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Introduction Negative Pressure Wound Therapy (NPWT) is a procedure used for nonhealing wounds. In NPWT, a special sealed dressing of large cell foam (>400 µm) or gauze is connected to a pump. Most commonly, negative pressures between −10 and −125 millimeters of mercury (mm Hg) are used. The mechanism of healing is unknown but maybe attributable to removal of the exudate and bacteria, and the stimulation of tissue repair through microdeformation. Reticulated foams with micron-size open cells, Capillary Suction Devices (CSD; 100 to 5 µm) exert capillary suction between 10 and 70 mm of Hg with a multilayered foam dressing. Materials and Methods Yorkshire pigs received 5 surgical excision wounds, 3 cm2, on each side of the back. The wounds were covered with a NPWT dressing (110 mm Hg negative pressure by a pump), CSD with capillary suctions of 30 mm Hg (CSD-30) and 70 mm Hg (CSD-70), and a conventional gauze dressing. The wounds were measured on day 2, and then every 4-5 days thereafter; the total fluid collected by the various dressing over time. Results By post-wound day 20, the wounds treated with CSD-70 and NPWT were 100% closed while the wounds treated with CSD-30 and gauze were 65% and 45%, respectively. This indicated comparable wound closure efficacies for CSD-70 and NPWT. The average total fluid uptake measured in grams dry weight were similar for CSD-70 and NPWT, 36 and 38 g, respectively, while the values were 24 g for CSD-30 and 12 g for gauze. However, the maximum fluid uptake observed at day 2 indicated that CSD-70 and CSD 30, 24 and 14 g, respectively, were superior to NPWT and gauze 12 and 7 g, respectively. Conclusion This data indicate comparable wound closure efficacies for CSD-70 and NPWT. It is felt that CSD is an effective, safe, and lower cost alternative to vacuum-assisted NPWT.

Phosphorylation of cardiac sarcomeric proteins plays a major role in the regulation of the physiological performance of the heart. Phosphorylation of thin filament proteins, such as troponin I and T, dramatically affects calcium sensitivity of the myofiber and systolic and diastolic functions. Phosphorylation of the regulatory protein tropomyosin (Tpm) results in altered biochemical properties of contraction; however, little is known about the physiological effect of Tpm phosphorylation on cardiac function. To address the in vivo significance of Tpm phosphorylation, here we generated transgenic mouse lines having a phosphomimetic substitution in the phosphorylation site of α-Tpm (S283D). High expression of Tpm S283D variant in one transgenic mouse line resulted in an increased heart:body weight ratio, coupled with a severe dilated cardiomyopathic phenotype resulting in death within 1 month of birth. Moderate Tpm S283D mice expression in other lines caused mild myocyte hypertrophy and fibrosis, did not affect lifespan, and was coupled with decreased expression of extracellular signal-regulated kinase 1/2 kinase signaling. Physiological analysis revealed that the transgenic mice exhibit impaired diastolic function, without changes in systolic performance. Surprisingly, we observed no alterations in calcium sensitivity of the myofibers, cooperativity, or calcium-ATPase activity in the myofibers. Our experiments also disclosed that casein kinase 2 plays an integral role in Tpm phosphorylation. In summary, increased expression of pseudo-phosphorylated Tpm impairs diastolic function in the intact heart, without altering calcium sensitivity or cooperativity of myofibers. Our findings provide the first extensive in vivo assessment of Tpm phosphorylation in the heart and its functional role in cardiac performance.

Introduction Negative Pressure Wound Therapy (NPWT) is a well-established procedure used for non-healing wounds including burn wounds. In NPWT, a special sealed dressing of large cell foam (>400 µm) or gauze is connected to a pump. The sealed dressing acts as a conduit to pressure transfer and aids in the passive flow of fluid. Most commonly, negative pressures between -10 and -125 millimeters of mercury (mm Hg) are used. The mechanism of healing is unknown but maybe due to removal of the exudate and bacteria, and the stimulation of tissue repair through micro deformation. Reticulated foams with micron-size open cells (100 to 5µm) exerts capillary suction between 10 and 60 mm of Hg with a multilayered foam dressing. The negative pressures produced are within the range of suction by mechanical pumps. Therefore, the long-term goal our research is to examine effectiveness of Capillary Suction Devices (CSD) as a topical device in both preventing and healing complicated wounds, burn and donor site wounds. Our hypothesis is that CSD is an effective, safe and lower cost alternative to vacuum-assisted NPWT. Methods Yorkshire pigs received 4 surgical posterior excision wounds, measuring 2 in. by 2 in. The wounds were covered with a NPWT dressing (110 mm Hg negative pressure by a pump), CSD with capillary suctions of 30 mm Hg (CSD-30) and 60+ mm Hg (CSD-60), and a conventional gauze dressing. The wounds were measured on 2 day and then every 4 to 5 days until wounds closed. We also measured and compared the total fluids collected with each dressing. Results By post-wound day 20, the wounds treated with CSD-60 and NPWT were 100% closed while the wounds treated with CSD-30 and gauze were 65 and 45%, respectively. This indicated comparable wound closure efficacies for CSD-60 and NPWT. The CSD-30 product was superior to gauze, but less efficacious than the tested NPWT protocol. The average total fluid uptake measured in grams dry weight were similar for CSD-60 and NPWT, 36 and 38g., respectively while the values were 24g for CSD-30 and 12g for gauze. However, the maximum fluid uptake observed at day 2 indicated that CSD-60 and CSD 30, 24 and 14g respectively, were superior to NPWT and gauze 12 and 7g respectively. Conclusions This data indicates comparable wound closure efficacies for CSD-60 capillary suction and NPWT. The CSD product with 30 mmHg suction was superior to gauze, but less efficacious than NPWT or CSD-60 protocol. The study results provide impetus for further research on the use of high suction CSD for replacement of pump mediated NPWT and the moderate suction CSD for preventing progression of high-risk wounds to an ulcerous stage. Applicability of Research to Practice This small study suggests that CSD maybe alternative in to NPWT. CSD provides a significant cost and weight advantage to the patient, as well is allowing for increased mobility.

This study examined the effect of human immunodeficiency virus (HIV) protease inhibitor therapy on lipoprotein production and catabolism in vivo. The HIV protease inhibitor ritonavir was given to C57BL/6 mice fed either a basal low-fat diet or a Western type high-fat diet. Fasted mice were injected with Triton WR1339 followed by hourly blood collection to monitor lipoprotein production. Results showed that ritonavir increased VLDL triglyceride production by 30% over a 4 h period when mice were fed the low-fat basal diet. The ritonavir effect was more pronounced under high-fat feeding conditions, with a 2-fold increase in VLDL triglyceride production rate. Ritonavir did not alter hepatic expression levels of diacylglycerol acyltransferase or microsomal triglyceride transfer protein, but increased hepatic apolipoprotein B (apoB) secretion rates under both low- and high-fat dietary conditions. In contrast to its effect on lipoprotein production, ritonavir did not alter triglyceride-rich lipoprotein clearance from circulation under either dietary condition. Taken together, these results indicate that the hyperlipidemic effect of HIV protease inhibitors is a direct result of increased hepatic lipoprotein production. The mechanism appears to be related to their role in preventing proteasome-mediated degradation of apoB and activated sterol regulatory element binding proteins in the liver.

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Human immunodeficiency virus type 1 (HIV-1) infection of an individual generates a population of related but distinct cocirculating viral variants (15). This high level of genetic diversity allows HIV-1 to adapt quickly to multiple selective pressures, including neutralizing antibodies, cytotoxic T lymphocytes, and antiretroviral drugs (17, 21, 32-34, 42). The highest level of diversity within the HIV genome is found in the env gene. The five hypervariable domains, named V1 through V5, form loops on the outside of the gp120 molecule (22). The V3 loop is involved in binding a chemokine receptor for viral entry after gp120 binds the CD4 receptor (40, 43). Changes in the V3 domain determine in part whether HIV utilizes CCR5 (R5 variants) or CXCR4 (X4 variants) chemokine coreceptors (6). The V1-V2 loop is thought to interact with the V3 loop within the gp120 trimer on the virion surface (5, 23). The V4 and V5 domains are on a different face of gp120 than V1-V2 and V3 are (22) and are not directly involved in receptor binding. The V1-V2 and V4-V5 regions of gp120 are highly variable in sequence and length within an infected individual and between infected individuals (19, 37, 41). The domains are heavily glycosylated, and changes in the location and number of N- and O-linked glycosylation sites in the V1-V2 and V4-V5 regions are associated with escape from neutralization (reviewed in reference 29). Removal of some of the glycosylation sites from the V1 and V2 regions results in increased immunogenicity of the domains (31) and has been shown in one study to redirect the immune response toward V3 rather than V1-V2 (7). Sequence changes observed in V1-V2 and V3 to V5, including insertions, deletions, and point mutations, have been linked to escape from neutralizing antibodies. The accumulation of nonsynonymous substitutions was associated with escape in two separate studies (11, 12), while changes in the glycosylation sites of gp120 have been described in terms of an evolving glycan shield that leads to escape from neutralizing antibodies in a third study (42). It has also been shown that mutations in env lead to escape from neutralizing antibodies through conformational masking of epitopes (21). Mutations in env have also been shown to affect cytotoxic T-lymphocyte (CTL) epitopes (11, 17), indicating that both neutralizing antibodies and CTL apply selective pressure on HIV-1 in vivo. High levels of env sequence diversity have been linked with both slower disease progression and more-effective immune responses against the virus, both in simian immunodeficiency virus and HIV (3, 8, 14, 16, 26), thereby implying a link between strong immune selection and slower disease progression. Heteroduplex assays are powerful methods for displaying the number and variety of variants within a viral population (reviewed in reference 4) without the potential selection bias inherent to sequence-based analyses. Heteroduplex mobility assays reveal the diversity within a sample through the visualization of heteroduplexes formed between different variants within the sample, while heteroduplex tracking assays (HTA) use a radiolabeled probe to display differences between the probe and the sample. We have previously used HTA to examine the diversity and changes in V1-V2 in monthly samples from subjects with low CD4 counts (18). In 12 of 21 of these subjects, at least one V1-V2 variant population was gained or lost over 5 to 9 months. Sequence analysis of the V1-V2 regions for several of the subjects revealed point mutations, recombination events, and deletions as the main mechanisms of sequence change. Delwart et al. analyzed the V3 to V5 variant populations in semiannual samples from subjects with different rates of progression, showing that subjects with faster CD4+ T-cell decline had slower diversification of the region from V3 to V5 and that progression of disease was correlated with reduced diversity (8). These latter results support the idea that the level of sequence diversity directly correlates with immune selection. In the present study, we have used HTA analysis to examine the V1-V2 and V4-V5 regions of HIV-1 env in semiannual plasma samples from subjects in the San Francisco Men's Health Study. Subjects with different rates of progression, as defined by rate of CD4 cell loss, were compared for rates of change in the env viral population using a newly developed HTA index algorithm that highlights periods of increasing env diversity.

A major complication associated with the use of protease inhibitors (PIs) in treatment of HIV-infected patients is lipid abnormalities including dyslipidemia, lipodystrophy, and liver steatosis. Previous studies revealed that these abnormalities are associated with PI-induced accumulation of activated sterol regulatory element binding proteins (SREBPs) in the nucleus of liver and adipose tissues, resulting in constitutive activation of lipid metabolism genes. This study used the mouse model to determine the potential of polyunsaturated fatty acid (PUFA) diet or leptin replacement therapy to alleviate these PI-induced metabolic abnormalities. Results showed that feeding C57BL/6 mice with a PUFA-rich diet failed to normalize plasma cholesterol and triglyceride levels in ritonavir-treated mice. The PUFA-rich diet also had no effect on ritonavir-induced interscapular fat accumulation and liver steatosis. In contrast, daily administration of leptin significantly reversed the elevated plasma cholesterol level induced by ritonavir. Leptin replacement therapy also significantly reduced the ritonavir-induced interscapular fat mass and improved liver steatosis. Taken together, these data suggest that PI-induced lipid abnormalities, especially dyslipidemia, lipodystrophy, and liver steatosis, may be reduced with leptin replacement therapy.

The mechanism by which human immunodeficiency virus (HIV) protease inhibitor therapy adversely induces lipodystrophy and hyperlipidemia has not been defined. This study explored the mechanism associated with the adverse effects of the prototype protease inhibitor ritonavir in mice. Ritonavir treatment increased plasma triglyceride and cholesterol levels through increased fatty acid and cholesterol biosynthesis in adipose and liver. Ritonavir treatment also resulted in hepatic steatosis and hepatomegaly. These abnormalities, which were especially pronounced after feeding a Western type high fat diet, were due to ritonavir-induced accumulation of the activated forms of sterol regulatory binding protein (SREBP)-1 and -2 in the nucleus of liver and adipose, resulting in elevated expression of lipid metabolism genes. Interestingly, protease inhibitor treatment did not alter SREBP mRNA levels in these tissues. Thus, the adverse lipid abnormalities associated with protease inhibitor therapy are caused by the constitutive induction of lipid biosynthesis in liver and adipose tissues due to the accumulation of activated SREBP in the nucleus.

The H,K-ATPase of the gastric parietal cell is the most critical component of the ion transport system mediating acid secretion in the stomach. To study the requirement of this enzyme in the development, maintenance, and function of the gastric mucosa, we used gene targeting to prepare mice lacking the alpha-subunit. Homozygous mutant (Atp4a(-/-)) mice appeared healthy and exhibited normal systemic electrolyte and acid-base status but were achlorhydric and hypergastrinemic. Immunocytochemical, histological, and ultrastructural analyses of Atp4a(-/-) stomachs revealed the presence of chief cells, demonstrating that the lack of acid secretion does not interfere with their differentiation. Parietal cells were also present in normal numbers, and despite the absence of alpha-subunit mRNA and protein, the beta-subunit was expressed. However, Atp4a(-/-) parietal cells had dilated canaliculi and lacked typical canalicular microvilli and tubulovesicles, and subsets of these cells contained abnormal mitochondria and/or massive glycogen stores. Stomachs of adult Atp4a(-/-) mice exhibited metaplasia, which included the presence of ciliated cells. We conclude that ablation of the H,K-ATPase alpha-subunit causes achlorhydria and hypergastrinemia, severe perturbations in the secretory membranes of the parietal cell, and metaplasia of the gastric mucosa; however, the absence of the pump appears not to perturb parietal cell viability or chief cell differentiation.

The sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca2+ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5' end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by approximately 45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca2+ uptake into the sarcoplasmic reticulum were reduced by approximately 35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca2+ sequestering activity, and that the deficit in Ca2+ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation.

Mutations in the gene encoding the thiazide-sensitive Na+-Cl− cotransporter (NCC) of the distal convoluted tubule cause Gitelman's syndrome, an inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria. These metabolic abnormalities are secondary to the deficit in NaCl reabsorption, but the underlying mechanisms are unclear. To gain a better understanding of the role of NCC in sodium and fluid volume homeostasis and in the pathogenesis of Gitelman's syndrome, we used gene targeting to prepare an NCC-deficient mouse. Null mutant (Ncc −/−) mice appear healthy and are normal with respect to acid-base balance, plasma electrolyte concentrations, serum aldosterone levels, and blood pressure.Ncc −/− mice retain Na+ as well as wild-type mice when fed a Na+-depleted diet; however, after 2 weeks of Na+ depletion the mean arterial blood pressure of Ncc −/− mice was significantly lower than that of wild-type mice. In addition, Ncc −/−mice exhibited increased renin mRNA levels in kidney, hypomagnesemia and hypocalciuria, and morphological changes in the distal convoluted tubule. These data indicate that the loss of NCC activity in the mouse causes only subtle perturbations of sodium and fluid volume homeostasis, but renal handling of Mg2+ and Ca2+ are altered, as observed in Gitelman's syndrome.

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.