[Recognize tuberculosis--prevent outbreak]. / Tunnista tuberkuloosi--ehkäise epidemia.

INTRODUCTION: The number of tuberculosis cases in Finland has decreased. Cases among immigrants have, however, increased, and the disease may not be recognized early enough. METHODS: We describe four group exposures to tuberculosis that occurred in Finland, the index patient coming from a country with a high incidence rate of tuberculosis. RESULTS: Over 900 persons were exposed to tuberculosis. Coordination of the surveys was hampered by the fact that several healthcare operators participated in the study. Three index patients had drug-resistant tuberculosis, one of which was multidrug resistant. CONCLUSIONS: Extensive operations of tracking and prevention could have been avoided, if the symptoms would have been recognized earlier.

Elicitation of T-cell responses by structural and non-structural proteins of coxsackievirus B4.

Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within the family Picornaviridae. To investigate target proteins recognized by T-cells in human enterovirus B infections, virus-encoded structural [VP0 (VP4 and VP2), VP1, VP3] and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in Escherichia coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognized enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of the 19 (79 %) donor lines. Non-structural 2C protein was recognized in 11 of the 19 (58 %) lines, and 11 of the 19 (58 %) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.

T-cell reactivity to insulin peptide A1-12 in children with recently diagnosed type 1 diabetes or multiple beta-cell autoantibodies.

Insulin-specific immune responses appear early in preclinical type 1 diabetes (T1D), and bovine insulin in cow's milk-based infant formulas has been suggested to be of importance in induction of the primary response to insulin in humans. To characterize insulin-specific T-cell reactivity we studied T-cell responses to 10 insulin peptides derived from bovine (BI) and human insulin (HI) in 42 children with recently diagnosed T1D, 47 children with multiple autoantibodies and 111 autoantibody-negative control children with risk-associated HLA alleles. Proliferation responses detected in antigen-stimulated peripheral blood mononuclear cells did not differ between the three groups when the comparison was performed without considering HLA genotypes. However, significant differences were observed, when children with the high-risk genotype HLA (DRB1*03)-DQA1*05-DQB1*02/DRB1*0401-DQA1*03-DQB1*0302 were analyzed separately. The responses to the peptides including amino acids A1-12 derived from B1 and H1 were significantly higher in children with T1D (P=0.008, P=0.004, for B1 and H1, respectively) and in children with diabetes-associated autoantibodies (P=0.002 and P=0.001, respectively) than in control children. Positive responses (stimulation indices SI> or =3) were seen more frequently in T1D children than in controls (4/7 vs 2/19; P=0.03 and 4/7 vs 1/19; P=0.01 for B1 and H1, respectively). T-cell response to the insulin peptide A1-12 is enhanced in clinical and preclinical T1D associated with the high-risk HLA-genotype emphasizing the importance of this epitope.

Reduced CD4+T cell activation in children with type 1 diabetes carrying the PTPN22/Lyp 620Trp variant.

The 620Trp variant of the LYP protein, encoded by the lymphoid tyrosine phosphatase 22 gene (PTPN22), is associated with autoimmunity. In this study we aimed at characterising the role of this variant on lymphocyte activation. We analysed cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) and CD4(+)T cells in a cohort of clinically non-diabetic, multiple autoantibody-positive children, healthy controls and in children with type 1 diabetes (T1D). We found a decreased proliferation and IL-2 production of CD4(+)T cells after anti-CD3/anti-CD28 stimulation (p=0.04 for IL-2) among T1D patients. In addition, a profoundly decreased intracellular calcium flux in CD4(+)T cells after PHA stimulus was detected among 620Trp carriers. In contrast, no effect of this polymorphism on tuberculin and tetanus toxoid induced PBMC proliferation and cytokine secretion was observed in autoantibody positive children, healthy controls and children with newly-diagnosed T1D. In conclusion, the LYP 620Trp variant is associated with reduced activation, proliferation and IL-2 production in CD4(+)T cells among T1D patients. In accordance with our previous findings on the key role of this variant on disease progression, this mechanism is likely to contribute to the development of beta-cell specific autoimmunity.

Circulating CD4+CD25 high regulatory T cells and natural killer T cells in children with newly diagnosed type 1 diabetes or with diabetes-associated autoantibodies.

Type 1 diabetes is a T-cell-mediated autoimmune disease in which insufficient regulatory mechanisms are perceived to be involved in the pathogenesis. We used flow cytometry to analyze the proportion of CD4(+)CD25(high) regulatory T cells and natural killer T (NKT) cells in peripheral blood obtained from 25 children with newly diagnosed type 1 diabetes, 21 nondiabetic subjects positive for two or more diabetes-associated autoantibodies, and from 39 autoantibody-negative age- and HLA-matched control subjects. CD4(+)CD25(high) T cells were also stained for additional markers HLA-DR, CD69, and CD62L. As NKT cell markers, we used CD161, V beta 11, and V alpha 24. The frequency of CD4(+)CD25(high)HLA-DR(-) T cells was significantly higher in multiple autoantibody-positive children than in controls (P = 0.021). We also detected a significantly higher level of CD4(+)CD25(high)HLA-DR(-) and CD4(+)CD25(high)CD69(-) T cells among children expressing three to four autoantibodies when compared to the controls (P = 0.004 and P = 0.048, respectively). The proportions of CD161(+)V beta 11(+) or V alpha 24(+)V beta 11(+) NKT cells were similar in all three groups of children studied. Interestingly, children with only two autoantibodies had a higher level of CD161(+)V beta 11(+) NKT cells than the controls (P = 0.002). Our data might be interpreted as indicative of an intensified regulatory response of regulatory T cells and NKT cells during the preclinical phase of the disease.

GAD65- and proinsulin-specific CD4+ T-cells detected by MHC class II tetramers in peripheral blood of type 1 diabetes patients and at-risk subjects.

In type 1 diabetes the major loss of insulin producing beta-cells is caused by autoreactive T-cells specific for antigens expressed by the pancreatic islets. In this study we have analyzed the prevalence of glutamate decarboxylase 65 (GAD65)- and proinsulin-specific CD4(+) T-cells in type 1 diabetes patients, at-risk subjects and in HLA-matched control children. Peripheral blood mononuclear cells were cultured in the presence of two different GAD65 peptides (555-567, 557I and 274-286) or with a proinsulin (B24-C36) peptide for 10-11days. The autoreactive T-cells were detected using antigen specific-MHC class II tetramers by flow cytometry. Our results show that 11 of 18 (61%) type 1 diabetes patients and 7 of the 20 (35%) at-risk subjects were positive for one of the three GAD65 or proinsulin-containing tetramers, whereas only 2 of 21 (9.5%) controls had tetramer binding cells (p = 0.0007 type 1 diabetes vs. controls and p = 0.0488 at-risk subjects vs. controls, Chi-square test). Type 1 diabetes patients responded to all three peptides. At-risk subjects recognized also the GAD65 555-567 557I peptide, while none of the controls responded to it. In conclusion, type 1 diabetes patients and at-risk subjects have a significantly higher prevalence of GAD65- and proinsulin-specific CD4(+) T-cells than the control subjects.

Epitopes recognized by CBV4 responding T cells: effect of type 1 diabetes and associated HLA-DR-DQ haplotypes.

The present study aimed at characterizing the epitopes recognized by coxsackievirus B4 (CBV4)-specific T-cell lines established from 23 children with type 1 diabetes (T1D) and 29 healthy children with T1D risk-associated HLA genotypes. Responsiveness to VP1 region was dependent on the specific infection history as 55% of the T-cell lines from donors with neutralizing antibodies to CBV serotypes responded to VP1 peptides compared to none of the T-cell lines from other donors (P = 0.01). The pattern of recognized peptides was dependent of the HLA genotype. Forty-two percent of the T-cell lines from donors carrying the HLA-(DR4)-DQB1*0302 haplotype responded to VP1 peptides 71-80 compared to none of the T-cell lines from donors without this haplotype (P = 0.02). No evidence for the existence of diabetes-specific epitopes was found. Only few epitopes were exclusive recognized by T cells from diabetic children, and in each case only one or two T-cell lines were responding.

Diagnostic potential of parechovirus capsid proteins.

To study humoral and cellular immunity against human parechovirus type 1 (HPEV1), the viral capsid proteins VP0, VP1, and VP3 were expressed and purified as glutathione S-transferase (GST)-tagged recombinant proteins. The fusion proteins were used to raise antisera in rabbits. VP0 and VP1 antisera specifically detected HPEV1-infected cells in culture by immunoperoxidase staining and immunofluorescence. Furthermore, antisera against the VP0 and VP1 proteins had neutralizing effects against HPEV1 infection. When the HPEV1 antibody titers of 20 adults and 55 children were determined by a microneutralization test, the prevalence of HPEV1 antibodies in the adult population was 96%, while 50% of children were seropositive. Selected sera were used to evaluate HPEV1 fusion proteins as antigens in an enzyme immunoassay. The VP3 capsid protein appeared to be suitable for the purpose, with specificity of 100% and sensitivity of 96% compared to the neutralization test. Furthermore, T-cell responses to the purified HPEV1 and HPEV1 capsid fusion proteins were studied in 20 adults. Sixty percent of the subjects had T-cell proliferation responses to purified HPEV1, and 90% of the subjects also had positive T-cell responses to at least one of the GST capsid proteins.

Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses.

To determine the usefulness of nasal swabs as a simple method for detection of respiratory viruses, we compared nasal swabs and nasopharyngeal aspirates obtained at the same time from the opposite nostrils of 230 children with upper respiratory infection. The sensitivity of nasal swabs was comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses except respiratory syncytial virus.

T cell epitopes in coxsackievirus B4 structural proteins concentrate in regions conserved between enteroviruses.

The present study aimed to characterize systematically the target epitopes of T cell responses in CBV4 structural proteins. These were studied by synthesizing 86 overlapping 20-aa-long peptides covering the known sequence of CBV4 structural proteins and analyzing the proliferation responses of 18 CBV4-specific T cell lines against these peptides. Recognized peptides differed depending on the HLA-DR genotype of the T cell donor. They were concentrated to the VP4 and VP2 regions as six of seven common peptide epitopes located in this region, whereas there was only one in the VP3 region and none in the VP1 region. Peptides from conserved areas were recognized more often (on average, 15% of them stimulated each T cell line) than those derived from variable areas (3%) (P < 0.0001, Fisher's exact test). Some conserved peptides inducing T cell responsiveness in most subjects were identified, a knowledge which can be useful in the development of new synthetic vaccines.