CD106 (VCAM-1) in testicular immunoregulation.

The expression of CD18, CD49d/CD29, CD44, CD54 and CD106 was studied in the testis of normal mice at various ages, in the cryptorchid testis, in the testis of estrogen-treated mice and in the testis of non-obese diabetic (NOD) mice, using immunocytochemistry to see which of these lymphocyte and endothelial adhesion proteins may be involved in lymphocyte regulation in the testis. CD18-, CD49d/CD29-, CD44- and CD54-expressing cells were not found in the normal > 10-week-old BALB/c mouse testis. Leydig cells expressed CD106 strongly at this age. In contrast to the > 10-week-old testis, only very few interstitial cells of the 2-week-old normal mice expressed CD106. The expression of CD106 increased gradually with age so that at 6 weeks of age the expression of CD106 was moderate in the interstitial tissue. In the experimentally abdominal testis, CD106 was expressed in the interstitial tissue as strongly as in the contralateral scrotal testis. CD44- and CD18-expressing cells were occasionally present in the interstitial tissue of the abdominal testis, but not in the contralateral scrotal testis. CD54 was present in the epithelium of the ductuli efferentes. In the testis of the estrogen-treated mice, CD106 was expressed in the interstitial tissue as strongly as in the normal mice. Occasional CD44- and CD18-expressing cells were found in the testicular capsule. In the testis of adult NOD mice, CD106 was present in the interstitial tissue, but none of the other studied proteins. Immunoblotting of CD106 from the adult testis under reducing conditions demonstrated a single broad band with a M(r) of 51-65 kDa. This is a novel isoform of CD106. In a modified Stamper-Woodruff assay, lymphocytes bound to the testicular interstitial tissue. In co-incubations of native Leydig cells and lymphocytes, anti-CD106 antibodies prevented formation of Leydig cell-lymphocyte rosettes more than isotype-matched irrelevant control antibodies, suggesting that Leydig cell lymphocyte binding occurs through CD106-CD49d interactions. In lymphocyte cultures in the presence of anti-CD3, anti-CD28, the M(r) > 5 K fraction of testis extract (containing CD106 as shown by immunoblotting) and anti-CD106 or control antibody, anti-CD106 did not consistently affect T cell 3H-TdR incorporation. The present results suggest that CD106 expressed by the Leydig cells may act as an adhesion-promoting molecule or a co-stimulatory factor for T cells migrating to the testis.

The role of Fas ligand in the development of insulitis in nonobese diabetic mice.

The role of Fas ligand-induced lymphocyte apoptosis in the development of insulitis was studied in nonobese diabetic (NOD) mice. Fas ligand with a molecular weight of 45 kD appeared in the pancreas of NOD mice during the onset of insulitis as demonstrated by western blot analysis. In situ DNA end-labeling (ISEL) of the pancreases of NOD mice demonstrated positive cells in the islets of Langerhans, with an age-dependent increase in the density and frequency of animals with islets containing ISEL-positive cells. In the pancreatic islets of BALB/c mice, no ISEL-positive cells were observed in any of the age groups studied. In ultrastructural analysis degenerating cells with condensed or fragmented nuclei and plasma membranes detached from neighboring cells were observed both in and around the islets. In some cases, these cells were being phagocytosed by the neighboring islet cells. Degenerating cells with characteristics of lymphocytes were seen in contact with healthy lymphocytes around the islets. Neutralizing Fas ligand antibodies affected 3H-thymidine incorporation of CD3+CD28+ pancreatic lymphocytes from 12- to 30-week-old NOD mice in one of three cultures. There was no difference in the effect of neutralizing Fas ligand antibodies between pancreatic and blood lymphocytes. The present results on an increase in density of apoptotic cells in pancreatic islets of NOD mice simultaneously with the onset of insulitis and appearance of Fas ligand suggest that the pancreas infiltrating lymphocytes may be destroyed by Fas ligand-induced apoptosis.

Dry sausage fermented by Lactobacillus rhamnosus strains.

The ability of three probiotic Lactobacillus rhamnosus strains GG, E-97800 and LC-705 and one commercial Pediococcus pentosaceus starter strain (control) to produce dry sausage was studied. During the fermentation process the numbers of inoculated lactic acid bacteria increased from approx. 7 log10 to 8-9 log10 cfu/g and the pH values decreased from 5.6 to 4.9-5.0. The sensory test indicated that the dry sausages fermented by L. rhamnosus LC-705 were inferior to the control sausages. The presence of inoculated experimental strains as predominant organisms in the dry sausages was recognised on the basis of their genetic fingerprints by ribotyping. The concentrations of biogenic amines remained low during the ripening process. These results indicated that the studied Lactobacillus rhamnosus strains, especially strains GG and E-97800, are suitable for use as probiotic starter cultures in fermenting dry sausage.

Screening of commercial meat starter cultures at low pH and in the presence of bile salts for potential probiotic use.

The survival of lactic acid bacterial strains from eight meat starter cultures under conditions similar to those in the gastrointestinal tract was determined. The conditions in stomach were simulated by using pH 1-5 phosphate-buffered saline. The conditions in small intestine were simulated by using MRS broth over a pH range 4-7 and two bile salt concentrations (0.15% and 0.30%). The survival capacity at pH 3 was strain dependent. 0.30% bile salts were critical for screening bile salt tolerant strains at pH 6. Strains of Lactobacillus sake (RM10) and Pediococcus acidilactici (P2) had the best survival capacities under acidic conditions and at the higher concentration of bile salts.

Flavour profiles of dry sausages fermented by selected novel meat starter cultures.

Probiotic or bioprotective Lactobacillus rhamnosus strains GG, LC-705 and E-97800 as well as Pediococcus pentosaceus E-90390 and Lactobacillus plantarum E-98098 were studied for their ability to act as main fermenting organisms in the manufacturing process of dry sausages. In the preliminary tests, their abilities to produce lactic acid and biogenic amines, histamine or tyramine, were studied in MRS broth and analysed by high-performance liquid chromatography. The strains produced higher or equal amounts of lactic acid compared to control and were amine negative. During the actual fermentation process of dry sausages the numbers of inoculated bacteria increased from the level 6.5-7.0 log cfu/g to 8.0-9.0 log cfu/g. The most fast growing strains were P. pentosaceus E-90390 and the control while the growth of L. plantarum E-98098 and L. rhamnosus LC-705 were the slowest. The pH value of the sausages decreased from 5.6 to 4.9-5.0. The presence of these experimental strains as major organisms in the sausages after fermentation and ripening was confirmed on the bases of their genetic fingerprints. The flavour profiles of the experimental sausages produced by these probiotic or protective strains were similar with that produced by the commercial meat starter culture and commercial North European dry sausage recipe.

Inhaled budesonide influences cellular and biochemical abnormalities in pulmonary sarcoidosis.

In a randomized, double-blind study 19 patients with newly-detected pulmonary sarcoidosis were treated with either inhaled budesonide, 800 micrograms twice daily (n = 9), or placebo (n = 10) for 8-10 weeks. Before and after treatment, chest roentgenograms, lung function tests, bronchoalveolar lavage (BAL) and biochemical tests were performed. Angiotensin converting enzyme (ACE) activity and beta2-microglobulin (beta 2M) concentrations were measured in serum. The same tests, as well as albumin and hyaluronan were measured in the BAL-fluid. The total cell number in BAL-fluid, differential counts and lymphocyte subpopulations were determined (total T- and B-lymphocytes, T-helper/inducer (OKT-4) and T-suppressor/cytotoxic (OKT-8) lymphocytes). No significant changes in chest roentgenograms or lung function tests were observed during the short study time. However, a decrease in serum ACE (p less than 0.1) and beta 2 M (p less than 0.05) as well as in BAL-hyaluronan (p less than 0.01) was found in the budesonide-treated patients as well as a decrease in the percentage of BAL T-lymphocytes (p less than 0.05) and the T4/T8 ratio (p less than 0.1). No significant changes were seen in the placebo group. The findings suggest that treatment with inhaled budesonide influences biochemical and cellular findings in patients with early sarcoidosis in the same way as treatment with systemic corticosteroids. The results may also explain clinical effects seen in sarcoidosis patients treated with inhaled corticosteroids. However, further studies are required to determine the long-term clinical benefits of inhaled steroids in pulmonary sarcoidosis.

First-phase insulin response in young healthy children at genetic and immunological risk for Type I diabetes.

AIMS/HYPOTHESIS: A reduced first-phase insulin response to intravenous glucose is perceived as a sign of far-advanced deterioration of beta-cell function during the development of Type I (insulin-dependent) diabetes mellitus, but data on insulin responses at the onset of diabetes-related autoimmunity are lacking. We studied the first-phase insulin responses of small children soon after observed seroconversion to autoantibody positivity. METHODS: In the Type I Diabetes Prediction and Prevention Study newborn infants are screened for HLA-DQB1-associated genetic risk for Type I diabetes and those with increased risk are followed-up for the emergence of islet-cell antibodies. If antibodies are detected, autoantibodies to three other antigens (insulin, GAD65 and IA-2) are also measured. To measure first-phase insulin responses, intravenous glucose tolerance tests were carried out in 52 (1 to 5-year-old) children who had recently seroconverted to islet-cell antibody positivity. RESULTS: The first-phase insulin response was subnormal (<38 mU/l, the 5(th) percentile of insulin responses of 20 islet-cell antibody negative healthy children at this age) in 22 of the 52 children (42%). Stepwise multiregression analysis showed that islet-cell antibody greater than 20 JDFU (p=0.0005), insulin autoantibodies (p=0.0009) and an increasing number of positive autoantibodies (p=0.0011) were independent predictors of low first-phase insulin response. CONCLUSION/INTERPRETATION: A decreased first-phase insulin response could be an early phenomenon in the course of prediabetes in young children, implying a rapid autoimmune destruction or loss of function of beta cells as well as possible metabolic compensation mechanisms, since 11 out of the 22 high risk children remain nondiabetic for a considerable period of time despite low insulin responses.

The normal population distribution of PRNP codon 129 polymorphism.

OBJECTIVES: The common prion protein gene (PRNP) codon 129 polymorphism modifies the susceptibility to and the phenotype of prion diseases. However, no truly representative normal population-based data, or data stratified according to age or gender are available on the distribution of this polymorphism. MATERIAL AND METHODS: Allelic variation of codon 129 in three Finnish populations representing different age groups, and among Finnish, British and Irish blood donors were examined. RESULTS: The PRNP codon 129 genotype distribution in the total Finnish sample was 49% for methionine-methionine (MM), 42% for methionine-valine (MV) and 9% for valine-valine (VV), for the UK blood donors 42% for MM, 47% for MV and 11% for VV, and for the Irish blood donors 34% for MM, 56% for MV, and 10% for VV. CONCLUSIONS: The genotype frequencies were almost identical in all three Finnish populations of different ages, with no gender differences, and did not differ from corresponding figures for the Finnish blood donors. However, the PRNP codon 129 genotype distribution in Finland differed significantly from that of the British and the Irish blood donors and the previously published blood donor data on other Western Europeans and Americans.

Enterovirus antibody levels during the first two years of life in prediabetic autoantibody-positive children.

AIMS/HYPOTHESIS: We evaluated the role of enterovirus infections in the pathogenesis of Type I (insulin-dependent) diabetes mellitus by monitoring enterovirus antibody levels in prediabetic children who turned positive for diabetes-associated autoantibodies in a prospective birth cohort study. METHODS: Serial serum samples taken during prospective observation starting at birth were analysed for IgG and IgA class antibodies against enterovirus antigens including purified coxsackievirus B4, echovirus 11, poliovirus 1 and a synthetic enterovirus peptide antigen using enzyme immunoassay. Maternal samples taken at the end of the third month of pregnancy were also studied. Analyses were done from 21 childen who developed autoantibodies and from 104 autoantibody-negative control children who were matched for the time of birth, gender and HLA susceptibility alleles. For comparison, adenovirus antibodies were also analysed from all samples collected. RESULTS: IgG class enterovirus antibody levels were high in maternal samples and in cord blood in both case and control children. After birth the IgG levels decreased reaching a nadir at the age of 6 months. No IgA class antibodies were detected at birth but started to emerge postnatally. Antibody levels did not differ between the autoantibody positive and the control children during the first 6 months of life. From 6 months to 24 months of age, the autoantibody positive children had higher IgG and IgA levels against coxsackievirus B4, echovirus 11 and the synthetic enterovirus peptide antigens than control children but poliovirus 1 and adenovirus antibodies were closely similar in the two groups. The difference between children with autoantibodies and control children was predominantly seen among boys and among those with the HLA-DQB1*0302/x genotype. CONCLUSIONS/INTERPRETATION: Our data show that children who seroconverted for diabetes-associated auto-antibodies develop stronger humoral immune responses to coxsackievirus B4, echovirus 11 and a synthetic enterovirus peptide antigen than children who remained negative for autoantibodies. Poliovirus antibodies induced by uniform vaccinations did not differ between the prediabetic and control children suggesting that the regulation of antibody responses to enteroviruses is not disturbed. Accordingly, the results imply a stronger enterovirus exposure in prediabetic children supporting the role of enteroviruses in the pathogenesis of Type I diabetes.

No evidence of abnormal regulation of antibody response to coxsackievirus B4 antigen in prediabetic children.

Enterovirus infections are a potential environmental trigger of the autoimmune process leading to clinical type 1 diabetes. It has been suggested that the risk of virus-induced beta-cell damage might be connected with a defect in humoral immune responsiveness to enteroviruses. In the present study we assessed whether such a defect in IgG responsiveness to coxsackievirus B4 antigen existed in young children who developed diabetes-associated autoantibodies during prospective observation from birth until the age of 18 months. IgG levels and maturation of antibody avidity were analysed in 21 children with autoantibodies and 41 control children who had experienced an equal number of enterovirus infections and were additionally matched for age, sex and HLA-DQB1 risk alleles for type 1 diabetes but had not produced diabetes-associated autoantibodies. IgG levels to coxsackievirus B4 were high in cord serum reflecting the presence of maternal antibodies. Mean IgG levels gradually decreased but began to increase after the age of 6 months, showing no significant difference between autoantibody positive and control children. The avidity of antibodies was strong in cord serum and decreased gradually during the first year of life when maternal antibodies disappeared. The avidity indices, which varied considerably from child to child, did not differ between the autoantibody-positive and -negative subjects. In conclusion, our data suggest that children affected by a beta-cell damaging autoimmune process show normal responses to coxsackievirus B4 antigens.

Rotavirus infections and development of diabetes-associated autoantibodies during the first 2 years of life.

Rotavirus, the most common cause of childhood gastroenteritis, has been implicated as one of the viral triggers of diabetes-associated autoimmunity. To study the possible association between rotavirus infections and the development of diabetes-associated autoantibodies, we measured the prevalence of rotavirus antibodies in serum samples collected at 3-6-month intervals up to the age of 2 years from 177 children selected from consecutive newborns because they carried HLA-DQB1 alleles associated with increased risk for type 1 diabetes. Twenty-nine of the children developed at least two of four diabetes-associated autoantibodies (ICA, IAA, GADA or IA-2A) during the first 2 years of life (the cases), whereas 148 children remained autoantibody-negative matched with the cases for date of birth, gender, living region and HLA-DQB1 alleles. The temporal association between the development of the first-appearing diabetes-associated autoantibody and rotavirus infections was studied by analysing whether the cases had a diagnostic increase in rotavirus antibody titre more often during the 6-month period that preceded seroconversion to autoantibody positivity than the controls. By the age of 12 months one of the 13 case children (7%), who had a serum sample drawn at that age and who had developed at least one type of diabetes-associated autoantibodies, had experienced a rotavirus infection, while 12 of the 61 (20%) autoantibody-negative control children had had a rotavirus infection. By 18 months, four of the 22 autoantibody-positive cases (18%) and 18 of the 89 controls (20%) had rotavirus antibodies, and by the age of 24 months the respective numbers were five of the 27 cases (19%) and 32 of the 113 (28%) controls. A rotavirus infection occurred during the 6 months preceding the sample which was positive for an autoantibody in four of the 25 periods (16%) for which both necessary samples were available, while the controls had a rotavirus infection during 55 of the 370-such periods (15%). Accordingly, our data suggest that rotavirus infections are unlikely triggers of beta-cell autoimmunity in young children with genetic susceptibility to type 1 diabetes.