Activated lymphocytes in patients with newly diagnosed type 1 (insulin-dependent) diabetes mellitus

The expression of activation antigens (transferrin receptor, IL-2 receptor and Ia antigen) on circulating T lymphocytes from Japanese children with Type 1 diabetes was studied using five monoclonal antibodies (Ab), OKT9, anti-Tac Ab, OKIa1, anti-human HLA-DR Ab and OKT3. For detecting Ia positive T cells, the dual staining technique using OKT3 and anti-Ia antibody was employed. Four out of six patients (67%) with newly diagnosed Type 1 diabetes showed a raised level of either OKT9 or Tac positive cells when examined at diagnosis. These patients, however, rapidly lost these activation antigens after the insulin therapy was started. In contrast, in 32 long-standing patients, only 2 (6%) had a high percentage of OKT9 positive cells and none of them demonstrated Tac positive cells. One out of six newly diagnosed patients or three out of 21 long-standing patients had a significantly high percentage of Ia-positive T cells compared with normal subjects. In poorly controlled long-standing patients whose HbA1 value was higher than 14%, none of them had an increased number of activated lymphocytes. Therefore, it is unlikely that insulin deficiency and hyperglycemia were responsible for the changes observed in these studies. Activated lymphocytes might be related to activation of the immune system involved in pathogenesis of Type 1 diabetes.     

Analysis ofHLA-DR and -DQ gene polymorphisms in sudanese patients with type 1 (insulin-dependent) diabetes

In this study we report for the first time, the molecular analysis of HLA-DR and -DQ gene frequencies in a large cohort of well charaterized type 1 (insulin-dependent) diabetes mellitus (IDDM) patients (n=72), and ethnically matched controls (n=59) collected in subSaharan Africa. High molecular mass DNA was prepared and analyzed in Southern blots with DRBI, DQA1, and DQB1 probes. By identifying DR and DQ allele-specific restriction fragment length polymorphisms (RFLPs), we have shown a strong positive association between IDDM and the Asp 57^– DQB1 allele *0201 (DQw2). A rare DR4, DQw2 haplotype was also identified at high frequency in the IDDM cohort. We can now confirm that the association between Asp 57^–DQB1 alleles and IDDM, previously reported in ethnically diverse cohorts collected in Western Europe, North America, and South Asia, is also present in an IDDM cohort collected in Africa.     

PCR-in situ hybridization detection of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes of patients with HTLV-1-associated neurologic disease.

PCR-in situ hybridization (PCR-ISH) was developed and utilized to determine the distribution of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes (PBL) from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). PCR-ISH of HTLV-1 tax DNA in PBL from patients with HAM/TSP revealed that 1 in 5,000 to 1 in 10,000 PBL contained virus. PCR-ISH was sensitive, because a positive signal was consistently demonstrated from the HTLV-1-infected cell lines HUT-102 (which contains four to six copies of HTLV-1 proviral DNA per cell) and MT-1 (which contains one to three copies of HTLV-1 proviral DNA per cell). Also, intracellular amplification by PCR-ISH significantly increased sensitivity compared with conventional ISH and was shown to be specific for HTLV-1 tax DNA. These results are in contrast to solution-phase PCR amplification in which greater than 1% of cells were estimated to be infected. The discordance between these results is discussed and may indicate that more than one copy of HTLV-1 tax proviral DNA is present in an individual PBL.     

Cellular immunity in insulin-dependent diabetes mellitus (type 1)

Cell-mediated immunity was investigated with T-cell blastic transformation stimulated by phytohaemagglutinin and/or insulin in patients with diabetes mellitus type 1. T-cell blastic transformation was determined in the whole blood by the intake of labelled thymidine intake by the lymphocytic DNA. Healthy individuals and patients with diabetes mellitus type 2 served as control groups. It was found that T-cell blastic transformation stimulated with phytohaemagglutinin is markedly diminished in patients with diabetes mellitus type 1 and to a lesser degree in patients with diabetes mellitus type 2. Insulin increased T-cell blastic transformation in insulin-dependent diabetic patients but has no effect in diabetes mellitus type 2. The obtained results suggest that induction and central phases of the cell-mediated immunological response are diminished in diabetes mellitus independently on its type. Such disorders may have different etiology depending on the type of diabetes mellitus.     

Blood glucose discrimination training in insulin-dependent diabetes mellitus (IDDM) patients

Self-management of insulin-dependent diabetes mellitus (IDDM) is dependent on a negative feedback loop of blood glucose (BG) fluctuations, which in turn directs treatment decisions to maintain normal BG. Although this feedback is typically accomplished by self-monitoring of blood glucose (SMBG), SMBG has limitations, and patients often rely on what their BG "feels" like. Two studies were performed to evaluate whether patients could learn to more accurately "feel"/discriminate their BG on the basis of internal cues or internal plus external BG cues. In Study I, BG Awareness Training significantly improved pre- to posttreatment BG estimation accuracy, relative to a control group. Study II replicated BG Awareness Training efficacy in improving BG estimation accuracy. Improvement in estimation accuracy was related only to initial accuracy; those who were initially less accurate improved the most. This improvement was represented in a 31% reduction in dangerous BG estimation errors and a 9% increase in accurate estimates. Resulting estimations were, however, still significantly less accurate than SMBG at the end of training.     

Blood glucose discrimination training in insulin-dependent diabetes mellitus (IDDM) patients

Self-management of insulin-dependent diabetes mellitus (IDDM) is dependent on a negative feedback loop of blood glucose (BG) fluctuations, which in turn directs treatment decisions to maintain normal BG. Although this feedback is typically accomplished by self-monitoring of blood glucose (SMBG), SMBG has limitations, and patients often rely on what their BG feels like. Two studies were performed to evaluate whether patients could learn to more accurately feel/discriminate their BG on the basis of internal cues or internal plus external BG cues. In Study I, BG Awareness Training significantly improved pre- to posttreatment BG estimation accuracy, relative to a control group. Study II replicated BG Awareness Training efficacy in improving BG estimation accuracy. Improvement in estimation accuracy was related only to initial accuracy; those who were initially less accurate improved the most. This improvement was represented in a 31% reduction in dangerous BG estimation errors and a 9% increase in accurate estimates. Resulting estimations were, however, still significantly less accurate than SMBG at the end of training.This research was supported by NIH grants AM282880, AM24177, AM22125, and RR00847 and by the Ames Company. The authors express their appreciation for the contribution made by trainers Leslie Butterfield and Linda Zimbelman, by the nursing staff at the University of Virginia's Clinical Research Center and the Diabetes and Nutrition Unit, and by Dr. James May from the Medical College of Virginia in soliciting subjects. We would also like to thank Andrea Snyder for her assistance.     

Erythrocyte membrane acetylcholinesterase in type 1 (insulin-dependent) diabetes mellitus.

1. The erythrocyte membrane acetylcholinesterase activity is significantly (P less than 0.001) decreased in insulin-dependent diabetes mellitus. 2. The activity is negatively correlated (r = -0.97) with the fasting blood glucose level. 3. Insulin treatment restores the activity to normal. 4. The Km of the enzyme for acetylthiocholine iodide was unchanged; however, the Vmax. was decreased, suggesting a decrease in the number of active enzyme molecules in diabetes.     

Detection of triple-helix related structures adopted by poly(dG)-poly(dC) sequences in supercoiled plasmid DNA.

Negative superhelical strain induces the poly(dG)-poly(dC) sequence to adopt two totally different types of triple-helices, either a dG.dG.dC triplex in the presence of Mg(+)+ at both neutral and acidic pHs or a protonated dC+.dG.dC triplex in the absence of Mg(+)+ ions at acidic pH (1). To examine whether there are still other types of non-B DNA structures formed by the same sequence, we constructed supercoiled plasmid DNAs harboring varying lengths of the poly(dG) tract, and the structures adopted by each supercoiled plasmid DNA were studied with a chemical probe, chloroacetaldehyde. The potential of a poly(dG)-poly(dC) sequence to adopt non-B DNA structures depends critically on the length of the tract. Furthermore, in the presence of Mg(+)+ and at a mildly acidic pH, in addition to the expected dG.dG.dC triplex detected for the poly(dG) tracts of 14 to 30 base pairs (bp), new structures were also detected for the tracts longer than 35 bp. The structure formed by a poly(dG) tract of 45 bp revealed chemical reaction patterns consistent with a dG.dG.dC triplex and protonated dC+.dG.dC triple-helices fused together. This structure lacks single-stranded stretches typical of intramolecular triplexes.     

Altered energetic properties in skeletal muscle of men with well-controlled insulin-dependent (type 1) diabetes

This study asked whether the energetic properties of muscles are changed by insulin-dependent diabetes mellitus (or type 1 diabetes), as occurs in obesity and type 2 diabetes. We used (31)P magnetic resonance spectroscopy to measure glycolytic flux, oxidative flux, and contractile cost in the ankle dorsiflexor muscles of 10 men with well-managed type 1 diabetes and 10 age- and activity-matched control subjects. Each subject performed sustained isometric muscle contractions lasting 30 and 120 s while attempting to maintain 70-75% of maximal voluntary contraction force. An altered glycolytic flux in type 1 diabetic subjects relative to control subjects was apparent from significant differences in pH in muscle at rest and at the end of the 120-s bout. Glycolytic flux during exercise began earlier and reached a higher peak rate in diabetic patients than in control subjects. A reduced oxidative capacity in the diabetic patients' muscles was evident from a significantly slower phosphocreatine recovery from a 30-s exercise bout. Our findings represent the first characterization of the energetic properties of muscle from type 1 diabetic patients. The observed changes in glycolytic and oxidative fluxes suggest a diabetes-induced shift in the metabolic profile of muscle, consistent with studies of obesity and type 2 diabetes that point to common muscle adaptations in these diseases.     

Reduction of human T-cell leukemia virus type 1 (HTLV-1) proviral loads in rats orally infected with HTLV-1 by reimmunization with HTLV-1-infected cells

Human T-cell leukemia virus type 1 (HTLV-1) persistently infects humans, and the proviral loads that persist in vivo vary widely among individuals. Elevation in the proviral load is associated with serious HTLV-1-mediated diseases, such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, it remains controversial whether HTLV-1-specific T-cell immunity can control HTLV-1 in vivo. We previously reported that orally HTLV-1-infected rats showed insufficient HTLV-1-specific T-cell immunity that coincided with elevated levels of the HTLV-1 proviral load. In the present study, we found that individual HTLV-1 proviral loads established in low-responding hosts could be reduced by the restoration of HTLV-1-specific T-cell responses. Despite the T-cell unresponsiveness for HTLV-1 in orally infected rats, an allogeneic mixed lymphocyte reaction in the splenocytes and a contact hypersensitivity response in the skin of these rats were comparable with those of naive rats. HTLV-1-specific T-cell response in orally HTLV-1-infected rats could be restored by subcutaneous reimmunization with mitomycin C (MMC)-treated syngeneic HTLV-1-transformed cells. The reimmunized rats exhibited lower proviral loads than untreated orally infected rats. We also confirmed that the proviral loads in orally infected rats decreased after reimmunization in the same hosts. Similar T-cell immune conversion could be reproduced in orally HTLV-1-infected rats by subcutaneous inoculation with MMC-treated primary T cells from syngeneic orally HTLV-1-infected rats. The present results indicate that, although HTLV-1-specific T-cell unresponsiveness is an underlying risk factor for the propagation of HTLV-1-infected cells in vivo, the risk may potentially be reduced by reimmunization, for which autologous HTLV-1-infected cells are a candidate immunogen.     

Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis

The aim of the present study was to investigate the presence of Leishmania (Viannia) subgenus DNA in peripheral blood from patients with cutaneous lesions due to American cutaneous leishmaniasis. The buffy coats from 68 blood samples were analyzed by polymerase chain reaction using the MP1L/MP3H primers. The parasite DNA was detected in 2 (3.4%) out of 59 patients who had amastigotes present in samples taken from lesions. The presence of Leishmania (Viannia) DNA in the blood of these patients indicates hematogeneous parasite dissemination.     

High frequency of HLA-DQB1 non-Asp(57) alleles in Kuwaiti children with insulin-dependent diabetes mellitus

The prevalence of polymorphic amino acids at position 57 of the HLA DQB1 in Kuwaiti children with insulin-dependent diabetes mellitus (IDDM) and nondiabetic controls has been determined using a polymerase chain reaction-sequence-specific primers (PCR-SSP) method. Using this approach, 34/55 (62%) IDDM children were found to be homozygous Ala/Ala and 19/55 (35%) were heterozygous with various combinations. Amongst the IDDM children with heterozygous genotype at codon 57 of HLA DQB1, 6/55 (11%) had Asp/Ala, 8/55 (15%) had Ala/Val, 4/55 (7%) had Ala/Ser and 1/55 had Asp/Val allelic combinations. When considered collectively, the nonaspartate (NA) alleles were represented in 87% of the IDDM cases and only 13% cases had Asp(57) allele in different heterozygous combinations, while none of the IDDM subjects had a homozygous Asp genotype. In nondiabetic controls, homozygous non-Asp (NA) alleles were represented in 44% subjects, 37% of the controls were heterozygous (NA/A) and 19% had a homozygous (A/A) genotype. These differences between the IDDM group and the control group were found to be statistically significant. Our data report one of the highest frequency of NA/NA residues at this locus compared with that from different world populations (Sardinians, Norwegians, US Caucasians, US Blacks and Chinese).     

Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences

Internal transcribed spacer (ITS-1) sequences from 97 accessions representing 23 species of Lactuca and related genera were determined and used to evaluate species relationships of Lactuca sensu lato (s.l.). The ITS-1 phylogenies, calculated using PAUP and PHYLIP, correspond better to the classification of Feráková than to other classifications evaluated, although the inclusion of sect. Lactuca subsect. Cyanicae is not supported. Therefore, exclusion of subsect. Cyanicae from Lactuca sensu Feráková is proposed. The amended genus contains the entire gene pool (sensu Harlan and De Wet) of cultivated lettuce (Lactuca sativa). The position of the species in the amended classification corresponds to their position in the lettuce gene pool. In the ITS-1 phylogenies, a clade with L. sativa, L. serriola, L. dregeana, L. altaica, and L. aculeata represents the primary gene pool. L. virosa and L. saligna, branching off closest to this clade, encompass the secondary gene pool. L. virosa is possibly of hybrid origin. The primary and secondary gene pool species are classified in sect. Lactuca subsect. Lactuca. The species L. quercina, L. viminea, L. sibirica, and L. tatarica, branching off next, represent the tertiary gene pool. They are classified in Lactuca sect. Lactucopsis, sect. Phaenixopus, and sect. Mulgedium, respectively. L. perennis and L. tenerrima, classified in sect. Lactuca subsect. Cyanicae, form clades with species from related genera and are not part of the lettuce gene pool.     

Characterization and sequencing of prototypic human T-lymphotropic virus type 1 (HTLV-1) from an HTLV-1/2 seroindeterminate patient

Serological screening for human T-lymphotropic virus type 1 (HTLV-1) parallels the standard screening process for human immunodeficiency virus (HIV), in which samples found positive by enzyme-linked immunosorbent assay (ELISA) are confirmed with a modified Western blot procedure. There are a significant number of cases in which HTLV-1/2 ELISA-positive specimens demonstrate an incomplete banding pattern on this Western blot. Individuals providing these atypical antibody responses are categorized as seroindeterminate for HTLV-1/2. Although HTLV-1 genomic sequences are readily detectable in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies have repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1. As a result, identification of the agent responsible for this indeterminate reactivity has been of interest. We have generated an HTLV-1-positive B-cell line (SI-1 B) from one of these seroindeterminate individuals. Previous screening for HTLV-1 in PBL from this patient had been routinely negative by primary PCR; however, HTLV-1 tax had been periodically detected by nested PCR. DNA sequence data generated with genomic DNA from the SI-1 B cell line and HTLV-1-specific primers demonstrated the presence of a full-length viral genome with >97% homology to the Cosmopolitan form of HTLV-1. A 12-bp deletion was identified in the 3'-gag/5'-prot region, which would predict translation of altered or nonfunctional proteins from these genes. We propose that this HTLV-1/2-seroindeterminate patient is infected with a prototypic form of HTLV-1 at an extremely low viral load and that this finding may explain HTLV-1/2 seroindeterminate reactivity in at least a subset of these individuals.     

High circulating frequencies of tumor necrosis factor alpha- and interleukin-2-secreting human T-lymphotropic virus type 1 (HTLV-1)-specific CD4+ T cells in patients with HTLV-1-associated neurological disease

Significantly higher frequencies of tumor necrosis factor alpha- and interleukin-2-secreting human T-lymphotropic virus type 1 (HTLV-1)-specific CD4(+) T cells were present in the peripheral blood mononuclear cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients than in those of asymptomatic carriers with similar provirus loads. The data suggest that HTLV-1-specific CD4(+) T cells play a role in the pathogenesis of HAM/TSP.     

Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.