Selection and T-cell antigenicity of synthetic long peptides derived from SARS-CoV-2.

The pandemic caused by SARS-CoV-2 has led to the successful development of effective vaccines however the prospect of variants of SARS-CoV-2 and future coronavirus outbreaks necessitates the investigation of other vaccine strategies capable of broadening vaccine mediated T-cell responses and potentially providing cross-immunity. In this study the SARS-CoV-2 proteome was assessed for clusters of immunogenic epitopes restricted to diverse human leucocyte antigen. These regions were then assessed for their conservation amongst other coronaviruses representative of different alpha and beta coronavirus genera. Sixteen highly conserved peptides containing numerous HLA class I and II restricted epitopes were synthesized from these regions and assessed in vitro for their antigenicity against T-cells from individuals with previous SARS-CoV-2 infection. Monocyte derived dendritic cells were generated from these peripheral blood mononuclear cells (PBMC), loaded with SARS-CoV-2 peptides, and used to induce autologous CD4+ and CD8+ T cell activation. The SARS-CoV-2 peptides demonstrated antigenicity against the T-cells from individuals with previous SARS-CoV-2 infection indicating that this approach holds promise as a method to activate anti-SAR-CoV-2 T-cell responses from conserved regions of the virus which are not included in vaccines utilising the Spike protein.

Directing T-Cell Immune Responses for Cancer Vaccination and Immunotherapy.

Cancer vaccination and immunotherapy revolutionised the treatment of cancer, a result of decades of research into the immune system in health and disease. However, despite recent breakthroughs in treating otherwise terminal cancer, only a minority of patients respond to cancer immunotherapy and some cancers are largely refractive to immunotherapy treatment. This is due to numerous issues intrinsic to the tumour, its microenvironment, or the immune system. CD4+ and CD8+ αß T-cells emerged as the primary effector cells of the anti-tumour immune response but their function in cancer patients is often compromised. This review details the mechanisms by which T-cell responses are hindered in the setting of cancer and refractive to immunotherapy, and details many of the approaches under investigation to direct T-cell function and improve the efficacy of cancer vaccination and immunotherapy.

Biovacc-19: A Candidate Vaccine for Covid-19 (SARS-CoV-2) Developed from Analysis of its General Method of Action for Infectivity.

This study presents the background, rationale and method of action of Biovacc-19, a candidate vaccine for corona virus disease 2019 (Covid-19), now in advanced preclinical development, which has already passed the first acute toxicity testing. Unlike conventionally developed vaccines, Biovacc-19's method of operation is upon nonhuman-like (NHL) epitopes in 21.6% of the composition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)'s spike protein, which displays distinct distributed charge including the presence of a charged furin-like cleavage site. The logic of the design of the vaccine is explained, which starts with empirical analysis of the aetiology of SARS-CoV-2. Mistaken assumptions about SARS-CoV-2's aetiology risk creating ineffective or actively harmful vaccines, including the risk of antibody-dependent enhancement. Such problems in vaccine design are illustrated from past experience in the human immunodeficiency viruses domain. We propose that the dual effect general method of action of this chimeric virus's spike, including receptor binding domain, includes membrane components other than the angiotensin-converting enzyme 2 receptor, which explains clinical evidence of its infectivity and pathogenicity. We show the nonreceptor dependent phagocytic general method of action to be specifically related to cumulative charge from insertions placed on the SARS-CoV-2 spike surface in positions to bind efficiently by salt bridge formations; and from blasting the spike we display the NHL epitopes from which Biovacc-19 has been down-selected.

COMPARISON OF INTRAOPERATIVE KETAMINE VS. FENTANYL USE DECREASES POSTOPERATIVE OPIOID REQUIREMENTS IN TRAUMA PATIENTS UNDERGOING CERVICAL SPINE SURGERY.

BACKGROUND: Postoperative airway compromise following cervical spine surgery is a potentially serious adverse event. Residual effects of anesthesia and perioperative opioids that can cause both sedation and respiratory depression further increase this risk. Ketamine is an N-methyl-d-aspartate (NMDA) receptor antagonist that provides potent analgesia without noticeable respiratory depression. We investigated whether intraoperative ketamine administration could decrease perioperative opioid requirements in trauma patients undergoing cervical spine surgery. METHODS: We retrospectively reviewed anesthesia records identifying cervical spine surgeries performed between March 2014 and February 2015. All patients received a balanced anesthetic technique utilizing sevoflurane 0.5 minimum alveolar concentration (MAC) and propofol infusion (50-100 mcg/kg/min). For intraoperative analgesia, one group of patients received ketamine (N=25) and a second group received fentanyl (N=27). Cumulative opioid doses in the recovery room and until 24 hours postoperatively were recorded. RESULTS: Fewer patients in the ketamine group (11/25 [44%] vs. 20/27 [74%], respectively; p = 0.03) required analgesics in the recovery room. Additionally, the total cumulative opioid requirements in the ketamine group decreased postoperatively at both 3 and 6 hours (p = 0.01). CONCLUSION: Ketamine use during cervical spine surgery decreased opioid requirements in both the recovery room and in the first 6 hours postoperatively. This may have the potential to minimize opioid induced respiratory depression in a population at increased risk of airway complications related to the surgical procedure.

Supernatants of tumours treated with chemotherapy can alter tumour growth and development in vivo.

BACKGROUND: Tumour-derived supernatants are comprised of bioactive substances that have the capacity to transform host systems rendering them more supportive of tumour growth. Certain chemotherapies are able to alter the make-up of these supernatants. MATERIALS AND METHODS: We explored the effects that vaccination with supernatants derived from tumours may have on tumour growth in a BALB/c model. RESULTS: A number of cytokines were detected in the supernatants capable of increasing B-cell lymphoma 2 (BCL2) protein expression in cancer cells; of note, significantly higher levels of granulocyte-macrophage colony stimulating factor (GM-CSF) were detected in chemotherapy-treated supernatants compared to controls. Vaccinating mice with supernatants from untreated tumours significantly impeded the growth of sub-cutaneous-implanted tumours. However, this anticancer effect was significantly diminished if the supernatants used were from cancer cells treated with gemcitabine. CONCLUSION: The study lends in vivo support to the idea that tumours produce bioactive components that can influence host biology and that certain chemotherapies can negate these.

Enhancing the activity of cannabidiol and other cannabinoids in vitro through modifications to drug combinations and treatment schedules.

Cannabinoids are the bioactive components of the Cannabis plant that display a diverse range of therapeutic qualities. We explored the activity of six cannabinoids, used both alone and in combination in leukaemic cells. Cannabinoids were cytostatic and caused a simultaneous arrest at all phases of the cell cycle. Re-culturing pre-treated cells in drug-free medium resulted in dramatic reductions in cell viability. Furthermore, combining cannabinoids was not antagonistic. We suggest that the activities of some cannabinoids are influenced by treatment schedules; therefore, it is important to carefully select the most appropriate strategy in order to maximise their efficacy.

Dendritic cell phenotype can be improved by certain chemotherapies and is associated with alterations to p21(waf1/cip1.).

INTRODUCTION: Dendritic cells (DCs) possess the capacity to elicit immune responses against harmful antigens and have been used in DC-vaccines to stimulate the immune system to engage cancer cells. However, a lack of an appreciation of the quality of the DC that is used and/or the monocyte from which it is derived has limited their successful incorporation into treatment strategies. METHODS: In the current study, we explored the relationship between cytokine receptor expression on the monocytes and its subsequent development into DCs. The significance of p21 expression in DCs during differentiation was also studied, as was the effect that manipulating this with chemotherapy may have on DC quality. RESULTS: DCs separated into two groups based on their ability to respond to a maturation stimulus. This quality correlated with a particular receptor profile of granulocyte-macrophage colony-stimulating factor and interleukin 4 expressed on the monocytes from which they were derived. DC quality was also associated with p21 expression, and artificially increasing their levels in DCs by using some chemotherapy improved function. CONCLUSIONS: Overall, these studies have highlighted a role for common chemotherapy in activating p21 in DCs, which is a prerequisite for good DC function.

The immunomodulatory drug lenalidomide restores a vitamin D sensitive phenotype to the vitamin D resistant breast cancer cell line MDA-MB-231 through inhibition of BCL-2: potential for breast cancer therapeutics.

1α,25-Dihydroxyvitamin D3, (1,25-D3) the biologically active form of vitamin-D, is well established as a cancer cell growth inhibitor in addition to maintaining bone mineralization. In breast cancer cells, inhibitory effects on angiogenesis, and metastasis have been observed together with enhancement of apoptosis and induction of cell cycle arrest. There is a correlation between vitamin-D receptor expression on breast cancer cells and patient survival. However vitamin-D resistance and hypercalcaemia are key limiting factors in clinical use. The IMiD(®) immunomodulatory drug lenalidomide, (Revlimid(®), CC-5013) used in myeloma, can also modulate apoptotic and growth signalling. We studied whether lenalidomide treated breast cancer cells would acquire sensitivity to 1,25-D3 with resulting growth inhibition. The cell lines MCF-12A, MCF-7 and MDA-MB-231, representing non-tumorogenic, tumorogenic, and vitamin-D resistant lines respectively were treated with lenalidomide and/or 1,25-D3(at 100 nM). Whereas lenalidomide alone had no effect on cell growth, a 50% inhibition of cell growth by 1,25-D3 was achieved with additional 1 µM lenalidomide in resistant cells. This effect was through apoptosis measured by PARP cleavage and annexin-V expression. An apoptosis protein array showed that the 1,25-D3 and lenalidomide combination increased pro-apoptotic proteins (phosphorylated p53) and decreased BCL-2 expression. BCL-2 inhibition is proposed as a mechanism of action for the combined drugs in the MDA-MB-231 cell line. In vitamin D resistant cell lines MCF-7VDR and HBL-100 where the combination does not affect BCL-2-no inhibitory effect is observed. These results demonstrate the potential for the combinatorial use of lenalidomide and 1,25-D3 for vitamin D refractory tumours.

Associations between Helicobacter pylori infection, co-morbid infections, gastrointestinal symptoms, and circulating cytokines in African children.

BACKGROUND: Refugee children have complex medical needs and often have multiple infections. The relationship between infection, gastrointestinal symptoms, and systemic inflammation is poorly understood. We investigated these parameters in refugee children with a high prevalence of Helicobacter pylori, helminth, and malaria infection. MATERIALS AND METHODS: African refugee children were recruited at resettlement health screening. Data were collected on demography, gastrointestinal symptoms, co-morbid infection, and serum for peripheral cytokine levels. Helicobacter pylori infection was diagnosed by a fecal-based immunoassay. RESULTS: Data from 163 children were analyzed, of which 84.0% were positive for H. pylori. Infected children were significantly older (9.2 years +/- 3.7 vs 7.1 years +/- 3.9, p = .01). Half the cohort (84/163, 51.5%) described gastrointestinal symptoms but these were not strongly associated with co-morbid infections. Helicobacter pylori-infected children had significantly lower circulating log-interleukin-8 (IL-8) (odds ratio 0.61, 95% confidence interval (CI) 0.40, 0.94, p = .025). Helminth infections were common (75/163, 46%) and associated with elevated log-IL-5 (beta: 0.42, 95% CI 0.077, 0.76). Children with malaria (15/163, 9.2%) had elevated log-tumor necrosis factor-alpha (TNFalpha) and log-IL-10 (beta: 0.67, 95% CI 0.34, 1.0 and beta: 1.3, 95% CI 0.67, 1.9, respectively). IL-10 : IL-12 ratios were increased in H. pylori-infected children with malaria or helminth infections. Symptoms were generally not associated with levels of circulating peripheral cytokines irrespective of co-morbid infection diagnosis. CONCLUSIONS: There is a high prevalence of asymptomatic H. pylori infection in recently resettled African refugee children. Gastrointestinal symptoms were not predictive of H. pylori nor of helminth infections. Serum cytokines, particularly IL-5, IL-10, and TNFalpha, were significantly elevated in children with malaria and helminth infections but not in those with H. pylori infection.

Handlebar hernia: traumatic abdominal wall hernia with multiple enterotomies. A case report and review of the literature.

Handlebar hernia, caused by low-energy impact against a handlebar, is a localized abdominal wall hernia that is blunt enough not to penetrate the skin but severe enough to cause the disruption of abdominal wall musculature. In 1964, Roberts (Br J Surg 1964;51:153) reported the first case of a traumatic abdominal wall hernia that occurred in a 9-year-old boy after a fall upon a bicycle handle. Since then, 12 other cases have been reported. To the best of our knowledge, the case described here is the first documented case of an incarcerated handlebar hernia associated with a small bowel perforation and mesenteric disruption.

p16(INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: Experience with squamous lesions in 189 consecutive cervical biopsies.

AIM: To test the usefulness of p16(INK4a) immunostaining for improving the diagnostic accuracy of cervical punch biopsies referred to a routine laboratory setting during the investigation of women with abnormal Papanicolaou smears. METHODS: A total of 188 consecutive and unselected colposcopically directed cervical biopsies and a single contemporaneous cervical polyp were accessioned prospectively over a 3-month period, step-serially sectioned and examined by H&E and immunostained for p16(INK4a). The clinical context, results of concurrent Papanicolaou smears/ThinPrep slides and Digene hybrid capture tests for high-risk human papillomavirus (HPV) subtypes, as well as follow-up cervical smears/ThinPrep, biopsies and loop excisions of transformation zones or cone biopsies were all correlated with the morphological and immunohistochemical findings. RESULTS: Seventy-seven biopsies (40.7%) displayed a high-grade squamous intraepithelial lesion (HGSIL; cervical intraepithelial neoplasia [CIN] 2-3), 27 (14.3%) showed a low grade squamous intraepithelial lesion (HPV +/- CIN1) and 85 (45%) showed a range of non-dysplastic (inflammatory or reactive) changes. Diffuse strong parabasal immunostaining for p16(INK4a), suggestive of integrated high-risk HPV DNA into the host genome, was observed in 81 biopsies (42.9%, including the cervical polyp) and correlated (>90%) with HGSIL in the H&E sections. Only one case revealed irreconcilable discordance between the histological features and this strong parabasal immunostaining pattern. Focal and weaker midzonal or superficial p16(INK4a) immunostaining, suggestive of episomal HPV infection, was noted in 19 biopsies (10%) and these biopsies exhibited a range of histological changes but predominantly low grade squamous intraepithelial lesion (LGSIL). No staining of the squamous epithelium was seen in 89 biopsies (47.1%). Again, only one case revealed irreconcilable discordance between the histological features and this negative immunostaining pattern. On review of all cases where discordant results were noted between the H&E appearances and expected p16(INK4a) immunostaining, we found 26 cases (13.7%) in which this discordance prompted justifiable modification of the original diagnosis. CONCLUSIONS: Thus, within a routine diagnostic laboratory, p16(INK4a) immunostaining appears to be a very useful adjunctive test in the examination of colposcopically directed cervical biopsies, in the diagnostic cascade of women investigated for abnormal Papanicolaou smears. It is possible, as further data accumulate concerning the importance of integration of high-risk HPV DNA into the host cell genome and the reliability with which this can be identified by p16(INK4a) immunostaining, that this will become the diagnostic 'lesion of interest', replacing the subjective histological grading of cervical dysplasia, in the management of such patients; i.e., the discriminatory watershed between continued surveillance and active intervention.