Acute Effects of Dietary Nitrate on Exercise Tolerance, Muscle Oxygenation, and Cardiovascular Function in Patients With Peripheral Arterial Disease.

Previous studies have used supplements to increase dietary nitrate intake in clinical populations. Little is known about whether effects can also be induced through vegetable consumption. Therefore, the aim of this study was to assess the impact of dietary nitrate, through nitrate-rich vegetables (NRV) and beetroot juice (BRJ) supplementation, on plasma nitrate and nitrite concentrations, exercise tolerance, muscle oxygenation, and cardiovascular function in patients with peripheral arterial disease. In a randomized crossover design, 18 patients with peripheral arterial disease (age: 73 ± 8 years) followed a nitrate intake protocol (∼6.5 mmol) through the consumption of NRV, BRJ, and nitrate-depleted BRJ (placebo). Blood samples were taken, blood pressure and arterial stiffness were measured in fasted state and 150 min after intervention. Each intervention was followed by a maximal walking exercise test to determine claudication onset time and peak walking time. Gastrocnemius oxygenation was measured by near-infrared spectroscopy. Blood samples were taken and blood pressure was measured 10 min after exercise. Mean plasma nitrate and nitrite concentrations increased (nitrate; Time × Intervention interaction; p < .001), with the highest concentrations after BRJ (494 ± 110 µmol/L) compared with NRV (202 ± 89 µmol/L) and placebo (80 ± 19 µmol/L; p < .001). Mean claudication onset time and peak walking time did not differ between NRV (413 ± 187 s and 745 ± 220 s, respectively), BRJ (392 ± 154 s and 746 ± 176 s), and placebo (403 ± 176 s and 696 ± 222 s) (p = .762 and p = .165, respectively). Gastrocnemius oxygenation, blood pressure, and arterial stiffness were not affected by the intervention. NRV and BRJ intake markedly increase plasma nitrate and nitrite, but this does not translate to improved exercise tolerance, muscle oxygenation, and/or cardiovascular function.

Systematic review and meta-analysis of skin substitutes in the treatment of diabetic foot ulcers: Highlights of a Cochrane systematic review.

Skin substitutes are increasingly used in the treatment of various types of acute and chronic wounds. The aim of this study was to perform a systematic review and meta-analysis to evaluate the effectiveness of skin substitutes on ulcer healing and limb salvage in the treatment of diabetic foot ulcers. Randomized clinical trials were searched and assessed following the methodology of The Cochrane Collaboration. We included 17 trials, totaling 1655 randomized participants. Risk of bias was variable among included trials. Thirteen trials compared the skin substitutes with standard care. The pooled results showed that that skin substitutes can, in addition to standard care, increase the likelihood of achieving complete ulcer closure compared with standard care alone after 6-16 weeks (risk ratio 1.55, 95% confidence interval [CI] 1.30-1.85). Four of the included trials compared two types of skin substitutes but no particular product showed a superior effect over another. Two trials reported on total incidence of lower limb amputations. Pooling the results of these two trials yielded a statistically significantly lower amputation rate among patients treated with skin substitutes (risk ratio 0.43, 95% CI 0.23-0.81), although the absolute risk difference was small (-0.06, 95% CI -0.10 to -0.01). This systematic review provides evidence that skin substitutes can, in addition to standard care, increase the likelihood of achieving complete ulcer closure compared with standard care alone in the treatment of diabetic foot ulcers. However, effectiveness on the long term, including lower limb salvage and recurrence, is currently lacking and cost-effectiveness is unclear.

Skin grafting and tissue replacement for treating foot ulcers in people with diabetes.

BACKGROUND: Foot ulceration is a major problem in people with diabetes and is the leading cause of hospitalisation and limb amputations. Skin grafts and tissue replacements can be used to reconstruct skin defects for people with diabetic foot ulcers in addition to providing them with standard care. Skin substitutes can consist of bioengineered or artificial skin, autografts (taken from the patient), allografts (taken from another person) or xenografts (taken from animals). OBJECTIVES: To determine the benefits and harms of skin grafting and tissue replacement for treating foot ulcers in people with diabetes. SEARCH METHODS: In April 2015 we searched: The Cochrane Wounds Specialised Register; the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library); Ovid MEDLINE; Ovid MEDLINE (In-Process & Other Non-Indexed Citations); Ovid EMBASE and EBSCO CINAHL. We also searched clinical trial registries to identify ongoing studies. We did not apply restrictions to language, date of publication or study setting. SELECTION CRITERIA: Randomised clinical trials (RCTs) of skin grafts or tissue replacements for treating foot ulcers in people with diabetes. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed the quality of the included studies. MAIN RESULTS: We included seventeen studies with a total of 1655 randomised participants in this review. Risk of bias was variable among studies. Blinding of participants, personnel and outcome assessment was not possible in most trials because of obvious differences between the treatments. The lack of a blinded outcome assessor may have caused detection bias when ulcer healing was assessed. However, possible detection bias is hard to prevent due to the nature of the skin replacement products we assessed, and the fact that they are easily recognisable. Strikingly, nearly all studies (15/17) reported industry involvement; at least one of the authors was connected to a commercial organisation or the study was funded by a commercial organisation. In addition, the funnel plot for assessing risk of bias appeared to be asymmetrical; suggesting that small studies with 'negative' results are less likely to be published.Thirteen of the studies included in this review compared a skin graft or tissue replacement with standard care. Four studies compared two grafts or tissue replacements with each other. When we pooled the results of all the individual studies, the skin grafts and tissue replacement products that were used in the trials increased the healing rate of foot ulcers in patients with diabetes compared to standard care (risk ratio (RR) 1.55, 95% confidence interval (CI) 1.30 to 1.85, low quality of evidence). However, the strength of effect was variable depending on the specific product that was used (e.g. EpiFix® RR 11.08, 95% CI 1.69 to 72.82 and OrCel® RR 1.75, 95% CI 0.61 to 5.05). Based on the four included studies that directly compared two products, no specific type of skin graft or tissue replacement showed a superior effect on ulcer healing over another type of skin graft or tissue replacement.Sixteen of the included studies reported on adverse events in various ways. No study reported a statistically significant difference in the occurrence of adverse events between the intervention and the control group.Only two of the included studies reported on total incidence of lower limb amputations. We found fewer amputations in the experimental group compared with the standard care group when we pooled the results of these two studies, although the absolute risk reduction for amputation was small (RR 0.43, 95% CI 0.23 to 0.81; risk difference (RD) -0.06, 95% CI -0.10 to -0.01, very low quality of evidence). AUTHORS' CONCLUSIONS: Based on the studies included in this review, the overall therapeutic effect of skin grafts and tissue replacements used in conjunction with standard care shows an increase in the healing rate of foot ulcers and slightly fewer amputations in people with diabetes compared with standard care alone. However, the data available to us was insufficient for us to draw conclusions on the effectiveness of different types of skin grafts or tissue replacement therapies. In addition, evidence of long term effectiveness is lacking and cost-effectiveness is uncertain.

[Acute abdominal symptoms in patients with a gastric band]. / Acute buikklachten bij patiënten met een maagband.

Three women aged 32, 35 and 37 years, respectively, presented with upper abdominal symptoms such as pain, nausea and vomiting. Their history revealed that an adjustable silicone gastric band had been implanted for the treatment of obesity one, four and seven years previously. Their symptoms were not immediately recognised as being possible complications of the gastric band and this caused treatment delay. In the first two patients ischaemic lesions were found at laparotomy. Their surgical treatment was successful. The third patient, however, was referred to the surgeon who had placed the gastric band, but she died in the mean time. Lack of recognition of symptoms caused by gastric banding and delay in diagnostic and therapeutic intervention may lead to very serious complications, or even death. Good diagnostic imaging and prompt therapeutic intervention can prevent the progression from herniation of the stomach through the silicone gastric band to irreversible ischaemia and eventually necrosis and perforation.

Expression of glutamine synthetase and carbamoylphosphate synthetase i in a bioartificial liver: markers for the development of zonation in vitro.

BACKGROUND: Mechanisms underlying hepatic zonation are not completely elucidated. In vitro test systems may provide new insights into current hypotheses. In this study, zonally expressed proteins, i.e. glutamine synthetase (GS; pericentral) and carbamoylphosphate synthetase (CPS; periportal), were tested for their expression patterns in the bioartificial liver of the Academic Medical Center (AMC-BAL). METHODS: Distribution and organization of porcine hepatocytes inside the AMC-BAL as well as GS and CPS expression were analyzed (immuno-)histochemically in time. Ten zonally expressed proteins were analyzed by RT-PCR on cell isolate and bioreactor samples. General metabolic and hepatocyte-specific functions were determined as well. RESULTS: Viable hepatocyte layers of approximately 150 microm were observed around gas capillaries, whereas inside the matrix, single cells or small aggregates were present. GS protein and mRNA levels were upregulated in time. GS protein was preferentially expressed in hepatocytes adjacent to oxygen-supplying capillaries and in previously CPS-positive hepatocytes. No shift towards a periportal or pericentral phenotype was observed from RT-PCR analysis. CONCLUSION: Induction of GS expression inside the AMC-BAL is not dependent of (low) oxygen tensions and hepatic nuclear factor 4alpha transcript levels. GS expression might be related to (1) low substrate levels and/or autocrine soluble factors, or (2) to cytoskeleton interactions, putatively associated with the beta-catenin signaling pathway.

Evaluation of a new immortalized human fetal liver cell line (cBAL111) for application in bioartificial liver.

BACKGROUND/AIMS: Clinical use of bioartificial livers (BAL) relies heavily on the development of human liver cell lines. The aim of this study was to assess the potential of the recently developed human fetal liver cell line cBAL111 for application in the AMC-BAL. METHODS: Laboratory-scale AMC-BAL bioreactors were loaded with 20 or 200 million cBAL111 cells and were cultured for 3 days. Parameters for hepatocyte-specific function and general metabolism were determined daily using tests with culture medium or 100% human serum. The bioreactors were also analyzed for mRNA levels of liver-specific genes and histology. RESULTS: cBAL111 eliminated ammonia at a rate up to 49% of that in primary porcine hepatocytes (PPH), despite a low (1.1%) urea production. Transcript levels of glutamine synthetase (GS) were 570% of that in human liver, whereas genes of the urea cycle showed low expression. GS expression was confirmed immunohistochemically, and glutamine was produced by the cells. cBAL111 eliminated galactose (90.1% of PPH) and lidocaine (0.1% of PPH) and produced albumin (6% of PPH). Human serum did not increase function of cBAL111. CONCLUSIONS: cBAL111 showed liver-specific functionality when cultured inside the AMC-BAL and eliminated ammonia mainly by the activity of GS, and not through the urea cycle.

Enhanced oxygen availability improves liver-specific functions of the AMC bioartificial liver.

Long-term culturing of primary porcine hepatocytes (PPH) inside the Academic Medical Center (AMC)-bioartificial liver is characterized by increased anaerobic glycolysis. Recommendations to increase oxygen availability were proposed in a previous numerical study and were experimentally evaluated in this study. Original bioreactors as well as new configuration bioreactors with 2.2-fold thinner nonwoven matrix and 2-fold more capillaries were loaded with PPHs and oxygenated with different gas oxygen pressures resulting in medium pO(2) (pO(2-med)) of either 135-150 mm Hg or 235-250 mm Hg. After 6 days culturing, new configuration bioreactors with pO(2-med )of 250 mm Hg showed significantly reduced anaerobic glycolysis, 60% higher liver-specific functions, and increased transcript levels of five liver-specific genes compared to the standard bioreactor cultures. Changed bioreactor configuration and increasing pO(2-med) contributed equally to these improvements. Histological examination demonstrated small differences in cell organization. In conclusion, higher metabolic stability and liver-specific functionality was achieved by enhanced oxygen availability based on a prior modeling concept.

Time-related analysis of metabolic liver functions, cellular morphology, and gene expression of hepatocytes cultured in the bioartificial liver of the Academic Medical Center in Amsterdam (AMC-BAL).

A comprehensive understanding of the mechanisms that underlie hepatic differentiation inside a bioartificial liver (BAL) device is obtained when functional, histological, and gene expression analyses can be combined. We therefore developed a novel cell-sampling technique that enabled us to analyze adherent hepatocytes inside a BAL device during a 5-day culture period, without the necessity of terminating the culture. Biochemical data showed that hepatocyte-specific functions were relatively stable, despite an increase in glycolytic activity. Quantitative reverse transcriptase polymerase chain reaction analysis of hepatic genes cytochrome p450 3A29, albumin, glutamine synthetase, alpha-1 antitrypsin, and carbamoyl-phosphate synthetase, but also de-differentiation marker pi-class glutathione S transferase showed stable messenger ribonucleic acid (mRNA) levels from day 1 to 5. In contrast, mRNA levels of alpha-fetoprotein, pro- and anti-apoptotic genes Bax-alpha and Bcl-X(L), metabolic genes lactate dehydrogenase and uncoupling protein 2, and cytoskeleton genes alpha- and beta-tubulin and beta-actin increased in 5 days. Histological analysis revealed viable tissue-like structures with adaptation to the in vitro environment. We conclude that hepatocytes show a tendency for de-differentiation shortly after seeding but thereafter remain acceptably differentiated during 5 days of culture. Furthermore, partly impaired mitochondrial function is suggestive for local hypoxic regions and may trigger the observed metabolic changes. Anti-apoptotic activity seems to balance pro-apoptotic activity. This new cell-sampling technique facilitates the analysis of dynamic processes of hepatocyte culture inside a BAL.

Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver.

The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)-BAL during a 7-day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2-fold more ammonia and produced 3-fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5-fold higher than MPHs and 6.6-fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue-like structures inside the AMC-BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading.

Three-dimensional numerical modeling and computational fluid dynamics simulations to analyze and improve oxygen availability in the AMC bioartificial liver.

A numerical model to investigate fluid flow and oxygen (O(2)) transport and consumption in the AMC-Bioartificial Liver (AMC-BAL) was developed and applied to two representative micro models of the AMC-BAL with two different gas capillary patterns, each combined with two proposed hepatocyte distributions. Parameter studies were performed on each configuration to gain insight in fluid flow, shear stress distribution and oxygen availability in the AMC-BAL. We assessed the function of the internal oxygenator, the effect of changes in hepatocyte oxygen consumption parameters in time and the effect of the change from an experimental to a clinical setting. In addition, different methodologies were studied to improve cellular oxygen availability, i.e. external oxygenation of culture medium, culture medium flow rate, culture gas oxygen content (pO(2)) and the number of oxygenation capillaries. Standard operating conditions did not adequately provide all hepatocytes in the AMC-BAL with sufficient oxygen to maintain O(2) consumption at minimally 90% of maximal uptake rate. Cellular oxygen availability was optimized by increasing the number of gas capillaries and pO(2) of the oxygenation gas by a factor two. Pressure drop over the AMC-BAL and maximal shear stresses were low and not considered to be harmful. This information can be used to increase cellular efficiency and may ultimately lead to a more productive AMC-BAL.

Bioartificial liver: its pros and cons.

Both the large variety of liver functions for maintaining body homeostasis and the proven effectivity of whole liver transplantation in the therapy of acute liver failure (ALF), are important reasons to presume that cell-free liver support systems will not be able to adequately support the failing liver. Accordingly, bioartificial liver (BAL) systems have shown their efficacy in experimental ALF models in small and large animals, and have shown to be suitable and safe in phase 1 studies in humans with ALF. However, the optimal BAL system is still under development. Important issues are the source of the cellular component and the configuration of the BAL system with regard to cell attachment, mass transfer characteristics and oxygenation at site. The deficiency of all BAL systems to excrete bile effectively is another important topic for improvement. The great challenge for the future is to develop a well-functioning and safe human hepatic cell line which can replace the widely used porcine (xenogeneic) hepatocytes. Theoretically, a combination of a cell-free liver support system and a BAL system might be optimal for the treatment of ALF patients in the near future.

Mild hypothermic preservation for transport purposes of the AMC bioartificial liver charged with porcine hepatocytes.

BACKGROUND: Preservation conditions play a crucial role during transport of a bioartificial liver (BAL) from the laboratory to the hospital. We assessed the possibility to preserve the AMC-BAL loaded with freshly isolated porcine hepatocytes at mild hypothermic temperatures. METHODS: Two laboratory-scale AMC-bioreactors were loaded with 1 billion freshly isolated porcine hepatocytes per experiment (n=6). Bioreactors in the control group were kept for three days at 37 degrees C. Bioreactors in the transport group were kept at 37 degrees C during day 1, at 15 degrees C during day 2, and again at 37 degrees C during day 3. In addition, long-term mild hypothermic preservation periods of 45 and 110 hr at 15 degrees C and 26 degrees C, respectively, were assessed. The effect of mild hypothermic preservation on hepatocytes inside the bioreactors was tested by determination of cell damage parameters, as well as metabolic and hepatocyte-specific functions. RESULTS: A 24-hour period of mild hypothermic preservation did not reduce any hepatocyte-specific function. LDH release was significantly higher only at day 2. Albumin production at day 2 and lidocaine elimination at day 3 were significantly higher with glucose consumption and lactate production being significantly lower at both test days. Long-term mild hypothermic preservation had a drastic negative effect on cellular viability and hepatocyte-specific function. CONCLUSIONS: Mild hypothermic preservation at temperatures as low as 15 degrees C and for a duration of 24 hr is a feasible method to preserve BAL systems loaded with freshly isolated porcine liver cells and will simplify the logistics of BAL transport from the laboratory to the hospital.