Fibrin Network and Platelets Densities in Platelet-Rich Fibrin (PRF) Membranes Produced from Plastic Tubes Without Additives: A New Approach to PRF Clinical Use.

Background/Purpose: The present study aimed to investigate plastic tubes without additives as alternatives to glass and silica-coated plastic tubes, in the production of PRF membranes. Materials and Methods: Nine blood samples were collected from eight volunteers (n = 8) separated into three groups, according to tube material: glass, silica-coated plastic, and plastic without additives. In each group, the samples were centrifuged using different relative centrifugation forces: L-PRF (700 g/12 min), A-PRF (200 g/14 min), and A-PRF + (200 g/8 min). The generated membranes were evaluated by histomorphometry, considering the fibrin network, platelet aggregates, and cellular morphology, by light microscopy. The ultrastructural cellular morphology integrity was evaluated by transmission electron microscopy. Results: The L-PRF (p < 0.019) and A-PRF (p < 0.001) membranes showed a significantly lower fibrin network density in plastic tubes without additives compared to glass and silica-coated plastic tubes. Plastic tubes without additives revealed a significantly higher platelet percentage, regardless of the protocol (p < 0.005). In all groups, TEM analysis showed preserved normal morphological ultrastructure, maintaining the integrity of cellular components. Conclusion: Plastic tubes without additives offer a viable alternative for producing PRF membranes. They exhibited a higher platelet density and demonstrated fibrin network and cellular morphology similar to those of glass and silica-coated plastic tubes, irrespective of the centrifugation protocol.

Roles of viable but non-culturable state in the survival of Campylobacter jejuni.

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

Subversion strategies of lysosomal killing by intracellular pathogens.

Many pathogenic organisms need to reach either an intracellular compartment or the cytoplasm of a target cell for their survival, replication or immune system evasion. Intracellular pathogens frequently penetrate into the cell through the endocytic and phagocytic pathways (clathrin-mediated endocytosis, phagocytosis and macropinocytosis) that culminates in fusion with lysosomes. However, several mechanisms are triggered by pathogenic microorganisms - protozoan, bacteria, virus and fungus - to avoid destruction by lysosome fusion, such as rupture of the phagosome and thereby release into the cytoplasm, avoidance of autophagy, delaying in both phagolysosome biogenesis and phagosomal maturation and survival/replication inside the phagolysosome. Here we reviewed the main data dealing with phagosome maturation and evasion from lysosomal killing by different bacteria, protozoa, fungi and virus.

Trypanosoma cruzi P21 recombinant protein modulates Toxoplasma gondii infection in different experimental models of the human maternal-fetal interface.

Introduction: Toxoplasma gondii is the etiologic agent of toxoplasmosis, a disease that affects about one-third of the human population. Most infected individuals are asymptomatic, but severe cases can occur such as in congenital transmission, which can be aggravated in individuals infected with other pathogens, such as HIV-positive pregnant women. However, it is unknown whether infection by other pathogens, such as Trypanosoma cruzi, the etiologic agent of Chagas disease, as well as one of its proteins, P21, could aggravate T. gondii infection. Methods: In this sense, we aimed to investigate the impact of T. cruzi and recombinant P21 (rP21) on T. gondii infection in BeWo cells and human placental explants. Results: Our results showed that T. cruzi infection, as well as rP21, increases invasion and decreases intracellular proliferation of T. gondii in BeWo cells. The increase in invasion promoted by rP21 is dependent on its binding to CXCR4 and the actin cytoskeleton polymerization, while the decrease in proliferation is due to an arrest in the S/M phase in the parasite cell cycle, as well as interleukin (IL)-6 upregulation and IL-8 downmodulation. On the other hand, in human placental villi, rP21 can either increase or decrease T. gondii proliferation, whereas T. cruzi infection increases T. gondii proliferation. This increase can be explained by the induction of an anti-inflammatory environment through an increase in IL-4 and a decrease in IL-6, IL-8, macrophage migration inhibitory factor (MIF), and tumor necrosis factor (TNF)-α production. Discussion: In conclusion, in situations of coinfection, the presence of T. cruzi may favor the congenital transmission of T. gondii, highlighting the importance of neonatal screening for both diseases, as well as the importance of studies with P21 as a future therapeutic target for the treatment of Chagas disease, since it can also favor T. gondii infection.

Rottlerin impairs early and late steps of Toxoplasma gondii infection in human trophoblast cells and villous explants.

Congenital toxoplasmosis, caused by the opportunistic protozoan parasite T. gondii, can cause stillbirths, miscarriages and fetal abnormalities, as well as encephalitis and chorioretinitis in newborns. Available treatment options rely on antiparasitic drugs that have been linked to serious side effects, high toxicity and the development of drug-resistant parasites. The search for alternative therapeutics to treat this disease without acute toxicity for the mother and child is essential for the advancement of current therapeutic procedures. The present study aimed to unravel the mode of the anti-T. gondii action of Rottlerin, a natural polyphenol with multiple pharmacological properties described. Herein, we further assessed the antiparasitic activity of Rottlerin against T. gondii infection on the human trophoblastic cells (BeWo cells) and, for the first time, on human villous explants. We found that non-cytotoxic doses of Rottlerin impaired early and late steps of parasite infection with an irreversible manner in BeWo cells. Rottlerin caused parasite cell cycle arrest in G1 phase and compromised the ability of tachyzoites to infect new cells, thus highlighting the possible direct action on parasites. An additional and non-exclusive mechanism of action of Rottlerin involves the modulation of host cell components, by affecting lipid droplet formation, mitochondrial function and upregulation of the IL-6 and MIF levels in BeWo cells. Supporting our findings, Rottlerin also controlled T. gondii proliferation in villous explants with low toxicity and reduced the IL-10 levels, a cytokine associated with parasite susceptibility. Collectively, our results highlighted the potential use of Rottlerin as a promising tool to prevent and/or treat congenital toxoplasmosis.

Copper sulfate affects Nile tilapia (Oreochromis niloticus) cardiomyocytes structure and contractile function.

Copper sulfate (CuSO(4))is an inorganic chemical product worldwide used as an algaecide and a fungicide in aquaculture and agriculture and being discharged into freshwater environments where it can affect the freshwater fauna, especially fishes. The impact of copper on fish cardiac function was analyzed in two groups of Nile tilapias, Oreochromis niloticus (control group and group exposed to 1 mg l(-1) of CuSO(4) for 96 h). Structural and ultra-structural changes were studied and related to perturbations of the inotropic and chronotropic responses of ventricle strips. Fish of Cu exposed group did not show significant alterations in the medium diameter and in the percentage of collagen in the cardiac myocytes when evaluated through the light microscope. However, the ultrastructural analysis revealed cellular swelling followed by mitochondrial swelling. The myofibrils did not show significant variations among groups. Force contraction was significantly decreased, and rates of time to tension increase (contraction) and decrease (relaxation) were significantly augmented after copper exposure. The results suggest that the copper sulfate impairs the oxidative mitochondrial function and consequently alters the cardiac performance of this species.

Isolation, in vitro propagation, genetic analysis, and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil.

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.

The role of cytoskeleton, components of inositol phospholipid signaling pathway and iron in Ehrlichia canis in vitro proliferation.

Ehrlichia canis, etiologic agent of Canine Monocytic Ehrlichiosis, is an obligatory intracellular bacterium that parasitizes monocytes and macrophages. In this study we analyzed the role of the cytoskeleton specifically actin microfilaments and microtubules, components of inositol phospholipid signaling pathway such as phospholipase C (PLC), protein kinase (PTK) and calcium channels as well as the role of iron in the E. canis proliferation in DH82 cells. Different inhibitory compounds were used for each component: Cytochalasin D (inhibits actin polymerization), Nocodazole (inhibits microtubule polymerization), Neomycin (PLC inhibitor), Genistein (PTK inhibitor), Verapamil (calcium channel blocker) and Deferoxamine (iron chelator). We observed a significant decrease in the total number of bacteria in infected cells treated suggesting that these cellular components analized are essentials to E. canis proliferation.