Engineering receptor-binding domain and heptad repeat domains towards the development of multi-epitopes oral vaccines against SARS-CoV-2 variants.

The inability of existing vaccines to cope with the mutation rate has highlighted the need for effective preventative strategies for COVID-19. Through the secretion of immunoglobulin A, mucosal delivery of vaccines can effectively stimulate mucosal immunity for better protection against SARS-CoV-2 infection. In this study, various immunoinformatic tools were used to design a multi-epitope oral vaccine against SARS-CoV-2 based on its receptor-binding domain (RBD) and heptad repeat (HR) domains. T and B lymphocyte epitopes were initially predicted from the RBD and HR domains of SARS-CoV-2, and potential antigenic, immunogenic, non-allergenic, and non-toxic epitopes were identified. Epitopes that are highly conserved and have no significant similarity to human proteome were selected. The epitopes were joined with appropriate linkers, and an adjuvant was added to enhance the vaccine efficacy. The vaccine 3D structure constructs were docked with toll-like receptor 4 (TLR-4) and TLR1-TLR2, and the binding affinity was calculated. The designed multi-epitope vaccine construct (MEVC) consisted of 33 antigenic T and B lymphocyte epitopes. The results of molecular dockings and free binding energies confirmed that the MEVC effectively binds to TLR molecules, and the complexes were stable. The results suggested that the designed MEVC is a potentially safe and effective oral vaccine against SARS-CoV-2. This in silico study presents a novel approach for creating an oral multi-epitope vaccine against the rapidly evolving SARS-CoV-2 variants. These findings offer valuable insights for developing an effective strategy to combat COVID-19. Further preclinical and clinical studies are required to confirm the efficacy of the MEVC vaccine.

Enhancing the solubility and potency of tetrahydrocurcumin as an anti-cancer agent using a ß-cyclodextrin inclusion complex approach.

Curcuminoids originating from turmeric roots are renowned for their diverse pharmacological applications, particularly as a natural anticancer agent. Unfortunately, harnessing the full potential of curcumin derivatives in cancer therapy has been impeded by its inherent limitations, specifically instabilities owing to poor solubility, leading to low systemic bioavailability under normal physiological circumstances. To circumvent this, a novel organic-based drug delivery system employing physically adsorbed ß-cyclodextrin (ßCD) as an excipient was developed in this study. This resulted in improved aqueous dispersion coupled with anticancer enhancements of tetrahydrocurcumin (THC) at a molar ratio of 2:1. Encapsulation of this agent was confirmed by physicochemical characterisation using UV-vis spectroscopy, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and both in vitro and in vivo approaches. Through the presence of an inclusion complex, a higher aqueous dispersion (65-fold) resulting in a higher drug content and an elevated release profile was achieved. Athymic nude (Nu/Nu) mice exposed to this treatment displayed improvements in tumour regression compared to stand-alone agents, consistent with in vitro cytotoxicity assays with an SI value > 10. The inclusion complex further enhanced apoptosis, as well as anti-migration and anti-invasion rates. Mechanistically, this formulation was consistent in terms of caspase 3 activation. Furthermore, the inclusion complex exhibited reduced systemic toxicity, including reduced inflammation in vital organs as examined by hematoxylin and eosin (H&E) staining. This study also revealed a notable sequential reduction in serum levels of tumour markers, including carcinoembryonic antigen (CEA) and mouse Cytochrome P450 1A2 (CYP1A2), correlating with a significant decrease in tumour bulk volume upon treatment commencement. These compelling findings highlight the potential of this formulation to empower insoluble or poorly soluble hydrophobic agents, thus offering promising prospects for their effective utilisation in colorectal cancer (CRC) chemotherapy.

Stability and expression of K-ras mimotopes in freeze-dried recombinant Lactococcus lactis NZ3900-fermented milk powder during storage in vacuum packaging.

AIMS: This study aims to evaluate the storage stability of the freeze-dried recombinant Lactococcus lactis NZ3900-fermented milk powder expressing K-ras (Kristen rat sarcoma viral oncogene homolog) mimotopes targeting colorectal cancer in vacuum packaging. METHODS AND RESULTS: The freeze-dried L. lactis-fermented milk powder stored in 4-ply retortable polypropylene (RCPP)-polyamide (PA)-aluminium (AL)-polyethylene terephthalate (PET) and aluminium polyethylene (ALPE) was evaluated throughout 49 days of accelerated storage (38°C and 90% relative humidity). The fermented milk powder stored in 4-ply packaging remained above 6 log10 CFU g-1 viability, displayed lower moisture content (6.1%), higher flowability (43° angle of repose), water solubility (62%), and survivability of L. lactis after simulated gastric and intestinal digestion (>82%) than ALPE packaging after 42 days of accelerated storage. K-ras mimotope expression was detected intracellularly and extracellularly in the freeze-dried L. lactis-fermented milk powder upon storage. CONCLUSIONS: This suggests that fermented milk powder is a suitable food carrier for this live oral vaccine.

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus).

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

Digitising diabetes education for a safer Ramadan: Design, delivery, and evaluation of massive open online courses in Ramadan-focused diabetes education.

AIMS: Ramadan-focused diabetes education is critical to facilitate safer Ramadan fasting amongst Muslim people living with diabetes. We present the design, delivery, and evaluation of two parallel massive open online courses (MOOCs) in Ramadan-focused diabetes education for people with diabetes and HCPs. METHODS: Two Ramadan-focused diabetes education MOOCs were developed and delivered for Ramadan 2023: one for HCPs in English, and another for people with diabetes in English, Arabic and Malay. A user-centred iterative design process was adopted, informed by user feedback from a 2022 pilot MOOC. Evaluation comprised a mixed-methods evaluation of pre- and post-course user surveys. RESULTS: The platform was utilised by people with diabetes and their family, friends and healthcare professionals. Overall, a total of 1531 users registered for the platform from 50 countries, 809 started a course with a 48% subsequent completion rate among course starters. Qualitative analysis showed users found the course a user-friendly and authoritative information source. In the HCP MOOC, users reported improved post-MOOC Ramadan awareness, associated diabetes knowledge and ability to assess and advise patients in relation to their diabetes during Ramadan (p<0.01). CONCLUSIONS: We demonstrate the potential of MOOCs to deliver culturally tailored, high-quality, scalable, multilingual Ramadan-focused diabetes education to HCPs and people with diabetes.

Cardiovascular disease risk assessment and multidisciplinary care in prostate cancer treatment with ADT: recommendations from the APMA PCCV expert network.

PURPOSE: Androgen deprivation therapy (ADT) is the mainstay approach for prostate cancer (PCa) management. However, the most commonly used ADT modality, gonadotropin-releasing hormone (GnRH) agonists, has been associated with an increased risk of cardiovascular disease (CVD). METHODS: The PCa Cardiovascular (PCCV) Expert Network, consisting of multinational urologists, cardiologists and oncologists with expertise in managing PCa, convened to discuss challenges to routine cardiovascular risk assessment in PCa management, as well as how to mitigate such risks in the current treatment landscape. RESULTS: The experts identified several barriers, including lack of awareness, time constraints, challenges in implementing risk assessment tools and difficulties in establishing multidisciplinary teams that include cardiologists. The experts subsequently provided practical recommendations to improve cardio-oncology care for patients with PCa receiving ADT, such as simplifying cardiovascular risk assessment, individualising treatment based on CVD risk categories, establishing multidisciplinary teams and referral networks and fostering active patient engagement. A streamlined cardiovascular risk-stratification tool and a referral/management guide were developed for seamless integration into urologists' practices and presented herein. The PCCV Expert Network agreed that currently available evidence indicates that GnRH antagonists are associated with a lower risk of CVD than that of GnRH agonists and that GnRH antagonists are preferred for patients with PCa and a high CVD risk. CONCLUSION: In summary, this article provides insights and guidance to improve management for patients with PCa undergoing ADT.

Real or MADE-up: Investigating Mask-Associated Dry Eye (MADE) as an emerging syndrome.

Mask-Associated Dry Eye (MADE) has emerged as a consequence of widespread face mask usage during the Covid-19 pandemic. This review critically assesses the available evidence. A comprehensive search on PUBMED and EMBASE was conducted to identify studies on MADE, which were then categorized based on their design. A total of 26 studies were critically appraised, with the majority exhibiting poor study design quality. Investigating the impact of mask use on ocular surface irritation faced challenges due to global mask mandates, pandemic-related behavioural changes, and a lack of validated methods to measure the response to mask wear. Among the 15 studies, 12 reported a statistically significant reduction in TBUT associated with mask wear; however, the median decrease of 1.3 s was considered clinically insignificant. Results from Schirmer's test in 8 studies varied, with 5 studies reporting a decrease, contradicting the hypothesis of misdirected airflow. Out of 7 studies on corneal and conjunctival staining, 6 indicated a worsening associated with mask wear. Five studies investigating OSDI scores reported an increase following mask wear, but 3 studies repeated the survey too soon. Limited evidence suggests that masks may cause mild ocular surface irritation, but the quality and certainty of this evidence remain low. Methodological limitations were prevalent across the majority of studies, and the observed changes were minimal. Therefore, it is unlikely that significant ocular surface pathology will develop in the majority of mask wearers. Currently, there is insufficient data to support the establishment of a new syndrome.

Multi-disciplinary community-based group intervention for fibromyalgia: a pilot randomized controlled trial.

Fibromyalgia is characterized by widespread pain, fatigue, sleep disturbances, mood disturbances, and cognitive impairment. Most individuals with fibromyalgia experience poorly managed symptoms and increased healthcare service use. Multicomponent therapies, with a focus on nonpharmacological modalities, are increasingly supported in the literature. However, given the limited resources available, implementation in smaller communities remains a challenge. This research tested a community-based multidisciplinary group intervention for individuals diagnosed with FM living in a small urban centre. The primary outcome was perceptions of quality of care and secondary outcomes included disease-related functioning, anxious and depressive symptoms, pain beliefs, and health service utilization. A pilot randomized control trial was conducted in which 60 patients diagnosed with fibromyalgia were randomized into a 10-week community-based multidisciplinary group intervention program or usual care. Treatment components included twice-weekly exercise sessions and weekly education sessions (e.g., pain education, cognitive behavioral strategies for stress, nutrition, peer support). The trial (NCT03270449) was registered September 1 2017. Statistically significant post-intervention improvements were found in the primary outcome, perceived quality of care (Cohen's d = 0.61, 0.66 for follow up care and goal setting, respectively). Secondary outcomes showing statistically significant improvements were disease-related daily functioning (Cohen's d = 0.70), depressive symptoms (Cohen's d = 0.87), and pain beliefs (Cohen's d = 0.61, 0.67, 0.82 for harm, disability and control, respectively). No adverse events were reported. Community-based multidisciplinary group interventions for fibromyalgia show promise for improving satisfaction with quality of care, disease-related functioning, and depression, and fostering more adaptive pain beliefs.

Effectiveness and Safety of Tofacitinib in Canadian Patients With Rheumatoid Arthritis: Primary Results From a Prospective Observational Study.

OBJECTIVE: The Canadian Tofacitinib for Rheumatoid Arthritis Observational (CANTORAL) is the first Canadian prospective, observational study assessing tofacitinib. The objective was to assess effectiveness and safety for moderate to severe rheumatoid arthritis (RA). Coprimary and secondary outcomes are reported from an interim analysis. METHODS: Patients initiating tofacitinib from October 2017 to July 2020 were enrolled from 45 Canadian sites. Coprimary outcomes (month 6) included the Clinical Disease Activity Index (CDAI)-defined low disease activity (LDA) and remission. Secondary outcomes (to month 18) included the CDAI and the 4-variable Disease Activity Score in 28 joints (DAS28) using the erythrocyte sedimentation rate (ESR)/C-reactive protein (CRP) level to define LDA and remission; the proportions of patients achieving mild pain (visual analog scale <20 mm), and moderate (≥30%) and substantial (≥50%) pain improvements; and the proportions of patients achieving a Health Assessment Questionnaire disability index (HAQ DI) score greater or equal to normative values (≤0.25) and a HAQ DI score greater or equal to minimum clinically important difference (MCID) (≥0.22). Safety was assessed to month 36. RESULTS: Of 504 patients initiating tofacitinib, 44.4% received concomitant methotrexate. At month 6, 52.9% and 15.4% of patients were in CDAI-defined LDA and remission, respectively; a similar proportion of patients achieved outcomes by month 3 (first post-baseline assessment). By month 3, 27.2% and 41.7% of patients, respectively, were in DAS28-ESR-defined LDA and DAS28-CRP <3.2; 14.7% and 25.8% achieved DAS28-ESR remission and DAS28-CRP <2.6. By month 3, mild pain and moderate and substantial pain improvements occurred in 29.6%, 55.6%, and 42.9% of patients, respectively; 19.9% and 53.7% of patients achieved a HAQ DI score greater than or equal to normative values and a HAQ DI score greater than or equal to MCID, respectively. Outcomes were generally maintained to month 18. Incidence rates (events per 100 patient-years) for treatment-emergent adverse events (AEs), serious AEs, and discontinuations due to AEs were 126.8, 11.9, and 14.5, respectively, and AEs of special interest were infrequent. CONCLUSION: Tofacitinib was associated with early and sustained improvement in RA signs and symptoms in real-world patients. Effectiveness and safety were consistent with the established tofacitinib clinical profile.

Intraoperative subconjunctival steroid reduces the incidence of pseudophakic macular oedema: a cohort study of 20,066 consecutive phacoemulsification surgeries.

BACKGROUND/OBJECTIVES: Pseudophakic macular oedema remains the most common sight-threatening complication following cataract surgery. This study aims to assess the effect of intraoperative subconjunctival steroids on the rate of pseudophakic cystoid macular oedema. METHODS: A retrospective, observational database study of 20 066 consecutive phacoemulsification surgeries. The incidence of pseudophakic cystoid macular oedema was compared in eyes that did and did not receive intraoperative subconjunctival steroid injection during routine cataract surgery. RESULTS: Intraoperative subconjunctival injection of dexamethasone or betamethasone sodium phosphate significantly reduced the odds of developing pseudophakic cystoid macular oedema across the cohort (odds ratio: 0.67; 95% confidence interval: 0.46-0.98, p = 0.039). The effect of subconjunctival steroids on pseudophakic macular oedema remained independently associated on multivariate logistic regression analysis (p = 0.028). CONCLUSION: This study demonstrates that administration of intraoperative subconjunctival steroid injection is associated with a reduced incidence of pseudophakic cystoid macular oedema in routine, uncomplicated cataract surgery.

Immunomodulatory action of Lactococcuslactis.

Fermented foods are gaining popularity due to health-promoting properties with high levels of nutrients, phytochemicals, bioactive compounds, and probiotic microorganisms. Due to its unique fermentation process, Lactococcus lactis plays a key role in the food business, notably in the manufacturing of dairy products. The superior biological activities of L. lactis in these functional foods include anti-inflammatory and immunomodulatory capabilities. L. lactis boosted growth performance, controlled amino acid profiles, intestinal immunology, and microbiota. Besides that, the administration of L. lactis increased the rate of infection clearance. Innate and acquired immune responses would be upregulated in both local and systemic compartments, resulting in these consequences. L. lactis is often employed in the food sector and is currently being exploited as a delivery vehicle for biological research. These bacteria are being eyed as potential candidates for biotechnological applications. With this in mind, we reviewed the immunomodulatory effects of different L. lactis strains.

Biophysical Evaluation of Water-Soluble Curcumin Encapsulated in ß-Cyclodextrins on Colorectal Cancer Cells.

Curcumin (CUR), a curcuminoid originating from turmeric root, possesses diverse pharmacological applications, including potent anticancer properties. However, the use of this efficacious agent in cancer therapy has been limited due to low water solubility and poor bioavailability. To overcome these problems, a drug delivery system was established as an excipient allowing improved dispersion in aqueous media coupled with enhanced in vitro anticancer effects. Different analyses such as UV-vis spectroscopy, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), solubility and dissolution assays were determined to monitor the successful encapsulation of CUR within the inner cavity of a ß-cyclodextrin (ß-CD) complex. The results indicated that water solubility was improved by 205.75-fold compared to pure CUR. Based on cytotoxicity data obtained from MTT assays, the inclusion complex exhibited a greater decrease in cancer cell viability compared to pure CUR. Moreover, cancer cell migration rates were decreased by 75.5% and 38.92%, invasion rates were decreased by 37.7% and 35.7%, while apoptosis rates were increased by 26.3% and 14.2%, and both caused caspase 3 activation toward colorectal cancer cells (SW480 and HCT116 cells). This efficacious formulation that enables improved aqueous dispersion is potentially useful and can be extended for various chemotherapeutic applications. Preliminary toxicity evaluation also indicated that its composition can be safely used in humans for cancer therapy.

Current progress in treatment technologies for plastic waste (bisphenol A) in aquatic environment: Occurrence, toxicity and remediation mechanisms.

Bisphenol-A (BPA) is a type of endocrine disrupting compound (EDC) that is being widely used in the production of polycarbonate and epoxy resins. In the last few years, human exposure to BPA has been extensively high due to the continuous increment in the Annual Growth Rate (AGR) of the BPA global market. The presence and transportation of BPA in the environment could cause serious damage to aquatic life and human health. This paper reviewed the literature on the exposure and toxicity mechanisms of BPA and advanced analytical techniques for the detection of BPA in the environment and human beings. The study indicated that BPA can cause damaging effects on numerous tissues and organs, including the reproductive system, metabolic dysfunction, respiratory system, immune system and central nervous system. On the basis of reported studies on animals, it appears that the exposure of BPA can be carcinogenic and responsible for causing a variety of cancers like ovarian cancer, uterine cancer, prostate cancer, testicular cancer, and liver cancer. This review paper focused mainly on the current progress in BPA removal technologies within last ten years (2012-2022). This paper presents a comprehensive overview of individual removal technologies, including adsorption, photocatalysis/photodegradation, ozonation/advance oxidation, photo-fenton, membranes/nanofilters, and biodegradation, along with removal mechanisms. The extensive literature study shows that each technology has its own removal mechanism and their respective limitations in BPA treatment. In adsorption and membrane separation process, most of BPA has been treated by electrostatic interaction, hydrogen boning and π-π interations mechanism. Whereas in the degradation mechanism, O* and OH* species have played a major role in BPA removal. Some factors could alter the removal potential and efficiency of BPA removal. This review paper will provide a useful guide in providing directions for future investigation to address the problem of BPA-containing wastewater treatment.

Endolysins against Streptococci as an antibiotic alternative.

Multi-drug resistance has called for a race to uncover alternatives to existing antibiotics. Phage therapy is one of the explored alternatives, including the use of endolysins, which are phage-encoded peptidoglycan hydrolases responsible for bacterial lysis. Endolysins have been extensively researched in different fields, including medicine, food, and agricultural applications. While the target specificity of various endolysins varies greatly between species, this current review focuses specifically on streptococcal endolysins. Streptococcus spp. causes numerous infections, from the common strep throat to much more serious life-threatening infections such as pneumonia and meningitis. It is reported as a major crisis in various industries, causing systemic infections associated with high mortality and morbidity, as well as economic losses, especially in the agricultural industry. This review highlights the types of catalytic and cell wall-binding domains found in streptococcal endolysins and gives a comprehensive account of the lytic ability of both native and engineered streptococcal endolysins studied thus far, as well as its potential application across different industries. Finally, it gives an overview of the advantages and limitations of these enzyme-based antibiotics, which has caused the term enzybiotics to be conferred to it.

Current Advances and Trends in KRAS Targeted Therapies for Colorectal Cancer.

Kirsten Rat Sarcoma (KRAS) gene somatic point mutations is one of the most prominently mutated proto-oncogenes known to date, and accounts for approximately 60% of all colorectal cancer cases. One of the most exciting drug development areas against colorectal cancer is the targeting of undruggable kinases and kinase-substrate molecules, although whether and how they can be integrated with other therapies remains a question. Current clinical trial data have provided supporting evidence on the use of combination treatment involving MEK inhibitors and either one of the PI3K inhibitors for patients with metastatic colorectal cancer to avoid the development of resistance and provide effective therapeutic outcome rather than using a single agent alone. Many clinical trials are also ongoing to evaluate different combinations of these pathway inhibitors in combination with immunotherapy for patients with colorectal cancer whose current palliative treatment options are limited. Nevertheless, continued assessment of these targeted cancer therapies will eventually allow patients with colorectal cancer to be treated using a personalized medicine approach. In this review, the most recent scientific approaches and clinical trials targeting KRAS mutations directly or indirectly for the management of colorectal cancer are discussed.

Understanding multidisciplinary care for people with rheumatic disease in British Columbia, Canada, through patients, nurses and physicians voices: a qualitative policy evaluation.

BACKGROUND: In 2011, the province of British Columbia (BC) moved to allow patients with complex rheumatic disease to be seen by nurses along with their rheumatologist by introducing a 'Multidisciplinary Care Assessments' (MCA) billing code (G31060). OBJECTIVE: To describe multidisciplinary care introduced as part of MCAs across BC and investigate the perceived impact of this intervention, the addition of nurses to the care team, on patient care from the perspective of patients, nurses, and rheumatologists. METHODS: We conducted semi-structured interviews, informed by a qualitative evaluation approach with patients, nurses, and rheumatologists from September 2019 - August 2020. Interviews investigated 1) the experiences of all stakeholders with adopting the multidisciplinary care billing code, 2) the perceived role of the nurse in the care team, and 3) the perceived impact of multidisciplinary care on patient experience and outcomes. We purposefully sampled practices for maximum variation of geographical location (rural vs. urban), size of practice (i.e., patient caseload), and number of nurses employed. RESULTS: We interviewed 21 patients, 13 nurses, and 12 rheumatologists from across BC. Our analysis identified variation in the way rheumatologists adopted multidisciplinary care across BC. Our analysis showed some heterogeneity in the way the MCA was delivered in rheumatology practices; however, patient education was identified as the core role of nurses across practices. We identified six core themes describing the impact of this model of care, all representing improvements in the way practices functioned, from improved efficiency to access, patient experience, time management, clinician experience, and patient health outcomes. Contextual factors that influenced the presence of these themes were related to the time the nurses spent with patients and the professional roles they performed. CONCLUSION: Our results suggest nurse care can complement physician care by extending contact time for patients and promoting the efficient use of health care professionals' skills, time, and resources. These data may encourage future uptake of the billing code to help ensure the policy delivers maximum benefits to patients given the wide range of perceived benefits described by clinicians and patients.

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells.

BACKGROUND: KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. METHODS: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. RESULTS: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 µg/mL, with minimal cytotoxicity effect on NHDF cells. DISCUSSION: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.

Long-term effectiveness and safety of infliximab and golimumab in ankylosing spondylitis patients from a Canadian prospective observational registry.

BACKGROUND: The objectives of this study were to describe the profile of ankylosing spondylitis (AS) patients treated with either infliximab (IFX) or subcutaneous golimumab (GLM) treatment in Canadian routine care setting along with assessing long-term effectiveness and safety. METHODS: AS patients who were eligible for treatment with IFX or subcutaneous GLM as per their respective Canadian product monographs were enrolled into the BioTRAC registry from 2005 to 2017. The study visits occurred at baseline and every 6 months thereafter. Effectiveness was assessed by changes in clinical outcomes and acute phase reactants. Safety was evaluated by assessing the incidence of adverse events (AEs) and drug survival rates. RESULTS: A total of 389 IFX- and 421 GLM-treated patients were enrolled. A significant decrease in disease duration at baseline was observed in the IFX cohort, from a median of 8.0 in 2005-2008 to 1.0 years in 2009-2015 (p < 0.001). A reduction in baseline BASFI score (p = 0.011) and proportion of patients in ASDAS very high disease activity (p = 0.004) was also observed over time. Meanwhile, in the GLM cohort, most disease parameters remained similar from 2010 to 2017. Treatment with both agents significantly improved all disease parameters over time with similar efficacy between the two agents. The incidence of AEs and SAEs were 136 and 131 events/100 PYs and 10.5 and 8.45 events/100 PYs for IFX- and GLM-treated patients, respectively. CONCLUSION: Both IFX and GLM treatment in AS significantly reduced disease activity in most outcome measures in a similar fashion and were well tolerated in Canadian routine care. TRIAL REGISTRATION: NCT00741793 .

Cytotoxic and apoptosis-inducing effects of wildtype and mutated Hydra actinoporin-like toxin 1 (HALT-1) on various cancer cell lines.

BACKGROUND: Hydra actinoporin like toxin -1 (HALT-1), is a small 18.5 kDa pore forming toxin derived from Hydra magnipapillata which has been shown to elicit strong haemolytic and cytolytic activity when in contact with cell membranes. Due to its cytotoxic potency, HALT-1 was further investigated for its potential as a toxin moiety candidate in immunotoxin developmental efforts, ideally as a form of targeted therapy against cancer. METHODS: In this study, wtHALT-1 (wild type) and its Y110A mutated binding domain counterpart (mHALT-1) were produced and evaluated for their cytotoxic and apoptotic effects on various cancer cell lines. A total of seven different tumour and non-tumour cell lines including HeLa, HepG2, SW-620, MCF-7, CCD841CoN, NHDF and HCT116 were used. Immunofluorescence assays were used to observe membrane binding and localization changes between both HALT-1 recombinant proteins based on 6xHis-tag detection. RESULT: Based on MTT data, mHALT-1 demonstrated a significant reduction of 82% ±  12.21% in cytotoxic activity across all cell lines after the membrane recognition domain had been mutated in comparison to the wtHALT-1. Annexin V FITC/PI assay data also indicated that HeLa, HepG2 and MCF-7 demonstrated an apoptosis-mediated cell death after being treated with wtHALT-1. Additionally, a notable difference between wtHALT-1 and mHALT-1 binding affinity was clearly observed where emission of green fluorescence along the cell membrane was observed only in wtHALT-1 treated cells. DISCUSSION: These results suggest that mHALT-1 (Y110A) can be potentially developed as a toxin-moiety candidate for the development of future immunotoxins against various human cell-based diseases.

In silico-guided sequence modifications of K-ras epitopes improve immunological outcome against G12V and G13D mutant KRAS antigens.

BACKGROUND: Somatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions. METHODS: In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA. RESULTS: In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls. DISCUSSION: In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.