Regulation of salmon gonadotrophin-releasing hormone gene expression by sex steroids in rainbow trout brain.

Salmon gonadotrophin-releasing hormone (sGnRH) is the major form of gonadotrophin-releasing hormone in the brain of Salmonids and is encoded by two different genes: sGnRH1 and sGnRH2. In the present study, we examined the expression patterns of these two genes during development and throughout the reproductive cycle of the female rainbow trout (Oncorhynchus mykiss), and also investigated the feedback action of sex steroids on brain mRNA levels. Both genes are expressed as early as 14 days postfertilisation and show a similar expression pattern during early life stages. In the adult female, sGnRH1 and sGnRH2 mRNAs are both present in neurones located in the ventral forebrain. This gene expression in the brain appears to be low during early vitellogenesis, and increases during oocyte maturation to reach a maximum after ovulation. The expression of sGnRH1 was not modified by in vivo steroid treatments in any experiment; however, testosterone and 5alpha-dihydrotestosterone down-regulate brain sGnRH2 gene in immature and adult ovariectomised females. Oestradiol treatment decreases sGnRH2 mRNA levels in the brain of adult ovariectomised females only. In the triploid fish brain, none of the steroids affect brain sGnRH mRNA levels. Our results suggest that, unlike sGnRH1, the sGnRH2 gene is under a strongly androgenic inhibitory control in the immature and adult female rainbow trout.

Characteristics and outcome of 49 patients with symptomatic cryoglobulinaemia.

OBJECTIVE: To describe a population of patients with symptomatic cryoglobulinaemia, comparing manifestations and outcome as a function of hepatitis C virus (HCV) status. PATIENTS AND METHODS: A retrospective study on 179 patients who tested positive for cryoglobulins, seen between 1978 and 1998 in an internal medicine department. RESULTS: Among 179 cryoglobulin-positive patients, only 49 (18 men, 31 women; mean age 59.96+/-12 yr) had clinical manifestations attributable to cryoglobulinaemia. Thirty-three had HCV infection, 20 had systemic autoimmune diseases, two had haematological diseases, one had human immunodeficiency virus and HCV co-infection, one had HCV and HBV co-infection and six had essential mixed cryoglobulinaemia. The clinical manifestations and cryoglobulin levels in HCV(+) and HCV(-) patients did not differ significantly. Only arthralgias and elevated transaminases were significantly more frequent in HCV(+) patients (P<0.02 and <0.05, respectively). Five-year survival rates were comparable for HCV(+) and HCV(-) patients. Eight patients died (six HCV(+), two HCV(-)), with a median time between diagnosis and death of 38.7 months. CONCLUSION: Clinical manifestations of cryoglobulinaemia, except arthralgias, were comparable for HCV(+) and HCV(-) patients. When systemic manifestations are present, the prognosis is poor despite intensive or prolonged therapy.

Two different messenger RNAs for salmon gonadotropin-releasing hormone are expressed in rainbow trout (Oncorhynchus mykiss) brain.

Two different precursor genes encoding the decapeptide salmon GnRH (sGnRH) are present in most salmonid species. In rainbow trout, a precedent Southern blot study revealed the existence of two different sGnRH genes and, recently, two different genes and their complementary DNAs that encode the identical peptide sGnRH were isolated from ovary and testis. Our study confirms the existence of two different mRNAs encoding sGnRH (sGnRH mRNA-I and sGnRH mRNA-II) in the brain of rainbow trout and, for the first time, full-length complementary DNA sequences are given. Central and peripheral distributions of the two messengers are described and seem to indicate different regulation of their expression. sGnRH mRNA-I is found essentially in the olfactory bulbs and telencephalon, whereas sGnRH mRNA-II is more widely expressed in the brain. Our observations allow speculation on the respective roles of two genes encoding the same decapeptide.

Nutritional status after short-term dietary supplementation in hospitalized malnourished geriatric patients.

AIM: To examine the evolution of different parameters of the nutritional status after short-term oral protein-energy supplementation in moderately malnourished geriatric patients. METHODS: Seventeen hospitalized malnourished elderly patients and 12 healthy adults received dietary supplements for 10 days. A group of six malnourished elderly subjects served as controls. Spontaneous oral intakes, biological and biophysical markers of the nutritional status were measured. Fat-free mass (FFM) was assessed using Dual energy X-ray absorptiometry (DXA), bio-impedance analysis (BIA) and anthropometry. RESULTS: In elderly subjects, the supplementation significantly increased both dietary intake (energy +32%, protein +65%) and FFM (+1.3 kg, P<0.001) as assessed using DXA. BIA and anthropometric data correlated with DXA measurements in the elderly (BIA: r=0.68--0.80, anthropometry: r=0.80--0.89), but failed to reflect accurately the changes measured in FFM. Supplementation had no notable effect on biological markers in any of the groups. IGF-I and hand-grip strength were not significantly influenced by the supplementation despite trends towards an improvement. CONCLUSIONS: Monitoring short-term changes in nutritional status in malnourished elderly individuals is a problem in routine clinical management. Our data put in the limelight the changes in IGF-I values related to dietary supplementation, and, chiefly, suggest a prime role for the assessment of dietary intake and FFM, as assessed by DXA, as indicators of short-term efficacy of refeeding. Nevertheless larger studies are necessary to confirm the clinical and prognostic significance of the changes.

Expression of sGnRH mRNA in gonads during rainbow trout gametogenesis.

The salmon gonadotropin-releasing hormone (sGnRH) is the major form of GnRH decapeptide expressed in the salmonid brain and it acts as a gonadotropin releaser. In rainbow trout, sGnRH-1 and sGnRH-2 mRNA forms were found in brain and gonads. We analyzed the expression of both forms in trout gonads at different stages of gametogenesis. Northern blot demonstrated that sGnRH-2 mRNA was the major sGnRH form in testis and ovary. In testis but not in ovary, brain or pituitary, alternatively spliced sGnRH-2 transcripts which coded for prepro-sGnRH with a truncated GnRH-associated peptide due to a premature stop codon in retained intron 2 were detected. In testis, sGnRH mRNA was highly expressed before the onset of spermatogenesis, it disappeared at stage II and then increased progressively up to stage VI. In ovary, the expression of sGnRH was high in immature pre-vitellogenic fish and progressively decreased throughout vitellogenesis. At ovulation it reached its maximum and came down again after stripping. The decrease of sGnRH mRNA expression during the period of active spermatogonial proliferation in testis and increase during meiosis occurrence in testis and ovary suggest an anti-proliferative and meiosis-stimulating effect of sGnRH during rainbow trout gametogenesis.

Stage-dependent and alternative splicing of sGnRH messengers in rainbow trout testis during spermatogenesis.

The gonadotropin releasing hormone (GnRH) has long been considered as a neuropeptide involved in the control of the reproductive cycle. However, the presence of GnRH and its receptors in various tissues, including ovary and testis, suggests a role as autocrine/paracrine factor. In the present study, we report the expression of the sGnRH-1 and sGnRH-2 genes encoding salmon GnRH in rainbow trout testis throughout testicular development and spermatogenesis. We demonstrate that both sGnRH mRNA are expressed prior of sexual differentiation. In adult, northern blot analysis indicates that sGnRH-2 transcripts are expressed in the testis at higher levels than sGnRH-1 messengers. Moreover, we observed that the expression of sGnRH-2, and not sGnRH-1, messengers was stage-dependent. sGnRH-2 mRNA expression decreases at the onset and progressively rebounds at the end of spermatogenesis. In addition, we demonstrate that a complex stage-dependent and differential splicing of the sGnRH-2 messengers occurs throughout spermatogenesis. We isolated five transcripts corresponding to sGnRH-2 messengers. Two of them may encode a novel and shortened GnRH-associated peptide containing 18 residues instead of 46. Our data provide new insight in the putative role of GnRH and GAP peptides as autocrine/paracrine factors of spermatogenesis.

Transgenic rainbow trout expressed sGnRH-antisense RNA under the control of sGnRH promoter of Atlantic salmon.

A recombinant vector containing antisense DNA complementary to Atlantic salmon (Salmo salar) sGnRH cDNA driven by specific promoter Pab derived from a corresponding sGnRH gene was introduced into rainbow trout (Oncorhynchus mykiss) eggs. This resulted in transgenic animals that had integrated one copy of the transgene into their genome and transmitted it through the germline. Antisense-sGnRH mRNA (AS) was expressed mainly in the brain of transgenic AS(+) fish. Levels of sGnRH endogenous mRNA in the brain were lower in 11-month-old AS(+) fish compared with nontransgenic AS(-) individuals from the same F2 progeny. sGnRH levels significantly decreased in the pituitary of transgenic males and females around the maturation period and in the brain of AS(+) immature females compared with controls. No reliable statistical difference was found in the levels of FSH and LH between AS(+) and AS(-) groups either in immature or mature fish. The majority of transgenic fish reached maturity at the same time as did nontransgenic individuals, although the maturation of AS(+) animals seemed to be more asynchronous. For the first time, the influence of antisense messengers on endogenous mRNA in transgenic fish and the corresponding protein is described.

Short-term protein and energy supplementation activates nitrogen kinetics and accretion in poorly nourished elderly subjects.

BACKGROUND: An increase in protein intake exerts a stimulating effect on protein kinetics in children, young adults, and healthy elderly persons. However, there are few data on the response to such dietary changes in malnourished elderly subjects, despite important medical implications in this population. OBJECTIVE: The objective of this study was to determine the metabolic response to short-term nutritional supplementation in moderately malnourished elderly subjects. DESIGN: The influence of 10 d of supplementation (1.67 MJ/d and 30 g protein/d) on body composition, resting energy expenditure, and whole-body protein kinetics was studied in 17 malnourished elderly patients and 12 healthy young adults. A control group of 6 malnourished elderly patients received no supplementation. RESULTS: Supplemented elderly subjects had a significantly greater fat-free mass gain than did unsupplemented elderly subjects (1.3 and 0.1 kg, respectively; age effect, P < 0.05; diet effect, P < 0.02) and a significantly greater increase in fasting rate of protein synthesis than did young supplemented subjects (0.6 and 0.2 g*kg FFM(-1)*11 h(-1); age effect, P < 0.05). The net protein balance in the supplemented elderly subjects in the fed state was positively correlated with protein intake (r(2) = 0.46) and in the fasted state was negatively correlated with protein intake (r(2) = 0.27). The sum of these regressions is a line with increasingly positive net diurnal protein balance produced by increasing protein intake. CONCLUSION: These data provide evidence of a short-term anabolic response of protein metabolism to dietary supplementation in malnourished elderly patients that is likely to improve muscle strength and functional status.

[Procalcitonin, a new marker for bacterial infections]. / Intérêt de la procalcitonine, nouveau marquer de l'infection bactérienne.

Procalcitonin (PCT), the precursor protein of the hormone calcitonin, appears to be an early marker of the presence of severe systemic infection. High serum concentrations are associated with severe systemic bacterial, parasitic or fungal infections. In contrast, PCT is generally not induced by severe viral infections or inflammatory reactions of non-infectious origin. Hence, PCT can be used for differential diagnosis of bacterial and viral meningitis. PCT may be helpful in the differentiation between infectious and non-infectious origin of systemic inflammatory response syndrome (SIRS) and acute respiratory distress syndrome (ARDS), pancreatitis, cardiogenic shock and acute rejection of organ transplants. PCT monitoring may be useful in patients with high risk of bacterial infection (major surgery, trauma, immunocompromised patients). PCT is a very stable molecule in vitro, and its measurement requires only 20 ml of plasma or serum and can be done within 2 hours.

Assessment of net postprandial protein utilization of 15N-labelled milk nitrogen in human subjects.

The nutritional quality of milk proteins, evaluated both in terms of digestibility and postprandial oxidation and retention in human subjects, was investigated in this study. Five healthy adult volunteers were given 480 ml 15N-labelled milk (i.e. 190 mmol N). 15N was subsequently determined at the ileal level, using a naso-intestinal intubation technique, as well as at the faecal level. Plasma and urine were sampled for 8 h after meal ingestion. Dietary exogenous N recovered at the terminal ileum after 8 h reached 8.6 (SE 0.8) mmol while the amount collected in the faeces was 6.5 (SE 0.7) mmol after 5 d. The true ileal and faecal digestibilities were 95.5 (SE 0.4)% and 96.6 (SE 0.4)% respectively. The appearance of [15N]amino acids in the plasma was rapid and prolonged. The measurement of 15N in the body urea pool and in the N excreted in the urine allowed us to calculate the deamination occurring after [15N]milk protein absorption. The net postprandial protein utilization (i.e. NPPU = (Nabsorbed-Ndeaminated)/Ningested), calculated as an index of protein quality 8 h after milk ingestion, was 81.0 (SE 1.9)%. Our data confirm that milk protein has a high oro-ileal digestibility in man and demonstrate that milk protein has a high NPPU, an index corresponding to a period in which the dietary protein retention is maximal.

Characterisation of serotonin transport mechanisms in rainbow trout peripheral blood lymphocytes: role in PHA-induced lymphoproliferation.

In this study, we investigated the serotonin transport mechanisms in rainbow trout (Oncorhynchus mykiss) peripheral blood Lymphocytes. We have observed that the transport of serotonin is a membrane transport process that have the properties of a secondary active transport system. The binding isotherm of [3H]-paroxetine, a serotonin transport blocker, demonstrated a high-affinity binding site with a positive type of cooperativity, Hill coefficient being higher than unity. Known specific inhibitors of the mammalian serotonin transporter significantly inhibited the uptake process in fish lymphocytes. In order to demonstrate the physiological relevance of the serotonin transporter in T-cell activation, we conducted experiments on lymphocytes activated or not by phytohemagglutinin (PHA), a T-cell mitogen. We have observed that addition of PHA for 24hrs, increased the Vmax but not the Km of this transporter. Serotonin uptake inhibitors diminished the PHA-activated proliferation of fish lymphocytes. The intracellular concentrations of cAMP were found to regulate the serotonin uptake and the PHA-stimulated proliferation as the agents known to augment cAMP stimulated serotonin uptake, and inhibited the lymphoproliferation. Inhibitory effects of increased cAMP on the proliferation were reversed by the addition of the nanomolar concentrations of 8-OH-DPAT, a 5-HT1A receptor agonist which is known to diminish the intracellular cAMP concentrations, suggesting that serotonin also regulates PHA-induced proliferation via 5-HT1A membrane receptors in an autocrine manner. These results all together demonstrate that fish lymphocytes possess an active serotonin transporter that is implicated in the proliferation of these immunocompetent cells.

Net postprandial utilization of [15N]-labeled milk protein nitrogen is influenced by diet composition in humans.

The aim of this study was to follow the fate of dietary nitrogen to assess the postprandial utilization of purified milk protein and to determine the acute influence of energy nutrients. For this purpose, a [15N]-labeling dietary protein approach was used. Twenty-five subjects swallowed an ileal tube and ingested [15 N]-milk protein alone or supplemented with either milk fat or sucrose. The absorption and postprandial deamination of dietary protein was monitored for 8 h. Sucrose delayed the absorption of protein longer than fat, but the ileal digestibility did not differ among groups (94.5-94.8%). Sucrose, but not fat, significantly reduced the postprandial transfer of [15N]-milk nitrogen to urea. Consequently, the net postprandial protein utilization (NPPU) of milk protein calculated 8 h after meal ingestion was 80% when ingested either alone or supplemented with fat and was significantly greater with sucrose (NPPU = 85%). This study shows that energy nutrients do not affect the nitrogen absorption but modify the metabolic utilization of dietary protein in the phase of nitrogen gain. Our method provides information concerning the deamination kinetics of dietary amino acids and further allows the detection of differences of dietary protein utilization in acute conditions. The diet composition should be carefully considered, and protein quality must be determined under optimal conditions of utilization.

Elevated concentrations of soluble E-selectin and vascular cell adhesion molecule-1 in NIDDM. Effect of intensive insulin treatment.

OBJECTIVE: To evaluate the effects of a 14-day intensive insulin therapy and short-term improvement of glycemic control on serum levels of soluble forms of adhesion molecules, i.e., intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), and E-selectin (sE-selectin) in NIDDM patients with poor glycemic control. RESEARCH DESIGN AND METHODS: A total of 16 NIDDM patients were compared with 23 healthy subjects (control group) and investigated before and after intensive insulin treatment. RESULTS: On day 0, sE-selectin and sVCAM-1 levels were significantly higher in NIDDM patients than in nondiabetic control subjects (median 87, range 63-115; median 544, range 408-797 vs. 58, 43-80; 443, 395-573 ng/ml, respectively) (P < 0.008 in both cases). On day 15, the fall in sE-selectin levels was significant (P < 0.0001) and at a lesser extent in sVCAM-1 levels (64, 48-85; 506, 417-678 ng/ml, respectively); these levels reached values that no longer differed from those of control subjects (P = 0.23 and 0.15, respectively). Moreover, the fall in sE-selectin was positively associated with the change in LDL cholesterol and the improvement of glycemia. CONCLUSIONS: In poorly controlled NIDDM patients, sE-selectin levels are increased and significantly fall to normal after short-term improvement of glycemic control. This suggests that assaying sE-selectin makes it possible to detect endothelium activation and to follow its reversal with euglycemia.

Large-scale analysis of hepatitis C virus serological typing assay: effectiveness and limits.

The HCV (hepatitis C virus) Serotyping 1-6 Assay (Murex Laboratories) was evaluated on 303 French HCV-infected patients. Serological typing results were compared to the genotypes obtained from sequence analyses of the 5' noncoding regions of the virus genome from 46 HCV-infected patients, and assay specificity was found to be high (97.6%). The serological typing assay, run in 257 consecutive HCV-infected patients, yielded an assay sensitivity lower (70.6%) than that previously reported. This finding was attributed mainly to nonreactive sera from human immunodeficiency virus (HIV)-positive patients (P < 0.001) and perhaps reflected cryoglobulin positivity in others. No anti-type 6 reactivity was detected, and the overall serological type distribution values for types 1 to 5 were 67.3, 7.9, 16.4, 6.6, and 0.9%, respectively. A higher prevalence of type 4 was noted among HIV-infected patients (P < 0.001). In addition, serotype 2 was significantly more frequent in cryoglobulinemia positive than in cryoglobulinemia-negative patients (P < 0.05). Although an initial high level (7%) of mixed serological typing reactivities was found, after predilution of serum only two mixed infections could be confirmed (0.9%). It is suggested, therefore, that mixed reactivities have to be interpreted carefully and retested with prediluted serum, particularly when the optical density of the reactivity is > 2.5 or remains > 0.4 after competition with all type-specific peptides. The high specificity and relatively good sensitivity even in immunocompromised patients obtained with this assay indicate that it can be used routinely. Because response to treatment is linked to HCV type, this assay could be used to identify HCV serotype to guide therapeutic decisions.

Hepatitis C virus genotypes implicated in mixed cryoglobulinemia.

Recent reports suggest that hepatitis C virus (HCV) might be a causative agent of mixed cryoglobulinemia. To determine whether the HCV genotype is a factor implicated in the onset of cryoglobulinemia, genotyping by direct sequencing of polymerase chain reaction products of the 5' non coding region was carried out among 45 HCV-infected patients. Genotypes 1 and 2 were found more prevalent in symptomatic cryoglobulinemia patients. Due to the presence of genotypes 4 and 5 found in this panel of French patients (9.3%), HCV genotyping based on sequence determination is recommended.

Prevalence of cryoglobulins and hepatitis C virus infection in HIV-infected patients.

PURPOSE AND METHODS: In order to evaluate the prevalence of positive hepatitis C virus (HCV) serology and cryoglobulinemia in human immunodeficiency virus (HIV)-infected patients, the prevalence and the clinical significance of cryoglobulinemia were prospectively studied in a cohort of 86 HIV-infected subjects seen as outpatients. They were compared to a control group consisting of 101 HIV-HCV+ patients being followed at the same hospital. RESULTS: HCV serology was positive in 53/86 (61.6%) patients, 25 (47.2%) of whom had detectable cryoglobulins in their sera although only 1 had clinical symptoms consistent with cryoglobulinemia. Cryoglobulinemia was also detected in 9/33 (27.3%) HCV- patients, with only one of them presenting clinical symptoms. Although the mean cryoglobulin concentration was lower for HIV+ patients than in controls (268 versus 585 mg/l, p < 0.01), their prevalence (39.5% and 27.2%, respectively) was higher (p < 0.03). CONCLUSION: Cryoglobulinemia is frequently detected in HIV-infected patients, regardless of their HCV serology, but is poorly correlated with clinical symptoms.

[15N]-labeled pea flour protein nitrogen exhibits good ileal digestibility and postprandial retention in humans.

The aim of the present study was to evaluate postprandial absorption of pea protein as well as exogenous nitrogen retention in humans. For this purpose, after fasting overnight, seven healthy adults (4 males and 3 females) ingested [15N]-labeled pea protein (195 mmol N). Ileal effluents were collected for 8 h at 30-min intervals using a nasointestinal intubation technique. Urine and plasma samples were collected for 24 h. The [15N]-enrichment was determined in the intestinal samples, in the plasma amino acids and urea as well as in the urinary urea and ammonia fractions. The true gastroileal absorption of pea protein was 89.4 +/- 1.1%. This absorption was correlated with a significant increase (P < 0.05) in [15N]-enrichment in the plasma amino acids and in the nitrogen incorporated into the body urea pool for 1 h following pea ingestion. The enrichment remained significantly higher than the basal values in these pools 24 h after pea ingestion. The recovery of total urinary exogenous nitrogen after 22 h was 31.1 +/- 9.3 mmol N. Moreover, the kinetics of [15N]-labeled pea amino acids deamination reached a plateau of 39 mmol. Under these conditions, pea nitrogen retention represented 78% of the absorbed dietary nitrogen in healthy humans. The present results demonstrate the good true nitrogen digestibility and retention of pea protein in humans.