Integrated omics analysis of sweat reveals an aberrant amino acid metabolism pathway in Vogt-Koyanagi-Harada disease.

Vogt-Koyanagi-Harada (VKH) disease is an autoimmune disease leading to visual impairment. Its pathogenic mechanisms remain poorly understood. Our purpose was to investigate the distinctive protein and metabolic profiles of sweat in patients with VKH disease. In the present study, proteomics and metabolomics analysis was performed on 60 sweat samples (30 VKH patients and 30 normal controls) using liquid chromatography tandem mass spectrometry. Parallel reaction monitoring (PRM) analysis was used to validate the results of our omics analysis. In total, we were able to detect 716 proteins and 175 metabolites. Among them, 116 proteins (99 decreased and 17 increased) were observed to be significantly different in VKH patients when compared to controls. Twenty-one differentially expressed metabolites were identified in VKH patients, of which 18 included choline, L-tryptophan, betaine and L-serine were reduced, while the rest were increased. Our multi-omics strategy reveals an important role for the amino acid metabolic pathway in the pathogenesis of VKH disease. Significant differences in proteins and metabolites were identified in the sweat of VKH patients and, to some extent, an aberrant amino acid metabolism pathway may be a pathogenic factor in the pathogenesis of VKH disease.

Identification of susceptibility SNPs in CTLA-4 and PTPN22 for scleritis in Han Chinese.

The aim of this study was to determine the association between 13 single nucleotide polymorphisms (SNPs) in the cytotoxic T lymphocyte-associated antigen-4 (CTLA4) and protein tyrosine phosphatase non-receptor type 22 (PTPN22) genes with scleritis in a Chinese Han population. We recruited 432 scleritis patients and 710 healthy controls. Four tag SNPs of CTLA4 and nine tag SNPs of PTPN22 were selected using Haploview. Genotyping was performed with the Sequenom MassArray® iPLEX GOLD Assay. Genotype and allele frequency differences were analyzed by χ2 test and Bonferroni correction. Haplotype analysis was performed to further evaluate the association of these two genes with scleritis. In this study, CTLA4/rs3087243 G allele frequency and GG genotype frequency were significantly increased in scleritis patients compared to healthy controls [corrected P-value (Pc) = 0·02, odds ratio (OR) = 1·475, 95% confidence interval (CI) = 1·175-1·851; Pc = 0·04, OR = 1·546, 95% CI = 1·190-2·008, respectively]. None of the tested SNPs in the PTPN22 gene showed an association with scleritis. Haplotype analysis revealed a lower frequency of a CTLA4 TCAA haplotype (order of SNPs: rs733618, rs5742909, rs231775, rs3087243) (Pc = 4·26 × 10-3 , OR = 0·618, 95% CI = 0·540-0·858) and a higher frequency of a PTPN22 TTATACGCG haplotype (order of SNPs: rs3789604, rs150426536, rs1746853, rs1217403, rs1217406, rs3789609, rs1217414, rs3789612, rs2488457) (Pc = 2·83 × 10-4 , OR = 1·457, 95% CI = 1·210-1·754) in scleritis patients when compared to healthy controls. In conclusion, our findings indicate that CTLA4 and PTPN22 might confer genetic susceptibility to scleritis in a Chinese Han population.

ERAP1/ERAP2 and RUNX3 polymorphisms are not associated with ankylosing spondylitis susceptibility in Chinese Han.

Previous studies show that endoplasmic reticulum-associated aminopeptidase (ERAP1/ERAP2) and runt-related transcription factor 3 (RUNX3) gene polymorphisms are associated with AS (ankylosing spondylitis) in European Caucasians. However, contradictory results were reported in different Asian populations. The purpose of this study was to determine whether eleven candidate single nucleotide polymorphisms (SNPs) in ERAP1/ERAP2 and six in RUNX3 genes confer susceptibility to AS with or without acute anterior uveitis (AAU) [AS+ AAU+ or AS+ AAU- ] in Chinese Han. Therefore, a case-control association study was performed in 882 AS+ AAU- , 884 AS+ AAU+ and 1727 healthy controls. Genotyping was performed using the iPLEXGold genotyping assay. A meta-analysis was performed to assess the association of polymorphisms of ERAP1 with AS susceptibility in Asian populations. No association was found between SNPs of ERAP1/ERAP2/RUNX3 and susceptibility of AS with or without AAU. A case-control study between patients with human leucocyte antigen HLA-B27-positive and healthy controls also failed to demonstrate an association of the tested SNP with AS with or without AAU. Moreover, a meta-analysis showed that there was no association of rs30187, rs27037, rs27980, rs27434 and rs27582 in ERAP1 with AS in Chinese Han. Taken together, 17 SNPs in ERAP1/ERAP2 and RUNX3 genes did not confer disease susceptibility to AS in Chinese Han.

The Role of Mitochondria-Associated Reactive Oxygen Species in the Amyloid ß Induced Production of Angiogenic Factors b y ARPE-19 Cells.

OBJECTIVE: This study aimed to investigate the mechanisms whereby Amyloidbeta (Aß) induces the production of angiogenic factors by a human retinal pigment epithelial cell line (ARPE-19) cells. METHODS: ARPE-19 cells obtained from the American Type Culture Collection (ATCC) were utilized in this study. The expression level of vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and complement activation fragments C3a and C5a were measured by Real-time quantitative PCR (RT-PCR) and Enzyme-linked immunosorbent assay (ELISA). The production of mitochondria-associated reactive oxygen species (ROS) was measured by flow cytometry. RESULTS: The expression of VEGF, IL-8, MCP-1, C3a and C5a was significantly increased in Aß-treated ARPE-19 cells. Mitochondria-associated ROS production was also significantly increased when exposed to Aß. Inhibition of mitochondrial ROS with Diphenyleneiodonium chloride (DPI) markedly decreased the Aß induced production of VEGF, IL-8, MCP-1, C3a and C5a by ARPE-19 cells. Anti-C3a or anti-C5a neutralizing antibodies did not have a detectable influence on the secretion of VEGF, IL-8 and MCP-1 by ARPE-19 cells upon stimulation with Aß. CONCLUSION: Our results support the hypothesis that Aß is involved in the pathogenesis of choroidal neovascularization (CNV) formation by promoting the production of the angiogenic cytokines VEGF, IL-8 and MCP-1 by RPE cells. Mitochondrial ROS was shown to play a role in the regulation of Aß induced expression of these cytokines.

Higher Expression of NOD1 and NOD2 is Associated with Vogt-Koyanagi-Harada (VKH) Syndrome But Not Behcet's Disease (BD).

NOD1 and NOD2 have been found to play a significant regulatory role in autoimmune disease. To analyze the role of NOD1 and NOD2 in the pathogenesis of Vogt- Koyanagi-Harada (VKH) syndrome and Behcet's disease (BD). We analyzed the expression of NOD1 and NOD2 from PBMCs by RT-PCR and Western Blot. PBMCs and DCs were cultured with NOD receptor ligands iE-DAP (NOD1) or MDP (NOD2) and cells and supernatants were analyzed by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA). DCs and CD4+T cells were co-cultured with or without stimulation and cells and supernatants were analyzed by FCM and ELISA. A higher expression of NOD1 and NOD2 was observed in patients with active VKH syndrome as compared with controls. However, no significant differences were found between BD patients and controls. Activation of NOD1 and NOD2 with iE-DAP or MDP markedly increased the level of IL-6, TNF-α and IL-1ß in PBMCs and DCs and induced the expression of CD40, CD80, CD83, CD86 and HLA-DR on DCs. Activation of NOD1 and NOD2 in DCs promoted the differentiation and proliferation of CD4(+)T cells. In conclusion, activation of NOD1 or NOD2 increased the production of pro-inflammatory cytokines in PBMCs and promoted the maturation and activation of human DCs in association with stimulation of Th1 and Th17 cells. Our results suggest that over-expression of NOD1 and NOD2 may be involved in the pathogenesis of VKH syndrome.

TLR3 and TLR4 But not TLR2 are Involved in Vogt-Koyanagi- Harada Disease by Triggering Proinflammatory Cytokines Production Through Promoting the Production of Mitochondrial Reactive Oxygen Species.

Vogt-Koyanagi-Harada (VKH) disease is considered to be an autoimmune disease possibly triggered by an abnormal response to infection. Activation of TLRs signaling pathways by microbial products can drive inflammatory responses and adaptive immunity. In the present study, we investigated the role of TLRs in the pathogenesis of VKH disease. We showed that the expression of TLR3 and TLR4, but not TLR2, was significantly increased in monocyte-derived macrophages (MDMs) from VKH patients with active uveitis compared to controls. VKH patients with active uveitis showed an elevated level of IL-1ß, IL-6, IL-8, TNF-α and reactive oxygen species (ROS) in MDMs. IL-1ß, IL-6, IL-8 and TNF-α production could be significantly upregulated and downregulated by a ROS activator or inhibitor, respectively. Downregulation of the NLRP3 inflammasome significantly inhibited the production of IL-1ß but not IL-6, IL-8 and TNF-α. The phosphorylation levels of p38 and ERK1/2 were significantly higher in MDMs from active VKH patients compared to controls. Inhibition of p38 or ERK1/2 significantly decreased IL-1ß, IL-6, IL-8 and TNF-α expression. These results suggest that the increased expression of TLR3/4 in MDMs may be involved in the pathogenesis of VKH disease by the induction of inflammatory cytokines which is mediated by enhanced production of ROS.

Activation of the liver X receptor inhibits Th17 and Th1 responses in Behcet's disease and Vogt-Koyanagi-Harada disease.

Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome are two intraocular inflammatory diseases that are caused by an aberrant T lymphocyte response. Th17 cells, mainly producing the cytokine IL-17, and Th1 cells, characterized by the production of the index cytokine IFN-γ, are the CD4(+) T lymphocyte subsets implicated in the pathogenesis of both BD and VKH. Suppressing the excessive response of these Th17 and Th1 cells has been reported to be an effective therapeutic approach to treat these patients and continuous efforts are being undertaken to find new methods to modulate the function of these cells. Evidence is emerging that the Liver X receptor (LXR) is an important regulator of inflammatory and immune responses and the study reported here was designed to investigate the role of LXR activation in BD and VKH. Here we demonstrate that the frequency of Th17 and Th1 cells along with the relevant cytokines IL-17, IFN-γ and corresponding transcriptional factors RORC, T-bet were all decreased following LXR activation by the agonist GW3965. LXR controlled the expression of inflammatory cytokines through an effect on NF-kappa B (NFκb) phosphorylation. Data from our study provide evidence for an association between a decreased LXR expression and disease activity in both BD and VKH, due to the fact that a lower LXR activation may result in an enhanced Th1 and Th17 immune response. Our study suggests that enhancing LXR activation may offer a potential therapeutic approach targeting aberrant immune responses by inhibiting Th1 and Th17 cell responses.

Activation of the aryl hydrocarbon receptor affects activation and function of human monocyte-derived dendritic cells.

Aryl hydrocarbon receptor (AhR) is well known for mediating the toxic effects of dioxin-containing pollutants, but has also been shown to be involved in the natural regulation of the immune response. In this study, we investigated the effect of AhR activation by its endogenous ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the differentiation, maturation and function of monocyte-derived DCs in Behçet's disease (BD) patients. In this study, we showed that AhR activation by FICZ and ITE down-regulated the expression of co-stimulatory molecules including human leucocyte antigen D-related (HLA-DR), CD80 and CD86, while it had no effect on the expression of CD83 and CD40 on DCs derived from BD patients and normal controls. Lipopolysaccharide (LPS)-treated dendritic cells (DCs) from active BD patients showed a higher level of interleukin (IL)-1ß, IL-6, IL-23 and tumour necrosis factor (TNF)-α production. FICZ or ITE significantly inhibited the production of IL-1ß, IL-6, IL-23 and TNF-α, but induced IL-10 production by DCs derived from active BD patients and normal controls. FICZ or ITE-treated DCs significantly inhibited the T helper type 17 (Th17) and Th1 cell response. Activation of AhR either by FICZ or ITE inhibits DC differentiation, maturation and function. Further studies are needed to investigate whether manipulation of the AhR pathway may be used to treat BD or other autoimmune diseases.

A possible role for interleukin 37 in the pathogenesis of Behcet's disease.

Interleukin 37 has been found to play a significant regulatory role in the innate immune response. It is not yet known whether IL-37 has also been involved in the development of Behcet's disease (BD), a chronic systemic inflammatory disease. To examine the role of IL-37 in the pathogenesis of BD, a number of experiments were performed. IL-37 expression in peripheral blood mononuclear cells (PBMCs) from BD patients and normal controls was measured by RT-PCR and flow cytometry. Monocyte-derived Dendritic Cells (DCs) were cultured with or without IL-37 and levels of cytokines in the culture supernatants were measured by ELISA. The DC surface markers, reactive oxygen species (ROS) production and mitogen-activated protein kinase (MAPK) activation were measured by flow cytometry. The effect of IL-37-treated DCs on the development of CD4(+) T cells was measured by ELISA and flow cytometry. The results show that both IL-37 mRNA level and protein expression were significantly decreased in PBMCs from active BD patients compared to normal controls. DCs stimulated with rIL-37 showed a decreased expression of IL-6, IL-1ß and TNF-α, and a higher production of IL-27. rIL-37 significantly inhibited the production of ROS by DCs and reduced the activation of ERK1/2, JNK and P38 MAPK in DCs. rIL-37-treated DCs remarkably inhibited Th17 and Th1 cell responses as compared to control DCs. rIL-37 did not affect the expression of DC surface markers (CD40, CD86, CD80 and HLA-DR) or IL-10 production by DCs. We conclude that a decreased IL-37 expression in active BD patients may trigger the production of pro-inflammatory cytokines and ROS in association with activation of Th1 and Th17 cells by DCs.

Neospora caninum and Toxoplasma gondii in brain tissue of feral rodents and insectivores caught on farms in the Netherlands.

We investigated the presence of both Neospora caninum and Toxoplasma gondii in 250 brain tissue samples from 9 species of feral rodents and insectivores caught on 10 organic farms in the Netherlands in 2004. Collected samples were conserved in 4% paraformaldehyde solution and analysed by real-time PCR. For N. caninum, 31 samples originating from 6 species tested positive (12.4%): common shrews (33.3%), wood mice (17.6%), harvest mice (16.7%), house mice (15.4%), white-toothed shrews (10.8%) and common voles (4.2%). For T. gondii, the overall contamination level was 4%, and only three species were found to be positive: house mice (9.0%), common voles (4.2%) and white-toothed shrews (2.0%). Most N. caninum infected samples (27/31; 87%) were found on farms where dogs were present. Due to the observation that rodents and insectivores can contract both parasites, they might function as indicator species for the parasitic load on farms.

Food safety in free-range and organic livestock systems: risk management and responsibility.

Animal production systems that offer outdoor access to the animals have become increasingly popular in the Western world due to the growing general discontent of consumers with conventional bioindustrial farming practices. These open production systems offer improved animal welfare but may create new problems for animal health, resulting in increased food safety risks from bacterial, viral, or parasitic infections or environmental contaminants. Examples of these new problems include increased Toxoplasma gondii infections in pigs and high dioxin levels in eggs from free-range hens. In this review, the relation between positive and negative points of free-range and organic livestock production systems is discussed with reference to production in The Netherlands. We investigated how proponents of more animal welfare friendly systems deal with potential negative issues in public and whether any risk communication is used. Generally, we found that the existence of a dilemma is disputed or avoided in communication with the consumer. This avoidance could be detrimental for public trust in alternative animal production systems, should problems occur. To prevent future problems, it will be necessary to communicate about the relevant types and sources of the food safety risks to the consumers. The responsibility for protecting food safety should be properly divided among the various parties involved: producers, processors, governments, nongovernmental organizations, and consumers.

No association of CTLA-4 polymorphisms with susceptibility to Behçet disease.

BACKGROUND: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a key negative regulator of T lymphocytes and has been shown to be associated with a number of autoimmune diseases. The present study was performed to assess the association between CTLA-4 polymorphisms and Behçet disease (BD) in Chinese patients. METHODS: Two hundred and twenty-eight BD patients and 207 controls were analysed for four single nucleotide polymorphisms (SNPs) (-1661A/G, -318C/T, +49G/A and CT60G/A) in the CTLA-4 gene by PCR-restriction fragment length polymorphism (RFLP) analysis. The association between SNP +49A/G and BD in Chinese population as well as other ethnic groups was analysed by meta-analysis. RESULTS: No association could be detected between CTLA-4 SNPs or haplotypes and BD. Also, no association was observed between CTLA-4 polymorphisms and BD subgroups, stratified by clinical features. A meta-analysis showed that there was no heterogeneity between studies (p = 0.60, I(2) = 0%) and that CTLA-4 SNP +49 was not associated with BD (overall effect: Z = 0.26, p = 0.79). CONCLUSION: This study and a meta-analysis failed to demonstrate any association between the tested CTLA-4 polymorphisms and BD.

Inhibitory effect of rapamycin and dexamethasone on production of IL-17 and IFN-gamma in Vogt-Koyanagi-Harada patients.

AIMS: To evaluate the effect of rapamycin (RAPA) and dexamethasone (DEX) on the production of IL-17 and IFN-gamma by peripheral blood mononuclear cells (PBMCs) from Vogt-Koyanagi-Harada (VKH) patients and healthy individuals. METHODS: Blood samples were drawn from 10 active VKH patients and 10 healthy individuals. PBMCs were cultured with or without anti-CD3 and anti-CD28 antibodies in the presence or absence of different concentrations of RAPA or DEX for 72 h. IL-17 and IFN-gamma concentrations in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of IL-17 and IFN-gamma was significantly increased in active VKH patients compared with that in healthy controls. Both RAPA and DEX were able to significantly inhibit the production of IL-17 and IFN-gamma by PBMCs from patients and healthy controls. RAPA was able to completely inhibit IL-17 production at a dosage of 10 ng/ml but only partially suppressed IFN-gamma production even at a much higher concentration (1000 ng/ml). DEX inhibited the production of both IL-17 and IFN-gamma by approximately 70%. CONCLUSIONS: This study indicates that both RAPA and DEX inhibit the production of IL-17 and IFN-gamma by PBMCs. RAPA is much stronger in inhibiting the production of IL-17 than DEX.

Patient ignorance is the main reason for treatment delay in primary rhegmatogenous retinal detachment in The Netherlands.

AIMS: Treatment delay of progressive vision-threatening conditions should be minimal. In this study, the treatment delay of patients with a rhegmatogenous retinal detachment (RRD) undergoing retinal detachment surgery was quantified, and causes for this delay were evaluated. MATERIALS AND METHODS: Consecutive patients (n=205) presenting with a primary RRD between June 2006 and June 2007 at the tertiary referral center (TRC) were interviewed. Five categories of delay were discerned in the following: 'patient delay,' 'general practitioner's delay,' 'referring ophthalmologist's delay,' 'delay at the TRC' and 'delay before surgery at the TRC'. In addition, overall delay was calculated. RESULTS: In total, 186 eyes were included in the analysis. Median overall delay between the patients' first symptoms and RRD surgery was 10 days. Almost 60% of this overall delay time was due to patient delay and the delay of the general practitioner. More than 50% of patients had a delay owing to unawareness of the symptoms. The median patient delay was significantly lower in patients with a vitreous hemorrhage and in patients with a history of a RRD in the fellow eye. CONCLUSIONS: The major reason for patient delay with a RDD was the patients' unawareness and unfamiliarity with the symptoms of a retinal detachment.

Aqueous flare and cells in Fuchs syndrome.

PURPOSE: To quantitatively evaluate aqueous flare and cells in patients with Fuchs syndrome. METHODS: The medical records of 40 patients (47 eyes) diagnosed with Fuchs syndrome between February 2006 and January 2007 at the Uveitis Study Center of Sun Yat-sen University were retrospectively reviewed. Aqueous flare and cells were clinically evaluated and quantified with laser flare-cell meter. Statistical analysis was performed to investigate the relationship between flare values and cell counts, and clinical parameters including patients' age, sex, duration of disease, best-corrected visual acuity, keratic precipitate, iris depigmentation, intraocular pressure, and posterior subcapsular lens opacities. RESULTS: Aqueous flare values (photon counts/ms) were significantly higher in Fuchs syndrome (9.40+/-5.85) than in normal controls (5.77+/-1.89, P=0.000). Aqueous cell counts (cells/0.5 mm(3)) were also significantly higher in Fuchs syndrome (5.09+/-4.84) than in normal controls (1.14+/-1.03, P=0.000). The flare values were positively correlated with the cell counts (r=0.331, P=0.001). Both flare values and cell counts were higher in eyes with keratic precipitates scored 2+ or 3+ as compared to those with a 1+ score. Higher flare values and cell counts were also observed in eyes with a 2+ or 3+ iris depigmentation score as compared to those with a 1+ score. No difference was found between flare values and cell counts and other parameters. CONCLUSION: Breakdown of blood-aqueous barriers and increased cell counts are present in the affected eyes in patients with Fuchs syndrome. These changes are positively associated with the degree of keratic precipitates and iris depigmentation.

Longitudinal quantification of aqueous flare and cells in Vogt-Koyanagi-Harada disease.

AIMS: To quantitatively evaluate the changes of aqueous flare and cells in eyes with Vogt-Koyanagi-Harada (VKH) disease. METHODS: This prospective study included 35 initial-onset VKH patients (70 eyes) and 46 recurrent VKH patients (92 eyes) following immunotherapy. Aqueous flare and cells were quantified using the laser flare-cell meter before treatment, 2 weeks, 1, 3, 6 and 9 months after treatment. RESULTS: Before treatment, mean aqueous flare (ph/ms) in initial-onset and recurrent VKH eyes were 8.1 (SD 4.1) vs 43.6 (20.7) (p = 0.000). Following treatment, recurrent VKH eyes showed a significantly higher flare value than initial-onset VKH eyes at 2 weeks, 1, 3 and 6 months. Prior to treatment, mean cell counts (cells/0.5 mm3) in initial-onset and recurrent VKH eyes were 2.0 (1.9) vs 39.4 (23.1) (p = 0.000). Following treatment, recurrent VKH eyes showed significantly higher cell counts than initial-onset VKH eyes at 2 weeks, 1 and 3 months. CONCLUSIONS: Our study shows that recurrent VKH patients displayed a more striking and long-lasting breakdown of the BAB and more severe inflammation than initial-onset VKH patients. Our study also indicates that the disruption of BAB lasted longer than aqueous cells either in initial-onset or in recurrent VKH patients.

Effect of flock size on dioxin levels in eggs from chickens kept outside.

To decrease dioxin uptake by the general population the European Union (EU) has set limits to the dioxin content of many foodstuffs including eggs. Eggs from free foraging chickens are known to have a higher dioxin content compared with confined laying hens, and the question is whether these eggs can adhere to current EU regulations. The aim of the study was to investigate parameters that are involved in the contamination of eggs from chickens raised under organic conditions. Samples from 34 organic farms including soil and earthworm samples were collected between September and December of the year 2003. Dioxin levels were assayed by gas chromatography-mass spectrometry. Various parameters were collected by on farm interviews. Egg dioxin content varied between 0.4 and 8.1 pg of toxic equivalents (TEQ)/g of egg fat with a mean of 2.2 pg of TEQ/g of egg fat. Nine out of 34 farms exceeded the EU limit of 3 pg of TEQ/g of egg fat. In addition, dioxin-like polychlorinated biphenyls (DL-PCB) were measured, and 8 samples exceeded the limit for the sum of dioxins and DL-PCB. Overall, egg samples from 10 farms were noncompliant with the dioxin or total TEQ limits. No statistically significant relation could be observed between egg dioxin levels and the concentration observed in soil or earthworms. A statistically significant association was observed between flock size and egg dioxin and DL-PCB content. This effect is most likely attributable to the fact that flock size is related to the time chickens spend outside. Restricting outdoor run use on one of the farms resulted in a decrease of the egg dioxin content to a level that was within the EU limits. This demonstrates that the most likely contamination source is the soil or soil organisms but that the behavior of the hens determines the extent of the contamination. Following the completion of this study, a dioxin monitoring protocol has been set up in the Netherlands to prevent marketing of eggs with raised dioxin levels.

Questionnaire survey of disease prevalence and veterinary treatments in organic layer husbandry in the Netherlands.

Disease prevalence and veterinary treatments in organic animal production differ from those in conventional systems. In order to gather information about current practices in organic layer husbandry, 33 organic egg producers of 16 small, 12 medium-sized, and 5 large farms were asked to complete a questionnaire on disease prevalence and treatments. On these farms, the mean mortality was 9% and the mean laying percentage was 79%. In contrast with the regulations for organic farming, the use of chemotherapeutics was not recorded on 30% of the farms. One third of the farmers were not aware of the type of vaccination given to their hens, and on the other farms hens had been vaccinated as pullets against nine or more viral and bacterial diseases. Several health problems were mentioned, such as feather pecking, red mites, helminths, infectious bronchitis, colibacillosis, and coccidiosis. On 19 farms, diseases were treated with homeopathic, phytotherapeutic, or other alternative medicines; on 10 of these farms chemotherapeutics were also used. On 4 farms only chemotherapeutics were used, on 10 farms no products were used, and on some farms up to seven products were used. Although quite a large number of organic layer farmers in the Netherlands used homeopathic, phytotherapeutic, or other alternative medicines, the use of chemotherapeutics is currently inevitable to prevent animal suffering or distress in organic husbandry.

Selective blockade of phosphodiesterase types 2, 5 and 9 results in cyclic 3'5' guanosine monophosphate accumulation in retinal pigment epithelium cells.

AIM: To investigate which phosphodiesterase (PDE) is involved in regulating cyclic 3'5' guanosine monophosphate breakdown in retinal pigment epithelium (RPE) cells. METHODS: cGMP content in the cultured RPE cells (D407 cell line) was evaluated by immunocytochemistry in the presence of non-selective or isoform-selective PDE inhibitors in combination with the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). mRNA expression of PDE2, PDE5 and PDE9 was studied in cultured human RPE cells and rat RPE cell layers using non-radioactive in situ hybridisation. RESULTS: In the absence of PDE inhibitors, cGMP levels in cultured RPE cells are very low. cGMP accumulation was readily detected in cultured human RPE cells after incubation with Bay60-7550 as a selective PDE2 inhibitor, sildenafil as a selective PDE5 inhibitor or Sch51866 as a selective PDE9 inhibitor. In the presence of PDE inhibition, cGMP content increased markedly after stimulation of the particulate guanylyl cyclase. mRNA of PDE2,PDE5 and PDE9 was detected in all cultured human RPE cells and also in rat RPE cell layers. CONCLUSIONS: PDE2, PDE5 and PDE9 have a role in cGMP metabolism in RPE cells.