BACKGROUND

The calcineurin inhibitors cyclosporine (INN, cyclosporin) and tacrolimus have a narrow therapeutic index and show considerable interindividual variability in their pharmacokinetics. The low oral bioavailability of calcineurin inhibitors is thought to result from the actions of the metabolizing enzymes cytochrome P450 (CYP) 3A4 and CYP3A5 and the multidrug efflux pump P-glycoprotein, encoded by MDR-<em>1</em>.

OBJECTIVE

Our objective was to determine the role of genetic polymorphisms in CYP3A4, CYP3A5, and MDR-<em>1</em> with respect to interindividual variability in cyclosporine and tacrolimus pharmacokinetics.

METHODS

Kidney transplant recipients receiving cyclosporine (n = <em>1</em><em>1</em>0) or tacrolimus (n = 64) were genotyped for CYP3A4*<em>1</em>B and *3, CYP3A5*3 and *6, and MDR-<em>1</em> C3435T. Dose-adjusted trough levels were determined and correlated with the corresponding genotype.

RESULTS

Tacrolimus dose-adjusted trough levels were higher in CYP3A5*3/*3 patients (n = 45) than in *<em>1</em>/*3 plus *<em>1</em>/*<em>1</em> patients (n = <em>1</em>7), as follows: median and range, 94 (34-398) ng/mL per mg/kg versus 6<em>1</em> (37-<em>1</em>63) ng/mL per mg/kg (P <.000<em>1</em>, Mann-Whitney test). CYP3A4*<em>1</em>B allele carriers (n = <em>1</em>0) had lower tacrolimus dose-adjusted trough levels compared with those in patients with the wild-type (*<em>1</em>/*<em>1</em>) genotype (n = 54): median and range, 57 (40-<em>1</em>63) ng/mL per mg/kg versus 89 (34-398) ng/mL per mg/kg) (P =.003, Mann-Whitney test). No evidence was found supporting a role for the MDR-<em>1</em> C3435T polymorphism in tacrolimus dose requirement. None of the polymorphisms studied correlated with cyclosporine dose-adjusted predose concentrations.

CONCLUSIONS

As a group, patients with the CYP3A5*3/*3 genotype require less tacrolimus to reach target predose concentrations compared with CYP3A5*<em>1</em> allele carriers, whereas CYP3A4*<em>1</em>B carriers require more tacrolimus to reach target trough concentrations compared with CYP3A4*<em>1</em> homozygotes.

We present a liquid chromatography-mass spectrometry (LC-MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water-methanol gradient on a C<em>1</em>8 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 to <em>1</em>000 m/z at <em>1</em> Hz and <em>1</em>00,000 resolution in negative ion mode on a stand alone orbitrap ("Exactive"). The median limit of detection, across 80 metabolite standards, was 5 ng/<em>mL</em> with the linear range typically>>or=<em>1</em>00-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker's yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected <em>1</em>37 known compounds, whose (<em>1</em>3)C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL2<em>1</em>5C), we observed accumulation of an ion of m/z <em>1</em>28.035<em>1</em>, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL2<em>1</em>5C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.

To develop and validate an international set of classification criteria for primary Sjögren's syndrome (SS) using guidelines from the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR). These criteria were developed for use in individuals with signs and/or symptoms suggestive of SS.

We assigned preliminary importance weights to a consensus list of candidate criteria items, using multi-criteria decision analysis. We tested and adapted the resulting draft criteria using existing cohort data on primary SS cases and non-SS controls, with case/non-case status derived from expert clinical judgment. We then validated the performance of the classification criteria in a separate cohort of patients.

The final classification criteria are based on the weighted sum of 5 items: anti-SSA/Ro antibody positivity and focal lymphocytic sialadenitis with a focus score of ≥<em>1</em> foci/4 mm2 , each scoring 3; an abnormal ocular staining score of ≥5 (or van Bijsterveld score of ≥4), a Schirmer's test result of ≤5 mm/5 minutes, and an unstimulated salivary flow rate of ≤0.<em>1</em> <em>ml</em>/minute, each scoring <em>1</em>. Individuals with signs and/or symptoms suggestive of SS who have a total score of ≥4 for the above items meet the criteria for primary SS. Sensitivity and specificity against clinician-expert-derived case/non-case status in the final validation cohort were high, i.e., 96% (95% confidence interval [95% CI] 92-98%) and 95% (95% CI 92-97%), respectively.

Using methodology consistent with other recent ACR/EULAR-approved classification criteria, we developed a single set of data-driven consensus classification criteria for primary SS, which performed well in validation analyses and are well-suited as criteria for enrollment in clinical trials.

BACKGROUND

Previous studies have associated reduced hemoglobin levels with increased adverse events in heart failure. It is unclear, however, whether this relation is explained by underlying kidney disease, treatment differences, or associated comorbidity.

RESULTS

We examined the associations between hemoglobin level, kidney function, and risks of death and hospitalization in persons with chronic heart failure between <em>1</em>996 and 2002 within a large, integrated, healthcare delivery system in northern California. Longitudinal outpatient hemoglobin and creatinine levels and clinical and treatment characteristics were obtained from health plan records. Glomerular filtration rate (GFR; <em>mL</em>.min(-<em>1</em>).<em>1</em>.73 m(-2)) was estimated from the Modification of Diet in Renal Disease equation. Mortality data were obtained from state death files; heart failure admissions were identified by primary discharge diagnoses. Among 59,772 adults with heart failure, the mean age was 72 years and 46% were women. Compared with that for hemoglobin levels of <em>1</em>3.0 to <em>1</em>3.9 g/dL, the multivariable-adjusted risk of death increased with lower hemoglobin levels: an adjusted hazard ratio (HR) of <em>1</em>.<em>1</em>6 and 95% confidence interval (CI) of <em>1</em>.<em>1</em><em>1</em> to <em>1</em>.2<em>1</em> for hemoglobin levels of <em>1</em>2.0 to <em>1</em>2.9 g/dL; HR, <em>1</em>.50 and 95% CI, <em>1</em>.44 to <em>1</em>.57 for <em>1</em><em>1</em>.0 to <em>1</em><em>1</em>.9 g/dL; HR, <em>1</em>.89 and 95% CI, <em>1</em>.80 to <em>1</em>.98 for <em>1</em>0.0 to <em>1</em>0.9; HR, 2.3<em>1</em> and 95% CI, 2.<em>1</em>8 to 2.45 for 9.0 to 9.9; and HR, 3.48 and 95% CI, 3.25 to 3.73 for <9.0 g/dL. Hemoglobin levels>> or = <em>1</em>7.0 g/dL were associated with an increased risk of death (adjusted HR, <em>1</em>.42; 95% CI, <em>1</em>.24 to <em>1</em>.63). Compared with those with a GFR>> or = 60 <em>mL</em> . min(-<em>1</em>).<em>1</em>.73 m(-2), persons with a GFR <45 <em>mL</em>.min(-<em>1</em>).<em>1</em>.73 m(-2) had an increased mortality risk: adjusted HR, <em>1</em>.39 and 95% CI, <em>1</em>.34 to <em>1</em>.44 for 30 to 44; HR, 2.28 and 95% CI, 2.<em>1</em>9 to 2.39 for <em>1</em>5 to 29; HR, 3.26 and 95% CI, 3.05 to 3.49 for (<em>1</em>5; and HR, 2.44 and 95% CI, 2.28 to 2.6<em>1</em> for those on dialysis. Relations were similar for the risk of hospitalization. The findings did not differ among patients with preserved or reduced systolic function, and hemoglobin level was an independent predictor of outcomes at all levels of kidney function.

CONCLUSIONS

Very high >> or = <em>1</em>7 g/dL) or reduced ((<em>1</em>3 g/dL) hemoglobin levels and chronic kidney disease independently predict substantially increased risks of death and hospitalization in heart failure, regardless of the level of systolic function. Randomized trials are needed to evaluate whether raising hemoglobin levels can improve outcomes in chronic heart failure.

Patients with coronary heart disease or equivalent risk received a single dose of 30, <em>1</em>00, 300, or 500 mg of unformulated D-4F (n = 8, each dose) or placebo (n = 8) under fasting conditions. An additional <em>1</em>0 patients received 500 mg (n = 8) or placebo (n = 2) with a low-fat meal. There were no significant trends in any safety parameter. D-4F was detectable in plasma at all doses with a T(max) of 30 min, <em>1</em> h, and 2 h for 30, <em>1</em>00, and>> or = 300 mg, respectively. The area under the curve((0-t)) was 27.8<em>1</em> ng/hr/<em>ml</em> and 54.7<em>1</em> ng/hr/<em>ml</em> for the 300 mg and 500 mg dose groups, respectively, and <em>1</em>7.96 ng/hr/<em>ml</em> for the 500 mg dose given with food. HDL from each time point for each subject was tested for its ability to inhibit LDL-induced monocyte chemotactic activity in cultures of human aortic endothelial cells. The values obtained were normalized to <em>1</em>.0 for LDL alone to obtain the HDL inflammatory index. This index significantly improved at 4 h at the 300 mg dose and at 2 h at the 500 mg dose compared with placebo (P < 0.05). There were no changes in plasma lipid or lipoprotein levels. We conclude that unformulated D-4F has low bioavailability that is improved under fasting conditions, and that a single dose of D-4F is safe and well tolerated and may improve the HDL anti-inflammatory index.

The present studies were undertaken to assess the mechanisms responsible for cortisol-induced insulin resistance in man. The insulin dose-response characteristics for suppression of glucose production and stimulation of glucose utilization and their relationship to monocyte and erythrocyte insulin receptor binding were determined in six normal volunteers after 24-h infusion of cortisol and 24-h infusion of saline. The infusion of cortisol (2 microgram kg-<em>1</em> min-<em>1</em>) increased the plasma cortisol concentration approximately 4-fold (37 +/- 3 vs. <em>1</em>4 +/- <em>1</em> microgram/dl; P less than 0.0<em>1</em>) to values observed during moderately severe stress in man. This hypercortisolemia increased postabsorptive plasma glucose (<em>1</em>26 +/- 2 vs. 97 +/- 2 mg/dl; P less than 0.0<em>1</em>) and plasma insulin (<em>1</em>6 +/- 2 vs. <em>1</em>0 +/- 2 microU/<em>ml</em>; P less than 0.0<em>1</em>) concentrations and rates of glucose production (2.4 +/- 0.<em>1</em> vs. 2.<em>1</em> +/- -0.<em>1</em> mg kg-<em>1</em> min-<em>1</em>; P less than 0.0<em>1</em>) and utilization (2.5 +/- 0.<em>1</em> vs. 2.<em>1</em> +/- 0.<em>1</em> mg kg-<em>1</em> min -<em>1</em>; P less than 0.0<em>1</em>). Insulin dose-response curves for both suppression of glucose production (half-maximal response at 8<em>1</em> +/- <em>1</em>9 vs. 3<em>1</em> +/ 5 microU/<em>ml</em>; P less than 0.05) and stimulation of glucose utilization (half-maximal response at <em>1</em>04 +/- 9 vs. 64 +/- 7 microU/<em>ml</em>; P less than 0.0<em>1</em>) were shifted to the right, with preservation of normal maximal responses to insulin. Neither monocyte nor erythrocyte insulin binding was decreased. However, except at near-maximal insulin receptor occupancy, the action of insulin on glucose production and utilization per number of monocyte and erythrocyte insulin receptors occupied was decreased. These results indicate that the cortisol-induced insulin resistance in man is due to the decrease in both hepatic and extrahepatic sensitivity to insulin. Assuming that insulin binding to monocytes and erythrocytes reflects insulin binding in insulin-sensitive tissues, this decrease in insulin action can be explained on the basis of a postreceptor defect.

Insulin resistance, a hallmark of type 2 diabetes, is associated with oxidative stress. However, the role of reactive oxygen species or specific antioxidant enzymes in its development has not been tested under physiological conditions. The objective of our study was to investigate the impact of overexpression of glutathione peroxidase <em>1</em> (GPX<em>1</em>), an intracellular selenoprotein that reduces hydrogen peroxide (H(2)O(2)) in vivo, on glucose metabolism and insulin function. The GPX<em>1</em>-overexpressing (OE) and WT male mice (n = 80) were fed a selenium-adequate diet (0.4 mg/kg) from 8 to 24 weeks of age. Compared with the WT, the OE mice developed (P < 0.05) hyperglycemia (<em>1</em><em>1</em>7 vs. <em>1</em>49 mg/dl), hyperinsulinemia (4<em>1</em>9 vs. <em>1</em>,350 pg/<em>ml</em>), and elevated plasma leptin (5 vs. <em>1</em>6 ng/<em>ml</em>) at 24 weeks of age. Meanwhile, these mice were heavier (37 vs. 27 g, P < 0.00<em>1</em>) and fatter (37% vs. <em>1</em>7% fat, P < 0.0<em>1</em>) than the WT mice. At 30-60 min after an insulin challenge, the OE mice had 25% less (P < 0.05) of a decrease in blood glucose than the WT mice. Their insulin resistance was associated with a 30-70% reduction (P < 0.05) in the insulin-stimulated phosphorylations of insulin receptor (beta-subunit) in liver and Akt (Ser(473) and Thr(308)) in liver and soleus muscle. Here we report the development of insulin resistance in mammals with elevated expression of an antioxidant enzyme and suggest that increased GPX<em>1</em> activity may interfere with insulin function by overquenching intracellular reactive oxygen species required for insulin sensitizing.

BACKGROUND

Changes in acid-base balance caused by infusion of a 0.9% saline solution during anesthesia and surgery are poorly characterized. Therefore, the authors evaluated these phenomena in a dose-response study.

METHODS

Two groups of <em>1</em>2 patients each who were undergoing major intraabdominal gynecologic surgery were assigned rando<em>ml</em>y to receive 0.9% saline or lactated Ringer's solution in a dosage of 30 <em>ml</em> x kg(-<em>1</em>) x h(-<em>1</em>). The pH, arterial carbon dioxide tension, and serum concentrations of sodium, potassium, chloride, lactate, and total protein were measured in 30-min intervals. The serum bicarbonate concentration was calculated using the Henderson-Hasselbalch equation and also using the Stewart approach from the strong ion difference and the amount of weak plasma acid. The strong ion difference was calculated as serum sodium + serum potassium - serum chloride - serum lactate. The amount of weak plasma acid was calculated as the serum total protein concentration in g/dl x 2.43.

RESULTS

Infusion of 0.9% saline, but not lactated Ringer's solution, caused a metabolic acidosis with hyperchloremia and a concomitant decrease in the strong ion difference. Calculating the serum bicarbonate concentration using the Henderson-Hasselbalch equation or the Stewart approach produced equivalent results.

CONCLUSIONS

Infusion of approximately 30 <em>ml</em> x kg(-<em>1</em>) x h(-<em>1</em>) saline during anesthesia and surgery inevitably leads to metabolic acidosis, which is not observed after administration of lactated Ringer's solution. The acidosis is associated with hyperchloremia.

OBJECTIVE

To describe the pharmacokinetics of 0.5 mg of intravitreal ranibizumab (Lucentis) and to compare it with that of <em>1</em>.25 mg of intravitreal bevacizumab (Avastin), using the same rabbit model.

METHODS

Experimental animal study.

METHODS

Twenty-eight Dutch-belted rabbits.

METHODS

One eye of each of 20 rabbits was injected with 0.5 mg of intravitreal ranibizumab. Both eyes of each of 4 rabbits were enucleated at days <em>1</em>, 3, 8, <em>1</em>5, and 29. Ranibizumab concentrations were measured in aqueous fluid, whole vitreous, and serum. A further 8 rabbits were used to measure serum and fellow ranibizumab at additional time points of 3 and 8 hours.

METHODS

Ranibizumab concentrations in the aqueous, vitreous, and serum.

RESULTS

Although vitreous concentrations of ranibizumab declined in a monoexponential fashion with a half-life of 2.88 days, concentrations of >0.<em>1</em> microg/ml ranibizumab were maintained in the vitreous humor for 29 days. Ranibizumab concentrations in the aqueous humor of the injected eye reached a peak concentration of <em>1</em>7.9 microg/ml, 3 days after drug administration. Elimination of ranibizumab from the aqueous humor paralleled that found in the vitreous humor, with a half-life value of 2.84 days. No ranibizumab was detected in the serum or the fellow eye.

CONCLUSIONS

In the rabbit, the vitreous half-life of 0.5-mg intravitreal ranibizumab is 2.88 days, shorter than the half-life of <em>1</em>.25-mg intravitreal bevacizumab of 4.32 days. No ranibizumab was detected in the serum or the fellow uninjected eye; whereas small amounts of intravitreal bevacizumab have been detected in the serum and fellow uninjected eye.

Studies were done to determine whether the minimal model approach and the glucose clamp measure equivalent indices of insulin action. Euglycemic glucose clamps (glucose, G: 85 mg/dl) were performed at two rates of insulin (I) infusion (<em>1</em>5 and 40 mU/min per m2) in <em>1</em>0 subjects (body mass index, BMI, from 2<em>1</em> to 4<em>1</em> kg/m2). Insulin sensitivity index (SI) from clamps varied from 0.<em>1</em>5 to 3.<em>1</em>5 (mean: <em>1</em>.87 +/- 0.36 X <em>1</em>0(-2) dl/[min per m2] per microU/<em>ml</em>), and declined linearly with increasing adiposity (versus BMI: r = -0.97; P less than 0.00<em>1</em>). SI from modeling the modified frequently sampled intravenous tolerance test varied from 0.66 to 7.34 X <em>1</em>0(-4) min-<em>1</em> per microU/<em>ml</em>, and was strongly correlated with SIP(clamp) (r = 0.89; P less than 0.00<em>1</em>). SI and SIP(clamp) were similar (0.046 +/- 0.008 vs. 0.037 +/- 0.007 dl/min per microU/<em>ml</em>, P greater than 0.35); the relation had a slope not different from unity (<em>1</em>.05 P greater than 0.70) and passed through the origin (P greater than 0.40). However, on a period basis, SI exceeded SIP(clamp) slightly, due to inhibition of hepatic glucose output during the FSIGT, not included in SIP(clamp). These methods are equivalent for assessment of overall insulin sensitivity in normal and insulin-resistant nondiabetic subjects.

BACKGROUND

Vitamin D deficiency has been associated with many chronic illnesses, but little is known about its relationship with chronic obstructive pulmonary disease (COPD).

OBJECTIVE

Serum 25-hydroxyvitamin D (25-OHD) levels were measured in 4<em>1</em>4 (ex)-smokers older than 50 years and the link between vitamin D status and presence of COPD was assessed. The rs704<em>1</em> and rs4588 variants in the vitamin D-binding gene (GC) were genotyped and their effects on 25-OHD levels were tested.

RESULTS

In patients with COPD, 25-OHD levels correlated significantly with forced expiratory volume in <em>1</em> s (FEV(<em>1</em>)) (r=0.28, p<0.000<em>1</em>). Compared with 3<em>1</em>% of the smokers with normal lung function, as many as 60% and 77% of patients with GOLD (Global Initiative for Obstructive Lung Disease) stage 3 and 4 exhibited deficient 25-OHD levels <20 ng/<em>ml</em> (p<0.000<em>1</em>). Additionally, 25-OHD levels were reduced by 25% in homozygous carriers of the rs704<em>1</em> at-risk T allele (p<0.000<em>1</em>). This correlation was found to be independent of COPD severity, smoking history, age, gender, body mass index, corticosteroid intake, seasonal variation and rs4588 (p<0.000<em>1</em>). Notably, 76% and <em>1</em>00% of patients with GOLD stage 3 and 4 homozygous for the rs704<em>1</em> T allele exhibited 25-OHD levels <20 ng/<em>ml</em>. Logistic regression corrected for age, gender and smoking history further revealed that homozygous carriers of the rs704<em>1</em> T allele exhibited an increased risk for COPD (OR 2.<em>1</em><em>1</em>; 95% CI <em>1</em>.20 to 3.7<em>1</em>; p=0.009).

CONCLUSIONS

Vitamin D deficiency occurs frequently in COPD and correlates with severity of COPD. The data warrant vitamin D supplementation in patients with severe COPD, especially in those carrying at-risk rs704<em>1</em> variants.

We developed a method using isotope dilution on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination in urine of nine environmental phenolic compounds: Bisphenol A; 4-tert-octylphenol; o-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; benzophenone-3 (2-hydroxy-4-metoxybenzophenone); and triclosan (2,4,4'-trichloro-2'-hydroxyphenyl ether). A unique fully automated column-switching system, constructed using <em>1</em> autosampler, 2 HPLC pumps, and a <em>1</em>0-port switching valve, was designed to allow for concurrent SPE-HPLC operation with peak focusing. The phenols present in <em>1</em>00 microL of urine were retained and concentrated on a C<em>1</em>8 reversed-phase size-exclusion SPE column. Then, the phenols were "back-eluted" from the SPE column and diluted through a mixing Tee before being separated from other urine matrix components using a pair of monolithic HPLC columns. The phenols were detected by negative ion-atmospheric pressure chemical ionization-MS/MS. The efficient preconcentration of the phenols by the SPE column, analyte peak focusing by the dilution, and minimal ion suppression in the LC/MS interface by the buffer-free mobile phases resulted in limits of detection as low as 0.<em>1</em>-0.4 ng/<em>mL</em> for most analytes. The method was validated on spiked pooled urine samples and on urine samples from 30 adults with no known occupational exposure to environmental phenols. The method can be used for quick and accurate analysis of large numbers of samples in epidemiologic studies for assessing the prevalence of human exposure to environmental phenols.

OBJECTIVE

Gastric gastrointestinal stromal tumors (GISTs) are rare neoplasms that require excision for cure. Although the feasibility of minimally invasive resection of gastric GIST has been established, the long-term safety and efficacy of these techniques are unclear. We hypothesized that complete resection of gastric GISTs using a combination of laparoscopic or laparoendoscopic techniques results in low perioperative morbidity and an effective long-term control of the disease.

METHODS

Between August <em>1</em>996 and June 2005, 50 consecutive patients undergoing laparoscopic or laparoendoscopic resection of gastric GISTs were identified in a prospectively collected database. Outcome measures included patient demographics and outcomes, operative findings, morbidity, and histopathologic characteristics of the tumor. Patient and tumor characteristics were analyzed to identify risk factors for tumor recurrence.

RESULTS

Fifty patients, mean age 60 years (range, 34-84 years), underwent 47 local and 3 segmental laparoscopic gastric resections. GI bleeding and dyspepsia were the most common symptoms. Mean tumor size was 4.4 cm (range, <em>1</em>.0-8.5 cm) with the majority of the lesions located in the proximal stomach. Mean operative time was <em>1</em>35 minutes (range, 49-295 minutes), the mean blood loss was 85 <em>mL</em> (range, <em>1</em>0-450 <em>mL</em>), and the mean length of hospitalization was 3.8 days (range <em>1</em>-<em>1</em>0 days). There were no major perioperative complications or mortalities. All lesions had negative resection margins (range, 2-45 mm). Nine patients had <em>1</em>0 or more mitotic figures per 50 high power fields, while <em>1</em><em>1</em> had ulceration and/or necrosis of the lesion. At a mean follow-up of 36 months, 46 (92%) patients were disease free, <em>1</em> patient was alive with disease, <em>1</em> patient with metastases died of a cardiac event, and 2 (4%) patients died of metastatic disease. No local or port site recurrences have been identified. Patient age, tumor size, mitotic index, tumor ulceration, and necrosis were statistically associated with tumor recurrence. The presence of <em>1</em>0 or more mitotic figures per 50 high power fields was an independent predictor of disease progression (P = 0.006).

CONCLUSIONS

A laparoscopic approach to surgical resection of gastric GIST is associated with low morbidity and short hospitalization. As found in historical series of open operative resection, the tumor mitotic index predicts local recurrence. The long-term disease-free survival of 92% in our study establishes laparoscopic resection as safe and effective in treating gastric GISTs. Given these findings as well as the advantages afforded by minimally invasive surgery, a laparoscopic approach may be the preferred resection technique in most patients with small- and medium-sized gastric GISTs.

OBJECTIVE

To evaluate the health benefits of an exclusively human milk-based diet compared with a diet of both human milk and bovine milk-based products in extremely premature infants.

METHODS

Infants fed their own mothers' milk were randomized to <em>1</em> of 3 study groups. Groups HM<em>1</em>00 and HM40 received pasteurized donor human milk-based human milk fortifier when the enteral intake was <em>1</em>00 and 40 <em>mL</em>/kg/d, respectively, and both groups received pasteurized donor human milk if no mother's milk was available. Group BOV received bovine milk-based human milk fortifier when the enteral intake was <em>1</em>00 <em>mL</em>/kg/d and preterm formula if no mother's milk was available. Outcomes included duration of parenteral nutrition, morbidity, and growth.

RESULTS

The 3 groups (total n = 207 infants) had similar baseline demographic variables, duration of parenteral nutrition, rates of late-onset sepsis, and growth. The groups receiving an exclusively human milk diet had significantly lower rates of necrotizing enterocolitis (NEC; P = .02) and NEC requiring surgical intervention (P = .007).

CONCLUSIONS

For extremely premature infants, an exclusively human milk-based diet is associated with significantly lower rates of NEC and surgical NEC when compared with a mother's milk-based diet that also includes bovine milk-based products.

Systematically synthesized derivatives of <em>ML</em>-9, <em>1</em>-(5-chloronaphthalenesulfonyl)-<em>1</em>H-hexahydro-<em>1</em>,4-diazepine, were found to inhibit both Ca2+-calmodulin-dependent and -independent smooth muscle myosin light chain kinases with a similar concentration dependence, and their inhibitions were of the competitive type with respect to ATP. Moreover, <em>ML</em>-9 as well as ATP or ADP exhibited an effective protection to inactivation of smooth muscle myosin light chain kinase by the nucleotide affinity label 5'-p-fluorosulfonylbenzoyladenosine, suggesting that <em>ML</em>-9 binds at or near the ATP-binding site on the kinase molecule. These derivatives, which were structurally unrelated to ATP and exhibited more hydrophobic properties detected by reverse-phase high-performance liquid chromatography, exhibited more potent inhibition toward smooth muscle myosin light chain kinase, indicating that the hydrophobic properties of these derivatives positively correlated well with their potencies of inhibiting the catalytic activity for the enzyme. These findings suggest that the ATP-binding site at the active center of smooth muscle myosin light chain kinase is located in a hydrophobic environment. The potent vaso-relaxing effect of <em>ML</em>-9 on rabbit vascular strips and on saponin-treated skinned smooth muscle cells was discussed in relation to the in vivo inhibition by this drug of smooth muscle myosin light chain kinase.

Tracer methodology has been applied extensively to the estimation of endogenous glucose production (Ra) during euglycemic glucose clamps. The accuracy of this approach has been questioned due to the observation of significantly negative estimates for Ra when insulin levels are high. We performed hyperinsulinemic (300 microU/<em>ml</em>)-euglycemic glucose clamps for <em>1</em>80 min in normal dogs and compared the standard approach, an unlabeled exogenous glucose infusate (cold GINF protocol, n = <em>1</em>2), to a new approach in which a tracer (D-[3-3H]glucose) was added to the exogenous glucose used for clamping (hot GINF protocol, n = <em>1</em>0). Plasma glucose, insulin and glucagon concentrations, and glucose infusion rates were similar for the two protocols. Plasma glucose specific activity was 20 +/- <em>1</em>% of basal (at <em>1</em>20-<em>1</em>80 min) in the cold GINF studies, and 44 +/- 3 to <em>1</em>87 +/- 5% of basal in the hot GINF studies. With the one-compartment, fixed pool volume model of Steele, Ra for the cold GINF studies was -2.4 +/- 0.7 mg X min-<em>1</em> X kg-<em>1</em> at 25 min and remained significantly negative until <em>1</em><em>1</em>0 min (P less than .05). For the hot GINF studies, Ra was never significantly less than zero (P greater than .05) and was greater than in the cold GINF studies at 20-90 min (P less than .05). There was substantially less between-(78%) and within- (40%) experiment variation for the hot GINF studies compared with the cold GINF studies. An alternate approach (regression method) to the application of the one-compartment model, which allows for a variable and estimable effective distribution volume, yielded Ra estimates that were suppressed 60-<em>1</em>00% from basal. In conclusion, the one-compartment, fixed pool volume model of glucose kinetics is inadequate for the estimation of Ra during euglycemic glucose clamps. Two new strategies for estimating Ra from the one-compartment model, the hot GINF protocol and the regression method calculation, yielded more accurate and physiologically plausible estimates of Ra than currently used methodology.

Escherichia coli strains recovered from Crohn's disease (CD) lesions are able to adhere to and invade cultured intestinal epithelial cells. We analyzed the behavior within macrophages of adherent invasive E. coli (AIEC) strains isolated from patients with CD. All the <em>1</em>5 AIEC strains tested were able to replicate extensively within J774-A<em>1</em> cells: the numbers of intracellular bacteria increased 2.2- to 74.2-fold at 48 h over that at <em>1</em> h postinfection. By use of murine peritoneal macrophages and human monocyte-derived-macrophages, the reference AIEC strain LF82 was confirmed to be able to survive intracellularly. Transmission electron micrographs of AIEC LF82-infected macrophages showed that at 24 h postinfection, infected cells harbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles occurring after 8 h postinfection. No lactate dehydrogenase (LDH) release, no sign of DNA fragmentation or degradation, and no binding to fluorescein isothlocyanate-labeled annexin V were observed with LF82-infected J774-A<em>1</em> cells, even after 24 h postinfection. LF82-infected J774-A<em>1</em> cells secreted 2.7-fold more tumor necrosis factor alpha (TNF-alpha) than cells stimulated with <em>1</em> microg of lipopolysaccharide (LPS)/<em>ml</em>. No release of interleukin-<em>1</em>beta was observed with LPS-prestimulated J774-A<em>1</em> cells infected with AIEC LF82. These findings showed that (i) AIEC strains are able to survive and to replicate within macrophages, (ii) AIEC LF82 replication does not induce any cell death of the infected cells, and (iii) LF82-infected J774-A<em>1</em> cells release high levels of TNF-alpha. These properties could be related to some features of CD and particularly to granuloma formation, one of the hallmarks of CD lesions.

BACKGROUND

Recurrence rates after primary repair of ventral and incisional hernias range from 25% to 52%. Recurrence after open surgery is less likely if mesh is used, but the wide fascial dissection and required flap creation increase complication rates. Laparoscopic techniques offer an alternative.

METHODS

To assess the safety and efficacy of laparoscopic ventral and incisional herniorrhaphy, we reviewed the records of all our patients who underwent such a procedure from November <em>1</em>993 to August <em>1</em>999. A laparoscopic approach was attempted in all patients considered to require a mesh repair. Patient demographic characteristics, operative details, and outcomes were recorded.

RESULTS

Of 4<em>1</em>5 patients scheduled to undergo laparoscopic ventral or incisional herniorrhaphy, conversion to an open procedure was necessary in 8. All the remaining 407 patients (205 men and 202 women; mean age 53.2 years; range <em>1</em>3 to 88 years) were included in the study. Mean fascial defect size was <em>1</em>00.<em>1</em> cm2 (range <em>1</em> to 480 cm2). In 97% of patients, expanded polytetrafluoroethylene mesh was used. Mean operating time was 97 minutes (range <em>1</em><em>1</em> to 270 minutes). Mean estimated blood loss was 35 <em>mL</em> (range <em>1</em>0 to <em>1</em>50 <em>mL</em>). Average hospital stay was <em>1</em>.8 days (range 0 to <em>1</em>7 days). There were 53 complications (<em>1</em>3.0%), including cellulitis of a trocar site, infection requiring mesh removal, prolonged suture pain, persistent seroma, intestinal injury, hematoma or postoperative bleeding, prolonged ileus, urinary retention, respiratory distress, fever, intraabdominal abscess, and trocar site herniation. There were no deaths. During a mean followup time of 23 months (range <em>1</em> to 60 months), there were <em>1</em>4 hernia recurrences (3.4%), 6 in patients in whom only a stapling device (no sutures) had been used to secure the mesh to the abdominal wall.

CONCLUSIONS

Laparoscopic repair was completed in 98.<em>1</em>% of patients in whom it was attempted. The complication rate was acceptable. A short hospital stay and minimal blood loss were documented. The recurrence rate was 3.4%. Laparoscopic ventral and incisional hernia repair appear to be safe and effective.

BACKGROUND

The transversus abdominis plane (TAP) block is a novel approach for blocking the abdominal wall neural afferents via the bilateral lumbar triangles of Petit. We evaluated its analgesic efficacy in patients during the first 24 postoperative hours after abdominal surgery, in a randomized, controlled, double-blind clinical trial.

METHODS

Thirty-two adults undergoing large bowel resection via a midline abdominal incision were randomized to receive standard care, including patient-controlled morphine analgesia and regular nonsteroidal antiinflammatory drugs and acetaminophen (n = 16), or to undergo TAP block (n = 16) in addition to standard care (n = 16). After induction of anesthesia, 20 mL of 0.375% levobupivacaine was deposited into the transversus abdominis neuro-fascial plane via the bilateral lumbar triangles of Petit. Each patient was assessed by a blinded investigator in the postanesthesia care unit and at 2, 4, 6, and 24 h postoperatively.

RESULTS

The TAP block reduced visual analog scale pain scores (TAP versus control, mean +/- sd) on emergence (1 +/- 1.4 vs 6.6 +/- 2.8, P < 0.05), and at all postoperative time points, including at 24 h (1.7 +/- 1.7 vs 3.1 +/- 1.5, P < 0.05). Morphine requirements in the first 24 postoperative hours were also reduced (21.9 +/- 8.9 mg vs 80.4 +/- 19.2 mg, P < 0.05). There were no complications attributable to the TAP block. All TAP patients reported high levels of satisfaction with their postoperative analgesic regimen.

CONCLUSIONS

The TAP block provided highly effective postoperative analgesia in the first 24 postoperative hours after major abdominal surgery.

M2-polarized tumor-associated macrophages (TAMs) are key regulators of the link between inflammation and cancer. A negative correlation between infiltration intensity of M2-polarized TAMs and prognosis of pancreatic cancer has been reported. Epithelial-mesenchymal transition (EMT) is an important biological process in the progression of primary tumors toward metastasis. Inflammation-induced EMT has been previously shown, therefore, we hypothesized M2-polarized TAMs could induce EMT in pancreatic cancer. Toll-like receptor 4 (TLR4) signaling has an active role in tumor progression during chronic inflammation and the receptor is primarily expressed on macrophages. Activation of TLR4 on M2-polarized TAMs stimulates an increase in the cytokine interleukin-<em>1</em>0 (IL-<em>1</em>0); consequently, another aim was to investigate the potential role of TLR4/IL-<em>1</em>0 signaling in the EMT of pancreatic cancer. Treatment with IL-4 (20 ng/<em>ml</em>) for 24 h successfully induced the polarization of macrophage cell line RAW 264.7 to M2 phenotype, IL-<em>1</em>0(high), IL-<em>1</em>2(low), and IL-23(low), and high expression of CD204 and CD206. A coculture system allowed investigation of the roles of M2-polarized TAMs and TLR4/IL-<em>1</em>0 signaling in the EMT of Panc-<em>1</em> and BxPC-3 pancreatic cancer cell lines. Our results showed that coculture with M2-polarized TAMs increased fibroblastic morphology, upregulated mesenchymal markers vimentin and snail at the mRNA and protein levels, and increased proliferation, migration, and metalloproteinase (MMP)2 and MMP9 proteolytic activity in pancreatic cancer cells. Simultaneously, coculture with M2-polarized TAMs decreased the expression of the epithelial marker E-cadherin. Coculture with pancreatic cancer cells increased TLR4 mRNA and protein expression in M2-polarized TAMs. Application of TLR4 siRNA and neutralizing antibodies against TLR4 and IL-<em>1</em>0 markedly inhibited E-cadherin reduction and the upregulation of snail and vimentin. Furthermore, activation of TLR4 signaling by lipopolysaccharide profoundly increased the EMT of pancreatic cancer cells. In conclusion, M2-polarized TAMs promoted EMT in pancreatic cancer cells partially through TLR4/IL-<em>1</em>0 signaling, suggesting novel therapeutic strategies and enhancing our understanding of M2-polarized TAMs.

Pharmacologically-induced activation of replication competent proviruses from latency in the presence of antiretroviral treatment (ART) has been proposed as a step towards curing HIV-<em>1</em> infection. However, until now, approaches to reverse HIV-<em>1</em> latency in humans have yielded mixed results. Here, we report a proof-of-concept phase Ib/IIa trial where 6 aviremic HIV-<em>1</em> infected adults received intravenous 5 mg/m2 romidepsin (Celgene) once weekly for 3 weeks while maintaining ART. Lymphocyte histone H3 acetylation, a cellular measure of the pharmacodynamic response to romidepsin, increased rapidly (maximum fold range: 3.7–7.7 relative to baseline) within the first hours following each romidepsin administration. Concurrently, HIV-<em>1</em> transcription quantified as copies of cell-associated un-spliced HIV-<em>1</em> RNA increased significantly from baseline during treatment (range of fold-increase: 2.4–5.0; p = 0.03). Plasma HIV-<em>1</em> RNA increased from <20 copies/<em>mL</em> at baseline to readily quantifiable levels at multiple post-infusion time-points in 5 of 6 patients (range 46–<em>1</em>03 copies/<em>mL</em> following the second infusion, p = 0.04). Importantly, romidepsin did not decrease the number of HIV-specific T cells or inhibit T cell cytokine production. Adverse events (all grade <em>1</em>–2) were consistent with the known side effects of romidepsin. In conclusion, romidepsin safely induced HIV-<em>1</em> transcription resulting in plasma HIV-<em>1</em> RNA that was readily detected with standard commercial assays demonstrating that significant reversal of HIV-<em>1</em> latency in vivo is possible without blunting T cell-mediated immune responses. These finding have major implications for future trials aiming to eradicate the HIV-<em>1</em> reservoir.

BACKGROUND

clinicaltrials.gov NTC02092<em>1</em><em>1</em>6.

BACKGROUND

Prophylaxis with co-trimoxazole (trimethoprim-sulphamethoxazole) is recommended for people with HIV infection or AIDS but is rarely used in Africa. We assessed the effect of such prophylaxis on morbidity, mortality, CD4-cell count, and viral load among people with HIV infection living in rural Uganda, an area with high rates of bacterial resistance to co-trimoxazole.

METHODS

Between April, 200<em>1</em>, and March, 2003, we enrolled, and followed up with weekly home visits, 509 individuals with HIV-<em>1</em> infection and their <em>1</em>522 HIV-negative household members. After 5 months of follow-up, HIV-positive participants were offered daily co-trimoxazole prophylaxis (800 mg trimethoprim, <em>1</em>60 mg sulphamethoxazole) and followed up for a further <em>1</em>.5 years. We assessed rates of malaria, diarrhoea, hospital admission, and death.

RESULTS

Co-trimoxazole was well tolerated with rare (<2% per person-year) adverse reactions. Even though rates of resistance in diarrhoeal pathogens were high (76%), co-trimoxazole prophylaxis was associated with a 46% reduction in mortality (hazard ratio 0.54 [95% CI 0.35-0.84], p=0.006) and lower rates of malaria (multivariate incidence rate ratio 0.28 [0.<em>1</em>9-0.40], p<0.000<em>1</em>), diarrhoea (0.65 [0.53-0.8<em>1</em>], p<0.000<em>1</em>), and hospital admission (0.69 [0.48-0.98], p=0.04). The annual rate of decline in CD4-cell count was less during prophylaxis than before (77 vs 203 cells per microL, p<0.000<em>1</em>), and the annual rate of increase in viral load was lower (0.08 vs 0.90 log(<em>1</em>0) copies per mL, p=0.0<em>1</em>).

CONCLUSIONS

Daily co-trimoxazole prophylaxis was associated with reduced morbidity and mortality and had beneficial effects on CD4-cell count and viral load. Co-trimoxazole prophylaxis is a readily available, effective intervention for people with HIV infection in Africa.

BACKGROUND

We investigated the effects of candesartan (an angiotensin II antagonist) alone, enalapril alone, and their combination on exercise tolerance, ventricular function, quality of life (QOL), neurohormone levels, and tolerability in congestive heart failure (CHF).

RESULTS

Seven hundred sixty-eight patients in New York Heart Association functional class (NYHA-FC) II to IV with ejection fraction (EF) <0.40 and a 6-minute walk distance (6MWD) <500 m received either candesartan (4, 8, or <em>1</em>6 mg), candesartan (4 or 8 mg) plus 20 mg of enalapril, or 20 mg of enalapril for 43 weeks. There were no differences among groups with regard to 6MWD, NYHA-FC, or QOL. EF increased (P=NS) more with candesartan-plus-enalapril therapy (0.025+/-0.004) than with candesartan alone (0.0<em>1</em>5+/-0.004) or enalapril alone(0.0<em>1</em>5+/-0.005). End-diastolic (EDV) and end-systolic (ESV) volumes increased less with combination therapy (EDV 8+/-4 <em>mL</em>; ESV <em>1</em>+/-4 <em>mL</em>; P<0.0<em>1</em>) than with candesartan alone (EDV 27+/-4 <em>mL</em>; ESV <em>1</em>8+/-3 <em>mL</em>) or enalapril alone (EDV 23+/-7 <em>mL</em>; ESV <em>1</em>4+/-6 <em>mL</em>). Blood pressure decreased with combination therapy (6+/-<em>1</em>/4+/-<em>1</em> mm Hg) compared with candesartan or enalapril alone (P<0.05). Aldosterone decreased (P<0.05) with combination therapy (23.2+/-5.3 pg/<em>mL</em>) at <em>1</em>7 but not 43 weeks compared with candesartan (0.7+/-7.8 pg/<em>mL</em>) or enalapril (-0.8+/-<em>1</em><em>1</em>. 3 pg/<em>mL</em>). Brain natriuretic peptide decreased with combination therapy (5.8+/-2.7 pmol/L; P<0.0<em>1</em>) compared with candesartan (4. 4+/-3.8 pmol/L) and enalapril alone (4.0+/-5.0 pmol/L).

CONCLUSIONS

Candesartan alone was as effective, safe, and tolerable as enalapril. The combination of candesartan and enalapril was more beneficial for preventing left ventricular remodeling than either candesartan or enalapril alone.

OBJECTIVE

Adefovir dipivoxil possesses potent in vitro and in vivo antiviral activity in wild-type hepatitis B. This study assessed the safety and efficacy of adefovir dipivoxil alone and in combination with lamivudine compared with ongoing lamivudine therapy in patients with chronic hepatitis B with compensated liver disease and lamivudine-resistant hepatitis B virus (HBV).

METHODS

Fifty-nine hepatitis B e antigen (HBeAg)-positive patients with genotypic evidence of lamivudine-resistant HBV, serum alanine aminotransferase (ALT) level>> or =1.2 times the upper limit of normal, and serum HBV DNA level>> or =6 log(10) copies/mL despite ongoing treatment with lamivudine were randomized to adefovir dipivoxil 10 mg, lamivudine 100 mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. The primary end point was the time-weighted average change from baseline in serum HBV DNA level (DAVG) up to week 16.

RESULTS

Rapid reductions in serum HBV DNA level were seen by 4 weeks in all recipients of adefovir dipivoxil; DAVG(16) was -0.07 in the lamivudine group compared with -2.45 and -2.46 log(10) copies/mL in the adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups, respectively (P < 0.001). Median change from baseline in serum HBV DNA level at week 48 was 0.0, -3.59, and -4.04 log(10) copies/mL in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. ALT level normalized in 10 of 19 (53%) and 9 of 18 (47%) recipients of adefovir dipivoxil/lamivudine and adefovir dipivoxil, respectively, compared with 1 of 19 (5%) recipients of lamivudine. Three patients receiving adefovir dipivoxil or adefovir dipivoxil/lamivudine and none receiving lamivudine monotherapy were HBeAg negative at week 48 and one became hepatitis B surface antigen negative.

CONCLUSIONS

These data, limited to patients with compensated liver disease, indicate that adefovir dipivoxil alone or in combination with ongoing lamivudine therapy provides effective antiviral therapy in patients with lamivudine-resistant HBV.