The longitudinal loss of islet autoantibody responses from diagnosis of type 1 diabetes occurs progressively over follow-up and is determined by low autoantibody titres, early-onset, and genetic variants.

The clinical usefulness of post-diagnosis islet autoantibody levels is unclear and factors that drive autoantibody persistence are poorly defined in type 1 diabetes (T1D). Our aim was to characterise the longitudinal loss of islet autoantibody responses after diagnosis in a large, prospectively sampled UK cohort. Participants with T1D [n = 577] providing a diagnosis sample [range -1.0 to 2.0 years] and at least one post-diagnosis sample (<32.0 years) were tested for autoantibodies to glutamate decarboxylase 65 (GADA), islet antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A). Select HLA and non-HLA SNPs were considered. Non-genetic and genetic factors were assessed by multivariable logistic regression models for autoantibody positivity at initial sampling and autoantibody loss at final sampling. For GADA, IA-2A, and ZnT8A, 70.8%, 76.8%, and 40.1%, respectively, remained positive at the final sampling. Non-genetic predictors of autoantibody loss were low baseline autoantibody titres (P < 0.0001), longer diabetes duration (P < 0.0001), and age-at-onset under 8 years (P < 0.01--0.05). Adjusting for non-genetic covariates, GADA loss was associated with low-risk HLA class II genotypes (P = 0.005), and SNPs associated with autoimmunity RELA/11q13 (P = 0.017), LPP/3q28 (P = 0.004), and negatively with IFIH1/2q24 (P = 0.018). IA-2A loss was not associated with genetic factors independent of other covariates, while ZnT8A loss was associated with the presence of HLA A*24 (P = 0.019) and weakly negatively with RELA/11q13 (P = 0.049). The largest longitudinal study of islet autoantibody responses from diagnosis of T1D shows that autoantibody loss is heterogeneous and influenced by low titres at onset, longer duration, earlier age-at-onset, and genetic variants. These data may inform clinical trials where post-diagnosis participants are recruited.

Islet autoantibody profiles associated with higher diabetes risk in Lithuanian compared with English schoolchildren.

During a 15-year period, the incidence of type 1 diabetes has doubled in Lithuania, while increasing by a third in England; however, England still has a higher incidence. Analysis of sera collected from non-diabetic schoolchildren from Lithuania and England more than 20 years ago showed a similar number of multiple autoantibody-positive schoolchildren between the populations, but a higher prevalence of islet antigen-2 autoantibodies (IA-2A) in English schoolchildren. We aimed to use recently developed, more specific islet autoantibody tests to characterize differences in humoral autoimmunity between these two general population cohorts in greater detail. Samples from 88 Lithuanian and 133 English schoolchildren previously found islet autoantibody-positive were selected for measurement of additional islet autoantibodies by radioimmunoassay. Samples were tested for autoantibodies to zinc transporter 8 (ZnT8A), GAD (96-585), the protein tyrosine phosphatase region of islet antigen-2 (PTPA) and the related IA-2ßA, while autoantibodies to IA-2A were reassayed using the current harmonized method. IA-2-related autoantibodies PTPA (0·13 versus 0·45%, P = 0·027) and IA-2ßA (0 versus 0·35%, P < 0·001), but not IA-2A measured using the harmonized method, were less common in Lithuanian compared to English schoolchildren. Lithuanian schoolchildren who were islet autoantibody-positive were positive for fewer biochemical autoantibodies compared with English schoolchildren (P = 0·043). Background rates of islet autoimmunity in childhood differ subtly between countries, which have different incidences of type 1 diabetes. The optimal screening strategy (age and combination of markers) for detection of islet autoimmunity may vary between countries, dependent upon the pattern of autoantibodies found in the general population.

A quarter of patients with type 1 diabetes have co-existing non-islet autoimmunity: the findings of a UK population-based family study.

Individuals with type 1 diabetes (T1D) are at increased risk of coeliac disease (CD), autoimmune thyroiditis and autoimmune gastritis, but the absolute risks are unclear. The aim of this study was to investigate the prevalence of autoantibodies to tissue transglutaminase (TGA), thyroid peroxidase (TPOA) and gastric H+ /K+ -ATPase (ATPA) and their genetic associations in a well-characterized population-based cohort of individuals with T1D from the Bart's-Oxford family study for whom islet autoantibody prevalence data were already available. Autoantibodies in sera from 1072 patients (males/females 604/468; median age 11·8 years, median T1D duration 2·7 months) were measured by radioimmunoassays; HLA class II risk genotype was analysed in 973 (91%) using polymerase chain reaction with sequence specific primers (PCR-SSP). The prevalence of TGA (and/or history of CD), TPOA and ATPA in patients was 9·0, 9·6 and 8·2%, respectively; 3·1% had two or more autoantibodies. Females were at higher risk of multiple autoimmunity; TGA/CD were associated with younger age and TPOA with older age. ATPA were uncommon in patients under 5 years, and more common in older patients. Anti-glutamate decarboxylase autoantibodies were predictive of co-existing TPOA/ATPA. TGA/CD were associated with human leucocyte antigen (HLA) DR3-DQ2, with the DR3-DQ2/DR3-DQ2 genotype conferring the highest risk, followed by DR4-DQ8/DR4-DQ8. ATPA were associated with DR3-DQ2, DRB1*0404 (in males) and the DR3-DQ2/DR4-DQ8 genotype. TPOA were associated with the DR3-DQ2/DR3-DQ2 genotype. Almost one-quarter of patients diagnosed with T1D aged under 21 years have at least one other organ-specific autoantibody. HLA class II genetic profiling may be useful in identifying those at risk of multiple autoimmunity.

Validation of a frailty index in older cancer patients with solid tumours.

BACKGROUND: Frailty is an indicator of physiological reserve in older people. In non-cancer settings, frailty indices are reliable predictors of adverse health outcomes. The aims of this study were to 1) derive and validate a frailty index (FI) from comprehensive geriatric assessment (CGA) data obtained in the solid tumour chemotherapy setting, and 2) to explore whether the FI-CGA could predict chemotherapy decisions and survival in older cancer patients with solid tumours. METHODS: Prospective cohort study of a consecutive series sample of 175 cancer patients aged 65 and older with solid tumours. A frailty index was calculated using an accumulated deficits model, coding items from the comprehensive geriatric assessment tool administered prior to chemotherapy decision-making. The domains of physical and cognitive functioning, nutrition, mood, basic and instrumental activities of daily living, and comorbidities were incorporated as deficits into the model. RESULTS: The FI-CGA had a right-skewed distribution, with median (interquartile range) of 0.27 (0.21-0.39). The 99% limit to deficit accumulation was below the theoretical maximum of 1.0, at 0.75. The FI-CGA was significantly related (p < 0.001) to vulnerability as assessed by the Vulnerable Elders Survey-13 and to medical oncologists' assessments of fitness or vulnerability to treatment. Baseline frailty as determined by the FI-CGA was also associated with treatment decisions (Treatment Terminated, Treatment Completed, No Planned Treatment) (p < 0.001), with the No Planned Treatment group significantly frailer than the other two groups. CONCLUSION: The FI-CGA is a potentially useful adjunct to cancer clinical decision-making that could predict chemotherapy outcomes in older patients with solid tumours.

The first 142 amino acids of glutamate decarboxylase do not contribute to epitopes recognized by autoantibodies associated with Type 1 diabetes.

AIMS: Glutamate decarboxylase (GAD) antibodies are the most widely used predictive marker for Type 1 diabetes, but many individuals currently found to be GAD antibody-positive are unlikely to develop diabetes. We have shown previously that radioimmunoassays using N-terminally truncated 35 S-GAD65 (96-585) offer better disease specificity with similar sensitivity to full-length 35 S-GAD65 (1-585). To determine whether assay performance could be improved further, we evaluated a more radically truncated 35 S-GAD65 (143-585) radiolabel. METHODS: Samples from people with recent-onset Type 1 diabetes (n = 157) and their first-degree relatives (n = 745) from the Bart's-Oxford family study of childhood diabetes were measured for GAD antibodies using 35 S-labelled GAD65 (143-585). These were screened previously using a local radioimmunoassay with 35 S-GAD65 (1-585). A subset was also tested by enzyme-linked immunosorbent assay (ELISA), which performs well in international workshops, but requires 10 times more serum. Results were compared with GAD antibody measurements using 35 S-GAD65 (1-585) and 35 S-GAD65 (96-585). RESULTS: Sensitivity of GAD antibody measurement was maintained using 35 S-GAD65 (143-585) compared with 35 S-GAD65 (1-585) and 35 S-GAD65 (96-585). Specificity for Type 1 diabetes was improved compared with 35 S-GAD65 (1-585), but was similar to 35 S-GAD65 (96-585). Relatives found to be GAD antibody-positive using these truncated labels were at increased risk of diabetes progression within 15 years, compared with those positive for GAD(1-585) antibody only, and at similar risk to those found GAD antibody-positive by ELISA. CONCLUSIONS: The first 142 amino acids of GAD65 do not contribute to epitopes recognized by Type 1 diabetes-associated GAD antibodies. Low-volume radioimmunoassays using N-terminally truncated 35 S-GAD65 are more specific than those using full-length GAD65 and offer practical alternatives to the GAD antibody ELISA for identifying children at increased risk of Type 1 diabetes.

An increased frequency of NK cell receptor and HLA-C group 1 combinations in early-onset type 1 diabetes.

AIMS/HYPOTHESIS: Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptor (KIR) genes and their HLA class I ligands. Alterations in NK cell activity have been associated with type 1 diabetes. The aim of this study was to determine whether KIR-HLA class I gene frequency: (1) is altered in a current population with type 1 diabetes compared with healthy controls; and (2) has changed over the half century in which the incidence of type 1 diabetes has increased rapidly. METHODS: KIR-HLA class I gene frequencies were compared in 551 individuals diagnosed with type 1 diabetes ≤ 15 years of age (394 in a current cohort and 157 from the historical 'Golden Years' cohort) and 168 healthy controls. The overall balance of activation and inhibition was analysed using KIR-HLA genotype models. RESULTS: Children with type 1 diabetes who were positive for KIR2DS2/KIR2DL2 and KIR2DL3 were more often homozygous for HLA-C group 1 and this effect was strongest in children diagnosed with diabetes before the age of 5 years (p = 0.003, corrected p [p (corr)] = 0.012) and (p = 0.001, p (corr) = 0.004), respectively. Children with type 1 diabetes have fewer inhibitory KIRs with their corresponding ligands compared with healthy controls (p = 1.9 × 10(-4)). This pattern of NK activation has not changed significantly in individuals with type 1 diabetes over the last half century. CONCLUSIONS/INTERPRETATION: Activating combinations of KIR-HLA genes are more frequent in young children with type 1 diabetes diagnosed in the first 5 years of life, suggesting that NK cell responses may be altered in this group.

Progression to diabetes in relatives with islet autoantibodies. Is it inevitable?

OBJECTIVE: A large cohort of family members with islet cell antibodies (ICA) > or = 20 Juvenile Diabetes Foundation units (JDF U) was examined to determine whether there was a subgroup at low risk of progression to diabetes; whether risk of progression changed over time; and whether rate of progression to diabetes varied according to age, islet autoantibodies, and genetic markers of susceptibility. RESEARCH DESIGN AND METHODS: Individuals with ICA > or = 20 JDF U were identified from 4,423 family members recruited to prospective family studies in the U.K. Subjects were followed for up to 18 years. Antibodies to insulin, GAD, and IA-2 were measured in the first sample, and HLA class II typing was performed. RESULTS: Of 147 family members with ICA > or = 20 JDF U on at least one occasion, 29 developed type 1 diabetes after a median of 3.2 years (maximum 18.1). The cumulative risk of developing diabetes within 15 years was 47% (95% CI 28-67) for all family members with ICA > or = 20 JDF U, 2.8% (0-8.2) for those with ICA alone, and 66% (44-87) for those with at least one additional autoantibody marker. There were no differences in age, HLA class II type, or levels of ICA, insulin autoantibodies, or IA-2 antibodies between those who developed diabetes within 5 years of testing and those who developed diabetes after this time. GAD antibody levels we ..., however, higher in those who progressed more slowly. CONCLUSIONS: Family members with ICA alone are at low risk of progression to diabetes. Rapid development of disease after ICA detection could not be distinguished from delayed development on the basis of autoantibodies or markers of genetic susceptibility, and those with multiple antibodies remained at high risk throughout long-term follow-up. This suggests that all family members with multiple islet autoantibodies are destined to develop autoimmune diabetes.

Bidentate carbenoid ester coordination in ruthenium(II) Schiff-base complexes leading to excellent levels of diastereo- and enantioselectivity in catalytic alkene cyclopropanation.

Exceptionally high stereoselectivity (ee < or = 98%, dr < or = 99:1) in the cyclopropanation of alkenes with ethyl diazoacetate using a non-planar ruthenium(II) Schiff-base precatalyst is a result of eta 2C,O binding of the carbenoid ester intermediate, according to DFT calculations.

Development of radiographic and microscopic techniques for the characterization of bacterial transport in intact sediment cores from Oyster, Virginia.

The objective of this study was to ascertain the physical and mineralogical properties responsible for the retention of bacteria in subsurface sediments. The sediment core chosen for this study was a fine-grained, quartz-rich sand with minor amounts of Fe and Al hydroxides. A bacterial transport experiment was performed using an intact core collected from a recent excavation of the Butler's Bluff member of the Nassawadox formation in the borrow pit at Oyster, VA. and a 14C-labeled bacterial strain OYS2-A was selected for its relatively low adhesion. After the bacterial breakthrough was observed in the effluent, the intact core was dissected to determine the internal distribution of the injected bacteria retained in the sediment. The sediment was dried, epoxy fixed, and thin sectioned. The distribution of 14C activity in the thin sections was mapped using a phosphor screen and X-ray film. The remainder of the core was subsampled and the 14C activity of the subsamples was determined by liquid scintillation counting. The phosphor imaging technique was capable of directly imaging the distribution of radiolabeled bacteria in thin sections, because of its high sensitivity and linear response over a large activity range. The phosphor imaging signal intensity was utilized as a measure of bacterial concentration. The distribution of bacteria at the millimeter scale in the thin sections was compared to the grain size, porosity, and mineralogy as measured by scanning electron microscopy (SEM) and energy dispersive spectrum (EDS) analyses. No apparent correlation was observed between the retention or collision efficiency of bacteria in the sediment and the amount of Fe and Al hydroxides. This apparent lack of correlation can be qualitatively explained by combination of several factors including a nearly neutral surface charge of the bacterial strain, and texture of the Fe and Al hydroxides in the sediment. The combination of phosphor imaging with SEM-EDS proved to be a robust method for relating the physical and mineralogical microscopic properties of poorly indurated sediment to the distribution of adsorbed bacteria, allowing bacterial retention mechanisms to be unambiguously unraveled.

Autoantibodies to islet antigen-2 are associated with HLA-DRB1*07 and DRB1*09 haplotypes as well as DRB1*04 at onset of type 1 diabetes: the possible role of HLA-DQA in autoimmunity to IA-2.

AIMS/HYPOTHESIS: To further our understanding of antigen presentation by HLA class II molecules, we have examined the influence of HLA class II genotype on expression of autoantibodies to islet antigen-2 (IA-2A). METHODS: HLA class II genotype and IA-2A were determined within 3 months of diagnosis in 618 patients with type 1 diabetes (median age 11 years [range 0.7-20.9]). Antibodies to the juxtamembrane region of IA-2 were measured by a radiobinding assay in 481 of 484 IA-2A-positive patients. RESULTS: IA-2A prevalence was highest in patients carrying at least one HLA-DRB1*04-DQA1*0301 (385 of 450; 86%), DRB1*07-DQA1*(0201 or 0301) (58 of 64; 91%) or DRB1*09-DQA1*0301 haplotype (18 of 19; 95%). Multiple regression showed that IA-2A were strongly associated with the number of these haplotypes carried; only 69 of 132 (52%) patients carrying none of these haplotypes had IA-2A, compared with 322 of 391 (82%) patients with one and 93 of 95 (98%) with two of these haplotypes (p < 0.001). IA-2 juxtamembrane antibodies were less frequent in IA-2A-positive patients with one (35%) or two (36%) DRB1*03-DQB1*02 or DRB1*07-DQB1*02 haplotypes than in those negative for these haplotypes (52%) (p = 0.002), but showed an independent positive association with IA-2A level (p < 0.001). CONCLUSIONS/INTERPRETATION: HLA class II alleles strongly influence the prevalence of IA-2A. The high IA-2A prevalence in patients carrying DRB1*04, DRB1*07 and DRB1*09 alleles in linkage disequilibrium with DQA1*0301 or the closely related DQA1*0201 suggests the humoral response to IA-2 may be driven by HLA-DQA1 genes.

An association analysis of the HLA gene region in latent autoimmune diabetes in adults.

AIMS/HYPOTHESIS: Pathophysiological similarities between latent autoimmune diabetes in adults (LADA) and type 1 diabetes indicate an overlap in genetic susceptibility. HLA-DRB1 and HLA-DQB1 are major susceptibility genes for type 1 diabetes but studies of these genes in LADA have been limited. Our aim was to define patterns of HLA-encoded susceptibility/protection in a large, well characterised LADA cohort, and to establish association with disease and age at diagnosis. MATERIALS AND METHODS: Patients with LADA (n = 387, including 211 patients from the UK Prospective Diabetes Study) and non-diabetic control subjects (n = 327) were of British/Irish European origin. The HLA-DRB1 and -DQB1 genes were genotyped by sequence-specific PCR. RESULTS: As in type 1 diabetes mellitus, DRB1 0301_DQB1 0201 (odds ratio [OR] = 3.08, 95% CI 2.32-4.12, p = 1.2 x 10(-16)) and DRB1 0401_DQB1 0302 (OR = 2.57, 95% CI 1.80-3.73, p = 4.5 x 10(-8)) were the main susceptibility haplotypes in LADA, and DRB1 1501_DQB1 0602 was protective (OR = 0.21, 95% CI 0.13-0.34, p = 4.2 x 10(-13)). Differential susceptibility was conferred by DR4 subtypes: DRB1 0401 was predisposing (OR = 1.79, 95% CI 1.35-2.38, p = 2.7 x 10(-5)) whereas DRB1 0403 was protective (OR = 0.37, 95% CI 0.13-0.97, p = 0.033). The highest-risk genotypes were DRB1 0301/DRB1 0401 and DQB1 0201/DQB1 0302 (OR = 5.14, 95% CI 2.68-10.69, p = 1.3 x 10(-8); and OR = 6.88, 95% CI 3.54-14.68, p = 1.2 x 10(-11), respectively). These genotypes and those containing DRB1 0401 and DQB1 0302 associated with a younger age at diagnosis in LADA, whereas genotypes containing DRB1 1501 and DQB1 0602 associated with an older age at diagnosis. CONCLUSIONS/INTERPRETATION: Patterns of susceptibility at the HLA-DRB1 and HLA-DQB1 loci in LADA are similar to those reported for type 1 diabetes, supporting the hypothesis that autoimmune diabetes occurring in adults is an age-related extension of the pathophysiological process presenting as childhood-onset type 1 diabetes.

Relative predispositional effects of HLA class II DRB1-DQB1 haplotypes and genotypes on type 1 diabetes: a meta-analysis.

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

Cloning of a partial cDNA for rat interleukin-12 (IL-12) and analysis of IL-12 expression in vivo.

Experimental models of autoimmunity in the rat may feature selective activation of either the Th1 or Th2 subset of helper T cells. Interleukin-12 (IL-12) is a key cytokine in the development of Th1 responses. In order to study IL-12 in the rat we used polymerase chain reaction (PCR) primers based on murine IL-12 to amplify a partial cDNA from rat tissue. The product was cloned and sequenced: it shows 94% nucleotide identity with the murine gene and 94% identity of predicted amino acid sequence. Primers based on the rat IL-12 sequence were used to analyse IL-12 expression in vivo using semi-quantitative PCR. We studied RNA from lymphoid tissues of two rat strains which differ in their response to mercuric chloride (HgCl2): Brown Norway (BN) rats develop autoimmunity with a predominant Th2 response; Lewis rats are resistant. Interleukin-12 expression was higher in Lewis than BN, and higher in spleen than lymph node. After HgCl2, IL-12 expression increased in BN towards the time when the autoimmune response autoregulates. Variation in baseline levels of IL-12 expression may account for the Th2 predisposition of BN rats compared to Lewis rats; IL-12 may play a role in the autoregulation of the Th2 response induced by HgCl2.

Diabetes and gender.

It is often assumed that there is little or no sex bias within either Type I (insulin-dependent) or Type II (non-insulin-dependent) diabetes mellitus. This review considers evidence that sex effects of interest and importance are present in both forms of the disease. Type I diabetes is the only major organ-specific autoimmune disorder not to show a strong female bias. The overall sex ratio is roughly equal in children diagnosed under the age of 15 but while populations with the highest incidence all show male excess, the lowest risk populations studied, mostly of non-European origin, characteristically show a female bias. In contrast, male excess is a consistent finding in populations of European origin aged 15-40 years, with an approximate 3:2 male:female ratio. This ratio has remained constant in young adults over two or three generations in some populations. Further, fathers with Type I diabetes are more likely than affected mothers to transmit the condition to their offspring. Women of childbearing age are therefore less likely to develop Type I diabetes, and--should this occur--are less likely to transmit it to their offspring. Type II diabetes showed a pronounced female excess in the first half of the last century but is now equally prevalent among men and women in most populations, with some evidence of male preponderance in early middle age. Men seem more susceptible than women to the consequences of indolence and obesity, possibly due to differences in insulin sensitivity and regional fat deposition. Women are, however, more likely to transmit Type II diabetes to their offspring. Understanding these experiments of nature might suggest ways of influencing the early course of both forms of the disease.

Levamisole induces interleukin-18 and shifts type 1/type 2 cytokine balance.

Immune responses can be classified, according to the predominant cytokines involved, into type 1 (featuring interferon-gamma, IFN-gamma) and type 2 (featuring interleukin-4, IL-4); imbalance between type 1 and type 2 cytokine compartments has been implicated in many human diseases. Levamisole is a drug with an unknown mode of action that has been used to boost immunity in infectious diseases including leprosy, and in some cancers. To test the hypothesis that levamisole acts by inducing a shift to a type 1 immune response, we used Brown Norway (BN) rats, which are markedly biased to type 2 responses. BN rats treated with levamisole showed a dose-dependent rise in serum IFN-gamma and fall in serum immunoglobulin E (IgE) level. Detailed analysis of cytokine gene expression showed upregulation of IFN-gamma and downregulation of IL-4 messenger RNA. This coincided with marked upregulation of IL-18, a recently characterized cytokine with potent activity in stimulating IFN-gamma production. IL-12 was not induced. Further, the type 2 response induced in BN rats by mercuric chloride was markedly attenuated when rats were pretreated with levamisole: there was a 2-log reduction in maximum serum IgE level and marked attenuation of IL-4 gene upregulation. These data indicate that levamisole acts by resetting the immune balance towards a type 1 response via induction of IL-18. Our findings provide a direction for development of more specific immunomodulating therapy.

Oxpentifylline inhibits tumor necrosis factor-alpha mRNA transcription and protects against arthritis in mercuric chloride-treated brown Norway rats.

The phosphodiesterase inhibitor oxpentifylline (OXP) has a number of potentially important immunomodulatory actions which include a selective inhibition of the Th1 subset of CD4+ cells in vitro and inhibition of tumor necrosis factor (TNF)-alpha mRNA transcription. In vivo, it has a dramatic protective effect against experimental allergic encephalomyelitis. In this animal model, tissue injury is associated with both a Th1 response and with TNF-alpha production, either of which could be targets for the protective action of OXP. In an attempt to clarify the relative importance of the Th cell subsets and TNF-alpha in pathogenesis, we investigated the effect of OXP on a Th2 model of T cell-dependent disease, mercuric chloride (HgCl2)-induced autoimmunity in the Brown Norway rat. The effects of OXP on the Th1:Th2 response, TNF-alpha mRNA transcription in spleen and ankle joints, and on the incidence and severity of arthritis and cecal vasculitis have been examined and the effects in vivo have been compared with those of a soluble TNF receptor-IgG1 fusion protein (sTNFR) that neutralizes rat TNF-alpha. In two separate experiments, OXP significantly enhanced unstimulated levels of splenic interleukin-4 (IL-4) mRNA (median 62%, of an artificial IL-4 mRNA construct, vs. 36.5% in controls) and in one experiment, exaggerated the total IgE response to HgCl2. OXP inhibited HgCl2-induced TNF-alpha mRNA transcription in spleen and ankle joints. In three separate experiments, OXP had a significant protective effect against arthritis, with the mean incidence reduced from 100% to 30% and mean peak score reduced from 7.2 to 2.59 (experiments 1 and 2). The protection against arthritis was indistinguishable from that produced by sTNFR. There was no such protection against cecal vasculitis with either OXP or sTNFR. These results demonstrate that OXP induces a shift towards a Th2 response, inhibits TNF-alpha mRNA transcription locally in joint and systemically in spleen, and has a protective effect against arthritis similar to that produced by sTNFR in the HgCl2-treated BN rat. We conclude that TNF-alpha is a critical cytokine in the pathogenesis of arthritis but not cecal vasculitis in this model, and that inhibition of TNF-alpha transcription is the most important mode of action of OXP in this situation. OXP may be a potential therapeutic agent in the treatment of other arthritides, such as human rheumatoid arthritis, in which TNF-alpha has been implicated in pathogenesis.

Low-dose mercuric chloride induces resistance in brown norway rats to further mercuric chloride by up-regulation of interferon-gamma.

Mercuric chloride induces autoimmunity in Brown Norway rats with polyclonal B-cell activation, hyper-IgE and multiple autoantibodies. Pre-treatment with low-dose HgCl2 (one-tenth of the standard dose) induces resistance to later full-dose HgCl2; we have studied the mechanism of this resistance. Brown Norway rats given low-dose HgCl2 showed only a modest increase in serum IgE level, three logs lower than rats given standard-dose HgCl2, and no up-regulation of splenic interleukin (IL)-4 mRNA. There was up-regulation of splenic interferon (IFN)-gamma gene expression and a progressive rise in serum IFN-gamma. Neither IL-12 nor IL-18 were induced, but there was up-regulation of IL-12 receptor beta2-chain (IL-12Rbeta2) expression. IL-10 and transforming growth factor (TGF)-beta expression did not change. Serum IgE and splenic IL-4 mRNA expression remained static when these rats were rechallenged, confirming resistance. Thereafter IFN-gamma expression gradually fell, after which IL-4 expression and serum IgE rose slightly. Our observations suggest that low-dose HgCl2 confers protection in Brown Norway rats to further HgCl2 by up-regulation of IFN-gamma, associated with enhanced IL-12Rbeta2 expression. The immunological response to HgCl2 in susceptible rat strains is more complex than previously appreciated and is dose dependent, with low doses inducing a T helper '(Th)1' type of response in contrast to the 'Th2' type response associated with standard doses.

Effects of type 2 cytokines on glomerular epithelial cells.

Visceral glomerular epithelial cells (GECs) are involved in the maintenance of the filtration barrier and may play a role in immune responses. Cytokines may act on GECs and we wished to test this in vitro. Vascular endothelial growth factor (VEGF) is a specific product of the GEC that may play a role in glomerular permeability. We have investigated whether GECs in culture express receptors for interleukin (IL)-4, 10 and 13 (often grouped together as type 2 cytokines) and whether these cytokines alter GEC VEGF production. Type 2 cytokines were compared to transforming growth factor-beta (TGF-beta) and IL-1beta which are known to upregulate VEGF production. GECs were grown from human nephrectomy specimens and cultured with and without the addition of exogenous cytokines. Messenger RNA data demonstrated the presence of IL-4 receptor alpha, IL-10 receptor 1 and 2, and IL-13 receptors alpha1 and alpha2. However, at the protein level by flow cytometry, only IL-13 alpha2 could be consistently demonstrated. IL-4, IL-10 and IL-13 inhibited production of VEGF but did not affect the pattern of isoform expression. In contrast, TBF-beta and IL-1beta caused an increase in VEGF production. These effects were not explained by effects on proliferation. Our data provide evidence that GECs express receptors for type 2 cytokines and that these cytokines can act directly on GECs, to decrease VEGF production.

Soluble L-selectin levels in type I diabetes mellitus: a surrogate marker for disease activity?

L-selectin (CD62L) is a cell adhesion molecule which plays a key role in the initiation of leucocyte migration from blood vessels to sites of local inflammation. The aim of this study was to investigate T-lymphocyte expression of CD62L antigen and serum levels of soluble L-selectin (sL-selectin) in subjects with clinical and preclinical type I diabetes to determine whether they could provide surrogate markers for disease activity. CD62L selectin expression on memory T lymphocytes was studied by cytometric analysis in 22 patients with newly diagnosed type I diabetes, 20 first-degree relatives of patients with type I diabetes, 14 patients with Graves' disease, and 22 healthy controls. sL-selectin levels were measured by enzyme-linked immunosorbent assay (ELISA) in enlarged groups of subjects in these categories, as well as in patients with long-standing type I diabetes, treated Graves' disease and type II (non-insulin dependent) diabetes. L-selectin levels were also related to islet autoantibodies, human leucocyte antigen (HLA) genotype and L-selectin T668C gene polymorphisms. L-selectin expression on memory T lymphocytes was reduced in newly diagnosed diabetes and islet autoantibody positive siblings compared with controls. sL-selectin levels were significantly raised in newly diagnosed type I diabetes compared with controls, with intermediate levels in family members, both with and without islet autoantibodies, and in long-standing type I diabetes. Levels were also raised in patients with untreated Graves' disease. Patients with type II diabetes had sL-selectin levels which did not differ from controls. sL-selectin levels correlated with the presence of diabetes-associated HLA alleles in both family members and controls; levels also fell with increasing age in family members. Multiple regression analysis showed that HLA genotype and age were independent determinants of sL-selectin levels. sL-selectin levels are raised at the time of diagnosis of type I diabetes and Graves' disease and appear to be modulated by disease activity, but levels are determined predominantly by HLA-associated genetic susceptibility and age. sL-selectin may provide a late marker of autoimmune destruction of islets and sequential measurement may be useful in monitoring disease activity and the effect of interventions preceding type I diabetes.

Role of beta1 and beta2 subunits of the interleukin-12 receptor in determining T helper 1/T helper 2 responses in vivo in the rat.

Interleukin-12 (IL-12) responsiveness, and hence capacity to mount a T helper type 1(Th1) immune response, may be regulated via differential expression of the IL-12 receptor beta2 subunit at least in vitro in human and murine cells. To test whether a similar phenomenon operates in vivo in the rat we cloned and sequenced partial cDNAs for rat IL-12Rbeta1 and IL-12Rbeta2 subunits and analysed expression of these genes in vivo in two rat strains with different Th1/Th2 bias. After treatment with mercuric chloride (HgCl2), Brown-Norway rats develop Th2-biased autoimmunity whereas Lewis rats do not develop autoimmunity, instead becoming resistant to Th1-biased diseases to which they are normally susceptible. We report close sequence homology between the segments of the rat IL-12R genes sequenced and corresponding mouse genes (95.6% and 92% for IL-12Rbeta1 and IL-12Rbeta2, respectively). Both Brown-Norway and Lewis rats express both beta1 and beta2 subunits of IL-12 receptor in vivo in spleen; Brown-Norway rats express the beta2 subunit at a lower level than Lewis rats. After HgCl2 treatment, IL-12Rbeta1 expression was not altered but there was down-regulation of IL-12Rbeta2 expression in both strains. We conclude that relative under-expression of IL-12Rbeta2 by Brown-Norway rats contributes to their Th2 bias, and that down-regulation of IL-12Rbeta2 after HgCl2 administration in Lewis rats underlies subsequent resistance to induction of Th1-biased diseases.