Serological autoimmune profile of systemic lupus erythematosus in deep and non-deep endometriosis patients.

OBJECTIVE: Several studies have reported a high prevalence of autoimmune diseases such as systemic lupus erythematosus (SLE) in endometriosis patients. The aim of this study was to evaluate the SLE autoimmune antibody profile in patients with deep (DE) and non-deep endometriosis (Non-DE). MATERIALS AND METHODS: Four groups of premenopausal patients were evaluated: patients with DE (n = 50); patients with ovarian endometriomas (Non-DE; n = 50); healthy patients without endometriosis (C group; n = 45); and SLE patients without endometriosis (SLE group; N = 46). Blood samples were obtained and the standard SLE autoimmune profile was evaluated in all patients. Pain symptoms related to endometriosis and clinical SLE manifestations were also recorded. RESULTS: The DE group presented a statistically significant higher proportion of patients with antinuclear antibodies (ANA) (20%) compared to the Non-DE group (4%) and C group (2.2%). Levels of complement were more frequently lower among DE and Non-DE patients although differences did not reach statistical significance. Similarly, anti-dsDNA antibodies and anticoagulant lupus were positive in more patients of the DE group but did not reach statistical significance. The DE group complained of more arthralgia and asthenia compared to the Non-DE and C groups. CONCLUSIONS: The results of this study showed higher positivity of ANA and greater arthralgia and asthenia in patients with DE compared with Non-DE patients and healthy controls, suggesting that they may have a higher susceptibility to autoimmune diseases and present more generalized pain.

First Complete Genome Sequences of Zika Virus Isolated from Febrile Patient Sera in Ecuador.

Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, EcEs062_16 and EcEs089_16, isolated from the sera of febrile patients in Esmeraldas City, in the northern coastal province of Esmeraldas, Ecuador, in April 2016. These are the first complete ZIKV genomes to be reported from Ecuador.

Total circulating microparticle levels are increased in patients with deep infiltrating endometriosis.

STUDY QUESTION: Are the levels of total circulating cell-derived microparticles (cMPs) and circulating tissue factor-containing microparticles (cMP-TF) increased in patients with endometriosis? SUMMARY ANSWER: The levels of total cMP, but not cMP-TF, were higher in patients with endometriosis, and these were attributed to higher levels in patients with deep infiltrating endometriosis (DIE). WHAT IS KNOWN ALREADY: Previous studies have reported elevated levels of total cMP in inflammatory conditions as well as higher levels of other inflammatory biomarkers in endometriosis. Increased expression of tissue factor (a transmembrane receptor for Factor VII/VIIa) in eutopic and ectopic endometrium from patients with endometriosis has been described. There is no previous data regarding total cMP and cMP-TF levels in patients with endometriosis. STUDY DESIGN, SIZE, DURATION: A prospective case-control study including two groups of patients was carried out. The E group included 65 patients with surgically confirmed endometriosis (37 with DIE lesions) and the C group comprises 33 women without surgical findings of any form of endometriosis. Patients and controls were recruited during the same 10-month period. Controls were the next patient without endometriosis undergoing surgery, after including two patients with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Venous blood samples for total cMP and cMP-TF determinations were obtained at the time of surgery, before anesthesia at a tertiary care center. To assess total cMP, an ELISA functional assay was used and cMP-TF activity in plasma was measured using an ELISA kit. MAIN RESULTS AND THE ROLE OF CHANCE: Total cMP levels in plasma were higher in the E group compared with the C group (P < 0.0001). The subanalysis of endometriosis patients with DIE or with ovarian endometriomas without DIE showed that total cMP levels were higher in the DIE group (P = 0.001). There were no statistically significant differences in cMP-TF levels among the groups analyzed. LIMITATIONS, REASONS FOR CAUTION: This is a preliminary study in which the sample size was arbitrarily decided, albeit in keeping with previous studies analyzing cMP in other inflammatory diseases and other biomarkers in endometriosis. The control group included patients with other pathologies as well as healthy controls, and blood samples were taken at different phases of the cycle. WIDER IMPLICATIONS OF THE FINDINGS: Elevated total cMP levels in DIE patients may reflect an inflammatory and/or procoagulant systemic status in these patients. Further studies are warranted to confirm our findings and to assess the role of cMP levels in the pathophysiology of DIE. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by a grant from FIS-PI11/01560 and FIS-PI11/00977 within the 'Plan Nacional de I + D + I' and co-funded by the 'ISCIII-Subdirección General de Evaluación' and 'Fondo Europeo de Desarrollo Regional (FEDER)' and by the grant 'Premi Fi de Residència Emili Letang 2015' from the Hospital Clínic of Barcelona. The authors have no competing interests to disclose.

Increased levels of dioxin-like substances in adipose tissue in patients with deep infiltrating endometriosis.

STUDY QUESTION: Are the levels of biologically active and the most toxic dioxin-like substances in adipose tissue of patients with deep infiltrating endometriosis (DIE) higher than in a control group without endometriosis? SUMMARY ANSWER: DIE patients have higher levels of dioxins and polychlorinated biphenyls (PCBs) in adipose tissue compared with controls without endometriosis. WHAT IS KNOWN ALREADY: Some studies have investigated the levels of dioxin-like substances, in serum samples, in patients with endometriosis, with inconsistent results. STUDY DESIGN, SIZE, DURATION: Case-control study including two groups of patients. The study group (DIE group) consisted of 30 patients undergoing laparoscopic surgery because of DIE. In all patients, an extensive preoperative work-up was performed including clinical exploration, magnetic resonance imaging (MRI) and transvaginal sonography. All patients with DIE underwent a confirmatory histological study for DIE after surgery. The non-endometriosis control group (control group), included the next consecutive patient undergoing laparoscopic surgery in our center due to adnexal benign gynecological disease (ovarian or tubal procedures other than endometriosis) after each DIE patient, and who did not present any type of endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: During the surgical procedure 1-2 g of adipose tissue from the omentum were obtained. Dioxin-like substances were analyzed in adipose tissue in DIE patients and controls without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: The total toxic equivalence and concentrations of both dioxins and PCBs were significantly higher in patients with DIE in comparison with the control group (P < 0.05), mainly due to the significantly higher values of the two most toxic dioxins (2,3,7,8-tetrachlorodibenzo-p-dioxin [2,3,7,8-TCDD] and 1,2,3,7,8-pentachlorodibenzo-p-dioxin [1,2,3,7,8-PeCDD]) (P < 0.01 for each compound). The levels of furan 2,3,4,7,8-PeCDF were statistically higher in the DIE group compared with controls. Only four congeners of PCBs had toxic equivalence values and concentrations that were statistically higher in patients with DIE, but these included the most toxic and carcinogenic PCB-126 (PCB-114 P < 0.05; PCB-156 P < 0.05; PCB-189 P = 0.04; PCB-126 P < 0.01). LIMITATIONS, REASONS FOR CAUTION: Since few patients were recruited, the study is only exploratory. Our results need to be confirmed in larger and more heterogeneous population studies since environmental and even genetic factors involved in determining dioxins and PCBs widely vary in different countries. Furthermore, the strict eligibility criteria used may preclude generalization of the results to other populations and the surgery-based sampling frame may induce a selection bias. Finally, adipose tissue was obtained only from the omentum, and not from other adipose tissue of the body. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest a potential role of dioxin-like substances in the pathogenesis of DIE. Further studies are warranted to confirm our findings. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: Not applicable.

Mixoma intramuscular: A propósito de tres casos / Intramuscular myxoma: Report of three cases

El mixoma intramuscular es un raro tumor benigno de tejidos blandos, de origen mesenquimal, que se presenta como masa de crecimiento lento, indolora, habitualmente en el muslo. El diagnóstico histológico es necesario antes de la resección, siendo la exéresis su tratamiento. No existen casos de malignización y su recurrencia se debe a resección incompleta. Presentamos tres casos de mixoma intramuscular tratados en nuestro centro entre los años 2004 y 2011. De este modo aprovechamos para realizar una revisión de la presentación clínica, diagnóstico, tratamiento y resultados funcionales de los mismos. En todos los casos se llevó a cabo el mismo protocolo diagnóstico y terapéutico, comenzando con la exploración física del paciente y realizando una ecografía y RMN como pruebas complementarias. Posteriormente una biopsia incisional del tumor para estudio anatomopatológico y finalmente la exéresis íntegra.

Intramuscular myxoma is a rare soft tissue benign tumor, arising from the mesenchyma; it presents as a slow-growing painless mass located usually in the thigh. Histologic diagnosis is necessary before resection and treatment consists of exeresis. There are no cases of malignization and recurrence results from incomplete resection. We report herein three cases of intramuscular myxoma treated at our center from 2004 to 2011. At the same time, we conducted a review of the clinical presentation, diagnosis, treatment and functional results. The same diagnostic and therapeutic protocol was used in all cases. It began with the patient’s physical exam, and ultrasound and MRI as complementary tests. An incisional biopsy of the tumor was taken for anatomopathological studies and, finally, complete exeresis was performed.

Intravenous ferrous sucrose versus placebo in addition to oral iron therapy for the treatment of severe postpartum anaemia: a randomised controlled trial.

OBJECTIVE: The aim of the study was to evaluate the effectiveness of intravenous iron versus placebo added to standard oral iron therapy in the treatment of severe postpartum anaemia. DESIGN: A randomised, double-blind, parallel-group, placebo-controlled clinical trial was performed in a single centre. SETTING: Hospital Clinic of Barcelona, Barcelona, Spain. POPULATION: A cohort of 72 women with severe postpartum anaemia (6.0-8.0 g/dl) treated with oral ferrous sulphate (two tablets of 525 mg). METHODS: Women were randomised to receive either intravenous ferrous sucrose (200 mg/24 hours for two consecutive days) or intravenous placebo, in addition to standard iron therapy. Clinical and laboratory data were obtained at 1, 2, and 6 weeks. MAIN OUTCOME MEASURES: Haemoglobin and haematocrit at 1, 2, and 6 weeks. Other haematological and clinical parameters, psychological status, and adverse side effects were also evaluated. RESULTS: Haemoglobin and haematocrit values were comparable in women receiving intravenous iron or placebo in addition to oral iron therapy at any of the time points. At 6 weeks, haemoglobin level (mean ± SD) was 12.2 ± 1.0 versus 12.2 ± 0.9 g/dl, with a mean difference of -0.03 (95% CI -0.6 to 0.6), in the placebo and in the intravenous iron groups, respectively. No differences were found between clinical symptoms of anaemia, psychological status, and adverse side effects between groups. CONCLUSIONS: Intravenous iron added to oral iron therapy did not show significant benefits over placebo, neither in haemoglobin rise nor in symptoms or adverse side effects.

[Intramuscular myxoma. Report of three cases]. / Mixoma intramuscular. A propósito de tres casos.

Intramuscular myxoma is a rare soft tissue benign tumor, arising from the mesenchyma; it presents as a slow-growing painless mass located usually in the thigh. Histologic diagnosis is necessary before resection and treatment consists of exeresis. There are no cases of malignization and recurrence results from incomplete resection. We report herein three cases of intramuscular myxoma treated at our center from 2004 to 2011. At the same time, we conducted a review of the clinical presentation, diagnosis, treatment and functional results. The same diagnostic and therapeutic protocol was used in all cases. It began with the patient's physical exam, and ultrasound and MRI as complementary tests. An incisional biopsy of the tumor was taken for anatomopathological studies and, finally, complete exeresis was performed.

Differential sensitivity of rat hepatocyte CYP isoforms to self-generated nitric oxide.

Early loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study investigates the influence of endogenously generated NO (or NO-derived species) on the relative expression of cytochrome P450 (CYP) isoforms in rat hepatocytes. Our results support the view that loss of P450 holoenzyme in culture is the ultimate consequence of a NO driven process, activated during the common hepatocyte isolation procedure, that leads to an accelerated and selective degradation of specific CYP apoproteins. Under conditions in which NO and peroxynitrite formation is operative, changes in the level of specific CYP isoforms result in a significant alteration of the CYP apoprotein profile that after 24 h of culture is quite different from that found in the liver of uninduced rats. This process is reverted by the early and efficient inhibition of NO synthesis, which allows for (1) maintenance of total P450 holoenzyme content, (2) preservation of the initial constitutive CYP pattern in culture and (3) the early expression of the normal inducibility in response to model inducers.

Co-regulation between cyclo-oxygenase-2 and inducible nitric oxide synthase expression in the time-course of murine inflammation.

Many in vitro studies have used cell cultures to focus on the relationships between cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) isoforms. We have investigated the time-course of regulation and the role of COX-2 and iNOS in a model of experimental inflammation in mice, the air pouch injected with zymosan. This study demonstrates that there is an early acute phase (4 h) mediated mainly by eicosanoids, with high levels of prostaglandin E2 (PGE2) produced by cyclo-oxygenase-1. In addition, in the later phase (from 12 h) there is a participation of nitric oxide (NO) and PGE2 accompanied by co-induction of both iNOS and COX-2. These enzymes were detected in migrating leukocytes as well as in macrophages lining the air pouch. Administration of NS398 or indomethacin inhibited PGE2 levels and COX activity, but also nitrite levels and iNOS activity, which was accompanied by a reduction in iNOS expression. Aminoguanidine inhibited nitrite levels and iNOS activity in addition to exerting inhibitory effects on the COX pathway. Treatment of animals with dexamethasone reduced nitrite and PGE2 concentrations in air pouch exudates, as well as iNOS and COX-2 expression in migrating cells. Our results indicate that PGE2 and NO may play in vivo mutual modulatory roles in the inflammatory response caused by zymosan injection into the mouse air pouch, a suitable model to study drugs acting on those pathways.

Removal of amphipathic epitopes from genetically engineered antibodies: production of modified immunoglobulins with reduced immunogenicity.

Several approaches have been developed to reduce the human immune response to nonhuman antibodies. However, chimeric antibodies and humanized antibodies often have decreased binding affinity. We described a new approach for reducing the immunogenicity of chimeric antibodies while maintaining the affinity. This approach seeks to prevent the recognition of murine immunogenic peptides from the antibody variable region by human lymphocytes. Putative immunogenic epitopes in the variable region are identified and subjected to site directed mutagenesis to make them human and/or to break the amphipathic motifs. The R3 antibody, which blocks the epidermal growth factor (EGF) receptor, was used as a model system to test this approach. Four segments containing possible amphipathic epitopes were found in the heavy variable domain using the program AMPHI. Six amino acids within two of these segments were substituted by the corresponding residues from a homologous human sequence. No mutations were made in the murine light variable domain. Experiments in monkeys suggested that the "detope" R3 antibody was less immunogenic than its chimeric analogue. A search for possible amphipathic epitopes in the Kabat database revealed the presence of conserved patterns in the different families of variable region sequences, suggesting that the proposed method may be of general applicability.

Nitric oxide synthase and cyclo-oxygenase pathways in the inflammatory response induced by zymosan in the rat air pouch.

1. We have studied the participation of nitric oxide (NO) in an animal model of inflammation, the rat air pouch stimulated with zymosan. 2. Saline or zymosan was injected into 6-day rat air pouches at different time points and measurements were made of cell migration, levels of nitrite/nitrate (NO2/NO3-), prostaglandin E2 (PGE2), leukotriene B4 (L.TB4) and secretory phospholipase A2 (sPLA2) in exudates. Nitric oxide synthase (NOS) activity was determined in high speed supernatants from cells present in pouch exudates. Western blot analysis was also performed on these samples. 3. Zymosan injection induced a time-dependent increase in leukocyte infiltration, NO2/NO3- levels and cellular NOS activity that reached a peak by 8 h. Western blot analysis showed the same time course for induction of NOS protein. Colchicine administration to rats inhibited cellular infiltration and decreased the levels of NO metabolites and cellular NOS activity zymosan-injected air pouch at 8 h. NOS activity was present in polymorphonuclear leukocytes (PMNs) and monocytes, but not in the lymphocytes present in exudates. This enzyme is calcium-independent and needs NADPH for activity. PGE2 levels in exudates showed a time course inverse to that of NOS activity and NO metabolites, with maximum levels of PGE2 observed at 4 h after zymosan injection. 4. Administration of NG-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine to rats significantly reduced cellular NOS activity, NO2/NO3- levels and chemiluminescence, whereas they were without effect on cell migration and degranulation, eicosanoid levels and sPLA2 activity. 5. Treatment of animals with dexamethasone inhibited cellular NOS activity, NO2/NO3- levels, chemiluminescence and the increase in the levels of PGE2 and LTB4, with only a weak effect on elastase release. 6. Administration of the selective cyclo-oxygenase-2 (COX-2) inhibitor NS398 to rats strongly reduced PGE2 levels in exudates without affecting NO metabolites or NOS activity at 4 h after zymosan injection. 7. Our data indicate that NOS is induced in the zymosan-stimulated rat air pouch model of inflammation. This enzyme is expressed in the cells migrating into the air pouch and caused an increased production of NO metabolites in exudates. The results also suggest the presence of an earlier phase in which eicosanoids play the main role, with participation of COX-2 activity, and a later phase mediated by NO. The endogenous release of NO does not modify prostaglandin biosynthesis in this in vivo model.

Effect of DNA methylation on protein-DNA interactions upstream of the gamma-glutamyl transpeptidase gene.

We have used exonuclease III and DNase I protection assays to study proteins which bind to potentially regulatory elements located in the 5'-flanking region of the gamma-glutamyl transpeptidase gene. With both liver and kidney nuclear extracts, exonuclease III barriers were located at -566 in the coding strand and at -585 in the noncoding strand, and footprints were found from -595 to -566 and from -600 to -562, respectively. When the DNA was methylated in the CG dinucleotides, the exonuclease III barriers disappeared and the footprints were greatly reduced. The transcription factor Sp1 bound to this DNA region but did not seem to be involved in the binding activity. Since the gamma-glutamyl transpeptidase presents different levels of methylation in liver and kidney, associated with different levels of expression, these results suggest that the binding activity could play a role in the control of the expression of the gamma-glutamyl transpeptidase gene in liver and kidney.

Tissue-specific methylation in the 5' flanking region of the gamma-glutamyl transpeptidase gene.

We have studied the relationship between the methylation and the expression of the gamma-glutamyl transpeptidase gene in adult rat liver and kidney. In the liver, where the level of expression is very low, the 5' flanking region of the gene appeared fully methylated, whereas in the kidney, where the gene is expressed at the highest level, it is undermethylated. In addition, kidney chromatin showed a DNase I hypersensitive site located near the origin of transcription. These results support a strong correlation between DNA undermethylation, DNase I sensitivity and tissue-specific gene expression.

Induction of altered hepatic foci in rats by the administration of hypolipidemic peroxisome proliferators alone or following a single dose of diethylnitrosamine.

The effect of feeding hypolipidemic peroxisome proliferators on the induction of altered hepatic foci (AHF) in Fischer rats was studied in order to determine whether such agents can induce or promote the development of AHF. In the first study, rats were fed ciprofibrate (10 mg/kg/day) for 1 yr. AHF, neoplastic nodules, and hepatocellular carcinomas were induced. The presence of putative gamma-glutamyltranspeptidase (GGT) activity was numerically the most common marker, although it was absent in larger foci and nodules. A deficiency in canalicular ATPase and glucose-6-phosphatase provided the best markers for the larger foci and nodules. In the second study, rats were subjected to partial hepatectomy, and half of the animals were then intubated with diethylnitrosamine (10 mg/kg). One wk later, rats were fed Wy-14,643 at concentrations of 0, 0.05, and 0.1% in the diet for 6 mo. At 6 mo, the number and volume of foci were increased by the feeding of Wy-14,643 after partial hepatectomy alone and were greatly increased when Wy-14,643 was fed after partial hepatectomy/diethylnitrosamine administration. Canalicular adenosine triphosphatase and glucose-6-phosphatase deficiencies were the most common markers of AHF, and AHF of these phenotypes occupied practically all of the focal volume. The larger AHF did not express GGT, and those foci exhibiting GGT were much less common and occupied very little volume. The absence of the GGT protein itself, as opposed to an inhibition of GGT activity, was verified by immunohistochemical staining using an antibody to GGT. These studies show that hypolipidemic peroxisome proliferators can stimulate an increase in AHF following a single dose of diethylnitrosamine and a mitotic stimulus, and they thus can act as promoters in two-stage liver carcinogenesis. GGT is a poor marker for identifying AHF induced by peroxisome proliferators during the early, premalignant phase of hepatocarcinogenesis.

Characterization and sequence of a cDNA clone of gamma-glutamyltranspeptidase.

We have isolated several cDNA's complementary to gamma-glutamyltranspeptidase (GGT) mRNA by screening a rat kidney library constructed in lambda gtll with antibodies specifically reactive to the enzyme protein. The clone selected an mRNA that was translated into a 62 Kd peptide, corresponding to the GGT precursor. The longest clone isolated was 1842 bp long with an open reading frame coding for 565 amino acids. The length of the mRNA coding for GGT was estimated to be 2.2 kb long. The amino acid sequence derived from the nucleotide sequence matched the short sequences determined by us as well as by other authors.

Biochemical and immunologic studies on the native and deglycosylated forms of gamma-glutamyltranspeptidase of rat kidney.

We have deglycosylated the enzyme gamma-glutamyl transpeptidase by treatment of the protein with anhydrous hydrofluoric acid at 0 degree C. After deglycosylation, the heavy and light subunits showed a molecular weight of 43 and 23 Kd respectively. Whereas the antiserum against the native enzyme recognized both proteins, the antiserum against the deglycosylated enzyme failed to recognize the native enzyme, indicating that some of the determinants of the native enzyme are masked by the carbohydrate moiety.

Isozyme pattern of thymidine kinase during liver regeneration.

Liver regeneration after partial hepatectomy is characterized by an active phase of cell proliferation that is associated with a marked increase in thymidine kinase activity. Using non-denaturing gel electrophoresis and different substrate specificity, two isozymes could be detected. One was identified as the adult isozyme while the other was the fetal one. Both forms were present during liver regeneration. When the regenerative process was completed, the total enzymatic activity dropped to normal values and the fetal isozyme was displaced by the adult type.