Neutralizing antibodies reduce MxA protein induction in interferon-beta-1a-treated MS patients.

BACKGROUND: Neutralizing antibodies (NAb) during interferon-beta (IFNbeta) treatment of MS are associated with reduced clinical and MR efficacy. NAb inhibit the IFN- inducible MxA gene expression and neutralize the capability of IFNbeta to inhibit virus growth in vitro. Presently, there is no clear concept of the biologic importance of IFNbeta antibodies; most of the tests applied for the detection of NAb in previous publications are not widely available, and the results are not fully comparable. METHODS: A 1-year prospective study of the development of binding antibodies (BAb) and NAb and their relationship to IFN-inducible MxA protein levels in peripheral blood leukocytes in 20 IFNbeta-1a-treated patients with relapsing-remitting MS was conducted. RESULTS: In seven of nine NAb-positive patients, IFNbeta-1a was unable to induce MxA protein. BAb were detected in 11 patients, and they preceded or paralleled the development of NAb in all the patients. The titer of NAb correlated positively with BAb titer and negatively with MxA expression level. There was also a weaker but clear correlation between BAb titers and MxA levels. CONCLUSIONS: NAb, in most but not all cases, inhibited the in vivo function of IFNbeta. Analysis of MxA protein in lymphocytes together with analysis of NAb is a promising marker for evaluating the biologic effects of IFNbeta treatment in MS patients.

Induction of interferon-gamma and IL-4 production by mitogen and specific antigens in peripheral blood lymphocytes of Type 1 diabetes patients.

A functional imbalance in cytokine production resulting in dominance of Th1 over Th2 type response has been suggested to play a critical role in the pathogenesis of type 1 diabetes. In this study the cellular responses to pokeweed mitogen and a panel of specific antigens were analysed by measuring the production of IFN-gamma and IL-4 cytokines at the levels of mRNA expression (expression index=antigen/medium) and protein secretion in culture supernatants. Two enterovirus preparates were included due to the suggested significance of these viruses in the aetiology of type 1 diabetes. The study included 22 children with newly-diagnosed type 1 diabetes, 15 children with longer duration of disease and 20 healthy children. Comparisons were made between age- and sex-matched groups. Newly diagnosed diabetic patients had significantly higher IFN-gamma mRNA expression index (p<0.02) but also higher IL-4 mRNA expression index (p<0.05) in tetanus toxoid stimulated peripheral blood mononuclear cells compared to healthy controls. Also the diabetic patients studied 3-72 months after the diagnosis of type 1 diabetes showed a tendency to higher IFN-gamma mRNA expression index compared to controls (0.05

Cytokine expression in unstimulated PBMC of children with type 1 diabetes and subjects positive for diabetes-associated autoantibodies.

The aim of this study was to evaluate possible changes in the circulating levels of interferon (IFN)-gamma, interleukin (IL)-4 and transforming growth factor (TGF)-beta in association with the autoimmune process leading to type 1 diabetes. Expression levels of mRNAs specific for each cytokine were determined in peripheral blood mononuclear cells (PBMC) by a multiplex reverse transcription-polymerase chain reaction (RT-PCR) followed by hybridization reactions with lanthanide-labelled probes and detection by time-resolved fluorometry. Newly diagnosed diabetic children had lower levels of IFN-gamma, IL-4 and TGF-beta 1 signals compared to their age- and sex-matched controls (P < 0.02, P < 0.005 and P < 0.005, respectively) and also the autoantibody-positive subjects had significantly lower levels of IL-4 and TGF-beta 1 in comparison with their matched controls (P = 0.0013 and P = 0.012). No significant differences were observed when comparing matched pairs of diabetic children and autoantibody-positive subjects. Our results suggest a systemic bias towards reduced production of T-helper cell type 2 cytokines (IL-4 and TGF-beta 1) during the autoimmune process, but there was also a reduced level of IFN-gamma expression in the periphery at the onset of clinical diabetes.

Lack of induction by rhinoviruses of systemic type I interferon production or enhanced MxA protein expression during the common cold.

To study whether MxA protein expression is systemically upregulated during rhinovirus infection, blood specimens were collected from 40 patients with common cold and MxA expression in mononuclear cells analyzed by flow cytometry. None of the patients with a confirmed rhinovirus infection (n = 15) or with an infection of unknown etiology (n = 20) had elevated expression of the MxA protein (median fluorescence intensity, 549 and 582, respectively) when compared to healthy controls (n = 11, median 590). Patients with influenza infections had significantly elevated values (n = 5, median 750), and interferon could be detected only in serum samples from influenza patients. In conclusion, expression of MxA in blood lymphocytes and an apparently systemic type I interferon response is not induced during rhinovirus infection or during most other cases of common cold in young adult patients.

Comparison of enterovirus-specific cellular immunity in two populations of young children vaccinated with inactivated or live poliovirus vaccines.

Enterovirus-specific cellular immunity was studied in Estonian and in Finnish children at the age of 9 months. The aim was to evaluate the level of responsiveness in two neighbouring countries with different poliovirus immunization practices and striking differences in the incidence of insulin-dependent diabetes mellitus (IDDM), a disease in which early enterovirus infections are an aetiological risk factor. The Estonian children immunized with live attenuated polio vaccine had stronger T cell responses to coxsackievirus B4 and poliovirus type 1 when compared with Finnish children immunized with inactivated polio vaccine (median stimulation indices 10.4 and 6.3 in Estonian children and 1.9 and 2.9 in Finnish children, respectively; P < 0.05). Lymphocytes stimulated by poliovirus type 1 antigen expressed interferon-gamma (IFN-gamma) mRNAs, which strongly correlated with the level of proliferation responses. Lymphocytes of Estonian children had a tendency towards stronger expression of IFN-gamma upon poliovirus challenge when compared with Finnish children. The number of children who had experienced coxsackievirus B infections, as determined by the presence of neutralizing antibodies, did not differ between Estonian and Finnish children. The results show that Finnish children have weaker cellular immunity against enteroviruses at the age of 9 months compared with Estonian children at the same age. This is most probably due to the difference in polio vaccination schedules; in Estonia live poliovirus vaccine is used and given at earlier ages than the inactivated vaccines in Finland. This leads to stronger T cell immunity which cross-reacts with other enterovirus serotypes. This may explain the lower incidence of IDDM in Estonia by providing effective protection against diabetogenic enterovirus strains in Estonian children.

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry.

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.

Interferon-gamma production in antigen specific T cell response: quantitation of specific mRNA and secreted protein.

Interferon gamma (IFN-gamma) production as a measure of cellular sensitization was studied by detection of the cytokine in culture supernatant by enzyme immunoassay (EIA) and by measuring cellular mRNA using the reverse transcriptase polymerase chain reaction (RT-PCR) method. These assays were compared to the standard lymphocyte proliferation assay as a marker of T cell responsiveness to foreign antigens. When blood donors seropositive for herpes simplex virus (HSV) were compared to seronegative donors, all measurements of cellular sensitization separated the groups without overlap. There were significant correlations between the IFN-gamma mRNA titre and the secreted IFN-gamma (r = 0.57, P = 0.03), and the proliferative response and the secreted IFN-gamma (r = 0.78, P = 0.001), as well as between the IFN-gamma mRNA titre and the proliferative response (r = 0.78, P < 0.001). When tetanus toxoid (TT) responses were studied in immunized subjects, a wide range of responsiveness could be seen and correlation between various measurements was poor. However, constant individual levels of the cytokine production were demonstrated. Six people who had received their last TT booster vaccination more than 5 years ago were revaccinated and repeatedly studied. An increase in the levels of produced IFN-gamma could be seen in all subjects and two who lacked a lymphocyte proliferation response developed it after revaccination.

Expression of MxA protein in blood lymphocytes discriminates between viral and bacterial infections in febrile children.

Interferons (IFNs), which are induced by viruses, form an essential part of host's defense systems against viral infections. The antiviral actions of IFNs are mediated by several IFN-inducible gene products, one of which is Mx protein. To evaluate whether MxA protein expression in lymphocytes could function as an indicator of endogenous IFN production in children with acute febrile illness, we analyzed MxA protein levels in peripheral blood lymphocytes by flow cytometry in the acute phase of the disease. Children with a laboratory-confirmed viral infection [respiratory syncytial virus (RSV) in 21, adenovirus in 10, rotavirus in 5, and influenza, herpes simplex, or EBV in 7 other cases] had significantly higher (p < 0.002) MxA protein levels (median fluorescences in different virus groups ranged from 707 to 765) compared with children with a bacterial infection (n = 12, median fluorescence 548). To characterize further MxA protein expression during infections, cells from 41 patients were stimulated in vitro with exogenous IFN-alpha, and the level of MxA protein expression was determined. The rise in MxA staining levels was significantly higher in the group with bacterial infections compared with those with viral infection (p < 0.005), further indicating that the MxA protein levels were already elevated in vivo in patients with viral infections. This study suggests that elevated MxA protein expression levels can be used in the differential diagnosis of bacterial versus viral disease in febrile children.

Effect of polymorphism in the insulin gene region on IDDM susceptibility and insulin secretion. The Childhood Diabetes in Finland (DiMe) Study Group.

In addition to the major genetic determinants of insulin-dependent diabetes mellitus (IDDM) in the major histocompatibility complex (MHC) on chromosome 6, there are also minor genetic risk markers, e.g. in the insulin gene region on chromosome 11p15.5 (IDDM2). We studied the significance of the-23 HphI polymorphism in the insulin gene region (-23 HphI INS) in the Finnish population in combination with HLA genotyping data. The frequency of the-23 HphI INS +/+ genotype was higher in diabetic subjects with a low risk HLA DQB1 genotype than in control subjects (P = 0.05). Diabetic children in multiple-case families also had a higher frequency of the INS risk genotype than the controls (P < 0.05), and this difference was independent of the HLA genotype. Furthermore, we studied siblings positive for islet cell antibodies (ICAs) and/or insulin autoantibodies (IAAs) to evaluate the impact of the-23 HphI INS +/+ genotype on their beta-cell function assessed by sequential intravenous glucose tolerance tests and on their progression to IDDM. When analysing siblings with a low-risk HLA DQB1 genotype, those with the-23 HphI INS +/+ genotype had lower first phase insulin responses (P < 0.02) on several occasions than the remaining sibling. Six siblings (26.1%) in the former group progressed to clinical disease during the observation period, whereas none in the latter group presented with IDDM (P = 0.01). These observations suggest that the-23 HphI INS +/+ polymorphism is associated with an increased risk of IDDM in subjects without predisposing genes in the MHC region. The enhanced susceptibility may be related to a reduced insulin secretory capacity.