Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep.

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health.

Low copper levels measured in the aortic wall of New Zealand patients with non-syndromic ascending thoracic aortic aneurysm.

OBJECTIVES: Studies in animals have shown causal relationships between copper (Cu) deficiency and the development of thoracic aortic aneurysms (TAAs) [1, 2]. Cu deficiency is widespread in New Zealand (NZ) soils; the high soil pH from the use of lime fertilizers reduces the bioavailability of Cu for grazing animals and growing plants; this, in turn, reduces Cu availability in the NZ human food chain. Our study is a pilot study to explore associations between Cu and TAA. We measured Cu levels in aneurysmal aortic tissues in patients undergoing Bentall procedures and non-aneurysmal aortic tissue from coronary artery bypass graft patients. METHODS: Aortic samples were collected from 2 groups of patients during elective open-heart surgery over 4 months between November 2017 and February 2018. The groups were a TAA group, patients with non-syndromic aortic aneurysm and without the bicuspid aortic valve or known infectious or inflammatory condition (ANEURYSM; n = 13), and a control coronary artery bypass graft group (CONTROL; n = 44). Standardized digested dry tissue weighed samples were analysed from both groups. Tissue extraction of trace elements was carried out using HCl-H2O2 digestion and a highly sensitive analytical technique, inductively coupled plasma mass spectrometry-used to measure elemental concentrations. RESULTS: Cu concentration (mean ± SD) was significantly lower in ANEURYSM (3.34 ± 0.16 µg/g) when compared to the CONTROL group tissues (4.33 ± 0.20 µg/g) (dry weight; mean ± SD; Student's t-test, P < 0.05). Over 46% of the Aneurysm patients were Maori and live in a geographically Cu-deficient NZ territory. CONCLUSIONS: Cu deficiency may play a role in the development or progression of non-syndromic ascending aortic aneurysms in NZ. Maori patients are more at risk as they commonly live in rural NZ, dependent on locally grown nutritional sources. Further studies are required to confirm this exciting finding and to establish cause and effect relationship.

Comparison of three oral selenium compounds in cancer patients: Evaluation of differential pharmacodynamic effects in normal and malignant cells.

BACKGROUND: Selenium (Se) compounds have demonstrated therapeutic synergism in combination with anticancer treatments whilst reducing normal tissue toxicities in a range of experimental models. While reduction in some toxicities of chemotherapy and radiation has been confirmed in randomised clinical trials, they have not been powered to evaluate improved anticancer efficacy. A lack of data on the clinical potencies of the main nutritionally-relevant forms of Se and the relationship between their pharmacokinetic (PK) profiles and pharmacodynamic (PD) effects in cancer patients has hampered progress to date. The primary objective of this study was to determine the dose and form of Se that can be most safely and effectively used in clinical trials in combination with anti-cancer therapies. STUDY METHODS: In a phase I randomised double-blinded study, the PD profile of sodium selenite (SS), Se-methylselenocysteine (MSC) and seleno-l-methionine (SLM) were compared in two cohorts of 12 patients, one cohort with chronic lymphocytic leukaemia (CLL) and the other with solid malignancies. All 24 patients were randomised to receive 400 µg of elemental Se as either SS, MSC or SLM, taken orally daily for 8 weeks. PD parameters were assessed before, during and 4 weeks after Se compound exposure in plasma and peripheral blood mononuclear cells (PBMCs). RESULTS: No significant sustained changes were observed in plasma concentrations of vascular endothelial growth factor-α (VEGF-α), expression of proteins associated with endoplasmic reticulum stress (the unfolded protein response) or in intracellular total glutathione in PBMCs, in either disease cohort or when grouped by Se compound. CONCLUSIONS: At the 400 µg dose level no substantial changes in PD parameters were noted. Extrapolating from pre-clinical data, the dose examined in this cohort was too low to achieve the Se plasma concentration (≥ 5 µM) expected to elicit significant PD effects. Recruitment of a subsequent cohort at higher doses to exceed this PK threshold is planned.

Transcriptional changes in response to ketamine ester-analogs SN 35210 and SN 35563 in the rat brain.

BACKGROUND: Ketamine ester analogs, SN 35210 and SN 35563, demonstrate different pharmacological profiles to ketamine in animal models. Both confer hypnosis with predictably rapid offset yet, paradoxically, SN35563 induces a prolonged anti-nociceptive state. To explore underlying mechanisms, broad transcriptome changes were measured and compared across four relevant target regions of the rat brain. RESULTS: SN 35563 produced large-scale alteration of gene expression in the Basolateral Amygdala (BLA) and Paraventricular Nucleus of the Thalamus (PVT), in excess of 10x that induced by ketamine and SN 35210. A smaller and quantitatively similar number of gene changes were observed in the Insula (INS) and Nucleus Accumbens (ACB) for all three agents. In the BLA and PVT, SN 35563 caused enrichment for gene pathways related to the function and structure of glutamatergic synapses in respect to: release of neurotransmitter, configuration of postsynaptic AMPA receptors, and the underlying cytoskeletal scaffolding and alignment. CONCLUSION: The analgesic ketamine ester analog SN 35563 induces profound large-scale changes in gene expression in key pain-related brain regions reflecting its unique prolonged pharmacodynamic profile.

Comparative Safety and Pharmacokinetic Evaluation of Three Oral Selenium Compounds in Cancer Patients.

Selenium (Se) compounds have demonstrated anticancer properties in both preclinical and clinical studies, with particular promise in combination therapy where the optimal form and dose of selenium has yet to be established. In a phase I randomised double-blinded study, the safety, tolerability and pharmacokinetic (PK) profiles of sodium selenite (SS), Se-methylselenocysteine (MSC) and seleno-l-methionine (SLM) were compared in patients with chronic lymphocytic leukaemia and a cohort of patients with solid malignancies. Twenty-four patients received 400 µg of elemental Se as either SS, MSC or SLM for 8 weeks. None of the Se compounds were associated with any significant toxicities, and the total plasma Se AUC of SLM was markedly raised in comparison to MSC and SS. DNA damage assessment revealed negligible genotoxicity, and some minor reductions in lymphocyte counts were observed. At the dose level used, all three Se compounds are well-tolerated and non-genotoxic. Further analyses of the pharmacodynamic effects of Se on healthy and malignant peripheral blood mononuclear cells will inform the future evaluation of higher doses of these Se compounds. The study is registered under the Australian and New Zealand Clinical Trials Registry No: ACTRN12613000118707.

The Interaction of Selenium with Chemotherapy and Radiation on Normal and Malignant Human Mononuclear Blood Cells.

Selenium, a trace element with anticancer properties, can reduce harmful toxicities of chemotherapy and radiotherapy without compromising efficacy. However, the dose-response relationship in normal versus malignant human cells is unclear. We evaluated how methylseleninic acid (MSA) modulates the toxicity and efficacy of chemotherapy and radiation on malignant and non-malignant human mononuclear blood cells in vitro. We specifically investigated its effects on endoplasmic reticulum stress induction, intracellular glutathione concentration, DNA damage and viability of peripheral blood mononuclear cells and THP1 monocytic leukaemia cells in response to radiation, cytosine arabinoside or doxorubicin chemotherapy. MSA, at lower concentrations, induced protective responses in normal cells but cytotoxic effects in malignant cells, alone and in conjunction with chemotherapy or radiation. However, in normal cells higher concentrations of MSA were directly toxic and increased the cytotoxicity of radiation but not chemotherapy. In malignant cells higher MSA concentrations were generally more effective in combination with cancer treatments. Thus, optimal MSA concentrations differed between normal and malignant cells and treatments. This work supports clinical reports that selenium can significantly reduce dose-limiting toxicities of anticancer therapies and potentially improve efficacy of anticancer treatments. The optimal selenium compound and dose is not yet determined.

Development of a qPCR Method to Measure Mitochondrial and Genomic DNA Damage with Application to Chemotherapy-Induced DNA Damage and Cryopreserved Cells.

DNA damage quantitation assays such as the comet assay have focused on the measurement of total nuclear damage per cell. The adoption of PCR-based techniques to quantify DNA damage has enabled sequence- and organelle-specific assessment of DNA lesions. Here we report on an adaptation of a qPCR technique to assess DNA damage in nuclear and mitochondrial targets relative to control. Novel aspects of this assay include application of the assay to the Rotor-Gene platform with optimized DNA polymerase/fluorophore/primer set combination in a touchdown PCR protocol. Assay validation was performed using ultraviolet C radiation in A549 and THP1 cancer cell lines. A comparison was made to the comet assay applied to peripheral blood mononuclear cells, and an estimation of the effects of cryopreservation on ultraviolet C-induced DNA damage was carried out. Finally, dose responses for DNA damage were measured in peripheral blood mononuclear cells following exposure to the cytotoxic agents bleomycin and cisplatin. We show reproducible experimental outputs across the tested conditions and concordance with published findings with respect to mitochondrial and nuclear genotoxic susceptibilities. The application of this DNA damage assay to a wide range of clinical and laboratory-derived samples is both feasible and resource-efficient.

Distinct gene expression profiles directed by the isoforms of the transcription factor neuron-restrictive silencer factor in human SK-N-AS neuroblastoma cells.

Neuron-restrictive silencer factor (NRSF) and its isoforms are differentially regulated in rodent models of self-sustaining status epilepticus (SSSE). NRSF isoforms regulate genes associated with SSSE, including the proconvulsant tachykinins, brain-derived neurotrophic factor and multiple ion channels. NRSF isoforms may direct distinct gene expression patterns during SSSE, and the ratio of each isoform may be a causative factor in traumatic damage to the central nervous system. Here, we analysed global gene expression changes by microarray in human SK-N-AS neuroblastoma cells following the over-expression of NRSF and a truncated isoform, HZ4. We used bioinformatics software to analyse the microarray dataset and correlated these data with epilepsy candidate gene pathways. Findings were validated by reverse transcriptase-polymerase chain reaction. We demonstrated that NRSF and HZ4 direct overlapping as well as distinct gene expression patterns, and that they differentially modulated gene expression patterns associated with epilepsy. Finally, we revealed that NRSF gene expression may be modulated by the anticonvulsant, phenytoin. We have interpreted our data to reflect altered gene expression directed by NRSF that might be relevant for SSSE.

Connexin36 knockout mice display increased sensitivity to pentylenetetrazol-induced seizure-like behaviors.

OBJECTIVE: Large-scale synchronous firing of neurons during seizures is modulated by electrotonic coupling between neurons via gap junctions. To explore roles for connexin36 (Cx36) gap junctions in seizures, we examined the seizure threshold of connexin36 knockout (Cx36KO) mice using a pentylenetetrazol (PTZ) model. METHODS: Mice (2-3months old) with Cx36 wildtype (WT) or Cx36KO genotype were treated with vehicle or 10-40mg/kg of the convulsant PTZ by intraperitoneal injection. Seizure and seizure-like behaviors were scored by examination of video collected for 20min. Quantitative real-time PCR (QPCR) was performed to measure potential compensatory neuronal connexin (Cx30.2, Cx37, Cx43 and Cx45), pannexin (PANX1 and PANX2) and gamma-aminobutyric acid type A (GABA(A)) receptor α1 subunit gene expression. RESULTS: Cx36KO animals exhibited considerably more severe seizures; 40mg/kg of PTZ caused severe generalized (≥grade III) seizures in 78% of KO, but just 5% of WT mice. A lower dose of PTZ (20mg/kg) induced grade II seizure-like behaviors in 40% KO vs. 0% of WT animals. There was no significant difference in either connexin, pannexin or GABA(A) α1 gene expression between WT and KO animals. CONCLUSION: Increased sensitivity of Cx36KO animals to PTZ-induced seizure suggests that Cx36 gap junctional communication functions as a physiological anti-convulsant mechanism, and identifies the Cx36 gap junction as a potential therapeutic target in epilepsy.

SCG3 transcript in peripheral blood is a prognostic biomarker for REST-deficient small cell lung cancer.

PURPOSE: Specific markers of circulating tumor cells may be informative in managing lung cancer. Because the RE-1 silencing transcription factor (REST/NRSF) is a transcriptional repressor that is inactivated in neuroendocrine lung cancer, we identified REST-regulated transcripts (CHGA, CHGB, SCG3, VGF, and PCSK1) for evaluation as biomarkers in peripheral blood. EXPERIMENTAL DESIGN: Transcripts were screened across lung cancer and normal cell lines. Candidates were assessed by reverse transcription-PCR and hybridization of RNA extracted from the peripheral blood of 111 lung cancer patients obtained at clinical presentation and from 27 cancer-free individuals. RESULTS: Expression profiling revealed multiple chromogranin transcripts were readily induced on REST depletion, most notably SCG3 was induced >500-fold. The SCG3 transcript was also overexpressed by 12,000-fold in neuroendocrine compared with nonneuroendocrine lung cancer cells. In peripheral blood of lung cancer patients and cancer-free individuals, we found that SCG3 was more tumor-specific and more sensitive than other chromogranin transcripts as a biomarker of circulating tumor cells. Overall, 36% of small cell lung cancer (SCLC) and 16% of non-SCLC patients scored positively for normalized SCG3 transcript. This correlated with worse survival among SCLC patients with limited disease (n = 33; P = 0.022) but not extensive disease (n = 29; P = 0.459). Interestingly, the subcohort of 6 SCLC patients with resistance to platinum/etoposide chemotherapy all scored positively for peripheral blood SCG3 transcript (P = 0.022). CONCLUSIONS: SCG3 mRNA, a component of the REST-dependent neurosecretory transcriptional profile, provides a sensitive prognostic biomarker for noninvasive monitoring of neuroendocrine lung cancer.