Total joint replacement of hip or knee as an outcome measure for structure modifying trials in osteoarthritis.

OBJECTIVE: The Group for the Respect of Ethics and Excellence in Science (GREES) organized a working group to assess the value of time to joint surgery as a potential therapeutic failure outcome criterion for osteoarthritis (OA) of the hip or knee in the assessment of potential structure modifying agents. METHODS: PubMed was searched for manuscripts from 1976 to 2004. Relevant studies were discussed at a 1-day meeting. RESULTS: There are no accepted guidelines for 'time to' and 'indications for' joint replacement surgery. A limited number of trials have examined joint replacement surgery within the study population. Several parameters, particularly joint space narrowing (interbone distance), correlate with surgical intervention. However, at the level of the knee, none of the parameters have positive predictive value for joint replacement surgery better than 30%. In contrast, lack of significant joint space narrowing has a strong negative predictive value for joint replacement surgery (>90%), that remains after controlling for OA pain severity. CONCLUSION: At this time, GREES cannot recommend time to joint surgery as a primary endpoint of failure for structure modifying trials of hip or knee OA-as the parameter has sensitivity but lacks specificity. In contrast, in existing trials, a lack of progression of joint space narrowing has predictive value of >90% for not having surgery. GREES suggests utilizing joint space narrowing (e.g., >0.3-0.7 mm) combined with a lack of clinically relevant improvement in symptoms (e.g., >/=20-25%) for 'failure' of a secondary outcome in structure modifying trials of the hip and knee.

Citrate infusion test in the diagnosis of hypocalcemia due to a mutation in the calcium-sensing receptor gene.

The calcium-sensing receptor (Ca-R) is a G-protein-coupled surface receptor that plays a crucial role in calcium homeostasis via parathyroid hormone secretion. Mutations of this receptor can cause a gain in, or loss of, function, leading to hypo- or hypercalcemia, respectively. We report here a family with hypocalcemia in whom a heterozygous missense mutation in exon 4 was demonstrated, predicting a proline to leucine substitution (P221L) in the extracellular part of the Ca-R. Clinical symptoms were limited to fatigue. When serum calcium was further lowered via a citrate infusion, a significant increase in circulating iPTH was observed, although with lower peak values than in normal controls, suggesting a gain in function of the Ca-R. Treatment with calcium supplements and calcitriol led to prohibitive hypercalciuria without normalizing serum calcium. The aims of this case report are: (1) to present a mutation in the Ca-R with a gain in function at a codon where previously loss of function was described, and (2) to suggest that measuring circulating iPTH during a citrate infusion in the presence of familial hypocalcemia is an additional test to diagnose this particular form of hypoparathyroidism.

Non-secreting atypical parathyroid adenoma.

Parathyroid tumors can be divided in adenomas and carcinomas, usually detected by hypercalcemia. We report a case of parathyroid adenoma in a young man, who complained of a pressure in the left neck region. Physical examination revealed a firm mass in the neck, without lymphnodes. Although Ca (9.7 mg/dl), phosphorus (3.3 mg/dl) and intact-PTH (49 pg/ml) were normal, imaging techniques (computed tomography scan and sestamibi substraction scan) suggested that the mass could arise from the parathyroid gland. Histology and immune staining for chromogranin and parathyroid hormone confirmed the parathyroid nature of the mass. Histological criteria defined the lesion as an atypical parathyroid adenoma. We review the pathology, diagnosis and treatment of parathyroid adenomas in its non-secreting atypical form.

Glycosylated rat prolactin: isolation and structural characterization.

Isolation of glycosylated 26 kDa rat prolactin and subsequent proper carbohydrate characterization has so far not been reported. In the present work the hormone isoform was isolated to 95% homogeneity by preparative electrophoretic separation on Mini Prep Cell of rat pituitary homogenate. The isoform was then investigated by 2-mercaptoethanol gradient electrophoresis, Cleveland's sequential SDS-PAGE, digestion with endoproteinase Asp-N and N-glycanase. The glycosidic part of the isoform was examined in O-profiling and its monosaccharide composition obtained by FACE and HPAE-PAD analysis. The outcome of the experimental data is: 1) in contrast to unglycosylated 23 kDa rat prolactin, intra-chain S-S bridging is not affected in 26kDa rat prolactin, neither by transiting through a thiol gradient nor in sequential nonreducing/reducing SDS-PAGE; 2) the conformational availability of Asp residues involved in the endoproteinase Asp-N attack is the same in 23- and 26 kDa rat prolactin; the glycan moiety apparently does not cause steric hindrance at this level; 3) no glycosidic N-linkage could be detected, only O-linkage(s); 4) 26 kDa rat prolactin is no glycosyl-phosphaditylinositol-anchored protein; 5) in O-profiling an oligosaccharide chain of Mr +/- 1.4 kDa was recorded; 6) the monosaccharide composition obtained in FACE is peculiar in the sense that next to Fuc, Man, GalNac, GlcNac and NeuAc also Rib was determined; 7) HPAE-PAD analysis identified NeuAc subtypes; 8) in vitro, glycosylation of rat prolactin modulates immune recognition through steric hindrance of the access to the epitope sites.

[Postmenopausal osteoporosis in women]. / L'ostéoporose d'involution chez la femme.

Postmenopausal osteoporosis is now considered a major public health problem in aging women, due to the burden related to the consequent fractures. Over recent years, several pharmacological approaches were developed for the prevention and treatment of osteoporosis. Besides regular physical exercise and calcium rich diet, calcium supplementation can be suggested to both genders, after seventy years as well as systematic vitamin D supplementation in order to cope with the frequent lack observed in our country. Hormone replacement therapy is the first choice in prevention of postmenopausal osteoporosis. Based on a careful evaluation of the needs of a postmenopausal woman as well as on the risk/benefit ratio derived from her individual risk factors, selective estrogen receptor modulators (raloxifene) or second generation bisphosphonates (alendronate) can be considered as alternative to estrogens. Due to its prohibitive cost, nasal calcitonin should be only considered for very specific cases. In osteoporosis confirmed by bone densitometry or by occurrence of fractures, bisphosphonate (alendronate) reduces subsequent fracture rate. Fluoride salts can, in some cases, improve spinal symptomatic osteoporosis. The use of etidronate, a molecule from the past, should be avoided as much as possible and, at any rate, strictly restricted to its legal indication including women with several vertebral crush fractures and severely decreased bone mineral density.

[How to evaluate osteoporosis: bone densitometry and biochemical tests]. / Comment j'explore ... l'ostéoporose: mesure de la masse osseuse et analyses biologiques.

The use of bone densitometry, by dual energy x-ray absorptiometry, allows the identification, at an early stage, of subjects who will, 15-20 years later, present a fracture event. Furthermore, this technique is the most appropriate to confirm the efficacy of preventive or curative therapeutic approaches of osteoporosis. A balanced utilisation of biochemical tests is mandatory to exclude a secondary cause of osteoporosis, to help in the choice of the most appropriate therapeutic strategy and to evaluate the early response of patients to the administered drugs.

Calcitonin reserve in different stages of atrophic autoimmune thyroiditis.

The objective of this study was to determine the calcitonin (CT) hormone reserve in different severity of atrophic autoimmune thyroiditis (AAT). Forty-eight female patients with AAT were divided into four groups based on basal and peak thyrotropin (TSH) values (after oral thyrotropin-releasing hormone [TRH], free triiodothyronine (FT3) and free thyroxine (FT4) ranging from normal in group 1 to overt hypothyroidism in group 4. All had thyroid antibodies. The control group comprised euthyroid females of comparable age, without thyroid antibodies. Basal CT and CT response to calcium infusion (area under the curve) were investigated as parameters of CT reserve. Basal CT was lower in groups 2 to 4 of patients with AAT (compared to controls), but the difference was not significant. Stimulated CT levels were lower (p < 0.05) in all groups of patients compared to controls, with markedly reduced CT-secretory reserve in group 4. Thyroid antibody concentrations and, basal and postinfusion calcium levels were not significantly different among the various groups. In conclusion CT deficiency (especially stimulated values) occurs in AAT and is more severe in hypothyroid patients than in earlier stages of AAT.

Routing, processing and export of rat pituitary prolactin: identification of a 36 kDa disulphide-bridged oligomeric preprolactin.

To gain an insight in the routing, processing and export of rat prolactin, rat pituitary cells were cultured in serum-free medium in the presence of cycloheximide, carbonyl cyanide m-chlorophenylhydrazone, Brefeldin A and monensin. The potential influence of these perturbants, whose well documented effects are the altering of protein synthesis and transport, was studied on rat prolactin molecular size isoforms appearing in cellular extracts and in culture medium. The outcome of the culture experiments as recorded in vertical SDS-PAGE, thiol gradient electrophoresis and sequential SDS-PAGE followed by prolactin specific immunoblotting and densitometry, was as follows: (1) at the cellular level we were able to characterize a novel 36 kDa protein as a disulphide-bridged oligomeric precursor prolactin, which is presumably rapidly transformed in the cis/medial Golgi; to designate monomeric rat prolactin as an early Golgi protein and t o advance evidence that the main processing of the glycosylated rat prolactin is a cis/medial Golgi event; (2) in release none of the perturbants disturbed the relative distribution of monomeric and glycosylated rat prolactin, the main molecular size isoforms currently secreted by untreated pituitary cells, or induced the appearance of transformed molecular size isoforms; (3) the secretion mode indicates that rat prolactin is released via the regulated pathway in the presence of the perturbants used.

Cafeteria diet-induced obesity is associated with a low spontaneous growth hormone secretion and normal plasma insulin-like growth factor-I concentrations.

Pituitary growth hormone (GH) secretion has been shown to be blunted in human and animal obesity. With respect to human obesity, cafeteria diet-induced obesity might be an appropriate model to study spontaneous GH secretion. In 6 cafeteria diet-overfed obese male Wistar rats and 6 control rats with chronically implanted catheters, GH levels were measured every 15 min over 6 h by standard RIA. A significantly lower GH secretion, reflected by the integrated GH concentration, was found in the obese rats (median 16.46, [range 10.55-19.13] ng/ml x 6 h vs 35.63 [range 21.90-41.50] ng/ml x 6 h, P < 0.05). The GH secretion in the obese rats was significantly negatively correlated with the body fat percentage, assessed by dual X-ray absorptiometry (Rho = -0.95, P < 0.05). Median plasma insulin-like growth factor-I (IGF-I) concentration was comparable between the two groups, while the median insulin concentration was significantly higher in the obese group (1.95 [range 1.76-3.55] ng/ml vs 1.21 [range 0.86-2.13] ng/ml, P < 0.05). No significant correlation existed between GH secretion and the plasma insulin concentration. In conclusion, cafeteria diet-induced obesity is associated with a low spontaneous GH secretion and normal plasma IGF-I concentration. The hyperinsulinemia present in this model probably explains the normal IGF-I concentrations, but not the GH hyposecretion.

Two years of replacement therapy in adults with growth hormone deficiency.

OBJECTIVES: Although several studies have shown beneficial short-term effects of recombinant human growth hormone (rhGH) therapy in adult GH deficient (GHD) patients, few data are available on large groups of patients treated for more than one year. In addition, the optimal dose of rhGH for each patient and the baseline parameters that predict which patients will benefit most from therapy or will have adverse events are not entirely elucidated. DESIGN: 148 adult GHD patients were enrolled in a multicentre 2-year rhGH replacement study which was placebo controlled for the first six months. rhGH (Genotropin/Genotonorm Pharmacia & Upjohn) was given in a dose of 0.25 IU/kg/week sc (1.5 IU/m2/day). MEASUREMENTS: Every 3-6 months body composition was measured using body impedance analysis and general well being was assessed using the Nottingham Health Profile (NHP) and social self-reporting questionnaire. At the same time patients had a full clinical examination and blood was sampled for glucose, HbA1c, IGF-1, creatinine, full blood count, thyroid hormones and liver function tests. RESULTS: With rhGH therapy IGF-1 levels increased from -2.00 +/- 2.60 SDS to 1.47 +/- 2.6 SDS after six months (P < 0.001), continued to rise despite no change in dose to 1.84 +/- 2.8 SDS after one year and remained constant thereafter (1.98 +/- 2.4 after 2 years). 56% of patients ultimately attained supranormal IGF-1 levels (+2 SD), 22% had levels below the mean, of which 9% were below -2 SD. Within 3 months lean body mass (LBM) increased by +5.09% (P < 0.001), total body water (TBW) by +5.40% (P < 0.001), while body fat (BF) dropped by -10.89% (P < 0.001) and waist circumference by -1.42% (P < 0.004). These effects were maintained during the first year of therapy, but the effect was attenuated after 24 months: LBM, +3.91% (P < 0.001); TBW, +3.28%, P < 0.001, BF, -6.42% (P < 0.001) and waist -2.22% (P < 0.009). Individual differences in response were large and could not be predicted by any of the baseline parameters, except for a better response in males. Treatment resulted in a large and progressive improvement on the NHP scale, especially energy, emotions and sleep, but a similar change was also found in patients during placebo treatment. With rhGH the number of full days of sick leave/6 months decreased from 12.17 +/- 3.90 days (SEM) to 7.15 +/- 3.50 days after six months (P = 0.009), 2.93 +/- 1.55 days after 12 months (P = 0.01), 0.39 +/- 0.17 days after 18 months (P < 0.001) and 3.3 +/- 2.51 days after 24 months (P = 0.026). Similarly, the hospitalization rate went down from 14.9 to 7% after 6 months and remained at this level thereafter (P = 0.12). About one third of patients on rhGH experienced fluid-related adverse events, most often within the first 3 months. They usually disappeared spontaneously or responded well to dose reduction. Cumulative dropout rates were 29% after 1 year and 38% after two years. Two thirds of these patients stopped treatment because of insufficient subjective improvement. Neither drop-outs nor fluid retention could not be predicted by any of the baseline parameters. CONCLUSIONS: We confirmed in a large group of patients the beneficial effects of rhGH therapy on body composition, metabolic parameters and general well-being and found a consistent drop in number of sick days and hospitalization rate. These effects were maintained during two years of therapy, except for an attenuation in body composition changes after 24 months. The high incidence of fluid-related adverse events suggests that it may be better to start with lower doses of rhGH and to increase the dose more slowly over a number of weeks. The finding of suboptimal high or low IGF-1 levels in many patients reinforces guidelines not to give rhGH in a weight-dependent dose but to titrate it individually for each patient.

The weight gain and ultimate adiposity in cafeteria diet-induced obesity is unrelated to the central serotoninergic tonus.

Early detection of subjects with a propensity to obesity might be of great help for setting up preventive intervention studies. In this study we tested whether the development of obesity in Wistar rats, given ad libitum cafeteria foods, could be predicted by a low prolactin (PRL) response to 5-hydroxytryptophan (5HTP), as an index of low hypothalamic serotoninergic tonus. Basal and 5HTP-stimulated (50 mg/kg body weight i.p.) PRL were measured by RIA in 15 young male Wistar rats, whose pelleted diet was afterwards supplemented with cafeteria foods. In the tested animals an increase of PRL between 4 and 56 times the basal value was observed 60 min after the 5HTP injection. After 2 months of feeding, marked inter-individual differences in weight gain between the cafeteria fed animals were observed. After 10 months of feeding, median body fat percentage, assessed by dual X-ray absorptiometry, of the overfed rats was significantly higher than that of control animals: median (range): 41.2% (28.9 - 51.5%) vs 25.1 (18.0 - 32.2%) (p < 0.0001). The PRL response at the start of the experiment was neither correlated with the monthly weight increases, nor with the fat mass percentage at the end of the experiment, suggesting that a pre-existing low hypothalamic serotoninergic tonus is probably not involved in the overeating and ultimate overweight of cafeteria diet fed animals.

Effect of N omega-nitro-L-arginine methyl ester, a nitric oxide synthesis inhibitor, on stress- and morphine-induced prolactin release in male rats.

1. The effect of the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was investigated on stress- and morphine-induced prolactin (PRL) secretion in vivo in male rats, by use of a stress-free blood sampling and drug administration method by means of a permanent indwelling catheter in the right jugular vein. 2. Three doses of L-NAME were tested (1, 10 and 30 mg kg-1) and were given intraperitoneally one hour before blood sampling; control rats received saline. After the first blood sample, rats received an initial intravenous injection of morphine (3, 6 or 12 mg kg-1) or were subjected to immobilization stress. In the case of a morphine administration, rats received a second dose of morphine (3, 6 or 6 mg kg-1, respectively) 90 min later, when tolerance had developed, while rats subjected to immobilization stress received 6 mg kg-1 morphine 90 min after onset of stress. 3. L-NAME had no effect on basal plasma PRL concentration. However, it potentiated acute morphine-induced PRL secretion and attenuated the subsequent tolerance in a dose-dependent way. Immobilization stress-induced PRL secretion was inhibited dose-dependently by L-NAME, as was its subsequent tolerance to morphine; however, in this case, in a reversed dose-dependent way. 4. When the highest dose of morphine (12 mg kg-1) was combined with the highest dose of L-NAME pretreatment (30 mg kg-1), all rats showed a dramatic potentiation of the morphine-induced PRL rise compared to controls. Moreover, all of these rats died within 90 min after the administration of morphine. 5. These results show that NO plays a role in the acute opioid action on PRL release during stress as well as in the development of tolerance to the opioid effect, and some possible mechanisms are discussed.

Molecular heterogeneity and glycosylation modulation of rat pituitary prolactin isoforms synthesized and secreted in vitro in postnatal ontogeny, gestation, lactation and weaning.

The modulation of both the molecular size heterogeneity and the relative distribution of rat prolactin variants, synthesized and secreted in vitro by rat pituitary cells in the course of postnatal ontogeny and in gestation, lactation and weaning was investigated by SDS-PAGE, immunoblotting, radioimmunological techniques and O-sialoendopeptidase digestion. The outcome of the experiments is as follows: 1) from day 1 of postnatal life 20-, 23-, 26-, 40-44 kDa and oligomeric rat prolactin isoforms were stored and secreted; 2) perinatal life is characterized by a high degree of variability of prolactin size isoforms and their respective repartition in storage and release; in addition to the major variants, transient ones of M, 25-, 28-, 33- and 36 kDa were secreted and/or stored; 3) O-sialoglycoprotease digestion of pituitary cell lysate gave good evidence for 25 kDa prolactin being a glycoform; 4) at 1 month of age 16 kDa rat prolactin appeared and persisted over the whole postnatal span (1 day-->1 year) but only in stored form; 5) the physiology of gestation was essentially characterized by the M(r)-modulation of the glycoform (26 kDa-->26.3 kDa) and the virtual absence of stored 26 kDa rat prolactin at week 1 of pregnancy; 6) in lactation and weaning uncommon multiple banding was observed in secreted oligomeric prolactin; 7) in pregnancy, lactation and weaning the differential distribution of released and stored prolactin isoforms displayed a considerable intra- and intervariability; 8) in the vast array of size isoforms observed in all our experiments monomeric 23 kDa prolactin was always the dominating variant. In conclusion, the molecular size heterogeneity and the differential distribution of secreted and stored rat pituitary prolactin is considerably influenced by age and physiological stimuli. The nature of polymeric prolactin and of the transient variants is presently unclear, and the exact physiological role of molecular heterogeneity modulation is unknown, both in humans and rat, but the patterns of change we observed in definite stages of life, suggest that this phenomenon is important in the maturation of the hypothalamus-pituitary axis and in the metabolic and hormonal changes accompanying gestation.

The weight gain and ultimate adiposity in cafeteria diet - induced obesity is unrelated to the central serotoninergic tonus.

Early detection of subjects with a propensity to obesity might be of great help for setting up preventive intervention studies. In this study we tested whether the development of obesity in Wistar rats, given ad libitum cafeteria foods, could be predicted by a low prolactin (PRL) response to 5-hydroxytryptophan (5HTP), as an index of low hypothalamic serotoninergic tonus. Basal and 5HTP-stimulated (50 mg/kg body weight i.p.) PRL were measured by RIA in 15 young male Wistar rats, whose pelleted diet was afterwards supplemented with cafeteria foods. In the tested animals an increase of PRL between 4 and 56 times the basal value was observed 60 min after the 5HTP injection. After 2 months of feeding, marked inter-individual differences in weight gain between the cafeteria fed animals were observed. After 10 months of feeding, median body fat percentage, assessed by dual X-ray absorptiometry, of the overfed rats was significantly higher than that of control animals: median (range): 41.2% (28.9-51.5%) vs 25.1 (18.0-32.2%) (p <0.0001). The PRL response at the start of the experiment was neither correlated with the monthly weight increases, nor with the fat mass percentage at the end of the experiment, suggesting that a pre-existing low hypothalamic serotoninergic tonus is probably not involved in the overeating and ultimate overweight of cafeteria diet fed animals.

Secretion of 23 kDa and glycosylated prolactin by rat pituitary cell culture in serum-free media: a comparative morphological, cyto- and immunochemical study.

The secretion of 23 kDa prolactin by rat pituitary cells has been thoroughly investigated, but secretion of glycosylated rat prolactin is not currently known. This is mainly due to the lack of an antiserum which is solely specific for glycosylated rat prolactin and therefore we studied the basal secretion of this variant by an indirect method. Rat pituitary cells were cultured in total culture medium and three different serum-free media (DMEM, keratinocyte-serum-free medium, protein-free hybridoma medium) and secretion of 23 kDa and glycosylated rat prolactin was recorded by radioactive techniques and immunoblotting. The pituitary cell quality was monitored by electron microscopy, cell activation-and cell death assessment. In short-range culture (2 days) the pituitary cell quality and behaviour was very good and comparable in total culture medium, DMEM and keratinocyteserum-free medium, i.e. numerous secretory granules, moderate amount of ER, cristae well in place in the mitochondriae. In medium-range culture (8 days) only cells cultured in total culture medium and DMEM presented a parallel behaviour: migration of cells toward each other, marked degranulation, massive array of ER. The inner membrane of the mitochondria was no longer folded into cristae leaving an unoccupied central space. At day 2 of the culture span secretion of 23 kDa rat prolactin was very comparable in all media used; hereafter, secretion of 23 kDa rat prolactin in total culture medium and DMEM assumed the well known pattern of peaking and slowing down, whereas in the other serumfree media it steadily decreased over the culture span. Pertaining to the important novel point of glycosylated rat prolactin secretion, it was low in comparison to the one of 23 kDa rat prolactin and it assumed a near steady pattern in all media used. 26 kDa rat prolactin was identified as the preferentially secreted glycoform, and the 23 kDa isoform as the major secretory product of rat pituitary lactotroph cells.

5-Hydroxytryptophan-stimulated prolactin levels in cafeteria diet fed rats: an in vivo evaluation of the central serotonergic tonus.

Hyperphagia in rats fed a cafeteria diet might be related to the palatability of the diet or to diet-induced changes in central neurotransmitters regulating the feeding behavior. In this study the central serotonergic tonus in adult male Wistar rats was evaluated in vivo after 6 weeks of feeding a cafeteria diet by the prolactin response to the administration of 5-hydroxytryptophan (5HTP), the immediate serotonin precursor. Blood was taken just before, 30, 60 and 90 min after the ip injection of 50 mg/kg 5HTP for the determination of prolactin concentrations were comparable between cafeteria fed rats and control rats, fed normal laboratory chow (12.7 +/- 5.4 vs 7.7 +/- 4.5 ng/ml). The 5HTP-stimulated prolactin secretion in the cafeteria diet fed rats, determined by the peak value (95.8 +/- 17.2 vs 119.1 +/- 27.0 ng/ml) as well as by the integrated area under the curve (5478 +/- 774 vs 5916 +/- 2275 ng/ml. 90 min) was not significantly lower than in the control rats. In conclusion, our results did not show a significantly decreased 5HTP-induced prolactin release in cafeteria-fed rats, suggesting that a low hypothalamic serotonergic tonus is probably not involved in the overeating of this dietary-induced obesity model.