Natural killer cell subsets in cerebrospinal fluid of patients with multiple sclerosis.

Changes in blood natural killer (NK) cells, important players of the immune innate system, have been described in multiple sclerosis (MS). We studied percentages and total cell counts of different effector and regulatory NK cells in cerebrospinal fluid (CSF) of MS patients and other neurological diseases to gain clearer knowledge of the role of these cells in neuroinflammation. NK cell subsets were assessed by flow cytometry in CSF of 85 consecutive MS patients (33 with active disease and 52 with stable MS), 16 with other inflammatory diseases of the central nervous system (IND) and 17 with non-inflammatory neurological diseases (NIND). MS patients showed a decrease in percentages of different CSF NK subpopulations compared to the NIND group. However, absolute cell counts showed a significant increase of all NK subsets in MS and IND patients, revealing that the decrease in percentages does not reflect a real reduction of these immune cells. Remarkably, MS patients showed a significant increase of regulatory/effector (CD56(bright) /CD56(dim) ) NK ratio compared to IND and NIND groups. In addition, MS activity associated with an expansion of NK T cells. These data show that NK cell subsets do not increase uniformly in all inflammatory neurological disease and suggest strongly that regulatory CD56(bright) and NK T cells may arise in CSF of MS patients as an attempt to counteract the CNS immune activation characteristic of the disease.

Anti-myelin antibodies play an important role in the susceptibility to develop proteolipid protein-induced experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is an autoimmune disorder in which activated T cells cross the blood-brain barrier (BBB) to initiate an inflammatory response that leads to demyelination and axonal damage. The key mechanisms responsible for disease initiation are still unknown. We addressed this issue in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. It is widely known that EAE manifests only in certain strains when immunized with myelin proteins or peptides. We studied the differential immune responses induced in two mouse strains that are susceptible or resistant to EAE induction when they are immunized with the 139-151 peptide of proteolipid protein, an encephalitogenic peptide capable of inducing EAE in the susceptible strain. The adequate combination of major histocompatibility complex alleles and myelin peptides triggered in susceptible mice a T helper type 17 (Th17) response capable of inducing the production of high-affinity anti-myelin immunoglobulin (Ig)G antibodies. These were not detected in resistant mice, despite immunization with the encephalitogenic peptide in junction with complete Freund's adjuvant and pertussis toxin, which mediate BBB disruption. These data show the pivotal role of Th17 responses and of high-affinity anti-myelin antibodies in EAE induction and that mechanisms that prevent their appearance can contribute to resistance to EAE.

[Metabolic, clinical and body composition changes in Mexican adults undergoing bariatric surgery]. / Cambios metabólicos, clínicos y de composición corporal en adultos mexicanos sometidos a cirugía bariátrica.

BACKGROUND: morbid obesity is a major public health problem that is increasing. Currently, there are a limited number of studies carried out in the Mexican population that describe the effects of bariatric surgery. OBJECTIVE: to establish in people undergoing a bariatric procedure the metabolic and body composition difference before and after bariatric surgery. MATERIAL AND METHODS: an observational, analytical, and longitudinal study was carried out in 50 patients with morbid obesity who underwent laparoscopic Sleeve Gastrectomy (LSG) and Laparoscopic Roux-en-Y gastric bypass (LRYGB). Body composition and metabolic markers in blood were measured. Differences in the metabolic profile before and after surgery were analyzed in the entire study group and a subanalysis was performed by bariatric surgical technique, determining the percentage of remission of comorbidities. RESULTS: after the intervention, there is a significant decrease in all metabolic and body composition markers, except HDL cholesterol, which showed a tendency to increase without being significant. Women with LRYGB have a greater decrease in fat-free mass. LRYGB decreased the prevalence of fatty liver, gastroesophageal reflux, insulin resistance, and hypercholesterolemia more, while LSG decreased the prevalence of hypertension, osteoarthritis, hypothyroidism, and hypertriglyceridemia more. CONCLUSION: bariatric surgery induces metabolic changes that could contribute to improving comorbidities associated with obesity. In general, metabolic improvement is greater in LRYGB compared to LSG.

Allergen profile of Protophormia terraenovae, other species of calliphoridae, and Lumbricus terrestris in anglers allergic to maggots in Cáceres, Spain.

BACKGROUND: Our group previously found that up to 7% of amateur anglers in Caceres, Spain may be allergic to the larvae of Protophormia terraenovae (order Diptera, family Calliphoridae) used as live bait for fishing. OBJECTIVE: To identify the pattern of major allergens in P terraenovae and other species of Calliphoridae. MATERIALS AND METHODS: Extracts of P terraenovae, Calliphora vomitoria, Lucilia sericata and Lumbricus terrestris were characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and IgE-immunoblotting techniques in individual sera from 24 patients with a positive skin test result and/or specific IgE determination (enzyme-linked immunosorbent assay [ELISA]) to P terraenovae. ELISA and IgE-immunoblotting inhibition studies were also performed to identify potential cross-reactive allergens between these species. RESULTS: IgE-immunoblotting with P terraenovae showed a band of 15.3 kDa recognized by 15 patients, in addition to 2 further allergens of 22.8 kDa and 69 kDa. For C vomitoria, 5 bands of 73, 46, 40, 28, and 14 kDa were observed. For L sericata, 2 major allergens of 73 kDa and 14 kDa were observed. In the case of L terrestris, IgE from 13 patients recognized 1 allergen of around 15.5 kDa. IgE-immunoblotting and ELISA inhibition revealed the presence of cross-reactivity, mainly between L terrestris and P terraenovae. CONCLUSIONS: P terraenovae appears to have species-specific allergens and allergens shared with C vomitoria and L sericata. Striking immunological cross-reactivity was observed between P terraenovae and L terrestris. An allergen of 15-16 kDa could be involved in this phenomenon.

Gallbladder plication as a rare complication of endoscopic sleeve gastroplasty: A case report.

BACKGROUND: Endoscopic sleeve gastroplasty (ESG) is a minimally invasive procedure used in the treatment of obesity, with a complication rate of less than 2% of cases. There have been only two reported cases worldwide of gallbladder injuries as a major complication of ESG. CASE SUMMARY: We present the case of a 34-year-old patient who developed a complication after ESG. The patient experienced epigastric and right hypochondrium pain 12 h after the procedure, and a positive Murphy's sign was identified on physical examination. Laboratory results showed a leukocyte count of 17 × 103/µL, and computed tomography indicated the presence of free fluid in the pelvic cavity and perihepatic recesses as well as a possible suture in the wall of the Hartmann's pouch toward the anterior surface of the stomach. A diagnostic laparoscopy was performed, revealing plication of the Hartmann's pouch wall to the anterior stomach wall. Laparoscopic cholecystectomy and lavage were carried out. The patient had a stable recovery and was discharged 72 h after surgery, tolerating oral intake. CONCLUSION: Gallbladder plication should be suspected if signs and symptoms consistent with acute cholecystitis occur after ESG.

Intraoperative endoscopy prevents technical defect related leaks in laparoscopic Roux-en-Y gastric bypass: A randomized control trial.

BACKGROUND: Postoperative anastomotic leaks, bleeding and stenosis are major causes of morbidity after laparoscopic Roux-en-Y gastric bypass (LRYGB). Retrospective studies suggest that intraoperative endoscopy reduces the incidence of these complications. METHODS: We conducted a prospective randomized controlled trial in a single institution between March 2013 and January 2016. Patients were assigned to one of two groups: LRYGB with Intraoperative Endoscopy (IOE) or LRYGB without IOE. Patient selection criteria were morbidly obese patients, 18 years or older who were candidates to LRYGB. The primary outcome was the frequency of technical defect related anastomotic leaks. Secondary outcomes were operative time, length of hospital stay, anastomotic related complications, reoperations and 30-day mortality. RESULTS: 50 patients were randomly assigned in the IOE group and 50 in the control group. The IOE group had statistically significant lower rate of anastomotic leak (0 vs. 8%, p = .0412), and lower need for reoperation (0 vs. 8%, p = .0412). The IOE group had longer operative time (194.10 vs. 159 min, p < .001), and shorter mean length of hospital stay (2.44 vs. 3.46 days, p = .025). No differences were found in the rate of bleeding of the anastomosis, narrow anastomosis and 30-day mortality. CONCLUSION: This study specifically provides evidence that air leak test performed by intraoperative endoscopy is superior to simple visual inspection in preventing technical defect related leaks after laparoscopic Roux-en-Y gastric bypass.

Adipocyte Size and Leptin Receptor Expression in Human Subcutaneous Adipose Tissue After Roux-en-Y Gastric Bypass.

The molecular mechanisms implicated in pronounced weight loss and metabolic benefits after bariatric surgery are still unknown. Adipocyte phenotype and metabolism have not been entirely explored. However, some features of adipocyte function have been studied, such as adipocyte size and inflammation, which are both reduced after bariatric surgery. Adipocyte fat metabolism, which is partly regulated by leptin, is likely modified, since adipocyte area is decreased. Here, we show that leptin receptor expression is increased, while adipocyte size is decreased 8 months after Roux-en-Y gastric bypass. Thus, adipocyte function is possibly modified by improved leptin signaling after bariatric surgery.

Effect of nitric oxide on the somatostatinergic system in the rat exocrine pancreas.

Nitric oxide (NO) and somatostatin (SS) are two important mediators of the exocrine and endocrine pancreas, exerting opposite effects on this organ. There is strong evidence suggesting an interaction between pancreatic NO and SS. The aim of this study was to determine whether L-arginine (L-Arg), the substrate for NO synthase (NOS), and Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, regulate pancreatic somatostatin-like immunoreactivity (SSLI) content and the SS mechanism of action in pancreatic acinar cell membranes. L-Arg (150 mg/kg, intraperitoneally (i.p.)), L-NAME (50 mg/kg, i.p.) or L-NAME plus L-Arg were injected twice daily at 8 h intervals for 8 days. L-Arg decreased pancreatic SSLI content as well as the number of SS receptors in pancreatic acinar cell membranes whereas L-NAME increased both parameters. The stable SS analogue SMS 201-995 induced a significantly lower inhibition of forskolin-stimulated adenylyl cyclase activity in pancreatic acinar cell membranes from L-Arg-treated rats whereas an increased inhibition was observed in pancreatic acinar membranes from L-NAME-treated rats. These results indicate that the NO system may contribute to the regulation of the pancreatic somatostatinergic system.

Lactational changes in the rat exocrine pancreas somatostatin receptors and modulation of guanylate cyclase.

Hyperplasia of the pancreatic tissue during late lactation (third week) and lasting for at least the first two weeks after weaning has been observed by several authors. Since the tetradecapeptide somatostatin (SS) inhibits pancreatic growth and its plasma levels are elevated during these periods, the aim of the present study was to determine the possible implication of the somatostatinergic system in the pancreatic changes cited above. Thus, the present study investigated 125I-Tyr(11)-somatostatin (125I-Tyr(11)-SS) binding and the effects of SS on guanylate cyclase activity as well as pancreatic somatostatin-like immunoreactivity (SSLI) levels in pancreatic acinar membranes from control, lactating and weaning rats. SS receptors were identified using 125I-Tyr(11)-SS and isolated pancreatic acinar membranes in vitro. There was an increase in the number of SS receptors after the third week of lactation (244 +/- 6 vs. 155 +/- 12 fmol/mg protein, P < 0.01) and the first two weeks after weaning (327 +/- 8 vs. 164 +/-10 fmol/mg protein, P < 0.001). No change in the affinity of the receptor site was detected at either study time. In addition, SS-stimulated guanylate cyclase activity was markedly increased at the third week of lactation (119%) and at the second week after weaning (158%) when compared with the control group. In contrast, basal guanylate cyclase activity was not modified at either study period. Thus, SS-stimulated guanylate cyclase activity is increased in pancreatic acinar membranes at late lactation and at the second week after beginning weaning probably due to an increase in the number of SS receptors. Significant decreases in SSLI content were observed at the third week of lactation (69%) and the second week after weaning (37%) when compared with the respective controls. The present results suggest that pancreatic acinar cell growth observed at the third week of lactation and the second week after weaning is associated with up-regulation of SS receptors which would represent a mechanism promoted by the cell that would negatively regulate the mitogenic activity of the increased number of pancreatic growth factors observed during both periods.

Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning.

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.

Thiolic antioxidants protect from nitric oxide-induced toxicity in fetal midbrain cultures.

Nitric oxide (NO) may act as a neuroprotector or neurotoxic agent in dopamine neurons, depending on cell redox status. We have investigated the effect of several thiolic antioxidants, glutathione (GSH), its cell permeable analog GSH ethyl ester (GSHEE), and the GSH synthesis precursor L-N-acetyl cysteine (L-NAC), as well as non-thiolic antioxidants like ascorbic acid (AA) and uric acid, on NO-induced toxicity in fetal midbrain cultures. The cultures were treated for 8-24 h with neurotoxic doses of the NO donor diethylamine/nitric oxide complex sodium DEA/NO (200-400 micro M) and/or antioxidants. Thiolic antioxidants, at equimolar concentrations, added at the same time or previous to DEA/NO, protected from cell death, from tyrosine hydroxylase (TH) positive cell number decrease and from intracellular GSH depletion, induced by DEA/NO, without increasing intracellular GSH content. In these conditions, S-nitrosothiol compound formation was detected in the culture media. Protection disappeared when antioxidants were supplied 30 min after NO treatment. Nevertheless, non-thiolic antioxidants, AA and uric acid, with similar peroxynitrite scavenging activity to thiolic antioxidants, and free radical-scavenging enzymes as catalase and Cu/Zn-superoxide dismutase, which prevent extracellular peroxynitrite ion formation, and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), which prevents intracellular peroxynitrite ion formation, did not rescue cell cultures from neurotoxicity induced by NO. In addition, AA exacerbated DEA/NO-induced toxicity in a dose-dependent manner from 200 micro M AA. The present results suggest that only antioxidants with thiol group exert neuroprotection from NO-induced toxicity in fetal midbrain cultures, probably by direct interaction of NO and thiol groups, resulting in NO blocking. On the other hand, some classical antioxidants, like AA, exacerbate neurotoxicity due to NO.

Nitric oxide induces differentiation in the NB69 human catecholamine-rich cell line.

The nitric oxide (NO) donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), induced differentiation of human neuroblastoma NB69 cells to a dopamine phenotype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 microM SNAP in serum-free-defined medium for 24 h. SNAP treatment did not increase the number of necrotic or apoptotic cells. However, a decrease in the number of viable cells was observed at 750 microM SNAP. In addition, a decrease in (3)H-thymidine uptake was detected at the highest dose of SNAP. An increase in the antiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapoptotic Bax and Bcl-xS protein levels were also detected by Western blot analysis after SNAP treatment. At low doses (50-125 microM), SNAP induced an increase in catecholamine levels, (3)H-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250-750 microM). The TH protein content, analyzed by Western blot, remained unchanged in SNAP-treated cells throughout the range of doses studied, when compared with the control group. SNAP produced a dose-dependent decrease in the glutathione (GSH) content of the culture medium, without altering intracellular GSH. In addition, cGMP levels and nitrite concentration, measured in the supernatant of SNAP-treated cells, increased in a dose-dependent manner, as compared to control levels. The guanylate cyclase inhibitor lH-[1,2, 4]oxadiazolo[4,3a]quinoxaline-l-one (ODQ) did not revert the SNAP-induced effect on (3)H-dopamine uptake to control values. These results suggest that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the post-transcriptional level through a cGMP-independent mechanism.

Hippocampal somatostatin receptors and modulation of adenylyl cyclase activity in histamine-treated rats.

In the present study, the effects of an intracerebroventricular (i.c.v.) dose of histamine (0.1, 1.0 or 10.0 micrograms) on the hippocampal somatostatin (SS) receptor/effector system in Wistar rats were investigated. In view of the rapid onset of histamine action, the effects of histamine on the somatostatinergic system were studied 2 h after its administration. Hippocampal SS-like immunoreactivity (SSLI) levels were not modified by any of the histamine doses studied. SS-mediated inhibition of basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity was markedly increased in hippocampal membranes from rats treated with 10 micrograms of histamine (23% +/- 1% vs. 17% +/- 1% and 37% +/- 2% vs. 23% +/- 1%, respectively). In contrast, neither the basal nor the FK-stimulated enzyme activities were affected by histamine administration. The functional activity of the hippocampal guanine-nucleotide binding inhibitory protein (Gi protein), as assessed by the capacity of the stable GTP analogue 5'-guanylylimidodiphosphate (Gpp[NH]p) to inhibit FK-stimulated AC activity, was not modified by histamine administration. These data suggest that the increased response of the enzyme to SS was not related to an increased functional activity of Gi proteins. In fact, the increased AC response to SS in hippocampal membranes from histamine (10 micrograms)-treated rats was associated with quantitative changes in the SS receptors. Equilibrium binding data obtained with [125I]Tyr11-SS indicate an increase in the number with specific SS receptors (541 +/- 24 vs. 365 +/- 16 fmol/mg protein, P < 0.001) together with a decrease in their apparent affinity (0.57 +/- 0.04 vs. 0.41 +/- 0.03 nM, P < 0.05) in rat hippocampal membranes from histamine (10 micrograms)-treated rats as compared to control animals. With the aim of determining if these changes were related to histamine binding to its specific receptor sites, the histaminergic H1 and H2 receptor antagonists mepyramine and cimetidine, respectively, were administered 1 h before histamine injection. The pretreatment with mepyramine or cimetidine induced an increase in the number and affinity constant of the SS receptors whereas the simultaneous pretreatment with both histamine antagonists prevented the histamine-induced changes in SS binding to its receptors. Since the hippocampal SS receptor/effector system is modulated by histamine, it is tempting to speculate that in the hippocampus, SS could be involved as a mediator of the histamine effects on behaviors such as learning and memory.

Influence of fluoxetine and p-chloroamphetamine on the somatostatin receptor-adenylyl cyclase system in the rat frontoparietal cortex.

There is evidence that suggests a reciprocal functional link between the serotonergic and the somatostatinergic system in the rat frontoparietal cortex. However, to date, the role of endogenous 5-hydroxytryptamine (serotonin) on the regulation of the somatostatin (SS) receptor-adenylyl cyclase (AC) system remains unclear. In the present study, the administration of fluoxetine (10 mg/kg i.p.), a 5-hydroxytryptamine uptake inhibitor in a single dose or administered daily for 14 days increased the number of specific [125I]Tyr11-SS receptors, with no change in the receptor affinity, in rat frontoparietal cortical membranes. However, the capacity of SS to inhibit forskolin (FK)-stimulated AC activity in these membranes was lower than in the control groups. The ability of the stable GTP analogue 5'-guanylylimidodiphosphate (Gpp(NH)p) to inhibit FK-stimulated AC activity in frontoparietal cortical membranes was also decreased in rats acutely and chronically treated with fluoxetine. p-Chloroamphetamine (5 mg/kg i.p.), which leads to a lasting reduction of 5-hydroxytryptamine innervation, administered on days 1, 3 and 5 and the rats sacrificed 1 or 3 weeks after the first injection, decreased the number of SS receptors without changing the receptor affinity. In this experimental group, SS also caused a significantly lower inhibition of FK-stimulated AC activity. p-Chloroamphetamine had no effect on the ability of Gpp(NH)p to inhibit FK-stimulated AC activity in frontoparietal cortical membranes at all the time periods studied. The present results suggest that under normal circumstances some SS receptors are under a tonic stimulatory control through the serotonergic system.

Growth inhibition of experimental pancreatic cancers and sustained reduction in epidermal growth factor receptors during therapy with hormonal peptide analogs.

Reduction in receptors for epidermal growth factor (EGF) in cancers appears to be one of the principal mechanisms through which peptide hormone analogs can inhibit tumor growth. In this study, hamsters with nitrosamine-induced pancreatic cancers were treated for 8 weeks with bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095, somatostatin analog RC-160 or the luteinizing hormone-releasing hormone antagonist Cetrorelix, using sustained delivery systems releasing 20, 35 and 20 microg analog/ day respectively. To establish the pattern of changes in the number and affinity of EGF receptors on tumors, groups of animals were sacrificed at regular intervals during therapy. Chronic treatment with RC-3095 or Cetrorelix resulted in an early (day 10) and sustained reduction (71% or 69% respectively) in EGF receptors on pancreatic tumors. In contrast, RC-160 decreased receptor concentration by 60% only after 20 days. Among the histological characteristics of proliferation, the decrease in argyrophilic nucleolar organizer regions, but not apoptotic and mitotic indices, showed a correlation with the fall in EGF receptors. The concentration of the receptors returned to the control level 4 days after cessation of chronic treatment with RC-3095. The effect of single injections of RC-3095, RC-160 and Cetrorelix on EGF receptors was also investigated. RC-160 decreased the number of EGF receptors on pancreatic cancers by 31% 3 h after administration, but the receptors had returned to normal level at 6 h. RC-3095 and Cetrorelix caused a 67% and 59% decline, respectively, in EGF receptors only 6 h after injection and the concentration of receptors remained low for 24 h. Thus, the pattern of downregulation of EGF receptors in pancreatic cancers appears to depend on the peptide used for therapy. Since the antitumor effect may be the result of the fall in EGF receptors in cancers, information on the time course of changes in these receptors during treatment with these analogs may lead to an improvement in therapeutic regimens.