Biomedical subjects

T L Daniels

Publications and source records attributed to T L Daniels.

6 recordsLinked to original sources

PubMed2001

A novel monoclonal antibody specific for the N-terminal end of GAD65.

We describe a novel monoclonal antibody raised towards the N-terminus of the 65 kDa isoform of glutamate decarboxylase (GAD65). This N-GAD65 mAb is highly specific for GAD65 and can be used in a wide range of applications such as Western blot analysis, immunoprecipitation and immunocytochemistry. Full-length cDNAs coding for N-GAD65 mAb heavy and light chains were cloned and characterized by nucleotide sequence analysis. Both heavy and light chain-specific cDNAs are functional and are members of mouse heavy chain subgroup IgG1 and kappa chain group 2, respectively. Comparing the N-GAD65 mAb gene sequences with GenBank shows that the cDNAs were not reported previously.

Amino Acid Sequence↗

PubMed2001

Preservation of enzyme activity and antigenicity after mutagenesis of the membrane anchoring domain of GAD65.

The smaller isoform of glutamic acid decarboxylase, GAD65, is an important autoantigen implicated in the pathogenesis of type 1 diabetes whereas the larger isoform, GAD67 appears to play no major role. The primary difference between the two isoforms resides in the N-terminal part of the molecule including the GAD65 membrane-anchoring domain. The aim of this study was to generate mutants of the membrane targeting domain spanning amino acids 24 to 31 of GAD65 to determine effects on enzyme activity and antibody recognition. Three GAD65 mutants were generated by substituting two, nine or eleven nucleotides coding for the membrane targeting with the corresponding bases of GAD67. SDS-PAGE and Western blotting wildtype (wt) and mutated GAD65 ascertained that they were of similar size and recognized GAD65-specific antibodies. No difference in enzymatic activity was found between the mutants and wt GAD65. GAD65 antibody positive sera from type 1 diabetes patients immunoprecipitated mutated GAD65 whether two, nine or eleven nucleotides were replaced. Mono-or polyclonal antibodies to the N-terminal region demonstrated that the mutated GAD65 with two or nine nucleotides replaced was immunoprecipitated markedly better than wt whereas no difference was detected using antibodies specific for the PLP-binding site in the middle part of GAD65 or the C-terminal region. Taken together, these data suggest that no major conformational changes have been introduced by mutating the membrane-anchoring domain of GAD65.

Autoimmunity↗

PubMed2000

GAD65-specific autoantibodies enhance the presentation of an immunodominant T-cell epitope from GAD65.

GAD65 autoantibodies (GAD65Ab) are highly prevalent in type 1 diabetes, but their functional role in the pathogenesis of the disease and their relationship to T-cell reactivity to GAD65 is still unclear. We tested the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. T-cell hybridoma T33.1, which recognizes the GAD65 274-286 epitope in the context of HLA-DRB 1*0401, was incubated with antigen-presenting cells exposed to recombinant human GAD65 alone or complexed with GAD65Ab' or GAD65Ab- sera. Stimulation of the T33.1 hybridoma was greatly enhanced by multiple GAD65Ab+ sera. The enhancement effect was most prominent with sera from patients with high GAD65 autoantibody levels. Sera from GAD65Ab- subjects had no effect. The correlation between T-cell stimulation and GAD65Ab levels was not absolute, suggesting that other variables such as autoantibody recognition of different regions of GAD65 and variable effects on processing of the 274-286 epitope may contribute. Uptake of antibody-complexed GAD65 was Fc receptor (FcR)-mediated because the enhancement of presentation was inhibited by monoclonal antibodies against FcR. Our results support the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. Increased antigen uptake and heterogeneity in the autoantibody specificity may provide a mechanism for antibody-facilitated T-cell response influencing the progression of type 1 diabetes.

Adolescent↗

PubMed1997

Women with functional hypothalamic amenorrhea but not other forms of anovulation display amplified cortisol concentrations.

OBJECTIVE: To test the hypothesis that increased cortisol secretion is specific to women with decreased GnRH drive and not found in eumenorrheic women or those with other causes of anovulation. DESIGN: Cortisol concentrations in blood were determined at 30-minute intervals for 24 hours in three well-characterized groups: women with functional hypothalamic amenorrhea, those with other causes of anovulation, and eumenorrheic women. SETTING: Academic medical center. PATIENT(S): Women aged 20 through 35 years, with well-defined reproductive states. INTERVENTION(S): Venous blood samples were obtained from, and psychometric inventories were completed by, the participants. MAIN OUTCOME MEASURE(S): Twenty-four-hour cortisol levels, 24-hour LH pulse patterns, and serial P levels were measured in women with functional hypothalamic amenorrhea, eumenorrheic women, and those with other causes of anovulation. RESULT(S): Cortisol secretion was higher in women with functional hypothalamic amenorrhea (n = 19) than in those with other causes of anovulation (n = 19) or eumenorrheic women (n = 19). Six women who recovered from functional hypothalamic amenorrhea had cortisol levels comparable to those of eumenorrheic women and those with other causes of anovulation. CONCLUSION(S): These data underscore the association between increased hypothalamic-pituitary-adrenal activity and reduced GnRH drive and support the concept that functional hypothalamic amenorrhea develops in response to stress-induced alterations in central neural function that modify hypothalamic function.

Adult↗

PubMed1997

Can polycystic ovary syndrome exist without concomitant hypothalamic dysfunction?

Polycystic ovary syndrome describes a conformational ovarian state that may be the final common manifestation of several pathogenic pathways. Because the ovarian thecal and stromal hyperplasia characteristic of polycystic ovarian (PCO) morphology depends upon relative LH excess while follicular arrest requires a relative deficiency of FSH, it is likely that the PCO morphology cannot be expressed or maintained without both prior and concomitant exposure to the characteristic alterations in hypothalamic-pituitary secretion. Although increased LH and decreased FSH secretion are hypothesized to be integral to the expression of this morphological state and, in this limited sense, causal, this dependence does not necessarily mean that hypothalamic alterations are the primary etiology of this syndrome. However, recognition of the relationship between gonadotropin secretory alterations and the development of the PCO state does have treatment implications.

Female↗

PubMed1997

Resistance of gonadotropin releasing hormone drive to sex steroid-induced suppression in hyperandrogenic anovulation.

Women with hyperandrogenic anovulation (HAA) exhibit increased GnRH drive, as evidenced by a faster LH pulse frequency that slows in response to progestin-induced opioidergic tone. To determine whether increased GnRH-LH drive in HAA reflects altered sex steroid exposure caused by chronic anovulation or is an intrinsic hypothalamic attribute, we compared the pulsatile LH response to oral contraceptive (OC)-induced suppression in seven women with HAA, with that of seven eumenorrheic women (EW). LH levels were determined at 10-min intervals for 12 h after 19-21 days of OC use and 5-7 days after cessation. Testosterone, androstenedione, estradiol, FSH, and LH levels were determined at weekly intervals before, during, and after OC use. LH pulse number/12 h was higher (P < 0.001) in HAA during and after OCs, when compared with that of EW. Mean LH was increased in HAA before, during, and after OCs. Testosterone, androstenedione, and estradiol levels were higher in HAA before OCs, but they decreased to similar levels during OC use in both groups. FSH concentrations were similar before and during OCs but rose more after cessation of OCs in EW. These findings indicate that GnRH drive in HAA is resistant to OC-induced suppression and, therefore, could be an intrinsic hypothalamic attribute.

Adult↗