Targeted nNOS gene transfer into the cardiac vagus rapidly increases parasympathetic function in the pig.

Nitric oxide (NO) derived from neuronal nitric oxide synthase (nNOS) facilitates cardiac vagal neurotransmission and bradycardia in vitro. Here we provide evidence of rapid (within 9 h) protein expression and increased vagal responsiveness in vivo following targeted gene transfer of nNOS into the cardiac vagus of the pig. Right vagi were injected with vector encoding nNOS (Ad.nNOS) or saline, while left vagi received an injection of vector encoding enhanced green fluorescent protein (Ad.eGFP). Enhanced nNOS protein expression was detected exclusively in the right vagus nerve, with no evidence of iNOS expression. This was associated with increased baroreflex sensitivity and greater heart rate responsiveness to right vagal stimulation. In contrast, responsiveness of left vagi, or sham-injected right vagi remained constant over the same time period. Basal heart rate was unchanged following gene transfer, suggesting no change in vagal tone. These results support the pre-/post-ganglionic synapse as a site for NO-mediated facilitation of vagal bradycardia in the pig. In addition they demonstrate in vivo that functional gene expression induced with adenoviral vectors occurs earlier than first thought, and may therefore, provide a novel intervention to acutely modulate the neural control of cardiac excitability.

Targeting neuronal nitric oxide synthase with gene transfer to modulate cardiac autonomic function.

Microdomains of neuronal nitric oxide synthase (nNOS) are spatially localised within both autonomic neurons innervating the heart and post-junctional myocytes. This review examines the use of gene transfer to investigate the role of nNOS in cardiac autonomic control. Furthermore, it explores techniques that may be used to improve upon gene delivery to the cardiac autonomic nervous system, potentially allowing more specific delivery of genes to the target neurons/myocytes. This may involve modification of the tropism of the adenoviral vector, or the use of alternative viral and non-viral gene delivery mechanisms to minimise potential immune responses in the host. Here we show that adenoviral vectors provide an efficient method of gene delivery to cardiac-neural tissue. Functionally, adenovirus-nNOS can increase cardiac vagal responsiveness by facilitating cholinergic neurotransmission and decrease beta-adrenergic excitability. Whether gene transfer remains the preferred strategy for targeting cardiac autonomic impairment will depend on site-specific promoters eliciting sustained gene expression that results in restoration of physiological function.

Neuronal nitric oxide synthase gene transfer promotes cardiac vagal gain of function.

Nitric oxide (NO) generated from neuronal nitric oxide synthase (NOS-1) in intrinsic cardiac ganglia has been implicated in parasympathetic-induced bradycardia. We provide direct evidence that NOS-1 acts in a site-specific manner to promote cardiac vagal neurotransmission and bradycardia. NOS-1 gene transfer to the guinea pig right atrium increased protein expression and NOS-1 immunolocalization in cholinergic ganglia. It also increased the release of acetylcholine and enhanced the heart rate (HR) response to vagal nerve stimulation (VNS) in vitro and in vivo. NOS inhibition normalized the HR response to VNS in the NOS-1-treated group compared with the control groups (enhanced green fluorescent protein and sham) in vitro. In contrast, an acetylcholine analogue reduced HR to the same extent in all groups before and during NOS inhibition. These results demonstrate that NOS-1-derived NO acts presynaptically to facilitate vagally induced bradycardia and that upregulation of NOS-1 via gene transfer may provide a novel method for increasing cardiac vagal function.

Effect of deproteination on bone mineral morphology: implications for biomaterials and aging.

Bone mineral morphology is altered by processing and this is rarely considered when preparing bone as a bioimplant material. To examine the degree of transformation, a commercial, coarsely particulate bone mineral biomaterial produced by prolonged deproteination, defatting, dehydration, and heating (donor material) was compared with similar particles of human bone (recipient material) prepared optimally by low-temperature milling. The two powders were freeze-substituted and embedded without thawing in Lowicryl K4M before sectioning for transmission electron microscopy (TEM) (other aliquots were processed by traditional TEM methods). To maximize resolution, electron micrographs were image-enhanced by digitization and printed as negatives using a Polaroid Sprint Scan 45. In addition to their morphology, the particles were examined for antigenicity (specific by reference to fluorescein isothiocyanate [FITC]-conjugated fibronectin, and nonspecific by reference to general FITC-conjugated immunoglobulins). Results showed that the optimally prepared human bone fragments stained discretely for fibronectin with negligible background autofluorescence. In contrast, the bioimplant fragments stained extensively with this and any other FITC-conjugated antibody and, unlike fresh bone, it also autofluoresced a uniform yellow. This difference was also expressed structurally and, although the bioimplant mineral consisted of rhomboidal plates up to 200 nm across and 10 nm thick, the optimally prepared bone mineral was composed of numerous clusters of 5-nm-wide sinuous calcified filaments of variable density and indeterminate length (which became straight needles 50 nm long and 5 nm thick following traditional chemical TEM fixation/staining). It was concluded that the inorganic phase of bone is both morphologically and immunologically transmutable and that, in biomaterials, the transformation is apparently so great that a broad indigenous antigenicity is unmasked, increasing the likelihood of resorption or rejection. This marked change may also provide preliminary insight into a more modest natural aging phenomenon with the localized lateral fusion of calcified filaments into less flexible, more immunologically reactive fenestrated plates.

Impaired glucose tolerance precedes but does not predict insulin-dependent diabetes mellitus: a study of identical twins.

Non-diabetic identical twins of insulin-dependent diabetic patients were studied within five years of the diagnosis of their index twin in order to determine whether changes in intermediary metabolism precede the onset of insulin-dependent diabetes mellitus. Two studies were performed: a cross-sectional study of 12 non-diabetic twins and a prospective study of a separate group of 41 non-diabetic twins. Of the 12 twins tested in the cross-sectional study six developed insulin-dependent diabetes and six did not; the six who developed diabetes were given an oral glucose load a mean of 10 months before diagnosis; they then had normal fasting blood glucose levels but worse glucose tolerance than control subjects (120 min post-load (mean +/- SD) blood glucose 8.5 +/- 3.5 vs 4.9 +/- 0.9 mmol/l respectively, p less than 0.05). However, blood lactate, pyruvate, alanine, glycerol, 3-hydroxybutyrate and serum insulin levels were similar. In contrast, the six twins in this cross-sectional study who did not develop diabetes and are now unlikely to do so, as a group, had no significant changes compared with the control subjects though one had impaired glucose tolerance. To determine the predictive value of impaired glucose tolerance a separate group of 41 non-diabetic twins was studied prospectively for 8 to 22 years having a total of 147 glucose tolerance tests in this period; in this group six developed diabetes. Eight of the 41 had impaired glucose tolerance; impaired glucose tolerance was found in four of the six who developed diabetes as compared with only four of the 35 who did not (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

B-cell responses to intravenous glucose and glucagon in non-diabetic twins of patients with type 1 (insulin-dependent) diabetes mellitus.

The B-cells of patients with recently diagnosed Type 1 (insulin-dependent) diabetes may have no response to glucose when the response to glucagon is present but attenuated. This observation suggests that the recognition of glucose is more severely affected than that for non-glucose stimulants. To determine whether a similar selective decrease in glucose response was present before the onset of diabetes we studied two groups of non-diabetic identical twins of patients with recently diagnosed Type 1 diabetes: one group with complement-fixing islet cell antibodies who were at high risk of developing diabetes (four of the five have already developed diabetes) and a group without such antibodies at low risk of developing diabetes. In addition, a group of patients with chronic pancreatitis were studied to control for non-specific damage to the B-cell. Responses to i.v. glucose and i.v. glucagon were compared. Patients with chronic pancreatitis has similar responses to both glucose and glucagon and the responses did not differ from control subjects. The B-cells of the immune positive group showed evidence of pathology because the insulin and C-peptide responses to both stimuli were reduced when compared to either their control subjects or the immune negative twin group. However, the B-cell response to both glucose and glucagon in the immune positive twins was similar. Because the B-cell response to glucose was not less than that to glucagon, a selective destruction of the glucose recognition system cannot be a characteristic of all twins throughout the period before they develop Type 1 diabetes.

The significance of the concordance rate for type 1 (insulin-dependent) diabetes in identical twins.

We studied prospectively 49 non-diabetic identical twins of recently-diagnosed Type 1 (insulin-dependent) diabetic patients for up to 24 years (median 9 years). During this time 15 developed Type 1 diabetes. Actuarial analysis indicates that by 12 years 34% of the twins will have developed Type 1 diabetes and that thereafter only another 2% will do so. Inevitable bias in ascertainment of the twins makes it likely that the true figure is less. We conclude that factors which are not genetically determined must be important in the pathogenesis of the disease. The rates of developing Type 1 diabetes in the co-twins declines sharply in the years after diagnosis of the index twin, which suggests that the initiation of the process leading to Type 1 diabetes occurs within a finite, and not a prolonged, period.

Increased proinsulin levels as an early indicator of B-cell dysfunction in non-diabetic twins of type 1 (insulin-dependent) diabetic patients.

Glucose tolerance and insulin secretion were studied in two groups of non-diabetic identical twins of recently-diagnosed Type 1 (insulin-dependent) diabetic patients: (1) a group of 5 twins with islet cell antibodies, and (2) a group of 6 twins without. Despite similar fasting glucose, insulin and C-peptide concentrations both groups of twins had significantly higher fasting proinsulin concentrations than the control group (p less than 0.05). The twins with complement-fixing islet cell antibodies had reduced glucose tolerance and clearance, whilst the twins without islet cell antibodies did not. Neither group of twins showed any abnormality in insulin, C-peptide or proinsulin response to oral or intravenous glucose. We conclude that increased fasting proinsulin levels precede abnormalities of insulin secretion, and are an early indication of minor B-cell damage in these twins irrespective of their risk of developing diabetes.

Long-term use of an injectable contraceptive: effect of depot-norethisterone oenanthate on carbohydrate metabolism.

In order to determine the metabolic effects of long-term use of the injectable contraceptive norethisterone oenanthate, plasma glucose and serum insulin concentrations were studied in two groups of women who had used the method continuously for at least five years. Group 1 comprised 24 subjects, from whom only fasting blood samples were taken. Despite similar plasma glucose concentrations to those of the controls, the subjects had significantly increased serum insulin concentrations (164.5 (39.9) v 120.3 (34.3) pmol/l, p less than 0.01). In addition the insulin:glucose ratios were also significantly increased (34.3 (8.5) v 24.6 (6.7), p less than 0.01), consistent with decreased insulin sensitivity. Group 2 comprised 13 of the original 24 subjects who also had an oral glucose tolerance test. Basal plasma glucose concentrations were similar in the subjects and their controls, whilst the significantly increased insulin:glucose ratios (35.0 (7.7) v 28.7 (5.6), p less than 0.05) were consistent with the results of the larger group. Following oral glucose challenge, plasma glucose concentrations, serum insulin concentrations and insulin:glucose ratios were similar in the subjects and their controls throughout the test. Thus, long-term use of norethisterone oenanthate injections is associated with a decrease in peripheral insulin sensitivity. However, these changes are not associated with any evidence of oral glucose intolerance.

Evidence of beta cell dysfunction which does not lead on to diabetes: a study of identical twins of insulin dependent diabetics.

Ten non-diabetic identical twins of insulin dependent diabetics were studied to see whether they showed changes in insulin secretion. The twins were selected because more than 11 years had elapsed since the diagnosis of the diabetic twin and they were therefore unlikely to develop diabetes, and they had had islet cell antibodies. Despite similar glucose concentrations to the controls the twins had greater total immunoreactive insulin responses to both oral (mean 3280 (SD 699) versus 2338 (1110) pmol/dl at 180 minutes; p less than 0.05) and intravenous (1346 (690) versus 699 (294) pmol/dl at 30 minutes; p less than 0.05) glucose challenge. The C peptide responses to intravenous glucose were also increased consistent with increased insulin secretion. In addition, basal serum proinsulin concentrations in the twins were increased (2.1 (1.2) versus 1.0 (0.3) pmol/dl; p less than 0.01) and remained so throughout both tests. These twins, who were unlikely to develop insulin dependent diabetes, showed evidence of beta cell dysfunction which does not progress to diabetes.