HbA1c variability and long-term glycemic control are linked to peripheral neuropathy in patients with type 1 diabetes.

BACKGROUND: HbA1c variability has been linked to retinopathy, renal disease and autonomic neuropathy in patients with type 1 diabetes mellitus (T1D) and type 2 diabetes mellitus (T2D). Although the same relationship has been demonstrated for diabetic peripheral neuropathy (DPN) in patients with T2D, data for T1D are still lacking. METHODS: Patients older than 17 years of age with ≥ 10 years of T1D duration and follow-up were included. All patients underwent nerve conduction studies and neurological examination. Laboratorial data was retrospectively extracted from chart review. Mean HbA1c (mHbA1c) over 10 years was calculated, as well as HbA1c variability estimated by standard deviation (HbA1c-SD) and coefficient of variation (HbA1c-CV). RESULTS: Fifty patients with T1D were included (30 females and 21 non-caucasians), with mean age and T1D duration of 25.6 ± 5.0 and 17.9 ± 6.1 years, respectively. The frequency of DPN was 24%. Higher mHbA1c (10.4 ± % vs 8.1 ± %; p < 0.001), HbA1c-SD (1.8 ± 0.8 vs 0.9 ± 0.4; p < 0.001), and HbA1c-CV (1.7 ± 0.8 vs 1.2 ± 1.1; p = 0.006) were observed in patients with DPN compared to others. SD-HbA1c and HbA1c-CV were associated with DPN, diagnosed by either clinical or NCS criteria, independent of mHbA1c, age and gender. CONCLUSIONS: Not only long-term glycemic control, but also its variability is associated with DPN in patients with T1D. Larger studies are required to confirm this finding.

Prospective evaluation of glutamine and phospholipids levels in first degree relatives of patients with Type 1 Diabetes from a multiethnic population.

BACKGROUND: A dysregulation in the metabolism of lipids may be an early marker of autoimmunity in Type 1 Diabetes (T1D). It would be of general importance to identify metabolic patterns that would predict the risk for T1D later in life. The aim of this study was to perform a prospective evaluation of glutamine and phospholipids levels in Brazilian first degree relatives (FDR) of patients with T1D in a mean interval of 5 years. FINDINGS: Brazilian FDR (n = 30) of patients with T1D were evaluated and blood was sampled to measure the levels of glutamine and phospholipids in the fasting serum by quantitative colorimetric method. The tests were repeated after a mean interval of 5 years and compared to a control group (n = 20). The FDR presented lower levels of phospholipids than controls (p = 0.028), but not of glutamine (p = 0.075). Phospholipids levels decreased over time (p = 0.028) in FDR and were associated with Glutamic acid decarboxylase autoantibody (GADA) titers (p = 0.045), autoantibody positivity (p = 0.008) and PTPN22 polymorphisms (p = 0.014). CONCLUSIONS: In this Brazilian multiethnic population, there was a significant decrease in phospholipids levels in FDR in patients with T1D during a 5-year prospective follow-up, as well as a significant association between these metabolite, GADA and PTPN22 polymorphisms. For Glutamine no difference was found. These findings suggest that a dysregulation in the metabolism of lipids may precede the onset of the autoimmunity in T1D.

Should thyroid-stimulating hormone goals be reviewed in patients with type 1 diabetes mellitus? Results from the Brazilian Type 1 Diabetes Study Group.

AIMS: To investigate if thyroid-stimulating hormone (TSH) levels are associated with any differences in glycaemic control or diabetes-related complications in individuals with Type 1 diabetes. METHODS: This observational, cross-sectional and multicentre study included patients with Type 1 diabetes for ≥ 5 years, with a recent TSH measurement and without a known previous thyroid disease. Patients were divided into three groups according to TSH levels: 0.4-2.5 mU/l; 2.5-4.4 mU/l; and ≥ 4.5 mU/l. RESULTS: We included 1205 individuals with a mean ± sd age of 23.8 ± 11.3 years. Seven patients had TSH levels <0.4 mU/l and were excluded from the comparison between groups. HbA1c levels, systolic and diastolic blood pressure, LDL cholesterol and disease duration were similar in all groups (P = 0.893, P = 0.548, P = 0.461, P = 0.575 and P = 0.764, respectively). The rates of diabetic retinopathy and GFR < 60/mL/min/1.73 m(2) differed between groups (P = 0.006 and P < 0.001, respectively) and were lower in those with lower TSH levels. Multivariate analysis confirmed these associations. The frequencies of retinopathy and GFR < 60 mL/min/1.73 m(2) were higher not only in patients with TSH ≥ 4.5 mU/l (odds ratio 1.878 and 2.271, respectively) but also in those with TSH levels of 2.5-4.4 mU/l (odds ratio 1.493 and 2.286, respectively), when compared with patients with TSH levels of 0.4-2.5 mU/l. CONCLUSIONS: TSH levels of 0.4-2.5 mU/l are associated with a lower risk of diabetic retinopathy and renal failure in individuals with Type 1 diabetes, independently of glycaemic control and duration of the disease.

Pancreatic autoantibodies, HLA DR and PTPN22 polymorphisms in first degree relatives of patients with type 1 diabetes and multiethnic background.

AIM: To evaluate the prevalence of pancreatic auto-antibodies (PAb) as well as its relationship with HLA DR B1 and PTPN22 polymorphisms in first degree relatives (FDR) of Brazilian patients with Type 1 diabetes (T1D) and multiethnic background. METHODS: FDR of patients with T1D were interviewed and blood was sampled for PAb measurement, HLA DRB1 and PTPN22 genotyping. Genotyping was also performed in index cases. RESULTS: In FDR (n=78), 16.7% presented at least one PAb. These individuals had a higher prevalence of HLA DRB1* 03 than others (p=0.03), without differences in PTPN22 genotyping. While the genetic profile was similar in FDR with PAb and their index cases, those without PAb had a lower frequency of HLA DR B1 * 03 than their correspondent patients (p=0.009). CONCLUSION: In this multiethnic population, a significant proportion of FDR of T1D patients had PAb, which was associated with HLA DR B1 * 03 but not with the PTPN22 polymorphism.

Viruses of hemorrhagic fever with renal syndrome (HFRS) grouped by immunoprecipitation and hemagglutination inhibition.

Nine viruses antigenically related to Hantaan virus that were isolated from various rodents in several countries and from a patient were examined by immunoprecipitation and by hemagglutination inhibition (HI). The immunoprecipitation tests were done with a monoclonal antibody that reacted with glycoprotein G2 and an antibody against a nucleocapsid protein. The viruses were classified into four subtypes on the basis of the molecular weights of their precipitated polypeptides and the pattern of results of the HI tests.

Persistent infection of rats with haemorrhagic fever with renal syndrome virus and their antibody responses.

Newborn (within 24 h after birth), 1-week-old and 6-week-old (adult) rats were inoculated with a Hantaan-related virus (B-1) and attempts were made to isolate the virus from various organs. Virus-specific antigens were detected in various organs of newborn rats. Moreover virus could be isolated from almost all their organs even 25 weeks after infection. In contrast, in rats infected at 6 weeks of age the virus could be isolated from various organs but its concentration decreased progressively with time. The levels of haemagglutination-inhibiting (HI) and neutralizing antibodies to B-1 virus in the sera were measured. In adult rats, HI antibodies were first detected 2 weeks after infection and their titre rose to a maximum after 5 weeks. On the other hand, in newborn rats the levels of antibodies remained low until 5 or 6 weeks after infection and started to increase to a high level more than 9 weeks after infection. Furthermore, in rats infected soon after birth IgM antibodies predominated for a long time and these antibodies also neutralized infectivity at a high level. These data suggest that the induction of a high titre of neutralizing antibodies mainly of the IgM type does not result in virus clearance in newborn rats and that cellular immunity may be important for virus clearance in vivo.

Characterization of glycoproteins of viruses causing hemorrhagic fever with renal syndrome (HFRS) using monoclonal antibodies.

Viruses causing hemorrhagic fever with renal syndrome (HFRS) encode two glycoproteins, G1 and G2. For determination of the biological functions of these glycoproteins, we isolated 15 hybridomas secreting monoclonal antibodies directed against the glycoproteins of the B-1 and Hantaan viruses (HV). From results of neutralizing and hemagglutination inhibition (HI) tests, and studies on the antigenic reactivities of the antibodies with other HV-related viruses by immunofluorescence, we classified these hybridoma clones into two groups producing antibodies to the G1 proteins of the B-1 virus, six groups producing antibodies to G2 proteins of the B-1 virus, and four groups producing antibodies to the G2 protein of HV. Of the antibodies to G2 produced by 12 clones, groups A and B had high HI activity with HV-related virus cross-reactivity and moderate neutralizing activity, group C had moderate HI activity with virus specificity but low neutralizing activity, group G had high neutralizing activity and low HI activity, and five other groups had little or no HI or neutralizing activity. Group A reacting with G1 protein had low level of both neutralizing and HI activity, while group B had no HI activity. One clone of monoclonal antibody had high neutralizing activity and no HI activity, but it did not react with either polypeptide by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by the immunoblotting method. These data suggest that both glycoproteins are the targets of neutralizing antibodies. Furthermore, the results indicate that the antigenic determinants with hemagglutination activity are mainly on the G2 protein, and that the domains related to neutralizing activity and to HI activity are separate.

Haemagglutination-inhibition test for haemorrhagic fever with renal syndrome using virus antigen prepared from infected tissue culture fluid.

Haemagglutinating (HA) antigens of four strains of virus related to that causing haemorrhagic fever with renal syndrome (HFRS) were prepared from infected tissue culture fluids by ultracentrifugation. The titres of the precipitated antigens were increased considerably by acetone extraction and sonication. Acetone extraction completely inactivated infectious virus in the antigen preparations. The antigens were pH-dependent, with pH optima at 5.8. Good correlations were observed in human and rat sera between the titres obtained by the haemagglutination-inhibition (HI) test and an indirect fluorescent antibody test. Moreover, strong cross-reactions among these strains were demonstrated by the HI test. The HI test has not been used previously with HFRS viruses because of the danger involved in preparing HA antigen, but these results indicate that a safe method is available for serological identification of HFRS.

Isolation of haemorrhagic fever with renal syndrome virus from leukocytes of rats and virus replication in cultures of rat and human macrophages.

Newborn rats were inoculated intraperitoneally with haemorrhagic fever with renal syndrome (HFRS)-related virus (B-1 strain), and virus isolation from their various organs was attempted between 1 and 25 weeks after inoculation. Virus could be isolated repeatedly from lung, brain, spleen and kidney and also from peripheral blood. When virus isolation was carried out on fractionated peripheral blood cells, virus was associated mainly with the macrophage fraction and to a lesser extent with granulocytes. Virus replicated well in peritoneal exudate cells of normal rats and it grew in the adherent mononuclear cells from normal human peripheral blood. These data suggest that macrophages, permissive for HFRS-related virus replication, might contribute to the spread of viral infection in vivo.

Antigenic differences between two viruses, isolated in Japan and Korea, that cause hemorrhagic fever with renal syndrome.

Hantaan virus (HV) 76-118, isolated from Apodemus agrarius coreae in Korea, and hemorrhagic fever with renal syndrome (HFRS) virus B-1, isolated from a rat in Japan, were examined for polypeptide compositions and for differences in immune responses in rats. In immunoprecipitation experiments, a major polypeptide of ca. 50 kilodaltons (K) was detected with antisera against HV 76-118 in cell extracts from Vero E6 cells infected with HFRS virus B-1, whereas three major polypeptides of 74 K (glycosylated), 57 K (glycosylated), and 50 K were detected with antisera against HFRS virus B-1. On the other hand, two polypeptides with molecular weights of 55,000 (glycosylated) and 50,000 were detected with either antiserum in cell extracts infected with HV 76-118. In neutralizing antibody tests with antisera prepared in rats, a remarkable difference in antibody titer (5 to 30 times higher to the homologous virus than to the heterologous virus) was observed between the two viruses. However, this difference was not so marked (1 to 4 times higher to the homologous virus) in the immunofluorescent antibody test. Twenty hybrid cell lines producing mouse monoclonal antibodies against HV 76-118 were isolated by fusion of spleen cells from BALB/c mice immunized against HV (strain 76-118) with mouse myeloma cells. The specificity of these monoclonal antibodies was established by immunofluorescent antibody, neutralizing antibody, and fluorescent antibody to membrane antigen tests and by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These hybrid cell lines were classified into three groups based principally on the IF staining pattern of the HV-infected cells: (i) antibodies which showed a discrete patch pattern in the cytoplasm by the immunofluorescent antibody test, reacting with the membrane antigen of infected cells and immunoprecipitating a 55-K glycoprotein from HV 76-118-infected cell lysates and a 57-K glycoprotein from the heterologous (strain B-1) HFRS virus-infected cell lysates. Among these, depending on the neutralizing antibody activity and the reaction with the heterologous antigen, three subgroups designated I-A, I-B, and I-C were established; (ii) antibodies which showed large granular dots in the cytoplasm, neither having neutralizing antibody activity nor immunoprecipitating any antigen; (iii) antibodies which showed defined granular dots throughout the cytoplasm, reacting with a 50-K polypeptide of both virus strains. These antibodies also classified into two subgroups based on the reactivity with the B-1 strain.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of hemorrhagic fever with renal syndrome (HFRS) virus from a tumor specimen in a rat.

A strain of hemorrhagic fever with renal syndrome (HFRS) virus was isolated in a cell culture from a tumor specimen in a rat kept in a medical institution in which there was a case of HFRS. Positive immunofluorescent reaction with sera from HFRS patients was recognized at the second passage and the number of cells containing antigen increased in the third passage. This virus, named B-1 strain, was identified as the HFRS virus by immunofluorescent tests with sera from patients.