Immunocompromised children and young people are at no increased risk of severe COVID-19.

OBJECTIVES: We aimed to prospectively describe the incidence and clinical spectrum of SARS-CoV-2 infection in immunocompromised paediatric patients in the UK. METHODS: From March 2020 to 2021 weekly questionnaires were sent to immunocompromised paediatric patients or their parents. Information, including symptom presentation and SARS-CoV-2 PCR test results, was collected from 1527 participants from 46 hospitals. Cross-sectional serology was investigated in February and March 2021. RESULTS: Until the end of September 2020, no cases were reported. From September 28th 2020 to March 2021 a total of 38 PCR-detected SARS-CoV-2 infections were reported. Of these, four children were admitted to hospital but none had acute severe COVID-19. Increasing age in association with immunodeficiency increased reporting of SARS-CoV-2 infection. Worsening of fever, cough, and sore throat were associated with participants reporting SARS-CoV-2 infection. Serology data included 452 unvaccinated participants. In those reporting prior positive SARS-CoV-2 PCR, there were detectable antibodies in 9 of 18 (50%). In those with no prior report of infection, antibodies were detected in 32 of 434 (7•4%). CONCLUSIONS: This study shows SARS-CoV-2 infections have occurred in immunocompromised children and young people with no increased risk of severe disease. No children died.

Patients with tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) are hypersensitive to Toll-like receptor 9 stimulation.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder characterized by recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNFRSF1A, which encodes tumour necrosis factor receptor 1 (TNF-R1). Our aim was to understand the influence of TRAPS mutations on the response to stimulation of the pattern recognition Toll-like receptor (TLR)-9. Peripheral blood mononuclear cells (PBMCs) and serum were isolated from TRAPS patients and healthy controls: serum levels of 15 proinflammatory cytokines were measured to assess the initial inflammatory status. Interleukin (IL)-1ß, IL-6, IL-8, IL-17, IL-22, tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), interferon (IFN)-γ, monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor (TGF)-ß were significantly elevated in TRAPS patients' sera, consistent with constitutive inflammation. Stimulation of PBMCs with TLR-9 ligand (ODN2006) triggered significantly greater up-regulation of proinflammatory signalling intermediates [TNF receptor-associated factor (TRAF 3), IL-1 receptor-associated kinase-like 2 (IRAK2), Toll interacting protein (TOLLIP), TRAF6, phosphorylated transforming growth factor-ß-activated kinase 1 (pTAK), transforming growth factor-ß-activated kinase-binding protein 2 (TAB2), phosphorylated TAK 2 (pTAB2), IFN-regulatory factor 7 (IRF7), receptor interacting protein (RIP), nuclear factor kappa B (NF-κB) p65, phosphorylated NF-κB p65 (pNF-κB p65) and mitogen-activated protein kinase kinase (MEK1/2)] in TRAPS patients' PBMCs. This up-regulation of proinflammatory signalling intermediates and raised serum cytokines occurred despite concurrent anakinra treatment and no overt clinical symptoms at time of sampling. These novel findings further demonstrate the wide-ranging nature of the dysregulation of innate immune responses underlying the pathology of TRAPS and highlights the need for novel pathway-specific therapeutic treatments for this disease.

Effects of pro-inflammatory cytokines on cannabinoid CB1 and CB2 receptors in immune cells.

AIMS: To investigate the regulation of cannabinoid receptors CB1 and CB2 on immune cells by pro-inflammatory cytokines and its potential relevance to the inflammatory neurological disease, multiple sclerosis (MS). CB1 and CB2 signalling may be anti-inflammatory and neuroprotective in neuroinflammatory diseases. Cannabinoids can suppress inflammatory cytokines but the effects of these cytokines on CB1 and CB2 expression and function are unknown. METHODS: Immune cells from peripheral blood were obtained from healthy volunteers and patients with MS. Expression of CB1 and CB2 mRNA in whole blood cells, peripheral blood mononuclear cells (PBMC) and T cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression of CB1 and CB2 protein was determined by flow cytometry. CB1 and CB2 signalling in PBMC was determined by Western blotting for Erk1/2. RESULTS: Pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α (the latter likely NF-κB dependently) can upregulate CB1 and CB2 on human whole blood and peripheral blood mononuclear cells (PBMC). We also demonstrate upregulation of CB1 and CB2 and increased IL-1ß, IL-6 and TNF-α mRNA in blood of patients with MS compared with controls. CONCLUSION: The levels of CB1 and CB2 can be upregulated by inflammatory cytokines, which can explain their increase in inflammatory conditions including MS.

Natural and disease-specific autoantibodies in chronic obstructive pulmonary disease.

Autoimmunity may contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). Studies have identified disease-specific autoantibodies (DSAAbs) in COPD patients, but natural autoantibodies (NAAbs) may also play a role. Previous studies have concentrated on circulating autoantibodies, but lung-associated autoantibodies may be most important. Our aim was to investigate NAAbs and DSAAbs in the circulation and lungs of COPD smoking (CS) patients compared to smokers (S) without airway obstruction and subjects who have never smoked (NS). Immunoglobulin (Ig)G antibodies that bind to lung tissue components were significantly lower in the circulation of CS patients than NS (with intermediate levels in S), as detected by enzyme-linked immunosorbent assay (ELISA). The levels of antibodies to collagen-1 (the major lung collagen) detected by ELISA were also reduced significantly in CS patients' sera compared to NS. The detection of these antibodies in NS subjects indicates that they are NAAbs. The occurrence of DSAAbs in some CS patients and S subjects was indicated by high levels of serum IgG antibodies to cytokeratin-18 and collagen-5; furthermore, antibodies to collagen-5 eluted from homogenized lung tissue exposed to low pH (0·1 M glycine, pH 2·8) were raised significantly in CS compared to S and NS. Thus, this study supports a role in COPD for both NAAbs and DSAAbs.

Noninvasive tissue oxygen saturation determined by near-infrared spectroscopy following peripheral nerve block.

BACKGROUND: Noninvasive physiologic measurement of cutaneous tissue oxygenation using near-infrared spectroscopy (NIRS) has become increasingly common in cardiovascular and plastic surgery. The aim of this study was to determine whether clinically available NIRS-based monitors could detect changes in tissue oxygen saturation (rSO(2)) following a variety of peripheral nerve blocks. We hypothesize that peripheral nerve blocks will produce detectable changes in cutaneous tissue oxygenation levels that can be measured by noninvasive NIRS-based oximetry. METHODS: Forty adult patients scheduled for pre-operative peripheral nerve block placement were enrolled. Prior to block placement, NIRS sensors were placed on the operative and nonoperative (control) limb. Baseline tissue oxygen saturation values were obtained prior to dosing of the nerve block, and measurements were recorded every 5 min thereafter. RESULTS: Initial rSO(2) values were higher in the operative vs. control limbs prior to nerve block placement. Tissue oxygen saturation increased in the blocked, but not control, limbs with time. Subgroup analysis suggested statistically significant differences in rSO(2) values in blocked vs. control limbs for cervical paravertebral, infraclavicular, and femoral nerve blocks. CONCLUSIONS: Our results demonstrated sustained increases in tissue rSO(2) values following peripheral nerve block placement, in addition to higher initial rSO(2) values in operative limbs prior to block placement. Further investigations are necessary to define the expected baseline rSO(2) values in operative and control limbs. Future efforts utilizing NIRS-based detection of tissue ischemia should consider the small but significant changes in rSO(2) resulting from a successful nerve block.

Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E.

Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>>>>>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.

Systemic cytokine levels and the effects of etanercept in TNF receptor-associated periodic syndrome (TRAPS) involving a C33Y mutation in TNFRSF1A.

OBJECTIVE: To investigate the levels of the pro-inflammatory cytokines IL-6, TNF-alpha, IL-1beta, IL-8, IL-10 and IL-12p70 in the plasma of patients with TNF receptor-associated periodic syndrome (TRAPS) in relation to CRP levels and treatment with etanercept. METHODS: Cytokine concentrations were measured in sequential plasma samples obtained from eight patients with a C33Y mutation in TNFRSF1A and diagnosed with TRAPS, using cytokine bead array. The TRAPS samples were compared with samples from normal controls and rheumatoid arthritis patients. RESULTS: Levels of IL-6 were significantly elevated in C33Y TRAPS patients and these correlated with CRP levels in some of the patients. IL-8 levels were also significantly elevated in the TRAPS patients. However, neither TNF-alpha nor IL-1beta demonstrated a similar increase. This differed from the patients with rheumatoid arthritis, for whom levels of IL-6, IL-8, TNF-alpha, IL-1beta and IL-10 were significantly elevated. The levels of detectable TNF-alpha in the TRAPS patients' plasma were elevated during etanercept treatment. CONCLUSIONS: The cytokine profile of C33Y TRAPS differs from that of a typical autoimmune inflammatory condition such as rheumatoid arthritis, as only IL-6 and IL-8 were elevated in C33Y TRAPS patients, as distinct from a generalized elevation of pro-inflammatory cytokines. However, only some of the C33Y patients tested showed a relationship between elevated IL-6 and CRP. This is consistent with clinical observations that there is marked heterogeneity between individuals with TRAPS, including those in the same family cohort. Although etanercept has a therapeutic effect in some TRAPS patients, it induces increased plasma concentrations of TNF-alpha, possibly by increasing TNF-alpha stability.

Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche.

BACKGROUND/AIMS: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. METHODS: Systematic serial 5-7 microm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the "stem cell" marker ABCG2 transporter protein. RESULTS: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. CONCLUSIONS: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.

Distinct antigenic features of linear epitopes at the N-terminus and C-terminus of 65 kDa glutamic acid decarboxylase (GAD65): implications for autoantigen modification during pathogenesis.

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N- and C-termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the alpha-helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

Inactivation of the Fas gene by Alu insertion: retrotransposition in an intron causing splicing variation and autoimmune lymphoproliferative syndrome.

Mutations in the Fas (apo-1, CD95) gene result in autoimmune lymphoproliferative syndrome (ALPS). These mutations are dominated by small deletions and point mutations that result in splicing errors or missense changes. We report here a novel mutation caused by retrotransposon insertion, which results in loss of exon 8 and ALPS. A father and son suffering from recurrent lymphadenopathy were examined for resistance to Fas-mediated apoptosis. A functional defect was detected and RT-PCR analysis revealed two different copies of Fas mRNA, one normal and a second shorter version lacking exon 8. DNA analysis of the genomic region between exons seven and nine in the longer copy revealed two PCR products, one being 331 base pairs (bp) longer than expected. Sequencing revealed that intron 7 had undergone an insertion event with an Alu element (99.31% homology with Alu-Sb1) of 331 bp. This element included a 34-bp Poly A tract that was flanked on each side by a perfect 17 bp direct duplication of the target site. Both patients were heterozygous for the mutated allele that produced Fas mRNA lacking exon 8, although not due to loss of a splice junction. The structure of the insertion suggests that the Alu element may have integrated by retrotransposition, and represents the first report of a retrotransposon causing ALPS.

S antigen specific effector T cell activation detected by cytokine flow cytometry.

BACKGROUND/AIMS: Effector T cell activation is particularly important in the initiation of autoimmune uveitis. This pilot study seeks to demonstrate activation of human peripheral effector T cells in response to the uveitis candidate autoantigen, retinal S antigen (SAg), using cytokine flow cytometry (CFC). METHODS: Peripheral blood mononuclear cell (PBMC) suspensions from uveitis patients and controls were stimulated with bovine SAg. Activation responses were detected by CFC. RESULTS: Electronic gating enabled analysis of CD69+, IFN-gamma+ CD4+ lymphocytes. An SAg specific response was detectable in four of 13 patients and four of eight controls. CONCLUSION: SAg specific, peripheral, effector T cell activation can be detected by CFC. Similar levels of responsiveness were seen in patient and control groups. More detailed cytokine profiling may demonstrate functional differences between the groups.

Differential expression of TGF-beta isoforms by normal and inflammatory bowel disease intestinal myofibroblasts.

Intestinal strictures are frequent in Crohn's disease but not ulcerative colitis. We investigated the expression of transforming growth factor (TGF)-beta isoforms by isolated and cultured primary human intestinal myofibroblasts and the responsiveness of these cells and intestinal epithelial cells to TGF-beta isoforms. Normal intestinal myofibroblasts released predominantly TGF-beta(3) and ulcerative colitis myofibroblasts expressed both TGF-beta(1) and TGF-beta(3), whereas in myofibroblast cultures from fibrotic Crohn's disease tissue, there was significantly lower expression of TGF-beta(3) but enhanced release of TGF-beta(2). These distinctive patterns of TGF-beta isoform release were sustained through several myofibroblast passages. Proliferation of Crohn's disease myofibroblasts was significantly greater than that of myofibroblasts derived from normal and ulcerative colitis tissue. In contrast to cells from normal and ulcerative colitis tissue, neutralization of the three TGF-beta isoforms did not affect the proliferation of Crohn's disease intestinal myofibroblasts. Studies on the effect of recombinant TGF-beta isoforms on epithelial restitution and proliferation suggest that TGF-beta(2) may be the least effective of the three isoforms in intestinal wound repair. In conclusion, the enhanced release of TGF-beta(2) but reduced expression of TGF-beta(3) by Crohn's disease intestinal myofibroblasts, together with their enhanced proliferative capacity, may lead to the development of intestinal strictures.

Genetic analysis as a valuable key to diagnosis and treatment of periodic Fever.

We describe 2 Dutch patients with recurrent fever attacks undiagnosed for more than 40 years. The diagnosis of periodic fever was made when molecular analysis revealed novel mutations in the tumor necrosis factor (TNF) receptor gene (TNFRSF1A), establishing the diagnosis of TNF receptor-associated periodic syndrome. This syndrome is an autosomal dominant disorder characterized by recurring episodes of fever, arthralgia, and skin lesions that is caused by mutations in the 55-kd TNFRSF1A gene. This finding has facilitated treatment for TNF receptor-associated periodic syndrome because blocking of TNF signaling seems to alleviate the symptoms. Use of a short course of recombinant p75TNFR:Fc fusion protein (etanercept) induced prolonged remission in one patient.

Retroviral activity in Behçet's syndrome and systemic lupus erythematosus detected by a PCR-based reverse transcriptase assay.

The objective of this study was to assess retroviral activity in Behçet's syndrome (BS) and systemic lupus erythematosus (SLE) patients. Serum and peripheral blood mononuclear cells (PBMC) were obtained from patients and normal volunteers and PBMC cultured with and without phytohaemagglutinin stimulation. Reverse transcriptase (RT) activity in serum and culture supernatants was measured using a sensitive polymerase chain reaction-based assay. An RT activity above the levels in normal controls was detected in a minority of patients with BS (2/15) and SLE (1/13) and was typically present in all three types of sample. Elevated levels of RT activity were not detected in follow-up samples from the two BS patients. Our findings indicate that elevated RT activity is present in only a minority of patients with BS and SLE. Simultaneously elevated activity in all three sample types implicates PBMC as the source of this retroviral activity.

The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors.

We report here the molecular characterisation of the Arabidopsis MALE STERILITY1 gene, which is a critical sporophytic controlling factor for anther and pollen development. Homozygous ms1 mutants do not produce viable pollen, but are otherwise phenotypically normal. Degeneration of pollen occurs soon after microspore release from the tetrads, at which time the tapetum also appears abnormally vacuolated. The MS1 gene is expressed at low levels in anthers from closed buds, with expression in the tapetum at the stage of microspore release. No expression is seen in open flowers. The deduced MS1 protein sequence shows strong homology to the PHD-finger motif found in known transcription factors from humans, yeast and higher plants. Six alleles of ms1 have been identified; all result in premature termination of the MS1 protein and loss of the PHD-finger motif. MS1 is likely to play a key role in regulating transcription during specific stages of male gametogenesis and anther development. As such, MS1 provides a valuable tool for the manipulation of male sterility in higher plants.

Quantitative analysis of IL-10 and IFN-gamma mRNA levels in normal cervix and human papillomavirus type 16 associated cervical precancer.

Human papillomavirus type 16 is a major factor in cervical carcinogenesis. Inappropriate cytokine synthesis may direct the local immune response away from a type-1 (cellular) pattern and may subsequently contribute to the development and progression of precancer. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) using a competitive mimic was carried out to determine type-1 (interferon gamma (IFN-gamma)) and type-2 (interleukin-10 (IL-10)) cytokine mRNA levels in whole cervical specimens (without microdissection) from seven normal and nine HPV-16 positive CIN formalin-fixed paraffin-embedded tissues. Microdissection was used to measure separately the epithelial and sub-epithelial levels of IFN-gamma and IL-10 mRNAs in 11 specimens of normal cervix and 25 HPV-16 positive CIN (nine CIN 1, seven CIN 2 and nine CIN 3). IFN-gamma mRNA was lower in CIN than normal (p=0.04). IL-10 mRNA level in CIN was significantly higher (p=0.005) than in normal cervix (before microdissection). Epithelial IFN-gamma mRNA showed a significant decrease in all grades of CIN (median=3.58) compared with normal (7.74) (p<0.05), but there was no significant difference between the grades. A significant decrease in sub-epithelial IFN-gamma mRNA was found in CIN 1(9.81), CIN 2 (3.82) and CIN 3 (4.62) compared with normal cervix (27.35) (p<0.05). Also, sub-epithelial IFN-gamma mRNA was significantly lower in CIN 2 and CIN 3 than in CIN 1 (p=0.005 and 0.0005, respectively). IL-10 was detected in the epithelium of only one of 11 normal and one of 25 CIN, but sub-epithelial IL-10 was significantly higher in CIN 2 (0.08) and CIN 3 (0.26) than in normal (0.00) (p=0.036 and 0.0032, respectively). There was no significant difference in the sub-epithelial level of IL-10 between normal and CIN 1 (0.00) (p=0.96). Our results suggest that reduced epithelial and sub-epithelial IFN-gamma, as well as increased sub-epithelial IL-10 synthesis may play a role in the development and progression of HPV-16 associated cervical precancer.

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection.

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV-infected patients and controls, focusing on the antigen-specific CD8(+) T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT-PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV-specific CD8(+) cells in the two compartments. T cell receptor usage in the liver of HCV-infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8(+) cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8(+) cells. A greater proportion of the liver tetramer-positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8(+) cells in the liver. In the course of chronic HCV infection, HCV-specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

Differential gene expression during meningeal-meningococcal interaction: evidence for self-defense and early release of cytokines and chemokines.

Using microarray technology, we studied the early differential expression of 3,528 genes in human meningothelial cells in response to meningococcal challenge. Thirty-two genes were up-regulated, and four were down-regulated. Those up-regulated included the tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8 (but not IL-1beta) genes, suggesting that meningeal cells may be a local and early source of these cytokines. Also, a trend in up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes was observed. This is the first evidence that meningothelial cells may mount cytoprotective responses to pathogenic bacteria.

Intravitreal invading cells contribute to vitreal cytokine milieu in proliferative vitreoretinopathy.

AIM: To examine the contribution of infiltrating cells in the local production of cytokines within the vitreous of patients with proliferative vitreoretinopathy (PVR). METHODS: The presence of mRNA coding for IL-6, IL-8, IL-1beta, IL-1alpha, TNFalpha, IFNgamma, IL-12, and HPRT was investigated in 25 vitreous samples from patients with PVR, 11 vitreous samples from patients with retinal detachment (RD) not complicated by PVR, and 10 vitreous samples from patients with macular hole (MH). A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using an internal competitor was used to investigate these samples. From these samples, 15 PVR, 8 RD, and 8 MH were analysed for the protein levels of the same cytokines using enzyme linked immunosorbent assay (ELISA). Spearman correlation was used to test any association between mRNA and cytokine protein levels, as an indicator of the contribution these cells make to the intravitreal cytokine milieu. RESULTS: A strong correlation was found between mRNA and their respective cytokine levels (protein products) for IL-6, IL-8, IL-1beta, IL-1alpha, TNFalpha, IFNgamma (Spearman r = 0.83, 0.73, 0.67, 0.91, 0.73, and 0.73 respectively), but not for IL-12. The median levels of IL-6, IL-8, IL-1beta, and IFNgamma mRNA and their respective cytokines were significantly higher (p <0.05) in patients with PVR than in those with macular hole. There was no statistically significant difference in the median levels of IL-1alpha mRNA between PVR and MH but the cytokine IL-1alpha was detected at a significantly higher level in PVR compared with MH patients. Between PVR and RD patients, there was no statistically significant difference in mRNA levels for all the investigated cytokines (p >0.05) except for IL-6 where there was a statistical significance (p= 0.038). In contrast, the median levels of IL-6, IL-8, and IL-1beta cytokines were significantly higher (p <0.05) in patients with PVR than in those with RD, whereas for IL-1alpha and IFNgamma no significant statistical difference was detected between PVR and RD patients (p >0.05). When results of RD and MH patients were compared, a statistical difference was only detected in mRNA levels of INFgamma (p = 0.008). However, no difference was detected for INFgamma (protein product) or for any of the other cytokines between RD and MH patients. CONCLUSION: Levels of both protein and mRNA encoding IL-6, IL-8, IL-1beta, and IFNgamma is significantly increased in vitreous samples from patients with PVR. The strong correlation between ELISA detectable cytokines (protein products) and their respective mRNA levels suggest that intravitreal, invasive cells are the major source of these cytokines, with the exception of IL-12. Cells invading the vitreous do not appear to locally produce IL-12 mRNA. This would appear to implicate cells peripheral to the vitreal mass as the major source of this cytokine.