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.

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.

Association and dissociation of aggregated IgG from rat peritoneal macrophages.

Stable aggregated IgG (A-IgG) of various sizes, having high biological activity, were incubated at 4 degree C with adhering peritoneal macrophages from normal rats and the kinetics of A-IgG binding to the cell surface were studied. Equilibrium constants were high (2.8-11.7 X 10(8) M-1) and varied as a function of aggregate size. The maximum number of A-IgG bound per cell varied from 230,000 for A-IgG9 to 90,000 for A-IgG74. Binding was 50% inhibited by near physiological concentrations of monomeric IgC. These data suggest that A-IgG are bound at multiple sites by attachment of Fc frgments to Fc receptors present on the macrophage surface with larger A-IgG being more avidly bound. Dissociation was slower for larger A-IgG while no clear trend was seen relating associating rates and aggregate size. Thus, differences in the avidity of binding of A-IgG are due primarily to slower dissociation of larger A-IgG. Dissociationissociation of A-IgG was slower from cells exposed initially to higher doses of A-IgG and dissociation did not follow simple first order kinetics. Thus, the avidity of binding appears to be heterogeneous in a population of similar sized A-IgG. As expected, association was dose-dependent, more rapid than dissociation, and followed pseudo first order kinetics. Based on all of the above data, it is proposed that binding of A-IgG proceeds in two steps. First, A-IgG are loosely bound to perhaps a single Fc receptor. Then, depending upon the availability and mobility of Fc receptors, additional Fc fragments are attached and the A-IgG becomes more firmly attached. Thus binding is slow, but once attached A-IgG are avidly held.

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.

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.

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.

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.

Cats and goat whey associated with Toxoplasma gondii infection in pigs.

In organic livestock production systems, farm-management factors are thought to play an important role in the on-farm prevalence of Toxoplasma gondii. Serological results and the results of an HACCP analysis were combined to determine important risk factors for the prevalence of this protozoan parasite. Mathematical analysis demonstrated that feeding goat whey to pigs and the presence of a high number of cats were positively correlated to T. gondii seroprevalence in pigs. Not covering roughage and the farmers' assumption that pigs can come into contact with cat feces also showed a positive relationship. In order to decrease the risk of T. gondii infecting their pigs, farmers should limit the access and number of cats on their farms and refrain from feeding goat whey to their pigs.

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.

Retinal pigment epithelium-immune system interactions: cytokine production and cytokine-induced changes.

Vision is dependent on proper function of several intraocular structures. Immune responses to eliminate invading pathogens from the eye may threat vision by causing damage to these structures. Therefore, immunological defence of the eye should be carefully balanced between efficacy and maintenance of functional integrity. The eye is equipped with several regulatory mechanisms to prevent certain immune and inflammatory responses and is, therefore, regarded as an immune privileged site. The retinal pigment epithelium (RPE) contributes to the immune privileged status of the eye as part of the blood-eye barrier and by the secretion of immunosuppressive factors inside the eye. RPE cells, however, may also play an important role in the development of immune and inflammatory responses in the posterior part of the eye. During the last decade it has become clear that RPE cells are highly sensitive to a variety of inflammatory cytokines. Under inflammatory conditions, RPE cells produce a myriad of cytokines that may activate the resident ocular cells or attract and activate leukocytes. Cytokine stimulation of RPE cells causes profound effects, including nitric oxide secretion, cell surface expression of MHC class II and adhesion molecules and abrogation of barrier function. This article provides a comprehensive review of the literature concerning RPE cells and cytokines.

Upregulation of T-bet expression in peripheral blood mononuclear cells during Vogt-Koyanagi-Harada disease.

AIM: To test the hypothesis that T-bet expression is altered in patients with Vogt-Koyanagi-Harada (VKH) disease. METHODS: Peripheral blood was withdrawn from 16 VKH patients before and after immunosuppressive treatment and from 16 healthy individuals. IFN-gamma, IL-2, and IL-4 in the serum and the supernatants of peripheral blood mononuclear cells (PBMC) cultured with or without phytohaemagglutinin (PHA) were measured by ELISA. T-bet mRNA and protein expression in PBMC cultured with or without PHA was detected by RT-PCR and western blot, respectively. RESULTS: The level of IFN-gamma, but not IL-2 and IL-4, was significantly higher in the supernatants of stimulated PBMC in patients than in controls. A significantly increased T-bet mRNA was found in VKH patients during an active uveitis episode, but not in quiescent patients, compared to controls. T-bet protein was detectable in VKH patients during an active uveitis episode, but not in quiescent patients nor in the healthy controls. Stimulation of PBMC with PHA resulted in a marked upregulation of T-bet mRNA and protein expression for both patients and controls with no significant difference between the two groups. CONCLUSIONS: Upregulation of T-bet may be associated with the development of a Th1 mediated immune response in VKH disease.

Long-term changes in corneal endothelial morphology after discontinuation of low gas-permeable contact lens wear.

PURPOSE: Low gas-permeable contact lens wear of polymethyl methacrylate or hydroxyethyl methacrylate material is known to cause morphologic abnormalities in the corneal endothelial cell layer. These lenses were widely prescribed and successfully worn until their use was actively discouraged in the late 1980s and early 1990s. This study was designed to investigate whether discontinuation of low gas-permeable contact lens wear leads to an improvement of corneal endothelial cell morphology. METHODS: At the time of discontinuation and at least 5 years after discontinuation of low gas-permeable contact lens wear, noncontact specular photographs of the central corneal endothelium were made in 66 patients (14 male and 52 female, mean age 37.7 +/- 8.4, range 24.6-69.0). By computer analysis of endothelial photographs, parameters for polymegethism and pleomorphism were calculated, as well as cell density. RESULTS: Mean follow-up time between photographs was 6.8 years (SD 1.1). Sixty-one patients were refitted with rigid high gas-permeable contact lenses or high-water-content soft lenses, and 5 patients switched to spectacle wear. A small but significant recovery of the corneal endothelial cell morphology was found for the mean coefficient of variation of cell area, from 37.5 to 35.7 (P = 0.022), and for the coefficient of variation of the number of sides, from 13.1 to 12.4 (P = 0.004). The mean percentage of hexagonal cells increased from 54.2 to 56.2 (P = 0.013). Although the corneal endothelial cell morphology improved significantly on cessation of LGP contact lens wear, the values did not return to levels observed in normal, non-contact lens wearing individuals. During follow-up, the mean endothelial cell density decreased significantly (P = 0.001) from 2994 to 2890 (a 3.5% cell loss in 6.8 years), which is similar to the known normal age-related cell loss of 0.6% per year in non-contact lens wearing healthy individuals. CONCLUSION: Endothelial polymegethism and pleomorphism caused by PMMA or HEMA contact lens wear is partly reversible.

Immunological factors in the pathogenesis and treatment of age-related macular degeneration.

Recent findings indicate that immunological factors are involved not only in the pathogenesis of age-related macular degeneration (AMD), but also in its treatment. Earlier data showing the presence of inflammatory cells in affected areas of AMD retinas support this statement. Although a possible role for autoimmunity was initially suggested, it has never reached general acceptance. Microorganisms have also been implied in the pathogenesis of AMD. Both serum antibacterial antibody levels and positive DNA tests from neovascular membranes have pointed to a possible role for Chlamydia pneumoniae in the pathogenesis of AMD. New data is providing evidence for the hypothesis that deposits between Bruch's membrane and the retinal pigment epithelium (RPE) cell layer may act as a stimulus for the local activation of the complement system. This may lead to a further growth of the deposits due to the strong chemotactic activity of certain complement activation products (such as C5a) with an influx of inflammatory cells. The buildup of cells and extracellular deposits may lead to local ischemia resulting in the activation of RPE cells. These activated RPE cells are thought to release angiogenic stimuli leading to choroidal neovascularization, which is the most serious complication of AMD. The fact that immunosuppressive drugs such as triamcinolone acetonide and anecortave acetate are capable of inhibiting choroidal neovascularization is consistent with an inflammatory component in the pathogenesis of AMD. Specific immunotherapy directed at certain cytokines or growth factors is now being investigated at both the animal and patient levels. Various clinical trials involving engineered antibodies are now being applied to block angiogenic factors such as the vascular endothelial growth factor (VEGF). An approach using gene therapy to influence angiogenesis by inducing the production of the pigment epithelium-derived factor (PEDF) was able to block neovascularization in an experimental murine model. Besides trying to block ongoing processes in AMD, retinal transplantation is now also being investigated as a treatment option. The fact that the retina is possibly an immunoprivileged tissue in combination with experimental data showing that the subretinal space is an immunoprivileged site is an indication that transplantation would not suffer from the rejection process. A larger obstacle is the question whether transplanted retinal tissue will regain its functional properties.

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.

Serological and polymerase chain reaction-based analysis of aqueous humour samples in patients with AIDS and necrotizing retinitis.

OBJECTIVE: To evaluate the measurement of intraocular antibody production and detection of DNA by the polymerase chain reaction (PCR) for diagnosis of the causative microorganism in patients with AIDS and necrotizing retinitis. METHODS: Paired serum and aqueous humour samples obtained from 28 patients with AIDS and necrotizing retinitis, seen between January 1987 and March 1992, were analysed for intraocular antibody production against cytomegalovirus (CMV), varicella zoster virus, herpes simplex virus, Epstein-Barr virus, and Toxoplasma gondii. Specific antibody titres in the inflamed eye and in the circulation were related to total immunoglobulin G content in the aqueous humour and serum. In addition, PCR analysis was performed in 15 samples. Results were compared to the final diagnosis, which was based on the subsequent clinical course. Results were also related to parameters describing the immune state of the patients: CD4 count, time between diagnosis of an AIDS-defining illness and retinitis, and time of survival following the diagnosis of retinitis. RESULTS: In 11 (39%) out of 28 patients we found local intraocular antibody production which correlated with the final diagnosis (one out of two cases with acute retinal necrosis, three out of five cases with toxoplasma retinitis, and eight out of 21 patients with CMV retinitis). In all 13 patients with CMV retinitis PCR analysis detected CMV DNA. In one patient with the clinical diagnosis of Toxoplasma retinitis, Toxoplasma DNA could be determined, whereas in the same sample CMV DNA was also found. In yet another patient with Toxoplasma retinitis only CMV DNA could be detected. A relationship between results of local antibody determination with either CD4 counts, or the time interval between AIDS-defining illness and retinitis, or survival time after diagnosis of retinitis could not be established. CD4 counts were higher than 50 x 10(6)/l in eight out of 19 patients with CMV retinitis. No complications of paracentesis were seen. CONCLUSIONS: Detection of intraocular antibody production and PCR analysis are quick and safe procedures and helpful tools for diagnosis of the involved pathogen in AIDS patients with a necrotizing retinitis. Negative results of local antibody production, even in the presence of detectable viral DNA, could not be related to the parameters of a more deteriorated immune status of these patients.

Cytomegalovirus (CMV) strain differences between the eye and blood in AIDS patients with CMV retinitis.

OBJECTIVE: To investigate possible differences in cytomegalovirus (CMV) strain distribution between the eye and blood in AIDS patients with CMV retinitis. METHODS: CMV DNA sequences from aqueous humour and peripheral blood leukocytes (PBL), obtained from 13 AIDS patients with CMV retinitis, were compared. DNA was isolated and the CMV IE-1 sequence (part of the immediate early-1 gene) and the a-sequence (located in the a-region) were amplified by polymerase chain reaction (PCR). The PCR products of the a-sequence were analysed by Southern blotting for amplified fragment-length polymorphisms. The level of divergence between the a-sequences of aqueous humour- and PBL-derived CMV was studied in two patients by cloning these sequences followed by sequence analysis. RESULTS: CMV DNA could be detected in all aqueous humour samples and in 10 out of 13 paired blood samples. In the 10 patients, with CMV DNA detectable in both aqueous humour and PBL, seven cases showed differences between the amplified products of both compartments. Sequence analysis in two patients revealed that the aqueous humour and PBL of the same patient can harbour both identical, similar and highly divergent CMV a-sequences. CONCLUSION: These results indicate that despite the haematogenous spread of CMV, the eye, being a relatively shielded organ, may contain CMV strains different from those found in the blood.

Characteristics of soluble immune complexes prepared from oligovalent DNP conjugates and anti-DNP antibodies.

The stability of soluble immune complexes was investigated after isolation by gel filtration and sucrose gradient ultracentrifugation. Soluble immune complexes were formed between specific goat anti-dinitrophenol (DNP) antibodies and DNP conjugated to a large (19 S) carrier, namely bovine thyroglobulin. The composition and molecular weight of these complexes were determined by ultracentrifugation on calibrated sucrose density gradients and the use of different isotopic markers for antigen and antibody. A good separation of immune complexes containing one, two, or three antigen molecules per complex was obtained by ultracentrifugation while gel filtration was less effective. Ultracentrifugational analysis of fractions isolated by these two procedures showed that large immune complexes containing more than one antigen were relatively labile, whereas small immune complexes containing one antigen were stable. The stability of large immune complexes was dependent on dilution. Because dilution affects the size and composition of soluble immune complexes, it is important to emphasize that for the investigation of a causal relationship between the biological properties and the size and composition of immune complexes, analysis of the immune complexes should be performed in the same dilutions in which they will be used experimentally.