Evaluation of the genotoxic and mutagenic potentials of homeopathic Candida albicans solutions

Background: Candida spp is naturally found in humans’ flora of skin, gastrointestinal and genitourinary tracts and, in general, up to 75% of the population does not have any symptom [1]. However, oral candidiasis is very common among HIV patients and patients undergoing chemotherapy. The treatment of oral candidiasis is necessary once the disease causes discomfort and dysphagia, resulting in poor nutrition, slow recovery, and prolonged hospital stay [2,3]. Preliminary results obtained by our group with a new biotherapic prepared from Candida albicans (Candida 30x) showed a great potential to reduce the candida yeast adhesion rate when the epithelial cells were pre-treated. This study is currently being developed with the evaluation of mutagenic and genotoxic potentials of several homeopathic solutions. Aims: The goal of this study was to assess the genotoxic and mutagenic potentials of different homeopathic potencies of C. albicans. Methodology: One part of C. albicans yeast obtained from Brazilian patient’s blood [4] was diluted in 9 parts of sterile water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution (1x). Then, 1 ml of this solution was diluted in 9 ml of solvent, submitted to 100 succussions, obtaining 2x potency. This procedure was successively repeated to obtain 30x potency, according to Brazilian Homeopathic Pharmacopoeia [5]. By the same technique, water vehicle was prepared until 30x to be used as control. All samples were prepared in sterile and aseptic conditions, using laminar flow cabinet, class II and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x of C. albicans and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: In the Inductest no decrease in survival fraction of bacteria and no increase in the formation of lysogenic induction were detected independently of the homeopathic potency employed. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: Afterwards, we can conclude that these samples are not able to induce DNA damage in the cells tested. So, the use of this medicine does not present any side effects related to mutagenesis and genotoxicity.

Comparison of the activation capabilities of hemosorbents by adhesion rate of blood cells in vitro

Background: The relevance of the work lies in the search for new hemocontact drugs with hemocompatibility and a pronounced activation effect on the cellular and humoral blood systems for their possible use in clinical practice during low-volume hemoperfusion.The aim of this work was to assess the activation capabilities of three granular hemosorbents by the rate of adhesion of blood cellular elements to the surface of granules in vitro.Materials and methods. When using the method of low-volume hemoperfusion (LVH) in the clinic it is important to take into account the activation properties of solid-phase granular drugs. Blood-contact interaction was carried out in bench conditions with the use of donated blood in rotary mode. Blood samples were taken before the experiment and after 5, 20, 40 and 60 minutes. Changes in blood cell and subcellular populations were evaluated using the Sysmex XT 1800i hematological analyzer (26 parameters), which made it possible to indirectly judge the activation of blood cells. 30 experiments were conducted. To analyze the activation functions of the hemocontact preparations the speed-time adhesive profile of blood cells on the sorbent was used.Results. The effect of using the preparations Silochrome S-120 and SPS in comparison with SСT-6A HP as contact hemoactivators can be more pronounced, since the activation potential of these sorbents for blood cells is much higher. Silochrome S-120 has the highest activation capabilities compared to SPS and SKT-6A HP.Conclusion. Adhesion rate indicators can be indicators of the activation of blood cells upon contact with foreign surfaces and serve as a criterion for assessing the activation capabilities of these surfaces when using the LVH method in the clinic.

Bacterial Adhesion on Prosthetic and Orthotic Material Surfaces

Prosthetic and orthotic parts, such as prosthetic socket and inner sides of orthoses, are often in contact with human skin, giving bacteria the capability to adhere and form biofilms on the materials of those parts which can further cause infections. The purpose of this study was to determine the extent of bacterial adhesion of Staphylococcus aureus and Staphylococcus epidermidis on twelve different prosthetic and orthotic material surfaces and how roughness, hydrophobicity, and surface charge of this materials affect the adhesion. The roughness, contact angle, zeta potential of material surfaces, and adhesion rate of Staphylococcus aureus and Staphylococcus epidermidis were measured on all twelve prosthetic and orthotic materials, i.e., poly(methyl methacrylate), thermoplastic elastomer, three types of ethylene polyvinyl acetates (pure, with low-density polyethylene and with silver nanoparticles), silicone, closed-cell polyethylene foams with and without nanoparticles, thermo and natural cork, and artificial and natural leather. The greatest degree of adhesion was measured on both closed-cell polyethylene foams, followed by artificial thermo cork and leather. The lowest adhesion extent was observed on ethylene-vinyl acetate. The bacterial adhesion extent increases with the increasing surface roughness. Smaller deviations of this rule are the result of the surface’s hydrophobicity and charge.

In vitro hemocompatibility investigation for the development of low-flow centrifugal blood pumps with less platelet clogging

Low-flow blood pumps rated under 1 L/min are emerging for new medical applications, such as hemofiltration in acute use. In those pumps, platelet adhesion and aggregation have to be carefully considered because of clogging risk in the filter part. To find an acceptable hemocompatibility that can be applied to low-flow centrifugal blood pump design, the platelet aggregation index, clogging on a micromesh filter, and the hemolysis index were investigated using a low-flow blood pump designed for hemofiltration use. We conducted circulation testing in vitro using fresh porcine blood and two centrifugal pumps with different impeller inlet shapes. The Negative Log Platelet Aggregation Threshold Index (NL-PATI), which reflects the ability of residual platelets to aggregate, and flow rate were measured during reflux for 60 min, and the Normalized Index of Hemolysis (NIH (g/20 min)) was calculated. In addition, blood cell clogging after reflux was observed on the micromesh filter by SEM, and the adhesion rate was calculated. Our results showed that the platelet clogging on the micromesh filter occurred when the average NL-PATI was greater than 0.28 and the average NIH (g/20 min) was greater than 0.01. In contrast, platelet clogging on the micromesh was suppressed when NL-PATI was less than 0.17 and the NIH (g/20 min) was less than 0.003. These values might be used as acceptable hemocompatibility of low-flow centrifugal blood pumps with suppressed platelet clogging for hemofiltration pumps.

Proposal of a Modified Comprehensive Technique to Avoid Local Scarring and Adhesion Formation for Isolated Gastrocnemius Recession: a Comparison Study.

BackgroundPoor cosmesis is one of the complications following surgical gastrocnemius recession by Strayer procedure. Our present study reported a modified comprehensive technique avoiding skin adhesion in releasing gastrocnemius contracture. A comparison cohort was conducted with inclusion cases via comprehensive modified technique and conventional Strayer procedure. MethodsFrom July 2017 to December 2019, 72 consecutive patients (84 feet) were retrospectively reviewed with 38 cases（42 feet）treated utilizing conventional Strayer procedure and 34 cases（42 feet）treated by modified comprehensive technique. All patients were followed up for minimum 12 months (mean 13.6, range 12 to 18 months). The ankle dorsiflexion range of motion and medical complications (scar adhesion) of above patients were recorded and evaluated. ResultsThe mean ankle dorsiflexion significantly improved from 14.5º±2.5º degrees preoperatively to -18.7º±3.2º degrees in modified procedure group postoperatively (P<0.05), while dorsiflexion improved from 15.2º±3.0º to -19.1º±3.9º in conventional Strayer procedure group. 1 of 42 (2.4%) in modified procedure group developed scar adhesion while 12 of 42 (28.6%) in conventional Strayer procedure group, which showed a statistically significant difference in postoperative complication (scar adhesion) rate between two groups (P<0.05). ConclusionsGastrocnemius recession with modified comprehensive technique can completely release the gastrocnemius aponeurosis and achieve satisfactory recovery of ankle dorsiflexion angle, especially effectively avoid postoperative local scar adhesion with a superior cosmetic appearance.Level of Clinical Evidence: 3

P–403 Sodium tungstate increases embryo adhesion through a direct effect on endometrial cells

Study question Does sodium tungstate treatment induce a change in endometrial cells’ capacity to implant trophoblasts? Summary answer Administration of sodium tungstate to endometrial cells increases trophoblast adhesion. What is known already Sodium tungstate (ST) has shown its capacity to modulate the activity of cytokines, such as leptin, an activator of an obligatory signalling cascade in the embryo-implantation process. STAT3, a signal transducer molecule critical for the embryo implantation process, is also known to be activated by ST. Still, ST’s effect on implantation using biological systems has never been studied. Embryo implantation process and endometrium roles are complicated to study in vivo due to a lack of animal models and appropriate techniques. In vitro techniques using immortalised cell lines allows a first approach to study early implantation stages, such as embryo adhesion. Study design, size, duration An in vitro study was carried out using a human endometrial carcinoma cell line (HEC–1-A) treated with sodium tungstate for 24 and 48h, and choriocarcinoma cell spheroids (JAr). Different times of treatment and concentrations were studied. Each experiment was performed in triplicate. Participants/materials, setting, methods Confluent endometrial HEC–1-A cultures were treated with ST at concentrations (0–150mM) and withaferin A (1mM), negative control for embryo adhesion. After the treatment period, HEC–1-A cultures were washed with ST-free culture medium to eliminate ST. Immediately, 15 JAr trophoblast spheroids were added to cultures and coincubated with gentle agitation for 30, 60 and 90 minutes. An inverted light microscope was used to count adhered and floating spheroids, and determine the trophoblast adherence ratio. Main results and the role of chance HEC–1-A cells treated with ST showed normal morphology and growth at all doses except 150mM. At the highest dose tested, the cells’ culture was still viable (negative blue trypan staining) and maintained morphology, but the adhesion to the plate surface was affected. Doses from 0.15 to 15mM were used to perform adhesion assays. HEC–1-A cells treated with ST for 24h showed an increased capacity to adhere JAr trophoblast spheroids. Adhesion rates reached significant differences at doses of 1.5 and 15mM after 60 and 90 minutes of coincubation. After 90 minutes, untreated cells reached 32.8% adhesion rate, while 1.5 and 15mM ST-treated cells reached 54.6% and 53.4% respectively (p &lt; 0.05 ST vs untreated). Thus, the increment of trophoblast adhesion rate induced by ST reached 66%. Lower adhesion rates were observed after 60 minutes of coincubation but were also significant with a relative increase of 49.1% at 1.5mM and 50.5% at 1.5mM when compared with untreated cells (p &lt; 0.05) Longer treatments (48h) showed similar trends to 24h-treatments, but with a lower extent of ST effect on HEC–1-A receptivity. Maximum adhesion rates were also observed at 90 minutes of coincubation and 1.5 and 15mM doses. The Mean adhesion rate increase was &gt;40% with both doses. Limitations, reasons for caution: The current study is the first approach to evaluate sodium tungstate effect on endometrium using an in vitro model. Future research using in vivo models should be performed to assess sodium tungstate effect on endometrium receptivity and its potential as a fertility treatment. Wider implications of the findings: We conclude that the direct effect of sodium tungstate on endometrial cells increases embryo adhesion rate. These results open a new research line to a potential treatment in human reproduction management with sodium tungstate to solve the unmet need of inducing embryo implantation. Trial registration number Not applicable

Plasmodium vivax Gametocytes Adherence to Bone Marrow Endothelial Cells

In a Plasmodium vivax infection, it was shown a proportionally increased on gametocyte distribution within the bone marrow aspirant, suggesting a role of this organ as a reservoir for this parasite stage. Here, we evaluated the ex vivo cytoadhesive capacity of P. vivax gametocytes to bone marrow endothelial cells (HBMEC) and investigated the involvement of some receptors in the cytoadhesion process by using transfected CHO cells (CHO-ICAM1, CHO-CD36 and CHO-VCAM), wild type (CHO-K1) or deficient in heparan and chondroitin sulfate (CHO-745). Ex-vivo cytoadhesion assays were performed using a total of 44 P. vivax isolates enriched in gametocyte stages by Percoll gradient in the different cell lines. The majority of isolates (88.9%) were able to adhere to HBMEC monolayer. ICAM1 seemed to be the sole receptor significantly involved. CD-36 was the receptor with higher adhesion rate, despite no significance was noticed when compared to CHO-745. We demonstrated that gametocyte P. vivax adheres ex vivo to bone marrow endothelial cells. Moreover, P. vivax gametocytes display the ability to adhere to all CHO cells investigated, especially to CHO-ICAM1. These findings bring insights to the comprehension of the role of the bone marrow as a P. vivax reservoir and the potential impact on parasite transmission to the vector.

Investigation of Commensal Escherichia coli Populations of Cormorant Hatchlings in the Absence of Anthropogenic Impacts in Remote Areas of West Mongolia

To increase our understanding of bacterial intestinal colonization in animal populations lacking substantial anthropogenic influence we studied the diversity of E. coli in cormorants from the pristine West-Mongolian steppe. E. coli were isolated from individual birds of two cormorant colonies located on small islands in lakes at least 100 km away from human settlements. Diversity of the isolates was studied using pulsed-field gel electrophoresis (PFGE). 137 isolates of cormorant colony-1 and 75 isolates of cormorant colony-2 resulted in 60 and 33 PFGE types, respectively. Representative strains of each PFGE type were analyzed via PCR in terms of phylogroups and extraintestinal virulence-associated genes (exVAGs). Bacterial adhesion to the chicken intestinal cell line CHIC-8E11 and antimicrobial resistance was also determined. Most isolates belonged to phylogroup B1 (68.3%) followed by B2 and E with B2 harboring the highest total number of exVAGs per isolate. Unexpectedly, a PFGE type with relatively few exVAGs displayed the highest isolation frequency, also showing a high adhesion rate. Comparative analysis of exVAGs to other E. coli populations of wildlife origin revealed that the secreted autotransporter toxin encoding sat gene was only present in cormorants. Overall, E. coli in cormorants maintained a high diversity under minimal anthropogenic influences, which likely enables intestinal colonization.

Streptococcus mutans adherence to conventional and self-ligating brackets: an in vitro study

ABSTRACT Introduction: Although self-ligating brackets presumably provide better hygiene conditions, no consensus has been reached so far. Objective: Therefore, the objective of this study was to evaluate, in an in vitro experimental design, the adherence of Streptococcus mutans (SM) in self-ligating and conventional brackets of different manufacturers and ligature types. Methods: Four commercial brands of maxillary premolar metal brackets were tested (Abzil®; Morelli®; 3M Unitek®; and GAC®). Each one was subdivided into three groups, which varied according to the type of ligature and bracket model (metallic, elastic, and self-ligating), totalizing twelve groups, composed of six brackets each. Previously sterilized brackets were initially immersed in saliva for one hour, and subsequently washed and added in a bacterial suspension, maintained in aerobiosis for 72 hours. The adhered bacteria were then separated and quantified by colony forming units (CFU/mL) counting after 48 hours of growth. The groups were compared by Kruskal-Wallis and Dunn post-hoc tests (p< 0.05). Results: Regardless of the commercial brand, self-ligating brackets had significantly less CFU/mL. However, according to comparisons performed within each commercial brand, only Abzil® self-ligating brackets had significantly lower biofilm adhesion. Among all of the self-ligating models, GAC® brackets presented the highest bacterial adhesion rate. Conclusions: Self-ligating brackets are likely to present lower rates of biofilm adhesion. Particularly, Abzil® and GAC® self-ligating brackets are less likely to accumulate biofilm. Although such results are derived from an in vitro study, practitioners might acknowledge findings concerning bacterial adhesion as one of the relevant features to be considered during bracket selection.

A microfluidic platform for in situ investigation of biofilm formation and its treatment under controlled conditions

Background Studying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions. Results Using Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium (modified M9 minimal medium) was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium (tryptic soy broth rich medium ). However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials. Conclusion The platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.