle of the assay system is flow cytometric analysis of CD46 antibody binding to the inner acrosomal membrane. The technique is a simple and objective method of analysis, allowing fluorescent analysis of a large segment (5000 spermatozoa) of the spermatozoa population under investigation, with concomitant isolation of the live fraction of the spermatozoa population. Four concentrations of progesterone (1, 25, 50, and 100 microg/ml) were examined for their effects on spermatozoa capacitated for 4 and 24 h. In addition, motility parameters were examined by the CellSoft 2000 automated semen analyser system. Analysis of variance revealed that progesterone had no effect on either the spontaneous acrosome reaction or the ionophore-induced acrosome reaction at both 4 h and 24 h of spermatozoa capacitation times. Further, no effects on sperm motility parameters or on spermatozoa viability could be attributed to progesterone. We therefore conclude that progesterone has no objectively measurable effects on either the sperm acrosome reaction or sperm motility parameters, as measured in normal sperm populations.

Gene usage and regulation of Gsα gene expression in thyroid cells.

The TSH receptor is a G-protein-coupled seven transmembrane segment receptor. The interaction between TSH and its receptor mediates signal transduction by activating adenylyl cyclase through Gsα. There are four forms of Gsα (two short [45 kDa] and two large [52 kDa]), arising from alternative splicing of exon 3 of the Gsα gene. Gsα-1 and -2 contain exon 3, whereas exon 3 is spliced out in Gsα-3 and -4. The inclusion of a serine residue at the 3' splice junction of exon 3 distinguishes Gsα-2 and -4 from Gsα-1 and -3. The expression of different Gsα forms appears to be tissue-specific. In this study, we have examined the Gsα splice variants in 26 human thyroid tumor specimens and rat thyroid tissues as well as a rat FRTL-5 cell line. Furthermore, we have studied the regulation of the Gsα gene expression by TSH and cAMP in FRTL-5 cells. We found that Gsα-1 and -4 mRNA were present in both human and rat thyroid cells, although Gsα-4 was more abundant in human thyroid cells as compared to rat thyroid and FRTL-5 cells. The Gsα mRNA can be easily amplified by RT-PCR regardless of tumor type and stage, suggesting that Gsα gene expression in thyroid tumors may not be markedly affected by dedifferentiation of thyroid cells.Both TSH and 8-bromo-cAMP, a cAMP analog, can stimulate the Gsα gene expression in FRTL-5 cells with maximal effect by 6 h and 1 h, respectively. The addition of cycloheximide to the culture of FRTL-5 cells abolished the effect of bTSH, but not that of 8-bromo-cAMP, on the expression of the Gsα gene. Cellular cAMP measurements showed that bTSH-stimulated cAMP production was significantly reduced to the basal level after addition of cycloheximide. These results suggest that regulation of the Gsα gene expression by TSH is mediated by a cAMP-dependent process and requires new protein synthesis.