TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth.

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) remains a deadly cancer worldwide with a need for new therapeutic approaches. A dysregulation in the equilibrium between pro- and anti-inflammatory responses with a predominant immunosuppressive inflammatory reaction in advanced stage tumors seem to contribute to tumor growth and metastasis. The current therapies do not include strategies against pro-tumorigenic inflammation in cancer patients. We have shown that the upregulated cell surface expression of Toll-like Receptor (TLR) 2 and of TLR9 inside PDAC cells maintain chronic inflammatory responses, support chemotherapeutic resistance, and mediate tumor progression in human pancreatic cancer. We further demonstrated intracellular TLR2 and TLR9 targeting using specific intrabodies, which resulted in downregulated inflammatory signaling. In this study, we tested, for the first time, an intrabody-mediated TLR blockade in human TLR2- and TLR9-expressing pancreatic cancer cells for its effects on inflammatory signaling-mediated tumor growth. Newly designed anti-TLR2- and anti-TLR9-specific intrabodies inhibited PDAC growth. Co-expression analysis of the intrabodies and corresponding human TLRs showed efficient retention and accumulation of both intrabodies within the endoplasmic reticulum (ER), while co-immunoprecipitation studies indicated both intrabodies interacting with their cognate TLR antigen within the pancreatic cancer cells. Cancer cells with attenuated proliferation expressing accumulated TLR2 and TRL9 intrabodies demonstrated reduced STAT3 phosphorylation signaling, while apoptotic markers Caspases 3 and 8 were upregulated. To conclude, our results demonstrate the TLR2 and TLR9-specific intrabody-mediated signaling pathway inhibition of autoregulatory inflammation inside cancer cells and their proliferation, resulting in the suppression of pancreatic tumor cell growth. These findings underscore the potential of specific intrabody-mediated TLR inhibition in the ER relevant for tumor growth inhibition and open up a new therapeutic intervention strategy for the treatment of pancreatic cancer.

HOW DOES CYCLIN D1 BEHAVIOR AS A BIOMARKER IN PAPILLIFEROUS THYROID CARCINOMAS AND MULTINODULAR GOITERS? / COMO A CICLINA D1 SE COMPORTA COMO BIOMARCADOR NOS CARCINOMAS PAPILÍFEROS DE TIREOIDE E BÓCIOS MULTINODULARES?

Introduction: Papillary carcinomas are the most prevalent and least aggressive thyroid carcinomas (PTC). In some cases, the diagnosis is doubtfull and the prognosis is poor. The search for tissue biomarkers that ensure both the diagnosis for indeterminate cases and the prognosis, identifying the most aggressive cases, has been studied in recent decades. Objective: To review the literature in search of cyclin D1 as a marker of papillary thyroid carcinomas and multinodular goiters and evaluate whether its expression correlates with the clinicopathological characteristics of papillary thyroid carcinomas. Methods: Narrative review carried out by collecting information for reading and analysis from online research on virtual platforms. Initially, a search was carried out from MESH descriptors related to the topic, using the following terms: "papillary thyroid carcinoma, cyclin D1, immunohistochemistry, diagnosis, prognosis." with AND or OR search, considering the title and/or abstract and those chosen were read in full. Results: The search included 77 articles that were compiled in this review. Conclusion: Cyclin D1 was expressed in the vast majority of PTCs, with diffuse distribution being predominant. There was no correlation between its expression and any clinicopathological characteristic of PTC.

Racional - Os carcinomas papilíferos são os mais prevalentes e menos agressivos de tireoide (CPT). Em alguns casos, o diagnóstico é duvidoso e o prognóstico ruim. A busca de biomarcadores teciduais que permitam assegurar tanto o diagnóstico para casos indeterminados, quanto o prognóstico, identificando os casos de maior agressividade, têm sido estudadas nas últimas décadas. Objetivo: Revisar na literatura na busca da ciclina D1 como marcador dos carcinomas papilíferos de tireoide e nos bócios multinodulares, e avaliar se a expressão dela apresenta correlação com as características clínico-patológicas dos carcinomas papilíferos de tireoide. Métodos: Revisão narrativa feita colhendo informações para leitura e análise a partir de pesquisa online em platoformas virtuais. Inicialmente foi realizada busca por descritores DECs relacionados ao tema, utilizando os seguintes termos: "carcinoma papilífero de tireoide, ciclina D1, imunoistoquímica, diagnóstico, prognóstico." com busca AND ou OR, considerando o título e/ou resumo e os escolhidos foram lidos na íntegra. Resultados: A busca incluiu 77 artigos que foram compilados nesta revisão.  Conclusão: A ciclina D1 foi expressa na grande maioria dos CPT sendo a distribuição difusa predominante. Não houve correlação entre a expressão dela com qualquer característica clinicopatológica dos CPT.

Platelet-derived growth factor (PDGF) cross-signaling via non-corresponding receptors indicates bypassed signaling in colorectal cancer.

Platelet-derived growth factor (PDGF) signaling, besides other growth factor-mediated signaling pathways like vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), seems to play a crucial role in tumor development and progression. We have recently provided evidence for upregulation of PDGF expression in UICC stage I-IV primary colorectal cancer (CRC) and demonstrated PDGF-mediated induction of PI3K/Akt/mTOR signaling in CRC cell lines. The present study sought to follow up on our previous findings and explore the alternative receptor cross-binding potential of PDGF in CRC. Our analysis of primary human colon tumor samples demonstrated upregulation of the PDGFRß, VEGFR1, and VEGFR2 genes in UICC stage I-III tumors. Immunohistological analysis revealed co-expression of PDGF and its putative cross-binding partners, VEGFR2 and EGFR. We then analyzed several CRC cell lines for PDGFRα, PDGFRß, VEGFR1, and VEGFR2 protein expression and found these receptors to be variably expressed amongst the investigated cell lines. Interestingly, whereas Caco-2 and SW480 cells showed expression of all analyzed receptors, HT29 cells expressed only VEGFR1 and VEGFR2. However, stimulation of HT29 cells with PDGF resulted in upregulation of VEGFR1 and VEGFR2 expression despite the absence of PDGFR expression and mimicked the effect of VEGF stimulation. Moreover, PDGF recovered HT29 cell proliferation under simultaneous treatment with a VEGFR or EGFR inhibitor. Our results provide some of the first evidence for PDGF cross-signaling through alternative receptors in colorectal cancer and support anti-PDGF therapy as a combination strategy alongside VEGF and EGF targeting even in tumors lacking PDGFR expression.

Translational development of ABCB5+ dermal mesenchymal stem cells for therapeutic induction of angiogenesis in non-healing diabetic foot ulcers.

BACKGROUND: While rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5+ mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers. METHODS: The angiogenic potential of ABCB5+ MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5+ MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial. RESULTS: Hypoxic incubation of ABCB5+ MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5+ MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5+ MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5+ MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5+ MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed. CONCLUSIONS: The present observations identify GMP-manufactured ABCB5+ dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784.

EVALUATION OF THE OMENTUM AS A STEM CELL DONOR ISCHEMIC MYOCARDIAL THROUGH CD34 IMMUNOHISTOCHEMICAL ANALYSIS / AVALIAÇÃO DO OMENTO COMO DOADOR DE CÉLULAS TRONCO NO MIOCÁRDIO ISQUÊMICO ATRAVÉS DA ANÁLISE IMUNOHISTOQUÍMICA DO CD34

Background: Cardiovascular diseases are the leading cause of death in the world. The use of hematopoietic precursor cells and recent advances made in heart graft bioengineering offer a new therapeutic modality for post-myocardial infarction (MI)  and cardiac tissue regeneration. CD34 is a marker expressed on all hematopoietic and endothelial precursor cells, and functions as a cell adhesion factor. The antibody corresponding to this marker is used in immunohistochemistry to assess the formation of new vessels and the presence of stem cells. Aim: To evaluate the efficacy of omentopexy as stem cell donor, on previously infarcted myocardium, using immunohistochemically analysis of CD34. Method: Myocardial infarction was generated in four pigs, by ligature of the 1st and 2nd marginal branches of the circumflex artery. In three animals, abrasion of the infarcted epicardium was performed followed by multiple myocardial perforations and the mobilization of the omentum from the abdominal cavity to the mediastinum, sutured on the infarcted area. In the fourth animal, omentopexy was not performed and only the abrasion and perforation of the infarcted area were performed. All hearts were removed for CD34 immunohistochemically evaluation. Results:  In the samples from the group submitted to omentopexy, there was a 60% increase in angiogenesis, and in the samples from the control animal there was minimal staining.  Four samples from different sites of each animal, totaling 16 histopathological samples were evaluated. All samples were immunolabelled for CD34. Conclusions: Omentopexy proved to be effective in seeding previously infarcted myocardium with stem angiogenic cells, seen through immunohistochemistry, using CD34 marker.

Racional: As doenças cardiovasculares são a principal causa de morte no mundo. O uso de células precursoras hematopoiéticas e os recentes progressos feitos na bioengenharia de enxertos cardíacos oferecem uma nova modalidade terapêutica para a regeneração do tecido cardíaco pós-infarto do miocárdio (IM). O CD34 é um marcador expresso em todas as células precursoras hematopoiéticas e endoteliais, e funciona como fator de adesão celular. O anticorpo que correspondente a este marcador é utilizado na imunohistoquímica para avaliar a formação de novos vasos e a presença de células-tronco. Objetivo: O estudo teve por objetivo avaliar a eficácia da omentopexia na neovascularização e na doação de células tronco de corações suínos previamente infartados, a partir da análise imunohistoquímica do CD34. Método: O infarto do miocárdio foi gerado em 4 suínos, por ligadura do 1°e 2° ramos marginais da artéria circunflexa. Em 3 animais realizou-se abrasão cuidadosa do epicárdio infartado seguido de múltiplas perfurações miocárdicas e a mobilização do omento da cavidade abdominal para o mediastino, envolvendo a área infartada e as perfurações. No quarto animal não foi realizado a omentopexia sendo realizado apenas a abrasão e perfuração da área infartada. Todos os animais foram eutanasiados ao 30º dia pós operatório e os corações retirados para avaliação macroscópica, microscópica e Imunohistoquímica do CD34. Resultados: Nas amostras do grupo submetido a omentopexia, ocorreu um aumento de 60% da angiogênese, sendo que nas amostras do animal controle houve marcação mínima. Foram avaliadas quatro amostras de diferentes sítios de cada coração dos animais, totalizando 16 amostras histopatológicas. Todas as amostras foram imunomarcadas para CD-34. Conclusões: O omento mostrou-se eficiente na indução de neovascularização pela presença de células tronco, vista através da marcação do CD34, demonstrando grande potencial como futura terapêutica para restaurar áreas de miocárdio isquêmico.

INVASIVE DUCTAL BREAST CARCINOMA: CORRELATION OF AXL AND Β-CATENIN IMMUNOHISTOCHEMICAL EXPRESSION WITH TUMOR AGGRESSIVITY / CARCINOMA MAMÁRIO DUCTAL INVASOR: CORRELAÇÃO DA EXPRESSÃO IMUNOISTOQUÍMICA DE AXL E Β-CATENINA COM A AGRESSIVIDADE TUMORAL

Introduction: Invasive ductal carcinoma corresponds to the most common histological type of the breast, coexisting with different forms of clinical evolution, histological grading, expression of certain tissue markers and genomic profiles that seek a better understanding of the disease. Objectives: To analyze the correlation of ß-catenin and AXL markers with tumor aggressiveness, with reference to overall survival, tumor progression and histopathological prognostic factors. Methods: A study of 101 samples of invasive ductal mammary carcinoma was performed. Those with a diagnosis of ductal type, initially submitted to biopsy or definitive surgical treatment, were included. For control purposes, 20 samples of intraductal carcinoma, 35 of breast fibroadenoma and 10 of breast tissue without any alteration were included. Those undergoing neoadjuvant chemotherapy, those without a tumor sample prior to chemotherapy, those lost to follow-up, and those with incomplete data, were excluded. Results: When the ß-catenin expression was analyzed, it was negative. As for AXL, different degrees of expression were observed without statistical significance between them. Conclusion: When analyzing invasive ductal breast adenocarcinoma in TMA, there was no correlation in the expression of ß-Catenin and AXL when compared to overall survival, tumor progression and histological grade.

Introdução: O carcinoma ductal invasor corresponde ao tipo histológico mais comum da mama coexistindo com formas diferentes de evolução clínica, graduação histológica, expressão de determinados marcadores teciduais e perfis genômicos que procuram melhor entendimento da doença. Objetivos: Analisar a correlação dos marcadores ß-catenina e AXL com a agressividade tumoral, tendo como referência a sobrevida global, progressão tumoral e fatores prognósticos histopatológicos. Métodos: Foi realizado estudo de 101 amostras de carcinoma mamário ductal invasor. Foram incluídas aquelas com diagnóstico do tipo ductal, submetidas inicialmente à biópsia ou tratamento cirúrgico definitivo. Incluiu-se para fins de controle 20 amostras de carcinoma intraductal, 35 de fibroadenoma mamário e 10 de tecido mamário sem qualquer alteração. Foram excluídos os submetidos à quimioterapia neoadjuvante, que não tivessem amostra tumoral prévia ao tratamento quimioterápico, que perderam o seguimento, e com dados incompletos. Resultados: Quando analisada a expressão da ß-catenina, foi negativa. Quanto ao AXL foram observados diferentes graus de expressão sem significância estatística entre eles. Conclusão: Quando analisados adenocarcinoma mamário do tipo ductal invasor em TMA não houve correlação na expressão de ß-catenina e AXL quando comparados a sobrevida global, progressão tumoral e grau histológico.

IMMUNOHISTOCHEMICAL ANALYSIS OF THE EXPRESSION OF ALCAM AND ALDH1 MARKERS IN PATIENTS WITH COLORRETAL ADENOCARCINOMA AND ASSOCIATION WITH CLINICOPATHOLOGICAL OUTCOMES / ANÁLISE IMUNOISTOQUÍMICA DA EXPRESSÃO DOS MARCADORES ALCAM E ALDH1 EM PACIENTES COM ADENOCARCINOMA COLORRETAL E ASSOCIAÇÃO COM DESFECHOS CLINICOPATOLÓGICOS

Background: Colorectal cancer has a high global mortality and tumor markers have emerged as diagnostic, management and prognostic indicators. New markers are being studied. Objective: To verify if there is a correlation between the immunohistochemical expression of ALCAM and ALDH1 proteins in colorectal adenocarcinoma tissue with epidemiological and clinicopathological characteristics, in particular their impact on disease progression and death. Method: Observational, single-center, analytical, retrospective study, through the investigation of patients undergoing surgical resection for colorectal cancer. 122 patients were evaluated. Regarding progression, it was shown that in individuals with positive ALCAM (n=40), 14/40 (35%) had progression, and for positive ALDH (n=54), 22/54 (40.7%). For death, the analysis of ALCAM positive (n=40), 24/40 (60%) died, and ALDH1 positive (n=54), 33/54 (61.1%). Conclusion: The immunohistochemical expression of ALCAM and ALDH1 markers was not associated with disease progression and death; it was also not possible to observe a correspondence relationship with the evaluated markers.

Racional: O câncer colorretal apresenta alta mortalidade global e marcadores tumorais têm surgido como sinalizadores de diagnóstico, manejo e prognóstico. Novos marcadores estão sendo estudados. Objetivo: Verificar se há correlação da expressão por imunoistoquímica das proteínas ALCAM e ALDH1 em tecido com adenocarcinoma colorretal com as características epidemiológicas e clinicopatológicas, em particular o seu impacto na progressão de doença e no óbito. Método: Estudo observacional, unicêntrico, analítico, retrospectivo, através da investigação de pacientes submetidos à ressecção cirúrgica por câncer colorretal. Foram avaliados 122 pacientes. Em relação a progressão, mostrou-se que nos indivíduos com ALCAM positiva (n=40), 14/40 (35%) tiveram progressão, e para ALDH positiva (n=54), 22/54 (40,7%). Para óbito, a análise da ALCAM positiva (n=40), 24/40 (60%) morreram, e ALDH1 positivo (n=54), 33/54 (61,1%). Conclusão: A expressão imunoistoquímica dos marcadores ALCAM e ALDH1 não apresentou associação com a progressão de doença e óbito; também não foi possível observar relação de correspondência com os marcadores avaliados.

IMMUNOHISTOCHEMICAL ANALYSIS OF THE CYCLIN D1 BIOMARKER IN PAPILLIFEROUS THYROID CARCINOMAS AND MULTINODULAR GOITERS / ANÁLISE IMUNOISTOQUÍMICA DO BIOMARCADOR CICLINA D1 NOS CARCINOMAS PAPILÍFEROS DE TIREOIDE E BÓCIOS MULTINODULARES

Background: Papillary thyroid carcinomas (PTC) are the most prevalent and least aggressive thyroid carcinomas. In some cases, the diagnosis is doubtfull and the prognosis is poor. The search for tissue biomarkers that make it possible to ensure both the diagnosis for indeterminate cases and the prognosis, identifying the most aggressive cases, has been studied in recent decades. Objective: To analyze the molecular marker cyclin D1 in PTC and multinodular goiter (BMN) and to verify the correlation of the marking with the clinical-pathological characteristics in carcinomas. Methods: 118 tissues from adult patients submitted to PTC thyroidectomy and 40 BMN were selected as a control group. Tissue immunostaining was performed with cyclin D1 with subsequent immunohistochemical analysis in both groups, evaluating the expression of the marker (intensity and distribution). In the PTC group, immunostaining data were also crossed with clinical and pathological data. Results: The majority (93.3%) expressed the staining of cyclin D1 with varying intensities (weak, moderate 3 and strong) and predominantly diffuse distribution (71.2%). The BMN control group expressed staining for cyclin D1 in 57.5%, with weak intensity (47.5%) and sparse distribution (37.5%). The difference between the groups (study and control) was statistically significant (p<0.001). In the CPT group, the clinical-pathological crossings did not show differences regarding age, sex, tumor type and size, lymph node status, focus, angiolymphatic invasion. Conclusion: Cyclin D1 was expressed in the vast majority of PTC with the predominant diffuse distribution. There was no correlation between the expression of cyclin D1 and any clinical-pathological characteristic of PTC.

Racional - Os carcinomas papilíferos são os mais prevalentes e menos agressivos de tireoide (CPT). Em alguns casos, o diagnóstico é duvidoso e o prognóstico ruim. A busca de biomarcadores teciduais que permitam assegurar tanto o diagnóstico para casos indeterminados, quanto o prognóstico, identificando os casos de maior agressividade, têm sido estudadas nas últimas décadas. Objetivo: Analisar a ciclina D1 nos CPT e nos bócios multinodulares (BMN) e verificar a correlação da marcação com as características clinicopatológicas. Métodos: Foram selecionados 118 tecidos de pacientes adultos submetidos àa tireoidectomia por CPT e 40 BMN como grupo controle. Realizou-se imunocoloração tecidual com ciclina D1 com subsequente análise imunoistoquímica em ambos grupos, avaliando-se a expressão do marcador (intensidade e distribuição). No grupo dos CPT os dados da imunocoloração foram também cruzados com os dados clinicopatológicos. Resultados: A maioria (93,3%) expressou a coloração da ciclina D1 com intensidades variadas (fraca, moderada e forte) e distribuição predominantemente difusa (71,2%). O grupo controle dos BMN, expressou coloração para ciclina D1 em 57,5%, com intensidade fraca (47,5%) e distribuição esparsa (37,5%). A diferença entre os grupos (estudo e controle) foi estatisticamente significante (p<0,001). No grupo dos CPT, os cruzamentos clinicopatológicos não evidenciaram diferenças quanto à idade, sexo, tipo e tamanho tumoral, estado linfonodal, focalidade e invasão angiolinfática. Conclusão: A ciclina D1 foi expressa na grande maioria dos CPT sendo a distribuição difusa predominante. Não houve correlação entre a expressão delacom qualquer característica clinicopatológica dos CPT.

CORRELATION OF IMMUNOHISTOCHEMICAL EXPRESSION OF BETA-CATENIN AND C-MYC WITH AGGRESSIVITY IN GASTRIC TUMORS / CORRELAÇÃO DA EXPRESSÃO IMUNOISTOQUíMICA DE BETA-CATENINA E C-MYC COM A AGRESSIVIDADE EM TUMORES DO ESTÔMAGO

­ Background: Biomarkers are macromolecules present in the body that may be related to neoplastic cells. None of them for diagnosis for secondary prevention were defined for gastric cancer. Objective: To investigate the immunohistochemical expression of beta-catenin and c-MYC proteins in gastric cancer and to correlate them with the aggressiveness of gastric tumors. Method: The sample consisted of histopathological slides, stained by H&E and paraffin blocks, and immunostained. Retrospective clinical data were collected. Clinical and epidemiological information was cross-referenced with the result obtained by immunostaining and its statistical analysis. Results: There was a predominance of men (69.1%), with a mean age of 63.8 years, with a predominance of lesions located in the antrum (54.5%), poorly differentiated (49.1%) with signet ring cells in 30 % of cases. On average, 18 lymph nodes were resected and in 30% no affected lymph nodes were detected. In 42.7% there was already distant metastasis, predominantly liver (61.7%). Stage IV was the classification of 43.6%, not being detected angiolymphatic invasion in 77.3% and perineural in 65.5%. Treatment was surgical and chemotherapy in 87.3%, with R0 resection in 79.1%. c-MYC was negative in 99.1% and beta-catenin was not expressed in 90.9%, being inconclusive in 6 cases. Conclusion: The immunohistochemical expression of these proteins in tissues with gastric cancer was not observed. The analyzed biomarkers, c-MYC and beta-catenin, showed no association with tumor aggressiveness in this cancer

Racional: Biomarcadores são macromoléculas presentes no organismo que podem estar relacionadas com células neoplásicas. Nenhum deles para diagnóstico para prevenção secundária foi definido para o câncer gástrico. Objetivo: Investigar a expressão imunoistoquímica das proteínas beta-catenina e c-MYC no câncer gástrico e correlacioná-las com a agressividade dos tumores do estômago. Método: A amostra consistiu em lâminas histopatológicas, coradas por H&E e blocos de parafina, e realizada imunomarcação. Dados clínicos retrospectivos foram coletados. As informações clinicoepidemiológicas foram cruzadas com o resultado obtido pela imunomarcação e sua análise estatística. Resultado: Houve predomínio de homens (69,1%), com idade média de 63,8 anos, predominando lesão localizada

Adenosinergic Pathway and Linked Suppression: Two Critical Suppressive Mechanisms of Human Donor Antigen Specific Regulatory T Cell Lines Expanded Post Transplant.

Regulatory T cells are an important component of an immune response shaping the overall behavior to potential antigens including alloantigens. Multiple mechanisms have been shown to contribute towards developing and sustaining a immunological regulatory response. One of the described contact dependent suppressive mechanisms regulatory cells have been shown to utilize is through the production of adenosine from extracellular ATP mediated by CD39 and CD73. In this study we demonstrate that the adenosinergic pathway plays a major role in the suppressive/regulatory effects antigen specific regulatory T cell enriched lines (ASTRLs) that have been of expanded ex vivo from stable kidney transplant patients. We have previously shown that these ASTRL cells are capable of suppressing alloimmune responses in vitro and significantly prolonging allograft survival in an animal model of kidney transplantation. For this study nineteen ASTRLs were expanded from 17 kidney transplant patients by repeated stimulation of recipient peripheral blood mononuclear cells with donor specific HLA-DR peptides. All 19 ASTRLs showed upregulation of numerous markers associated with regulatory cells and were able to inhibit donor antigen specific T cell proliferation in a dose dependent fashion. ASTRLs suppressed indirect and direct alloimmune responses compatible with our previous animal study findings. Upregulation of both CD39 and CD73 was observed post expansion and ASTRLs demonstrated extracellular hydrolysis of ATP, indicating functionality of the upregulated proteins. We also showed that inhibition of the adenosinergic pathway using inhibitors of CD39 resulted in abrogation of suppression and increased antigen specific T cell proliferation. This demonstrates that the main mechanism of action of the suppressive activity donor peptide driven ASTRLs generated from kidney transplant patients is the adenosinergic pathway. Furthermore this suggests the possibility that combining infusion of Tregs with other treatments, such as adenosine receptor agonists or increasing CD39 expression in the grafts may further enhance a regulatory response to the allograft and possibly achieve transplantation tolerance.

Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

Signal transduction and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although Stat3 deletion in tubular epithelial cells is known to protect mice from fibrosis, vFoxd1 cells remains unclear. Using Foxd1-mediated Stat3 knockout mice, CRISPR, and inhibitors of STAT3, we investigate its function. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury, whereas it is expanded to interstitial cells in fibrosis in mice and humans. Foxd1-mediated deletion of Stat3 protects mice from folic-acid- and aristolochic-acid-induced kidney fibrosis. Mechanistically, STAT3 upregulates the inflammation and differentiates pericytes into myofibroblasts. STAT3 activation increases migration and profibrotic signaling in genome-edited, pericyte-like cells. Conversely, blocking Stat3 inhibits detachment, migration, and profibrotic signaling. Furthermore, STAT3 binds to the Collagen1a1 promoter in mouse kidneys and cells. Together, our study identifies a previously unknown function of STAT3 that promotes kidney fibrosis and has therapeutic value in fibrosis.

PDE4D targeting enhances anti-tumor effects of sorafenib in clear cell renal cell carcinoma and attenuates MAPK/ERK signaling in a CRAF-dependent manner.

Clear cell renal cell carcinoma (ccRCC) is the most lethal form of kidney cancer and effective treatment regimens are yet to be established. Tyrosine kinase inhibitors (TKI) have widely been used as ccRCC therapeutics, but their efficacy is limited due to accompanying resistance mechanisms. Previous studies have provided substantial evidence for crosstalk between cAMP and the MAPK/ERK signaling pathway. Low levels of intracellular cAMP have been found in several human malignancies and some data suggest that elevation of cAMP expression can be achieved by phosphodiesterase 4 (PDE4) inhibition, resulting in cell growth arrest and/or cell death. The effects of crosstalk between cAMP and the MAPK/ERK pathway on the development progression in ccRCR, however, remain to be fully understood. In this study, we sought to explore the involvement of PDE4 in ccRCC and to assess its potential as a target for therapeutic intervention. We demonstrated that PDE4D is the predominant subtype of PDE4 expressed in healthy and cancerous renal cell lines, particularly in metastatic Caki-1 cells. We generated a CRISPR/Cas9-mediated PDE4D-KO Caki-1 cell model and showed that PDE4D depletion reduced cell proliferation and recovered cAMP expression in these cells. PDE4D-KO and/or PDE4 inhibition with the FDA approved PDE4 inhibitor, roflumilast, also attenuated MAPK/ERK signaling in a CRAF-dependent manner. Most interestingly, we showed that PDE4D-KO enhanced the effectiveness of the TKI, sorafenib, to stunt cell survival. In conclusion, we provide preliminary evidence of PDE4 involvement in ccRCC and suggest a rationale for dual tyrosine kinase/PDE4D targeting in patients with CRAF-dependent MAPK activation.

Allogeneic ABCB5+ mesenchymal stem cells for treatment-refractory chronic venous ulcers: a phase I/IIa clinical trial.

A significant number of chronic venous ulcers (CVUs) fail to heal despite of guideline-conform standard of care. Skin-derived ABCB5+ mesenchymal stem cells (MSCs) can dampen the sustained IL-1ß-driven inflammation present in chronic wounds. Based on their wound healing-facilitating effects in a mouse CVU model and an autologous first-in-human study, ABCB5+ MSCs have emerged as a potential candidate for cell-based advanced therapy of non-healing CVUs. In the present interventional, multicenter, single-arm, phase I/IIa clinical trial, subjects whose CVU had emerged as standard therapy-resistant received one or two topical applications of 1×106 allogeneic ABCB5+ MSCs/cm2 wound area in addition to standard treatment. Out of 83 treatment-emergent adverse events, only three were judged related to the cell product; they were mild or moderate and recovered without sequelae. Wound size markedly decreased from baseline to week 12, resulting in a median wound size reduction of 76% (full analysis set, N=31), 78% (per-protocol set, N=27) and 87% (subset of responders; n=21). In conclusion, the study treatment was well tolerated and safe. The treatment elicited a profound wound size reduction within 12 weeks, identifying ABCB5+ MSCs as a potential candidate for adjunctive therapy of otherwise incurable CVUs. These results justify the conduct of a larger, randomized, controlled trial to confirm clinical efficacy.

ARE STEM CELL MARKER EXPRESSION AND CD133 ANALYSIS RELEVANT TO DIFFERENTIATE COLORECTAL CANCER?

BACKGROUND: CD133 and AXL have been described as cancer stem cell markers, and c-MYC as a key regulatory cellular mechanism in colorectal cancer (CRC). AIM: Evaluate the prognostic role of the biomarkers CD133, AXL and c-MYC and their association with clinicopathologic characteristics in colorectal adenocarcinomas and adenomas. METHODS: A total of 156 patients with UICC stage I-IV adenocarcinomas (n=122) and adenomas (n=34) were analyzed. Tissue microarrays (TMA) from primary tumors and polyps for CD133, c-MYC and AXL expression were performed and analyzed for their significance with clinicopathologic characteristics. RESULTS: Poorly differentiated adenocarcinomas and disease progression were independent risk factors for poor overall survival. The median overall survival time was 30 months. Positive CD133 expression (35.9% of all cases), particularly of right-sided CRCs (44.8% of the CD133+ cases), was negatively correlated with death in the univariate analysis, which did not reach significance in the multivariate analysis. c-MYC (15.4% of all cases) was predominantly expressed in advanced-stage patients with distant (non-pulmonary/non-hepatic) metastasis. AXL expression was found only occasionally, and predominantly dominated in adenomas, with less penetrance in high-grade dysplasia. CONCLUSIONS: CD133 expression was not associated with inferior overall survival in CRC. While AXL showed inconclusive results, c-MYC expression in primary CRCs was associated with distant metastasis.

The Beneficial Role of Auricular Point Pressure in Insomnia and Anxiety in Isolated COVID-19 Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) causes psychological distress and can have a negative impact on the general mental health and rehabilitation in affected patients under currently implemented isolation guidelines. Auricular point pressure (APP) as well-established technique in traditional Chinese medicine may help to relieve sleep disturbance and anxiety in COVID-19 patients. METHODS: During the early phase of the epidemic/pandemic, patients were enrolled in this study (02/2020 until 03/2020 n = 84). They were strictly isolated on specific wards at the Hubei Provincial Hospital of Integrated Chinese and Western Medicine in Hubei. The retrospective cohort study design included two groups. Group A patients were treated with an auricular point pressure (APP) in addition to standard intensive care medicine while Group B participants (No-APP) received routine nursing measures alone. Treatment outcome was measured using the St. Mary's Hospital Sleep Questionnaire (SMH) Score and the 7-Item Generalized Anxiety Disorder Scale (GAD-7). Both scores were measured in each patient at baseline and on the discharge day. RESULTS: The SMH score and sleep status changed in APP patients at the end of the treatment period when compared with No-APP patients (P < 0.01). APP-treated patients demonstrated lower GAD-7 scores than No-APP controls (P < 0.01). Further, no significant differences in safety or adverse events between the APP and No-APP groups were observed. CONCLUSION: The results from our snapshot study during the early phase of the SARS-CoV-2 epidemic/pandemic suggest that auricular point pressure could be a simple and effective tool to relieve insomnia and situational anxiety in hospitalized patients suffering from COVID-19 and kept under disconcerting conditions of isolation.

ARE STEM CELL MARKER EXPRESSION AND CD133 ANALYSIS RELEVANT TO DIFFERENTIATE COLORECTAL CANCER?

BACKGROUND: A) CD133+ cytoplasmic B) AXL+ combined C) c-MYC+ nuclear. CD133 and AXL have been described as cancer stem cell markers, and c-MYC as a key regulatory cellular mechanism in colorectal cancer (CRC). AIM: Evaluate the prognostic role of the biomarkers CD133, AXL and c-MYC and their association with clinicopathologic characteristics in colorectal adenocarcinomas and adenomas. METHODS: A total of 156 patients with UICC stage I-IV adenocarcinomas (n=122) and adenomas (n=34) were analyzed. Tissue microarrays (TMA) from primary tumors and polyps for CD133, c-MYC and AXL expression were performed and analyzed for their significance with clinicopathologic characteristics. RESULTS: Poorly differentiated adenocarcinomas and disease progression were independent risk factors for poor overall survival. The median overall survival time was 30 months. Positive CD133 expression (35.9% of all cases), particularly of right-sided CRCs (44.8% of the CD133+ cases), was negatively correlated with death in the univariate analysis, which did not reach significance in the multivariate analysis. c-MYC (15.4% of all cases) was predominantly expressed in advanced-stage patients with distant (non-pulmonary/non-hepatic) metastasis. AXL expression was found only occasionally, and predominantly dominated in adenomas, with less penetrance in high-grade dysplasia. CONCLUSIONS: CD133 expression was not associated with inferior overall survival in CRC. While AXL showed inconclusive results, c-MYC expression in primary CRCs was associated with distant metastasis.

IS THERE A CLINICAL PATHOLOGICAL CORRELATION OF COLORECTAL ADENOCARCINOMA WITH THE IMMUNOHISTOCHEMICAL EXPRESSION OF OPN AND ABCB5?

BACKGROUND: OPN ABCB5. Studies with biomarkers in TMA (tissue microarray) have been showing important results regarding its expression in colon cancer. AIM: Correlate the expression profile of the OPN and ABCB5 biomarkers with the epidemiological and clinicopathological characteristics of the patients, the impact on the progression of the disease and the death. METHOD: A total of 122 CRC patients who underwent surgical resection, immunomarking and their relationship with progression and death events were evaluated. RESULT: The average age was 61.9 (±13.4) years. The cases were distributed in 42 (35.9%) in the ascending/transverse colon, 31 (26.5%) in the sigmoid, 27 in the rectum (23.1%), 17 (14.5%) in the descending colon. Most patients had advanced disease (stages III and IV) in 74 cases (60.9%). There was a predominance of moderately differentiated tumors in 101 samples (82.8%); despite this, the poorly differentiated subtype proved to be an independent risk factor for death in 70%. Metastasis to the liver proved to be an independent risk factor for death in 75% (18/24), as well as patients with primary rectal tumors in 81.5% (22/27). CONCLUSION: The immunohistochemical expression of the OPN and ABCB5 markers was not associated with epidemiological and clinicopathological characteristics. Regarding the progression of disease and death, it was not possible to observe a correspondence relationship with the evaluated markers.

Topical therapy with rhubarb navel plasters in patients with chronic constipation: Results from a prospective randomized multicenter study.

ETHNOPHARMACOLOGICAL RELEVANCE: Constipation is a functional gastrointestinal disorder and one of the most prevalent conditions encountered in primary care settings. Rhubarb navel dressings have been used for more than 2,000 years in Chinese medicine to treat constipation. However, the effect of topical rhubarb administration has still not been well recognized and this strategy is not yet established as an evidence-based approach. AIM OF THE STUDY: In this study, we performed a prospective multicentric randomized controlled trial to evaluate the efficacy and safety of rhubarb navel plasters for patients with chronic constipation. MATERIALS AND METHODS: A total of 374 patients from six teaching hospitals were prospectively included between 09/2016 and 10/2017 in the study based on Rome III criteria. All participants were randomly assigned (1:1) into verum/placebo group and given either Rheum officinale rhubarb powder or a placebo flour stick on the navel for 6 h/day/8 days. Primary outcome measures were the Cleveland Constipation Score (CCS) for the feces condition and Bristol Stool Scale (BSS) for stool consistency and 24 h defecation frequency. RESULTS: The groups demonstrated no statistical differences in demographic data, clinical diagnoses and concomitant medication at baseline. In patients treated with the verum CCS was 5.61 (day 8, 95% CI 5.15-6.07) compared to 8.62 (95% CI 8.07-9.18) in placebo-treated controls (P < 0.001). The mean change of CCS at the end of treatment (day 8 versus [vs] day 0) was 6.04 in verum-treated vs 2.73 in placebo-treated controls (P < 0.001). Also 24 h defecation frequency (BSS) showed superior results (day 5: 0.84 vs 0.62, 95% CI 0.67-0.80, P < 0.001; day 6: 0.82 vs 0.60, 95% CI 0.64-0.78, P < 0.01 and day 8: 0.82 vs 0.60, 95% CI 0.64-0.78, P < 0.01) and better BSS type classification during treatment than controls (P < 0.05). No significant differences in adverse events between both groups became obvious. CONCLUSION: Rhubarb navel plaster administration over an 8-day-treatment period resulted in significantly improved bowel function as demonstrated by the CCS, 24 h defecating frequency and BSS. Our results suggest that rhubarb navel plasters represent a feasible, safe and efficient application route for the treatment of patients suffering from chronic constipation.

Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data.

BACKGROUND AIM: Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation. METHODS: The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5+ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers. RESULTS: As of now, 12 wounds in nine patients have been treated with 5 × 105 autologous ABCB5+ MSCs per cm2 wound area, eliciting a median wound size reduction of 63% (range, 32-100%) at 12 weeks and early relief of pain. CONCLUSIONS: The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5+ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds.

Are stem cell marker expression and cd133 analysis relevant to differentiate colorectal cancer? / A expressão de marcadores de células-tronco e a análise do cd133 são relevantes na diferenciação do câncer colorretal?

ABSTRACT Background: CD133 and AXL have been described as cancer stem cell markers, and c-MYC as a key regulatory cellular mechanism in colorectal cancer (CRC). Aim: Evaluate the prognostic role of the biomarkers CD133, AXL and c-MYC and their association with clinicopathologic characteristics in colorectal adenocarcinomas and adenomas. Methods: A total of 156 patients with UICC stage I-IV adenocarcinomas (n=122) and adenomas (n=34) were analyzed. Tissue microarrays (TMA) from primary tumors and polyps for CD133, c-MYC and AXL expression were performed and analyzed for their significance with clinicopathologic characteristics. Results: Poorly differentiated adenocarcinomas and disease progression were independent risk factors for poor overall survival. The median overall survival time was 30 months. Positive CD133 expression (35.9% of all cases), particularly of right-sided CRCs (44.8% of the CD133+ cases), was negatively correlated with death in the univariate analysis, which did not reach significance in the multivariate analysis. c-MYC (15.4% of all cases) was predominantly expressed in advanced-stage patients with distant (non-pulmonary/non-hepatic) metastasis. AXL expression was found only occasionally, and predominantly dominated in adenomas, with less penetrance in high-grade dysplasia. Conclusions: CD133 expression was not associated with inferior overall survival in CRC. While AXL showed inconclusive results, c-MYC expression in primary CRCs was associated with distant metastasis.

RESUMO Racional: CD133 e AXL são descritos na literatura como marcadores de células-tronco tumorais, e c-MYC cumpre papel chave como mecanismo de regulação celular no câncer colorretal (CCR). Objetivo: Avaliar o papel prognóstico dos biomarcadores CD133, AXL e c-MYC e sua associação com características clinicopatológicas de adenocarcinomas e adenomas colorretais. Métodos: Um total de 156 pacientes com adenocarcinomas de estádio UICC I-IV (n=122) e adenomas (n=34) colorretais foram avaliados. Microarranjos teciduais (TMA) dos tumores primários e adenomas foram realizados em busca de expressão de CD133, c-MYC e AXL, com posterior análise de relação significativa com características clinicopatológicas. Resultados: Adenocarcinomas pobremente diferenciados e progressão de doença foram fatores de risco independentes para má sobrevida global. A taxa mediana de sobrevida global foi de 30 meses. Expressão positiva de CD133 (35,9% dos casos), particularmente em cânceres de cólon direito (44,8% dos casos CD133+), correlacionou-se negativamente com óbito na análise univariada, sem significância estatística na análise multivariada. c-MYC (15,4% dos casos) teve predomínio de expressão em pacientes com estádio avançado com metástases distantes (não-pulmonares/não-hepáticas). Expressão de AXL foi pouco encontrada, com predomínio em adenomas, com menor penetrância em displasia de alto grau. Conclusão: Expressão de CD133 não se associou com sobrevida global inferior em CCR. Enquanto AXL demonstrou resultados inconclusivos, expressão de c-MYC em tumores primários se associou-se à metástases à distância.