Targeting nicotinamide N-methyltransferase decreased aggressiveness of osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. METHODS: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. RESULTS: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy.

The effects of aerobic exercise and transcranial direct current stimulation on cognitive function in older adults with and without cognitive impairment: A systematic review and meta-analysis.

BACKGROUND: Aerobic exercise (AE) may slow age-related cognitive decline. However, such cognition-sparing effects are not uniform across cognitive domains and studies. Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation and is also emerging as a potential alternative to pharmaceutical therapies. Like AE, the effectiveness of tDCS is also inconsistent for reducing cognitive impairment in ageing. The unexplored possibility exists that pairing AE and tDCS could produce synergistic effects and reciprocally augment cognition-improving effects in older individuals with and without cognitive impairments. Previous research found such synergistic effects on cognition when cognitive training is paired with tDCS in older individuals with and without mild cognitive impairment (MCI) or dementia. AIM: The purpose of this systematic review with meta-analysis was to explore if pairing AE with tDCS could augment singular effects of AE and tDCS on global cognition (GC), working memory (WM) and executive function (EF) in older individuals with or without MCI and dementia. METHODS: Using a PRISMA-based systematic review, we compiled studies that examined the effects of AE alone, tDCS alone, and AE and tDCS combined on cognitive function in older individuals with and without mild cognitive impairment (MCI) or dementia. Using a PICOS approach, we systematically searched PubMed, Scopus and Web of Science searches up to December 2021, we focused on 'MoCA', 'MMSE', 'Mini-Cog' (measures) and 'cognition', 'cognitive function', 'cognitive', 'cognitive performance', 'executive function', 'executive process', 'attention', 'memory', 'memory performance' (outcome terms). We included only randomized controlled trials (RTC) in humans if available in English full text over the past 20 years, with participants' age over 60. We assessed the methodological quality of the included studies (RTC) by the Physiotherapy Evidence Database (PEDro) scale. RESULTS: Overall, 68 studies were included in the meta-analyses. AE (ES = 0.56 [95% CI: 0.28-0.83], p = 0.01) and tDCS (ES = 0.69 [95% CI: 0.12-1.26], p = 0.02) improved GC in all three groups of older adults combined (healthy, MCI, demented). In healthy population, AE improved GC (ES = 0.46 [95% CI: 0.22-0.69], p = 0.01) and EF (ES = 0.27 [95% CI: 0.05-0.49], p = 0.02). AE improved GC in older adults with MCI (ES = 0.76 [95% CI: 0.21-1.32], p = 0.01). tDCS improved GC (ES = 0.69 [90% CI: 0.12-1.26], p = 0.02), all three cognitive function (GC, WM and EF) combined in older adults with dementia (ES = 1.12 [95% CI: 0.04-2.19], p = 0.04) and improved cognitive function in older adults overall (ES = 0.69 [95% CI: 0.20-1,18], p = 0.01). CONCLUSION: Our systematic review with meta-analysis provided evidence that beyond the cardiovascular and fitness benefits of AE, pairing AE with tDCS may have the potential to slow symptom progression of cognitive decline in MCI and dementia. Future studies will examine the hypothesis of this present review that a potentiating effect would incrementally improve cognition with increasing severity of cognitive impairment.

Bioanalysis of EGFRm inhibitor osimertinib, and its glutathione cycle- and desmethyl metabolites by liquid chromatography-tandem mass spectrometry.

Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000 nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6 nM due to low accuracy at 2 nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80 mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels.

Bioanalysis of ibrutinib, and its dihydrodiol- and glutathione cycle metabolites by liquid chromatography-tandem mass spectrometry.

Ibrutinib is a targeted covalent inhibitor frequently used for the treatment of various lymphomas. In addition to oxidative metabolism, it is metabolized through glutathione coupling. The quantitative insight into this kind of metabolism is scarce, and tools for quantitation are lacking. The non-oxidative metabolism could prove a more prominent role when oxidative metabolism is impaired. Also, in-vitro studies could over-estimate the effect of CYP450-inhibition. To gain quantitative insight into this relatively unknown biotransformation pathway of the drug we have developed a validated simple, fast and sensitive bio-analytical assay for ibrutinib, dihydrodiol-ibrutinib, and the glutathione, cysteinylglycine and cysteine conjugates of ibrutinib in human plasma. The method emphasizes on simplicity, the thiol-conjugates were synthesized by a simple one step synthesis, followed by LC-purification. Sample preparation was done by a simple protein crash with acetonitrile containing labeled internal standards, evaporation of solvents, and reconstitution in eluent. Finally, the compounds were quantified using UHPLC-MS/MS. The assay was successfully validated in a 0.5-500nM calibration range for all compounds, and also a lower range of 0.05-50 nM was demonstrated for ibrutinib to accommodate for even the lowest trough levels. This assay has a considerably higher sensitivity than previous published assays, with the previous lowest LLOQ being 1.14 nM. Both, ibrutinib, dihydrodiol-ibrutinib and the cysteine conjugate were deemed stable under refrigerated or frozen storage conditions. At room temperature, the glutathione conjugate showed rapid degradation into the cysteinylglycine conjugate in plasma. Finally, the applicability of the assay was demonstrated in patient samples.

Proximal correlates of metabolic phenotypes during 'at-risk' and 'case' stages of the metabolic disease continuum.

OBJECTIVE: To examine the social and behavioural correlates of metabolic phenotypes during 'at-risk' and 'case' stages of the metabolic disease continuum. DESIGN: Cross-sectional study of a random population sample. PARTICIPANTS: A total of 718 community-dwelling adults (57% female), aged 18-92 years from a regional South Australian city. MEASUREMENTS: Total body fat and lean mass and abdominal fat mass were assessed by dual energy x-ray absorptiometry. Fasting venous blood was collected in the morning for assessment of glycated haemoglobin, plasma glucose, serum triglycerides, cholesterol lipoproteins and insulin. Seated blood pressure (BP) was measured. Physical activity and smoking, alcohol and diet (96-item food frequency), sleep duration and frequency of sleep disordered breathing (SDB) symptoms, and family history of cardiometabolic disease, education, lifetime occupation and household income were assessed by questionnaire. Current medications were determined by clinical inventory. RESULTS: 36.5% were pharmacologically managed for a metabolic risk factor or had known diabetes ('cases'), otherwise were classified as the 'at-risk' population. In both 'at-risk' and 'cases', four major metabolic phenotypes were identified using principal components analysis that explained over 77% of the metabolic variance between people: fat mass/insulinemia (FMI); BP; lipidaemia/lean mass (LLM) and glycaemia (GLY). The BP phenotype was uncorrelated with other phenotypes in 'cases', whereas all phenotypes were inter-correlated in the 'at-risk'. Over and above other socioeconomic and behavioural factors, medications were the dominant correlates of all phenotypes in 'cases' and SDB symptom frequency was most strongly associated with FMI, LLM and GLY phenotypes in the 'at-risk'. CONCLUSION: Previous research has shown FMI, LLM and GLY phenotypes to be most strongly predictive of diabetes development. Reducing SDB symptom frequency and optimising the duration of sleep may be important concomitant interventions to standard diabetes risk reduction interventions. Prospective studies are required to examine this hypothesis.

Testosterone modulates gene expression pathways regulating nutrient accumulation, glucose metabolism and protein turnover in mouse skeletal muscle.

Testosterone regulates energy metabolism and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. This study investigated the response of skeletal muscle to castration and testosterone replacement in 8-week-old male mice. Using microarray analyses of mRNA levels in gastrocnemius muscle, 91 genes were found to be negatively regulated by testosterone and 68 genes were positively regulated. The mRNA levels of the insulin signalling suppressor molecule Grb10 and the glycogen synthesis inhibitors, protein phosphatase inhibitor-1 and phosphorylase kinase-γ, were negatively regulated by testosterone. The insulin-sensitive glucose and amino acid transporters, Glut3 and SAT2, the lipodystrophy gene, Lpin1 and protein targeting to glycogen were positively regulated. These changes would be expected to increase nutrient availability and sensing within skeletal muscle, increase metabolic rate and carbohydrate utilization and promote glycogen accumulation. The observed positive regulation of atrogin-1 (Fbxo32) by testosterone could be explained by the phosphorylation of Akt and Foxo3a, as determined by Western blotting. Testosterone prevented the castration-induced increase in interleukin-1α, the decrease in interferon-γ and the atrophy of the levator ani muscle, which were all correlated with testosterone-regulated gene expression. These findings identify specific mechanisms by which testosterone may regulate skeletal muscle glucose and protein metabolism.

Testosterone, aging and quality of life.

Testosterone levels decline over the lifespan. Many symptoms of hypogonadism are similar to age-related changes in older males. A small number of studies have suggested that some of these symptoms may be reversed by testosterone. Among them, one trial has suggested that decline in bioavailable testosterone may be related to the development of functional decline. Testosterone replacement during rehabilitation may improve functional outcomes and plays an important role in the maintenance of sexual related quality of life. The overall improvement in well-being and/or health-related quality of life in older males needs to be determined with large placebo-controlled trials. This is particularly important in view of the substantial placebo effect on aging symptomatology.

Frailty and the aging male.

Frailty occurs in aging males for a variety of reasons. It is less common in males than females. Diseases which are particularly associated with frailty are diabetes mellitus, atherosclerosis, anemia and chronic obstructive pulmonary disease. Insulin resistance syndrome plays a pathogenetic role in the "fat-frail" syndrome. Sarcopenia occurs predominantly because of hormone deficiency and cytokine excess. Pain and anorexia are also associated with frailty. Stem cell research represents a potential promise for the treatment of frailty.

Defining 'relative' androgen deficiency in aging men: how should testosterone be measured and what are the relationships between androgen levels and physical, sexual and emotional health?

In men, bioavailable and free testosterone levels decline by about 1.0 and 1.2% per year, respectively, after the age of 40. The definition of clinically relevant androgen deficiency in the aging male remains uncertain. Clinical features common to both aging and androgen deficiency include decreased muscle mass and strength, and increased fatigue, increased fat mass, loss of libido, erectile dysfunction, impaired cognitive function and depression. It is, however, difficult to separate the effect on plasma testosterone of concomitant disease, compared with the effects of a decrease in testosterone levels alone. Testosterone supplementation has been shown to be effective in improving many of the clinical features of androgen deficiency in the older male, and is safe, at least in the short term. The maximum benefit occurs in those men with the lowest testosterone levels.

Systemic anti-tumor necrosis factor antibody treatment exacerbates endotoxin-induced uveitis in the rat.

Tumor necrosis factor is released in the circulation and aqueous humor during endotoxin-induced uveitis, and induces acute uveitis when injected intraocularly in rats. To elucidate the role of tumor necrosis factor in the development of endotoxin-induced uveitis we analysed the effect of neutralizing anti-tumor necrosis factor antibodies and of pentoxifylline, a drug that inhibits tumor necrosis factor synthesis. Lewis rats were treated with: (a) a single intracardial injection of polyclonal rabbit anti-murine tumor necrosis factor antiserum prior to foot pad injection of 200 micrograms lipopolysaccharide; (b) an intraperitoneal injection of 10 mg pentoxifylline 1 hr before, at the time of, and 3 hr after foot pad injection of lipopolysaccharide; or (c) an intravitreal injection of 20 to 500 micrograms pentoxifylline together with 1 microgram lipopolysaccharide. The ocular inflammation was examined by slit-lamp and evaluated for the presence of hyperemia, flare, miosis, infiltrating cells or hypopyon. Levels of tumor necrosis factor in serum and aqueous samples were determined using a bioassay. Systemic treatment with either anti-tumor necrosis factor antibodies or pentoxifylline resulted in a significant inhibition, 90 and 70% respectively, of serum tumor necrosis factor activity at 3 to 4 hr after lipopolysaccharide injection. Systemic pentoxifylline treatment had no influence on the severity of uveitis. Anti-tumor necrosis factor antibody treatment, in contrast, caused an exacerbation of endotoxin-induced uveitis at t = 20 hr; mean uveitis score 3.9 vs. 1.4 in controls; P < 0.01. Intraocular administration of pentoxifylline together with lipopolysaccharide also had an aggravating effect on uveitis, that was associated with increased levels of intraocular tumor necrosis factor. The results show that inhibition of serum tumor necrosis factor activity does not block the development of endotoxin-induced uveitis. In fact, anti-tumor necrosis factor antibody treatment exacerbates the intraocular inflammation. These findings suggest that tumor necrosis factor may have other than proinflammatory properties in this uveitis model.

Tumour necrosis factor-induced uveitis in the Lewis rat is associated with intraocular interleukin 6 production.

Lewis rats were injected with recombinant murine tumour necrosis factor-alpha either intravitreally (0.08-50 ng) or intracardially (1 microgram). The intraocular inflammatory response induced by tumour necrosis factor was examined by slit-lamp and protein extravasation into aqueous humor was determined. The phenotype of the inflammatory cells in the eye was analysed by immunohistochemistry. In addition, the kinetics of intraocular interleukin 6 production were determined. At 24 hr after intravitreal injection, a significant clinical uveitis was observed only in rats injected with 50 ng of tumour necrosis factor, when compared to saline-treated controls (P < 0.05). Maximal clinical uveitis and blood-aqueous barrier breakdown were already present at 4 hr after tumour necrosis factor injection. The uveitis was characterized by a massive cellular infiltrate in the anterior segment, consisting predominantly of polymorphonuclear cells and macrophages/monocytes, and to a lesser extent of T lymphocytes. Intraocular interleukin 6 mRNA expression and elevated levels of interleukin 6 in aqueous humor were detected 1 hr after tumor necrosis factor injection, reached a maximum at 3 to 4 hr after injection, and had declined again at 2 hr. Although intracardial injection of 1 microgram of tumour necrosis factor in Lewis rats induced a rise of circulating interleukin 6, it did not produce uveitis. The results obtained with intravitreally injected tumour necrosis factor indicate that intraocular TNF may play a pivotal role in the induction of uveitis in the rat. The transient intraocular production of interleukin 6 early during tumour necrosis factor-induced uveitis suggests that this cytokine may participate in the response induced by tumour necrosis factor.

Increases in human epidermal DR+CD1+, DR+CD1-CD36+, and DR-CD3+ cells in allergic versus irritant patch test responses.

In an attempt to differentiate an allergic patch test response from an irritant response, we evaluated by flow cytometry the percentages of various epidermal cell populations isolated from allergen and irritant-treated patch test sites. Nine allergic individuals were patch tested with various allergens (Rhus, dinitrochlorobenzene [DNCB], or nickel chloride) and a vehicle control for 48 h. Eight additional individuals were patch tested with irritating chemicals (sodium lauryl sulfate or nonanoic acid) and with a vehicle control for 48 h. Epidermal cells, isolated from suction blisters, were double labeled for CD1/HLA-DR, CD3/HLA-DR, or CD36/HLA-DR cell surface markers and analyzed by flow cytometry to determine the percentage of various cell populations. A mean increase of 0.91 +/- 0.3 in the percentage of DR+CD1+ Langerhans cells over the vehicle control patch test site was detected in allergen-positive patch test sites in allergic individuals, whereas a decrease of 0.19 +/- 0.2 in the percentage of DR+CD1+ Langerhans cells from the vehicle control patch test site was detected in irritant-treated patch test sites. Epidermal cells from allergen-positive patch test sites also exhibited an increase of 5.2 +/- 1.8 in percentage of DR+CD1- cells over the vehicle control patch test site compared to an increase change of 0.8 +/- 0.4 in epidermal cells isolated from irritant-treated patch test sites. We also found that DR+ cells that lacked the CD1 determinant expressed the macrophage/monocyte antigen CD36 (OKM5). Finally, a 2.3 +/- 0.8 increase in the percentage of DR-CD3+ cells over the vehicle control patch test site was observed in allergen-positive patch test sites compared to an increase of 0.2 +/- 0.2 observed in irritant-treated patch test sites. These results demonstrate a significant increase in DR+CD1+, DR+CD1-CD36+, and DR-CD3+ epidermal cells in allergen-positive patch test sites compared to irritant patch test sites.

Kinetics of intraocular tumor necrosis factor and interleukin-6 in endotoxin-induced uveitis in the rat.

PURPOSE: To determine the kinetics of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in serum and aqueous humor of rats with different susceptibilities to endotoxin-induced uveitis (EIU), after footpad injection of lipopolysaccharide (LPS). METHODS: Samples were collected from EIU-susceptible Lewis rats and EIU-resistant Brown Norway (BN) rats for up to 72 hours after LPS injection. Specific bioassays were used to measure TNF and IL-6 activity. Northern blot analysis was used to assess intraocular IL-6 mRNA expression. RESULTS: High levels of TNF and IL-6 were detected in serum of both rat strains early after LPS injection. A second rise in serum TNF was observed at 18 to 20 hours in Lewis rats only. In aqueous humor of Lewis rats, high levels of TNF and IL-6 were observed early after LPS injection (2 to 8 hours) and concomitant with maximal uveitis (18 to 24 hours). Low levels of TNF and IL-6 were found in aqueous humor of BN rats. Ocular IL-6 mRNA was detected at the same time as IL-6 activity was measured in aqueous humor. CONCLUSIONS: The results of this study indicate that both TNF and IL-6 may play a role in the pathogenesis of EIU. The early release of TNF in aqueous humor during EIU suggests that this cytokine may serve as an initial mediator of intraocular inflammation. Furthermore, Northern blot analysis indicates that IL-6 is produced locally during EIU.

Induction of autoantibodies to rat corneal protein 54.

Many studies have described the presence of circulating antibodies against corneal components in patients with corneal disease or uveitis, and in patients with skin or systemic disease with or without ocular involvement. The role of such antibodies in the underlying immunopathological process remains obscure. Here we describe the induction of autoantibodies against the rat cornea. Our attempts to induce corneal autoantibodies by various forms of keratitis and corneal trauma failed. However, circulating corneal autoantibodies could be detected by Western blotting after immunization of BN rats and Lewis rats with bovine corneal protein 54 (BCP 54). Rats immunized with rat corneal extracts (RaCE) or human serum albumin (HSA) as (auto) antigen did not develop corneal autoantibodies. During the study period (greater than 4 months), it was observed that the presence of circulating corneal autoantibodies did not elicit corneal inflammation. Severe keratitis did develop when BCP 54-immunized rats were challenged intracorneally with BCP 54, but the clinical signs were not significantly different from HSA-immunized rats after an intracorneal HSA challenge. Injection of corneal autoantibodies into the corneal stroma did not provoke keratitis. To the best of our knowledge this is the first study demonstrating corneal autoantibodies in rats without actual manipulation of the eye. This model may provide further insights in the role and significance of corneal autoantibodies in disease.

Endotoxin-induced uveitis in the rat. The significance of intraocular interleukin-6.

The potential role of interleukin-6 (IL-6) was studied as an inflammatory mediator of endotoxin (or lipopolysaccharide [LPS])-induced uveitis (EIU) in the rat. In young Lewis rats, levels of intraocular IL-6, but not serum IL-6, correlated with the severity of uveitis and with aqueous humor protein levels in response to foot pad injections of LPS (P less than 0.001). Adult Lewis rats did not develop uveitis and had no intraocular IL-6, although IL-6 was released systemically. Resistance to EIU and absence of IL-6 levels in the aqueous humor, despite the ability to release serum IL-6, also were observed in brown Norway rats, irrespective of age and weight. Intravitreal injection of as little as 1 ng of human recombinant IL-6 induced uveitis in young Lewis rats. In adult Lewis rats, and in young animals made tolerant to LPS, intravitreal IL-6 still caused substantial leakage of plasma proteins into the anterior chamber but no influx of inflammatory cells. As early as 2 hr after intravitreal injection of IL-6, immunohistochemical analysis showed invasion of the iris, corneal stroma, and anterior chamber by polymorphonuclear leukocytes (PMN) and expression of major histocompatibility complex (MHC) class II antigen in the retina by large cells that were macrophage-marker ED2 negative. This was followed by massive PMN infiltration of the retinal layers and vitreous. The MHC class II antigen expression of ciliary and iris epithelium occurred at a later stage (greater than 8 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of soluble protein BCP 11/24 from bovine corneal epithelium, different from the principal soluble protein BCP 54.

The water-soluble fraction of bovine corneal epithelium was analysed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Next to the principal soluble protein BCP 54, which has recently been identified as a corneal aldehyde dehydrogenase (ALDH), another abundant protein was observed, which we have denoted BCP 11/24, due to its estimated molecular weight of 11 kDa in SDS-PAGE and 24 kDa in high performance gel filtration under non-denaturing conditions. This protein was isolated and characterized by biochemical and immunochemical techniques. The isolation of BCP 11/24 was initially hampered by its tendency to bind non-covalently to BCP 54. BCP 11/24 behaves identically in reduced and unreduced SDS-PAGE and is probably not a glycoprotein. Isoelectric focusing indicated microheterogeneity of BCP 11/24, yielding bands with isoelectric points of 6.1, 5.9, 5.7 and 5.6. A rabbit antiserum directed against BCP 11/24, that did not recognize BCP 54, demonstrated that the distribution of BCP 11/24 in different ocular tissues as well as its light microscopic localization in corneal epithelium is strikingly similar to that of BCP 54. Together with its tendency to interact with BCP 54 in vitro, this suggests the possibility that BCP 11/24 is associated with BCP 54 in vivo, fulfilling a function which may be related to the activity of BCP 54 as a corneal ALDH. In contrast with BCP 54, however, BCP 11/24 was not detectable in corneal endothelium. The antiserum did not detect any immunologically related molecules in corneal epithelium extracts of sheep, human or rat origin, indicating that BCP 11/24 is probably not as highly conserved as BCP 54.

Analysis of IL-6 levels in human vitreous fluid obtained from uveitis patients, patients with proliferative intraocular disorders and eye bank eyes.

Several studies suggest a role for IL-6 in the pathogenesis of uveitis. Earlier we have shown that aqueous humour obtained from patients with uveitis contained raised levels of IL-6. In the study described here we investigated the IL-6 levels in vitreous fluid samples obtained from 75 uveitis patients with different uveitis entities. Vitreous samples from 14 patients with proliferative intraocular disorders (PID) and 29 eye bank eyes were used as controls. All the samples were tested in the IL-6 B9 bioassay as well as in a sensitive ELISA for IL-6. Raised IL-6 levels were detected in the vitreous fluid of uveitis patients as well as patients with PID, implicating IL-6 as a common inflammatory mediator. The highest mean level of IL-6 was found in the vitreous fluid of patients with acute retinal necrosis. The mean IL-6 levels measured by the ELISA were higher compared to the levels measured by the B9 bioassay. This may be caused by the presence of B9 bioassay inhibitory factors in the vitreous fluid of these patients.

Analysis of aqueous humour in ocular onchocerciasis.

The development of an onchocercal chorioretinopathy from the first detectable signs to a full blown oncho fundus is not fully understood. We investigated the intraocular humoral immune response against Onchocerca volvulus, human S-antigen, IRBP and crude retinal extract (using an ELISA) by examining paired aqueous humour and serum samples obtained from onchocerciasis patients (without [n = 10] and with ocular symptoms [n = 8]) and endemic controls [n = 14] from Sierra Leone (West Africa). A local intraocular anti-retinal IgG antibody production could not be demonstrated in onchocerciasis patients, whether they had ocular symptoms or not. A significantly higher level of O. volvulus antibodies and IgG was measured in the aqueous of onchocerciasis patients with ocular involvement, as compared to patients without ocular symptoms (Mann-Whitney ranksum test; p less than 0.001 and p less than 0.02 respectively). Since interleukin-6 (IL-6) plays an essential role in the differentiation of B cells into immunoglobulin producing plasma cells, we therefore measured this cytokine in paired aqueous and serum samples. Elevated IL-6 levels were found in the aqueous of two out of eight onchocerciasis patients tested. In view of these findings it seems improbable that retinal autoimmunity is a major factor in the pathogenesis of onchocercal chorioretinopathy. The high intraocular levels of antibodies against the parasite suggest a direct involvement of the parasite in the pathogenesis of onchocercal chorioretinopathy.