The role of lymphocyte transformation tests (LTT) in suspected severe delayed hypersensitivity reactions following COVID-19 vaccination.

Coronavirus (COVID-19) vaccines have proved to be effective in the pandemic response but can cause adverse events such as delayed hypersensitivity reactions (DHRs). Delayed-reading intradermal tests (IDT) to vaccines are limited by false-positive results and may reflect a cell-mediated rather than IgE-mediated immune response. Lymphocyte transformation test (LTT), which has been utilized in the diagnosis of drug allergy, may be helpful in suspected COVID-19 vaccine and/or its excipient-related DHRs. To investigate the use of LTT in two suspected cases of COVID-19 vaccine-induced DHRs, two patients with suspected DHRs to COVID-19 vaccination were tested by delayed-reading IDT and LTT against vaccines and their excipients. A 47-year-old man developed acute mixed-pattern hepatitis after the second dose of ChAdOx1 vaccine. LTT performed at 2 months post-vaccination revealed reactivity to the ChAdOx1 vaccine, polysorbate 80 and mildly to PEG 2050 but not BNT162b2 vaccine. Delayed-reading IDT returned negative to both vaccines and excipients. He tolerated BNT162b2 vaccination with no adverse events. A 36-year-old woman presented with subacute morbilliform eruption and hepatitis after the first dose of BNT162b2 vaccine. LTT performed 3 months later revealed reactivity to the BNT162b2 but not PEG 2050. Repeat LTT following subsequent natural Severe Acute Respiratory Coronavirus-2 (SARS-CoV-2) infection revealed reactivity to ChAdOx1 and NVX-CoV2373 vaccines but not polysorbate 80. Delayed-reading IDT remained negative. She proceeded with NVX-CoV2373 vaccination with no symptom recurrence. LTT may be a useful tool in suspected COVID-19 vaccine-related DHRs. Further evaluation with a larger patient cohort is required.

Development and initial validation of a modified lymphocyte transformation test (LTT) assay in patients with DRESS and AGEP.

BACKGROUND: The lymphocyte transformation test (LTT) is an in vitro assay used to diagnose drug induced hypersensitivity reactions by detecting the activation and expansion of drug-specific memory T cells to the suspected implicated drug. Traditionally radiolabelled thymidine (3H-thymidine) has been used but requires the handling and disposal of radioactive materials. OBJECTIVE: To examine safe alternatives to 3H-thymidine, test assay modifications for improved assay sensitivity and evaluate the modified LTT in patients with DRESS and AGEP. METHODS: Four proliferation detection assays (BRDU, CyQUANT™, MTT and XTT) were screened for LTT sensitivity. XTT the most sensitive and practical was selected for further evaluation Modifications like autologous serum (AS) and regulatory T cell depletion (T-REG) were tested for improved assay sensitivity. Finally, an initial evaluation of the XTT-LTT was performed in 8 patients with DRESS and 2 with AGEP including cytokine testing. RESULTS: Of the non-radioactive alternatives we tested, XTT a colorimetric assay was the most sensitive and practical to move to evaluation. The addition of AS increased background signal. Depletion of T-REGs improved sensitivity but cell sorting time and risk of contamination limited benefit. Of eight patients diagnosed with DRESS and 2 with AGEP tested with XTT-LTT assay results showed our assay matched clinical findings of implicated drugs in 8/10 patients when using a stimulation index (SI) ≥ 2 and 8/10 with analysis by ANOVA. All ten patients were correctly diagnosed by either analysis. CONCLUSION: XTT appears to be a safe, viable alternative to 3H-thymidine, with high sensitivity and allowing direct cytokine quantification on specific patient cells.

The Safety of an Adjuvanted Autologous Cancer Vaccine Platform in Canine Cancer Patients.

Canine cancer rates are similar to humans, though the therapeutic options might be limited. Inducing a patient's own immune system to have an anti-tumor response is an attractive approach to cancer therapy. In this safety study, autologous tumor vaccines produced specifically for each canine patient were combined with Advax™, a novel non-inflammatory immunomodulator and vaccine adjuvant and were tested for safety in a diverse range of patient presentations alone or in combination with other treatments. Canine patients had their tumor biopsied, debulked or resected and the tumor antigens were processed into an autologous vaccine formulated with Advax™ adjuvant with or without rhizavidin as an additional immune stimulant. Patients treated early in the trial received two intramuscular (IM) doses, 2 weeks apart. As the study progressed and no issues of safety were observed, the protocol was changed to weekly vaccinations for 4 weeks followed by monthly booster shots. Over the 150 I.M injections delivered to date, the vaccine was found to be very safe and no significant adverse reactions were observed. These results justify ongoing development and future controlled studies of this autologous vaccine approach.

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 µl whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

Establishing a panel of chemo-resistant mesothelioma models for investigating chemo-resistance and identifying new treatments for mesothelioma.

Mesothelioma is inherently chemo-resistant with only 50% of patients responding to the standard of care treatments, and consequently it has a very grim prognosis. The aim of this study was to establish a panel of chemo-resistant mesothelioma models with clinically relevant levels of resistance as tools for investigating chemo-resistance and identifying new treatments for mesothelioma. Chemo-resistant cell lines were established in vitro and characterized in vivo using syngeneic Fischer rats. Tumors derived from all chemo-resistant cell lines were immunohistochemically classified as mesothelioma. Homozygous deletion of p16(INK4A)/p14(ARF) and increased expression of several ATP-binding cassette transporters were demonstrated, consistent with findings in human mesothelioma. Further, the acquisition of chemo-resistance in vitro resulted in changes to tumor morphology and overall survival. In conclusion, these models display many features corresponding with the human disease, and provide the first series of matched parental and chemo-resistant models for in vitro and in vivo mesothelioma studies.

Streptavidin: a novel immunostimulant for the selection and delivery of autologous and syngeneic tumor vaccines.

Induction of antitumor immunity using autologous tumor proteins is an attractive approach to cancer therapy. However, better methods and stimulants to present these autologous proteins back to the immune system are needed. Here, we identify streptavidin as a novel carrier protein and stimulant, and test the efficacy of both syngeneic (rat) and autologous vaccines (dogs) using streptavidin in combination with reduced soluble tumor proteins. Initial syngeneic vaccine studies in the 9L rat glioma model were used to optimize vaccine dose and selectivity. Cytokine and blood analysis was used to monitor the response. Rats receiving two vaccinations of syngeneic tumor vaccine demonstrated a statistically significant (P < 0.05) survival advantage compared with controls (adjuvant only). Notably, vaccination also led to remission rates of between 30% and 60% in the aggressive 9L glioma model. Antibodies to streptavidin were detected in the serum of vaccinated rats; however, antibody levels did not correlate with the response. The cytokine TNF-α was upregulated in vaccine-treated rats, whereas ICAM1 was downregulated. After engraftment, vaccinated rats maintained CD4(+), CD8(+) T cells, and total lymphocyte levels closer to normal baseline than those in the controls. Twenty-five dogs treated with autologous vaccine preparations using streptavidin as a stimulant showed no adverse reactions, irrespective of additional chemotherapy and other medications. In this study, we developed a novel method for producing syngeneic and autologous vaccines using streptavidin selectivity and immunogenicity. These vaccines show efficacy in the 9L glioma rat model. Safety was also demonstrated in canine patients presenting with cancer treated with autologous vaccine.

SV40 TAg mouse models of cancer.

The discovery of a number of viruses with the ability to induce tumours in animals and transform human cells has vastly impacted cancer research. Much of what is known about tumorigenesis today regarding tumour drivers and tumour suppressors has been discovered through experiments using viruses. The SV40 virus has proven extremely successful in generating transgenic models of many human cancer types and this review provides an overview of these models and seeks to give evidence as to their relevance in this modern era of personalised medicine and technological advancements.

Investigating the effectiveness of the Mediterranean diet in pregnant women for the primary prevention of asthma and allergy in high-risk infants: protocol for a pilot randomised controlled trial.

BACKGROUND: Over recent decades there has been a substantial increase in asthma and allergic disease especially in children. Given the high prevalence, and the associated high disease burden and costs, there is a need to identify effective strategies for the primary prevention of asthma and allergy. A recent systematic review of the literature found strong supportive epidemiological evidence for a protective role of the Mediterranean diet, which now needs to be confirmed through formal experimental studies. This pilot trial in pregnant women aims to establish recruitment, retention and acceptability of a dietary intervention, and to assess the likely impact of the intervention on adherence to a Mediterranean diet during pregnancy. METHODS/DESIGN: This study was a pilot, two-arm, randomised controlled trial in a sample population of pregnant women at high risk of having a child who will develop asthma or allergic disease. DISCUSSION: The work ultimately aims to contribute to improving health outcomes through seeking to reduce the incidence of asthma and allergic problems. This pilot trial will prove invaluable in informing the subsequent planned large-scale, parallel group, randomised controlled trial.

Is informal caregiving independently associated with poor health? A population-based study.

BACKGROUND: Providing informal care has been linked with poor health but has not previously been studied across a whole population. We aimed to study the association between informal care provision and self-reported poor health. METHOD: We used data from the UK 2001 Census. The relationship between informal caregiving and poor health was modelled using logistic regression, adjusting for age, sex, marital status, ethnicity, economic activity and educational attainment. RESULTS: We included 44,465,833 individuals free from permanent sickness or disability. 5,451,902 (12.3%) participants reported providing informal care to another person. There was an association between provision of informal caregiving and self-reported poor health; OR 1.100, 95% CI 1.096 to 1.103. This association remained after adjustment for age, sex, ethnic group, marital status, economic activity and educational attainment. The association also increased with the amount of care provided (hours per week). CONCLUSIONS: Around one in eight of the UK population reports that he or she is an informal caregiver. This activity is associated with poor health, particularly in those providing over 20 h care per week.