IL-6-mediated endothelial injury impairs antiviral humoral immunity after bone marrow transplantation.

Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.

ASC Modulates CTL Cytotoxicity and Transplant Outcome Independent of the Inflammasome.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1ß and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.

Strain-specific antibody therapy prevents cytomegalovirus reactivation after transplantation.

Cytomegalovirus infection is a frequent and life-threatening complication that significantly limits positive transplantation outcomes. We developed preclinical mouse models of cytomegalovirus reactivation after transplantation and found that humoral immunity is essential for preventing viral recrudescence. Preexisting antiviral antibodies decreased after transplant in the presence of graft-versus-host disease and were not replaced, owing to poor reconstitution of donor B cells and elimination of recipient plasma cells. Viral reactivation was prevented by the transfer of immune serum, without a need to identify and target specific antigenic determinants. Notably, serotherapy afforded complete protection, provided that the serum was matched to the infecting viral strain. Thus, we define the mechanisms for cytomegalovirus reactivation after transplantation and identify a readily translatable strategy of exceptional potency, which avoids the constraints of cellular therapies.

Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.

INTRODUCTION: Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). METHODS: The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. RESULTS: The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. CONCLUSIONS: We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.

Human umbilical cord tissue-derived mesenchymal stromal cells attenuate remodeling after myocardial infarction by proangiogenic, antiapoptotic, and endogenous cell-activation mechanisms.

INTRODUCTION: Among the plethora of cells under investigation to restore a functional myocardium, mesenchymal stromal cells (MSCs) have been granted considerable interest. However, whereas the beneficial effects of bone marrow MSCs (BM-MSCs) in the context of the diseased heart are widely reported, data are still scarce on MSCs from the umbilical cord matrix (UCM-MSCs). Herein we report on the effect of UCM-MSC transplantation to the infarcted murine heart, seconded by the dissection of the molecular mechanisms at play. METHODS: Human umbilical cord tissue-derived MSCs (UCX®), obtained by using a proprietary technology developed by ECBio, were delivered via intramyocardial injection to C57BL/6 females subjected to permanent ligation of the left descending coronary artery. Moreover, medium produced by cultured UCX® preconditioned under normoxia (CM) or hypoxia (CMH) was collected for subsequent in vitro assays. RESULTS: Evaluation of the effects upon intramyocardial transplantation shows that UCX® preserved cardiac function and attenuated cardiac remodeling subsequent to myocardial infarction (MI). UCX® further led to increased capillary density and decreased apoptosis in the injured tissue. In vitro, UCX®-conditioned medium displayed (a) proangiogenic activity by promoting the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), and (b) antiapoptotic activity in HL-1 cardiomyocytes subjected to hypoxia. Moreover, in adult murine cardiac Sca-1+ progenitor cells (CPCs), conditioned medium enhanced mitogenic activity while activating a gene program characteristic of cardiomyogenic differentiation. CONCLUSIONS: UCX® preserve cardiac function after intramyocardial transplantation in a MI murine model. The cardioprotective effects of UCX® were attributed to paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis, augment proliferation, and activate a pool of resident CPCs. Overall, these results suggest that UCX® should be considered an alternative cell source when designing new therapeutic approaches to treat MI.

High-content siRNA screen reveals global ENaC regulators and potential cystic fibrosis therapy targets.

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.

Regulation of ENaC biogenesis by the stress response protein SERP1.

Cystic fibrosis lung disease is caused by reduced Cl(-) secretion along with enhanced Na(+) absorption, leading to reduced airway surface liquid and compromised mucociliary clearance. Therapeutic strategies have been developed to activate cystic fibrosis transmembrane conductance regulator (CFTR) or to overcome enhanced Na(+) absorption by the epithelial Na(+) channel (ENaC). In a split-ubiquitin-based two-hybrid screening, we identified stress-associated ER protein 1 (SERP1)/ribosome-associated membrane protein 4 as a novel interacting partner for the ENaC ß-subunit. SERP1 is induced during cell stress and interacts with the molecular chaperone calnexin, thus controlling early biogenesis of membrane proteins. ENaC activity was measured in the human airway epithelial cell lines H441 and A549 and in voltage clamp experiments with ENaC-overexpressing Xenopus oocytes. We found that expression of SERP1 strongly inhibits amiloride-sensitive Na(+) transport. SERP1 coimmunoprecipitated and colocalized with ßENaC in the endoplasmic reticulum, together with the chaperone calnexin. In contrast to the inhibitory effects on ENaC, SERP1 appears to promote expression of CFTR. Taken together, SERP1 is a novel cochaperone and regulator of ENaC expression.

Otosclerose ? resultados de estapedectomias e estapedotomias realizadas no Hospital Nossa Senhora da Conceição de Tubarão - SC

Introdução: A otosclerose é um processo patológicolocalizado na cápsula ótica do osso temporal que ocorreentre 0,5 a 1,0% da população, mais freqüentementeencontrada no sexo feminino, na faixa etária entre 20 e40 anos e em pessoas da raça branca. A cirurgia doestapédio constitui o tratamento consagrado para aotosclerose.Objetivo: Avaliar os resultados de estapedectomiase estapedotomias realizadas no Hospital Nossa Senhorada Conceição de Tubarão ? SC.Métodos: Realizamos um estudo de coorte históricalongitudinal em 34 pacientes submetidos a tratamentocirúrgico da otosclerose no período de janeiro de 2003 amarço de 2008.Resultados: Dos 34 pacientes incluídos no estudo, 9(26,5%) eram do gênero masculino e 25 (73,5%) dogênero feminino, a idade variou de 31 a 61 anos commédia 44.67 (± 8.95) anos. Em relação à etnia, 31(91,2%) pacientes eram caucasianos e 3 (8,8%) eramnão caucasianos. Houve melhora comprovada pelofechamento do gap aéreo-ósseo para menor ou igual a10 dB em 33 (97,1%) pacientes e apenas 1 (2,9%) nãoteve sucesso.Conclusão: A cirurgia estapediana mostrou-se umaótima opção terapêutica para a otosclerose, apresentandoalta taxa de sucesso, com melhora do gap aéreo-ósseoem quase todos os pacientes.

Introduction: The otosclerosis is a pathologicalprocess located in the otic capsule of temporal bone thatoccurs in 0.5 to 1.0% of the population, most frequentlyfound in females, age between 20 and 40 years old andin white people. The stapedius surgery is a renownedtreatment to otosclerosis.Objective: To evaluate the results of stapedectomiesand stapedotomies performed at Nossa Senhora daConceição Hospital in Tubarão ? SC, South Brazil.Methods: We performed a retrospective cohortstudy in 34 patients who have been undergone surgicaltreatment of otosclerosis from January 2003 to March2008.Results: Third four patients were included in thestudy, 9 (26.5%) were male and 25 (73.5%) female, theage ranged from 31 to 61 years old with an average44.67 ± 8.96 years old. In relation to ethnicity, 31 (91.2%)patients were Caucasian and 3 (8.8%) were noCaucasian. There was proved improvement by theclosing of the air-bone gap to less than or equal to 10 dBin 33 (97.1%) patients and only 1 (2.9%) had no success.Conclusion: The stapes surgery proved to be a goodtherapeutic option to otosclerosis, showing high successrate, with air-bone gap improvement in almost all ofpatients.

Avaliação da perda auditiva induzida por ruído em músicos de Tubarão-SC / Evaluation of noise-induced hearing loss in musicians from Tubarão-SC

Introdução: A perda auditiva induzida por ruído (PAIR) é considerada como uma das doenças ocupacionaismais prevalentes no mundo. Ela atinge profissionais de diversas áreas da sociedade, inclusive os músicos, que acabam sendo submetidos a elevados níveis de pressão sonora em ambientes de apresentação ou mesmo em seus ensaios. Objetivo: É determinar a prevalência de perda auditivainduzida por ruído em músicos que atuam na cidade de Tubarão (SC), assim como, determinar os principais fatores de risco e os sintomas associados a essaperda auditiva. Material e Métodos: Estudo observacional, descritivo, com delineamento transversal com 21 músicos entre 18 a 59 anos de idade, de diversos estilos musicais, que atuam em bandas na cidade de Tubarão (SC). Assim, entre agosto e outubro de 2007, foram realizados contatos com os músicos e aplicado um questionário.Após, os profissionais foram encaminhados ao consultório com horário agendado sendo realizado otoscopia e audiometria. Resultados: A prevalência de PAIR foi de 42,9%.Com um predomínio quase absoluto do gênero masculino, 95,2% profissionais. A média de idade foi de 29 anos,variando de 21 a 47 anos. Os sintomas auditivos mais freqüentes foram o zumbido (52,4%), a plenitude auricular(38,1%), a dificuldade de compreensão da fala (28,6%) e a tontura (23,8%).Conclusões: A prevalência de PAIR foi de 42,9%. A perda auditiva foi predominante em fatores como idadee tempo de exposição a sons de alta pressão sonora. É necessário a implementação de programas de prevenção e conservação auditiva entre os músicos de Tubarão -SC.

Introduction: Noise-induced hearing loss is considered as a common sick in the world. It gets professionals of different areas, including the musicians, who areexposed to high sound pressure levels in shows or during their practice. Aim: To determine the prevalence of cases suggestive of noise-induced hearing loss in musicians from Tubarão (SC), South Brazil and to point out the main riskfactors and the symptoms of hearing loss. Materials and Methods: Observational, descriptive, and transversal study with 21 musicians between 18 and 47 years old, of different music style, that played inTubarão (SC). So, among August and October of 2007 it was done a questionnaire with the musicians. After,they were sent to clinical to do otoscopy and audiometry. Results: The prevalence of hearing loss was of 42,9%. During the study period, we had 21 musicians,being 20 men (95,2%). The mean age was 29 years old, ranging from 21 to 47 years old. The more frequent hearingsymptoms were tinnitus (52,4%), aural fullness (38,1%), difficulty in speech understanding (28,6%), and dizziness (23,8%). Conclusions: The hearing loss noise-induced was 42,9%. Hearing loss was higher in factors like age andexposition time to noise with high sound pressure. It is necessary to implement prevention and conservation hearing programs among the musicians from Tubarão - SC.