Sialyl Tn-Expressing Bladder Cancer Cells Induce a Tolerogenic Phenotype in Innate and Adaptive Immune Cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature. Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.

Observações sôbre a terapêutica da ascaridiose com o hidroclorato e com o ciclamato de 2,3,5,6 – Tetrahidro - 6 fenil - imidazo (2,1-b) tiazol

Os autores estudam a ação do ciclamato e do cloridrato de 2, 3, 5, 6, - Tetrahidro-6-fenil-imidazo (2,1-b) tiazol no tratamento da ascaridiose. Foram tratados ao todo 84 indivíduos sendo 48 com o cloridrato e 36 com o ciclamato. O índice de cura com o primeiro foi de 83,9% e com o segundo de 76,4%. Nenhuma reação colateral foi observada com o emprêgo do cloridrato sendo que com o ciclamato 53,6 dos pacientes apresentaram sintomas discretos de intolerância. Os índices de cura assemelham-se aos observados com a piperazina que é a medicação clássica para terapêutica da ascaridiose.

Schistosomiasis mansoni of the bladder simulating bladder cancer: a case report

The relationship between bladder tumors and Schistosoma haematobium is well known, but only sporadic cases of bladder infection due to Schistosoma mansoni have been reported. In this case, a 48-year-old woman with macroscopic hematuria, dysuria and a palpable abdominal mass was investigated. Ultrasound showed a large exophytic mass in the bladder. Transurethral resection of the bladder revealed viable eggs of Schistosoma mansoni. The patient was treated clinically with oxamniquine and surgery was performed to resect the large mass. This case shows that schistosomiasis Mansoni in the bladder can simulate bladder cancer.

Thomsen-Friedenreich antigens in bladder cancer: evaluation of their prognostic value

RESUMO: Introdução. O cancro de bexiga é uma patologia comum que representa o 6° e o 5° cancro mais incidente em Portugal e na Itália, respetivamente. Em mais de metade dos casos ocorre reincidência durante o primeiro ano, requerendo acompanhamento clínico ao longo da vida. A instilação intravesical de Bacillus Calmette-Guérin (BCG) (uma estirpe atenuada do Mycobacterium bovis) representa uma imunoterapia eficaz no combate ao cancro de bexiga, no entanto, muitos aspetos da interação de BCG com as células tumorais bem como com as células do sistema imunitário permanecem por desvendar. As células tumorais de bexiga expressam frequentemente as formas sialiladas dos antigénios de Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). Contudo ainda se desconhece o significado da sua expressão na malignidade tumoral e se afeta a eficácia da terapêutica BCG. Objetivo do estudo. Investigar o papel dos antigénios sT e sTn no fenótipo maligno de células de cancro de bexiga bem como na resposta mediada pelo sistema imunitário à terapia com BCG. Metodologia. Para tal, foram utilizadas as linhas celulares de cancro da bexiga HT1376 e MCR, geneticamente modificadas por transdução com vetores codificantes para as sialiltransferases ST3GAL1 ou ST6GALNAC1, de forma a expressar homogeneamente os antigénios sT ou sTn respetivamente. Estes modelos celulares foram estudados após confronto com BCG. O nível de BCG internalizado foi avaliado por citometria de fluxo. O perfil global de expressão genética dos modelos celulares antes e após incubação com BCG foi analisado pela tecnologia de microarray. O perfil de citocinas secretadas pelos modelos celulares após incubação com BCG, bem como de macrófagos estimulados pelo secretoma de células de cancro de bexiga que por sua vez foram estimuladas previamente por BCG, foi estudado pelo sistema multiplex de “imuno-esferas”. Resultados. A análise do transcritoma dos modelos celulares revelou que grupos de genes envolvidos em funções específicas foram modulados em paralelo nos dois modelos celulares, após transdução, independentemente da sialiltransferase expressa. Ou seja, em células que expressavam a sialiltransferase ST3GAL1 ou ST6GALNAC1, os genes envolvidos na regulação da segregação cromossómica e na reparação do DNA foram consistentemente regulados negativamente. Genes descritos na literatura como marcadores para o cancro de bexiga foram também modulados. A incubação com BCG resultou numa tendência ao aumento da expressão de genes relevantes na preservação e estabilidade genómica e menor malignidade, no entanto, apenas em células que expressavam sT ou sTn. Entre as dez citocinas testadas, apenas a IL-6 e IL-8 foram expressas pelas linhas celulares de cancro da bexiga, com indução destas após estimulação com BCG, e principalmente em células que expressavam ST3GAL1 ou ST6GALNAC1. Em macrófagos, citocinas inflamatórias, tais como IL-1β, IL-6 e TNFα, e a citocina anti-inflamatória IL-10, foram induzidas apenas pelo secretoma de células de cancro da bexiga confrontadas com BCG, com maior relevância quando estas expressavam ST3GAL1 ou ST6GALNAC1, prevendo a estimulação de macrófagos semelhantes aos de tipo M1 e uma melhor resposta à terapia com BCG. Conclusões. O efeito geral da expressão destas sialiltransferases e dos produtos enzimáticos sT ou sTn nas células de cancro de bexiga conduz a um fenótipo de maior malignidade. Contudo, a maior avidez de estas na produção de citocinas inflamatórias após confronto com BCG, bem como a maior capacidade de estimulação de macrófagos, predirá uma resposta à terapia com BCG mais eficaz em tumores que expressem os antigénios de TF sialilados. Tais conclusões são totalmente concordantes com os nossos mais recentes dados clínicos obtidos em colaboração, que mostram que em doentes com cancro de bexiga que expressam sTn respondem melhor a terapia BCG. ----------ABSTRACT: Background. Bladder cancer is a common malignancy representing the 6th and the 5th most incident cancer in Portugal and in Italy, respectively. More than half of the cases relapse within one year, requiring though a lifelong follow-up. Intravesical instillation of Bacillus Calmette-Guérin (BCG) (an attenuated strain of Mycobacterium bovis) represents an effective immunotherapy of bladder cancer, although many aspects of the interaction of BCG with cancer cells and host immune cells remain obscure. Bladder cancer cells often express the sialylated forms of the Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). However, it’s still unknown the sense of such expression in tumour malignancy and in the BCG therapy efficacy. Aim of the study. To investigate the role of the sT and sTn antigens on the malignant phenotype of bladder cancer cells and the immune mediated response to BCG therapy. Experimental. We have utilized populations of the bladder cancer cell lines HT1376 and MCR, genetically modified by transduction with the sialyltransferases ST3GAL1 or ST6GALNAC1 to express homogeneously sT or sTn antigens. The level of BCG internalized was assessed by flow cytometry. The whole gene expression profile of BCG-challenged or unchallenged bladder cancer cell lines was studied by microarray technology. The profile of cytokines secreted by BCG-challenged bladder cancer cells and that of macrophages challenged by the secretome of BCG-challenged bladder cancer cells was studied by multiplex immune-beads assay. Results. Transcriptome analysis of the sialyltransferase-transduced cells revealed that groups of genes involved in specific functions were regulated in parallel in the two cell lines, regardless the sialyltransferase expressed. Namely, in sialyltransferase-expressing cells, genes involved in the proper chromosomal segregation and in the DNA repair were consistently down-regulated, while genes reported in literature as markers for bladder cancer were modulated. BCG-challenging induced a tendency to up-regulation of the genes preserving genomic stability and reducing malignancy, but only in cells expressing either sT or sTn. Among the ten cytokines tested, only IL-6 and IL-8 were expressed by bladder cancer cell lines and up-regulated by BCG-challenging, mainly in sialyltransferases-expressing cells. In macrophages, inflammatory cytokines, such as IL-1β, IL-6 and TNFα, and the antinflammatory IL-10 were induced only by the secretome of BCG-challenged bladder cancer cells, particularly when expressing either sialyltransferase, predicting the stimulation of M1-like macrophages and a better response to BCG therapy. Conclusions. The general effect of the expression of the two sialyltransferases and their products in the bladder cancer cells is toward a more malignant phenotype. However, the stronger ability of sialyltransferase expressing cells to produce inflammatory cytokines upon BCG-challenging and to stimulate macrophages predicts a more effective response to BCG in tumours expressing the sialylated TF antigens. This is fully consistent with our recent clinical data obtained in collaboration, showing that patients with bladder cancer expressing sTn respond better to BCG therapy.

Evolving hallmarks in urothelial bladder cancer: unveiling potential biomarkers

Urothelial bladder carcinoma (UBC), the most frequent type (90%) of bladder cancer and the second most common malignancy of the urogenital region, is a relatively well understood type of cancer, with numerous studies concerning pathogenetic pathways, natural history and bladder tumor biology being reported. Despite this, it continues to remain a challenge in the oncology field, mostly due to its relapsing and progressive nature, and to the heterogeneity in the response to cisplatin-containing regimens. Although the formulae based on clinical staging and histopathological parameters are classically used as diagnostic and prognostic tools, they have proven insufficient to characterize the individual biological features and clinical behaviour of the tumours. Understanding the pathobiology of the disease can add important information to these classical criteria, and contribute to accurately predict outcome and individualize therapy for UBC patients. In this line of investigation, we found that tumour angiogenesis and lymphangiogenesis, the process of invasion and metastasis and the energy metabolism reprogramming/tumour microenvironment encompass several potential biomarkers that seem to infl bladder cancer aggressiveness and chemoresistance. We particularly highlight the roles of lymphovascular invasion, and of RKIP, CD147 and MCT1 immunoexpressions, as relevant prognostic and/or predictive biomarkers, and as promising areas of therapeutic intervention, eliciting for the development of additional studies that can validate and further explore these biomarkers.

Konstruktion einer nicht-IGF-bindenden Insulin-like Growth Factor Binding Protein-2-Variante und Untersuchung ihrer Wirkung auf das Tumorzellwachstum

Magdeburg, Univ., Medizin. Fakultät, Diss., 2008

Untitled 2 Detail B

Detail of Untitled 2, 2011, pastel on paper.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and biological activity analysis of specific antibodies

Background: Plasmodium falciparum(P. falciparum) merozoite surfaceprotein 2 (MSP-2) is one of bloodstage proteins that are associated withprotection from malaria. MSP-2 consistsof a highly polymorphic centralrepeat region flanked by a dimorphicregion that defines the two allelicfamilies, 3D7 and FC27; N- and Cterminalregions are conserved domains.Long synthetic peptides (LSP)representing the two allelic familiesof MSP-2 and constant regions arerecognized by sera from donors livingin endemic areas; and specific antibodies(Abs) are associated with protectionand active in antibody dependentcellular inhibition (ADCI) in vitro.However, the fine specificity ofAb response to the two allelic familiesof MSP-2 is unknown. Methods: Peptidesrepresenting dimorphic regionof 3D7 and FC27 families and theirC-terminal (common fragment to thetwo families) termed 3D7-D (88 aa),FC27-D (48 aa) and C (40 aa) respectivelywere synthesized. Overlapping20 mer peptides covering dimorphicand constant regions of two familieswere also synthesized for epitopemapping. Human sera were obtainedfrom donors living in malaria endemicareas. SpecificDand CregionsAbs were purified from single or poolhuman sera. Sera from mice were obtainedafter immunization with thetwo families LSP mixture in three differentadjuvants: alhydrogel (Alum),Glucopyranosyl Lipid Adjuvant-Stableoil-in-water Emulsion (GLA-SE)and Virosome. For ADCI, P. falciparum(strain 3D7) parasite wasmaintained in culture at 0.5% parasitemiaand 4% hematocrit in air tightbox at love oxygen (2%) and 37 ºC.Results: We identified several epitopesfrom the dimorphic and constantregions of both families of MSP-2, inmice and humans (adults and children).In human, most recognizedepitopes were the same in differentendemic regions for each domain ofthe two families of MSP-2. In mice,the differential recognition of epitopewas depending on the strain of mouseand interestingly on the adjuvantused. GLA-SE and alum as adjuvantswere more often associated with therecognition of multiple epitopes thanvirosomes. Epitope-specific Abs recognizednative merozoites of P.falciparum and were active in ADCIto block development of parasite.Conclusion: The delineation of a limitednumber of epitopes could be exploitedto develop MSP-2 vaccinesactive on both allelic families ofMSP-2.

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Mutations at a single codon in Mad homology 2 domain of SMAD4 cause Myhre syndrome.

Myhre syndrome (MIM 139210) is a developmental disorder characterized by short stature, short hands and feet, facial dysmorphism, muscular hypertrophy, deafness and cognitive delay. Using exome sequencing of individuals with Myhre syndrome, we identified SMAD4 as a candidate gene that contributes to this syndrome on the basis of its pivotal role in the bone morphogenetic pathway (BMP) and transforming growth factor (TGF)-β signaling. We identified three distinct heterozygous missense SMAD4 mutations affecting the codon for Ile500 in 11 individuals with Myhre syndrome. All three mutations are located in the region of SMAD4 encoding the Mad homology 2 (MH2) domain near the site of monoubiquitination at Lys519, and we found a defect in SMAD4 ubiquitination in fibroblasts from affected individuals. We also observed decreased expression of downstream TGF-β target genes, supporting the idea of impaired TGF-β-mediated transcriptional control in individuals with Myhre syndrome.

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and fine specificity of human antibody response against non-polymorphic domains.

BACKGROUND: Two long synthetic peptides representing the dimorphic and constant C-terminal domains of the two allelic families of Plasmodium falciparum merozoite surface proteins 2 are considered promising malaria vaccine candidates. The aim of the current study is to characterize the immune response (epitope mapping) in naturally exposed individuals and relate immune responses to the risk of clinical malaria. METHODS: To optimize their construction, the fine specificity of human serum antibodies from donors of different age, sex and living in four distinct endemic regions was determined in ELISA by using overlapping 20 mer peptides covering the two domains. Immune purified antibodies were used in Western blot and immunofluorescence assay to recognize native parasite derivate proteins. RESULTS: Immunodominant epitopes were characterized, and their distribution was similar irrespective of geographic origin, age group and gender. Acquisition of a 3D7 family and constant region-specific immune response and antibody avidity maturation occur early in life while a longer period is needed for the corresponding FC27 family response. In addition, the antibody response to individual epitopes within the 3D7 family-specific region contributes to protection from malaria infection with different statistical weight. It is also illustrated that affinity-purified antibodies against the dimorphic or constant regions recognized homologous and heterologous parasites in immunofluorescence and homologous and heterologous MSP2 and other polypeptides in Western blot. CONCLUSION: Data from this current study may contribute to a development of MSP2 vaccine candidates based on conserved and dimorphic regions thus bypassing the complexity of vaccine development related to the polymorphism of full-length MSP2.