A Comparison of p53 Isoform Profiles and Apoptosis Induced by Camptothecin or a Herbal Khat Extract (Catha Edulis (Vahl) Forssk. ex Endl.) in Leukemic Cell Lines: Exploring Cellular Responses in Therapy Development

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics.publishedVersio

Camptothecin and khat (Catha edulis Forsk.) induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines

<p>Abstract</p> <p>Background</p> <p>An organic extract of the recreational herb khat (<it>Catha edulis </it>Forsk.) triggers cell death in various leukemia cell lines <it>in vitro</it>. The chemotherapeutics camptothecin, a plant alkaloid topoisomerase I inhibitor, was tested side-by-side with khat in a panel of acute myeloid leukemia cell lines to elucidate mechanisms of toxicity.</p> <p>Results</p> <p>Khat had a profound effect on MOLM-13 cells inducing mitochondrial damage, chromatin margination and morphological features of autophagy. The effects of khat on mitochondrial ultrastructure in MOLM-13 correlated with strongly impaired routine respiration, an effect neither found in the khat-resistant MV-4-11 cells nor in camptothecin treated cells. Enforced expression of anti-apoptotic Bcl-2 protein provided protection against camptothecin-induced cell death and partly against khat toxicity. Khat-induced cell death in MOLM-13 cells included reduced levels of anti-apoptotic Mcl-1 protein, while both khat and camptothecin induced c-FLIP_Lcleavage and procaspase-8 activation.</p> <p>Conclusion</p> <p>Khat activated a distinct cell death pathway in sensitive leukemic cells as compared to camptothecin, involving mitochondrial damage and morphological features of autophagy. This suggests that khat should be further explored in the search for novel experimental therapeutics.</p

Risk assessment and antibody responses to SARS-CoV-2 in healthcare workers

Background: Preventing infection in healthcare workers (HCWs) is crucial for protecting healthcare systems during the COVID-19 pandemic. Here, we investigated the seroepidemiology of SARS-CoV-2 in HCWs in Norway with low-transmission settings. Methods: From March 2020, we recruited HCWs at four medical centres. We determined infection by SARS-CoV-2 RT-PCR and serological testing and evaluated the association between infection and exposure variables, comparing our findings with global data in a meta-analysis. Anti-spike IgG antibodies were measured after infection and/or vaccination in a longitudinal cohort until June 2021. Results: We identified a prevalence of 10.5% (95% confidence interval, CI: 8.8–12.3) in 2020 and an incidence rate of 15.0 cases per 100 person-years (95% CI: 12.5–17.8) among 1,214 HCWs with 848 person-years of follow-up time. Following infection, HCWs (n = 63) mounted durable anti-spike IgG antibodies with a half-life of 4.3 months since their seropositivity. HCWs infected with SARS-CoV-2 in 2020 (n = 46) had higher anti-spike IgG titres than naive HCWs (n = 186) throughout the 5 months after vaccination with BNT162b2 and/or ChAdOx1-S COVID-19 vaccines in 2021. In a meta-analysis including 20 studies, the odds ratio (OR) for SARS-CoV-2 seropositivity was significantly higher with household contact (OR 12.6; 95% CI: 4.5–35.1) and occupational exposure (OR 2.2; 95% CI: 1.4–3.2). Conclusion: We found high and modest risks of SARS-CoV-2 infection with household and occupational exposure, respectively, in HCWs, suggesting the need to strengthen infection prevention strategies within households and medical centres. Infection generated long-lasting antibodies in most HCWs; therefore, we support delaying COVID-19 vaccination in primed HCWs, prioritising the non-infected high-risk HCWs amid vaccine shortage.publishedVersio

A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80–99 years, n = 89) and younger adults (23–77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1–89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72–0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72–76%) older adults respond after two vaccinations to alpha and delta, but only 58–62% to beta and gamma, compared to 96–97% of younger vaccinees and 68–76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.publishedVersio

SARS-CoV-2 specific immune responses in overweight and obese COVID-19 patients

Obesity is a known risk factor for severe respiratory tract infections. In this prospective study, we assessed the impact of being obese or overweight on longitudinal SARS-CoV-2 humoral and cellular responses up to 18 months after infection. 274 patients provided blood samples at regular time intervals up to 18 months including obese (BMI ≥30, n=32), overweight (BMI 25-29.9, n=103) and normal body weight (BMI 18.5-24.9, n=134) SARS-CoV-2 patients. We determined SARS-CoV-2 spike-specific IgG, IgA, IgM levels by ELISA and neutralising antibody titres by neutralisation assay. RBD- and spike-specific memory B cells were investigated by ELISpot, spike- and non-spike-specific IFN-γ, IL-2 and IFN-γ/IL-2 secreting T cells by FluoroSpot and T cell receptor (TCR) sequencing was performed. Higher BMI correlated with increased COVID-19 severity. Humoral and cellular responses were stronger in overweight and obese patients than normal weight patients and associated with higher spike-specific IgG binding titres relative to neutralising antibody titres. Linear regression models demonstrated that BMI, age and COVID-19 severity correlated independently with higher SARS-CoV-2 immune responses. We found an increased proportion of unique SARS-CoV-2 specific T cell clonotypes after infection in overweight and obese patients. COVID-19 vaccination boosted humoral and cellular responses irrespective of BMI, although stronger immune boosting was observed in normal weight patients. Overall, our results highlight more severe disease and an over-reactivity of the immune system in overweight and obese patients after SARS-CoV-2 infection, underscoring the importance of recognizing overweight/obese individuals as a risk group for prioritisation for COVID-19 vaccination

A Comparison of p53 Isoform Profiles and Apoptosis Induced by Camptothecin or a Herbal Khat Extract (Catha Edulis (Vahl) Forssk. ex Endl.) in Leukemic Cell Lines: Exploring Cellular Responses in Therapy Development

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

Background: Amphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by 3H-thymidine incorporation. Results: Cathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p- Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p- Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p- Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death. Conclusions: Single-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced Tlymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific singlecell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts

Stathmin Protein Level, a Potential Predictive Marker for Taxane Treatment Response in Endometrial Cancer

Stathmin is a prognostic marker in many cancers, including endometrial cancer. Preclinical studies, predominantly in breast cancer, have suggested that stathmin may additionally be a predictive marker for response to paclitaxel. We first evaluated the response to paclitaxel in endometrial cancer cell lines before and after stathmin knock-down. Subsequently we investigated the clinical response to paclitaxel containing chemotherapy in metastatic endometrial cancer in relation to stathmin protein level in tumors. Stathmin level was also determined in metastatic lesions, analyzing changes in biomarker status on disease progression. Knock-down of stathmin improved sensitivity to paclitaxel in endometrial carcinoma cell lines with both naturally higher and lower sensitivity to paclitaxel. In clinical samples, high stathmin level was demonstrated to be associated with poor response to paclitaxel containing chemotherapy and to reduced disease specific survival only in patients treated with such combination. Stathmin level increased significantly from primary to metastatic lesions. This study suggests, supported by both preclinical and clinical data, that stathmin could be a predictive biomarker for response to paclitaxel treatment in endometrial cancer. Re-assessment of stathmin level in metastatic lesions prior to treatment start may be relevant. Also, validation in a randomized clinical trial will be important

A Comparison of p53 Isoform Profiles and Apoptosis Induced by Camptothecin or a Herbal Khat Extract (Catha Edulis (Vahl) Forssk. ex Endl.) in Leukemic Cell Lines: Exploring Cellular Responses in Therapy Development

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics

Durable T-cellular and humoral responses in SARS-CoV-2 hospitalized and community patients

Background Neutralizing antibodies are important for protection against the pandemic SARS-CoV-2 virus, and long-term memory responses determine the risk of re-infection or boosting after vaccination. T-cellular responses are considered important for partial protection against novel variants of concern. Methods A prospective cohort of hospitalized (n = 14) and community (n = 38) patients with rt-PCR confirmed SARS-CoV-2 infection were recruited. Blood samples and clinical data were collected when diagnosed and at 6 months. Serum samples were analyzed for SARS-CoV-2-spike specific antibodies using ELISA (IgG, IgA, IgM), pseudotype neutralization and microneutralization assays. Peripheral blood mononuclear cells were investigated for virus-specific T-cell responses in the interferon-γ and interleukin-2 fluorescent-linked immunosorbent spot (FluroSpot) assay. Results We found durable SARS-CoV-2 spike- and internal protein specific T-cellular responses in patients with persistent antibodies at 6 months. Significantly higher IL-2 and IFN-γ secreting T-cell responses as well as SARS-CoV-2 specific IgG and neutralizing antibodies were detected in hospitalized compared to community patients. The immune response was impacted by age, gender, comorbidity and severity of illness, reflecting clinical observations. Conclusions SARS-CoV-2 specific T-cellular and antibody responses persisted for 6 months post confirmed infection. In previously infected patients, re-exposure or vaccination will boost long-term immunity, possibly providing protection against re-infection with variant viruses