The microstructure in the placenta is influenced by the functional diversity of HLA-G allelic variants.

The main expression sites of HLA-G are human extravillous trophoblast cells. The interaction of HLA-G with uterine NK cells promotes their maturation and differentiation into decidual NK (dNK) cells. dNK cells secrete chemokines, cytokines, and proangiogenic factors in favor of a vascular remodeling and an immune suppressive microenvironment of the decidua. HLA-G is the most polymorphic member of the oligomorphic non-classical HLA molecule family; yet, the impact of polymorphic differences is not comprehensively understood. sHLA-G levels in embryo culture medium correlate with successful pregnancy; however, it remains questionable if HLA-G allelic diversity impacts on the outcome of dNK cell development. We utilized synthetic sHLA-G*01:01, 01:03, and 01:04 molecules and transduced K652/mHLA-G*01:01, 01:03, and 01:04 cells to study the biological interaction between HLA-G alleles and primary NK cells of human term placenta. Despite its low frequency, HLA-G*01:04 and not the most prevalent allele HLA-G*01:01 appear to be strong catalysts of dNK cell proliferation. Concluding, this study illustrates novel insights into the impact and binding efficiency of the three most common variants of HLA-G on primary placental NK cells.

Diagnosis of right bundle branch block: a concordance study.

BACKGROUND: Right bundle branch block is one of the most common electrocardiographic abnormalities. Most cases of right bundle branch block are detected in asymptomatic patients in primary care, so a correct interpretation of electrocardiograms (ECGs) at this level is necessary. The objective of this research is to determine the degree of concordance in the diagnosis of incomplete and complete right bundle branch block between four primary care researchers and a cardiologist. METHODS: The research design is a retrospective cohort study of patients over 18 years of ages of patients over 18 years of ages who underwent an ECG for any reason and were diagnosed with right bundle branch block by their physician. The physicians participating, 4 primary care researchers and a cardiologist were specialized in interpreting electrocardiographic records. The diagnosis of incomplete and complete right bundle branch block was recorded and other secondary variables were analysed. In case of diagnostic discordance between the researchers, the ECGs were reviewed by an expert cardiologist, who interpreted them, established the diagnosis and analysed the possible causes for the discrepancy. RESULTS: We studied 160 patients diagnosed with right bundle branch block by their general practise. The patients had a mean age of 64.8 years and 54% of them were men. The concordance in the diagnosis of incomplete right bundle branch block showed a Fleiss' kappa index (k) of 0.71 among the five researchers and of 0.85 among only the primary care researchers. The k for complete right bundle branch block was 0.93 among the five researchers and 0.96 among only the primary care researchers. CONCLUSION: The interobserver agreement in the diagnosis of right bundle branch block performed by physicians specialized in ECG interpretation (primary care physicians and a cardiologist) was very good. The variability was greater for the diagnosis of incomplete right bundle branch block.

The nocebo effect challenges the non-medical infliximab switch in practice.

BACKGROUND: In clinical practice, non-medical switching of biological medication may provoke nocebo effects due to unexplained deterioration of therapeutic benefits. Indication extrapolation, idiosyncratic reactions, and interchangeability remain challenged in clinical practice after biosimilar approval by the European Medicines Agency. The principle of "first do no harm" may be challenged in a patient when switching from originator to biosimilar biological. AIM: To describe the 1-year results of a pragmatic study on infliximab biosimilar implementation in immune-mediated inflammatory disease patients on the basis of shared decision-making under effectiveness and safety monitoring. METHODS: Inflammatory bowel disease and rheumatology patients on infliximab originator were converted to infliximab biosimilar after providing informed consent. Nocebo response patients were monitored after switch back to originator. Linear mixed models were used to analyze continuous endpoints on effectiveness and laboratory outcomes to determine significance (P ≤ 0.05) of change over time after switching. RESULTS: After inviting 146 patients, a group of 125 patients enrolled in the project over time, respectively, 73 Crohn's disease, 28 ulcerative colitis, nine rheumatoid arthritis, ten psoriatic arthritis, and five ankylosing spondylitis patients. No statistically significant changes in effectiveness and safety were observed in any of the indications after a median of 4 infusions in 9 months of study. An overall nocebo response of 12.8% was found among the patients during a minimal observation period of 6 months after the transition to biosimilar infliximab. The overall nocebo response rate did not differ between the studied indications. CONCLUSIONS: In inflammatory bowel disease and rheumatological patients, similar effectiveness and safety were demonstrated on the transition into infliximab biosimilar. In our series, patient empowerment and registration of treatment outcomes delineated biosimilar transition, an approach that hypothetically could reduce nocebo response rates which are relevant to account for regarding biosimilar implementation.

Poor Muscle Strength and Low Physical Activity Are the Most Prevalent Frailty Components in Community-Dwelling Older Adults.

OBJECTIVE: To determine the prevalence of five physical frailty phenotype components and to assess the relationship between them and other clinical factors. METHOD: A population-based cross-sectional study was performed. Subjects 75 years and older were randomly selected from primary care databases (with sampling stratified by gender). Physical frailty phenotypes were assessed using Fried's criteria. Sociodemographic data, comorbidities, nutritional status, and functional capacity were assessed. RESULTS: 126 subjects were recruited (47% women). Prevalence rates were poor muscle strength: 50%; low physical activity: 29%; slow gait: 28%; exhaustion: 27%; and weight loss: 5%. Prefrailty and frailty prevalence rates were 35.7% and 29.4%, respectively. Poor muscle strength and low physical activity showed a close relationship and concordance (kappa = 0.92). Most frailty components were associated with outdoor activity, hours walked daily, and certain comorbidities. CONCLUSIONS: Poor muscle strength was the most prevalent frailty component and was closely associated with physical activity, suggesting that training programs may revert or prevent the frailty process.

The chronology of the earliest Upper Palaeolithic in northern Iberia: New insights from L'Arbreda, Labeko Koba and La Viña.

Since the late 1980s, northern Iberia has yielded some of the earliest radiocarbon dated Aurignacian assemblages in Western Europe, probably produced by anatomically modern humans (AMHs). This is at odds with its location furthest from the likely eastern entry point of AMHs, and has also suggested to some that the Châtelperronian resulted from cultural transfer from AMHs to Neanderthals. However, the accuracy of the early chronology has been extensively disputed, primarily because of the poor association between the dated samples and human activity. Here, we test the chronology of three sites in northern Iberia, L'Arbreda, Labeko Koba and La Viña, by radiocarbon dating ultrafiltered collagen from anthropogenically modified bones. The published dates from Labeko Koba are shown to be significant underestimates due to the insufficient removal of young contaminants. The early (c.44 ka cal BP [thousands of calibrated years before present]) Aurignacian chronology at L'Arbreda cannot be reproduced, but the reason for this is difficult to ascertain. The existing chronology of La Viña is found to be approximately correct. Together, the evidence suggests that major changes in technocomplexes occurred contemporaneously between the Mediterranean and Atlantic regions of northern Iberia, with the Aurignacian appearing around 42 ka cal BP, a date broadly consistent with the appearance of this industry elsewhere in Western Europe.

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Abdominal pain in patients with inflammatory bowel disease in remission: A prospective study on contributing factors.

BACKGROUND: Abdominal pain is highly prevalent in patients with inflammatory bowel disease (IBD) in remission, but the aetiology is incompletely understood. AIM: To investigate the association of clinical, lifestyle and psychosocial factors with abdominal pain in patients with IBD in remission. METHODS: We performed a prospective multicentre study enrolling consecutive patients with IBD. Data were collected between 1 January 2020 and 1 July 2021, using myIBDcoach, an established remote monitoring platform for IBD. Chronic abdominal pain in IBD in remission (IBDremissionPain+) was defined as abdominal pain score ≥3 (0-10 NRS) on ≥1/3 of all assessments, combined with faecal calprotectin <150 µg/g in 90 days around periodic assessments. Disease activity, lifestyle and psychosocial factors were assessed every 1-3 months during 18 months. Using linear mixed models, the association of these factors with abdominal pain over time was analysed. RESULTS: We included 559 patients, of whom 429 (76.7%) remained in biochemical remission. Of these, 198 (46.2%) fulfilled the criteria for chronic abdominal pain. IBDremissionPain+ patients were characterised by female sex, younger age, higher BMI, and shorter disease duration. They reported more often or higher levels of stress, fatigue, depressive and anxiety symptoms, and life events (all p < 0.001). In the multivariable analysis, sex, disease entity, fatigue, depressive symptoms and life events were associated with abdominal pain over time (all p < 0.05). CONCLUSION: In this cohort of patients with IBD in remission, abdominal pain was common and associated with psychosocial factors. A more holistic treatment approach for patients with IBD suffering from abdominal pain may improve quality of care and subjective wellbeing.

No evidence of association between functional polymorphisms located within IL6R and IL6ST genes and systemic sclerosis.

Systemic sclerosis (SSc) is a complex autoimmune disease which genetic component has not been yet completely understood. IL6 encodes a cytokine with a crucial role in the development of autoimmunity and fibrosis and its actions mainly are controlled by IL-6 receptor (IL-6R). We aimed to investigate whether the functional genetic variants rs8192284 and rs2228044 previously associated with several autoimmune diseases, located within the IL-6 receptor (IL-6R) subunits IL6R and IL6ST genes, respectively, are involved in the susceptibility to SSc and/or its major clinical subphenotypes. A Spanish cohort including 1013 SSc patients and 1375 controls was genotyped using the TaqMan® allelic discrimination technology. SSc patients were subdivided according to the major clinical forms, autoantibody status and presence of fibrotic lung affection. Our data showed no influence of the selected variants in global SSc susceptibility (rs8192284: P=0.67, odds ratios (OR)=0.98; rs2228044: P=0.99, OR=1.00). Similarly, the clinical/autoantibody subphenotype analyses did not yielded significant results. Our data suggest that the analyzed polymorphisms may not play a significant role in the SSc susceptibility.

Nurse endoscopists perform colonoscopies according to the international standard and with high patient satisfaction.

BACKGROUND AND STUDY AIMS: Colonoscopy is increasingly performed by nurse endoscopists. We aimed to assess the endoscopic quality and patient experience of these procedures. PATIENTS AND METHODS: This prospective multicenter study analyzed 100 consecutive colonoscopies each for 10 trained nurse endoscopists with respect to endoscopic quality and patient experience. Colonoscopies were performed under the supervision of a gastroenterologist, using the techniques and protocols of the participating hospitals. Patient experience was assessed using a questionnaire. RESULTS: Most nurse endoscopists were female (90 %; median age 43 [range 35 - 49]). Before the start of the study, they had performed a median of 528 colonoscopies (range 208 - 2103). For the 1000 patients, mean age was 56 ± 15 years; 55 % were women; and 96 % were in class I or II according to the American Society of Anesthesiologists' physical status classification system. Colonoscopies were performed for screening or surveillance in 42 %; for symptomatic indications in 58 % of patients. The unassisted cecal intubation rate was 94 %; the mean withdrawal time was 10 ±â€Š5 minutes. The adenoma detection rate was 26.7 %. In 229 of the colonoscopies (23 %), the nurse endoscopists required assistance from the supervising gastroenterologist. The complication rate was 0.2 %: one perforation and one cardiopulmonary complication. The questionnaire was completed by 734 /1000 patients (73 %) and of these 694 /734 (95 %) were satisfied with the endoscopic procedure. Among the respondents 530 /734 (72 %) had no specific preference for a physician or nurse endoscopist, whereas 113 /734 (15 %) preferred a physician endoscopist, and 91 /734 (12 %) preferred a nurse endoscopist. CONCLUSION: The nurse endoscopists performed colonoscopies according to the internationally recognized quality standards and with high patient satisfaction.

Fatigue and heat sensitivity in patients with multiple sclerosis.

OBJECTIVES: Fatigue is one of the most common and troubling symptoms of multiple sclerosis (MS), and heat is often reported as a trigger. Although it is assumed that this heat sensitivity is specific for MS, the evidence for disease specificity is limited. We studied the relationship between fatigue, heat sensitivity, and environmental temperature, and its specificity for MS. MATERIALS AND METHODS: We compared 88 MS patients with 76 patients with ulcerative colitis (UC), another chronic auto-immune disease. As most important outcome measures, heat sensitivity, physical fatigue, mental fatigue, environmental temperature, and ambient UV-light levels were determined. RESULTS: More patients with MS reported heat sensitivity for fatigue, compared to patients with UC (53.4% vs 35.5%, respectively, P = 0.016). However, heat-sensitive patients were equally fatigued as heat-insensitive patients. Climatological data, including day temperature and amount of ambient UV light, were not related to fatigue in both heat-sensitive and heat-insensitive patients with MS. CONCLUSIONS: Our findings support the assumption that heat sensitivity regarding fatigue has an MS-specific component. Although patients with MS experience a relationship between environmental temperature and fatigue, objective assessment by climatological data could not confirm this.

Somatic cells derived from haploid larvae are feasible as donors for nuclear transplant in zebrafish. Preliminary results.

Summary Somatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.

The STAT4 gene influences the genetic predisposition to systemic sclerosis phenotype.

The aim of this study was to investigate the possible role of STAT4 gene in the genetic predisposition to systemic sclerosis (SSc) susceptibility or clinical phenotype. A total of 1317 SSc patients [896 with limited cutaneous SSc (lcSSc) and 421 with diffuse cutaneous SSc (dcSSc)] and 3113 healthy controls, from an initial case-control set of Spanish Caucasian ancestry and five independent cohorts of European ancestry (The Netherlands, Germany, Sweden, Italy and USA), were included in the study. The rs7574865 polymorphism was selected as STAT4 genetic marker. We observed that the rs7574865 T allele was significantly associated with susceptibility to lcSSc in the Spanish population [P = 1.9 x 10(-5) odds ratio (OR) 1.61 95% confidence intervals (CI) 1.29-1.99], but not with dcSSc (P = 0.41 OR 0.84 95% CI 0.59-1.21). Additionally, a dosage effect was observed showing individuals with rs7574865 TT genotype higher risk for lcSSc (OR 3.34, P = 1.02 x 10(-7) 95% CI 2.11-5.31). The association of the rs7574865 T allele with lcSSc was confirmed in all the replication cohorts with different effect sizes (OR ranging between 1.15 and 1.86), as well as the lack of association of STAT4 with dcSSc. A meta-analysis to test the overall effect of the rs7574865 polymorphism showed a strong risk effect of the T allele for lcSSc susceptibility (pooled OR 1.54 95% CI 1.36-1.74; P < 0.0001). Our data show a strong and reproducible association of the STAT4 gene with the genetic predisposition to lcSSc suggesting that this gene seems to be one of the genetic markers influencing SSc phenotype.

Analysis of the influence of PTPN22 gene polymorphisms in systemic sclerosis.

OBJECTIVE: Two functional single nucleotide polymorphisms (SNP) in the PTPN22 gene (rs24746601 and rs33996649) have been associated with autoimmunity. The aim of this study was to investigate the role of the R263Q SNP for the first time and to re-evaluate the role of the R620W SNP in the genetic predisposition to systemic sclerosis (SSc) susceptibility and clinical phenotypes. METHODS: 3422 SSc patients (2020 with limited cutaneous SSc and 1208 with diffuse cutaneous SSc) and 3638 healthy controls of Caucasian ancestry from an initial case--control set of Spain and seven additional independent replication cohorts were included in our study. Both rs33996649 and rs2476601 PTPN22 polymorphisms were genotyped by TaqMan allelic discrimination assay. A meta-analysis was performed to test the overall effect of these PTPN22 polymorphisms in SSc. RESULTS: The meta-analysis revealed evidence of association of the rs2476601 T allele with SSc susceptibility (p(FDRcorrected)=0.03 pooled, OR 1.15, 95% CI 1.03 to 1.28). In addition, the rs2476601 T allele was significantly associated with anticentromere-positive status (p(FDRcorrected)=0.02 pooled, OR 1.22, 95% CI 1.05 to 1.42). Although the rs33996649 A allele was significantly associated with SSc in the Spanish population (p(FDRcorrected)=0.04, OR 0.58, 95% CI 0.36 to 0.92), this association was not confirmed in the meta-analysis (p=0.36 pooled, OR 0.89, 95% CI 0.72 to 1.1). CONCLUSION: The study suggests that the PTPN22 R620W polymorphism influences SSc genetic susceptibility but the novel R263Q genetic variant does not. These data strengthen evidence that the R620W mutation is a common risk factor in autoimmune diseases.

Ultraviolet radiation and handling medium osmolarity affect chimaerism success in zebrafish.

The effects of a predefined ultraviolet radiation dose (0.529 mW/cm2 for 30s) together with two different micromanipulation medium osmolarities (30 mOsm/kg vs 300 mOsm/kg) were tested on embryo survival at different developmental stages and on the somatic (skin) and germ-line chimaerism rates. Somatic (13%, 6/47 adults) and germ-line chimaerism (50% pigmented F1 larvae) were detected only in the UV-treated recipient embryos micromanipulated in a 300 mOsm/kg medium. From the results obtained, we concluded that the conditions cited above were the most suitable to improve somatic and germ-line chimaerism rates in zebrafish.

Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors.

This study shows that induction of tumor-specific CD4+ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4+ T cells were of the T helper cell 1 type. The peptide-specific CD4+ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8+ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.

Differential influence on cytotoxic T lymphocyte epitope presentation by controlled expression of either proteasome immunosubunits or PA28.

The proteasome is the principal provider of major histocompatibility complex (MHC) class I-presented peptides. Interferon (IFN)-gamma induces expression of three catalytically active proteasome subunits (LMP2, LMP7, and MECL-1) and the proteasome-associated activator PA28. These molecules are thought to optimize the generation of MHC class I-presented peptides. However, known information on their contribution in vivo is very limited. Here, we examined the antigen processing of two murine leukemia virus-encoded cytotoxic T lymphocyte (CTL) epitopes in murine cell lines equipped with a tetracycline-controlled, IFN-gamma-independent expression system. We thus were able to segregate the role of the immunosubunits from the role of PA28. The presence of either immunosubunits or PA28 did not alter the presentation of a subdominant murine leukemia virus (MuLV)-derived CTL epitope. However, the presentation of the immunodominant MuLV-derived epitope was markedly enhanced upon induction of each of these two sets of genes. Thus, the IFN-gamma-inducible proteasome subunits and PA28 can independently enhance antigen presentation of some CTL epitopes. Our data show that tetracycline-regulated expression of PA28 increases CTL epitope generation without affecting the 20S proteasome composition or half-life. The differential effect of these IFN-gamma-inducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response.

BANK1 functional variants are associated with susceptibility to diffuse systemic sclerosis in Caucasians.

OBJECTIVE: To investigate the possible association of the BANK1 gene with genetic susceptibility to systemic sclerosis (SSc) and its subphenotypes. METHODS: A large multicentre case-control association study including 2380 patients with SSc and 3270 healthy controls from six independent case-control sets of Caucasian ancestry (American, Spanish, Dutch, German, Swedish and Italian) was conducted. Three putative functional BANK1 polymorphisms (rs17266594 T/C, rs10516487 G/A, rs3733197 G/A) were selected as genetic markers and genotyped by Taqman 5 allelic discrimination assay. RESULTS: A significant association of the rs10516487 G and rs17266594 T alleles with SSc susceptibility was observed (pooled OR=1.12, 95% CI 1.03 to 1.22; p=0.01 and pooled OR=1.14, 95% CI 1.05 to 1.25; p=0.003, respectively), whereas the rs3733197 genetic variant showed no statistically significant deviation. Stratification for cutaneous SSc phenotype showed that the BANK1 rs10516487 G, rs17266594 T and rs3733197 G alleles were strongly associated with susceptibility to diffuse SSc (dcSSc) (pooled OR=1.20, 95% CI 1.05 to 1.37, p=0.005; pooled OR=1.23, 95% CI 1.08 to 1.41, p=0.001; pooled OR=1.15, 95% CI 1.02 to 1.31, p=0.02, respectively). Similarly, stratification for specific SSc autoantibodies showed that the association of BANK1 rs10516487, rs17266594 and rs3733197 polymorphisms was restricted to the subgroup of patients carrying anti-topoisomerase I antibodies (pooled OR=1.20, 95% CI 1.02 to 1.41, p=0.03; pooled OR=1.24, 95% CI 1.05 to 1.46, p=0.01; pooled OR=1.26, 95% CI 1.07 to 1.47, p=0.004, respectively). CONCLUSION: The results suggest that the BANK1 gene confers susceptibility to SSc in general, and specifically to the dcSSc and anti-topoisomerase I antibody subsets.

Off-label use of rituximab in 196 patients with severe, refractory systemic autoimmune diseases.

OBJECTIVES: To analyse the safety and efficacy of the off-label use of rituximab in patients with severe, refractory systemic autoimmune diseases. METHODS: In 2006, the Study Group on Autoimmune Diseases of the Spanish Society of Internal Medicine created the BIOGEAS project, a multicenter study devoted to collecting data on the use of biological agents in adult patients with systemic autoimmune diseases refractory to standard therapies (failure of at least two immunosuppressive agents). RESULTS: One hundred and ninety-six patients with systemic autoimmune diseases treated with rituximab have been included in the Registry (158 women and 38 men, mean age 43 years). Systemic autoimmune diseases included systemic lupus erythematosus (107 cases), inflammatory myopathies (20 cases), ANCA-related vasculitides (19 cases), Sjögren's syndrome (15 cases) and other diseases (35 cases). A therapeutic response was evaluable in 194 cases: 99 (51%) achieved a complete response, 51 (26%) a partial response and 44 (23%) were classified as non-responders. After a mean follow-up of 27.56+/-1.32 months, 44 (29%) out of the 150 responders patients relapsed. There were 40 adverse events reported in 33 (16%) of the 196 patients. The most frequent adverse events were infections, with 24 episodes being described in 19 patients. Thirteen (7%) patients died, mainly due to disease progression (7 cases) and infection (3 cases). CONCLUSIONS: Although not yet licensed for this use, rituximab is currently used to treat severe, refractory systemic autoimmune diseases, with the most favourable results being observed in Sjögren's syndrome, inflammatory myopathies, systemic lupus erythematosus and cryoglobulinemia.

Definition of three somatic adult cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization.

Zebrafish somatic nuclear transplant has only been attempted using preactivated eggs. In this work, three methods to carry out the nuclear transplant using adult cells before, during and after the egg activation/fertilization were developed in zebrafish with the aim to be used in reprogramming studies. The donor nucleus from somatic adult cells was inserted: (method A) in the central region of the egg and subsequently fertilized; (method B) in the incipient animal pole at the same time that the egg was fertilized; and (method C) in the completely defined animal pole after fertilization. Larval and adult specimens were obtained using the three methods. Technical aspects related to temperature conditions, media required, egg activation/fertilization, post-ovulatory time of the transplant, egg aging, place of the donor nucleus injection in each methodology are presented. In conclusion, the technical approach developed in this work can be used in reprogramming studies.

Micromanipulation medium osmolarity compromises zebrafish (Danio rerio) embryo and cell survival in chimaerism experiments.

In zebrafish chimaerism experiments, the cell injection can involve intra-embryonic cell lyses by osmolar effects. Moreover, the donor cells can be injured during manipulation due to osmolar changes into the transplant pipette. Therefore, the present study aimed to assess the effects of manipulation medium osmolarity on embryonic survival and donor cell viability.In Experiment I, 0.1 microl to 0.15 microl approximately of an isosmolar solution (300 mOsm) was injected into recipient embryos, which were kept at 300 (E1) or 30 mOsm (E2). Survival at day 1 was significantly higher in the E2 group than in E1 (E1: 68% vs E2: 81%, p < 0.05), but after 5 days embryo survival in the E1 group was slightly higher. In Experiment II, donor cells from zebrafish embryos were exposed (or not) to a possible osmolarity change (inner pipette medium: 300 mOsm vs external medium: 30 or 300 mOsm) using two different micropipette outer diameters, 40-50 and 60-70 microm. Cell mechanical damage was detected in the 40-50 microm pipette (p < 0.05), but not by the handling medium osmolarity. Results recommend the use of a 300 mOsm manipulation medium and bore-sized pipettes adjusted as closely as possible to the donor cell size.