Effect of HBO therapy on adipose-derived stem cells, fibroblasts and co-cultures: In vitro study of oxidative stress, angiogenic potential and production of pro-inflammatory growth factors in co-cultures1.

BACKGROUND: A key moderator of wound healing is oxygen. Wound healing is a dynamic and carefully orchestrated process involving blood cells, cytokines, parenchymal cells (i.e. fibroblasts and mesenchymal stem cells) and extracellular matrix reorganization. Human adipose derived stem cells as well as human fibroblasts produce soluble factors, exhibit diverse effects on inflammation and anti inflammation response and are involved in wound healing processes.Hyperbaric oxygen therapy is an effective adjunct treatment for ischemic disorders such as chronic infection or chronic wounds. In vitro effects of hyperbaric oxygen therapy on human cells were presented in many studies except for those on mono- and co-cultures of human adipose derived stem cells and fibroblasts. OBJECTIVE: The aim of this study was to investigate the effects of hyperbaric oxygen therapy on mono- and co-cultures of human adipose derived stem cells and fibroblasts. METHODS: Mono- and co-cultures from human adipose derived stem cells and fibroblasts were established. These cultures were exposed to hyperbaric oxygen therapy every 24 h for five consecutive days. Measuring experiments were performed on the first, third and fifth day. Therapy effects on the expression of VEGF, IL 6 and reactive oxygen species were investigated. RESULTS: After exposure to hyperbaric oxygen, cell culturess showed a significant increase in the expression of VEGF after 3 and 5 days. All cultures showed significantly reduced formation of reactive oxygen species throughout the experiments. The expression of IL-6 decreased during the experiment in mono-cultures of human adipose derived stem cells and co-cultures. In contrast, mono-cultures of human skin fibroblasts showed an overall significantly increased expression of IL-6. CONCLUSIONS: Hyperbaric oxygen therapy leads to immunmodulatory and proangiogenetic effects in a wound-like enviroment of adipose derived stem cells and fibroblasts.

[Cell salvage : Scientific evidence, clinical practice and legal framework]. / Maschinelle Autotransfusion : Wissenschaftliche Evidenz, klinische Praxis und rechtliche Rahmenbedingungen.

Cell salvage is an efficient method to reduce the transfusion of homologous banked blood, as documented by several meta-analyses detected in a systematic literature search. Cell salvage is widely used in orthopedics, trauma surgery, cardiovascular and abdominal transplantation surgery. The retransfusion of unwashed shed blood from wounds or drainage is not permitted according to German regulations. Following irradiation of wound blood, salvaged blood can also be used in tumor surgery. Cell salvage makes a valuable contribution to providing sufficient compatible blood for transfusions in cases of massive blood loss. Certain surgical procedures for Jehovah's Witnesses are only possible with the use of cell salvage. Another possible use is the washing of homologous banked blood, e. g. to prevent potassium-induced arrhythmia or sequestration of autologous platelets. Other advantages besides a good compatibility are the high vitality and functionality of the unstored autologous red blood cells. These have been declared a pharmaceutical product by the German transfusion task force in 2014, so that the autologous red blood cells are now under the control of the Pharmaceutical Products Act (AMG). The new hemotherapy guidelines, however, tolerate cell salvage only under strict rules, whereby the production of autologous blood during or after surgery is still possible without additional special permits. The new guidelines now require the introduction of a quality management system for cell salvage and regular quality controls. These quality controls include a control of the product hematocrit for every application, monthly controls of the protein and albumin elimination rates and the erythrocyte recovery rate for each cell salvage device. Testing for infection markers is not required. The application of cell salvage has to be reported to the appropriate authorities.

A new approach for fat removal in a discontinuous autotransfusion device-concept and evaluation.

BACKGROUND: Fat present during blood salvage in orthopaedic or cardiac surgery can pose a risk of fat embolism and should be eliminated before transfusion. Based on observations of central fat accumulation at the bottom of Latham bowls, a fat reduction program was developed using two volume displacements, where blood temporarily is removed and respun in the bowl to force the fat through the RBC sediment. MATERIALS AND METHODS: Pooled ABO-matched RBC and FFP were adjusted to a haematocrit of 10%, and human fat tissue added to a concentration of 1·25 vol%. In six experiments, blood was processed with the new-generation cell salvage device CS Elite in a newly developed fat reduction program in bowls of three sizes. Volumetric quantification of fat was performed after centrifugation of blood samples in Pasteur pipettes. From volumes, haematocrits and the concentrations of fat, RBC recovery and fat elimination rates were calculated. RESULTS: Fat removal rates of 93·2 ± 2·8, 97·0 ± 2·1 and 99·6 ± 0·3% were observed with a 70-ml, 125-ml and 225-ml bowl, respectively, and even higher rates when removal rates were calculated one cycle. At the same time, high RBC recovery and plasma elimination rates were maintained, not significantly different to the default program mode. CONCLUSION: Modifications in process parameters and sequence led to a fat reduction program that significantly improves fat removal with the Cell Saver Elite from 77·4 ± 5·1% in the default mode to an average of 98·6 ± 1·1%, yielding results equivalent to the continuous cell salvage system (C.A.T.S).

Performance of a new-generation continuous autotransfusion device including fat removal and consequences for quality controls.

BACKGROUND AND OBJECTIVES: Cell salvage plays a key role in blood conservation. To maintain high performance, quality management is recommended. Accordingly, a new-generation autotransfusion device was tested for its performance and compared with its predecessor. Two different calculations of quality parameters were applied. MATERIALS AND METHODS: In an experimental study, the continuous autotransfusion devices CATSmart and Continuous Autotransfusion System (C.A.T.S) plus were tested using banked blood adjusted to a haematocrit of 20% and anticoagulated with heparin 5 U/L. Test blood was processed using an emergency programme, a high-quality programme/smart wash programme and a low-volume wash programme. Samples were taken after the production of 200 mL of red blood cells (RBC) and after the final emptying of the separation chamber. In an additional set of tests, blood containing 1·25% fat was processed with both devices to examine fat removal. RESULTS: Both devices demonstrated an equally high performance with regards to product hematocrit (Hct); RBC recovery; and elimination rates of protein, heparin and fat. The high fat elimination rate (>99·8%) reported for C.A.T.S plus was confirmed for CATSmart, regardless of the used programme. Samples taken during the ongoing process show a higher haematocrit and RBC recovery rate than samples taken after the final emptying of the separation chamber. Interface sensors were not affected by fat in the blood. CONCLUSIONS: The new-generation autotransfusion device CATSmart is not inferior to its predecessor and shows high performance with regards to RBC recovery, plasma and fat elimination in all programme modes. Samples for quality controls should be taken during blood processing.

Non-Toxic Metabolic Management of Metastatic Cancer in VM Mice: Novel Combination of Ketogenic Diet, Ketone Supplementation, and Hyperbaric Oxygen Therapy.

The Warburg effect and tumor hypoxia underlie a unique cancer metabolic phenotype characterized by glucose dependency and aerobic fermentation. We previously showed that two non-toxic metabolic therapies - the ketogenic diet with concurrent hyperbaric oxygen (KD+HBOT) and dietary ketone supplementation - could increase survival time in the VM-M3 mouse model of metastatic cancer. We hypothesized that combining these therapies could provide an even greater therapeutic benefit in this model. Mice receiving the combination therapy demonstrated a marked reduction in tumor growth rate and metastatic spread, and lived twice as long as control animals. To further understand the effects of these metabolic therapies, we characterized the effects of high glucose (control), low glucose (LG), ketone supplementation (ßHB), hyperbaric oxygen (HBOT), or combination therapy (LG+ßHB+HBOT) on VM-M3 cells. Individually and combined, these metabolic therapies significantly decreased VM-M3 cell proliferation and viability. HBOT, alone or in combination with LG and ßHB, increased ROS production in VM-M3 cells. This study strongly supports further investigation into this metabolic therapy as a potential non-toxic treatment for late-stage metastatic cancers.

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer.

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM-M3 cells cultured in the presence and absence of ß-hydroxybutyrate (ßHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase-tagged syngeneic VM-M3 cells. Mice were fed a standard diet supplemented with either 1,3-butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies ßHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood ßHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM-M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM-M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use.

Therapeutic effects of stem cells and substrate reduction in juvenile Sandhoff mice.

Sandhoff Disease (SD) involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the ß-subunit gene of ß-hexosaminidase A and B (Hexb gene). Substrate reduction therapy, utilizing imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ), reduces ganglioside biosynthesis and levels of stored GM2 in SD mice. Intracranial transplantation of Neural Stem Cells (NSCs) can provide enzymatic cross correction, to help reduce ganglioside storage and extend life. Here we tested the effect of NSCs and NB-DGJ, alone and together, on brain ß-hexosaminidase activity, GM2, and GA2 content in juvenile SD mice. The SD mice received either cerebral NSC transplantation at post-natal day 0 (p-0), intraperitoneal injection of NB-DGJ (500 mg/kg/day) from p-9 to p-15, or received dual treatments. The brains were analyzed at p-15. ß-galactosidase staining confirmed engraftment of lacZ-expressing NSCs in the cerebral cortex. Compared to untreated and sham-treated SD controls, NSC treatment alone provided a slight increase in Hex activity and significantly decreased GA2 content. However, NSCs had no effect on GM2 content when analyzed at p-15. NB-DGJ alone had no effect on Hex activity, but significantly reduced GM2 and GA2 content. Hex activity was slightly elevated in the NSC + drug-treated mice. GM2 and GA2 content in the dual treated mice were similar to that of the NB-DGJ treated mice. These data indicate that NB-DGJ alone was more effective in targeting storage in juvenile SD mice than were NSCs alone. No additive or synergistic effect between NSC and drug was found in these juvenile SD mice.

[Cell salvage]. / Maschinelle Autotransfusion.

With increasing demands for blood transfusions, the costs and shortages, clinically relevant risks and doubts on the efficacy, blood conservation is an important issues. Among the available methods cell salvage is of great importance as it has proven effective and safe. The high availability and cost efficacy allows fast processing of at least half of the lost red blood cells. The method has wide applications in cardiac and vascular surgery, in abdominal and transplantation surgery, in orthopedics and emergency medicine, in massive hemorrhage and for Jehovah's Witnesses, and by the use of blood irradiation also in cancer surgery. Cell salvage provides autologous, washed, unstored red blood cells with unimpaired function and viability, avoiding immunological reactions and storage damage, for optimal hemotherapy. No restrictions in the indication for transfusion are necessary, thus allowing real therapy of anemia. The high quality of salvaged blood should be assured by a quality management including quality controls.

Calcium ATPase activities in synaptic plasma membranes of seizure-prone mice.

Audiogenic seizure (AGS)-susceptible DBA/2 (D2) mice have a significant reduction in brain Ca2+-ATPase activity compared to AGS-resistant C57BL/6 (B6) mice. This reduction is inherited together with AGS susceptibility in B6 X D2 recombinant inbred strains. The Ca2+-ATPase reduction occurs in microsomes and synaptosomes, but not in mitochondria. This enzyme activity is measured at a high Ca2+ concentration (2 mM) with no added Mg2+ or EGTA. We further studied this Ca2+-ATPase activity and a Mg2+-dependent (Ca2+ + Mg2+)-ATPase activity in synaptic plasma membranes (SPM) from the B6 and D2 strains. Using EGTA or CDTA to adjust free Ca2+ concentrations, we measured Ca2+-ATPase activities at Ca2+ concentrations from 0.8 microM to 436 microM. The Ca2+-ATPase activity is consistently lower in the D2 than in the B6 SPM over all Ca2+ concentrations. The basal Mg2+-ATPase activity measured at 2 mM MgCl2, is also lower in SPM of D2 than B6 mice. Calcium stimulates the basal Mg2+-ATPase activity to the same extent in the SPM of the B6 and the D2 mice. Maximum stimulation in both strains occurs at 150 microM added CaCl2 (buffered with 100 microM EGTA). Higher Ca2+ concentrations inhibit this ATPase activity similarly in both strains. The EGTA-EDTA washing of SPM significantly reduces by 50% of the (Ca2+ + Mg2+)-ATPase activities of both strains, whereas calmodulin treatment restored these activities. Neither of these treatments, however, has any noticeable effects on the Ca2+-ATPase activities of the strains.(ABSTRACT TRUNCATED AT 250 WORDS)