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KAWA-RACE study group., [Background] Kawasaki disease (KD) is an acute self-limited systemic vasculitis of unknown etiology affecting mainly children less than 5 years of age. Risk factors for cardiac involvement and resistance to treatment are insufficiently studied in non-Japanese children., [Objective] This study aimed to investigate the epidemiology, clinical features and risk factors for resistance to treatment and coronary artery lesions (CAL) in KD in Spain., [Methods] Retrospective study (May 2011-June 2016) of all patients less than 16 years of age diagnosed with KD included in KAWA-RACE network (84 Spanish hospitals)., [Results] A total of 625 cases were analyzed, 63% were males, 79% under 5 year-olds and 16.8% younger than 12 months. On echocardiographic examination CAL were the most frequent findings (23%) being ectasia the most common (12%). Coronary aneurysms were diagnosed in 9.6%, reaching 20% in infants under 12 months (p 900,000 cells/mm3, maximum temperature < 39.5°C, total duration of fever > 10 days and fever before treatment ≥ 8 days as independent risk factors for developing coronary aneurysms., [Conclusions] In our population, children under 12 months develop coronary aneurysms more frequently and children with KD with anemia and leukocytosis have high risk of cardiac involvement. Adding steroids early should be considered in those patients, especially if the treatment is not started before 8 days of fever. A score applicable to non-Japanese children able to predict the risk of aneurysm development and IVIG resistance is necessary., CC received a grant from Spanish Society of Paediatric Rheumatology (SERPE), 2015.

Objective The aim of this study was to evaluate the outcomes of paste-pinch-cut conjunctivoplasty and cautery conjunctivoplasty for the treatment of symptomatic conjunctivochalasis. Design This was a prospective cohort study. Participants Sixteen patients (32 eyes) with bilateral conjunctivochalasis that was symptomatic after medical therapy were enrolled in the study. Methods This was a single-centre, contralateral eye, prospective study. Paste-pinch-cut conjunctivoplasty was performed in the left eye, and thermal cautery conjunctivoplasty was performed in the right eye. The outcomes of each procedure were compared preoperatively and at the 1-month follow-up by using the Canadian Dry Eye Assessment (CDEA) scoring system, standard conjunctivochalasis grading, and corneal staining. Intraoperative discomfort and immediate postoperative discomfort were assessed by using a 10-point scale. Results The mean age of patients was 72.4 ± 8.67 years. Conjunctival redundancy was absent in 14 of 16 patients postoperatively. The mean CDEA score improved after both procedures (7.1 ± 2.8 preoperatively versus 4.5 ± 0.78 at the 1-month follow-up for cautery conjunctivoplasty, 7.4 ± 2.5 versus 4.9 ± 3.1 for paste-pinch-cut conjunctivoplasty). This improvement was statistically significant in the cautery conjunctivoplasty group ( p = 0.012). Mean intraoperative discomfort was 2.6 ± 2.1 with the use of paste-pinch-cut conjunctivoplasty and 3.5 ± 3.2 with the use of cautery conjunctivoplasty; however, the difference was not statistically significant. No intraoperative or postoperative complications were observed with either technique. Conclusions Paste-pinch-cut and thermal cautery conjunctivoplasty are both safe and effective surgical treatments for the repair of conjunctivochalasis, with patients reporting greater improvement in symptoms after the cautery technique.

Phenotypes of 38 samples of mononuclear (PBMC) cells from 11 different patients with pemphigus vulgaris (PV) at different stages of the disease were explored looking for a possible relationship between cell immunity, mucocutaneous or mucosal lesion intensity and capacity of serum autoantibodies to elicit the disease in mice. PBMC from 5 patients with mucocutaneous lesions and sera with IgG capable of inducing the disease in neonatal mice had a high proportion of mature monocytes with CD14low DRhigh, and co-expressing CD16 and CD11b. In addition, a high proportion of CD19+CD5+ activated B cells and a very low proportion of naive CD4+CD45RA+ and CD8+CD11b+ T lymphocytes was observed. Monocytes from these patients expressed inducible nitric oxide synthase (iNOS). In contrast, PBMC from 6 patients, with lesions restricted to mucosal membranes and IgG lacking the capacity to induce the disease in mice, contained a high proportion of CD14high DRlow co-expressing CD16 circulating macrophages, CD8+CD11b+ T cells, and a low proportion of activated B lymphocytes. The results suggest a possible association between proportion of different antigen presenting cells (monocytes with high HLA-DR and low CD14 expression and activated B lymphocytes, or differentiated monocytes/macrophages), type of PV and capacity of serum autoantibodies to elicit the disease in mice.

A set of new pyrimido[5,4-b]indole derivatives that are structurally related to some non-nucleside HIV-1 reverse transcriptase inhibitors were synthesized and biologically evaluated for their activity as inhibitors of wild and mutant HIV-1 RT types in an 'in vitro' recombinant HIV-1 RT screening assay, as well as anti-infectives in HLT4lacZ-1IIIB cells. Preliminary structure-activity relationships suggest that activity is promoted by simultaneous substitution in positions 2 and 4, especially when chains of alkyldiamine type are present, and by electron-releasing substituents (methoxy) in positions 7 and 8. The inactivity or the very low activity of title derivatives does not suggest interest in AIDS therapy.

Glucocorticoids and cAMP regulate, either in a synergistic or additive fashion, the transcription of multiple genes, although some antagonistic effects of dexamethasone on cAMP-activated transcription have been described. The increased glucocorticoid receptor (GR) mediated response of some cell types, as a result of augmented cAMP, has been considered to be mainly due to an increased stability of GR mRNA, although other plausible explanations should not be ruled out. We studied the possibility that GR transcription itself could be affected by cAMP levels. HeLa cells were transfected with human GR (hGR) promoter constructs and their transcriptional activity determined after inducing a cAMP increase with forskolin. We found that forskolin almost doubled the transcriptional activity of the promoter construct spanning −2995 to +38 of the hGR, whereas no significant variations were observed with shorter chimeras containing sequences downstream −979. Shift mobility showed binding of CREB in vitro to a putative cAMP responsive element located at −1000, suggesting that hGR may be upregulated by cAMP at the transcriptional level, thus adding a new mechanism ascribable to this second messenger, which in conjunction with the cAMP-induced GR mRNA increased stability, would lead to a more precise control of the amount of GR protein within the cell.

Activated and differentiated human monocytes with a CD14+CD16+ phenotype were found to contain a DNase activity associated with secretion granules. Activated cells were obtained from patients with autoimmune diseases. Activation and differentiation of monocytes were also achieved after incubation of PBMC from healthy subjects with protein A (SpA) or immunopotentiating peptides. DNase activity corresponded to a 66-kDa protein, similar to that described in granules from T lymphocytes, active preferentially on double-strand DNA. DNA fragmentation activity increased when NO donors were present; the activity was higher in the presence of Ca2+, and at low pH values. The Ca2+-dependent activity was inhibited by Zn2+. NO-dependent activity was additive with that of Ca2+-dependent and it was not inhibited by Zn2+. Dithiothreitol did not modify the effect of NO on DNase activity. Incubation of PBMC in the presence of NMLA, an inhibitor of NO synthases, decreased this DNase activity. Data reported clearly suggest a regulatory role of NO in granule-associated DNase activity.

We have found a common structural motif in human autoantigens, heat shock proteins and viral proteins. Peptides modelled after sequences present in those molecules were synthesized and immunomodulating properties tested. They share a core of 15 amino acid residues and a common pattern ('2-6-11' motif) characterized by requirements at fixed positions with respect to a Pro (position 6); an apolar residue or a Lys at position 2; and a Glu, Asp or Lys at position 11. Any of these peptides, when added to cultures of lymphomononuclear cells, caused the activation of monocytes manifested by a release of IL-1 alpha, IL-1 beta and TNF alpha. A release of INF gamma and IL-2 took also place; this release was abolished by anti-DR antibodies. Neither IL-4 nor IL-5 could be detected. This suggests a presentation by APCs and the appearance of cells with a Th1 phenotype. Monocytes and Th1 cells freshly obtained from 12 patients of Graves' disease, 8 of Hashimoto's disease and 8 of primary biliary cirrhosis exhibited activation features similar to those found in cells from healthy subjects incubated in the presence of peptides with a "2-6-11' motif and representing fragments of autoantigens. Their immunopotentiating properties suggest their involvement in the initiation or progression of the autoimmune response mediated by activated monocytes and Th1 cells.

The presence of inducible nitric oxide synthase (iNOS) in fresh monocytes from patients with Graves' disease was demonstrated for the first time. Immunophenotypic analysis showed a profile reflecting a state of activation and differentiation of monocytes. Incubation of lymphomononuclear cells from healthy volunteers in the presence of synthetic peptides with sequences related to thyroid autoantigens (TSH receptor, thyroid peroxidase, or thyroglobulin) led to a stimulation of monocytes manifested by a change in phenotype and expression of iNOS. This expression did not take place on isolated monocytes, unless products associated with Th1 activity were present in the medium. Active peptides contained a characteristic "2-6-11" motif already described [López-Moratalla et al. (1995) Biochim. Biophys. Acta 1265, 181-188]. These results are suggestive of a new role for autoantigens in the pathogenesis of Graves' disease: that of inducing the expression of iNOS and activating the monocyte possibly underlying the autoimmune response.

Peptides from 10 to 22 amino acids containing sequences encompassed by Staphylococcus aureus protein A were synthesized. Some of these peptides, when present in cultures of lymphomononuclear cells from healthy donors or from cancer patients (melanoma, breast carcinoma, non-Hodgkin lymphoma and renal cell carcinoma) promoted: (i) changes in the phenotype of the lymphomononuclear population, (ii) stimulation of monocytes (release of IL-1 and TNF-alpha), and (iii) an increase in cytotoxicity against K562, Daudi and HT-29 cells. Isolated monocytes responded also to those peptides with a release of IL-1 and TNF alpha and an increase of cytotoxicity against HT-29 cells. It was found that the active peptides had the following structural pattern: a length of at least 15 amino-acid residues with a proline at position 6, valine, leucine, isoleucine, glycine, alanine or lysine at position 2, and glutamic or aspartic acid at position 11. Replacement of Pro at position 6 with any other residue turned the peptide inactive. Replacement of residues at positions 2 and 11 with amino-acid residues other than those required for activity resulted in compounds with a marked decrease in the immunomodulating properties described, or lacking these properties altogether.

The ability to detect chromosomal or genetic defects in embryos in vitro, associated with assisted human reproduction techniques before his possible transfer to the uterus to complete its development was presented as an alternative to eugenic abortion. And an option for older women to procreate, to avoid pregnancy of embryos with chromosomal defects. Genetic diagnosis before implantation (PGD) and screening of embryos in vitro (by the acronym, PSC), offers the image of the disabled person as an individual excluded from society. It assumes a direct human experimentation without therapeutic purposes or to manipulate the embryo that is chosen or discarded according to diagnosis or for advancement in perinatal medicine. Because these techniques can have multiple embryos, eugenics has also generated a ″positive eugenics″ that seeks to select embryos according to a third party, having certain characteristics, sex, or lack of predisposition to disease. Several issues demand unavoidable ethical duty to report on this form of eugenics, in addition to be directly and intentionally directed to destroy human life in its early stages, and does not meet the minimum requirements of rigorous scientific research or biotechnology. There have been no previous animal tests to validate the techniques so that there are serious errors in diagnosis with false positives and false negatives. Recently it has been shown that some discarded embryos can eliminate their detected defects two days after the biopsy. Moreover, the study about what may or may not be diagnosed is retrospective and unrecoverable damage. And, of particular importance is the fact that it is not known with certainty the effects that an embryo biopsy may cause to those diagnosed.

In the period between puberty and maturity takes place the process of brain maturation. Hormone levels induce changes in neurons and direct the architecture and structural functionality thus affecting patterns of development of different brain areas. The onset of puberty brings with it the invasion of the female brain by high levels of hormones, cyclic surges of estrogen and progesterone in addition to steroids produced in situ. Control centers of emotions (amygdala), memory and learning (hippocampus) and sexual activity (hypothalamus) are modified according to the cyclical concentrations of both hormones. Sex hormones stimulate multimodal actions, both short and longer terms, because neurons in various brain areas have different types of receptors, membrane, cytoplasmic and nuclear. The composition of emergency contraceptive pill (postcoital pill) with high hormonal content raises the urgency of a thorough knowledge about the possible effect that the lack of control of the menstrual cycle in a time of consolidation of brain maturation, can bring in structuring and development of brain circuitry. Changes in the availability of sex steroids during puberty and adolescence underlie psychiatric disorders whose prevalence is typically feminine, such as depression, anxiety disorders. It is a fundamental ethical duty to present scientific data about the influence of estrogen in young female brain maturation, both for full information to potential users, and also to induce the appropriate public health measures.

It has been reported previously that a short synthetic immunomodulating peptide (Pa) and the neuropeptide beta-endorphin modulate the immune system. We have found now that NF-kappaB participates in the stimulation of monocytes by both peptides and we investigated the molecular mechanism by which these stimuli activate NF-kappaB. Pa and beta-endorphin induce accumulation of cyclic 3('),5(')-adenosine monophosphate (cAMP) in a calcium/calmodulin-dependent fashion since it was completely inhibited by the calmodulin antagonist W-7. The effect of these complexes seems to be mediated, at least in part, by nitric oxide (NO) synthesized by constitutive NO synthase since the NO synthase inhibitor N-methyl-L-arginine (NMLA) reduced the elevation of cAMP. Furthermore, the NO donor SIN-1 provoked nitration of G(S)alpha, leading to the cAMP elevation that was suppressed by the G(S)alpha-selective antagonist NF-449. Interestingly, the rapid degradation of NF-kappaB inhibitor IkappaBalpha induced by Pa- and beta-endorphin was reversed by a pretreatment with H-89 and cyclosporin A, inhibitors of protein kinase A (PKA) and protein phosphatase 2B (PP2B), respectively. These observations are consistent with the inhibition caused by W-7, NMLA, H-89, and cyclosporin A on NF-kappaB induction by these agonists, indicating the involvement of PKA and PP2B in the regulation of NF-kappaB in human monocytes.

A short immunomodulating peptide (Pa) containing a defined structural motif present in a number of extracellular matrix proteins and autoantigens was found to stimulate human monocytes. Pa-induced apoptosis of isolated monocytes, as indicated by internucleosomal DNA cleavage, increased annexin V binding capacity and cleavage of caspase substrates, such as poly(ADP)ribosylpolymerase. In addition, Bcl-2 protein levels were downregulated during Pa-induced cell death. Nuclear extracts of monocytes incubated with Pa showed higher neutral, Ca(2+)-dependent DNase activity than those obtained from nontreated monocytes. Caspase inhibitors prevented Pa-induced apoptosis, Bcl-2 depletion, and DNase activation. Treatment of monocytes with Pa activated c-Jun N-terminal kinases and p38 kinase, in an acidic sphingomyelinase- and caspase-dependent fashion. Pa-induced apoptosis was blocked by selective inhibitors of p38 kinase (SB203580) and acidic sphingomyelinase (SR33557). These results indicate that JNK and p38 kinase stimulation as well as monocyte apoptosis induced by Pa could depend, at least in part, on early activation of acidic sphingomyelinase.

Human monocyte subsets, isolated from cultures of mononuclear cells, or freshly obtained from patients with multiple sclerosis, Graves' disease or pemphigus vulgaris, differed in phenotype, apoptotic features, mRNA levels of arginase II (A-II) and the inducible form of nitric oxide synthase (iNOS). Liver-type arginase I mRNA was present in all subsets. Apoptosis was followed by the expression of T cell intracellular antigen (TIA) and the simultaneous detection of DNA stainability by propidium iodine and annexin V binding. Apoptosis was practically absent both in activated CD14(++)CD33(++)DR(++)CD25(++)CD69(++)CD71(++/+) CD16(-) cells, expressing A-II mRNA and having arginase activity, but not iNOS mRNA, and in not fully mature large CD14(++)CD16(+)CD23(+)DR(++) monocytes, expressing simultaneously both mRNAs and having both enzyme activities. However, differentiated small CD14(+/++)CD16(+)CD69(+)CD25(+/-)CD71(++)CD23(+) DR(++) monocytes, expressing high levels of iNOS mRNA, exhibited apoptotic signs. Amounts of NO synthesised by monocytes co-expressing iNOS and arginase changed with the addition of arginine or an iNOS inhibitor; in that case a correlation of NO production and apoptotic features was observed. Data suggest a regulatory role for endogenous NO in apoptosis of stimulated and differentiated monocytes, and also that iNOS and A-II, when simultaneously present, could control the production of NO as a consequence of their competition for arginine.

Evidence suggesting a relationship between neuroendocrine and immune systems is steadily growing. We demonstrate now that inducible nitric oxide synthase (iNOS) is expressed in human peripheral blood monocytes after incubation of lymphomononuclear cells in the presence of beta-endorphin, a neuropeptide released by the pituitary in response to mental or physical stress or by activated lymphocytes. beta-endorphin raised cAMP level in monocytes. The possible relationship between cAMP and iNOS expression on monocytes was investigated. Immunostaining for iNOS decreased, when besides beta-endorphin an inhibitor of protein kinase A (H-89) was added to the medium at the beginning of the incubation. The cAMP level raised by beta-endorphin was lowered by naloxone, which also reduced slightly iNOS expression. These results clearly point to the monocyte as a link between neuroendocrine and immune systems, an observation of potential relevance in our understanding of how stress and autoimmunity could be interconnected.

The authors have already described that a series of short peptides, modelled after sequences related to human extracelluar matrix (ECM) proteins and sharing some common structural features, activate Th1 clones through a process involving peptide presentation in HLA-DR proteins. Those peptides induce also LAK- and NK-dependent cytotoxicity as well as activation of monocytes/macrophages present in human peripheral blood mononuclear cell (PBMC) populations. The release of interleukin 2 (IL-2) and interferon γ (IFN-γ) by Th cells present in PBMC depleted of macrophages, or B cells is reported, after incubation in the presence of those peptides, fibronectin or Staphylococcus aureus protein A. The authors found that all the molecules tested needed at least the presence of a type of antigen-presenting cell (APC) to exert their stimulatory effect. Some peptides seem to be preferentially presented to Th cells by B cells, while others seem to depend on monocyte/macrophages for this presentation. The dependence on one or another APC seems to be due to differences in the sequences of these peptides. The immunomodulatory agents studied also gave rise to a clear increase in a DNase activity associated with secretion granules of PBMC. That there is a correlation between the release of IL-2 and this DNase activity when using a complete PBMC population, B cell-depleted PBMC or macrophage-depleted PBMC stimulated with the peptides tested has been found.

We analyzed the phenotypic characteristics of PBMC from 34 patients with Graves' disease (GD) at different stages of the disease to explore the sequence of immunological events associated with it. In all cases their monocytes were in a state of activation and differentiation more advanced than those of a group of 23 healthy individuals. Strikingly, some patients had CD14++DR- immature monocytes, which were absent in healthy individuals. CD14+CD16+DRhigh monocytes were more abundant in patients. We found a positive correlation between the CD14++DR- monocyte and CD4+CD45RA- helper cells and a negative correlation between the same monocyte subset and CD4+CD45RA+ naive cells. CD14+/++DRlow monocytes directly correlated with this latter T4 subset and CD14+ CD16+DRhigh with CD4+CD45RO+ memory lymphocytes. There was also a positive correlation between memory T4 cells and the subset of activated B lymphocytes (CD19+CD5+) and suppressor T8 cells (CD8+CD11b+). T8 cytotoxic cells (CD8+CD11b-) positively correlated with T4 naive cells. The circulating levels of T3 and TSI (thyroid-stimulating immunoglobulin) directly correlated with a decrease in naive cells and an increase in T8 suppressors. The results suggest that the imbalance suppression/cytotoxicity in GD may be due to a reiterated presentation of autoantigens, or mimetic antigens, to T helpers by mature monocytes and activated B cells.

Immunophenotypic analysis of peripheral blood leukocytes from patients with multiple sclerosis (MS) showed a profile reflecting a state of activation and differentiation of monocytes. A subset of CD16+ monocytes with high HLA-DR expression was more prominent in patients with MS than in healthy subjects. The presence of the inducible form of nitric oxide synthase (iNOS) in these differentiated and activated monocytes freshly obtained from patients with MS was demonstrated by immunocytochemistry and flow cytometry analysis with two different antibodies. Incubation of lymphomononuclear cells from healthy volunteers in the presence of an immunomodulating peptide (NVLGAPKKLNESQAV) led to stimulation and maturation of monocytes manifested by changes in phenotype and an increase in both iNOS mRNA and protein, as well as HLA-DR expression. In this case also iNOS was expressed mainly on subsets of CD16+ monocytes with high HLA-DR expression. NO produced by human monocytes seems to have a function in the upregulation of membrane HLA-DR. These results are suggestive of a role for monocytic iNOS in the autoimmune response underlying the pathogenesis of multiple sclerosis.

A method to evaluate interactions between biologically active agents is presented. Synergism, zero interaction, and antagonism were easily detected with the three-dimensional approach proposed herein. This method is compatible with a checkerboard design to diagnose the interaction between agents and obviate the need to test their mixtures in a fixed concentration ratio as proposed by Chou and Talalay. Dose-response curves for individual agents were obtained, and experimental data fitted to appropriate equations by nonlinear regression. If zero interaction was present, the predicted effect could be calculated for each combination using the classical isobole equation with any spreadsheet having a command to solve mathematical equations by iteration. This allowed the selection of appropriate concentrations for the combination of two or more agents. Interaction between agents could be assessed in two ways: by comparing experimental with expected effects, if zero interaction is present; or by analyzing the reduction or increase in total dose found as a consequence of the interaction. The applicability of both approaches is discussed and, for purposes of comparison with other methods, examples based on published data are analyzed and commented upon.

Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15-mers containing a characteristic 2-6-11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides.

Some fused pyridazino[4,5- b ]indoles (7) were synthesized. These new compounds present a planar topography and some resemblance to carbazeram, imadozan, and other pyridazlno agents with cardiotonic activity. These compounds also possess a complementary effect as inhibitors of platelet aggregation. 6-(3,5-Dimethylpyrazolyl)- 1,2,4-triazolo[4,3- b ]pyridazino[4,5- b ]indole (7a) has a good profile as an inodilatador with antiaggregate activity due to the inhibition of phosphodiesterase.

A series of peptides of 15 amino acids with sequences contained in human extracellular matrix (ECM) proteins (fibronectin, laminin A, laminin B1, tenascin, undulin, alpha 1-chain of type IV and VIII collagen and alpha 2-chain of type VIII collagen) have been synthesized. The selected structures conformed to the following pattern: (i) Pro at position 6, (ii) Leu, Lys, Ile, Val, Ala or Gly at position 2, (iii) Glu or Asp at position 11. Fibronectin and the indicated peptides, when present in cultures of lymphomononuclear cells from healthy donors, promoted stimulation of monocytes manifested by a release of IL-1 alpha, IL-beta, IL-6 and TNF alpha; an increase in the percentage of cells expressing CD14, CD16, CD11b and CD14/CD16; an increase in cytotoxicity against HT-29. Cytotoxicity against K562 and Daudi cells (targets of NK and LAK cells) was also observed together with an increase in the percentage of cells expressing CD56, CD56/CD16 (corresponding to NK cells), and CD56/CD8 (corresponding to NK-like lymphocytes), indicating a stimulation of lymphocytes. Activated monocytes and lymphocytes contained a large number of granules with DNAse activity. These results suggest that at least some of the immunological properties of ECM proteins could be accounted for by motifs fulfilling a characteristic sequence pattern shared by all of them.

Activation of human T helper 1 cells took place when lymphomononuclear cells from healthy donors were incubated in the presence of short synthetic peptides encompassing sequences present in extracellular matrix proteins. Active peptides conformed to a common structural pattern ("2-6-11 motif") [N.Lopez-Moratalla et al., Biochem. Biophys. Acta (1994) 1221, 153-158] conferring immnunomodulating properties. The release of IL-2 and IFNγ, as well as LAK and NK-dependent cytotoxicity induced by these peptides, could be blocked by anti-HLA-DR antibody. Activated CD4 + cells isolated from the mixed incubated population contained secretion granules with DNAase activity. These results suggest that these immunomodulating peptides presented by HLA-II play a key role in the differentiation of CD4 + T cells towards a Th1 functional phenotype.