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B Buckingham

Publications and source records attributed to B Buckingham.

At least 19 recordsLinked to original sources
PubMed2001

Semiquantitative polymerase chain reaction in RNase-producing tissues: Analysis of the developing pancreas.

BACKGROUND/PURPOSE: Many studies in pediatric surgical research use a quantitative analysis of gene expression in microscopic quantities of tissue. The authors describe an analysis of the beta-tubulin mRNA content of the embryonic pancreas, which contains abundant endogenous RNases. A detailed analysis of this RNase-containing system will provide a good template for analysis of other potentially simpler systems. METHODS: Embryonic mouse pancreases were harvested at serial gestational ages. DAPI nuclear staining allowed for counting of cells. cDNA was amplified using a fluoresceinated primer and the normalized fluorescence determined. Known numbers of molecules were amplified in parallel as a standard control. RESULTS: The number of cells increased from 38,000 to 2,700,000 between embryonic day 10.5 (E10.5) and E18.5. mRNA for beta-tubulin did not increase proportionately. Assuming a yield of 100% at E10.5 when no RNases are present, the yield of expected mRNA was 65.3% at E12.5, 13.8% at E15.5, and 0.9% at E18.5, presumably because of the appearance of RNases. CONCLUSIONS: Several parameters must be considered in performing semiquantitative reverse transcription polymerase chain reaction: (1) the yield of RNA based on the projected amount of mRNA, (2) the number of cells in the tissue, and (3) a known number of template molecules amplified in parallel.

Animals↗
PubMed2001

Familial isolated hypoparathyroidism caused by a mutation in the gene for the transcription factor GCMB.

Hypoparathyroidism is characterized by hypocalcemia, hyperphosphatemia, and absent or markedly reduced circulating concentrations of parathyroid hormone. The transcription factor GCMB is predominantly, if not exclusively, expressed in parathyroid cells and is critical for development of the parathyroid glands in mice. Thus, in the present study we examined the GCMB gene, mapped to 6p23-24, as a candidate for isolated hypoparathyroidism. We defined the boundaries of the five exons of the human GCMB gene and then identified a large intragenic mutation in the GCMB genes of the proband of an extensive kindred with isolated hypoparathyroidism. Her parents and several other unaffected relatives were heterozygous for the mutation. Despite an absence of any history of consanguinity, microsatellite analysis showed shared genotypes that flanked the GCMB gene over a span of 5 cM, suggesting that both of the proband's GCMB alleles had been derived from a single common ancestor. Analysis of additional, unrelated cases did not disclose the same mutation. We conclude that homozygous loss of function of the GCMB gene impairs normal parathyroid gland embryology and is responsible for isolated hypoparathyroidism in a subset of patients with this disease.

Alleles↗
PubMed1995

T-->A transversion 11 bp from a splice acceptor site in the human gene for steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia.

Congenial lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH. Affected individuals can make no adrenal or gonadal steroids. All affected individuals are phenotypic females irrespective of gonadal sex, and frequently die in infancy if mineralocorticoid and glucocorticoid replacements are not instituted. Recent data implicate the steroidogenic acute regulatory (StAR) protein in this disorder. We now describe a 46,XY patient of Vietnamese ancestry with lipoid CAH who had a somewhat milder form of the disease. Diagnosis was at 10 weeks of age, and low levels of plasma progesterone, corticosterone, 180H-corticosterone and androstenedione were detectable. Testicular RNA for StAR was reverse transcribed, amplified, cloned and sequenced, revealing a 185 bp deletion corresponding to all of exon 5. The corresponding mRNA did not encode active protein in transfected cells. Cloned genomic DNA from the patient revealed only a T-->A transversion in intron 4,11 bp from the splice acceptor site of exon 5. This transversion destroys an NcoI site; digestion of PCR-amplified genomic DNA from the patient and both parents confirmed that the patient was homozygous and the parents were heterozygous. Expression vectors for StAR minigenes were constructed containing all StAR exons plus introns 4, 5 and 6 either with or without the T-->A mutation in intron 4. RNase protection assays showed that expression of the vector with normal intron 4 yielded correctly spliced StAR mRNA in transfected COS-1 cells, while most, but not all StAR mRNA from the vector with the T-->A transversion in intron 4 was abnormally spliced. RNase protection of the patient's testicular RNA confirmed that most, but not all StAR mRNA was similarly spliced abnormally. Splicing errors appear to be a rare cause of genetic diseases, but subtle intronic mutations may be missed when genomic DNA is the only material available for study. The low level of normal StAR mRNA produced may account for the later clinical presentation and low levels of steroid hormones detected in this patient.

Adrenal Hyperplasia, Congenital↗
PubMed1992

Biochemical abnormalities in vitreous of humans with proliferative diabetic retinopathy.

Vitreous changes in diabetes can exacerbate proliferative diabetic retinopathy. These changes may be due to the effects of diabetes on vitreous collagen. Vitreous samples from 19 patients with proliferative diabetic retinopathy and 23 patients without diabetes were analyzed for collagen crosslinks, as well as for the early glycation products, glucitolyllysine and glucitolylhydroxylysine. Fluorometry was performed to measure advanced glycation end products. Vitreous collagen derived from diabetic patients was found to have significantly higher levels of the crosslink dihydroxylysinonorleucine (3.15 vs 1.24 mol/mol collagen, P<.01) than that of control subjects. Early glycation products were elevated in diabetic vitreous (1.65 vs 0.54 mol/mol collagen, P<.05). Levels of advanced glycation end products were 20 times higher in diabetic vitreous compared with the vitreous of controls. These diabetes-induced alterations of human vitreous may be of particular importance given the role of vitreous in proliferative diabetic retinopathy and vision loss.

Adult↗
PubMed1991

A naturally occurring 6-9-kilodalton interleukin-1 (IL-1) inhibitor prevents IL-1-mediated islet cytotoxicity but not IL-1-mediated suppression of insulin secretion.

Earlier studies have shown direct effects of interleukin-1 (IL-1) on isolated pancreatic islets. Coculture of isolated rat pancreatic islets with human rIL-1 beta for 6 days resulted in dose-dependent cytotoxicity (up to 100%) and suppression of insulin secretion (up to 88.5%). The cytotoxic effects of rIL-1 beta beta were blocked by the simultaneous presence of a naturally occurring 6-9-kilodalton (kDa) inhibitor of IL-1-induced T-cell proliferation. However, the ability of rIL-1 beta to suppress insulin secretion was not blocked by the 6-9-kDa inhibitor of IL-1 activity. This IL-1 inhibitor is produced by mononuclear cells and is resistant to pH 2, sensitive to heating at 56 degrees C for 30 min, has a pI of 4.5-5.6, and appears to be different from other recognized IL-1 inhibitors in both composition and mechanism of action. Unlike this IL-1 inhibitor, a monoclonal antibody specific for rIL-1 beta was able to neutralize both the islet cytotoxic and insulin modulatory effects of rIL-1 beta. These results demonstrate the use of an IL-1 inhibitor to prevent at least one mechanism of islet destruction, and suggest separate pathways for IL-1 mediated islet cytotoxicity and suppression of insulin secretion.

Animals↗
PubMed1990

Heterogeneity in the specificity of the islet cell cytoplasmic antibody response in insulin-dependent diabetes mellitus.

We assessed the heterogeneity in the islet cell cytoplasmic antibody (ICA) response of insulin-dependent diabetes mellitus (IDDM) patients via indirect immunofluorescence on frozen sections of human, bovine, and porcine pancreas. The three substrates detected comparable frequencies of ICA positives among the IDDM sera tested, whereas control sera were ICA negative on all three substrates. However, individual IDDM serum samples showed heterogeneity in ICA binding on the three pancreata. Of 28 sera tested on all three substrates, 22 were ICA positive on human pancreas, three were ICA positive on bovine pancreas, and two were ICA positive on porcine pancreas. Sensitivity of ICA epitopes to neuraminidase treatment and periodate oxidation suggests that glycoconjugates are recognized by serum ICA. Cholera toxin blocked ICA binding. However, the functional cholera toxin receptor ganglioside Gm1 is resistant to neuraminidase treatment and periodate oxidation. Therefore, it is unlikely that Gm1 is the ICA determinant. These data suggest that not all ICA antigens are equivalently expressed on islets from different pancreata and/or that each individual responds to a hierarchy of islet antigens such that restricted patterns of specific ICA binding are found.

Animals↗
PubMed1990

Relationship between the content of lysyl oxidase-dependent cross-links in skin collagen, nonenzymatic glycosylation, and long-term complications in type I diabetes mellitus.

Many abnormalities in collagen have been reported in insulin-dependent diabetes mellitus, some or all of which have been attributed to increased cross-linking. Although recent work has focused on the role of glucose-derived collagen cross-links in the pathogenesis of diabetic complications, relatively few studies have investigated the role of lysyl oxidase-dependent (LOX) cross-links. In the present study, LOX cross-links and nonenzymatic glycosylation were quantified in skin collagen from diabetic subjects. There was an increase in the difunctional cross-link dihydroxylysinonorleucine (DHLNL) as well as in one of its trifunctional maturation products, hydroxypyridinium. All other LOX crosslinks were normal. Nonenzymatic glycosylation was increased in diabetic skin collagen, and this increase was correlated with increases in DHLNL (P less than 0.001). The biochemical results were examined for correlations with clinical data from the same subjects. Increases in DHLNL content were associated with duration of diabetes (P less than 0.003), glycohemoglobin levels (P less than 0.001), hand contractures (P less than 0.05), skin changes (P less than 0.005), and microalbuminuria (P less than 0.01). In nondiabetic subjects age was not correlated with collagen cross-link content with the exception that his-HLNL increased with age (r = 0.79, P less than 0.02). In diabetic subjects, PA levels decreased with age (r = 0.51, P less than 0.02). With increased duration of diabetes, DHLNL content was increased (r = 0.55, P less than 0.003) and OHP was increased (r = 0.59, P less than 0.01), whereas PA levels were decreased (r = -0.48, P less than 0.04). Nonenzymatic glycosylation of collagen was also increased with increased duration of diabetes (hex-lys, r = 0.47, P less than 0.02; hex-hyl, r = 0.39, P less than 0.05). We conclude that: (a) lysyl oxidase-dependent cross-linking is increased in skin collagen in diabetes and (b) that these changes in skin collagen are correlated with duration of diabetes, glycemic control, and long-term complications.

Adolescent↗
PubMed1988

Interferon responsiveness of natural killer cells in type I human diabetes.

Abnormally low circulating numbers and function of NK cells are associated with new onset type I diabetes. Since alpha interferon is a stimulator of NK function, enriched T and non-T lymphocytes were incubated with 0, 100 and 1,000 units/ml of recombinant alpha interferon (rIFN alpha) and natural killing against K562 and pancreatic islet cell targets was measured. The killing of K562 (1:20 target:effector ratio) cells by non-T cells incubated with 0, 100 and 1,000 units/ml of rIFN alpha in patients was decreased to 27% (p less than 0.014 vs control), 34% (p less than 0.001) and 39% (p less than 0.003) when compared to killing by normal control non-T cells (48%, 74% and 58% respectively). T cell mediated killing of K562 cells in patients was decreased to 3.9% (p less than 0.03), 5.3% and 6.6% (p less than 0.003) when compared to that of controls (8.7%, 10-8% and 22.6% respectively). Non-T cell mediated killing of islet cells (1:20 target:effector ratio) following treatment of effector cells with 0, 100 and 1,000 units/ml of rIFN alpha in patients was 19%, 27%, and 26% which was comparable to control subjects killing of 31%, 18% and 37% respectively. Similar data were obtained using T-cells as effectors. These data indicate that in new onset type I diabetes; (a) NK cell functional activity is diminished in both T and non-T lymphocyte subpopulations and (b) NK activity is suboptimally enhanced with rIFN alpha.

Adolescent↗
PubMed1987

Natural killer cell and islet killer cell activities in human type 1 diabetes.

Peripheral blood mononuclear cells from 14 type 1 diabetic patients were examined for natural killer cell activity using the K562 cell line as 51Cr labeled targets. Mean cytotoxicity of K562 cells by unseparated mononuclear cells derived from new onset type 1 patients (12 +/- 1.6%) was lower (P less than .01) than that observed in non diabetic controls, (25 +/- 4.2%). Mean natural killer cell cytotoxicity mediated by enriched non-T cells from patients (41 +/- 5.8%) was also lower (P less than 0.03) than in the control group (56 +/- 3.7%). Specificity of these findings was evaluated by also examining other diabetic patient subgroups. Mean non T cell mediated natural killer cell activity in type 2 diabetic patients and type 1 patients with long term disease was 65 +/- 5.4% and 62 +/- 4.8% respectively (p less than 0.001 vs new onset type 1 patients). Longitudinal studies of new onset type 1 patients during the remission (honeymoon) phase revealed no improvement of impaired natural killer cell activity. In 30 new onset and 11 remission diabetic patients, mean non-T cell-mediated cytotoxicity was also measured using dispersed 51Cr labeled pancreatic islet target cells. Mean islet cytotoxicity mediated by cells from new onset patients was 34 +/- 2.4%, whereas in nondiabetic control subjects mean cytotoxicity was 25 +/- 1.8% (p less than 0.005). During remission, islet cytotoxicity returned to normal values in over half of the patients. There was no correlation between K562 and islet cell cytotoxicity in either of the latter two patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1986

Skin, joint, and pulmonary changes in type I diabetes mellitus.

Three hundred seventy-five patients with diabetes mellitus were examined for the presence of sclerodermalike skin changes, limited joint mobility, and vital capacity changes. Nineteen percent of patients had vital capacities 2 SDs below the mean of predicted values. There was no significant relationship between decreased vital capacities and duration of diabetes, sclerodermalike skin changes, limited joint mobility, smoking history, proteinuria, or retinopathy. Cutaneous involvement consisting of thickening, tightening, and/or a waxy quality of the skin was noted in 190 patients (51%). The severity of skin involvement correlated positively with the patients' duration of diabetes, age, severity of joint contractures, and diabetic retinopathy. Thus, sclerodermalike skin changes appear to reflect generalized connective tissue alterations in diabetes and may indicate increased risk for diabetic microvascular complications.

Adolescent↗
PubMed1986

Natural killer cell and islet killer cell activities in type 1 (insulin-dependent) diabetes.

Peripheral blood mononuclear cells from 20 Type 1 (insulin-dependent) diabetic patients were examined for natural killer cell activity using the K562 cell line as 51Cr labeled targets. Mean natural killer cell cytotoxicity mediated by enriched non-T cells from patients (37 +/- 4.0%) was lower (p less than 0.03) than in controls (56 +/- 3.7%). Specificity was evaluated by examining other patient subgroups. Mean non-T cell mediated natural killer cell activity in Type 2 (non-insulin-dependent) diabetic patients and Type 1 patients with long term disease was 65 +/- 5.4% and 62 +/- 4.8% respectively (p less than 0.003 vs new onset Type 1 patients). Longitudinal studies of new onset Type 1 patients during the remission (honeymoon) phase revealed persistently impaired natural killer cell activity in 3 of 4 patients. In 30 new onset and 11 remission Type 1 diabetic patients, mean non-T cell-mediated cytotoxicity was also measured using dispersed 51Cr labeled islet target cells. Mean islet cytotoxicity mediated by cells from new onset patients was 34 +/- 2.4%, whereas in non-diabetic control subjects mean cytotoxicity was 25 +/- 1.8% (p less than 0.005). During remission, islet cytotoxicity remained at similar or elevated levels in most patients. In patients evaluated simultaneously for K562 and islet cell cytotoxicity, natural killer cell activity was decreased, whereas islet killing was increased. These results suggest a dichotomy in natural killer cell and islet killer cell activities in new onset Type 1 diabetes that could have an important role in the pathogenesis of Type diabetes.

Adolescent↗
PubMed1986

Multiparameter immunologic studies in patients with newly diagnosed type 1 insulin-dependent diabetes mellitus.

Peripheral blood mononuclear cells from patients with Type 1 diabetes mellitus were examined for the proportion of monoclonal antibody-defined T-cell subsets, natural killer (NK) cells, and macrophages and the proliferative response to phytohemagglutinin (PHA), Concanavalin A (Con A), pokeweed mitogen (PWM) and in the autologous mixed lymphocyte reaction (AMLR) and allogeneic mixed lymphocyte reaction (MLR). The in vitro response of purified IL-2 on PHA- and PWM-induced proliferative response was also examined. Total T cells (Leu 1+), helper/inducer phenotype (Leu 3+) T cells, suppressor/cytotoxic phenotype (Leu 2+) T cells, surface Ig+ B lymphocytes and monoclonal antibody-defined monocytes (Mac +) in patient group were comparable to the control group. The Leu 7+ NK cells were, however significantly (p less than 0.05) decreased in the diabetic group. The NK function was also deficient in the diabetic group when compared to healthy non-diabetic controls. The proliferative responses to all 3 concentrations of PHA, PWM, and Con A, and in the MLR were similar in 2 groups. However, the proliferative response in the AMLR was significantly reduced (p less than 0.05) in the diabetic group. Exogenous purified IL-2 failed to induce any enhancement in the PHA- and PWM-induced proliferative response; this was in contrast to control group in which IL-2 enhanced proliferative response to both mitogens. This study demonstrates deficiency of the AMLR, Leu 7+ and of natural killer cell function and unresponsiveness of mitogen-activated T cells to purified IL-2. The significance of these findings is discussed.

Adolescent↗
PubMed1985

Complement activation in type 1 human diabetes.

Complement activation was quantitated in serum and plasma of diabetic and normal subjects by sensitive competitive equilibrium radioimmunoassays (RIA) for C3a, C4a, C5a, Factor B, and a newly described C5 neoantigen (termed C5 activation antigen, and abbreviated C5-AA) in a stable 54-kDa fragment of C5. Plasma C3a levels were significantly elevated in 8 of 16 patients with newly diagnosed Type 1 diabetes (P less than 0.0005) with the mean C3a concentration for these patients being more than 10-times greater than the mean value of normal controls. C4a levels were also elevated in 2 of these patients (P less than 0.02), but C5a levels, although higher than normal, were not significantly increased. In contrast, the levels of C5-AA in the serum of all patients (11/11) with chronic Type 1 diabetes were significantly higher than in control Type 2 patients (noninsulin-dependent diabetes) (P less than 0.0005) and 4 of 7 patients with new onset insulin-dependent diabetes mellitus also had significantly higher levels of C5-AA than the Type 2 patients (P less than 0.01). The levels of Factor B in the serum of 5 of 9 patients with new onset diabetes were significantly higher than normal (P less than 0.0025). Five recent onset Type 1 diabetes patients were evaluated longitudinally for C3a, C4a, and C5a: in 3 the levels of C3a were elevated during new onset disease decreasing into the normal range during remission; in 2 of these patients C4a was also significantly elevated and the levels decreased during remission; and in 3 patients the levels of C5a were not significantly elevated but they decreased during remission. Purified human complement proteins and complement hemolytic assays were used to measure complement activation in serum during incubation with rat pancreatic islet cells. With diluted normal human serum, less than 20% of C3 or Factor B were consumed during 30 min at 37 degrees C, while with new onset Type 1 diabetic patient sera up to 90% of C3 and Factor B were consumed in 5/6 sera and 4/6 sera, respectively. These findings suggest (a) that complement activation fragments C3a, C4a, and C5a are generated in vivo in new onset Type 1 diabetes; (b) that both the classical and the alternative complement pathways may be activated; and (c) that this may result in a measurable activation of C5 generating biologically and immunologically active C5a and other C5 activation fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗
PubMed1984

Autologous mixed lymphocyte reaction in man: XV. Cellular and molecular basis of deficient autologous mixed lymphocyte response in insulin-dependent diabetes mellitus.

The autologous mixed lymphocyte response (AMLR) and the allogeneic mixed lymphocyte response were deficient in a subset of patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM). Using a single set of HLA-identical twins, the cellular and molecular basis of deficient AMLR was investigated and appears to be due to a defect in both responder T cells and stimulator non-T cells. Interleukin-2 production was diminished in the patient but not in the healthy twin. The in vitro addition of purified interleukin-2 enhanced the depressed AMLR in the diseased twin. This suggests that the deficient AMLR in IDDM may be in part due to a deficiency in the production of interleukin-2.

Adolescent↗
PubMed1984

Deficiency of monoclonal antibody (Leu 7) defined NK cells in newly diagnosed insulin-dependent diabetes mellitus.

Peripheral blood from 11 newly diagnosed patients with insulin-dependent diabetes mellitus (IDDM) was studied for the proportion of monoclonal antibody (HNK 1, Leu 7) defined natural killer (NK) cells using a fluorescence-activated cell sorter analyzer. The proportion of Leu 7+ cells in patients with IDDM (7.0 +/- 4.0) was significantly (P less than 0.001) lower than in simultaneously studied healthy controls (16.8 +/- 7.0). A 2-yr-old boy with recent onset IDDM had a deficiency of Leu 7+ NK cells (6.1%), while his healthy identical twin had normal proportions of Leu 7+ cells (22.2%), when compared to a simultaneously studied healthy control. Two patients reexamined in remission and one other studied in remission alone, showed deficiency of Leu 7+ NK cells. This study demonstrates a quantitative deficiency of monoclonal antibody (Leu 7+) defined NK cells in newly diagnosed patients with IDDM that persists during remission of the disease and therefore appears to be independent of metabolic abnormality. The deficiency of NK cells may predispose genetically susceptible individuals to viral-induced islet cell injury, contributing to the pathogenesis of IDDM.

Adolescent↗
PubMed1984

A psychophysical analysis of acupuncture analgesia.

A psychophysical analysis of acupuncture analgesia was carried out in which low back pain patients made visual analogue scale (VAS) responses both to their chronic pain and to different levels of experimental heat pain (5 sec exposures to 43-51 degrees C) before and after electroacupuncture. VAS (sensory) responses to chronic pain, direct temperature matches to chronic pain, and VAS (sensory) responses to experimental pain were reduced in an internally consistent manner 1-2 h after treatment. The magnitude of this analgesic response was similar for dermatomes within the region of chronic pain and acupuncture needle placement (lower back) as well as for dermatomes remote from needle placement and chronic pain (forearm). Individual patients manifested either (1) a central-inhibitory pattern of analgesia wherein experimental and chronic pain in back regions and experimental pain in forearm regions were reduced, or (2) an origin-specific pattern wherein only the chronic low back pain was reduced. Patients tested several days after treatment all manifested the latter pattern of analgesia. VAS sensory and VAS affective analgesic responses to electroacupuncture treatment showed a delayed onset (1-24 h) to maximum effect and a duration of 10-14 days. Cumulative sensory and affective analgesic effects were observed at the end of 4 months of biweekly acupuncture treatments. The results of this analysis reveal the unique spatial and temporal properties of electroacupuncture analgesia and the extent to which it is mediated by at least two different mechanisms.

Acupuncture Therapy↗
PubMed1984

Immunological events in new onset diabetes.

Data from our laboratory are reviewed showing that both cell-mediated cytotoxicity, complement-dependent antibody-mediated cytotoxicity, antibody-dependent cellular cytotoxicity, and complement-augmented antibody-dependent cellular cytotoxicity may provide mechanisms which kill pancreatic islets during pathogenesis of insulin-dependent (type I) diabetes. Xenogenic test systems employing dispersed rat islet target cells were employed. Most of the findings were reversible during clinical remission of diabetes.

Animals↗