Biotech-Educated Platelets: Beyond Tissue Regeneration 2.0.

The increasing discoveries regarding the biology and functions of platelets in the last decade undoubtedly show that these cells are one of the most biotechnological human cells. This review summarizes new advances in platelet biology, functions, and new concepts of biotech-educated platelets that connect advanced biomimetic science to platelet-based additive manufacturing for tissue regeneration. As highly responsive and secretory cells, platelets could be explored to develop solutions that alter injured microenvironments through platelet-based synthetic biomaterials with instructive extracellular cues for morphogenesis in tissue engineering beyond tissue regeneration 2.0.

Platelets as a 'natural factory' for growth factor production that sustains normal (and pathological) cell biology.

Platelets have attracted substantial attention in the current decade owing to their unexpected pleiotropic properties and conflicted functions. In fact, platelets participate in both health (hemostasis) and disease (thrombotic diseases). Much of the plasticity of platelets comes from the fact that platelets are the reservoir and the 'natural factory' of growth factors (GFs), with pivotal functions in wound repair and tissue regeneration. By combining the platelets' plasticity and biotechnological processes, PlateInnove Biotechnology optimized the production of GFs in nanoparticle biointerfacing by platelet content, which opens an avenue of possibilities.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells.

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-ß, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.

Action and function of Faecalibacterium prausnitzii in health and disease.

Faecalibacterium prausnitzii, anaerobic bacteria, is one of the main components of gut microbiota and the most important butyrate-producing bacteria in the human colon. So far, this commensal bacterium has been considered as a bioindicator of human health, once when its population is altered (decreased), inflammatory processes are favored. Several reports in the literature highlighted that the amount of Faecalibacterium prausnitzii negatively correlates to the activity of inflammatory bowel disease and colorectal cancer. Therefore, counterbalancing dysbiosis using Faecalibacterium prausnitzii as a potential active component of probiotic formulations appears to be a promising therapeutic strategy for inflammatory bowel diseases and colorectal cancer. However, once this microbial is very sensitive to oxygen, the formulation development is a great challenge. In this review, we will focus our attention on Faecalibacterium prausnitzii biology, anti-inflammatory metabolites, modulators of this bacterium population and its impact on human health.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 ß-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

Plaquetas: papéis tradicionais e não tradicionais na hemostasia, na inflamação e no câncer / Platelets: traditional and nontraditional roles in hemostasis, inflammation and cancer

A principal e mais conhecida função plaquetária ainda está relacionada à parada de sangramento após um dano vascular. No entanto, plaquetas estão envolvidas em diversos processos, tais como iniciar e amplificar a inflamação, interagir com células da resposta imune, além de participar na progressão tumoral, angiogênese e metástase. Neste sentido, está claro que plaquetas apresentam funções no processo inflamatório e podem influenciar respostas imune, além de desordens plaquetárias autoimune erelacionadas a presença de auto-anticorpos após transfusões, comopor exemplo, na lesão pulmonar aguda associada à transfusão. Após muita especulação, recentes observações têm estabelecido novos paradigmas relacionando plaquetas à biologia molecular. Plaquetas humanas contêm fatores de spliceossomo, incluindo pequenos RNAs nucleares, proteínas de splicing e pre-mRNA endógenos. Outro ponto importante é o controle do número de plaquetas circulantes, resultado do equilíbrio entre a produção e destruição dessas células. Assim, é proposto um processo de morte programada da célula anucleada que determina seu tempo de vida. Esse processo é alvo de especulações desde a década de 60 e ainda permanece em discussão. A noção geral de que plaquetas funcionais são importantes para o sucesso de processos hematogênicos corroboram com inovações experimentais e também ligam a processos de interação plaquetas-células tumorais e seu microambiente que regula a progressão maligna. Plaquetas contribuem na sobrevivência e disseminação de células tumorais. Desta forma, discutimos aqui os mecanismos pelos quais as plaquetas atuam na imunidade, na inflamação e no câncer, uma vez que estas pequenas células são mais versáteis do que se pensava.

The principal and the most known function of platelets still remains stopping hemorrhage following vascular injury. However, platelets are involved in diverse processes such as triggering inflammation, participating in the immune response, besides tumor progression, angiogenesis, and metastasis. In this sense, it is becoming increasingly clear that platelets display inflammatory functions and can influence both innate and adaptive immune responses, such as autoimmune and alloimmune platelet disorders, and transfusion-related acute lung injury (TRALI). Despite much speculation recent observations have established new paradigms relevant to influence of platelets on molecular biology. Primary human platelets contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. Other point is, like all lineages of blood cells, the steady state number of mature platelets is the result of a balance between their production and destruction. Thus, it isproposed a programmed anuclear cell death delimits platelet lifespan is subject of speculation since the 1960s and has remainedelusive. The general notion that functional platelets are importantfor successful hematogenous tumor metastasis dates more than 4 decades and has been corroborated in numerous experimentalsettings. The dynamic crosstalk between tumors and their microenvironment is increasingly recognized as a key regulator ofmalignant progression. These contributions of platelets to tumorcell survival and spread suggest platelets as a new avenue forresearch. Here, we discuss the mechanisms by which plateletscontribute to immunity, inflammation, and cancer, since thesesmall cells are more versatile than we once thought.

Altered of apoptotic markers of both extrinsic and intrinsic pathways induced by hepatitis C virus infection in peripheral blood mononuclear cells.

BACKGROUND: Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U.S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection. METHODS: The present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined. RESULTS: HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). In group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05). CONCLUSIONS: HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.

A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction.

BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B(2) receptors (B(2)R). BbKI was examined on smooth muscle contraction and Ca(2+) mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 µm) increased [Ca(2+)](c) and contraction, as observed with BK (2.0 µm). Not blocked by B(1) receptors (B(1)R), the BbKI agonistic effect was blocked by the B(2)R antagonist, HOE-140 (6 µm), and the involvement of B(2)R was confirmed in B(2)R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca(2+) release from internal storages, as a consequence of its interaction with B(2)R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B(2)R involvement.

Magnetic bead technology for viral RNA extraction from serum in blood bank screening

Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. OBJECTIVE: Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. METHODS: Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. RESULTS: Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. CONCLUSION: The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.

Magnetic bead technology for viral RNA extraction from serum in blood bank screening.

UNLABELLED: Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. OBJECTIVE: Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. METHODS: Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. RESULTS: Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. CONCLUSION: The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.