Effect of 5 weeks of oral acetazolamide on patients with pulmonary vascular disease: A randomized, double-blind, cross-over trial.

BACKGROUND: The carbonic anhydrase inhibitor acetazolamide stimulates ventilation through metabolic acidosis mediated by renal bicarbonate excretion. In animal models, acetazolamide attenuates acute hypoxia-induced pulmonary hypertension (PH), but its efficacy in treating patients with PH due to pulmonary vascular disease (PVD) is unknown. METHODS: 28 PVD patients (15 pulmonary arterial hypertension, 13 distal chronic thromboembolic PH), 13 women, mean±SD age 61.6±15.0 years stable on PVD medications, were randomised in a double-blind crossover protocol to 5 weeks acetazolamide (250mg b.i.d) or placebo separated by a ≥2 week washout period. Primary endpoint was the change in 6-minute walk distance (6MWD) at 5 weeks. Additional endpoints included safety, tolerability, WHO functional class, quality of life, arterial blood gases, and hemodynamics (by echocardiography). RESULTS: Acetazolamide had no effect on 6MWD compared to placebo (treatment effect: mean change [95%CI] -18 [-40 to 4]m, p=0.102) but increased arterial blood oxygenation through hyperventilation induced by metabolic acidosis. Other measures including pulmonary hemodynamics were unchanged. No severe adverse effects occurred, side effects that occurred significantly more frequently with acetazolamide vs. placebo were change in taste (22/0%), paraesthesia (37/4%) and mild dyspnea (26/4%). CONCLUSIONS: In patients with PVD, acetazolamide did not change 6MWD compared to placebo despite improved blood oxygenation. Some patients reported a tolerable increase in dyspnoea during acetazolamide treatment, related to hyperventilation, induced by the mild drug-induced metabolic acidosis. Our findings do not support the use of acetazolamide to improve exercise in patients with PVD at this dosing. GOV IDENTIFIER: NCT02755298.

Reduced cancer mortality at high altitude: The role of glucose, lipids, iron and physical activity.

Residency at high altitude (HA) demands adaptation to challenging environmental conditions with hypobaric hypoxia being the most important one. Epidemiological and experimental data suggest that chronic exposure to HA reduces cancer mortality and lowers prevalence of metabolic disorders like diabetes and obesity implying that adaption to HA modifies a broad spectrum of physiological, metabolic and cellular programs with a generally beneficial outcome for humans. However, the complexity of multiple, potentially tumor-suppressive pathways at HA impedes the understanding of mechanisms leading to reduced cancer mortality. Many adaptive processes at HA are tightly interconnected and thus it cannot be ruled out that the entirety or at least some of the HA-related alterations act in concert to reduce cancer mortality. In this review we discuss tumor formation as a concept of competition between healthy and cancer cells with improved fitness - and therefore higher competitiveness - of healthy cells at high altitude. We discuss HA-related changes in glucose, lipid and iron metabolism that may have an impact on tumorigenesis. Additionally, we discuss two parameters with a strong impact on tumorigenesis, namely drug metabolism and physical activity, to underpin their potential contribution to HA-dependent reduced cancer mortality. Future studies are needed to unravel why cancer mortality is reduced at HA and how this knowledge might be used to prevent and to treat cancer patients.

Fixed-dose combination therapy with daclatasvir, asunaprevir, and beclabuvir for noncirrhotic patients with HCV genotype 1 infection.

IMPORTANCE: The antiviral activity of all-oral, ribavirin-free, direct-acting antiviral regimens requires evaluation in patients with chronic hepatitis C virus (HCV) infection. OBJECTIVE: To determine the rates of sustained virologic response (SVR) in patients receiving the 3-drug combination of daclatasvir (a pan-genotypic NS5A inhibitor), asunaprevir (an NS3 protease inhibitor), and beclabuvir (a nonnucleoside NS5B inhibitor). DESIGN, SETTING, AND PARTICIPANTS: This was an open-label, single-group, uncontrolled international study (UNITY-1) conducted at 66 sites in the United States, Canada, France, and Australia between December 2013 and August 2014. Patients without cirrhosis who were either treatment-naive (n = 312) or treatment-experienced (n = 103) and had chronic HCV genotype 1 infection were included. INTERVENTIONS: Patients received a twice-daily fixed-dose combination of daclatasvir, 30 mg; asunaprevir, 200 mg; and beclabuvir, 75 mg. MAIN OUTCOMES AND MEASURES: The primary study outcome was SVR12 (HCV-RNA <25 IU/mL at posttreatment week 12) in patients naive to treatment. A key secondary outcome was SVR12 in the treatment-experienced cohort. RESULTS: Baseline characteristics were comparable between the treatment-naive and treatment-experienced cohorts. Patients were 58% male, 26% had IL28B (rs12979860) CC genotype, 73% were infected with genotype 1a, and 27% were infected with genotype 1b. Overall, SVR12 was observed in 379 of 415 patients (91.3%; 95% CI, 88.6%-94.0%): 287 of 312 treatment-naive patients (92.0%; 95% CI, 89.0%-95.0%) and 92 of 103 treatment-experienced patients (89.3%; 95% CI, 83.4%-95.3%). Virologic failure occurred in 34 patients (8%) overall. One patient died at posttreatment week 3; this was not considered related to study medication. There were 7 serious adverse events, all considered unrelated to study treatment, and 3 adverse events (<1%) leading to treatment discontinuation, including 2 grade 4 alanine aminotransferase elevations. The most common adverse events (in ≥10% of patients) were headache, fatigue, diarrhea, and nausea. CONCLUSIONS AND RELEVANCE: In this open-label, nonrandomized, uncontrolled study, a high rate of SVR12 was achieved in treatment-naive and treatment-experienced noncirrhotic patients with chronic HCV genotype 1 infection who received 12 weeks of treatment with the oral fixed-dose regimen of daclatasvir, asunaprevir, and beclabuvir. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01979939.

Daclatasvir in combination with asunaprevir and beclabuvir for hepatitis C virus genotype 1 infection with compensated cirrhosis.

IMPORTANCE: Effective and well-tolerated, interferon-free regimens are needed for treatment of patients with chronic hepatitis C virus (HCV) infection and cirrhosis. OBJECTIVE: All-oral therapy with daclatasvir (nonstructural protein 5A [NS5A] inhibitor), asunaprevir (NS3 protease inhibitor), and beclabuvir (nonnucleoside NS5B inhibitor), with or without ribavirin, was evaluated in patients with HCV genotype 1 infection and compensated cirrhosis. DESIGN, SETTING, AND PARTICIPANTS: The UNITY-2 study was conducted between December 2013 and October 2014 at 49 outpatient sites in the United States, Canada, France, and Australia. Patients were treated for 12 weeks, with 24 weeks of follow-up after completion of treatment. Adult patients with cirrhosis were enrolled in 2 cohorts: HCV treatment-naive or HCV treatment-experienced. Statistical analyses were based on historical controls; there were no internal controls. INTERVENTIONS: All patients received twice-daily treatment with the fixed-dose combination of daclatasvir (30 mg), asunaprevir (200 mg), and beclabuvir (75 mg). In addition, patients within each cohort were stratified according to HCV genotype 1 subtype (1a or 1b) and randomly assigned (1:1) to receive double-blinded weight-based ribavirin (1000-1200 mg/d) or matching placebo. MAIN OUTCOMES AND MEASURES: Sustained virologic response at posttreatment week 12 (SVR12). RESULTS: One hundred twelve patients in the treatment-naive group and 90 patients in the treatment-experienced group were treated and included in the analysis. Enrolled patients were 88% white with a median age of 58 years (treatment-naive group) or 60 years (treatment-experienced group); 74% had genotype 1a infection. SVR12 rates were 98% (97.5% CI, 88.9%-100%) for patients in the treatment-naive group and 93% (97.5% CI, 85.0%-100.0%) for those in the treatment-experienced group when ribavirin was included in the regimen. With the fixed-dose combination alone, response rates were 93% (97.5% CI, 85.4%-100.0%) for patients in the treatment-naive group and 87% (97.5% CI, 75.3%-98.0%) for those in the treatment-experienced group. Three serious adverse events were considered to be treatment related and there were 4 adverse event-related discontinuations. Treatment-emergent grade 3 or 4 alanine aminotransferase elevations were observed in 4 patients, of which 1 had concomitant total bilirubin elevation. CONCLUSIONS AND RELEVANCE: In this open-label uncontrolled study, patients with chronic HCV genotype 1 infection and cirrhosis who received a 12-week oral fixed-dose regimen of daclatasvir, asunaprevir, and beclabuvir, with or without ribavirin, achieved high rates of SVR12.

On the antioxidant properties of erythropoietin and its association with the oxidative-nitrosative stress response to hypoxia in humans.

AIM: The aim of this study was to examine if erythropoietin (EPO) has the potential to act as a biological antioxidant and determine the underlying mechanisms. METHODS: The rate at which its recombinant form (rHuEPO) reacts with hydroxyl (HO˙), 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) and peroxyl (ROO˙) radicals was evaluated in-vitro. The relationship between the erythopoietic and oxidative-nitrosative stress response to poikilocapneic hypoxia was determined separately in-vivo by sampling arterial blood from eleven males in normoxia and following 12 h exposure to 13% oxygen. Electron paramagnetic resonance spectroscopy, ELISA and ozone-based chemiluminescence were employed for direct detection of ascorbate (A(˙-) ) and N-tert-butyl-α-phenylnitrone spin-trapped alkoxyl (PBN-OR) radicals, 3-nitrotyrosine (3-NT) and nitrite (NO2-). RESULTS: We found rHuEPO to be a potent scavenger of HO˙ (kr = 1.03-1.66 × 10(11) m(-1) s(-1) ) with the capacity to inhibit Fenton chemistry through catalytic iron chelation. Its ability to scavenge DPPH˙ and ROO˙ was also superior compared to other more conventional antioxidants. Hypoxia was associated with a rise in arterial EPO and free radical-mediated reduction in nitric oxide, indicative of oxidative-nitrosative stress. The latter was confirmed by an increased systemic formation of A˙(-) , PBN-OR, 3-NT and corresponding loss of NO2- (P < 0.05 vs. normoxia). The erythropoietic and oxidative-nitrosative stress responses were consistently related (r = -0.52 to 0.68, P < 0.05). CONCLUSION: These findings demonstrate that EPO has the capacity to act as a biological antioxidant and provide a mechanistic basis for its reported cytoprotective benefits within the clinical setting.

Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content.

Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt-ß-catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt-ß-catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.

Reticulon 4B (Nogo-B) facilitates hepatocyte proliferation and liver regeneration in mice.

UNLABELLED: Nogo-B, also known as reticulon 4B, promotes liver fibrosis and cirrhosis by facilitating the transforming growth factor ß (TGF-ß) signaling pathway in activated hepatic stellate cells. The aim of this study was to determine the role of Nogo-B in hepatocyte proliferation and liver regeneration. Partial hepatectomy (PHx, 70% resection) was performed in male wild-type (WT) and Nogo-A/B knockout mice (referred to as Nogo-B KO mice). Remnant livers were isolated 2 hours, 5 hours, and 1, 2, 3, 7, and 14 days after PHx. Hepatocyte proliferation was assessed by Ki67 labeling index. Quantitative real-time polymerase chain reaction was performed for genes known to be involved in liver regeneration. Hepatocytes isolated from WT and Nogo-B KO mice were used to examine the role of Nogo-B in interleukin-6 (IL-6), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and TGF-ß signaling. Nogo-B protein levels increased in the regenerating livers in a time-dependent manner after PHx. Specifically, Nogo-B expression in hepatocytes gradually spread from the periportal toward the central areas by 7 days after PHx, but receded notably by 14 days. Nogo-B facilitated IL-6/signal transducer and activator of transcription 3 signaling, increased HGF-induced but not EGF-induced hepatocyte proliferation, and tended to reduce TGF-ß1-induced suppression of hepatocyte proliferation in cultured hepatocytes. Lack of Nogo-B significantly induced TGF-ß1 and inhibitor of DNA binding expression 1 day after PHx and IL-6 and EGF expression 2 days after PHx. Lack of Nogo-B delayed hepatocyte proliferation but did not affect the liver-to-body ratio in the regenerative process. CONCLUSION: Nogo-B expression in hepatocytes facilitates hepatocyte proliferation and liver regeneration.

Acetazolamide and exercise hypoxia.

Acetazolamide is useful for acclimatizing to high altitude. How long it should be taken, and the physiological consequences of stopping it have not been thoroughly studied. We investigated the effect of acetazolamide cessation on exercise oxygenation at different altitudes and durations of use. Three groups were studied: group 1 acclimatized to 4,060 m for 6 days while taking acetazolamide 250 mg three times a day. On day 7 acetazolamide was stopped, then resumed on day 8. Standardized exercise oximetry was performed each day. The protocol for group 2 was identical to group 1, except acclimatization occurred over 14 days to 4 120 m. The protocol for group 3 was identical to group 2, except subjects acclimatized to 4,770 m. Multivariate regression revealed a negative effect of stopping acetazolamide on exercise oxygenation (p=0.028). At 4,100 m cessation of acetazolamide after one week resulted in a 11% drop in exercise oxygenation (p=0.008); after two weeks acclimatization to this altitude there was an non-significant drop in exercise oxygenation (2.5% p=0.064). At 4 770 m acetazolamide cessation resulted in an increase in exercise oxygenation (7% p=0.027). We conclude that exercise oxygenation after acetazolamide cessation is dependent both on duration of acclimatization/drug administration, and acclimatization altitude.

Adenosine inhibits chemotaxis and induces hepatocyte-specific genes in bone marrow mesenchymal stem cells.

UNLABELLED: Bone marrow-derived mesenchymal stem cells (MSCs) have therapeutic potential in liver injury, but the signals responsible for MSC localization to sites of injury and initiation of differentiation are not known. Adenosine concentration is increased at sites of cellular injury and inflammation, and adenosine is known to signal a variety of cellular changes. We hypothesized that local elevations in the concentration of adenosine at sites of tissue injury regulate MSC homing and differentiation. Here we demonstrate that adenosine does not induce MSC chemotaxis but dramatically inhibits MSC chemotaxis in response to the chemoattractant hepatocyte growth factor (HGF). Inhibition of HGF-induced chemotaxis by adenosine requires the A2a receptor and is mediated via up-regulation of the cyclic adenosine monophosphate (AMP)/protein kinase A pathway. This results in inhibition of cytosolic calcium signaling and down-regulation of HGF-induced Rac1. Because of the important role of Rac1 in the formation of actin stress fibers, we examined the effect of adenosine on stress fiber formation and found that adenosine inhibits HGF-induced stress fiber formation. In addition, we found that adenosine induces the expression of some key endodermal and hepatocyte-specific genes in mouse and human MSCs in vitro. CONCLUSION: We propose that the inhibition of MSC chemotaxis at sites of high adenosine concentration results in localization of MSCs to areas of cellular injury and death in the liver. We speculate that adenosine might initiate the process of differentiation of MSCs into hepatocyte-like cells.

No evidence for interstitial lung oedema by extensive pulmonary function testing at 4,559 m.

The aim of the present study was to better understand previously reported changes in lung function at high altitude. Comprehensive pulmonary function testing utilising body plethysmography and assessment of changes in closing volume were carried out at sea level and repeatedly over 2 days at high altitude (4,559 m) in 34 mountaineers. In subjects without high-altitude pulmonary oedema (HAPE), there was no significant difference in total lung capacity, forced vital capacity, closing volume and lung compliance between low and high altitude, whereas lung diffusing capacity for carbon monoxide increased at high altitude. Bronchoconstriction at high altitude could be excluded as the cause of changes in closing volume because there was no difference in airway resistance and bronchodilator responsiveness to salbutamol. There were no significant differences in these parameters between mountaineers with and without acute mountain sickness. Mild alveolar oedema on radiographs in HAPE was associated only with minor decreases in forced vital capacity, diffusing capacity and lung compliance and minor increases in closing volume. Comprehensive lung function testing provided no evidence of interstitial pulmonary oedema in mountaineers without HAPE during the first 2 days at 4,559 m. Data obtained in mountaineers with early mild HAPE suggest that these methods may not be sensitive enough for the detection of interstitial pulmonary fluid accumulation.

Chimeric mice reveal clonal development of pancreatic acini, but not islets.

Intestinal crypt stem cells establish clonal descendants. To determine whether the pancreas is patterned by a similar process, we used embryonic stem (ES) cell chimeric mice, in which male ES cells were injected into female blastocysts. Fluorescence in situ hybridization for the Y chromosome (Y-FISH) revealed clonal patterning of ES-derived cells in the adult mouse small intestine and pancreas. Intestinal crypts were entirely male or entirely female. Villi contained columns of male or female epithelial cells, consistent with upward migration of cells from the crypts which surround them. Within the exocrine pancreas, acini were entirely male or entirely female, consistent with patterning from a single stem/progenitor cell. Pancreatic islets contained a mixture of male and female cells, consistent with patterning from multiple progenitors. Male-female chimeric mice demonstrate that the adult mouse exocrine pancreatic acinus is patterned from a single stem/progenitor cell, while the endocrine pancreas arises from multiple progenitors.

Hepatocyte nuclear factor-1 as marker of epithelial phenotype reveals marrow-derived hepatocytes, but not duct cells, after liver injury in mice.

The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.

The hepatic stem cell niche: identification by label-retaining cell assay.

UNLABELLED: Label retention assays remain the state-of-the-art approach to identify the location of intraorgan epithelial stem cell niches, in situ and in vivo. They are commonly used in organs with rapid cell turnover but have not been applied to the liver, where cell turnover is very slow. We used a sublethal dose of acetaminophen administered coincident with bromodeoxyuridine to load possible hepatic stem cells in mice with label and then administered a second, sublethal chase of acetaminophen to accomplish "washout" of label from transit amplifying cell populations. CONCLUSION: Four possible hepatic stem cell niches are identified by this approach: the canal of Hering (proximal biliary tree), intralobular bile ducts, periductal "null" mononuclear cells, and peribiliary hepatocytes. These results confirm several different and often contradictory lines of investigation regarding the intrahepatic location of stem/progenitor cells and suggest that the liver has a multi-tiered, flexible system of regeneration rather than a single stem/progenitor cell location.

Physiological variations of stem cell factor and stromal-derived factor-1 in murine models of liver injury and regeneration.

BACKGROUND/AIMS: Stem cell factor (SCF) and stromal-derived factor-1 (SDF-1) regulate the regenerative response to liver injury, possibly through activation of liver progenitor 'oval' cells and recruitment of circulating, marrow-derived progenitors. METHODS: We performed a detailed analysis of SCF, SDF-1 and oval cell proliferation induced by tyrosinaemia, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or liver irradiation in mice by ELISA and immunofluorescence. RESULTS: Liver injury in the tyrosinaemia mouse is characterized by a dramatic decline in plasma SCF and absence of oval cell proliferation. In contrast, DDC induces bile duct (BD) and oval cell proliferation, and a modest decline in plasma SCF. Focal liver irradiation increases plasma SCF, but not oval cell density. In normal mouse liver, SCF is localized primarily to Kupffer cells, cholangiocytes and arterial smooth muscle, with little or no expression in hepatocytes. However, SCF appears in hepatocyte nuclei after injury, where its function is unknown. In all three models, SDF-1 is expressed exclusively in BD epithelium, indicating that tissue SDF-1 levels are proportional to the total mass of oval cells and cholangiocytes. However, increased plasma levels of SDF-1 in fumaryl acetoacetate hydroxylase-null mice were not accompanied by oval cell proliferation. CONCLUSION: Changes in SCF and SDF-1 varied with the nature of liver injury and were not directly related to oval cell proliferation.

Adherence to alendronate in male veterans.

UNLABELLED: In one Veterans Affairs' medical center, alendronate non-adherence was more likely in male veterans who smoke or report side effects, and less likely in men undergoing bone densitometry during therapy. Providers urgently need programs to increase adherence to osteoporosis medications. Initial programs should target patients with risk factors for non-adherence. INTRODUCTION: Adherence to osteoporosis therapy in men is unknown. We hypothesized that ca. 50% of men at one center would be adherent to alendronate and one or more patient-specific factors would associate with adherence. METHODS: We conducted a retrospective chart review study of male veterans to determine the rates and predictors of alendronate adherence over two years. We excluded women, men who received primary care elsewhere and those who took alendronate for indications other than low bone mass. We defined adherence as a medication possession ratio > or =80% in the first 24 months of therapy. RESULTS: Adherence in the first 12 and 24 months of therapy was 59% and 54%, respectively. In multivariate analyses, non-adherence was more likely in men using tobacco (OR 2.08, 95% CI 1.13, 3.84, p = 0.02) and reporting side effects (OR 2.06, 95% CI 1.14, 3.73, p = 0.02) and less likely in men undergoing bone density during therapy (OR 0.49, 95% CI 0.26, 0.90, p = 0.02). CONCLUSIONS: Alendronate non-adherence is more likely in male veterans who smoke or report side effects, and less likely in men having bone densitometry during therapy. Providers urgently need programs to increase adherence to osteoporosis medications. Initial programs should target patients with risk factors for non-adherence.

Alveolar PCO2 oscillations and ventilation at sea level and at high altitude.

This study examines the potential for a ventilatory drive, independent of mean PCO2, but depending instead on changes in PCO2 that occur during the respiratory cycle. This responsiveness is referred to here as "dynamic ventilatory sensitivity." The normal, spontaneous, respiratory oscillations in alveolar PCO2 have been modified with inspiratory pulses approximating alveolar PCO2 concentrations, both at sea level and at high altitude (5,000 m, 16,400 ft.). All tests were conducted with subjects exercising on a cycle ergometer at 60 W. The pulses last about half the inspiratory duration and are timed to arrive in the alveoli during early or late inspiration. Differences in ventilation, which then occur in the face of similar end-tidal PCO2 values, are taken to result from dynamic ventilatory sensitivity. Highly significant ventilatory responses (early pulse response greater than late) occurred in hypoxia and normoxia at sea level and after more than 4 days at 5,000 m. The response at high altitude was eliminated by normalizing PO2 and was reduced or eliminated with acetazolamide. No response was present soon after arrival (<4 days) at base camp, 5,000 m, on either of two high-altitude expeditions (BMEME, 1994, and Kanchenjunga, 1998). The largest responses at 5,000 m were obtained in subjects returning from very high altitude (7,100-8,848 m). The present study confirms and extends previous investigations that suggest that alveolar PCO2 oscillations provide a feedback signal for respiratory control, independent of changes in mean PCO2, suggesting that natural PCO2 oscillations drive breathing in exercise.

Limitations of green fluorescent protein as a cell lineage marker.

The enhanced green fluorescent protein (GFP) reporter has been widely adopted for tracking cell lineage. Here, we compare three transgenic mouse strains in which GFP is considered "ubiquitously expressed," with the GFP transgene under control of the chicken beta-actin (CBA) or human ubiquitin C (UBC) promoter. We compared the expression of GFP using flow cytometry, direct tissue fluorescence, and immunostaining with multiple commercially available anti-GFP antibodies. Mice of CBA-GFP strain 1Osb have strong but variegated expression of GFP in adult liver, kidney, small intestine, and blood. Mice of CBA-GFP strain Y01 have the highest proportion of GFP-positive peripheral blood cells yet limited GFP expression in liver, intestine, and kidney. UBC-GFP mice express GFP only weakly in solid organs and variably in blood. Direct fluorescent detection of GFP in formalin-fixed, paraffin-embedded tissue sections was the simplest approach, but it was useful only in high-expressing strains and potentially subject to artifact because of tissue autofluorescence. Immunofluorescence using either primary goat or primary rabbit antibodies was much more sensitive and allowed better discrimination of authentic signal from autofluorescence. Immunohistochemical staining was less sensitive than direct fluorescence or immunofluorescence and was subject to false-positive signal in the small intestine. In conclusion, there is considerable variability of expression within and between GFP transgenic strains. None of the tested strains gave truly ubiquitous GFP expression. A detailed analysis of GFP expression in one's tissues of interest must guide the choice of reporter mouse strain when GFP is used as a marker of cell lineage or donor origin. Disclosure of potential conflicts of interest is found at the end of this article.