Tissue Sterilization Research Articles

Machine learning models reveal microbial signatures in healthy human tissues, challenging the sterility of human organs

BackgroundThe presence of microbes within healthy human internal organs still remains under question. Our study endeavors to discern microbial signatures within normal human internal tissues using data from the Genotype-Tissue Expression (GTEx) consortium. Machine learning (ML) models were developed to classify each tissue type based solely on microbial profiles, with the identification of tissue-specific microbial signatures suggesting the presence of distinct microbial communities inside tissues.MethodsWe analyzed 13,871 normal RNA-seq samples from 28 tissues obtained from the GTEx consortium. Unaligned sequencing reads with the human genome were processed using AGAMEMNON, an algorithm for metagenomic microbial quantification, with a reference database comprising bacterial, archaeal, and viral genomes, alongside fungal transcriptomes. Gradient-boosting ML models were trained to classify each tissue against all others based on its microbial profile. To validate the findings, we analyzed 38 healthy living tissue samples (samples from healthy tissues obtained from living individuals, not deceased) from an independent study, as the GTEx samples were derived from post-mortem biopsies.ResultsTissue-specific microbial signatures were identified in 11 out of the 28 tissues while the signatures for 8 tissues (Muscle, Heart, Stomach, Colon tissue, Testis, Blood, Liver, and Bladder tissue) demonstrated resilience to in silico contamination. The models for Heart, Colon tissue, and Liver displayed high discriminatory performance also in the living dataset, suggesting the presence of a tissue-specific microbiome for these tissues even in a living state. Notably, the most crucial features were the fungus Sporisorium graminicola for the heart, the gram-positive bacterium Flavonifractor plautii for the colon tissue, and the gram-negative bacterium Bartonella machadoae for the liver.ConclusionThe presence of tissue-specific microbial signatures in certain tissues suggests that these organs are not devoid of microorganisms even in healthy conditions and probably they harbor low-biomass microbial communities unique to each tissue. The discoveries presented here confront the enduring dogma positing the sterility of internal tissues, yet further validation through controlled laboratory experiments is imperative to substantiate this hypothesis. Exploring the microbiome of internal tissues holds promise for elucidating the pathophysiology underlying both health and a spectrum of diseases, including sepsis, inflammation, and cancer.

Development of a sterilization process for amniotic membrane allograft tissue using supercritical carbon dioxide and NovaKill

Amniotic membrane is arguably one of the most popular biological wound dressings on the market today. Various growth factors and cytokines inherent to amniotic membrane tissue have been recognized as key mediators in wound healing and tissue regeneration, giving the tissue its clinical utility. Sterilization methodologies using irradiation are recognized as the gold standard in the field and routinely used to prepare tissue allografts, including amniotic membrane for transplantation. However, irradiation is not always compatible in preserving the physical structure or biochemical factors of biological materials and can potentially result in detrimental effects to the critical quality attributes of allograft tissues. Alternatively, a novel sterilization technique involving supercritical carbon dioxide (SCCO2) has been shown to have minimal effect on the inherent biophysical properties of sensitive biological tissues and tissue-derived products. At BioBridge Global, we have developed a process utilizing SCCO2 technology for the sterilization of an amniotic membrane tissue allograft product. This process, first and foremost, meets industry standards for sterilization while simultaneously maintaining the biochemical composition of the tissue. Our results show that upon SCCO2 sterilization, most of the growth factors tested were conserved, with many at quantities significantly greater than commercially available gamma and electron beam irradiated tissue. The SCCO2-sterilized amniotic membrane allograft is unique in that it is designed to overcome limitations associated with traditional tissue sterilization methodologies, namely, the conservation of key biological factors inherent to native amniotic membrane tissue. It is anticipated that by retaining these biological factors, clinical outcomes associated with the use of SCCO2-sterilized amniotic membrane will be improved.

Identification of Bacterial Endophytes Isolated from Different Medicinal Plants

Plants have very potential compounds to live on earth as they supply 90% of human calorie intake, 80% of protein intake directly, and perhaps the most vital sources of medicine with a vast diversity of microorganisms. As such it’s important to know those microorganisms, their kinds, the features they possess, and the significant compounds/metabolites they can produce. So, this study is based on identifying such microorganisms. To achieve this aim, isolation of endophytes has been done to know their biochemical activities and properties. Various identification procedures have been followed to get pure endophytic strains without any contamination. Surface sterilization of the plant tissue is a must in this progress, various surface sterilization techniques have been tried and finally, for 4/5 plant tissues, sodium hypochlorite and ethanol were given the best result and for 1/5 with the addition of mercuric chloride were the standardized method for surface sterilization. About 30 different bacterial endophytes have been isolated from five kinds of medicinal plants. 4% sodium hypochlorite and 75% ethanol were found effective in sterilizing the surface of Psidium guajava, Cassia occidentalis, Calotropis procera, and Hibiscus rosa-sinensa. While Mangifera indica required an addition of 0.1% mercuric chloride. 19 strains isolated were Gram-positive, 11 Gram-negative (5 were Lactose fermenters and 6 were not), and most of which were bacilli. All isolates have shown different biochemical results, 25 showed a positive result for oxidase, and 28 gave a positive result for catalase. Most of the endophytes identified in this work are Bacillus spp. However, this study highlighted the significance of surface sterilisation and most importantly the presence of potential endophytes capable of producing novel bioactive compounds usable in pharmaceutical/medicinal application.

Investigating the Influence of Temperature and Supplementation Timing on Antifungal Efficacy in Storage Medium for Corneal Transplantation.

Although antifungal supplementation reduces the fungal load in the corneal storage medium, consensus is lacking on the influence of dosing and temperature. The study aims to evaluate the impact of eye bank warming protocol and timing of antifungal supplements on efficacy in Optisol-GS and tissue. Corneoscleral rims contaminated with Candida albicans (C.albicans) were incubated in Optisol-GS, either without antifungal agents or with the addition of amphotericinB or voriconazole at various concentrations (2 ×, 5 ×, 10 ×, and 20 × MIC), at different time points, and under various preservation temperatures (2-8°C versus 2h-room temperature exposure). Antifungal efficacy was evaluated by counting viable yeast colonies cultured from Optisol-GS samples. Tissue sterility was determined through direct tissue culture and histological examination of the contaminated rims after a 14-day incubation period. Room temperature exposure did not increase colony growth at the same multiple MIC of antifungal agents. Although antifungal addition reduced C.albicans growth in a concentration-dependent manner, yeast growth was still observed in all Optisol-GS samples with a single supplementation after a 14-day incubation. Only groups with additional antifungal supplementation on either day2 or day6 showed a 99% or greater reduction of C.albicans growth in Optisol-GS samples and yielded negative results in direct tissue culture. The eye bank warming protocol did not compromise antifungal efficacy. To sustain the required concentration and effectively reduce C.albicans growth in Optisol-GS and contaminated tissue, additional antifungal supplementation on either day2 or day6 was necessary during a 2-week preservation period.

High pressure pasteurization: Simultaneous native tissue decellularization and sterilization

IntroductionTerminal sterilization is important for the clinical applicability of decellularized xenografts. High hydrostatic pressurization (HHP) process is a potential strategy for decellularization and decontamination of xenografts; however, its disinfection efficiency remains poorly elucidated. This study investigated the disinfection efficacy of the HHP process at physiologically relevant 36 °C against difficult-to-kill spore-forming bacteria. MethodsBacillus atrophaeus and Geobacillus stearothermophilus were suspended in a pressurization medium with or without antibiotic agents and pressurized under two different HHP procedures: repeated and sustained pressurization. ResultsThe sustained pressurizing conditions, exploited for the conventional tissue decellularization, did not effectively eliminate the bacteria; however, repeated pressurization greatly increased the disinfection effect. Moreover, the antibiotic-containing pressurization medium further increased the disinfection efficiency to the level required for sterilization. ConclusionsThe optimized high hydrostatic pressurization can be used to sterilize biological tissues during the decellularization process and is a promising strategy for manufacturing tissue-derived healthcare products.

Microorganisms inside healthy human organs, are the internal tissues sterile?

The recognition of microorganisms inhabiting the gut, urogenital system, skin, and lungs of humans has become widely accepted. Various studies provide evidence of microbial infiltration in tumor tissues, underscoring the potential role of the microbiome in cancer progression. Recent scientific inquiries challenge the traditional belief in the sterility of normal internal human tissues, presenting compelling findings about the existence of microbial communities within diverse internal organs. This perspective, though relatively unexplored, aligns with research in other species, such as mice. To delve deeper into the microbial presence in healthy human internal tissues, an analysis was conducted on 13,871 normal RNA-seq samples from 28 tissues obtained from the Genotype-Tissue Expression (GTEx) consortium. Acknowledging certain limitations within the GTEx analysis pipeline, it remains noteworthy that the GTEx consortium provides the most extensive and analytically robust dataset about RNA expression within healthy human tissues. Each sample's sequencing reads, not aligning with the human genome (assembly T2T-CHM13v2.0), underwent analysis using AGAMEMNON, an algorithm for aligning and quantifying microorganism abundance at various taxonomic levels. Reference database included 4034 bacterial, 489 archaea, and 11,259 viral genomes, along with 81 fungal transcriptomes. After cumulative sum scaling (CSS) normalization on species-level microbial abundances and retaining only species present in at least 10% of the samples for each tissue, gradient-boosting machine learning models were trained for each tissue. These models aimed to distinguish each tissue from all others based solely on their microbial profile. To validate the results, 38 healthy living tissue samples (from NCBI project PRJEB4337) were analyzed, as the GTEx samples were derived from post-mortem biopsies. The machine learning models exhibited robust performance for 11 out of 28 tissues (Muscle, Heart, Salivary Gland, Brain, Stomach, Colon, Small Intestine, Testis, Blood, Liver, and Bladder). Among these, 8 models demonstrated resilience to in silico contamination. The presence of resilient tissue-specific microbial signatures implies that microbial colonization is not a random occurrence. Testing the ML models on the external dataset of the 38 healthy living samples revealed high discriminatory performance for the Heart, Liver and Colon tissue, indicating the presence of a tissue-specific microbiome even in a living state for these tissues. Notably, the most crucial feature in the heart model was the fungus Sporisorium graminicola, for the colon model the gram-positive bacterium Flavonifractor plautii, and for the liver model was the gram-negative bacterium Bartonella machadoae. The presence of a distinct microbial signature within human internal organs, allowing differentiation of distinct organs through machine learning models, supports the assertion that even under healthy conditions, human internal organs host low-biomass microbial communities specific to each tissue. "BioCancer", “Bioradio 4”. University of Pennsylvania HEALTH SYSTEM (UPenn School of Medicine RedOnc), “Decision support systems to address Nosocomial Infections and Antimicrobial Resistance (MDR-CDSS).”. ‘Competitiveness, Entrepreneurship and Innovation’ [NSRF 2014 —2020], Greece. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

UV–visible spectral changes of gamma irradiated olive leaves extract

Olive leaves benefits are less well known, despite having potential health benefits that could make them a valuable addition to a daily diet, compared to olive oil, which is well known for the many benefits it provides. The antioxidant capacity of olive leaves extract (OLE) is higher than that of the most known powerful antioxidants, such as vitamins C and E or pure hydroxytyrosol. The aim of this study was first to maximize the bioactive compounds in extra virgin olive oil (EVOO) by enriching it with olive leaves; the EVOO acts as an extraction solvent, and a study of the macerating period was carried out to select the best macerating period to improve the stabilization of the bioactive compounds in the olive oil matrix. Once the best macerating period was selected (7 days), OLE was gamma-irradiated with a dose of 25 kGy to assess its oxidative stability. To this end, UV–visible spectroscopy was used to perform stability and quality analysis of the OLE during a macerating period of 21 days and after irradiation. The results of this study show an extremely significant (p < 0.0001) improvement in the quality parameters (K232, K270, and ΔK) and in total carotenoids and chlorophylls, assessed by K470 and K670, among all the macerating periods of OLE compared to the control group. Regarding the irradiated OLE compared to the irradiated EVOO, there was an extremely significant (p < 0.0001) difference in ΔK. In addition, there was a significant decrease in total carotenoids and chlorophylls in the irradiated EVOO compared to the irradiated OLE. In conclusion, olive leaves extract in olive oil could be used as a free radical scavenger in food irradiation and tissue sterilization to alleviate the detrimental effects of ionizing radiation.

Preservation of Mechanical and Morphological Properties of Porcine Cardiac Outflow Vessels after Decellularization and Wet Storage.

Widely used storage methods, including freezing or chemical modification, preserve the sterility of biological tissues but degrade the mechanical properties of materials used to make heart valve prostheses. Therefore, wet storage remains the most optimal option for biomaterials. Three biocidal solutions (an antibiotic mixture, an octanediol-phenoxyethanol complex solution, and a glycerol-ethanol mixture) were studied for the storage of native and decellularized porcine aorta and pulmonary trunk. Subsequent mechanical testing and microstructural analysis showed a slight increase in the tensile strength of native and decellularized aorta in the longitudinal direction. Pulmonary trunk elongation increased 1.3-1.6 times in the longitudinal direction after decellularization only. The microstructures of the tested specimens showed no differences before and after wet storage. Thus, two months of wet storage of native and decellularized porcine aorta and pulmonary trunks does not significantly affect the strength and elastic properties of the material. The wet storage protocol using alcohol solutions of glycerol or octanediol-phenoxyethanol mixture may be intended for further fabrication of extracellular matrix for tissue-engineered biological heart valve prostheses.

Comparison of the Effects of Tissue Processing on the Physicochemical Properties of Bone Allografts.

Purpose: To address the hypothesis that the tissue processing methods of solvent dehydration and freeze-drying would differentially affect the physicochemical characteristics of four commercially available bone allografts and the adhesion and differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSCs) on such substrates in vitro. Materials and Methods: The surface morphology, surface area, and elemental composition of four commercially available cancellous bone allografts were examined using SEM, Brunauer-Emmett-Teller (BET) gas adsorption, and inductively coupled plasma (ICP) analyses. SEM was also employed to compare the allograft surfaces to that of human bone exposed by in vitro osteoclastic resorption. The allografts were seeded with hBMSCs, and the number of adhered cells was assessed at 3 and 7 days. Alkaline phosphatase (ALP) activity was quantified as a measure of osteogenic differentiation after 21 days. Results: Marked differences were seen between the physicochemical characteristics of the solvent-dehydrated and freeze-dried allografts, as well as between their resulting bone microarchitectures and that of osteoclast-resorbed human bone. Increased hBMSC adhesion and differentiation were observed on the solvent-dehydrated allografts compared to freeze-dried allografts, which suggests a higher putative osteogenic potential. The latter was attributed to better preservation of the bone collagen microarchitecture integrity, which may provide not only a more complex substrate architecture, but also a more favorable microenvironment to allow nutrients and oxygen to flow to the adhered cells. Conclusion: Commercially available cancellous bone allografts significantly differ in their physicochemical characteristics, stemming from differences in tissue processing and sterilization methods undertaken by tissue banks. These differences impact the response of MSCs in vitro and may alter the biologic performance of the grafts in vivo. Therefore, it is important to consider these characteristics when choosing a bone substitute for clinical application, as the physicochemical properties of the grafts play a crucial role in their interactions with the biologic environment and subsequent incorporation into the native bone.

Influence of Non-Thermal Plasma (DBD) On Infertility Male Semen with Low Sperm Motility and Dna Damage

Non-thermal plasma(Dielectric barrier discharge) has many uses including living tissue sterilization, inactivation of the bacteria, excimer formation, angiogenesis, and surface treatment. This research aim is to use cold plasma as a tool to search the effect of the dielectric barrier discharge system at room temperature on human sperm motility and DNA integrity. This work was performed on 60 human semen samples suffering from low motility; each sample was prepared by centrifugation method, then each semen sample was divided into two sections, the first section is before significant exposure to the plasma system (DBD) and the second section is after treatment with the DBD system at ambient temperature. Before and after exposure to non-thermal Plasma (DBD), DNA integrity and sperm motility were assessed, patients suffered from asthenospermia has a high level of DNA fragmentation than fertile male, ( 24.16±4.14) p&lt;0.001 for untreated and after treatment the semen slide with dielectric barrier discharge the percentage decreases to (9.16±1.76) p&gt;0.006, and the comet slide was (35.44±4.15) then the percentage decline to (19.86 ±2.44) these results have shown that cold plasma improves sperm motility and decreases from DNA damage in patients with medium and high level of DNA damage.

Application of Tissue-Specific Extracellular Matrix in Tissue Engineering: Focus on Male Fertility Preservation.

In vitro spermatogenesis and xenotransplantation of the immature testicular tissues (ITT) are the experimental approaches that have been developed for creating seminiferous tubules-like functional structures in vitro and keeping the integrity of the ITTs in vivo, respectively. These strategies are rapidly developing in response to the growing prevalence of infertility in adolescent boys undergoing cancer treatment, by the logic that there is no sperm cryopreservation option for them. Recently, with the advances made in the field of tissue engineering and biomaterials, these methods have achieved promising results for fertility preservation. Due to the importance of extracellular matrix for the formation of vascular bed around the grafted ITTs and also the creation of spatial arrangements between Sertoli cells and germ cells, today it is clear that the scaffold plays a very important role in the success of these methods. Decellularized extracellular matrix (dECM) as a biocompatible, functionally graded, and biodegradable scaffold with having tissue-specific components and growth factors can support reorganization and physiologic processes of originated cells. This review discusses the common protocols for the tissue decellularization, sterilization, and hydrogel formation of the decellularized and lyophilized tissues as well as in vitro and in vivo studies on the use of the testis-derived dECM for testicular organoids.

Influence of X-rays and gamma-rays on the mechanical performance of human bone factoring out intraindividual bone structure and composition indices.

Doses of irradiation above 25 ​kGy are known to cause irreversible mechanical decay in bone tissue. However, the impact of irradiation doses absorbed in a clinical setting on the mechanical properties of bone remains unclear. In daily clinical practice and research, patients and specimens are exposed to irradiation due to diagnostic imaging tools, with doses ranging from milligray to Gray. The aim of this study was to investigate the influence of irradiation at these doses ranges on the mechanical performance of bone independent of inter-individual bone quality indices.Therefore, cortical bone specimens (n ​= ​10 per group) from a selected organ donor were irradiated at doses of milligray, Gray and kilogray (graft tissue sterilization) at five different irradiation doses. Three-point bending was performed to assess mechanical properties in the study groups.Our results show a severe reduction in mechanical performance (work to fracture: 50.29 ​± ​11.49 Nmm in control, 14.73 ​± ​1.84 Nmm at 31.2 ​kGy p ​≤ ​0.05) at high irradiation doses of 31.2 ​kGy, which correspond to graft tissue sterilization or synchrotron imaging. In contrast, no reduction in mechanical properties were detected for doses below 30 ​Gy. These findings are further supported by fracture surface texture imaging (i.e. more brittle fracture textures above 31.2 ​kGy).Our findings show that high radiation doses (≥31.2 ​kGy) severely alter the mechanical properties of bone. Thus, irradiation of this order of magnitude should be taken into account when mechanical analyses are planned after irradiation. However, doses of 30 ​Gy and below, which are common for clinical and experimental imaging (e.g., radiation therapy, DVT imaging, CT imaging, HR-pQCT imaging, DXA measurements, etc.), do not alter the mechanical bending-behavior of bone.

Critical media attributes in E-beam sterilization of corneal tissue

When ionizing irradiation interacts with a media, it can form reactive species that can react with the constituents of the system, leading to eradication of bioburden and sterilization of the tissue. Understanding the media's properties such as polarity is important to control and direct those reactive species to perform desired reactions. Using ethanol as a polarity modifier of water, we herein generated a series of media with varying relative polarities for electron beam (E-beam) irradiation of cornea at 25 kGy and studied how the irradiation media's polarity impacts properties of the cornea. After irradiation of corneal tissues, mechanical (tensile strength and modulus, elongation at break, and compression modulus), chemical, optical, structural, degradation, and biological properties of the corneal tissues were evaluated. Our study showed that irradiation in lower relative polarity media improved structural properties of the tissues yet reduced optical transmission; higher relative polarity reduced structural and optical properties of the cornea; and intermediate relative polarity (ethanol concentrations = 20-30% (v/v)) improved the structural properties, without compromising optical characteristics. Regardless of media polarity, irradiation did not negatively impact the biocompatibility of the corneal tissue. Our data shows that the absorbed ethanol can be flushed from the irradiated cornea to levels that are nontoxic to corneal and retinal cells. These findings suggest that the relative polarity of the irradiation media can be tuned to generate sterilized tissues, including corneal grafts, with engineered properties that are required for specific biomedical applications. Statement of significanceExtending the shelf-life of corneal tissue can improve general accessibility of cornea grafts for transplantation. Irradiation of donor corneas with E-beam is an emerging technology to sterilize the corneal tissues and enable their long-term storage at room temperature. Despite recent applications in clinical medicine, little is known about the effect of irradiation and preservation media's characteristics, such as polarity on the properties of irradiated corneas. Here, we have showed that the polarity of the media can be a valuable tool to change and control the properties of the irradiated tissue for transplantation.

Major approaches to antiseptic management for endodontic treatment in the COVID-19 pandemic: a guideline

Introduction: In the COVID-19 pandemic scenario, in addition to the pathogenesis of SARS-CoV-2, microbial coinfection increases the difficulties of diagnosis, treatment, the prognosis of COVID-19, as well as it can worsen comorbidities and affect the risk of the life of patients. COVID-19 has had a profound impact on dentistry. In addition to endodontic treatment, a management protocol was suggested. Objective: To present the importance of effectively performing endodontic asepsis in the context of the COVID-19 pandemic, to elucidate that infection by the SARS-CoV-2 virus can lead to coinfection, worsening the conditions for endodontic treatment. Methods: The research was carried out from July 2021 to August 2021 and developed based on Scopus, PubMed, Science Direct, Scielo, and Google Scholar, following the Systematic Review-PRISMA rules. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results: A total of 70 articles were found involving the endodontic treatment and COVID-19. A total of 58 articles were evaluated in full and 39 were included and evaluated in the present study. It was found that ozone has high antimicrobial action. N-acetylcysteine (NAC) has a potent effect against endodontic biofilms. Calcium hydroxide is more effective as a root canal disinfectant in primary teeth than formocresol and camphorphenol. The association of 2% chlorhexidine followed by ozone gas for 24 seconds promoted the complete elimination of Candida albicans and Enterococcus faecalis. Low-intensity laser therapy has the property of oral sterilization, facilitating tissue healing and sterilization. Combining antimicrobial photodynamic therapy with antimicrobial irrigants may provide a synergistic effect. Conclusion: There are effective treatments for the sterilization of endodontic tissues, to avoid as much as possible the coinfection with SARS-CoV-2 and the consequent worsening of the infectious condition, highlighting calcium hydroxide, ozone therapy, and laser therapy.

Photodynamic Therapy in the Extracellular Matrix of Mouse Lungs: Preliminary Results of an Alternative Tissue Sterilization Process

Lung transplantation is one of the most difficult and delicate procedures among organ transplants. For the success of the procedure and survival of the new organ, the sterilization step for acellular lungs prior to recellularization is important to ensure that they are free of any risk of transmitting infections from the donor to the recipient subject. However, there are no available information concerning the lung mechanical parameters after sterilizing photodynamic therapy. The aim of this study was to evaluate the extracellular matrix (ECM) and lung mechanical parameters of decellularized lungs undergoing sterilizing photodynamic therapy (PDT). Besides, we also analyzed the lung after controlled infection with C. albicans in order to evaluate the effectiveness of PDT. The lung mechanical evaluation parameters, resistance ( R L ) and elastance ( E L ), exhibited no significant differences between groups. In addition, there were no PDT-induced changes in lung properties, with maintenance of the viscoelastic behavior of the lung scaffold after 1 h exposure to PDT. The ECM components remained virtually unchanged in the acellular lungs of both groups. We also showed that there was a reduction in fungal infection population after 45 minutes of PDT. However, more studies should be performed to establish and verify the effectiveness of PDT as a possible means for sterilizing lung scaffolds. This manuscript was presented as a master thesis of Nadua Apostólico at the postgraduate program in rehabilitation sciences, University Nove de Julho—UNINOVE.

Manufacturing freshly cultured umbilical cord-derivedmesenchymal stromal cells (UC-MSCS) for a phase 1, multiple-dose clinical trial for COVID-19-induced acute respiratory distress syndrome (ARDS)

Background & Aim: The Cellular Immuno-Therapy for COVID-19 related ARDS (CIRCA-19) was a phase 1, single site, dose escalation trial using a 3+3+3 design to determine the safety and maximum feasible tolerated dose of intravenously delivered, freshly cultured UC-MSCs. Nine patients, each receiving repeated unit doses of UC-MSCs over 3 consecutive days, were enrolled into 3 dose panels: Panel 1: 25×106 cells/dose (cumulative dose: 75×106 MSCs);Panel 2: 50×106 cells/dose (cumulative dose: 150×106 MSCs);Panel 3: up to 90×106 cells/dose (cumulative dose: 270×106 MSCs). Methods, Results & Conclusion: UC-MSCs were isolated from cords of healthy term pregnancies delivered by C-section. Cords were mechanically and enzymatically digested, and UC-MSCs were propagated in xeno-free conditions for 2 weeks prior to cryopreservation in a cord specific cell bank. One fully validated cell bank was used in CIRCA-19 that was free of adventitious agents (HBV, HCV, HSV1/2, Parvo B19 and Retroviruses), had high viability (>95%) and MSC identity with positive expression (>95%) of CD73, CD90 and CD105 and negative expression ( 45%;DBT = 22h) and enhanced IDO expression (ΔΔCq?>18) when treated with IFN-γ. For the final product, UC-MSCs were thawed, plated and cultured for 24 to 120 h before harvesting to produce a batch of the final drug product formulated as 2.5×106 fresh UC-MSCs/mL suspended in PlasmaLyte A containing 5% Human Albumin, to be infused within 48h. Batches were tested for viability, endotoxin level, ACE-2 expression, tissue factor activity, sterility and mycoplasma. Sixteen batches of UC-MSCs were produced for a total of 41 cell doses (16 doses of 25M;13 doses of 50M;12 doses of 90M cells each). Twenty-seven of the 41 doses (9 doses of 25M, 50M and 90M cells each) were used to treat trial participants. The remaining doses were used for stability studies. All drug products had high viability (> 95%), endotoxin levels of 35;GAPHD Cq: 15±2;no detectable levels by western blotting) and had tissue factor activity levels between 250-310pM. UC-MSC drug product was stable for up to 96h (>80% viability) and had?>90% viability up to 48h in all 3 dose panels. This study demonstrates the feasibility of manufacture and delivery of a multi-dose fresh cell product in an emergent ICU setting.

A three-dimensional habitat for C. elegans environmental enrichment

As we learn more about the importance of gene-environment interactions and the effects of environmental enrichment, it becomes evident that minimalistic laboratory conditions can affect gene expression patterns and behaviors of model organisms. In the laboratory, Caenorhabditis elegans is generally cultured on two-dimensional, homogeneous agar plates abundantly covered with axenic bacteria culture as a food source. However, in the wild, this nematode thrives in rotting fruits and plant stems feeding on bacteria and small eukaryotes. This contrast in habitat complexity suggests that studying C. elegans in enriched laboratory conditions can deepen our understanding of its fundamental traits and behaviors. Here, we developed a protocol to create three-dimensional habitable scaffolds for trans-generational culture of C. elegans in the laboratory. Using decellularization and sterilization of fruit tissue, we created an axenic environment that can be navigated throughout and where the microbial environment can be strictly controlled. C. elegans were maintained over generations on this habitat, and showed a clear behavioral bias for the enriched environment. As an initial assessment of behavioral variations, we found that dauer populations in scaffolds exhibit high-frequency, complex nictation behavior including group towering and jumping behavior.

168. Efficacy of the Novel gwt1 Inhibitor APX2039 in a Rabbit Model of cryptococcus Meningitis

BackgroundCryptococcal meningitis (CM), caused primarily by Cryptococcus neoformans, is uniformly fatal if not treated. Treatment options are limited especially in resource-poor geographical regions, and mortality rates remain high despite current therapies. New oral treatment options are needed that demonstrate rapid reductions in CFU in CSF and brain tissue.APX2039 is a novel inhibitor of the fungal Gwt1 enzyme, which catalyzes an early step in glycosylphosphatidyl inositol (GPI) anchor biosynthesis. It is highly active against both C. neoformans and C. gattii and has previously demonstrated significant efficacy in a mouse delayed-treatment model of CM.CSF Fungal Burden in Rabbits MethodsMale New Zealand White rabbits were inoculated with C. neoformans H99 (1.4 ×106 CFU) directly into the cisterna magna. Rabbits were immunosuppressed with cortisone acetate at 7.5 mg/kg (i.m.), starting on Day -1 relative to inoculation and then administered drug daily throughout the 14-day experimental period. Treatment was initiated on Day 2 postinfection and continued through Day 14 consisting of: 50 mg/kg APX2039 PO (BID), 80 mg/kg fluconazole (FLU) PO (QD), c) 1 mg/kg amphotericin B deoxycholate (AMB) IV (QD); and vehicle control. CSF was removed via an intracisternal tap on Days 2, 7, 10 and 14 post-infection and CFU/ml was assessed. Animals were sacrificed on Day 14 and CFU/g brain tissue was assessed.ResultsAPX2039 demonstrated rapid reduction in CFU in both CSF and brain tissue. The range in CFU values in rabbit CSF is shown (Figure). Reductions in CFU were statistically different from the control group for all treatment groups. APX2039 was also different from both FLU and AMB and resulted in sterilization in CSF by Day 10. Brain harvested on Day 14 demonstrated a reduction in CFU/g tissue vs control of 1.8 log10 and 3.4 log10 for FLU and AMB, respectively, while a > 6 log10 reduction (tissue sterilization) was observed for APX2039.ConclusionAPX2039 demonstrated potent efficacy in a rabbit model of CM. The more rapid clearance in CSF than either AMB or FLU, as well as > 6 log10 reduction in brain CFU highlights the unique properties of this drug, warranting further investigation of this molecule for the treatment of CM.Disclosures Karen J. Shaw, PhD, Amplyx (Consultant)Forge Therapeutics (Consultant) Charles D. Giamberardino, Jr., MR, Box (Shareholder) John R. Perfect, MD, amplyx (Grant/Research Support)astellas (Grant/Research Support)astellas (Grant/Research Support)

KAJIAN ARTIKEL: POTENSI PLASMA NON TERMAL SEBAGAI KANDIDAT TERAPI MASTITIS SUBKLINIS

Subclinical mastitis is the occurrence of inflammation in the parenchymal tissue of mammaryglands due to bacterial contamination that does not show clinical symptoms lead to impacted oneconomic losses. The highest incidence of subclinical mastitis is the presence of Staphylococcusaureus bacteria contamination. Subclinical mastitis in Indonesia is reported to reduce milk productionby up to 53.5%. Pathogenesis of Staphylococcus aureus occurs in the presence of biofilm formation asbacterial immune system to protecting itself from disinfectants and antibiotics. In the last few decades,the development of plasma technology has been widely used in the medical field as cancer therapy,reducing pain, accelerating wound healing and tissue sterilization. There is no study that using theconcept of utilizing Non Termal Plasma in the field of veterinary science, especially in mastitistherapy. Non Termal Plasma is able to decontaminate Staphylococcus aureus through inhibition anddestruction of biofilms. The mechanism of action of Non Termal Plasma in repairing mammary glandtissue is through the destruction of DNA in the Reactive Oxygen Species (ROS) process by increasingthe growth factor TGF-1 which accelerates the process normal cell regeneration. The objective ofthis study is to reviewing the efficacy of plasma non termal as subclinical mastitis theraphy. This studywas conducted through a narrative review approach by collecting international and nationalliterature with interval time of publication within 2010 – 2020.

Effect of different human tissue processing techniques on SARS-CoV-2 inactivation-review.

The safety of the tissue transplant recipient is a top priority for tissue banks, and the emergence of the new coronavirus SARS-CoV-2 has raised significant concerns about the risks of releasing tissue for clinical use. In the present study, we conducted a literature review about the potential infectivity of SARS-CoV-2 in different biological tissues and the influence of various tissue processing and sterilization procedures on viral inactivation. The search revealed that SARS-CoV-2 binds to the human angiotensin-converting enzyme receptor to penetrate human cells. These receptors are present in skin cells, musculoskeletal tissue, amniotic membranes, cardiovascular tissue and ocular tissues, including the cornea. In general, we found that coronaviruses are stable at low temperatures, and inactivated upon exposure to extreme heat and pH. Notably, gamma irradiation, which has already been employed to inactivate SARS and MERS, could be useful for sterilizing skin, amnion and musculoskeletal tissues against SARS-CoV-2. We conclude that due to the limited information about the effects of physical and chemical tissue processing methods on viral neutralization, rigorous donor screening is still essential for tissue transplant recipient safety.