Samia Girgis

SummaryBy use of indirect immunofluorescence, this study demonstrated the presence of calcitonin gene-related peptide-like immunoreactive (CGRPI) fibers in the bladder of the rat. These fibers were abundant in the muscle layer, in which they ran parallel to the muscles, submucosa, and epithelium. No immunoreactive cells were detected. We also examined the origins of these fibers, using a method that combined biotinized retrograde tracer (biotin-wheat germ agglutinin) (B-WGA) and immunocytochemistry. Injection of the tracer into the bladder resulted in the demonstration of small to medium-sized labeled cells that contain CGRPI structures in single dorsal root ganglion cells mostly at the level of L6 and S1, but also a few at L2.Double-staining for CGRPI and immunoreactive P-like substance (SPI) indicated that there are cells in the dorsal root ganglia at the level of L6 and S1 that react to both, but that there are many CGRPI-positive cells that contain no demonstrable SPI; most of t...

Before interpreting the results of assays which claim to measure circulating levels, one must have full data on the sensitivity, the reproducibility and specificity of the methods and antisera used. Without this information figures are meaningless and may be regarded as fiction rather than science. Secondly, and of equal importance, one must have full details of those physiological and pharmacological factors which are known to affect the circulating levels. It is quite obvious to workers in the field that those laboratories which have paid attention to these matters, have achieved remarkable agreement in practice; those who have neglected these requirements have introduced quite unnecessary confusion in the literature.

The present study demonstrates synaptic contact between calcitonin gene-related peptide (CGRP)-like immunoreactive axon terminals and sympathetic neurons in the rat celiac ganglion. Our observations suggest that sensory ganglion neurons directly regulate the sympathetic activity via synapses, because CGRP immunoreactive (CGRPI) fibers in this ganglion are supplied by the sensory ganglia.

Familial dysautonomia (FD) patients have diminished sensory C-fibers. Calcitonin gene related peptide (CGRP) is a widely distributed neuropeptide and prominent neurotransmitter in C-fibers. We show that plasma CGRP levels measured by radioimmunoassay is significantly lower in 51 FD patients compared to controls (P<0.001). In 11/51 FD patients with FD crisis and in 19/51 FD patients with pneumonia, the mean CGRP levels rose significantly as compared to their baseline (P<0.003, P<0.001, respectively). The deficiency of CGRP in FD patients is consistent with their depletion of C-fibers, and may explain some of their symptoms, either directly or via modulation of sympathetic activity.

Plasma osteocalcin, a marker of osteoblastic activity, is reduced in starvation, malnutrition, and anorexia nervosa, resulting in low bone turnover osteoporosis. Contradictory findings about the role of leptin as a link between nutritional status and bone physiology have been reported. We demonstrate that leptin-deficient ob/ob and leptin-resistant db/db male mice have increased plasma osteocalcin, and that in male ob/ob mice osteocalcin is not decreased by starvation, unlike control mice. Intraperitoneal leptin administration increased plasma osteocalcin in male ob/ob mice, and prevented its fall during 24h fasting and 5 days of food restriction in normal male mice. This effect may be mediated via actions on the hypothalamic-pituitary-testicular or -growth hormone axes, or a direct action on osteoblasts. These studies support the hypothesis that the fall in leptin during starvation and weight loss is responsible for the associated reduction in osteoblast activity, and suggest a role for leptin in regulating bone turnover.

194 Brain Research, 330 (1985) 194-196ElsevierBRE 20685Coexistence of calcitonin gene-related peptide and substance P-like peptide insingle cells of the trigeminal ganglion of the rat: immunohistochemical analysisY. LEEl, Y. KAWAP, S. SHIOSAKA1, K. TAKAMIl, H. ...

This study was an examination of the ultrastructural characteristic features of calcitonin gene-related peptide (CGRP)-like immunoreactive neurons and their axon terminals in the nucleus of the tractus solitarius of the rat. Some axon terminals were identified as receiving synaptic inputs from non-immunoreactive axon terminals. This may suggest that part, if not all, CGRP containing afferents are affected presynaptically by other afferents.

1. The recently discovered peptide islet amyloid polypeptide shows considerable sequence homology with calcitonin-gene-related peptide, itself an alternative product of the calcitonin gene. The possibility that islet amyloid polypeptide might affect calcium homoeostasis and bone cell function was investigated. 2. Islet amyloid polypeptide messenger RNA was found to be expressed by human HTb 96 osteoblast-like cells in culture, and islet amyloid polypeptide immunoreactivity was present in the cell culture medium. 3. Infusion of islet amyloid polypeptide (150 pmol min-1 kg-1) caused a fall in serum calcium and phosphate concentrations in five patients with Paget's disease of the bone. This was similar to that caused by infusion of calcitonin (50 pmol min-1 kg-1). 4. These findings raise the possibility that islet amyloid polypeptide may act as a local factor within bone, produced by osteoblasts and regulating osteoclast function. The possibility of an action of islet amyloid polyp...

Katacalcin (KC) is situated on the C-terminal side of the procalcitonin molecule and is cleaved like calcitonin (CT) from this precursor peptide. Serum levels of KC were measured in 22 patients with C-cell carcinoma with a specific and sensitive radioimmunoassay (normal range, less than 0.1-0.15 ng/ml). Basal serum KC values in C-cell carcinoma patients were 0.32-290 ng/ml. There was a good correlation between KC and CT (r = 0.98, P less than 0.001). Serum KC, as well as CT, markedly increased after pentagastrin and calcium infusion. KC and CT were secreted in nearly equimolar amounts. During selective venous catheterization, KC and CT levels were increased in serum samples from veins draining tumor masses, which could be confirmed operatively. During the follow up, KC and CT measurements correlated well to the stage of disease. KC could be immunohistologically localized in C-cell carcinoma tissue. As a tumor marker, katacalcin is likely to be as useful as calcitonin in C-cell carcinoma.

Vitamin D deficiency in adults causes osteomalacia where there is a defect in bone mineralization resulting in an excess of unmineralised osteoid in the bone matrix. The aim of this study was to evaluate the markers of bone formation: total (TALP), bone-specific alkaline phosphatase (BSALP) and procollagen type I carboxyterminal peptide (PICP) in vitamin D deficiency. We studied 100 vitamin D deficient subjects and 82 gender-matched controls. Vitamin D deficiency was defined as serum 25-hydroxyvitamin D level of less than 7 ng/ml, and greater than 10 ng/ml for normal controls. Serum TALP assay was performed by a standard automated method, BSALP and PICP were measured by enzyme immunoassays (Metra Biosystems) and vitamin D by radioimmunoassay. There was significant difference in the TALP between female vitamin D deficient and control subjects (mean +/- sem = 99.8 +/- 8.2 vs 70.5 +/- 2.8 iu/l, p<0.001). Elevated serum TALP (>130 iu/l) was found in 20% (20/100) of the vitamin D deficient patients. There were no significant differences in BSALP or PICP between vitamin D deficient patients and gender-matched control subjects. There was no correlation between vitamin D and PICP in patients but in control subjects, a significant negative correlation (r= -0.431, p<0.0001) was found. In conclusion, although elevated TALP was observed in a minority of vitamin D deficient patients, it is a better marker than PICP. The lack of PICP response in vitamin D deficient subjects suggests the possibility of vitamin D deficiency leading to a block in osteoblast differentiation.

Calcitonin gene-related peptide (CGRP), a potent endogenous circulating vasodilator, is produced by the alternative splicing of the calcitonin/CGRP gene and is expressed mainly in neural and cardiovascular tissues. We recently reported a highly sensitive radioimmunoassay of CGRP, based on an antiserum recognizing the C-terminus of the molecule. We have also found that circulating immunoreactive CGRP is heterogeneous; thus we are unable to measure selectively the intact molecule with our one-site competitive approach. We therefore attempted to construct a two-site immunometric assay involving two antibodies, one that detects the C-terminus and another that recognizes the midregion of the molecule. To enhance assay sensitivity, we applied a colorimetric amplification system to this assay. This rapid, robust, and reproducible assay provides more nearly accurate estimates of circulating CGRP and offers a sensitive and more specific alternative to the radioimmunoassay, with advantages in...

Extracts of the corpuscules of Stannius enhance Eel gill calcium efflux and increase bone resorption in the Rat. Thus the hormone is hypocalcaemic in the Eel although hypercalcaemic in the Rat. We show here that a 15 min. pulse perfusion of a synthetic fragment of human PTH 1-34 had a similar effect on gill calcium fluxes to that of CS extract. Further, we found that Eel plasma contains a substance reacting with five different antibodies against bovine PTH. The concentration of this plasma material is enhanced seven-fold by parenteral calcium and rendered extremely low or absent by CS removal. A convenient name for this major calcium-regulating hormone is parathyrine of the corpuscles of Stannius (PCS) since this reflects both its glandular source and chemical structure.

An enzyme amplified immunoassay for rCGRP based on cofactor cycling has been found to be clearly superior to a comparable radioimmunoassay employing the same antiserum in terms of sensitivity, speed and convenience. Correlation between the two methods was very good. With the enzyme amplified immunoassay we have been able to demonstrate the existence of rCGRP in thyroid extract.

Tamoxifen preserves bone in postmenopausal women, but non-steroidal aromatase inhibitors accelerate bone loss and increase fracture risk. We aimed to study the effect on bone health in a subgroup of women included in the Intergroup Exemestane Study (IES), a large randomised trial that compared the switch to the steroidal aromatase inhibitor exemestane with continuation of tamoxifen in the adjuvant treatment of postmenopausal breast cancer. Results were analysed from 206 evaluable patients from the IES, in which postmenopausal women with histologically confirmed and completely resected unilateral breast cancer (that was oestrogen-receptor positive or of unknown status), who were disease-free after 2-3 years of treatment with tamoxifen were randomised to continue oral tamoxifen 20 mg/day or switch to oral exemestane 25 mg/day to complete a total of 5 years of adjuvant endocrine therapy. The primary endpoint was change in bone-mineral density (BMD) assessed by dual energy X-ray absorptiometry. Changes in biochemical markers of bone turnover were also analysed in this substudy, and the incidence of fractures in the entire study reported. The IES is registered on the Current Controlled Trials website . Within 6 months of switching to exemestane, BMD was lowered by 0.051 g/cm(3) (2.7%; 95% CI 2.0-3.4; p<0.0001) at the lumbar spine and 0.025 g/cm(3) (1.4%; 0.8-1.9; p<0.0001) at the hip compared with baseline. BMD decreases were only 1.0% (0.4-1.7; p=0.002) and 0.8% (0.3-1.4; p=0.003) in year 2 at the lumbar spine and hip, respectively. No patient with BMD in the normal range at trial entry developed osteoporosis. Bone resorption and formation markers increased at all time points in women receiving exemestane (p<0.001). With a median follow-up in all IES participants (n=4274) of 58 months, 162 (7%) and 115 (5%) patients in the exemestane and tamoxifen groups, respectively, had fractures (odds ratio 1.45 [1.13-1.87]; p=0.003). These results indicate that the increase in survival shown previously with the IES switch strategy is achieved at the expense of some detriment to skeletal health, so the risk-benefit ratio to women needs to be individually assessed.

Calcitonin gene-related peptide (CGRP) and calcitonin (CT) immunoreactivity were measured in hypothalamus, parahippocampal gyrus, pituitary and grey matter of the posterior and anterior spinal cord from five to six cases of Alzheimer-type dementia (ATD) and from five to six controls. CGRP was slightly increased and choline acetyltransferase decreased in the anterior grey of ATD spinal cord. No other significant differences were observed between the levels of the two peptides in the ATD and control tissues, even in the parahippocampal gyrus and posterior grey of the spinal cord which had reduced choline acetyltransferase activity in the ATD cases. These results show that CGRP and CT are not affected in ATD, either as a consequence of a direct effect on peptidergic neurons or secondary to the loss of choline acetyltransferase activity.

Messenger RNA for rat islet amyloid polypeptide (IAPP) has been identified not only in the pancreas but also, in lesser amounts, in preparations from the stomach and dorsal root ganglia. In the stomach, insulin mRNA was not detectable, ruling out possible contamination by pancreatic tissue. Because IAPP and calcitonin gene-related peptide (CGRP) are related and CGRP is present in both stomach and dorsal root ganglia, it was possible that 'IAPP' signals were in fact due to cross-hybridization with CGRP mRNA. A second IAPP probe was constructed which does not cross-react. This probe also detected mRNA in both tissues, confirming the expression of IAPP in both tissues. The regional distribution of IAPP mRNA in the stomach did not parallel that of gastrin mRNA. IAPP mRNA was present in the antrum, centrum and pylorus and, like gastrin, the highest amounts were in the pylorus. However, the ratio between the pylorus and centrum was 3.6:1 for IAPP and 156:1 for gastrin. The effects of dietary manipulation were determined; a period of 48 h of starvation reduced pancreatic IAPP mRNA by approximately 60%, whereas in the stomach there was no significant reduction. If the action of IAPP was hormonal, pancreas and stomach would not be acting in concert. A paracrine role for gastric IAPP therefore seems more likely.

We have used the polymerase chain reaction with mixed sequence primers to generate a probe for rat amylin and have used this to detect expression in various rat tissues. Amylin mRNA is found in greatest concentrations in the pancreas where a single mRNA species can be detected giving a hybridisation signal intensity approximately 10% that of insulin mRNA. When the beta cell population was depleted with streptozotocin, both amylin and insulin mRNAs were reduced to a similar extent. Consistent with its supposed role in the control of carbohydrate metabolism, amylin mRNA was also found in the stomach. Unlike the related peptide, CGRP, amylin mRNA is not present in the thyroid and is not widely distributed in the central nervous system. The only nervous tissue in which it could be detected was the dorsal root ganglion. Surprisingly, amylin mRNA was also found in the lung though only at very low levels.

The response of the islet amyloid polypeptide gene to chronic dexamethasone treatment in adult rats was investigated. After 12 daily injections, rats were severely underweight and fasting blood glucose levels were elevated. When pancreatic mRNA was analysed, a 16-fold elevation in islet amyloid polypeptide mRNA was observed with only a four-fold increase in insulin mRNA levels. Pancreatic islet amyloid polypeptide and insulin mRNA levels were also determined 12 days after streptozotocin treatment. In these rats, which were not severely diabetic, the reduction in islet amyloid polypeptide mRNA levels was sixfold less than the reduction in insulin mRNA levels. In both these models of diabetes the ratio of islet amyloid polypeptide to insulin mRNA levels was raised. This would not be expected if the physiological role of islet amyloid polypeptide is as a simple hyperglycaemic agent opposing insulin action or release.

The skeletal effects of low-dose methotrexate (MTX), in glucocorticoid-dependent asthmatics (GCDA), are unknown. We studied 9 patients from a total of 26 chronic GCDA who completed 28 weeks of MTX (15 mg weekly, intramuscularly). Prednisolone dose was not altered during the first 12 weeks, and was then reduced between 12 and 28 weeks. Mean (S.E.M.) age of the patients was 54 (4.0) years. They had normal bone mineral density (BMD), were not taking medication that affected bone metabolism (except prednisolone and inhaled corticosteroids) and all achieved at least 50% reduction in prednisolone dose at 28 weeks. Blood and urine samples were obtained at baseline, 12, 28 and 40 weeks for measurement of serum osteocalcin (OC) and bone alkaline phosphatase (Bone-ALP) as formation markers and urinary deoxypyridinoline (DPD) and N-terminal cross-linked telopeptide of type I collagen (NTX-I) as resorption markers. Concurrently with the changes in prednisolone dosage serum OC levels increased significantly at 28 weeks (p<0.008) (8.1+/-1.0 ng/ml) compared to baseline (4.7+/-0.6 ng/ml) and 12 weeks (5.1+/-0.6 ng/ml), but trended back by 40 weeks (6.6+/-0.6 ng/ml). No significant changes were observed for the other bone markers between baseline and the other time points. The beneficial effects of steroid reduction on bone metabolism do not appear to be impaired by concomitant MTX treatment at least over 12 weeks.

Before interpreting the results of assays which claim to measure circulating levels, one must have full data on the sensitivity, the reproducibility and specificity of the methods and antisera used. Without this information figures are meaningless and may be regarded as fiction rather than science. Secondly, and of equal importance, one must have full details of those physiological and pharmacological factors which are known to affect the circulating levels. It is quite obvious to workers in the field that those laboratories which have paid attention to these matters, have achieved remarkable agreement in practice; those who have neglected these requirements have introduced quite unnecessary confusion in the literature.

In addition to calcitonin and katacalcin, the human calcitonin gene encodes a novel peptide--calcitonin gene-related peptide (CGRP)--a potent vasodilator. A sensitive and specific radioimmunoassay was developed to study plasma levels of CGRP in normal subjects. CGRP circulates at five times the concentration of calcitonin, suggesting that it may be an important physiological regulator of vascular tone and blood flow.

Using fluorescence in situ hybridization (FISH) analysis, we examined the replication mode of the centromere region (homologous counterpart) and the aneuploidy level of chromosome 17 in the interphase nuclei of phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes from (1) patients with hepatocellular carcinoma (HCC); (2) patients with liver cirrhosis (LC) due to hepatitis C viral infection who are individuals at a higher increased risk for HCC; and (3) healthy control participants. We also compared the allelic-replication asynchrony and aneuploidy frequencies with serum alpha-fetoprotein (AFP) levels. We found a significant increase in centromeric replication asynchrony accompanied by a high frequency of aneuploidy in lymphocytes of HCC patients compared with those of LC patients and healthy control participants. These changes are similar to those previously observed in other types of malignancy (hematological, ovarian, prostate, and breast cancer). The cytogenetic alterations of aneuploidy and strong asynchronous replication displayed in the lymphocytes of HCC patients arose from malignancy, as they were associated neither with an increased risk for cancer nor with an infection. The cytogenetic cancer-associated markers observed in patients' lymphocytes appeared to be superior to serum AFP, the marker currently used for HCC. Thus, the cytogenetic cancer-associated markers may be potentially useful in noninvasive cancer detection.

In the pineal gland of gerbils, substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers were demonstrated immunohistochemically. After intrapineal injection of biotin-wheat germ agglutinin, origins of fibers were examined by the combined technique of tracing method and immunohistochemistry. It was confirmed that SP- and CGRP-fibers originated from the trigeminal ganglion, VIP-fibers from the pterygopalatine ganglion and NPY-fibers from the superior cervical ganglion.

Mucosal intra-epithelial nerve terminals have been found in the colon of the toad, Bufo marinus. Co-localized in the fibres are calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and substance P (SP)-LI. The intraepithelial nerves are likely to be terminals of primary afferent sensory neurones. Fibres with vasoactive intestinal peptide (VIP)-LI or neuropeptide Y (NPY)-LI also supply the mucosa but do not penetrate the epithelium.