Engineering of Injectable Antibiotic-laden Fibrous Microparticles Gelatin Methacryloyl Hydrogel for Endodontic Infection Ablation

This study aimed at engineering cytocompatible and injectable antibiotic-laden fibrous microparticles gelatin methacryloyl (GelMA) hydrogels for endodontic infection ablation. Clindamycin (CLIN) or metronidazole (MET) was added to a polymer solution and electrospun into fibrous mats, which were processed via cryomilling to obtain CLIN- or MET-laden fibrous microparticles. Then, GelMA was modified with CLIN- or MET-laden microparticles or by using equal amounts of each set of fibrous microparticles. Morphological characterization of electrospun fibers and cryomilled particles was performed via scanning electron microscopy (SEM). The experimental hydrogels were further examined for swelling, degradation, and toxicity to dental stem cells, as well as antimicrobial action against endodontic pathogens (agar diffusion) and biofilm inhibition, evaluated both quantitatively (CFU/mL) and qualitatively via confocal laser scanning microscopy (CLSM) and SEM. Data were analyzed using ANOVA and Tukey’s test (α = 0.05). The modification of GelMA with antibiotic-laden fibrous microparticles increased the hydrogel swelling ratio and degradation rate. Cell viability was slightly reduced, although without any significant toxicity (cell viability > 50%). All hydrogels containing antibiotic-laden fibrous microparticles displayed antibiofilm effects, with the dentin substrate showing nearly complete elimination of viable bacteria. Altogether, our findings suggest that the engineered injectable antibiotic-laden fibrous microparticles hydrogels hold clinical prospects for endodontic infection ablation.

Study on the Mechanism of Reducing Hepatotoxicity of Water-Grinding Realgar by Metabolomics, Morphology, and Chemical Analysis

Background. Realgar was usually selected as a substitute for arsenic trioxide to treat acute promyelocytic leukemia due to its higher effect without high cardiotoxicity. In traditional Chinese medicine (TCM), realgar is usually processed by the water-grinding method clinically, but the mechanism of realgar processing detoxification is still unclear. However, it is necessary to take safety and efficacy into account while evaluating a drug. Methods. Sixty male Wistar rats were divided into control group, realgar products-treated groups, and corresponding subgroups. Biochemistry analysis and histopathological examination were performed in the study, and plasma samples were collected from all the rats for metabolomics analysis. Results. No significant toxicity was observed in rats treated with 0.64 g/kg/day grinding realgar (G-r) and water-grinding realgar (WG-r). When the dose increased to 1.92 g/kg/day, the liver weight coefficients of the rats treated with G-r (HG-r: 3.65 ± 0.26%) and WG-r (HWG-r: 3.67 ± 0.14%) increased significantly and severe hepatic injury occurred in comparison to the control group (Group C: 3.00 ± 0.21%). After one week's withdrawal, the liver injury caused by the high dose of WG-r significantly recovered, while the liver damage caused by G-r was more difficult to recover. In metabolomics analysis, 14 metabolites were identified as the potential biomarkers in realgar-treated rats. These metabolites indicated that there were perturbations of the primary bile acid biosynthesis, arachidonic acid metabolism, linoleic acid metabolism, and glycerophospholipid metabolism in the realgar-treated groups. Conclusions. These results illustrate that, as a TCM processing method, water grinding had the effect of reducing toxicity, and the metabolomics method may be a valuable tool for studying the toxicity induced by TCM and the mechanism of TCM processing.

Essential oils of Eplingiella fruticosa populations: chemical, antioxidant, and cytotoxic analyses

This study investigates the variations in the chemical profiles and biological activities (antioxidant and cytotoxic) of Eplingiella fruticosa from the state of Sergipe, an endemic species from the Northeast region of Brazil. The essential oils were extracted from six populations by hydrodistillation and analyzed by GC/MS-FID. Cluster analysis was performed with the data of the constituents of the essential oils, and then a dissimilarity matrix, based on Euclidean distances, and a dendrogram, through the Ward clustering method, were constructed. The antioxidant activity of the essential oils was tested by different assays (DPPH, ABTS, β-carotene, and FRAP), and cytotoxic activity was tested by the SRB assay. The compounds found in greater amounts were α-pinene, β-pinene, 1,8-cineole, camphor, borneol, δ-elemene, α-cubebene, α-ylangene, (E)-caryophyllene, germacrene D, bicyclogermacrene, trans-calamenene, spathulenol, caryophyllene oxide, and viridiflorol. These compounds defined the formation of two groups. The first group was composed of the populations of São Cristóvão, Itaporanga, Japaratuba, and Malhada dos Bois municipalities and was characterized by the presence of the monoterpene camphor (8.39-11.27%) as the compound of greatest concentration in relation to the other municipal areas. The second group was composed of the populations of Moita Bonita and Pirambu municipalities and was characterized by the major presence of the sesquiterpene bicyclogermacrene (7.45% and 10.98%). The plants exhibited weak effects in terms of antioxidant activity; however, the essential oil showed significant toxicity for the lines A549 (51.00% cell viability) in the population of Japaratuba, and B16F10 (64.94% cell viability) in Malhada dos Bois. The observations of this study may open a way to optimize the use of the E. fruticosa populations in relation to their cytotoxic properties.

Donor Memory-like NK cells Persist and Induce Remissions in Pediatric Patients with Relapsed AML after Transplant

Pediatric and young adult (YA) patients with acute myeloid leukemia (AML) who relapse after allogeneic hematopoietic cell transplantation (HCT) have extremely poor prognosis. Standard salvage chemotherapy and donor lymphocyte infusions (DLI) have little curative potential. Previous studies showed that natural killer (NK) cells can be stimulated ex vivo with interleukin-12 (IL-12), IL-15, and IL-18 to generate memory-like (ML) NK cells with enhanced anti-leukemia responses. We treated nine pediatric/YA patients with post-HCT relapsed AML with donor ML NK cells on a phase I trial. Patients received fludarabine, cytarabine and filgrastim followed two weeks later by infusion of DLI and ML NK cells from the original HCT donor. ML NK cells were successfully generated from haploidentical, matched-related and matched-unrelated donors. Following infusion, donor-derived ML NK cells expanded and maintained ML multidimensional mass cytometry phenotype for over 3 months. Furthermore, ML NK cells exhibited persistent functional responses as evidenced by leukemia-triggered IFN-g production. Following DLI and ML NK cell adoptive transfer, 4 of 8 evaluable patients achieved complete remission at day 28. Two patients maintained a durable remission for over 3 months with one patient in remission for greater than two years. No significant toxicity was experienced. This study demonstrates that in a compatible immune environment post-HCT, donor ML NK cells robustly expand and persist with potent anti-leukemic activity in the absence of exogenous cytokines. ML NK cells in combination with DLI present a novel immunotherapy platform for AML that has relapsed after allogeneic HCT. This trial was registered at www.clinicaltrials.gov as #NCT03068819.

Characterising the Biological Activity of a Trichlorovinyl Cysteine-Containing Mycothiol Analogue

<p>Cancer is a disease characterised by the uncontrolled growth of mutated cells, and is one of the leading causes of death worldwide, with over a third of people diagnosed with cancer in their lifetime. Despite extensive investment of both time and money in cancer research, poor patient outcomes and quality of life, and the evolution of treatment resistant cancers indicates that continued research, and more efficacious therapies are required. A recent investigation identified a mycothiol analogue which displayed significant toxicity in the promyelocytic leukemia cell line (HL60). Designed as a negative control, no biological activity was expected from this compound and its cellular target and mode of action are unknown. This thesis describes the synthesis of a toxic trichlorovinyl cysteine-containing analogue of mycothiol, and the attempted synthesis of a propynylated and fluorescent derivative of this. The research also details immunomodulatory investigations, which were undertaken to probe the mode of action of the lead compound, and to determine whether its precursor, N-Boc-S-trichlorovinyl cysteine, induced toxicity through the same mechanism. The lead compound demonstrated mild immunomodulatory activity in splenocytes isolated from euthanised C57BL/6 mice, and enzyme linked immunosorbent assays revealed a likely Th2 mediated response, induced by the production of IL-4. The precursor however appears to promote a strong pro-inflammatory response, by inducing IL-17a production, which is widely considered a deleterious immune response in cancer. Whilst further work is required to determine the cellular target of the lead compound, the research described demonstrates the potential for this compound as an anti-cancer agent, while the precursor appears inappropriate for further development.</p>

Characterising the Biological Activity of a Trichlorovinyl Cysteine-Containing Mycothiol Analogue

<p>Cancer is a disease characterised by the uncontrolled growth of mutated cells, and is one of the leading causes of death worldwide, with over a third of people diagnosed with cancer in their lifetime. Despite extensive investment of both time and money in cancer research, poor patient outcomes and quality of life, and the evolution of treatment resistant cancers indicates that continued research, and more efficacious therapies are required. A recent investigation identified a mycothiol analogue which displayed significant toxicity in the promyelocytic leukemia cell line (HL60). Designed as a negative control, no biological activity was expected from this compound and its cellular target and mode of action are unknown. This thesis describes the synthesis of a toxic trichlorovinyl cysteine-containing analogue of mycothiol, and the attempted synthesis of a propynylated and fluorescent derivative of this. The research also details immunomodulatory investigations, which were undertaken to probe the mode of action of the lead compound, and to determine whether its precursor, N-Boc-S-trichlorovinyl cysteine, induced toxicity through the same mechanism. The lead compound demonstrated mild immunomodulatory activity in splenocytes isolated from euthanised C57BL/6 mice, and enzyme linked immunosorbent assays revealed a likely Th2 mediated response, induced by the production of IL-4. The precursor however appears to promote a strong pro-inflammatory response, by inducing IL-17a production, which is widely considered a deleterious immune response in cancer. Whilst further work is required to determine the cellular target of the lead compound, the research described demonstrates the potential for this compound as an anti-cancer agent, while the precursor appears inappropriate for further development.</p>

An institutional evaluation of radiotherapy with concurrent and adjuvant temozolomide exclusively in grade III gliomas

Background: Gliomas are the most common tumors that develop from glial cells in the brain. As per WHO classification, grade III (high-grade) gliomas are usually treated by surgery followed by radiotherapy. Concurrent and adjuvant chemotherapy with temozolomide (TMZ) is showing new hope in the management.Methods: Prospective study was conducted in 20 WHO grade III glioma patients at GSL medical college and general hospital for a period of 2 years (August 2019 to July 2021) to evaluate the role of concurrent and adjuvant TMZ with radiation in the treatment of grade III gliomas. The primary objectives of the study were to determine toxicity and response rates in our set of patients. The secondary objectives were to determine progression-free survival and overall survival.Results: Majority were males (80%) in the age group of 30 to 49 (75%). 40% of gliomas were in the temporal lobe. Headache was the most presenting symptom (60%) followed by seizures (35%).70 to 80 Karnofsky performance status/ KPS score was seen in 70% of cases. None of the patients in the study had grade 3 or 4 toxicities. 40% had a complete response according to MacDonald’s criteria. At a median follow-up of 10 months, 25% of patients had disease progression and 1yr overall survival was 95%.Conclusions: TMZ combined with radiation was well tolerated in our set of patients with grade III gliomas without any significant toxicity.

Evaluation of insecticidal potential of organochemicals on SF9 cell line

Background Organophosphates and Pyrethroids are the most widely used pesticides worldwide and are known to have significant toxicity on the nervous system of the target pest. Assessment for combined toxicity of Organophosphate and Pyrethroid on Sf9 (Spodoptera frugiperda) cells is less explored. The present study demonstrates and compares the two organochemicals whose trade names are Ammo and Profex, for its cytotoxic potential on the insect Sf9 cells. Ammo and Profex were selected as the test chemicals as toxicity of these insecticides at molecular and cellular level is poorly understood. Results The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that Ammo and Profex exhibited significant cytotoxicity to Sf9 cells in a time- and dose-dependent manner. In our study, the IC50 value was obtained by MTT assay and the sub-lethal concentrations (IC50/20-17.5 µg/ml, IC50/10-35 µg/ml, and IC50/5–70 µg/ml for Ammo and IC50/20-20 µg/ml, IC50/10-40 µg/ml, and IC50/5-80 µg/ml for Profex) were selected for further tests. Acridine orange/ethidium bromide staining proved the apoptotic cell death on exposure of both the insecticides confirming its toxic potential. Furthermore, antioxidant status was assessed using DCF-DA staining and both the insecticides resulted into an increased reactive oxygen species (ROS) generation. A dose- and time-dependent significant (p < 0.05) alterations in lipid peroxidase (LPO), glutathione (GSH) and catalase (CAT) activity were observed. Conclusion The results showed that both Ammo and Profex triggered apoptosis in Sf9 cells through an intrinsic mitochondrial pathway via the generation of ROS. Of the two insecticides, Ammo was found to be more toxic compared to Profex. The present study is important to evaluate the environmental safety and risk factors of Organochemicals’ exposure to crops and livestock.

Discovery of Novel Antituberculosis Compounds Using an Intra-Macrophage Assay

<p>The causative agent of tuberculosis (TB) is Mycobacterium tuberculosis, which affects 2 billion of the world population and kills 1.8 million people annually. It is among the top three infectious killers in the world human immuno deficiency virus, TB and Malaria. Every year among 300-400 new cases of TB are reported in New Zealand according to a recent WHO 2008 report. The current treatment regimen for TB is very long and results in significant toxicity, development of resistant strains and is unable to eliminate the latent bacilli. The above reasons demonstrate the growing need of research for novel antimycobacterial compounds and novel targets for the treatment of TB. Many in vitro and biochemical screens are available for testing against different mycobacterium strains but none of these screens can be considered comprehensive. The reason for this can be the lack of resemblance of the in vitro screen model with the biological systems. Hence we chose the intra-macrophage infection screening model to look for novel antimycobacterial prodrugs which are not active in an in vitro screen but selectively active inside macrophage cell lines. We were successful in establishing and validating such an intra-macrophage infection model using the non-pathogenic M. smegmatis. The model was validated using common anti-tuberculosis drugs. A preliminary high throughput screen was then set up using a mini-library demo model, followed by screening with an actual Lopac synthetic library.</p>