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Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.

Summary We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an optimized protocol. FVIII-expressing ECs were intraportally transplanted in monocrotaline-conditioned non-obese diabetic (NOD) severe combined immune-deficient (scid)-IL2rγ null HA mice generating a chimeric liver with functional human ECs. Transplanted cells engrafted and proliferated in the liver along sinusoids, in the long term showed stable therapeutic FVIII activity (6%). These results demonstrate that the hemophilic phenotype can be rescued by transplantation of ECs derived from HA FVIII-corrected iPSCs, confirming the feasibility of cell-reprogramming strategy in patient-derived cells as an approach for HA gene and cell therapy., Graphical Abstract, Highlights • Endothelial cells were differentiated from iPSCs obtained from hemophilic donors • LV transduced-hemophilic endothelial cells secreted FVIII in vitro and in vivo, Follenzi and colleagues show that endothelial cells can be obtained starting from blood CD34+ cells of both healthy and hemophilic donors passing through iPSC generation. During the differentiation process the cells can be genetically modified by LV to express FVIII; corrected endothelial cells secrete FVIII in vitro and ameliorate the hemophilic phenotype in a mouse model of the disease.

A deeper understanding of early disease mechanisms occurring in Parkinson’s disease (PD) is needed to reveal restorative targets. Here we report that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) obtained from healthy individuals or patients harboring LRRK2 PD-causing mutation can create highly complex networks with evident signs of functional maturation over time. Compared to control neuronal networks, LRRK2 PD patients’ networks displayed an elevated bursting behavior, in the absence of neurodegeneration. By combining functional calcium imaging, biophysical modeling, and DAn-lineage tracing, we found a decrease in DAn neurite density that triggered overall functional alterations in PD neuronal networks. Our data implicate early dysfunction as a prime focus that may contribute to the initiation of downstream degenerative pathways preceding DAn loss in PD, highlighting a potential window of opportunity for pre-symptomatic assessment of chronic degenerative diseases., Research from the authors’ laboratories is supported by the European Research Council-ERC (2012-StG-311736-PD-HUMMODEL), the MESOBRAIN project from the European Union’s Horizon 2020 research and innovation (grant agreement No. 713140), the Spanish Ministry of Economy and Competitiveness-MINECO (RTI2018-095377-B-100, FIS2016-78507-C2-2-P, PID2019-108842GB-C21) the Spanish Ministry of Economy and Competitiveness-MINECO/AEI/FEDER, UE (BFU2016-80870-P) and the Spanish Ministry of Economy and Competitiveness- AEI/10.13039/501100011033 (PID2019-108792GB-I00), Instituto de Salud Carlos III-ISCIII/FEDER (Red de Terapia Celular - TerCel RD16/0011/0024 and RD16/0011/0025), AGAUR (2017-SGR-899 and 2017-SGR-1061), and CERCA Program/Generalitat de Catalunya. M.P.E. was partially supported by a Beatriu de Pinós fellowship from the Agency for Management of University and Research Grants (AGAUR) of the Government of Catalonia (2017 BP 00133). L.B. is the recipient of a pre-doctoral fellowship FPI (BES-2017-080579) from the Spanish Ministry of Economy and Competitiveness (MINECO). G.C. was partially supported by pre-doctoral fellowship from Spanish Economy and Competitiveness-MINECO (BES-2014-069603).

Summary Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(−/−) cells, descendants from the tumor originating cell. These NF1(−/−)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(−/−) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available., Graphical Abstract, Highlights • We generated iPSCs from neurofibromatosis type 1 plexiform neurofibroma (PNF) cells • PNF-derived iPSCs were differentiated into neural crest and Schwann cells (SCs) • iPSC-differentiated NF1(−/−) SCs exhibit a high proliferation rate and form spheres • Sphere-forming SCs express the same markers as their primary PNF counterparts, In this article, Eduard Serra and colleagues describe the generation of iPSCs directly from plexiform neurofibromas (PNFs), benign Schwann cell (SC) tumors associated with neurofibromatosis type 1. iPSCs bearing the double inactivation of the NF1 gene were differentiated into SCs that exhibited a high proliferation rate, a poor myelination ability, and a tendency to form spheres, resembling PNF-derived SCs.

Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient., SCOPUS: ar.j, info:eu-repo/semantics/published

Summary Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration., Graphical Abstract, Highlights • Fibroblasts from two Sanfilippo C patients were reprogrammed to obtain iPSCs • iPSCs were successfully differentiated to neural cells that mimic the disease • Networks of patients’ neurons show altered activity and connectivity • Early functional phenotypes are prevented in gene-corrected patients’ neurons, iPSC technology has been used to model dozens of diseases. In this article, Raya, Grinberg, and colleagues take a step forward and show that this technology can also be used to model pre-symptomatic stages of human diseases. In particular, they used it to generate neurons from Sanfilippo C patients and found functional alterations in how these neurons interacted with one another, before the neurons themselves had any obvious alteration.

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. SummaryBackground The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level.

The generation of patient-specific induced pluripotent stem cells (iPSCPSCPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSCPSCPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCPSCPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCPSCPSCs can be obtained in 4–5 months.

The availability of induced pluripotent stem cells (iPSCs) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno-free cell culture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin (trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.

Previous reports have suggested that regulatory T cells (Treg) are abnormal in patients with systemic lupus erythematosus (SLE). In the present work, we quantified CD4+FOXP3+Treg cells in patients with SLE and found no quantitative alterations. However, we found a clear defect in suppression assays. Surprisingly, SLE-derived Treg cells exhibited a normal phenotype and functional capacity. Conversely, SLE-derived CD4+CD25−effector T cells resisted suppression by autologous and allogeneic regulatory cells. Our findings strongly suggest that the defect in T-cell suppression observed in SLE is because of effector cell resistance and not because of an abnormal regulatory function.

Objective To define the cytokine and chemokine profile in cerebrospinal fluid (CSF) from patients with neuropsychiatric systemic lupus erythematosus (NPSLE). Methods Forty-two SLE patients who had been hospitalized because of NP manifestations were studied. Patients were evaluated at hospitalization and 6 months later; a CSF sample was obtained at each evaluation. As controls, CSF from 6 SLE patients with septic meningitis, 16 SLE patients with no history of NP manifestations (non-NPSLE), and 25 patients with nonautoimmune diseases were also studied. Soluble molecules, including cytokines (interleukin-2 [IL-2], IL-4, IL-6, IL-10, tumor necrosis factor α [TNFα], and interferon-γ [IFNγ]) and chemokines (monocyte chemotactic protein 1 [MCP-1], RANTES, IL-8, monokine induced by IFNγ [MIG], and interferon-γ–inducible 10-kd protein [IP-10]), were measured with the use of cytometric bead array kits. Results CSF levels of the following molecules were significantly increased in NPSLE patients as compared with non-NPSLE and nonautoimmune diseases control patients, respectively: IL-6 (32.7 versus 3.0 and 2.96 pg/ml), IL-8 (102.8 versus 29.97 and 19.7 pg/ml), IP-10 (888.2 versus 329.7 [P not significant] and 133.6 pg/ml), RANTES (3.8 versus 2.5 and 2.2 pg/ml), MCP-1 (401.7 versus 257.9 [P not significant] and 136.9 pg/ml), and MIG (35.4 versus 11.4 and 3.5 pg/ml). Low levels of IL-2, IL-4, IL-10, TNFα, and IFNγ were found in all groups. All cytokines and chemokines, except TNFα, were significantly higher among the SLE patients with septic meningitis than among the NPSLE patients. Six months later and in the absence of NP manifestations, all elevated molecule levels, except RANTES, in patients with NPSLE had decreased significantly, and no differences were noted between the NPSLE and non-NPSLE groups. Conclusion A central nervous system response composed of IL-6 and chemokines, but not Th1/Th2 cytokines, is associated with NP manifestations in SLE patients.

CD55 and CD59 are glycophosphatidylinositol-anchored proteins with complement inhibitory properties. Lymphopenia in systemic lupus erythematosus (SLE) has been associated with autoantibodies targeting nuclear antigens. The aim of this study was to evaluate the surface density of CD55 and CD59 in T and B lymphocytes from patients with SLE and lymphopenia and its possible correlation with the presence of common SLE autoantibodies. Flow cytometric analyses were performed on CD55 and CD59 stained CD3 and CD19 cells from 40 SLE patients, 30 with lymphopenia and 10 without it, and 25 healthy controls. Autoantibodies were detected in the sera by enzyme linked immunosorbent assay. The mean fluorescence intensity of CD55 and CD59 in T and B cells was significantly diminished in SLE patients with lymphopenia when compared with healthy subjects. Interestingly, the opposite was found in T and B cells from non-lymphopenic SLE patients. Although there was no correlation between CD55 and CD59 surface density and the presence of any specificity of the autoantibodies tested, higher titres of anti-dsDNA, anti-SM and anti-ribosomal p antibodies were significantly associated with lymphopenia. The deficiency of CD55 and CD59 expression may play a role in the pathophysiology of lymphopenia, most likely by increasing the susceptibility of cells to complement mediated cytolysis.

Behcet’s disease is a multi-system inflammatory disorder of unknown etiology. The disease is more prevalent in Eastern Mediterranean countries and Japan where there is a linkage to HLA-B51. Mexican Mestizos are suitable subjects for studying the role of ethnicity in the susceptibility to Behcet’s disease. High-resolution HLA class I and class II typing was performed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) reverse dot blot and PCR-single-strand polymorphism in 32 patients with Behcet’s disease and 99 healthy ethnically-matched controls. A significant increased frequency of HLA-B * 44 ( P =0.02; OR = 2.78; CI 95% = 1.1–7.7), HLA-B * 52 ( P =0.02; OR = 5.33; CI 95% = 1.07–29.1), and HLA-B * 56 ( P =0.003; OR = 4.19; CI 95% = 3.37–5.21) as well as HLA-DRB1 * 01 and HLA-DRB1 * 13 ( P =0.007; OR = 3.36; CI 95% = 1.22–9.27) was found in Mexican patients with Behcet’s disease when compared to controls. The low frequency of native markers in Mexican Mestizo patients with Behcet’s disease suggests that genetic admixture between Eastern Mediterraneans and Orientals with Amerindians is a recent event that increased the risk of developing Behcet’s disease in the Mexican population.

Multidrug resistance-1 (MDR-1) is characterized by overfunction of P-glycoprotein (P-gp), a pump molecule that decreases intracellular drug concentration by effluxing them from the intracellular space. Broad ranges of structurally unrelated compounds are transported by P-gp, including antineoplastic agents, HIV protease inhibitors, prednisone, gold salts, methotrexate, colchicine as well as several antibiotics. In contrast, many other compounds such as calcium channel blockers (verapamil) and immunosupressors (cyclosporine-A) are able to inhibit P-gp function. The P-gp role in therapeutic failures has been extensively studied in cancer; however, there is little information regarding MDR-1 phenotype in autoimmune disorders. It has been reported that an increased number of lymphocytes are able to extrude P-gp substrates in rheumatoid arthritis, immune thrombocytopenic purpura and systemic lupus erythematosus, the patients with poor response to treatment being the ones that exhibit the highest values. This may be due, at least in part, to a simultaneous long-term usage of several drugs that induce P-gp function. Since abnormally activated cell compartments characterize autoimmune diseases, it is possible that those cells are the ones that exhibit drug resistance. The study of drug resistance mechanisms in autoimmunity may be helpful for the optimization of the current therapeutic schemes through their combination with low doses of P-gp inhibitors.

P-glycoprotein (P-gp) expression has been widely observed in normal and neoplastic cells. The physiologic role of P-gp involves hormone and metabolite secretion, bacterial product detoxification, and transport of several drugs to the extracellular space. Multidrug resistance-1 is characterized by drug extrusion through P-gp, reducing the intracellular levels of drugs and diminishing their pharmacological effects. Treatment of immune thrombocytopenic purpura (ITP) includes agents that are substrates of P-gp; hence, the objective of this study was to analyze the functional activity of P-gp in lymphocytes from patients with ITP.30 ITP patients (9 refractory, 5 dependent, 14 responders to treatment, and 2 with stable disease) and 25 healthy controls were studied. Peripheral blood mononuclear cells were isolated by gradient centrifugation and incubated with daunorubicin (a fluorescent drug extruded by P-gp). Functional activity of P-gp was analyzed by flow cytometry. Results were expressed as the percentage of lymphocytes able to extrude daunorubicin.ITP patients showed an increased number of lymphocytes with P-gp activity (mean=12.3%+/-16%) when compared to controls (mean=0.87%+/-0.72%) (p0.05). P-gp function was higher in the refractory group (median=9.4%) than in the treatment-dependent (median=5.4%), responder (median=6.4%), and stable disease (median=5.2%) groups, although no statistical differences were found among them.Enhanced P-gp activity in ITP may be related to an unfavorable clinical outcome and poor response to treatment. Furthermore, P-gp function might affect therapeutic requirements for disease control.

Little is known about the immune system of patients with systemic lupus erythematosus(SLE) during periods of silent disease. To address this issue we analysed lymphoid populations and cytokine productionof mononuclearcells obtained from SLE patients in remission.We studied 43 patients with inactive disease, 10 with active disease and 30 controls. Remission was defined as at least 1 year during which lack of clinical disease activity permitted withdrawal of all treatment. Remission length ranged from 1 to 30 years. Flow cytometry and ELISA were used to study lymphoid populations (CD4, CD8 and CD19) and cytokineproduction(IL-2, 4, 10, 12 and 18). Patientswith short remission periods (up to 15 years) exhibited an increased percentage of B cells; production of IL-2, IL-10 and IL-12 was decreased;productionof IL-18 was increased. Interestingly, patients from groups with long time of inactive disease had corrected most alterations, but had an impaired IL-18 expression. IL-12 production correlated strongly with the length of the remission period (r 0.7565). The immune system of patients with inactive lupus has partially corrected the disturbances present during disease activity.This is accomplishedgradually, sometimes until counter-regulatoryalterationsare developed. This may allow patients to remain without disease activity.

Resume Objectif. Ce travail a eu pour but d’evaluer l’efficacite therapeutique du leflunomide, prescrit en une prise hebdomadaire de 100 mg, chez des patients atteints de polyarthrite rhumatoide refractaire. Methodes. Les 16 patients inclus dans cette etude etaient âges de 18 a 72 ans et etaient atteints d’une polyarthrite rhumatoide evoluant depuis 2 a 32 ans. Huit patients ont ete traites par le leflunomide a la posologie d’une prise hebdomadaire de 100 mg et huit patients ont ete traites par le leflunomide selon les modalites conventionnelles comportant une prise quotidienne. Tous les patients ont ete evalues chaque mois pendant une annee. Nous avons utilise les criteres de reponse de l’ACR 1995 pour la polyarthrite rhumatoide. Resultats. Apres deux mois de traitement, il a ete observe une importante amelioration dans le groupe traite par leflunomide de maniere classique : sept des huit patients repondaient aux criteres ACR 20. En revanche, l’amelioration etait moins evidente dans le groupe traite par leflunomide hebdomadaire : trois des huit patients etaient repondeurs aux criteres ACR 20. Apres une periode de quatre a six mois, la reponse etait similaire entre les deux groupes ; une reponse ACR 50 etait observee chez les six patients traites de maniere quotidienne et chez six des huit patients traites de maniere hebdomadaire. Ce resultat a persiste jusqu’a la fin de l’etude. Il n’a pas ete observe de difference statistiquement significative entre les deux groupes et ce quel que soit le parametre etudie. Des effets indesirables sont survenus chez six des huit patients traites par le leflunomide de maniere quotidienne et deux de ces patients ont ete obliges d’arreter le leflunomide, l’un pour des signes gastro-intestinaux severes et l’autre pour une toxicite hepatique. Dans le groupe traite par le leflunomide hebdomadaire, deux patients ont eu des effets indesirables qui ont spontanement regresse. Conclusion. L’utilisation du leflunomide sous la forme d’une prise hebdomadaire de 100 mg permet d’obtenir un effet therapeutique similaire a celui du schema therapeutique conventionnel et pourrait representer une nouvelle option therapeutique dans la polyarthrite rhumatoide refractaire.

Aims. To assess the therapeutic effect of leflunomide in weekly dose of 100 mg in patients with refractory rheumatoid arthritis. Material and Methods. Sixteen patients were included (18-72 years, disease duration 2-32 years). Eight patients received a weekly dose of 100 mg of leflunomide and 8 the conventional dose. Current treatment was not modified. All patients underwent a monthly evaluation for one year, applying the 1995 ACR preliminary definition of improvement in rheumatoid arthritis. Results. After 2 months, the group treated with the conventional leflunomide dose evidenced a remarkable improvement (7/8 patients achieving ACR 20), while the group receiving the weekly dose, the improvement was not as clearly evident (3/8 patients achieving ACR 20). By the fourth to sixth month, the response was comparable on both groups (6/6 and 6/8 patients achieving ACR 50 in the daily and weekly dose, respectively) and prevailed through the end of the study. There were no statistical differences between groups at any evaluation. Side effects made itself clear in 6 patients in the daily leflunomide group, and 2 patients withdrawn leflunomide because severe gastrointestinal symptoms and hepatotoxicity, respectively. In the group of weekly leflunomide 2 patients presented side effects which disappeared spontaneously. Conclusion. The use of leflunomide in a weekly dose of 100 mg proved to have similar therapeutic benefit as that of the conventional scheme and might represent a new option for the treatment of refractory rheumatoid arthritis patients.

Objective, To identify bacterial DNA in synovial fluid cells of patients with active juvenile onset spondyloarthropathy (SpA). Methods. The main group of study constituted 22 patients with juvenile onset SpA. In addition, five patients with adult onset SpA and nine with rheumatoid arthritis (RA) were studied. Polymerase chain reaction (PCR) with either genus- or species-specific primers was performed on synovial fluid cells to detect DNA sequences of Chlamydia trachomatis, Yersinia enterocolitica, Salmonella sp., Shigella sp., Campylobacter sp. and Mycobacterium tuberculosis. The presence of antibacterial antibodies in sera and synovial fluid was also determined by enzyme-linked immunoassay. Results. The synovial fluid of nine patients with juvenile onset SpA, three with adult onset SpA and one with RA contained bacterial DNA. Five juvenile onset SpA samples had DNA of one single bacterium; two juvenile onset SpA and three adult onset SpA had DNA of two bacteria and two juvenile onset SpA had DNA of three bacteria. Overall, Salmonella sp. DNA was detected in seven synovial fluid samples, Shigella sp., Campylobacter sp. and M. tuberculosis were found in four samples each, and C. trachomatis was found in two. The bacterial DNA findings correlated with neither diagnosis nor disease duration. One RA synovial fluid had DNA of Campylobacter sp. Neither serum nor synovial fluid antibacterial antibodies correlated with DNA findings or clinical diagnosis. Conclusion. In this study, single and several combinations of bacterial DNA were identified in the synovial fluid of patients with long-term undifferentiated and definite juvenile onset SpA and adult onset SpA. Of relevance is that bacterial DNA corresponds to bacteria producing endemic disease in our population.

Immune imbalance in SLE increases the susceptibility to infectious diseases. The aim of this study was to analyze several mechanisms related to non-specific immunity in this autoimmune disorder. We studied in vivo CD11b expression, phagocytosis, and chemotaxis in polymorphonuclear cells (PMN) from SLE patients. All tests were also performed under hrIL-8 stimulating conditions and analyzed by flow cytometry. Intracellular leucocyte (monocytes and PMN) enzyme activity was evaluated using specific substrates for cathepsin B and D, collagenase, and oxidative burst by flow cytoenzymology. An exaggerated in vivo CD11b expression was observed on PMN from SLE patients without noticeably in vitro effect upon hrIL-8. Similarly both, phagocytosis and chemotaxis were diminished and showed no response to hrIL-8 stimulation. The opposite was found in PMN from controls. Intracellular enzyme activity was comparable between groups as far as cathepsin B and D are concerned. A tendency of decreased oxidative-burst induction was noted in monocytes and PMN from SLE patients, whereas collagenase activity was found clearly increased in both leucocyte subpopulations. Our results may represent a deficient ability of the innate immune mechanisms for the clearance of infectious agents, immune complexes, satisfactory resolution of inflammatory processes and tissue repair in SLE.

lipid-binding properties, and seems to be the actual target antigen for autoimmune type anticardiolipin (CL) antibodies that may bind to a neoepitope un

Resume Contexte. La multiresistance est caracterisee par une expression excessive de la P-glycoproteine, pompe membranaire qui expulse les medicaments de la cellule vers le milieu extracellulaire. Objectif. Rechercher dans la polyarthrite rhumatoide des correlations entre l'activite de la P-glycoproteine, la concentration d'ARN messager du TNF-α et l'etat clinique. Methodes. Nous avons etudie 16 malades, dont huit consideres refractaires au traitement (groupe R) et huit non refractaires (NR), ainsi que 24 sujets bien portants. L'activite de la P-glycoproteine fonctionnelle a ete mesuree par cytometrie en flux. Des cellules mononucleees du sang peripherique isolees par centrifugation a gradient ont ete incubees avec de la daunorubicine afin de permettre la detection de cellules ayant le phenotype correspondant au gene MDR-I (« MDR a pour multiple drug resistance ). Les concentrations d'ARN messager du TNF-α ont ete determinees par PCR quantitative. Resultats. Le pourcentage de lymphocytes caracterises par une forte activite P-glycoproteine etait plus eleve chez les malades que chez les sujets normaux ( p = 0,0001). Chez les malades refractaires, l'activite P-glycoproteine etait plus marquee et l'expression du gene TNF-α considerablement plus importante que chez les malades non refractaires ( p = 0,006). L'ARN messager du TNF-α n'etait pas decelable chez les sujets normaux. Conclusions. Une augmentation de l'activite P-glycoproteine semble etroitement liee a une evolution clinique defavorable et a une absence de reponse au traitement. Elle pourrait etre liee a une expression accrue de TNF-α et a l'administration au long cours de divers medicaments, notamment de glucocorticoides. Une activite accrue de la P-glycoproteine et une production excessive de TNF-α semblent liees a un mauvais pronostic dans la polyarthrite rhumatoide.

Objective. To evaluate interleukin-10 (IL-10) production in relatives of patients with systemic lupus erythematosus (SLE). Methods. Production of IL-10 was evaluated in 13 families in which several members had SLE. The constitutive IL-10 production in SLE patients (n = 16) was compared with that in healthy members of these multiplex families (n = 70), in 30 SLE patients who had no relatives with SLE, and in 46 healthy unrelated controls. Results. The level of IL-10 production did not differ between SLE patients who were members and those who were not members of multiplex families (mean ± SEM 4,384 ± 908 pg/ml and 4,709 ± 560 pg/ml, respectively), but was higher in both groups than in healthy unrelated controls (515 ± 88 pg/ml). The healthy members of the multiplex families constitutively produced large amounts of IL-10 (3,080 ± 311 pg/ml; P < 0.001 compared with healthy unrelated controls). This high IL-10 production was independent of age and sex, and was similar in first- and second-degree relatives of SLE patients. The IL-10 was produced both by monocytes and by a subpopulation of B lymphocytes in SLE patients and in their relatives. Conclusion. The dysregulation of IL-10 production previously identified in SLE patients is also present in healthy members of families with several cases of SLE, and it may contribute to the immunologic abnormalities affecting relatives of SLE patients.

Interleukin-10 (IL-10) is produced at a high level by B lymphocytes and monocytes of patients with systemic lupus erythematosus (SLE). In the present work, we analyzed whether this increased production of IL-10 contributed to the abnormal production of immunoglobulins (Ig) and of autoantibodies in SLE. The role of IL-10 was compared with that of IL-6, another cytokine suspected to play a role in these abnormalities. The spontaneous in vitro production of IgM, IgG, and IgA by peripheral blood mononuclear cells from SLE patients was weakly increased by recombinant IL (rIL)-6, but strongly by rIL-10. This production was not significantly affected by an anti-IL-6 mAb but was decreased by an anti-IL-10 mAb. We then tested the in vivo effect of these antibodies in severe combined immunodeficiency mice injected with PBMC from SLE patients. The anti-IL-6 mAb did not significantly affect the serum concentration of total human IgG and of anti-double-stranded DNA IgG in the mice. In contrast, the anti-IL-10 mAb strongly inhibited the production of autoantibodies, and, to a lesser extent, that of total human IgG. These results indicate that the Ig production by SLE B lymphocytes is largely IL-10 dependent, and that the increased production of IL-10 by SLE B lymphocytes and monocytes may represent a critical mechanism in the emergence of the autoimmune manifestations of the disease.

CD28 is a 44-kDa glycoprotein that contributes to T cell activation and proliferation. To elucidate the functional role of CD28 in T cell proliferation and IL-2 production in SLE, we studied its effects in cells from untreated patients with active (n = 10) or inactive disease (n = 10) as compared with normal subjects. Mean percentages of CD4+ CD28+ and CD8+ CD28+ T cells were decreased in SLE patients (P < 0.01). SLE patients had significantly decreased absolute CD8+ CD28+ T cells. To investigate whether CD28 antibody affects T cell proliferation, we stimulated peripheral blood T cells from SLE patients and normal controls with anti-CD28, anti-CD3 and/or Interleukin-2 (IL-2) during 3 days of culture. We found that T cells from SLE patients had significantly higher responses to CD28 than did cells from normal controls. This effect was higher in cells from patients with active disease than in those with inactive disease. Conversely, IL-2 had no significant effect on the proliferative response of SLE T cells. However, when it was used for co-stimulating with anti-CD28, there was an increase in the secretion of IL-2 which was greater in the cells of patients with active disease. Thus, on average, there was an 81% increase in the production of IL-2 in T cells from patients with active SLE, 48% in those from patients with inactive disease and 40% in T cells from healthy controls, as compared with the production in response to stimulus by anti-CD3 or with anti-CD3 and anti-CD28. Lymphocytes from patients with active disease showed increased gene expression of CD28 when compared with normal subjects. These data suggest that CD28 might play a central role in the defective immune response observed in SLE patients.

Objective. Interleukin-10 (IL-10) is a potent stimulator of B lymphocytes in vitro. In vivo dysregulation of IL-10 gene expression was therefore analyzed in patients with rheumatoid arthritis (RA), primary Sjogren's syndrome (SS), and systemic lupus erythematosus (SLE). Methods. Spontaneous production of IL-10 by peripheral blood mononuclear cells was measured using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay in untreated patients with either RA (n = 10), SS (n = 10), or SLE (n = 10), and in 15 normal control subjects. Results. IL-10 production was dramatically higher in RA, SS, and SLE patients than in controls. In each group, both B lymphocytes and monocytes, but not T lymphocytes, produced IL-10. Conclusion. IL-10 production is increased in RA, SS, and SLE. It may play a role in B lymphocyte hyperactivity and in the development of autoimmunity.

Sir, The ACR classifies neuropsychiatric (NP) manifestations into 19 syndromes [1]. From a classification perspective, the current approach of attributing NP manifestations in SLE patients may be appropriate; however, from a pathogenic standpoint, any misclassification will affect the advance in the knowledge of the pathogenic mechanisms, their diagnosis and treatment. Therefore, it is important to know whether the inflammatory profile in NP manifestations due to lupus, with and without associated factors, is similar. We studied 35 patients (ACR criteria) [2] with central NP manifestations (cNPSLE) with and without associated factors. They were evaluated at hospitalization and 6 months later, with a cerebrospinal fluid (CSF) sample at both times in which IgG ANA, anti-dsDNA, anti-ribosomal-P, aCL, anti-β2 glycoprotein-I and anti-N-methyl-d-aspartate receptor antibodies, as well as cytokines IL-2, -4, -6, -10, TNF-α, IFN-γ and -α and chemokines MCP-1, regulated on activation normal T cell expressed and secreted, IL-8, monokine induced by interferon gamma and gamma interferon inducible protein (IP-10) were tested. The study was approved by the Institutional Committee of Biomedical Research, and all patients provided informed consent. Of the 35 patients with cNPSLE (18 with and 17 without associated factors), the mean age was 30.6 ± 11.8 years and 86% were females. The NP manifestations present were 14 seizures, 8 acute confusional states, 8 cephalalgia, 3 cerebrovascular events, 1 psychosis and 1 pseudo-tumour cerebri. At hospitalization, patients with pure NP manifestations had greater disease activity (SLEDAI-2K) [3] (18.1 ± 8.0 vs 11.7 ± 9.7; P = 0.04). Six months later, disease activity decreased in both groups, but it was only in the group with pure cNPSLE that was statistically significant (14.8 ± 1.9; P < 0.001). The prevalence of all the antibodies studied at the time of hospitalization was similar in patients with pure NP manifestations and in those with associated factors (Table 1). Among the patients with paired CSF samples, no significant difference in the prevalence of antibodies at hospitalization and 6 months later was observed. In those patients who were found to be positive for each antibody at hospitalization, a non-significant decreasing trend in the levels of all the autoantibodies was observed in both groups. Table 1. Prevalence of antibodies and levels of cytokines and chemokines in CSF at hospitalization The levels of the studied cytokines and chemokines were similar in both groups, except for IP-10, which showed levels significantly higher in the group with pure NP manifestations (Table 1). In both groups, the level of all cytokines and chemokines decreased after 6 months. However, in patients with pure NP manifestations, a statistically significant decrease was observed only in IL-6 and IP-10 (85.4 ± 116.5 vs 2.9 ± 2.4 pg/ml; P = 0.02; and 2673.9 ± 2330.4 vs 723.3 ± 588.09 pg/ml; P = 0.01, respectively); whereas in those patients with NP manifestations with associated factors, only IP-10 decreased significantly after 6 months (1258.3 ± 1492. 2 vs 651.9 ± 682.2 pg/ml; P = 0.04). At present, no specific test exists that might define whether a given NP manifestation is due specifically to lupus activity or any other concomitant factor. Several inflammatory molecules have been found associated with NP manifestations in SLE [4–7]. Patients with pure NP manifestations seemed to have more intense inflammation as reflected by a significantly higher level of disease activity and of IP-10. These results may suggest that in contrast to patients with pure NP manifestations, the presence of associated factors may trigger the onset of NP manifestations at lower levels of inflammation, but the inflammatory profile in both lupus patients is similar that may be the result of a breach of the blood–brain barrier shared by both groups. The significant decrease observed in lupus activity 6 months after the outbreak of NP manifestations in patients with pure cNPSLE, but not with cNPSLE with associated factors, is consistent with this hypothesis [7]. The high levels of IP-10 seem to be indicative of disease activity in the CNS. Even though in NPSLE patients with associated factors, the levels of IP-10 were significantly lower than in patients with pure NP manifestations, the levels were still higher than that found in non-NPSLE patients. Therefore, IP-10 may be considered as a preponderant chemokine in the development of NPSLE. Our study has the following limitations: (i) the number of patients is not large enough to derive a definitive conclusion about the differences between central neuropsychiatric (cNP) manifestations pure and with associated factors; (ii) we studied the inflammatory profile of cNP manifestations in general, not specifically, hence we cannot reach any conclusion for any particular NP manifestation; (iii) the attribution of NP manifestations to SLE is complex, thus, some misclassification should be present; and (iv) we included patients with acute and severe cNP manifestation, therefore, our results could not be applied for mild or chronic manifestations. The results allow us to conclude that the inflammatory profile in the CSF of SLE patients with cNP manifestations with and without associated factors is similar; thus, the current rules for the attribution of cNPSLE manifestations seem to be valid, not only for classification purposes, but also for the study of their pathogenic mechanisms, their diagnosis and their treatment. Disclosure statement: The authors have declared no conflicts of interest.

In the present study, we report the clinical characteristics of a unique systemic lupus erythematosus (SLE) multiplex family with 6 of its members affected by the disease, 1 of them being male. Four patients showed nephropathy, 2 of them with late-onset SLE (52 and 55-year-old), one with cutaneous and articular involvement, and another one developing lupus after 5 years undergoing highly active antiretroviral therapy (HAART) due to acquired immunodeficiency syndrome. Notwithstanding the genetic load, the fact that 2 patients showed late-onset disease, and the extreme delay of the appearance of SLE after HAART in the proband suggest that not only genetic, but other--mainly environmental--factors are necessarily required for the development of SLE.

Background There is strong evidence that an individual's genetic background is an important predisposing factor to longevity. In the present study we analysed the frequency of HLA class I, class II, as well as the TNF-α -308 polymorphism that may be related to an increased life span in Mexican Mestizo healthy elders. Results HLA typing was performed by polymerase chain reaction sequence specific oligonucleotide (PCR SSO) reverse dot blot. The TNF-α -308 polymorphism was assessed by PCR restriction fragment length polymorphism. A significant increased frequency of HLA-DRB1*11 was found in elderly women whereas this allele was not present in elderly males. The TNF2 allele was also increased in the elder group when compared to young controls. The frequencies of the remaining alleles tested were not statistically different among groups. Conclusion These data suggest an ethnicity independent tendency of HLA-DRB1*11 in elder females to increase life span and a possible role of the TNF2 allele with the successful remodelling of senescent immune system.

Multidrug resistance (MDR) mechanisms have been widely studied in cancer. Among them, P-glycoprotein (P-gp) overfunction has been associated with resistance to several antineoplastic agents. The physiological role of P-gp involves hormone and metabolite secretion, bacterial product detoxification, and transport of several drugs to the extracellular space, thus inhibiting their toxic or therapeutic effects. The study of MDR-1 in diseases of autoimmune origin has just recently emerged. Corticosteroids remain the mainstay therapy for autoimmune diseases. As prednisone (PDN) is transported by P-gp, the aim of this study was to evaluate the P-gp function in lymphocytes from myasthenia gravis (MG) patients. Thirty MG patients and 25 healthy controls were studied. Peripheral blood mononuclear cells were isolated by gradient centrifugation and incubated with daunorubicin (DNR) (a fluorescent drug extruded by P-gp). Functional activity of P-gp was analyzed by flow cytometry. Results were expressed as percentage of gated lymphocytes able to efflux DNR. Overall, MG patients showed increased numbers of lymphocytes with functional P-gp activity when compared with controls (x = 4.92 +/- 5.26% vs. x = 0.7 +/- 0.48%, respectively) (P < 0.0001). When patients were classified as responders (n = 21) or refractory (n = 9) to treatment, the latter group exhibited higher values of functional P-gp (x = 10.18 +/- 6.39%) when compared to the responder group (x = 2.66 +/- 2.45%) (P = 0.0076). These data suggest, on the one hand, that drug resistance may be induced by long-term treatment or by high PDN doses and, on the other, emphasize the need for the study of P-gp antagonists in order to improve the current therapeutical schemes for the treatment of MG.

It is well known that infections in patients with diabetes mellitus are more severe, although there is controversy for increased susceptibility to them. Non-specific immune response mechanisms could be related to defense and/or susceptibility to pathogens. The aim of this study was to investigate the activity of several enzymes involved in the primary host defense mechanisms in non-insulin dependent diabetes mellitus (NIDDM). Twenty NIDDM females with a mean HbA(1c) level of 8.19% were included. No patient had clinical evidence of infection. As controls 20 healthy females were studied. The enzymes tested were dipeptidyl-peptidase I (DPP-I), cathepsin B and D, NADPH oxidase and superoxide dismutase (oxidative burst) and collagenase. Isolated leukocytes were incubated with the specific substrates in pyrogen free conditions. The intracellular enzyme activity was analyzed by flow cytometry. Collagenase enzymatic activity was similar in the three leukocyte subpopulations studied. Oxidative burst induction in monocytes was comparable between both groups. Enzyme activity of cathepsin B and D in all cell subsets, oxidative burst in PMN cells, and DPP-I in lymphocytes and monocytes from patients, was higher than those from healthy females (P

Objective To evaluate the safety and clinical efficacy of administering an anti–interleukin-10 (anti–IL-10) monoclonal antibody (mAb) to systemic lupus erythematosus (SLE) patients with active and steroid-dependent disease. In addition, we sought to assess the effects of in vivo IL-10 neutralization on biologic markers of SLE. Methods Treatment consisted of 20 mg/day intravenous administration of an anti–IL-10 murine mAb (B-N10) for 21 consecutive days, with a followup period of 6 months. Six patients were studied. Results Treatment was safe and well tolerated. All patients developed antibodies against B-N10. Cutaneous lesions and joint symptoms improved in all patients beginning during B-N10 administration and continuing to month 6. The SLE Disease Activity Index decreased from a mean ± SEM of 8.83 ± 0.91 on day 1 to 3.67 ± 0.67 on day 21 (P = 0.001), 1.50 ± 0.84 at month 2, and 1.33 ± 0.80 at month 6 (P < 0.001). At the end of followup, the disease was clinically inactive in 5 of the 6 patients. Prednisone administration was decreased from a mean ± SEM of 27.9 ± 5.7 mg/day on day 1 to 9.6 ± 2.0 mg/day at month 6 (P < 0.005). Activity of immune and endothelial cells rapidly decreased, as assessed by the early evolution of several biologic markers. Conclusion This is the first report of IL-10 antagonist administration to humans. The study shows the involvement of IL-10 in the pathogenesis of SLE, and indicates that the use of IL-10 antagonists may be beneficial in the management of refractory SLE.

A 39-year-old woman developed systemic lupus erythematosus with nephropathy after a holiday in Jamaica. She was prescribed with prednisone, azathioprine and aspirin. As she was obsessed with aesthetic procedures, she decided not to take the prescription. Instead, she took her bodybuilding trainer's advice of one intramuscular injection of stanozolol for 10 weeks in order to increase her gluteus area. One week after finishing the latter regimen, there was no disease activity. Whether lupus remission in this patient was spontaneous or a consequence of stanozolol administration will remain a riddle for this fortunate outcome.

Resume Une femme âgee de 39 ans a developpe une nephropathie dans le cadre d'un lupus erythemateux systemique apres un voyage en Jamaique. Un traitement par prednisone, azathioprine et aspirine fut prescrit. Comme cette patiente avait des preoccupations esthetiques, elle decida de ne pas prendre le traitement. A la place, elle prit les conseils de son entraineur de bodybuilding qui lui suggera l'administration intramusculaire de stanozolol pendant dix semaines pour augmenter son volume fessier. Une semaine apres avoir administre la derniere injection, il n'existait aucune activite de la maladie. Le fait que le lupus soit en remission spontanee ou grâce aux injections de stanozolol restera un mystere dans ce cas clinique dont l'issue fut favorable.

Lymphocytes from patients with systemic lupus erythematosus (SLE) exhibit abnormalities in early cell activation events as well as increased production of IL-10. We explored the possible role of IL-10 on defective cell activation events of SLE lymphocytes and first studied the in vitro effect of IL-10 on peripheral blood mononuclear cells (PBMNC) from healthy subjects. After 5 days of culture in the presence of exogenous IL-10, these cells demonstrated abnormal expression of CD69 as well as high intracellular pH and defective activation of the Na+/H+ anti-porter by PMA. We then investigated the effect of IL-10 blockade on PBMNC from SLE patients. SLE PBMNC cultured for 5 days with a blocking anti-IL-10 monoclonal antibody (mAb) partially corrected abnormalities in CD69 expression and intracellular pH; however, in 1/5 patients studied, no significant positive effect was observed. The effect of the anti-IL-10 mAb was apparently not related to protection against activation-induced cell death. We conclude that IL-10 in normal PBMNC induce some of the defects in early cell activation events seen in SLE lymphocytes. Accordingly, the blockade of IL-10 partially corrects these abnormalities in SLE cells. Our data further support the importance of IL-10 in immune dysfunction seen in SLE.

Background/Aims: In order to explore the role of cytokines in the pathogenesis of liver cirrhosis, we analyzed their gene expression in hepatic biopsies from patients with alcoholic liver cirrhosis, post-hepatitis C liver cirrhosis, and with idiopathic portal hypertension without cirrhosis. Methods: We assessed the gene expression of interleukins 1β, 2, 6, 8, and 10, as well as of tumor necrosis factor-α, transforming growth factor-β and interferon-γ by a quantitative polymerase chain reaction. Results: We found high levels of transforming growth factor-β in post-hepatitis C liver cirrhosis, high to moderate in alcoholic liver cirrhosis and low in non-cirrhotic specimens. Expression of interleukin-10, tumor necrosis factor-α, and interferon-γ genes was detected in most post-hepatitis C liver cirrhosis, but not in idiopathic portal hypertension or alcoholic liver cirrhosis biopsies. The interleukin1-β, 6 and 8 gene expression was significantly lower in alcoholic liver cirrhosis compared to post-hepatitis C liver cirrhosis, but higher compared to idiopathic portal hypertension specimens. Thus, post-hepatitis C liver cirrhosis samples showed a high degree of cytokine gene expression, whereas in alcoholic liver cirrhosis it tended to be moderate, and restricted to some cytokines (transforming growth factor-β, interleukin-1, 6 and 8, but not interleukin-10, tumor necrosis factor-α or interferon-γ). In contrast, most non-cirrhotic specimens showed a restricted and low cytokine gene expression. Conclusions: These data suggest that transforming growth factor-β may have an important role in liver fibrosis and inflammation. Interleukin-1β, 6, 8, tumor necrosis factor-α and interferon-γ, appear to participate in the pathogenesis of the mild to severe inflammatory phenomena seen in alcoholic and post-hepatitis C liver cirrhosis, respectively. Our data suggest that tumor necrosis factor-α does not participate in the hepatocellular damage of alcoholic liver cirrhosis, and indicate that neither interferon-γ nor interleukin-10, at least at the levels observed in post-hepatitis C liver cirrhosis, are able to counteract the fibrotic/inflammatory process seen in this condition.

The present study characterizes papillary thyroid carcinoma (PTC) in a Mexican patient sample and evaluates potential prognostic factors for recurrence. Clinical records of 229 patients with PTC were analyzed. Surgical specimens were rereviewed and DNA ploidy determined. Cox logistic regression was used to explore prognostic factors. Mean age +/- SD of the patients was 42 +/- 16 years, with a male/female ratio of 24:205. A thyroid mass was the initial manifestation in 99%. Extrathyroid invasion occurred in 45% and nodal metastases in 38%. Mean size +/- SD of the tumors was 3 +/- 2 cm. By flow cytometry 88% of the tumors were DNA euploid and 12% aneuploid. Complete tumor resection was achieved in 83% with an operative mortality of 0.4%. Postoperative hormone suppression was administered in 65% and remnant 131I thyroid ablation in 84%. The 10-year recurrence-free survival was 85%. In the group of patients with tumors totally removed and without distant metastases, none of the 14 evaluated variables demonstrated statistical significance as an independent prognostic factor for recurrence. However, the group of patients in whom a combination of the following factors was present--age > or = 40 years, tumor size > or = 3 cm, local invasion, and lymph node metastases--showed a higher incidence of tumor recurrence.

Patients with antiphospholipid syndrome, whether primary or secondary to systemic lupus erythematosus, may have thrombocytopenia. Their antibodies to anionic phospholipids might bind to phospholipids on the platelet wall but anionic phospholipids are asymmetrically located in the inner leaflet. In addition, antibodies to anionic phospholipids may require beta 2 glycoprotein I (beta 2GPI) as a cofactor in order to bind to phospholipids. In turn, beta 2GPI has high affinity for anionic phospholipids. Loss of this asymmetry occurs upon platelet activation and could thus permit such antibody-beta 2GPI-platelet interaction. We studied this by flow cytometry using purified beta 2GPI-FITC labelled and similarly labelled affinity-purified polyclonal antibodies to cardiolipin or phosphatidylserine (aPL) obtained from sera of patients with primary antiphospholipid syndrome. Five percent of resting platelets were bound by aPL in the presence of beta 2GPI. Such binding increased when we activated platelets with various agonists, reaching 31% with the concurrent use of thrombin and the calcium ionophore A23187. Platelet activation resulted in the expression of GMP140 but this did not correlate with aPL binding. This probably reflects that the expression of GMP140, which depends on their secretion of alpha granules, has different agonist responses and occurs at different times than do microvesicle formation and expression of prothrombinase activity which coincide with the loss of phospholipid asymmetry on the platelet wall. When we studied the binding of purified beta 2GPI we also found that it binds preferentially to activated platelets and that it seems to be a prerequisite for the binding of aPL onto them. Our findings indicate that aPL from patients with antiphospholipid syndrome may bind to activated platelets through beta 2GPI.

The planimetric, flow cytometric, and immunohistochemical characteristics of the macrofollicular variant of papillary thyroid carcinoma (MFVPTC) have not been reported before. The clinical, morphological, immunohistochemical, planimetric, and flow cytometric characteristics of six cases of the MFVPTC and six of the follicular variant of papillary thyroid carcinoma (FVPTC) were analyzed. Patients had undergone surgical treatment. The mean age was 38 (range 29–64 yr), and five were women. Tumors had a mean size of 3.2 cm (range 0.3–4.5 cm). Half were originally diagnosed as goiter. Macrofollicles had a mean diameter of 345.5 μm, perimeter of 1237 μm, and area of 13,779 μm2, with nuclear changes of PTC. Mean follow-up was 107 mo (range 12–277), and neither lymph node metastases nor recurrence were seen. Differences in diameter, perimeter, and area between the macrofollicular and follicular variants were found. Follicular neoplastic cells were thyroglobulin and S-100 protein positive in macrofollicles and normofollicles. All were negative to cytokeratin and to high-mol-wt keratin. All tumors were diploid. There were no significant differences in follow-up, DNA content, nor immunohistochemical reactivity. Differences in diameter (p

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson’s disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients’ genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type., AS-D was partially supported by a pre-doctoral fellowship from MEC. Additional support was provided by grants from MICINN (BFU2009- 13277, PLE2009-0144 and ACI2010-1117 to AR; RyC-2008- 02772 and BFU2010-21823 to AC; SAF2008-04360, to JA; SAF2009-07774 and PLE2009-0089, to JMC), FIS (PI10/00849 to MV; RD06/0010/0006 to JMC), NIH/NIA AG031782/ AG038072 (to AMC) and Fondazione Guido Berlucchi (to AC). The Cell Therapy Program was supported by the CMRB (Promt-0901 to JMC). The work was also part of a CIBERNED Cooperative Project (to JA, JL-B, MV, ET and AR).