c-Kit identifies a subpopulation of mesenchymal stem cells in adipose tissue with higher telomerase expression and differentiation potential.

The stromal vascular fraction (SVF) of adipose tissue is an easy to obtain source of adipose tissue-derived stem cells (ADSCs). We and others have achieved significant but suboptimal therapeutic effects with ADSCs in various settings, mainly due to low rates of differentiation into specific cell types and with the downside of undesired side effects as a consequence of the undifferentiated ADSCs. These data prompted us to find new stem cell-specific markers for ADSCs and/or subpopulations with higher differentiation potential to specific lineages. We found a subpopulation of human ADSCs, marked by c-Kit positiveness, resides in a perivascular location, and shows higher proliferative activity and self-renewal capacity, higher telomerase activity and expression, higher in vitro adipogenic efficiency, a higher capacity for the maintenance of cardiac progenitors, and higher pancreatogenic and hepatogenic efficiency independently of CD105 expression. Our data suggests that the isolation of ADSC subpopulations with anti-c-Kit antibodies allows for the selection of a more homogeneous subpopulation with increased cardioprotective properties and increased adipogenic and endodermal differentiation potential, providing a useful tool for specific therapies in regenerative medicine applications.

Intestinal persistence of a plasmid harbouring the OXA-48 carbapenemase gene after hospital discharge.

To study intestinal colonization by OXA-48-producing Klebsiella pneumoniae (KpO48) after hospital discharge, stool samples from 22 previously colonized subjects were collected. Time from discharge was 33-611 days, without readmissions. Eight subjects (36%) were identified as blaOXA-48 gene carriers. In all of them the hospital-acquired strain of KpO48 had been lost, and the gene was harboured by other strains of K. pneumoniae, Klebsiella oxytoca and/or Escherichia coli. Our findings show intestinal persistence for several months of a plasmid harbouring the OXA-48 carbapenemase gene in a significant proportion of individuals in the absence of antibiotic treatment.

Altered concentrations of appetite regulators may contribute to the development and maintenance of HIV-associated wasting.

OBJECTIVE: To examine the relation of circulating appetite neuropeptides, CCK-8 sulphate (CCK-8s) and beta-endorphin, and the tumour necrosis factor-alpha (TNF-alpha) and soluble TNF receptors (sTNFR) to the anorexia and wasting associated with HIV-infection. DESIGN: Cross-sectional analysis. SETTING: A university-based HIV/AIDS ambulatory clinic in Madrid, Spain. PARTICIPANTS: Thirty-six randomly selected AIDS patients without concomitant diseases or secondary infections were classified into two groups: 19 patients with wasting and 17 with normal body weight, and 18 healthy controls. MEASUREMENTS: Nutritional status was evaluated by anthropometry, laboratory parameters and self-report of appetite. Plasma levels of TNF-alpha and sTNFR proteins p55 (sTNFR-p55) and p75 (sTNFR-p75) were determined by enzyme immunoassay, whereas CCK-8s and beta-endorphin levels were measured by radioimmunoassay. RESULTS: AIDS patients with wasting had significantly higher plasma concentrations of CCK-8s, but lower levels of beta-endorphin when compared to well-nourished AIDS patients (P < 0.01) or controls (P < 0.001). Mean levels of TNF-alpha, and sTNFR-p55 and sTNFR-p75 were greater in AIDS patients with wasting than in asymptomatic AIDS patients or in controls. No significant association was observed between any of these circulating peptides and the parameters of malnutrition. CONCLUSIONS: An activation of the TNF system, together with reciprocal changes in plasma concentrations of two neuropeptides with opposing appetite regulation, that is increased concentrations of CCK-8s but lower levels of beta-endorphin, are associated with the presence of HIV wasting. We hypothesize that these changes may contribute to the development of HIV wasting by producing a pathological inhibition of appetite.

Alterations of anorectic cytokine levels from plasma and cerebrospinal fluid in idiopathic senile anorexia.

Plasma and cerebrospinal fluid (CSF) concentrations of two well-known satiety cytokine peptides have been measured in elderly persons with idiopathic anorexia and normal weight healthy subjects in a similar age range. Plasma and CSF levels of tumor necrosis factor alfa (TNF alpha) and interleukin-1 beta (IL-1 beta) were assayed by commercially available kits. Elderly under-weight anorectic patients had significantly lower levels of TNF alpha but unchanged concentrations of IL-1 beta in both plasma and CSF when compared to controls. In addition to significantly lower levels of TNF alpha in CSF, we found a positive correlation between the body mass index and CSF TNF alpha concentrations (r = 0.61, P < 0.05) in the anorectic group. CSF IL-1 beta concentrations showed a significant negative correlation with plasma albumin levels in senile anorectics (r = -0.66, P < 0.05) but not in controls. On the basis of our findings, we conclude that a decrease in plasma and CSF TNF alpha concentrations could have beneficial effects for the primary anorexia of aging both at central level to offset anorexia and at peripheral sites decreasing tissue catabolism.

Cell-mediated immune response and cytokine production in idiopathic senile anorexia.

Some aspects of humoral and cell-mediated immunity and the capacity of peripheral blood mononuclear cells (PBMCs) of fourteen elderly persons with idiopathic anorexia to produce several cytokines, such as tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 and interferon-gamma (IFN gamma), were studied and the results were compared with those obtained in a control group of ten age-matched, normal weight healthy subjects. In addition, spontaneous and induced production of these cytokines was also measured in cultures of PBMCs of fourteen healthy young individuals as a control group of age. A significant decrease in CD2 (pan T-cells) and CD4 (T-helper) lymphocyte subpopulations, but unchanged CD8 (T-suppressor) subset, and a reduced response in delayed cutaneous hypersensitivity tests were observed in senile underweight anorectic patients. Monocyte counts did not show significant differences between patients and control subjects. The spontaneous release by PBMCs of all the cytokines measured did not differ between the anorectic and either the elderly or young control group. A significant increase in IL-6 production after mitogen stimulation with tetradecanoylphorbol acetate (TPA) and phytohemagglutinin (PHA) after 24 and 48 h of culture, as well as a greater induced TNF alpha production after 48 h of incubation with the same mitogens, was found in the anorectic patients as compared with the elderly controls. However, stimulated production of both IL-1 beta with TPA and of IFN gamma with PHA did not differ significantly between anorectics and aged controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of gastrin-releasing peptide, neuropeptide Y, somatostatin and vasoactive intestinal peptide on interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha production by whole blood cells from healthy young and old subjects.

In the present study, we have investigated the effect in vitro of gastrin-releasing peptide (GRP, 10(-10) M), neuropeptide Y (NPY, 10(-10) M), somatostatin (10(-10) M) and vasoactive intestinal peptide (VIP, 10(-9) M) on the production of IL-1 beta, IL-6 and TNF alpha by peripheral whole blood cells from healthy young and old people. We have found that GRP, NPY, somatostatin and VIP stimulated the production of IL-1 beta in old subjects, and NPY, somatostatin and VIP in young ones. In addition, the production of IL-6 was enhanced by GRP, NPY and VIP in young and old people. The TNF alpha production was stimulated by NPY and somatostatin in young subjects, and by NPY, somatostatin and VIP in old ones, whereas GRP produced a decrease of TNF alpha in young persons. GRP in old subjects and VIP in young and old subjects stimulated in a great degree the LPS-induced IL-6 production by whole blood cells. On the contrary, GRP and VIP inhibited highly the LPS-induced TNF alpha production in young controls. Our results show that these neuropeptides, when added to whole blood cells at physiological concentrations, are able to stimulate the production of IL-1 beta, IL-6 and TNF alpha in a differential way according to the subject age.

Alterations in plasma and cerebrospinal fluid levels of neuropeptides in idiopathic senile anorexia.

Plasma and cerebrospinal fluid (CSF) concentrations of three well-known satiety neuropeptides, cholecystokinin (CCK), somatostatin and calcitonin gene-related peptide (CGRP), along with two powerful orexigenic neuropeptides, neuropeptide Y (NPY) and beta-endorphin have been measured in elderly persons with idiopathic anorexia and normal weight healthy subjects in a similar age range. Plasma and CSF immunoreactivity levels of the two main fractions of CCK (CCK8s and CCK33) after being separated by HPLC were measured by a radioimmunoassay (RIA) developed in our laboratory, whereas the other neuropeptides were assayed by commercially available RIA kits. Elderly underweight anorectic patients had significantly lower levels of beta-endorphin but increased concentrations of NPY in both plasma and CSF when compared to controls. In addition to significantly higher levels of CCK8s but not CCK33 in plasma, we found a trend to higher CSF concentrations of CCK8s and a positive correlation between the body mass index and either beta-endorphin (r = 0.58, P < 0.05) or CCK8s (r = 0.69, P < 0.01) concentrations in CSF in the anorectic group. CSF somatostatin concentrations were decreased significantly, but plasma somatostatin levels and plasma and CSF concentrations of CGRP were similar in senile anorectics and controls. Treatment of five anorectic patients with megestrol acetate, 480 mg daily for 6 months, reversed only the decrease in CSF beta-endorphin levels but did not normalize the body weight or the fat body mass. On the basis of our findings, we hypothesize that a decrease in CSF beta-endorphin concentration along with a rise in plasma levels of CCK8s might be accounted for the primary anorexia of aging.

Relationship between circulating levels of calcitonin gene-related peptide, nitric oxide metabolites and hemodynamic changes in human septic shock.

This study is aimed to investigate the relationship between plasma concentrations of nitrite and nitrate as a measure of ongoing nitric oxide (NO) production, the vasodilatory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), endotoxemia and hemodynamic changes in human septic shock. Thirteen patients with septic shock were studied within 6 h after the development of hypotension. Hemodynamic measurements and blood samples were recorded simultaneously at 2-h intervals from study admission. Eighteen normotensive patients with sepsis were included as control group of patients. On study entry, circulating levels of endotoxin did not relate to either CGRP or nitrite and nitrate plasma values. Septic shock patients had significantly higher plasma CGRP, and nitrite and nitrate concentrations, at each of the four time points, than patients with sepsis, as well as both groups of patients compared to normal subjects. No differences were found in plasma SP levels between the two groups of patients. For pooled data from all septic shock patients and measurements (n = 52), both plasma concentrations of CGRP and nitrite and nitrate were inversely correlated, independently from each other, to systemic vascular resistance. On study admission and at 2-h intervals, plasma CGRP concentrations correlated directly with nitrite and nitrate values. Our observations, thus, point to CGRP acting in concert with NO as important mediators responsible for hypotension in human septic shock.

Postprandial glucose-dependent insulinotropic polypeptide and insulin responses in patients with chronic pancreatitis with and without secondary diabetes.

OBJECTIVE: To study GIP and insulin release after a test meal in patients with chronic pancreatitis with and without secondary diabetes mellitus. METHODS: 28 patients with chronic pancreatitis were classified in groups I and II according to the presence or absence of secondary diabetes mellitus. Twelve healthy subjects were included as controls. After a test meal plasma GIP levels and serum insulin levels were determined at 0, 30, 60, 120 and 180 min. RESULTS: A significant diminished GIP response was found in the groups of patients with respect to the control group. No association could be detected with severity of pancreatic insufficiency. Higher values of GIP were demonstrated at 60 and 120 min in patients without diabetes than in patients with it. CONCLUSIONS: An abnormal GIP response is present in cases of chronic pancreatitis irrespective of the presence or severity of pancreatic insufficiency. This response is further affected if secondary diabetes mellitus is present.

Relationship of basal and postprandial intraduodenal bile acid concentrations and plasma cholecystokinin levels with abdominal pain in patients with chronic pancreatitis.

Abdominal pain in patients with chronic pancreatitis has been related to an increase in plasma cholecystokinin (CCK) levels. The aim of the study was to disclose the relation of the altered response with the low intraduodenal bile acids levels found in these patients. Twenty patients with chronic pancreatitis were classified into groups I (n = 11) and II (n = 9) according to the presence or absence of pain. Intraduodenal trypsin and bile acids concentrations and plasma CCK levels were measured before and 30, 60, and 90 min after a test meal. Comparisons between values in both groups were carried out. Correlation of intraduodenal trypsin and bile acids with plasma CCK was analyzed. Patients with pain exhibited significantly lower intraduodenal trypsin levels at 30 and 90 min and lower basal and postprandial intraduodenal bile acids levels than patients without pain. In patients with pain, basal and postprandial plasma CCK levels were significantly higher than in patients without pain. A negative correlation was demonstrated between intraduodenal bile acids and plasma CCK. In patients with chronic pancreatitis and pain, a reduction in intraduodenal postprandial trypsin and basal and postprandial bile acids concentrations, as well as an increase in basal and postprandial plasma CCK levels, was encountered. A negative correlation between intraduodenal bile acids and plasma CCK concentrations was detected that may be implicated in the pathogenesis of pain.

The effect of gold salts on substance P levels in rheumatoid arthritis.

The effect of gold salt therapy on substance P immunoreactivity levels in plasma and synovial fluid was studied in 42 patients with rheumatoid arthritis. Decreased levels of synovial fluid substance P, although not statistically significant, were found in rheumatoid patients who were currently receiving gold therapy when compared to either those patients previously treated or to those who never received this therapy. In addition, we found that patients who received more than 1000 mg of gold salts had significantly lower levels of substance P in synovial fluid than those treated with lower doses. Our results, therefore, seem to support the hypothesis that gold salts appear to be slow-acting neurotoxic drugs that significantly decrease the intrasynovial concentrations of substance P, a well-known inflammatory neuropeptide, in arthritis patients.

Neuropeptides and interleukin-6 in human joint inflammation relationship between intraarticular substance P and interleukin-6 concentrations.

Plasma and synovial fluid concentrations of interleukin-6 (IL-6), using an enzyme-linked immunosorbent assay, as well as immunoreactive levels of calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal peptide (VIP) were measured in 18 patients with rheumatoid arthritis and 20 with osteoarthritis of the knee. The concentrations of IL-6 were elevated in both plasma and synovial fluids from patients with rheumatoid arthritis whereas higher levels of substance P-, CGRP- and VIP-like immunoreactivities were found in the synovial fluid, but not in plasma, from patients with rheumatoid arthritis when compared with those in osteoarthritis. Furthermore, IL-6 and substance P levels in synovial fluid were significantly correlated both in rheumatoid arthritis and osteoarthritis patients. Our data seem to support the idea of an important role shared by neuropeptides and IL-6 in the pathogenesis of human inflammatory joint disease.

Intracellular glutathione deficiency is associated with enhanced nuclear factor-kappaB activation in older non-insulin dependent diabetic patients.

Diabetes mellitus may be associated with intracellular glutathione (GSH) deficiency. Since in vivo studies have shown that plasma intracellular GSH plays a key role in regulating the activation of nuclear factor kappaB (NF-kappaB), we have investigated the relationship between intracellular thiols (GSH, homocysteine, cysteine and cysteinyglycine) and NF-kappaB activity in the peripheral blood mononuclear cells (PBMC) of 63 elderly non-insulin dependent diabetes mellitus (NIDDM) patients (28 microalbuminurics and 35 normoalbuminurics) and 30 healthy age- and sex-matched subjects. In addition, we have measured plasma concentrations of these thiol compounds, serum concentrations of interleukin-6 (IL-6) and vascular cell adhesion molecule-1 (sVCAM-1), that are partly dependent on the NF-kappaB activation, as well as the serum levels of thiobarbituric acid reacting substances (TBARS), as index of lipid peroxidation. Diabetic patients with microalbuminuria (MAB) and normoalbuminuria had NF-kappaB activity 2.1- and 1.5-fold greater, respectively, than the control group. As compared to normoalbuminuric patients, patients with MAB had significantly higher levels of glycemia, plasma homocysteine, and serum concentrations of TBARS, IL-6 and sVCAM-1 (in all cases, p < 0.01), and significantly lower GSH content in the PBMC (p < 0.05). The intracellular GSH in PBMC correlated with NF-kappaB activation (r = -0.82; p < 0.0001), serum TBARS (r = -0.60; p < 0.001), and with fasting glycemia (r = -0.56; p < 0.001) in patients with MAB, whereas a weaker association between GSH levels in PBMC and NF-kappaB activation (r = -0.504, p < 0.001) was seen in patients without MAB. These results suggest that the decrease of intracellular GSH content in elderly NIDDM patients with MAB is strongly associated with enhanced NF-kappaB activation, which could contribute to the development of increased glomerular capillary permeability and its rapid progression.

An outbreak of Plasmodium vivax malaria among heroin users in Spain.

We report the first outbreak of induced malaria among heroin users in Spain, and the first one caused by Plasmodium vivax in Europe. Five drug addicts from Madrid, who had never travelled to endemic areas, were admitted to hospital with fever and splenomegaly. Four had P. vivax malaria with low parasitaemia, ranging from 1 to 3% red blood cells. The fifth case was considered a "seropositive contact" because he had fever and positive malaria indirect fluorescent antibody test but negative blood smear. The source of infection was a young drug addict, who had often travelled to Equatorial Guinea. Another heroin user with a diagnosis of malaria refused to be admitted to our hospital for further study. All had shared contaminated injection equipment. Treatment with chloroquine was effective and none had recrudescence of malaria during a mean follow-up of six months. Drug addicts with unexplained fever may have been infected by malaria transmitted by sharing injections.

Altered cerebrospinal fluid amino acid pattern in the anorexia of aging: relationship with biogenic amine metabolism.

To study the possible role of several amino acids on feeding in the anorexia of aging, we have measured plasma and cerebrospinal fluid (CSF) concentrations of 22 amino acids in 14 elderly persons with idiopathic anorexia and 10 healthy subjects with normal weight in a similar age range. Plasma and CSF amino acid concentrations and CSF homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) levels were all measured by HPLC methods. Elderly anorectic subjects had significantly lower levels of glutamic acid but increased concentrations of glutamine in both plasma and CSF compared to controls. Likewise, a significant increase of histidine, threonine, alanine, arginine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, ornithine and lysine was found in CSF, but not in plasma, from patients with anorexia. Besides, the CSF histidine/LNAA (large neutral amino acids) and tryptophan/LNAA ratios were elevated in anorectic patients as compared with controls of similar age. In addition, we found higher CSF concentrations of HVA and 5-HIAA, as well as a positive correlation between CSF LNAA and either HVA (r = 0.74, p = 0.002) or 5-HIAA (r = 0.61, p = 0.020) concentrations in elderly anorectics. CSF tryptophan correlated positively with 5-HIAA levels (r = 0.59, p = 0.026) and CSF tyrosine with HVA levels (r = 0.77, p = 0.002). Our results suggest that changes in the CSF concentration of amino acids could contribute to an increased biogenic amine metabolism in the central nervous system of elderly anorectic subjects, possibly increasing the synaptic liberation of biogenic amines involved in the appetite regulation.

Changes in the intracellular homocysteine and glutathione content associated with aging.

Since moderate hyperhomocysteinemia is an independent risk factor for vascular disease by mean of its oxidant effect and glutathione plays a main role as intracellular redox-regulating agent, we have studied for the first time the total intracellular content of homocysteine in aging. Plasma homocysteine concentration, total intracellular and plasma glutathione, and other related thiol compounds such as cysteine and the glutathione catabolite cysteinglycine were also studied. Forty three healthy elderly subjects and twenty seven healthy young ones were studied. The total intracellular peripheral blood mononuclear cell content was higher for homocysteine, cysteine and cysteinglycine, whereas that of the total glutathione was greatly decreased in elderly people with respect to young ones. Elderly subjects showed significantly higher levels than young ones of total plasma homocysteine and cysteinglycine, but not cysteine, whereas total plasma glutathione levels were increased. In addition, elderly subjects showed significantly decreased plasma vitamin E levels and increased concentrations of serum lipid peroxides measured as TBARS (reaction product of malondialdehyde with thiobarbituric acid). The intracellular glutathione content presented significantly negative correlation with serum TBARS, and intracellular and plasma homocysteine levels. These findings show an increase of homocysteine synthesis associated with aging, which in turn can produce an augmented oxidant effect on endothelium, and an impaired intracellular antioxidant capacity leading to an enhanced lipid peroxidation and decreased total intracellular glutathione content.

Changes in plasma concentrations of vasoactive neuropeptides in patients with sepsis and septic shock.

The aim of this work was to study the hypothesis that the release of vasoactive neuropeptides may be related to the hemodynamic changes and severity of disease in human sepsis and septic shock. Twenty-two patients diagnosed with sepsis and treated in medical wards with standard supportive therapy and twenty patients admitted to a medical intensive care unit because of septic shock were studied Twenty healthy volunteers in a similar age range were enrolled as control group. Blood samples were taken at onset and every 12 hours on the following day after hospital admission to measure plasma concentrations of calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and substance P (SP). Clinical and biochemical variables were measured simultaneously. The Acute Physiology and Chronic Health Evaluation (APACHE) II score was calculated on admission. From the day of admission, septic shock patients had significantly higher plasma CGRP-like immunoreactivity levels than patients with sepsis, as well as both groups of patients compared to control subjects. Plasma NPY-like immunoreactivity levels in patients with either sepsis or septic shock was significantly increased, and plasma SP-like immunoreactivity levels significantly reduced compared to those in controls. Plasma CGRP levels at study entry correlated with the APACHE II score (r = 0.71, p < 0.01), as well as with the cardiac index (r = 0.61, p < 0.05) and systemic vascular resistance index (r = -0.62, p < 0.05). Our data suggest that both CGRP and NPY, but not SP, are increasedly released into the circulation during the development of human sepsis and septic shock. In patients with sepsis the vasoconstriction mediated by the release of NPY appears to counterbalance the vasodilatory effect of CGRP. In septic shock patients, however, the release of NPY might be inadequately low to overcome the widespread CGRP-induced vasodilation.

Cardiac size and function in Paget's disease of bone.

We performed non-invasive assessment of cardiac size and function by clinical criteria, standard electrocardiography, chest X-ray, systolic time intervals and echo-cardiography in 27 patients with Paget's disease of bone and in 20 control subjects. The patients were divided into two groups on the basis of the degree of skeletal involvement (less than 15% in Group I and greater than 15% in Group II). No differences in heart size parameters of left ventricular performance were noted between Group I and controls. Cardiomegaly, increased left ventricular diastolic dimension and increased left ventricular mass indicative of ventricular hypertrophy were found in Group II compared to control subjects. In addition, patients with more extensive skeletal involvement had signs of depressed myocardial contractility, increased left ventricular volumes in diastole and systole and enlarged stroke volume, with no differences in echographic cardiac output compared to Group I and controls. The findings show an above normal incidence of cardiac enlargement and disturbed left ventricular performance in patients with Paget's disease and osseous lesions in greater than 15% of skeleton. The clinical implications of the altered cardiac function in patients with Paget's disease are briefly discussed.

Complete proximal tubular acidosis (Type 2, RTA) in chronic active hepatitis.

Fifteen patients with chronic active hepatitis (CAH) were tested in order to ascertain the site of tubular dysfunction in renal tubular acidosis (RTA) associated with CAH. Renal plasma flow and GFR were reduced in the patients compared to controls (P < 0.005). Underbasal and acidification conditions, twelve patients showed normal acid-base balance and net acid excretion, while three patients had basal hyperchloremic metabolic acidosis while passing alkaline urine and showing bicarbonaturia. A sustained acidification test showed adequate urinary acidification in these three patients. Bicarbonate loading carried out in two of the three patients showed a proximal tubular acidifying defect (Type 2 RTA), Distal RTA (Type 1 RTA) complicating CAH is widely known, but these data suggest that CAH can also involve the proximal convoluted tubule in isolation.

Angiographic findings in systemic necrotizing vasculitis.

Angiographic studies were carried out on 21 patients with systemic necrotizing vasculitis. Four basic arterial anomalies were found: (1) saccular microaneurysms appeared in 62% of the patients (2) arterial thrombosis was seen in 81% of patients; (3) arterial stenosis occurred in 81%; and (4) lumen irregularities occurred in 90%. Alterations in the renal vascular flow were also observed in accordance with changes in the cortical medullary differentiation, heterogeneous nephrogram, and prolonged washout. Two patients showed regression of microaneurysms after immunosuppressive therapy. We found angiography to be a low-risk technique of use for diagnosis and followup studies on vasculitis.