Linking tumor immune infiltrate and systemic immune mediators to treatment response and prognosis in advanced cervical cancer.

Cervical cancer (CC) poses a significant burden on individuals in developing regions, exhibiting heterogeneous responses to standard chemoradiation therapy, and contributing to substantial mortality rates. Unraveling host immune dynamics holds promise for innovative therapies and discovery of clinically relevant biomarkers. We studied prospectively locally advanced CC patients pre-treatment, stratifying them as responders (R) or non-responders (NR). R patients had increased tumor-infiltrating lymphocytes (TILs), while NR patients showed elevated PD-1 scores, CD8+ and PD-L2+ TILs, and PD-L1 immune reactivity. NR patients exhibited higher systemic soluble mediators correlating with TIL immune markers. R patients demonstrated functional polarization of CD4 T cells (Th1, Th2, Th17, and Treg), while CD8+ T cells and CD68+ macrophages predominated in the NR group. Receiver operating characteristic analysis identified potential CC response predictors, including PD-L1-immunoreactive (IR) area, PD-L2, CD8, FGF-basic, IL-7, IL-8, IL-12p40, IL-15, and TNF-alpha. Dysfunctional TILs and imbalanced immune mediators contribute to therapeutic insufficiency, shedding light on local and systemic immune interplay. Our study informs immunological signatures for treatment prediction and CC prognosis.

Systemic immune mediators reflect tumour-infiltrating lymphocyte intensity and predict therapeutic response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer (BC). Neoadjuvant chemotherapy has proven efficacy in its treatment, and a pathological complete response (pCR) to therapy is predictive of improved long-term survival. The immune response is key to successful neoadjuvant chemotherapy, as indicated by the relation between the percentage of stromal tumour-infiltrating lymphocytes (TILs) in pre-treated tumour tissue samples and the likelihood of achieving pCR. Here we studied systemic immune mediators from volunteer TNBC patients before undergoing neoadjuvant chemotherapy to determine the systemic response association with TIL intensity, treatment response and survival. Patients were classified into pCR responder or non-responder at time of surgery. We found higher levels of immune mediators before treatment began in patients that went on to be pCR responders versus non-pCR, with area under the curve (AUC) values of 0.64-0.80. We also observed a positive correlation between inflammatory systemic immune mediators and the percentage of TILs in pCR responder patients. Combining TILs and systemic immune mediator levels provided stronger AUC values (range of 0.72-0.82). Last, performing a progression-free survival analysis with several of the systemic cytokines that predict pCR, segregated the patients into long- and short-survival groups based on high and low production of the cytokines, respectively. Our study demonstrates that circulating cytokines, before treatment begins, predict pCR in TNBC patients treated with neoadjuvant chemotherapy. Moreover, they may act as a surrogate marker of high TILs or together with TILs to better predict pCR and survival.

Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Distinct systemic immune networks define severe vs. mild COVID-19 in hematologic and solid cancer patients.

Introduction: The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, has impacted health across all sectors of society. A cytokine-release syndrome, combined with an inefficient response of innate immune cells to directly combat the virus, characterizes the severe form of COVID-19. While immune factors involved in the development of severe COVID-19 in the general population are becoming clearer, identification of the immune mechanisms behind severe disease in oncologic patients remains uncertain. Methods: Here we evaluated the systemic immune response through the analysis of soluble blood immune factors and anti-SARS-CoV-2 antibodies within the early days of a positive SARS-CoV-2 diagnostic in oncologic patients. Results: Individuals with hematologic malignancies that went on to die from COVID-19 displayed at diagnosis severe leukopenia, low antibody production against SARS-CoV-2 proteins, and elevated production of innate immune cell recruitment and activation factors. These patients also displayed correlation networks in which IL-2, IL-13, TNF-alpha, IFN-gamma, and FGF2 were the focal points. Hematologic cancer patients that showed highly networked and coordinated anti-SARS-CoV-2 antibody production, with central importance of IL-4, IL-5, IL-12A, IL-15, and IL-17A, presented only mild COVID-19. Conversely, solid tumor patients that had elevated levels of inflammatory cytokines IL-6, CXCL8, and lost the coordinate production of anti-virus antibodies developed severe COVID-19 and died. Patients that displayed positive correlation networks between anti-virus antibodies, and a regulatory axis involving IL-10 and inflammatory cytokines recovered from the disease. We also provided evidence that CXCL8 is a strong predictor of death for oncologic patients and could be an indicator of poor prognosis within days of the positive diagnostic of SARS-CoV-2 infection. Conclusion: Our findings defined distinct systemic immune profiles associated with COVID-19 clinical outcome of patients with cancer and COVID-19. These systemic immune networks shed light on potential immune mechanisms involved in disease outcome, as well as identify potential clinically useful biomarkers.

A Genome-wide Association Study Identifies SERPINB10, CRLF3, STX7, LAMP3, IFNG-AS1, and KRT80 As Risk Loci Contributing to Cutaneous Leishmaniasis in Brazil.

BACKGROUND: Our goal was to identify genetic risk factors for cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. METHODS: Genotyping 2066 CL cases and 2046 controls using Illumina HumanCoreExomeBeadChips provided data for 4 498 586 imputed single-nucleotide variants (SNVs). A genome-wide association study (GWAS) using linear mixed models took account of genetic diversity/ethnicity/admixture. Post-GWAS positional, expression quantitative trait locus (eQTL) and chromatin interaction mapping was performed in Functional Mapping and Annotation (FUMA). Transcriptional data were compared between lesions and normal skin, and cytokines measured using flow cytometry and Bioplex assay. RESULTS: Positional mapping identified 32 genomic loci associated with CL, none achieving genome-wide significance (P < 5 × 10-8). Lead SNVs at 23 loci occurred at protein coding or noncoding RNA genes, 15 with eQTLs for functionally relevant cells/tissues and/or showing differential expression in lesions. Of these, the 6 most plausible genetic risk loci were SERPINB10 (Pimputed_1000G = 2.67 × 10-6), CRLF3 (Pimputed_1000G = 5.12 × 10-6), STX7 (Pimputed_1000G = 6.06 × 10-6), KRT80 (Pimputed_1000G = 6.58 × 10-6), LAMP3 (Pimputed_1000G = 6.54 × 10-6), and IFNG-AS1 (Pimputed_1000G = 1.32 × 10-5). LAMP3 (Padjusted = 9.25 × 10-12; +6-fold), STX7 (Padjusted = 7.62 × 10-3; +1.3-fold), and CRLF3 (Padjusted = 9.19 × 10-9; +1.97-fold) were expressed more highly in CL biopsies compared to normal skin; KRT80 (Padjusted = 3.07 × 10-8; -3-fold) was lower. Multiple cis-eQTLs across SERPINB10 mapped to chromatin interaction regions of transcriptional/enhancer activity in neutrophils, monocytes, B cells, and hematopoietic stem cells. Those at IFNG-AS1 mapped to transcriptional/enhancer regions in T, natural killer, and B cells. The percentage of peripheral blood CD3+ T cells making antigen-specific interferon-γ differed significantly by IFNG-AS1 genotype. CONCLUSIONS: This first GWAS for CL identified multiple genetic risk loci including a novel lead to understanding CL pathogenesis through regulation of interferon-γ by IFNG antisense RNA 1.

Biomass of unconventional plants from Brazilian semiarid as substrate for hydrolytic enzymes production by Aspergillus niger under solid and submerged fermentation

Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C

Biomass of unconventional plants from Brazilian semiarid as substrate for hydrolytic enzymes production by Aspergillus niger under solid and submerged fermentation

Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C a

Human Chondrocyte Activation by Toxins From Premolis semirufa, an Amazon Rainforest Moth Caterpillar: Identifying an Osteoarthritis Signature.

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model.

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Amblyomin-X, a recombinant Kunitz-type inhibitor, regulates cell adhesion and migration of human tumor cells.

In a tumor microenvironment, endothelial cell migration and angiogenesis allow cancer to spread to other organs causing metastasis.  Indeed, a number of molecules that are involved in cytoskeleton re-organization and intracellular signaling have been investigated for their effects on tumor cell growth and metastasis. Alongside that, Amblyomin-X, a recombinant Kunitz-type protein, has been shown to reduce metastasis and tumor growth in in vivo experiments. In the present report, we provide a mechanistic insight to these antitumor effects, this is,  Amblyomin-X modulates Rho-GTPases and uPAR signaling, and reduces the release of MMPs, leading to disruption of the actin cytoskeleton and decreased cell migration of tumor cell lines. Altogether, our data support a role for Amblyomin-X as a novel potential antitumor drug. ABBREVIATIONS: Amb-X: Amblyomin-X; ECGF: endotelial cell growth factor; ECM: extracellular matrix; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cell; LRP1: low-density lipoprotein receptor-related protein; MMP: matrix metalloproteinase; HPI-4: hedgehog pathway inhibitor 4; PAI-1: plasminogen activator inhibitor 1; PMA: phorbol 12-myristate-13-acetate; TFPI: tissue factor pathway inhibitor; uPA: urokinase plasminogen activator; uPAR: uPA receptor.

Human chondrocyte activation by toxins from premolis semirufa, an Amazon rainforest moth caterpillar: identifying an osteoarthritis signature

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Amblyomin-X, a recombinant Kunitz-type inhibitor, regulates cell adhesion and migration of human tumor cells

In a tumor microenvironment, endothelial cell migration and angiogenesis allow cancer to spread to other organs causing metastasis. Indeed, a number of molecules that are involved in cytoskeleton re-organization and intracellular signaling have been investigated for their effects on tumor cell growth and metastasis. Alongside that, Amblyomin-X, a recombinant Kunitz-type protein, has been shown to reduce metastasis and tumor growth in in vivo experiments. In the present report, we provide a mechanistic insight to these antitumor effects, this is, Amblyomin-X modulates Rho-GTPases and uPAR signaling, and reduces the release of MMPs, leading to disruption of the actin cytoskeleton and decreased cell migration of tumor cell lines. Altogether, our data support a role for Amblyomin-X as a novel potential antitumor drug.

Precarização do trabalho e o processo de derrocada do trabalhador / Labor precariousness and the worker's overthrow process

As mudanças globais vivenciadas atualmente fomentam um novo paradigma nas relações trabalhistas, a exemplo de formas precarizadas de trabalho e emprego, que por sua vez têm sido percebidas, a partir da proliferação das novas modalidades de contrato e do declínio da oferta de empregos típicos/permanentes, como uma das consequências mais visíveis da flexibilização do mercado de trabalho. Este artigo tem por objetivo apresentar os aspectos caracterizadores da precarização do trabalho e suas consequências para o trabalhador. A precarização, em suma, apresenta-se como um fenômeno que perpassa o dinâmico movimento de estruturação do trabalho e do emprego, posto que concerne tanto ao crescimento do desemprego e à ampliação do exército de reserva quanto às especificidades dos empregos disponíveis no mercado de trabalho, enfatizados pela instabilidade e efemeridade contratuais. Isso conduz à expansão do contingente de trabalhadores alienados de seus direitos e sujeitos a condições de trabalho instáveis, insatisfatórias e potencialmente adoecedoras

Current global changes foster a new paradigm in labor relationships, such as precarious forms of work and employment, which have been perceived, from the proliferation of new contract modalities and the decline of typical/permanent employment, as one of the most visible consequences of labor market flexibility. This article aims to present the characterizing aspects of work precariousness and its consequences for the worker. In short terms, precariousness is a phenomenon that runs through the dynamic movement of work and employment structuring, as it concerns not only to unemployment growth and the expansion of the reserve register but also to the specificities of the jobs available in the labor market, emphasized by the instability and contractual ephemerality. It leads to an increasing number of workers who are alienated from their rights and subjected to unstable, unsatisfactory and potentially ill conditions of labor

Promising pharmacological profile of a Kunitz-type inhibitor in murine renal cell carcinoma model.

Renal cell carcinoma (RCC), also called kidney cancer or renal adenocarcinoma, is highly resistant to current treatments. It has been previously reported that a Kunitz-type inhibitor domain-containing protein, isolated from the salivary glands of the Amblyomma cajennense tick, triggers apoptosis in murine renal adenocarcinoma cells (Renca) by inhibiting the proteasome and endoplasmic reticulum stress. Of note, Amblyomin-X is the corresponding recombinant protein identified in the cDNA library from A. cajennense salivary glands. Herein, using orthotopic kidney tumors in mice, we demonstrate that Amblyomin-X is able to drastically reduce the incidence of lung metastases by inducing cell cycle arrest and apoptosis. The in vitro assays show that Amblyomin-X is capable of reducing the proliferation rate of Renca cells, promoting cell cycle arrest, and down-regulating the expression of crucial proteins (cyclin D1, Ki67 and Pgp) involved in the aggressiveness and resistance of RCC. Regarding non-tumor cells (NIH3T3), Amblyomin-X produced minor effects in the cyclin D1 levels. Interestingly, observing the image assays, the fluorescence-labelled Amblyomin-X was indeed detected in the tumor stroma whereas in healthy animals it was rapidly metabolized and excreted. Taken the findings together, Amblyomin-X can be considered as a potential anti-RCC drug candidate.

Amblyomin-X induces ER stress, mitochondrial dysfunction, and caspase activation in human melanoma and pancreatic tumor cell.

During the last two decades, new insights into proteasome function and its role in several human diseases made it a potential therapeutic target. In this context, Amblyomin-X is a Kunitz-type FXa inhibitor similar to endogenous tissue factor pathway inhibitor (TFPI) and is a novel proteasome inhibitor. Herein, we have demonstrated Amblyomin-X cytotoxicity to different tumor cells lines such as pancreatic (Panc1, AsPC1BxPC3) and melanoma (SK-MEL-5 and SK-MEL-28). Of note, Amblyomin-X was not cytotoxic to normal human fibroblast cells. In addition, Amblyomin-X promoted accumulation of ER stress markers (GRP78 and GADD153) in sensitive (SK-MEL-28) and bortezomib-resistant (Mia-PaCa-2) tumor cells. The intracellular calcium concentration [Ca(2+)] i was slightly modulated in human tumor cells (SK-MEL-28 and Mia-PaCa-2) after 24 h of Amblyomin-X treatment. Furthermore, Amblyomin-X induced mitochondrial dysfunction, cytochrome-c release, PARP cleavage, and activation of caspase cascade in both human tumor (SK-MEL-28 and Mia-PaCa-2) cells. These investigations might help in further understanding of the antitumor properties of Amblyomin-X.

Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X.

The Kunitz-type recombinant protein, Amblyomin-X, is an antitumor recombinant molecule from a cDNA library prepared from the salivary glands of the tick Amblyomma cajennense. The primary target of this protein appears to be the proteasome. Amblyomin-X increased gene and protein expression of distinct subunits of the molecular motor dynein, which plays a key role in the intracellular transport. Herein, Amblyomin-X was specifically taken up by tumor cells through lipid-raft endocytic pathways, but not by fibroblasts. Moreover, dynein inhibitor, ciliobrevin A, decreased Amblyomin-X uptake by tumor cells. Furthermore, incubation of tumor cells with Amblyomin-X inhibited trypsin-like activity of the proteasome, which was restored upon pretreatment with ciliobrevin A. Only in tumor cells treated with Amblyomin-X, we identified proteins bounds to dynein that are related to aggresome formation, autophagy inhibition, and early and recycling endosome markers. In addition, Amblyomin-X was found to interact with dynein, increased Rab11A protein expression and Rab11A co-localization with the light-intermediate chain 2 (LIC2) of dynein. Thereby, the results provide new insights on the antitumor mechanism of Amblyomin-X and reveal an unsuspected role of cytoplasmic dynein in its uptake, intracellular trafficking and pro-apoptotic action.

Tick salivary gland as potential natural source for the discovery of promising antitumor drug candidates.

Nowadays, the relationship between cancer blood coagulation is well established. Regarding biodiversity and bioprospection, the tick biology has become quite attractive natural source for coagulation inhibitors, since its saliva has a very rich variety of bioactive molecules. For instance, a Kunitz-type FXa inhibitor, named Amblyomin-X, was found through transcriptome of the salivary gland of the Amblyomma cajennense. tick. This TFPI-like inhibitor, after obtained as recombinant protein, has presented anticoagulant, antigionenic, and antitumor properties. Although its effects on blood coagulation could be relevant for antitumor effect, Amblyomin-X acts by non-hemostatic mechanisms, such as proteasome inhibition and autophagy inhibition. Notably, cytotoxicity was not observed on non-tumor cells treated with this protein, suggesting some selectivity for tumor cells. Considering the current efforts in order to develop effective anticancer therapies, the findings presented in this review strongly suggest Amblyomin-X as a promising novel antitumor drug candidate.

Milk fermented with a 15-lipoxygenase-1-producing Lactococcus lactis alleviates symptoms of colitis in a murine model.

Inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, is characterized by extensive inflammation due to dysregulation of the innate and adaptive immune system whose exact etiology is not yet completely understood. Currently there is no cure for IBD, thus the search for new molecules capable of controlling IBD and their delivery to the site of inflammation are the goal of many researchers. The aim of this work was to evaluate the anti-inflammatory effect of the administration of milks fermented by a Lactococcus (L.) lactis strain producing 15-lipoxygenase-1 (15-LOX-1) using a trinitrobenzenesulfonic acid-induced IBD mouse model. The results obtained demonstrated that 15-LOX-1 producing L. lactis was effective in the prevention of the intestinal damage associated to inflammatory bowel disease in a murine model. The work also confirmed previous studies showing that fermented milk is an effective form of administration of recombinant lactic acid bacteria expressing beneficial molecules.