PO-13 - Production of a functional thrombopoietin by a human ovarian cancer cell line.

INTRODUCTION: Hemostatic abnormalities are frequently noticed in patients with malignant diseases. These complications include platelets disorders. The role of platelets in cancer extends beyond thrombocytosis and thrombosis, and also platelets promote cancer growth and metastatic dissemination. In the physiology, platelet production is regulated by thrombopoietin, which is mainly secreted by the liver. We, previously, reported that thrombopoietin could be secreted by the ovarian adenocarcinoma cell line, OVCAR-3. AIM: Our main purpose is to analyze the gene expression of thrombopoietin in ovarian cancer cells and to assess its functionality. MATERIALS AND METHODS: The thrombopoietin gene expression in ascitic cells from patients with ovarian carcinomatosis, as well as, in three cancer cell lines, including OVCAR-3 cells, performed using reverse transcription PCR, real-time PCR and gene sequencing, Normal human ovary and liver tissues are used as controls. The functionality of thrombopoietin on the basis of the viability of a thrombopoietin-dependent cell line (Ba/F3) using a co-culture method. RESULTS: Similarly to liver and ovary tissues, all cancer cells lines express the three TPO-1 (full length TPO), TPO-2 (12bp deletion) and TPO-3 (116pb deletion) variants. By flow cytometry, we show that thrombopoietin production by OVCAR-3 could be increased when cells are stimulated by activated protein C. Lastly, Our results confirm that activated protein C may act, in a paracrine fashion, to boost thrombopoietin production. CONCLUSIONS: We report, for the first time, that thrombopoietin secreted by ovarian cancer cells is functional. Hence, thrombopoietin produced by tumor cells may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.

HLA-G 14 bp insertion/deletion polymorphism and its association with sHLA-G levels in Behçet's disease Tunisian patients.

The purpose of this study was to investigate the HLA-G 3'UTR 14 bp polymorphism and sHLA-G levels in Tunisian patients with BD. The study included 119 patients with BD and 170 healthy blood donors (HD). HLA-G 14 bp polymorphism was genotyped by polymerase chain reaction. Serum levels of soluble HLA-G (sHLA-G) were measured using a commercial ELISA kit. A significant increased frequency of the -14 bp HLA-G allele was detected in patients with BD compared to HD (0.58 vs 0.49, p=0.023), and a significant increased frequency of HLA-G -14/-14 bp was observed in patients with BD compared to HD [0.37 vs 0.22, p=0.007, OR 2.04 (95% CI 1.21-3.42)]. The mean plasmatic concentration of sHLA-G levels were significantly increased in patients with active disease [231.63±286.4 U/mL] compared to those with inactive disease (103.14±77.8 U/mL, p=0.03) and HD (121.41±24.1 U/mL, p=0.04). Furthermore, our results showed that there is no association between HLA-G 14 bp polymorphism and sHLA-G plasma levels.

Phenotypic abnormalities of peripheral blood mononuclear cells in patients with Behçet's disease and association with HLA-B51 expression.

The aim of this study was to investigate the subclasses and the immunophenotypic profile of peripheral mononuclear cells in patients with Behçet's disease (BD) and to assess associations between the expression of HLA-B51 antigen and that of other cell markers. Thirty healthy volunteer blood donors and forty patients with BD were enrolled into this study. Phenotyping was performed using two color flow cytometry. HLA-B51 typing was performed using the complement dependent microlymphocytotoxicity assay. Unlike controls, patients with BD presented a modified immunophenotypic profile of lymphocytes. Compared to those in the remission phase, patients with active BD showed an increased mean of MFI ratio of CD56 on CD16+CD56+ cells (32.47 ± 14.26 versus 23.87 ± 10.3; p = 0.032), increased absolute numbers of CD4(-)CD8(bright) and CD4(+)CD8(+) cells (657.1 ± 463.6 cells/µL versus 319.24 ± 116.4 cells/µL; p = 0.017 and 40.77 ± 36.41 cells/µL versus 10.77 ± 9.78 cells/µL; p < 0.0001, respectively) and an elevated mean of MFI ratio of CD19 on B cells (252.3 ± 56.7 versus 205.67 ± 32.3; p = 0.021). However, expression of HLA-B51 was not associated with any specific immunophenotypic profile. In conclusion, abnormal immunophenotypic profile of peripheral lymphocytes was found in patients with BD, especially in active phase, reflecting an immune dysregulation. Moreover, HLA-B51 expression was not found to be related to the expression of other cell markers.

Anti-cardiolipin and anti-beta 2-glycoprotein I antibodies in celiac disease.

AIMS: To determine the frequency of anti-cardiolipin (aCL) and anti-ß2-glycoprotein I antibodies (aß2GPI) in celiac disease (CD) patients. PATIENTS AND METHODS: Sixty-three untreated CD patients and 40 healthy blood donors (HBD) were studied. IgG, IgA and IgM aCL and aß2GPI were detected by Elisa. RESULTS: The frequency of antiphospholipid antibodies (aPL) (aCL and/or aß2GPI) was significantly higher in CD patients (12 out of 63) than in HBD (two out of 40) (19% vs 5%, P=0.04). Six CD patients out of 63 (9.5%) and one HBD out of 40 (2.5%) had aCL. Ten CD patients (15.9%) and two HBD (5%) had aß2GPI. Only aß2GPI-IgA was significantly more frequent in CD patients than in HBD (14.3% vs 2.5%, P=0.048). In CD patients, aß2GPI-IgA (nine out of 63) was significantly more frequent (14.3%) than aß2GPI-IgG (1.6%) and IgM (1.6%) (P=0.008). In CD patients, the frequency of aCL-IgA and IgM was 6.3% (four out of 63) and aCL-IgG were not detected. Simultaneous presence of positive antibodies was found in four CD patients: one patient had four aPL, one had three aPL and two had two aPL. The four patients who had aCL-IgA had also aß2GPI-IgA and three of them had a titer higher than 50 units. Among nine patients with aß2GPI-IgA, four had a titer higher than 100 units. The highest titers were found in adults. CONCLUSIONS: aPL and particularly aß2GPI-IgA are frequent in CD. The significance of these antibodies has to be determined.

IgA anti-actin antibodies in celiac disease.

AIMS: The purpose of this study was to determine the sensitivity and specificity of IgA anti-actin antibodies (IgA-AAA) for celiac disease (CD), to investigate their usefulness as a marker of compliance in CD patients to the gluten-free diet (GFD), and to assess the relationship between their presence in the sera of CD patients and severity of intestinal mucosal damage. PATIENTS AND METHODS: A total of 182 patients with CD were studied: 63 patients were untreated; 50 patients were following a strict GFD; and 69 patients were non-compliant with a GFD. IgA-AAA was detected using a homemade enzyme-linked immunosorbent assay (ELISA). RESULTS: IgA-AAA showed a sensitivity of 41.3% and a specificity of 71.4% for a diagnosis of CD. In children, the frequency of IgA-AAA detection was lower in those following a strict GFD (23.1%) compared with untreated patients (39.4%) and those not complying with a GFD (32.5%). In patients following a strict GFD, IgA-AAA detection was significantly less frequent in children than in adults (23.1% vs. 58.3%, respectively; P<0.001). IgA-AAA was found in 17 out of 52 CD patients with total villous atrophy (32.7%), and in one out of 11 patients with subtotal villous atrophy (9%). CONCLUSION: IgA-AAA cannot replace anti-endomysium and anti-tissue transglutaminase antibodies in the diagnosis algorithm of CD, but it can serve as a reliable marker of severe intestinal mucosal damage in CD patients.

Uveitis responding on gluten free diet in a girl with celiac disease and diabetes mellitus type 1.

A 9-year old girl with a history of diabetes mellitus type 1, presented with visual loss of the left eye. The right eye examination was unremarkable. Slit-lamp examination revealed few small and fine keratic precipitates. We noted 2+ flare in the vitreous. There was no choroiditis, papillitis or retinal vasculitis. No aetiology was found. The patient was treated by topical and systemic corticosteroids without any improvement. Celiac disease was discovered by the presence of celiac antibodies in the work-up of joint pain and diabetes mellitus type 1. Antiendomysium antibodies and anti-transglutaminase antibodies were both positive. A small bowel biopsy confirmed celiac disease. A gluten free diet was set up and corticosteroids were tapered off. Recovery of the uveitis was obvious during gluten free diet and normalized within two months.

Angiogenic activity in sera of patients with systemic lupus erythematosus.

Angiogenesis plays a critical role in the pathogenesis of several connective tissue diseases. There is, however, relatively little information available on the role of angiogenesis in systemic lupus erythematosus (SLE). The aim of this study was to investigate the angiogenic activity in sera of patients with SLE and to determine the association between angiogenic activity and clinical complications. Sera from 66 Tunisian females with SLE and from 32 healthy blood donors were studied for their angiogenic activity using the in-vitro tube formation test on Matrigel. Samples were divided into five groups according to their angiogenic activity, which was scored from 0 (no angiogenesis) to 4 (high angiogenic activity). Samples from each group were then tested randomly to assess serum concentration of vascular endothelial growth factor (VEGF). No correlation was found between angiogenic activity scores and serum VEGF levels. Considering angiogenesis assessment in-vitro, sera of patients with SLE showed a much higher angiogenic activity than healthy controls since a high angiogenic score (score 4) is present in 43.9% of patients and in 6.3% of controls (P < 0.0002). This high angiogenic activity is not correlated with disease activity; however, SLE patients with anti-dsDNA antibodies and those with nephritis showed higher angiogenic activity compared with patients without these complications since score 4 is found in 50.9% and 67.9% versus 9.1% (P = 0.017) and 26.3% (P < 0.001), respectively. In conclusion, our study showed that high serum angiogenic activity in SLE was not correlated with the VEGF levels. We suggest the use of the 'in-vitro' tube formation test as a better tool to study the angiogenic potential of sera. We found that in patients with SLE, serum angiogenic activity is increased compared with healthy controls. This high angiogenic activity is associated with renal complications and with the presence of anti-dsDNA antibodies. These findings suggest an involvement of angiogenesis disturbance in the pathogenesis of SLE.

Prevalence of antineutrophil cytoplasmic antibodies during treatment with benzylthiouracil.

BACKGROUND: Drug induced antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis is a rare complication associated especially with propylthiouracil (PTU). Prevalence of ANCA in patients receiving PTU is well established. Few cases of vasculitis were also reported with benzylthiouracil (BTU). The objective of this study is to clarify the prevalence of ANCA in patients receiving BTU. METHODS: ANCA were investigated by indirect immunofluoresence and enzyme linked immunosorbant assay in 159 patients with Graves' disease (86 untreated and 73 treated with BTU). RESULTS: ANCA were positive in three (3.5%) untreated patients and 27 (37%) treated ones. Titres of ANCA varied between 1:20 and 1:200. There was a significant association between BTU treatment and ANCA (p<0.001). ANCA were directed against myeloperoxidase (MPO) in 28 (93.3%) patients. Median treatment duration was 24 months (ranges 0.5 to 144 months). There was no significant association between treatment duration and ANCA. Vasculitis was found in two (2.7%) treated patients. One patient has developed isolated cutaneous vasculitis and the other one a pulmonary vasculitis with diffuse alveolar haemorrhage. CONCLUSION: BTU therapy is characterised by a high prevalence of ANCA mainly but not exclusively directed against MPO. However, vasculitis remains a rare complication.

[Antineutrophil cytoplasmic antibodies and associated diseases]. / Anticorps anticytoplasme des polynucléaires neutrophiles et pathologies associées.

Antineutrophil cytoplasmic antibodies are classical serological markers of small-vessels vasculitis. However, they have been described in many other pathological situations. The aim of this study was to determine through our experience, the main antineutrophil cytoplasmic antibodies-associated diseases and to investigate antigen targets of these antibodies. Forty complete observations of antineutrophil cytoplasmic antibodies (ANCA) positive patients either by indirect immunofluorescence or by enzyme immunoassay were analysed. Only five (12.5%) patients have small-vessels vasculitis. Among these, antineutrophil cytoplasmic antibodies were detected only by Elisa in one patient and they were exclusively directed against bactericidal permeability increasing protein in another one. Our study confirms the presence of antineutrophil cytoplasmic antibodies in different diseases. It demonstrates that antineutrophil cytoplasmic antibodies should be investigated by Elisa when indirect immunofluorescence is negative. In small-vessels vasculitis, Proteinase 3 and myeloperoxidase are mainly but not exclusively the antigenic targets of antineutrophil cytoplasmic antibodies.

Detection of M2 antimitochondrial antibodies by dot blot assay is more specific than by enzyme linked immunosorbent assay.

AIMS: The objective of our study was, in one hand, to determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of ELISA and dot blot assay to investigate IgG M2 antimitochondrial antibodies (M2 AMA) and, on the other hand, to compare these results with those of indirect immunofluorescence technique (IIF). METHODS: Sera from patients suffering from primary biliary cirrhosis (PBC) (n=55), systemic lupus erythematosus (n=21), celiac disease (n=30) and blood donors (n=75) were analyzed. M2 AMA were detected by ELISA and dot blot using pyruvate dehydrogenase purified from porcine heart and by IIF on cryostat sections of rat liver-kidney-stomach. RESULTS: IIF was more sensitive (98%) than ELISA (93%) and dot blot (91%). The specificity of AMA for PBC using IIF, ELISA and dot blot reached 100%, 92% and 100%, respectively. The PPV of IIF, ELISA and dot blot was 100%, 93% and 100%, respectively. The NPV was 98% for IIF, 92% for ELISA and 91% for dot blot. CONCLUSION: Dot blot, using purified pyruvate dehydrogenase, had a higher specificity than ELISA and may be useful in confirming the specificity of AMA in cases of doubt with IIF.

B cell-ablative therapy: where are we now?

Based on their multifaceted functions, B cells participate in several pathological settings such as lymphoproliferative disorders, autoimmune diseases and graft rejection. B cell-ablative therapy has thus emerged as a mainstay in these diseases. A number of anti-B cell antibodies (Abs) have been generated, among which anti-CD20 Abs appear to be efficient. Rituximab (RTX) is one of these anti-CD20 monoclonal Abs. Originally approved for the treatment of non-Hodgkin lymphoma, RTX is now being administered in other malignant proliferations, applied to an increasing number of autoimmune diseases and required to prevent rejection of a graft. Although this medication is remarkably safe, a handful of laboratory tests have been proposed to monitor RTX-treated patients. The efficacy in different diseases, and the emergence of new anti-CD20 Abs raise many questions. Thus, their detailed understanding can lead to a better issue for inhibition of immune responses.

[Antihistones antibodies in systemic lupus erythematosus, comparison of three assays: Elisa, dot blot and immunoblot]. / Anticorps antihistones au cours du lupus érythémateux systémique, comparaison entre trois techniques: Elisa, dot blot et immunotransfert.

AIMS: The purpose of our study is to determine and compare the sensitivity of an enzyme linked immunosorbent assay (ELISA), a dot blot assay and an immunoblot assay for the detection of the IgG class antihistones antibodies in a population of systemic lupus erythematosus. The correlation between antihistones antibodies and the main clinical features of SLE or between antihistones antibodies and the presence of anti-double-stranded-DNA antibodies were analysed. MATERIALS AND METHODS: Serum samples from 126 systemic lupus erythematosus patients, classified according to the criteria of the American College of Rheumatology, were analysed for the presence of antihistones antibodies using a dot blot assay and an ELISA. Antihistones subfractions antibodies were assessed using the immunoblot technique on 88 out of the 126 sera. Serum samples from 50 blood-donors were analyzed as negative controls. RESULTS: The sensitivity of antihistones antibodies assessed by dot blot assay and ELISA was 69% and 54% respectively, and was lower than that of anti-double-stranded-DNA antibodies (83%). The sensitivity of the immunoblot assay for the detection of antihistones antibodies was 72%. Incidence of autoantibodies against histones H1, H2 A, H2B, H3 and H4 was 60%, 53%, 48%, 36% and 29.5% respectively. We found a correlation between the presence of antihistones antibodies, detected by the dot blot assay and ELISA, and the presence of anti-double-stranded-DNA antibodies. Antihistones antibodies detected by ELISA were correlated with renal disease in systemic lupus erythematosus; they showed a specificity, a positive and a negative predictive value for renal disease in systemic lupus erythematosus higher than those of anti-double-stranded-DNA antibodies. CONCLUSIONS: The sensitivity of the dot blot assay for the detection of antihistones antibodies is better than that of ELISA, but the latter technique could detect some cases negative by ELISA. Antihistones antibodies detected by ELISA have an important predictive value in the renal complications in systemic lupus erythematosus, better than that of AdsDNA. Antibodies to histone H1 were the most frequent antihistones autoandibodies in systemic lupus erythematosus and they were highly correlated with anti-double-stranded-DNA antibodies and renal disease.

Celiac disease in Tunisia: serological screening in healthy blood donors.

BACKGROUND: Recent epidemiological studies in Europe and in USA using antigliadin antibodies and antiendomysium antibodies for initial screening have shown that the overall prevalence of celiac disease (CD) is about 1:200 (0.5%). AIM: To screen for CD in healthy blood donors in Tunisia. PATIENTS AND METHODS: Sera from 2500 healthy blood donors (median age: 21 years, 70% men and 30% women) were screened for IgG-antigliadin antibodies and IgA-antigliadin antibodies with an enzyme-linked immunosorbent assay. All sera with positive antigliadin antibodies were tested for antiendomysium antibodies using human umbilical cord cryosections as substrate. RESULTS: Seven healthy blood donors (median age: 21 years; four men, three women) have antiendomysium antibodies. The prevalence of antiendomysium antibodies in healthy blood donors in Tunisia is 1:355 (0.28%). CONCLUSIONS: On the basis of a high specificity of the antiendomysium antibodies, it is likely that the seven blood donors identified in this study have CD. These data suggest that CD is frequent in Tunisia.

Tissue transglutaminase antibodies in celiac disease, comparison of an enzyme linked immunosorbent assay and a dot blot assay.

AIMS: The purpose of our study is to determine the sensitivity, specificity and predictive values of an enzyme linked immunosorbent assay (ELISA) and a dot blot assay for the detection of IgA class anti-tissue transglutaminase antibodies (IgA-AtTGA) and to compare these results with those of IgA class anti-endomysium antibodies (IgA-AEA), IgA class anti-reticulin antibodies (IgA-ARA) and IgA class anti-gliadin antibodies (IgA-AGA). PATIENTS: Serum samples from 143 patients (97 children, 46 adults) with untreated celiac disease (CD) confirmed by intestinal biopsy and 74 disease controls (64 children, 10 adults) were studied. Methods. - The anti-tissue transglutaminase antibodies were detected by dot blot assay and an ELISA using guinea pig tissue transglutaminase (gp-tTG) as antigen. The anti-endomysium antibodies were detected by an indirect immunofluorescence technique on cryostat sections of human umbilical cord. The anti-reticulin antibodies were also investigated by indirect immunofluorescence on cryostat sections of kidney, liver and stomach of rat. The anti-gliadin antibodies were determined by an ELISA. RESULTS: The sensitivity of an ELISA for the detection of anti-tissue transglutaminase antibodies was 86% in children and 87% in adults and the sensitivity of dot blot assay was 57% in children and 54% in adults. The specificity of an ELISA and dot blot for the detection for anti-tissue transglutaminase antibodies was, respectively, 96% and 88% lower than that of anti-endomysium antibodies (100%). The sensitivity of anti-gliadin antibodies was 97% in children and 91% in adults and their specificity was 85%. The sensitivity of anti-reticulin antibodies was 94% in children and 87% in adults. Their specificity was 100%. CONCLUSIONS: The sensitivity and specificity of an ELISA for the detection of anti-tissue transglutaminase antibodies were better than that of dot blot assay. However, this dot blot assay could screen four celiac patients who have not had anti-tissue transglutaminase antibodies by an ELISA. The sensitivity of anti-endomysium antibodies was better than that of anti-tissue transglutaminase antibodies, anti-reticulin antibodies and anti-gliadin antibodies but in children aged less than 2 years, the sensitivity of anti-gliadin antibodies was better than that of anti-tissue transglutaminase antibodies.

[Clinical and serological characteristics of systemic lupus erythematosus: 128 cases]. / Caractéristiques cliniques et sérologiques du lupus érythémateux systémique: à propos de 128 cas.

OBJECTIVE: To analyse the clinical and serological characteristics of systemic lupus erythematosus (SLE) in the center of Tunisia. METHODS: We studied 128 patients with SLE aged one to 73 years. Antinuclear antibodies (ANA) were detected by an immunofluorescence method. Anti-double-stranded DNA (anti-dsDNA) antibodies, anti-extractable nuclear antigen antibodies (anti-Sm, anti-SS-A, anti-SS-B and anti-RNP) and anti-cardiolipin (aCL of IgG, IgA and IgM isotypes) antibodies were detected by ELISA. RESULTS: Malar rash (71%) and anemia (71%) were the most common clinical manifestations. Arthritis was seen in 62.5%. Severe kidney damage was observed in 39%. Pericarditis and pleuritis were observed in only 23%. Neurological manifestations (16%) were uncommon. Clinical manifestations of anti-phospholipid syndrome (SAPL) were observed in 15%. ANA were detected in 100%, anti-dsDNA in 76%, anti-Sm in 55.5%, anti-SS-A in 64%, anti-SS-B in 33.6%, anti-RNP in 49%. aCL of IgG, IgA and IgM isotypes were detected in 63.5%, 49% and 40.6% of the patients respectively. The only significant positive clinical associations were those of arthritis with anti-dsDNA antibodies (p = 0.022) and malar rash with anti-SS-A antibodies (p = 0.002). CONCLUSIONS: This study suggests that tunisians with SLE present, in general, a mild form of disease predominantly manifested by cutaneous, musculoskeletal and hematologic involvement but low prevalence of major organ damage.

[Anti-endomysium, anti-reticulin and anti-gliadin antibodies, value in the diagnosis of celiac disease in the child]. / Anticorps anti-endomysium, anti-réticuline et anti-gliadine, intérêt dans le diagnostic de la maladie coeliaque chez l'enfant.

Coeliac disease is associated with gluten intolerance in genetically predisposed subjects. Environmental factors, particularly of viral origin, may also play a major role. In this study, the presence of IgA class anti-endomysium antibodies (AEA-IgA), IgA class anti-reticulin antibodies (ARA-IgA) and IgA class anti-gliadin antibodies (AGA-IgA) was investigated in 120 serum samples from 120 children (60 patients with coeliac disease and 60 control subjects). The AEA were detected by indirect immunofluorescence on sections of human umbilical cord. The ARA were also investigated by the same technique in rat kidney, liver and stomach. The AGA were determined by an enzyme-linked immunosorbent assay (ELISA). In the patients with coeliac disease, the sensitivity of AEA and ARA was 86% and 76% respectively. In both cases, the specificity was 100%. In children below two years of age, the sensitivity of AEA and ARA was too low, i.e., 57% and 35% respectively. In children aged between two and 15 years, the sensitivity of AEA and ARA was 95% and 89% respectively. The sensitivity of IgA class AGA was 86%, and their specificity was 83%. In this study population, these results show that IgA class AEA are interesting markers for the diagnosis of coeliac disease in the child, and could be used in screening for coeliac disease in a high-risk population.

Identification of visual arrestin (S-antigen) in retinal pigmented epithelial cells.

PURPOSE: Inactivation of photolyzed rhodopsin requires phosphorylation of this receptor and binding of the 48 kDa regulatory protein arrestin (S-antigen). Arrestin is also to cause an autoimmune disease, uveoretinits, that resembles uveitis in humans. In this study we demonstrate the presence of visual arrestin in retinal pigment epithelial cells (RPE) in culture. METHODS: Bovine RPE were isolated. Mouse and rat monoclonal and rabbit polyclonal antibodies against visual arrestin, and a synthetic peptide "GFLGELTSSEVATEVPFRLM" (a pathogenic sequence corresponding to residues 340 to 359 of human visual arrestin), and rabbit polyclonal antibody against the specific peptide "EDPDTAKESFQ" for bovine visual arrestin were used to detect arrestin in RPE cells. Using visual arrestin specific primer, RT-PCR of RNA from RPE was performed. RESULTS: By western blots analysis a 48 kDa protein, corresponding to visual arrestin was detected with both mAb and polyclonal antibodies in extracts of RPE cells. RT-PCR analysis of RNA from RPE cells confirmed the presence of arrestin mRNA of predicted 377 bp and exhibited 100% homology with visual arrestin 48 kDa. CONCLUSION: Visual arrestin proteins present in RPE may be involved in the desensitization of G-protein-coupled receptors in RPE cells and in arrestin uveopathogenesis.

Beta-arrestin-related proteins in ocular tissues.

PURPOSE: Proteins of the arrestin family contribute to the regulation of G-protein-mediated transduction. In this study, the presence of beta-arrestins in ocular tissues was investigated. METHODS: Mouse monoclonal and rabbit polyclonal antibodies were raised against the peptide Val-Asp-Thr-Asn-Ile-Leu-Glu-Leu-Asp-Thr-Asn-Asp-Asp-Asp-Ile, a sequence present in beta-arrestins 1 and 2 but absent from visual arrestin. These antibodies were used for the immunohistologic detection of beta-arrestins in parafin sections of rodent eyes fixed in Bouin's solution. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA from bovine retina, retinal pigmented epithelial (RPE) cells, lens epithelial cells, and human corneal fibroblasts was performed using beta-1 arrestin primers. RESULTS: In the eye, bet-arrestin staining predominated in RPE, inner segments of photoreceptors, synaptic spherules of rods, inner plexiform layer and ganglion cell fibers, epithelial cells from ciliary body, and vessels. RT-PCR amplified a 480 bp product, corresponding to the predicted length. The sequence of PCR products from bovine retina and RPE cells was identical with the bovine beta-arrestin mRNA. CONCLUSIONS: beta-arrestins were detected in several ocular tissues. In photoreceptor cells, their specific localization in the synaptic terminals and plexiform layer suggests a role of beta-arrestin in synaptic transmission. In other ocular tissues, the presence of beta-arrestin may be related either to adrenergic signal transduction or to signal transduction mediated by other G-protein-coupled receptors.

Copurification of selected glycolytic enzymes with retinal S-antigen (arrestin) by hydroxyapatite agarose chromatography of bovine retina.

PURPOSE: A variety of methods have been developed for the purification of S-antigen but a simple and rapid procedure based on hydroxyapatite-agarose (HA) adsorbtion is most widely used. In the present study, we investigated the nature of proteins purified with the aid of HA chromatography. METHODS: After elimination of retinal S-antigen by HA, the soluble extract of retinal tissue was readsorbed on HA. The proteins were thereafter desorbed by 10 to 500 mM phosphate buffer gradient. Two peaks obtained by SDS-PAGE were used for the generation of specific antisera for subsequent analysis by ELISA and Western blotting. RESULTS: Four proteins (two 48 kDa , one 50 kDa and one 46 kDa) were obtained in this manner. Partial amino acid sequencing permitted the identification of these proteins as alpha-enolase (48 kDa), gamma-enolase (48 kDa), Glucose-6-phosphate-Isomerase (50 kDa) and aspartate-amino-transferase (46 kDa). CONCLUSION: The selected glycolytic enzymes co-purified with retinal S-antigen by hydroxyapatite agarose chromatography of bovine retina.