Effects of nitrite and far-red light on coagulation.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it's activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Tumour-specific amplitude-modulated radiofrequency electromagnetic fields induce differentiation of hepatocellular carcinoma via targeting Cav3.2 T-type voltage-gated calcium channels and Ca2+ influx.

BACKGROUND: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.

Systemic Iron Deficiency in a Nonhuman Primate Model of Endometriosis.

Endometriosis is characterized by endometrial tissue development outside the uterus. Anemia and iron depletion do not commonly accompany endometriosis in women, despite chronic abdominal inflammation and heavy menstrual bleeding. The objective of this study was to examine iron kinetics associated with endometriosis by using a NHP model, to better understand the underlying mechanism of abnormal hematogram values in women with endometriosis. Hematologic data from 46 macaques with endometriosis were examined for signs of iron depletion. Bone marrow, liver, and serum were used to elucidate whether iron loss or inflammation best explained the hematologic findings. Additional serum markers and intestinal biopsies from NHP with and without endometriosis were evaluated for patterns in iron kinetics across the menstrual cycle and for relative dietary iron-absorbing capacity. Almost half of the NHP with endometriosis were anemic. Overall, NHP had decreased RBC counts, increased MCV, increased percentage of reticulocytes, decreased serum hepcidin, and decreased hepatic and bone marrow iron. Intestinal expression of ferroportin 1, a mediator of iron absorption, was increased, indicating that despite high dietary iron, intestinal iron absorption did not compensate for iron losses. We concluded that use of oral iron supplementation alone does not replenish iron stores in endometriosis. Consequently, iron stores should be evaluated in women with endometriosis, even without overt clinical signs of anemia.

The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML.

Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC50 values in the low micromolar range (mean IC50 37 µM [range 25.89-52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML.

Efficacy of an orange oil emulsion as an anthelmintic against Haemonchus contortus in gerbils (Meriones unguiculatus) and in sheep.

Haemonchus contortus is a blood-sucking abomasal parasite responsible for major losses to small ruminant producers worldwide. The recent increase in populations of anthelmintic resistant parasites has produced a demand for alternative control methods. An orange oil emulsion that has shown activity against plant parasitic nematodes and H. contortus in vitro was assessed for activity against H. contortus in a gerbil model and in the natural ovine host. In gerbil experiments, animals were infected with 600 infective third stage (L3) H. contortus larvae. In one experiment, gerbils were treated with 600 milligrams per kilogram bodyweight (mg/kg BW) orange oil once or daily for 5 days. In a second experiment, gerbils were treated with 1200 mg/kg BW orange oil once or daily for 5 days. On Day 9 post-infection, gerbils were killed, their stomachs removed, and the worms counted. The 600 mg/kg BW dosage caused 7% and 62.6% parasite reduction compared to a control group when given once or daily for 5 days, respectively. The 1200 mg/kg BW dosage of orange oil caused 25% and 87.8% parasite reduction compared to a control group when given once or daily for 5 days, respectively. The difference between the multiple treatment and control group were significant at both dosages (P<0.005). In the sheep trial, 18 lambs were orally inoculated with 10,000 L3 H. contortus. One month later, two groups of six lambs each were dosed with 600 mg/kg BW orange oil either once or daily for 3 days. Fecal egg counts were monitored daily starting on the first day of treatment (Day 0) and continuing for 14 days. Results showed that a single dose of the product caused high fecal egg count reduction (97.4%) compared to control sheep. Egg counts were significantly reduced by Day 2 (P<0.0001). Thus, the orange oil emulsion may potentially be useful in the control of ovine haemonchosis.