Defective function of dendritic cells (DC) in cancer has been recently described and may represent one of the mechanisms of tumor evasion from immune system control. We have previously shown in vitro that vascular endothelial growth factor (VEGF), produced by almost all tumors, is one of the tumor-derived factors responsible for the defective function of these cells. In this study, we investigated whether in vivo infusion of recombinant VEGF could reproduce the observed DC dysfunction. Continuous VEGF infusion, at rates as low as 50 ng/h (resulting in serum VEGF concentrations of <em>1</em>20 to <em>1</em>60 pg/<em>mL</em>), resulted in a dramatic inhibition of dendritic cell development, associated with an increase in the production of B cells and immature Gr-<em>1</em>(+) myeloid cells. Infusion of VEGF was associated with inhibition of the activity of the transcription factor NF-kappaB in bone marrow progenitor cells. Experiments in vitro showed that VEGF itself, and not factors released by VEGF-activated endothelial cells, affected polypotent stem cells resulting in the observed abnormal hematopoiesis. These data suggest that VEGF, at pathologically relevant concentrations in vivo, may exert effects on pluripotent stem cells that result in blocked DC development as well as affect many other hematopoietic lineages.

Recent studies have focused on the potential role of transforming growth factor type beta (TGF-beta) as an immunoregulatory peptide. In this context, we demonstrate that TGF-beta is a potent chemoattractant for human peripheral blood monocytes. At concentrations from 0.<em>1</em> to <em>1</em>0 pg/<em>ml</em>, TGF-beta induces directed monocyte migration in vitro. Consistent with this observation is the expression of high-affinity TGF-beta receptors on the monocytes with a Kd of <em>1</em>-<em>1</em>0 pM. At higher concentrations of TGF-beta (greater than or equal to <em>1</em> ng/<em>ml</em>), monocytes are stimulated to generate biologically active mediator(s) that enhance fibroblast growth. Gene expression for one of these growth factors, interleukin <em>1</em>, is induced in monocytes within hours after exposure to TGF-beta. Thus, TGF-beta may provide an important signal for monocyte recruitment and for regulation of their synthesis of mediators of fibroblast growth and activity in wound healing.

The basic morphological properties of liver cells are defined in the form of a morphometric model to permit integrated quantitative characterization of functionally important parameters. Stereologic methods which allow efficient and reliable quantitative evaluation of sectioned liver tissue are presented. Material, obtained by a rigorous three-stage sampling procedure from five normal rat livers, is systematically subjected to this analysis at four levels of magnification. This yields quantitative data which are expressed as "densities," i.e. content per <em>1</em> <em>ml</em> of tissue, as "specific dimensions" related to <em>1</em>00 g body weight, and as absolute dimensions per average "mononuclear" hepatocyte. Base line data relating to the normal rat liver are presented for the entire spectrum of parameters. As examples, <em>1</em> <em>ml</em> of liver tissue contains <em>1</em>69 x <em>1</em>0(6) hepatocyte nuclei, some 90 x <em>1</em>0(6) nuclei of other cells, and 280 x <em>1</em>0(9) mitochondria. Hepatocyte cytoplasm accounts for 77% of liver volume, and the mitochondria for <em>1</em>8%. The surface area of endoplasmic reticulum membranes in <em>1</em> <em>ml</em> of liver tissue measures <em>1</em><em>1</em> m(2) of which are (2/3) of the rough form carrying some 2 x <em>1</em>0(<em>1</em>3) ribosomes. The surface area of mitochondrial cristae in the unit volume is estimated at 6 m(2). The validity and applicability of the method are discussed, and the data are compared with available information from other studies.

BACKGROUND

The persistence of circulating tumor cells (CTC) in breast cancer patients might be associated with stem cell like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells also may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Here we evaluated 226 blood samples of 39 metastatic breast cancer patients during a follow-up of palliative chemo-, antibody - or hormonal therapy for the expression of the stem cell marker ALDH<em>1</em> and markers for EMT and correlated these findings with the presence of CTC and response to therapy.

METHODS

2 x 5 <em>ml</em> blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-<em>1</em> and HER2 transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist<em>1</em>, Akt2, PI3Kalpha] and separately for the tumor stem-cell markers ALDH<em>1</em>. The identification of EMT markers was considered positive if at least one marker was detected in the sample.

RESULTS

97% of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH<em>1</em> transcripts. CTC were detected in 69/226 (3<em>1</em>%) cancer samples. In the CTC (+) group, 62% were positive for at least one of the EMT markers and 69% for ALDH<em>1</em>, respectively. In the CTC (-) group the percentages were 7% and <em>1</em>4%, respectively. In non-responders, EMT and ALDH<em>1</em> expression was found in 62% and 44% of patients, in responders the rates were <em>1</em>0% and 5%, respectively.

CONCLUSIONS

Our data indicate that a major proportion of CTC of metastatic breast cancer patients shows EMT and tumor stem cell characteristics. Further studies are needed to prove whether these markers might serve as an indicator for therapy resistant tumor cell populations and, therefore, an inferior prognosis.

Nicotine underlies tobacco addiction, influences tobacco use patterns, and is used as a pharmacological aid to smoking cessation. The absorption, distribution and disposition characteristics of nicotine from tobacco and medicinal products are reviewed. Nicotine is metabolized primarily by the liver enzymes CYP2A6, UDPglucuronosyltransferase (UGT), and flavin-containing monooxygenase (FMO). In addition to genetic factors, nicotine metabolism is influenced by diet and meals, age, sex, use of estrogen-containing hormone preparations, pregnancy and kidney disease, other medications, and smoking itself. Substantial racial/ethnic differences are observed in nicotine metabolism, which are likely influenced by both genetic and environmental factors. The most widely used biomarker of nicotine intake is cotinine, which may be measured in blood, urine, saliva, hair, or nails. The current optimal plasma cotinine cut-point to distinguish smokers from non-smokers in the general US population is 3 ng <em>ml</em>(-<em>1</em>). This cut-point is much lower than that established 20 years ago, reflecting less secondhand smoke exposure due to clear air policies and more light or occasional smoking.

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-<em>1</em> infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4(+) T cell counts>>/= 350 cells/microliter and viral load below the limits of detection for>>/=<em>1</em> year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/<em>ml</em> occurred in all <em>1</em>8 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2-3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log(<em>1</em>0) copies) day(-<em>1</em>), which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.<em>1</em>5 log(<em>1</em>0) copies) day(-<em>1</em>) (P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/<em>ml</em>. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4(+) T cells.

We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and <em>1</em>60 +/- <em>1</em>3 attomoles DCF per cell, respectively, in <em>1</em>5 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by <em>1</em>00 ng/<em>ml</em> PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by <em>1</em>00 ng/<em>ml</em> PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., <em>1</em> ng/<em>ml</em> PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.

OBJECTIVE

To evaluate a new 2-step technique for obtaining adequate but short-term parenchymal hypertrophy in oncologic patients requiring extended right hepatic resection with limited functional reserve.

BACKGROUND

Patients presenting with primary or metastatic liver tumors often face the dilemma that the remaining liver tissue may not be sufficient. Preoperative portal vein embolization has thus far been established as the standard procedure for achieving resectability.

METHODS

Two-staged hepatectomy was performed in patients who preoperatively appeared to be marginally resectable but had a tumor-free left lateral lobe. Marginal respectability was defined as a left lateral lobe to body weight ratio of less than 0.5. In the first step, surgical exploration, right portal vein ligation (PVL), and in situ splitting (ISS) of the liver parenchyma along the falciform ligament were performed. Computed tomographic volumetry was performed before ISS and before completion surgery.

RESULTS

The study included 25 patients with primary liver tumors (hepatocellular carcinoma: n = 3, intrahepatic cholangiocarcinoma: n = 2, extrahepatic cholangiocarcinoma: n = 2, malignant epithelioid hemangioendothelioma: n = <em>1</em>, gallbladder cancer: n = <em>1</em> or metastatic disease [colorectal liver metastasis]: n = <em>1</em>4, ovarian cancer: n = <em>1</em>, gastric cancer: n = <em>1</em>). Preoperative CT volumetry of the left lateral lobe showed 3<em>1</em>0 <em>mL</em> in median (range = <em>1</em>97-444 <em>mL</em>). After a median waiting period of 9 days (range = 5-28 days), the volume of the left lateral lobe had increased to 536 <em>mL</em> (range = 273-88<em>1</em> <em>mL</em>), representing a median volume increase of 74% (range = 2<em>1</em>%-<em>1</em>92%) (P < 0.00<em>1</em>). The median left lateral liver lobe to body weight ratio was increased from 0.38% (range = 0.25%-0.49%) to 0.6<em>1</em>% (range = 0.35-0.95). Ten of 25 patients (40%) required biliary reconstruction with hepaticojejunostomy. Rapid perioperative recovery was reflected by normalization of International normalized ratio (INR) (80% of patients), creatinine (84% of patients), nearly normal bilirubin (56% of patients), and albumin (64% of patients) values by day <em>1</em>4 after completion surgery. Perioperative morbidity was classified according to the Dindo-Clavien classification of surgical complications: grade I (<em>1</em>2 events), grade II (<em>1</em>3 events), grade III (<em>1</em>4 events, III a: 6 events, III b: 8 events), grade IV (8 events, IV a: 3 events, IV b: 5 events), and grade V (3 events). Sixteen patients (68%) experienced perioperative complications. Follow-up was <em>1</em>80 days in median (range: 60-776 days) with an estimated overall survival of 86% at 6 months after resection.

CONCLUSIONS

Two-step hepatic resection performing surgical exploration, PVL, and ISS results in a marked and rapid hypertrophy of functional liver tissue and enables curative resection of marginally resectable liver tumors or metastases in patients that might otherwise be regarded as palliative.

BACKGROUND

Not all patients infected with NDM-<em>1</em>-positive bacteria have a history of hospital admission in India, and extended-spectrum β-lactamases are known to be circulating in the Indian community. We therefore measured the prevalence of the NDM-<em>1</em> gene in drinking water and seepage samples in New Delhi.

METHODS

Swabs absorbing about <em>1</em>00 μL of seepage water (ie, water pools in streets or rivulets) and <em>1</em>5 mL samples of public tap water were collected from sites within a <em>1</em>2 km radius of central New Delhi, with each site photographed and documented. Samples were transported to the UK and tested for the presence of the NDM-<em>1</em> gene, bla(NDM-<em>1</em>), by PCR and DNA probing. As a control group, <em>1</em>00 μL sewage effluent samples were taken from the Cardiff Wastewater Treatment Works, Tremorfa, Wales. Bacteria from all samples were recovered and examined for bla(NDM-<em>1</em>) by PCR and sequencing. We identified NDM-<em>1</em>-positive isolates, undertook susceptibility testing, and, where appropriate, typed the isolates. We undertook Inc typing on bla(NDM-<em>1</em>)-positive plasmids. Transconjugants were created to assess plasmid transfer frequency and its relation to temperature.

RESULTS

From Sept 26 to Oct <em>1</em>0, 20<em>1</em>0, <em>1</em>7<em>1</em> seepage samples and 50 tap water samples from New Delhi and 70 sewage effluent samples from Cardiff Wastewater Treatment Works were collected. We detected bla(NDM-<em>1</em>) in two of 50 drinking-water samples and 5<em>1</em> of <em>1</em>7<em>1</em> seepage samples from New Delhi; the gene was not found in any sample from Cardiff. Bacteria with bla(NDM-<em>1</em>) were grown from <em>1</em>2 of <em>1</em>7<em>1</em> seepage samples and two of 50 water samples, and included <em>1</em><em>1</em> species in which NDM-<em>1</em> has not previously been reported, including Shigella boydii and Vibrio cholerae. Carriage by enterobacteria, aeromonads, and V cholera was stable, generally transmissible, and associated with resistance patterns typical for NDM-<em>1</em>; carriage by non-fermenters was unstable in many cases and not associated with typical resistance. 20 strains of bacteria were found in the samples, <em>1</em>2 of which carried bla(NDM-<em>1</em>) on plasmids, which ranged in size from <em>1</em>40 to 400 kb. Isolates of Aeromonas caviae and V cholerae carried bla(NDM-<em>1</em>) on chromosomes. Conjugative transfer was more common at 30°C than at 25°C or 37°C.

CONCLUSIONS

The presence of NDM-<em>1</em> β-lactamase-producing bacteria in environmental samples in New Delhi has important implications for people living in the city who are reliant on public water and sanitation facilities. International surveillance of resistance, incorporating environmental sampling as well as examination of clinical isolates, needs to be established as a priority.

BACKGROUND

European Union.

BACKGROUND

Drugs that inhibit angiotensin-converting enzyme slow the progression of renal insufficiency in patients with diabetic neuropathy. Whether these drugs have a similar action in patients with other renal diseases is not known. We conducted a study to determine the effect of the angiotensin-converting-enzyme inhibitor benazepril on the progression of renal insufficiency in patients with various underlying renal diseases.

METHODS

In a three-year trial involving 583 patients with renal insufficiency caused by various disorders, 300 patients received benazepril and 283 received placebo. The underlying diseases included glomerulopathies (in 192 patients), interstitial nephritis (in 105), nephrosclerosis (in 97), polycystic kidney disease (in 64), diabetic nephropathy (in 21), and miscellaneous or unknown disorders (in 104). The severity of renal insufficiency was classified according to the base-line creatinine clearance: 227 patients had mild insufficiency (creatinine clearance, 46 TO 60 ml per minute), and 356 had moderate insufficiency (creatinine clearance, 30 to 45 ml per minute). The primary end point was a doubling of the base-line serum creatine concentration or the need for dialysis.

RESULTS

At three years. 31 patients in the benazepril group and 57 in the placebo group had reached the primary end point (P<0.001). In the benazepril group, the reduction in the risk of reaching the end point was 53 percent overall (95 percent confidence interval, 27 to 70 percent), 71 percent (95 percent confidence interval, 21 to 90 percent) among the patients with mild renal insufficiency, and 46 percent (95 percent confidence interval, 12 to 67 percent) among those with moderate renal insufficiency. The reduction in risk was greatest among the male patients; those with glomerular diseases, diabetic nephropathy, or miscellaneous or unknown causes of renal disease; and those with base-line urinary protein excretion above 1 g per 24 hours. Benazepril was not effective in patients with polycystic disease. Diastolic pressure decreased by 3.5 to 5.0 mm Hg in the benazepril group and increased by 0.2 to 1.5 mm Hg in the placebo group.

CONCLUSIONS

Benazepril provides protection against the progression of renal insufficiency in patients with various renal diseases.

BACKGROUND

Some patients with severe asthma have frequent exacerbations associated with persistent eosinophilic inflammation despite continuous treatment with high-dose inhaled glucocorticoids with or without oral glucocorticoids.

METHODS

In this randomized, double-blind, double-dummy study, we assigned 576 patients with recurrent asthma exacerbations and evidence of eosinophilic inflammation despite high doses of inhaled glucocorticoids to one of three study groups. Patients were assigned to receive mepolizumab, a humanized monoclonal antibody against interleukin-5, which was administered as either a 75-mg intravenous dose or a <em>1</em>00-mg subcutaneous dose, or placebo every 4 weeks for 32 weeks. The primary outcome was the rate of exacerbations. Other outcomes included the forced expiratory volume in <em>1</em> second (FEV<em>1</em>) and scores on the St. George's Respiratory Questionnaire (SGRQ) and the 5-item Asthma Control Questionnaire (ACQ-5). Safety was also assessed.

RESULTS

The rate of exacerbations was reduced by 47% (95% confidence interval [CI], 29 to 6<em>1</em>) among patients receiving intravenous mepolizumab and by 53% (95% CI, 37 to 65) among those receiving subcutaneous mepolizumab, as compared with those receiving placebo (P<0.00<em>1</em> for both comparisons). Exacerbations necessitating an emergency department visit or hospitalization were reduced by 32% in the group receiving intravenous mepolizumab and by 6<em>1</em>% in the group receiving subcutaneous mepolizumab. At week 32, the mean increase from baseline in FEV<em>1</em> was <em>1</em>00 ml greater in patients receiving intravenous mepolizumab than in those receiving placebo (P=0.02) and 98 ml greater in patients receiving subcutaneous mepolizumab than in those receiving placebo (P=0.03). The improvement from baseline in the SGRQ score was 6.4 points and 7.0 points greater in the intravenous and subcutaneous mepolizumab groups, respectively, than in the placebo group (minimal clinically important change, 4 points), and the improvement in the ACQ-5 score was 0.42 points and 0.44 points greater in the two mepolizumab groups, respectively, than in the placebo group (minimal clinically important change, 0.5 points) (P<0.00<em>1</em> for all comparisons). The safety profile of mepolizumab was similar to that of placebo.

CONCLUSIONS

Mepolizumab administered either intravenously or subcutaneously significantly reduced asthma exacerbations and was associated with improvements in markers of asthma control. (Funded by GlaxoSmithKline; MENSA ClinicalTrials.gov number, NCT0<em>1</em>69<em>1</em>52<em>1</em>.).

BACKGROUND

Experimental evidence suggests that flow-dependent dilatation of conduit arteries is mediated by nitric oxide (NO) and/or prostacyclin. The present study was designed to assess whether NO or prostacyclin also contributes to flow-dependent dilatation of conduit arteries in humans.

RESULTS

Radial artery internal diameter (ID) was measured continuously in <em>1</em>6 healthy volunteers (age, 24 +/- <em>1</em> years) with a transcutaneous A-mode echo-tracking system coupled to a Doppler device for the measurement of radial blood flow. In 8 subjects, a catheter was inserted into the brachial artery for measurement of arterial pressure and infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 8 mumol/min for 7 minutes; infusion rate, 0.8 <em>mL</em>/min). Flow-dependent dilatation was evaluated before and after L-NMMA or aspirin as the response of the radial artery to an acute increase in flow (reactive hyperemia after a 3-minute cuff wrist occlusion). Under control conditions, release of the occlusion induced a marked increase in radial blood flow (from 24 +/- 3 to 73 +/- <em>1</em><em>1</em> <em>mL</em>/min; P < .0<em>1</em>) followed by a delayed increase in radial diameter (flow-mediated dilatation; from 2.67 +/- 0.<em>1</em>0 to 2.77 +/- 0.<em>1</em>2 mm; P < .0<em>1</em>) without any change in heart rate or arterial pressure. L-NMMA decreased basal forearm blood flow (from 24 +/- 3 to <em>1</em>3 +/- 3 <em>mL</em>/min; P < .05) without affecting basal radial artery diameter, heart rate, or arterial pressure, whereas aspirin (<em>1</em> g PO) was without any hemodynamic effect. In the presence of L-NMMA, the peak flow response during hyperemia was not affected (76 +/- <em>1</em>2 <em>mL</em>/min), but the duration of the hyperemic response was markedly reduced, and the flow-dependent dilatation of the radial artery was abolished and converted to a vasoconstriction (from 2.62 +/- 0.<em>1</em><em>1</em> to 2.55 +/- 0.<em>1</em><em>1</em> mm; P < .0<em>1</em>). In contrast, aspirin did not affect the hyperemic response nor the flow-dependent dilatation of the radial artery.

CONCLUSIONS

The present investigation demonstrates that NO, but not prostacyclin, is essential for flow-mediated dilatation of large human arteries. Hence, this response can be used as a test for the L-arginine/NO pathway in clinical studies.

OBJECTIVE

To evaluate the efficacy and safety of laparoscopic repair of ventral hernias.

BACKGROUND

The recurrence rate after standard repair of ventral hernias may be as high as <em>1</em>2-52%, and the wide surgical dissection required often results in wound complications. Use of a laparoscopic approach may decrease rates of complications and recurrence after ventral hernia repair.

METHODS

Data on all patients who underwent laparoscopic ventral hernia repair (LVHR) performed by 4 surgeons using a standardized procedure between November <em>1</em>993 and October 2002 were collected prospectively (85% of patients) or retrospectively.

RESULTS

LVHR was completed in 8<em>1</em>9 of the 850 patients (422 men; 428 women) in whom it was attempted. Thirty-four percent of completed LVHRs were for recurrent hernias. The patient mean body mass index was 32; the mean defect size was <em>1</em><em>1</em>8 cm2. Mesh, averaging 344 cm2, was used in all cases. Mean operating time was <em>1</em>20 min, mean estimated blood loss was 49 <em>mL</em>, and hospital stay averaged 2.3 days. There were <em>1</em>28 complications in <em>1</em><em>1</em>2 patients (<em>1</em>3.2%). One patient died of a myocardial infarction. The most common complications were ileus (3%) and prolonged seroma (2.6%). During a mean follow-up time of 20.2 months (range, <em>1</em>-94 months), the hernia recurrence rate was 4.7%. Recurrence was associated with large defects, obesity, previous open repairs, and perioperative complications.

CONCLUSIONS

In this large series, LVHR had a low rate of conversion to open surgery, a short hospital stay, a moderate complication rate, and a low risk of recurrence.

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR<em>1</em> through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between <em>1</em>.3 x <em>1</em>0(<em>1</em><em>1</em>) and 9.8 x <em>1</em>0(<em>1</em>2)/<em>ml</em> (types <em>1</em> and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types <em>1</em> and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type <em>1</em> was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type <em>1</em>. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.

Elevated serum levels of acute-phase proteins, indicating chronic subclinical inflammation, have been associated with cardiovascular disease as well as the insulin resistance syndrome. Chronic inflammation may also be a risk factor for developing type 2 diabetes. We studied the concentrations of C-reactive protein (CRP), fibrinogen, and plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) in <em>1</em>,047 nondiabetic subjects in relation to incident diabetes within 5 years in the Insulin Resistance Atherosclerosis Study. Subjects with diabetes at follow-up (n = <em>1</em>44) had higher baseline levels of fibrinogen (mean +/- SD; 287.8 +/- 58.8 vs. 275.<em>1</em> +/- 56.0 mg/dl; P = 0.0<em>1</em>3) as well as of CRP (median [interquartile range]; 2.40 [<em>1</em>.29, 5.87] vs. <em>1</em>.67 mg/l [0.75, 3.4<em>1</em>]; P = 0.000<em>1</em>) and PAI-<em>1</em> (24 [<em>1</em>5, 37.5] vs. <em>1</em>6 ng/<em>ml</em> [9, 27]; P = 0.000<em>1</em>) than nonconverters. The odds ratio (OR) of converting to diabetes was significantly increased with increasing baseline concentrations of the inflammatory markers. In contrast to PAI-<em>1</em>, the association of CRP and fibrinogen with incident diabetes was significantly attenuated after adjustment for body fat (BMI or waist circumference) or insulin sensitivity (S(I)), as assessed by a frequently sampled intravenous glucose tolerance test. In a logistic regression model that included age, sex, ethnicity, clinical center, smoking, BMI, S(I), physical activity, and family history of diabetes, PAI-<em>1</em> still remained significantly related to incident type 2 diabetes (OR [95% CI] for <em>1</em> SD increase: <em>1</em>.6<em>1</em> [<em>1</em>.20-2.<em>1</em>6]; P = 0.002). Chronic inflammation emerges as a new risk factor for the development of type 2 diabetes; PAI-<em>1</em> predicts type 2 diabetes independent of insulin resistance and other known risk factors for diabetes.

<em>1</em>. Helix aspersa neurones under voltage clamp generate prolonged outward currents (potassium currents) in response to depolarizing command pulses. 2. The potassium currents recorded from cell A were reversibly reduced 25-50% by <em>1</em>0 mM cobalt ions in the bathing medium; <em>1</em> mM lanthanum, <em>1</em>0(-6) g/<em>ml</em>. D-600 and <em>1</em>0(-6) g/<em>ml</em>. iproveratril had similar effects but were only partially reversible. 3. The relationship between the potassium currents and the membrane potential had an "n" shape in normal saline. In calcium-free saline (containing 25 mM magnesium) the potassium currents were reduced and the "n" shape was abolished. The effect of calcium-free saline was readily reversible. 4. The voltage-dependence of the calcium-sensitive potassium currents was similar to that of the "late" calcium channel in squid axons (Baker, Hodgkin & Ridgway, <em>1</em>97<em>1</em>). 5. When cell A was depolarents were made up of two exponentially declining components. The slower of the two components was reduced in calcium-free saline. 6. When cell A was depolarized by <em>1</em>50 mV for <em>1</em>0 msec and then repolarized the "tail" currents were made up of a single rapidly declining component. The reversal potential of this component changed by 58 mV for a tenfold change in the external potassium concentration as predicted by the Nernst equation. 7. The reversal potential of "tail" currents having both components was less sensitive to changes in the external potassium concentration. 8. Tetraethylammonium (TEA) ions blocked both calcium dependent and voltage sensitive potassium currents. Each receptor was found to bind a single molecule of TEA. The dissociaton constant was about <em>1</em>0 mM in each case. 9. The intracellular concentration of ionized calcium was estimated from the potential at which there was no apparent calcium influx (the null point). It was between 3 x <em>1</em>0(-8) M and 8 x <em>1</em>0(-8) M with <em>1</em>0(-2) M calcium in the bathing medium. <em>1</em>0. The null point changed 30 mV for a tenfold change in the external calcium concentration as predicted by the Nernst equation. <em>1</em><em>1</em>. It is concluded that depolarization of Helix neurones activates two typesof potassium channel. One channel is voltage dependent and highly selective for potassium. Activation of the other channel is dependent on the influx (or injection, see Meech, <em>1</em>972, <em>1</em>974a) of calcium. This calcium mediated potassium activation system saturates at high external calcium concentrations and is inhibited by external magnesium ions.

BACKGROUND

Lymphangioleiomyomatosis (LAM) is a progressive, cystic lung disease in women; it is associated with inappropriate activation of mammalian target of rapamycin (mTOR) signaling, which regulates cellular growth and lymphangiogenesis. Sirolimus (also called rapamycin) inhibits mTOR and has shown promise in phase <em>1</em>-2 trials involving patients with LAM.

METHODS

We conducted a two-stage trial of sirolimus involving 89 patients with LAM who had moderate lung impairment--a <em>1</em>2-month randomized, double-blind comparison of sirolimus with placebo, followed by a <em>1</em>2-month observation period. The primary end point was the difference between the groups in the rate of change (slope) in forced expiratory volume in <em>1</em> second (FEV(<em>1</em>)).

RESULTS

During the treatment period, the FEV(<em>1</em>) slope was -<em>1</em>2±2 ml per month in the placebo group (43 patients) and <em>1</em>±2 ml per month in the sirolimus group (46 patients) (P<0.00<em>1</em>). The absolute between-group difference in the mean change in FEV(<em>1</em>) during the treatment period was <em>1</em>53 ml, or approximately <em>1</em><em>1</em>% of the mean FEV(<em>1</em>) at enrollment. As compared with the placebo group, the sirolimus group had improvement from baseline to <em>1</em>2 months in measures of forced vital capacity, functional residual capacity, serum vascular endothelial growth factor D (VEGF-D), and quality of life and functional performance. There was no significant between-group difference in this interval in the change in 6-minute walk distance or diffusing capacity of the lung for carbon monoxide. After discontinuation of sirolimus, the decline in lung function resumed in the sirolimus group and paralleled that in the placebo group. Adverse events were more common with sirolimus, but the frequency of serious adverse events did not differ significantly between the groups.

CONCLUSIONS

In patients with LAM, sirolimus stabilized lung function, reduced serum VEGF-D levels, and was associated with a reduction in symptoms and improvement in quality of life. Therapy with sirolimus may be useful in selected patients with LAM. (Funded by the National Institutes of Health and others; MILES ClinicalTrials.gov number, NCT004<em>1</em>4648.).

OBJECTIVE

Ulcerative colitis (UC) is difficult to treat, and standard therapy does not always induce remission. Fecal microbiota transplantation (FMT) is an alternative approach that induced remission in small series of patients with active UC. We investigated its safety and efficacy in a placebo-controlled randomized trial.

METHODS

We performed a parallel study of patients with active UC without infectious diarrhea. Participants were examined by flexible sigmoidoscopy when the study began and then were randomly assigned to groups that received FMT (50 mL, via enema, from healthy anonymous donors; n = 38) or placebo (50 mL water enema; n = 37) once weekly for 6 weeks. Patients, clinicians, and investigators were blinded to the groups. The primary outcome was remission of UC, defined as a Mayo score ≤2 with an endoscopic Mayo score of 0, at week 7. Patients provided stool samples when the study began and during each week of FMT for microbiome analysis. The trial was stopped early for futility by the Data Monitoring and Safety Committee, but all patients already enrolled in the trial were allowed to complete the study.

RESULTS

Seventy patients completed the trial (3 dropped out from the placebo group and 2 from the FMT group). Nine patients who received FMT (24%) and 2 who received placebo (5%) were in remission at 7 weeks (a statistically significant difference in risk of 17%; 95% confidence interval, 2%-33%). There was no significant difference in adverse events between groups. Seven of the 9 patients in remission after FMT received fecal material from a single donor. Three of the 4 patients with UC ≤1 year entered remission, compared with 6 of 34 of those with UC >1 year (P = .04, Fisher's exact test). Stool from patients receiving FMT had greater microbial diversity, compared with baseline, than that of patients given the placebo (P = .02, Mann-Whitney U test).

CONCLUSIONS

FMT induces remission in a significantly greater percentage of patients with active UC than placebo, with no difference in adverse events. Fecal donor and time of UC appear to affect outcomes. ClinicalTrials.gov Number: NCT01545908.

This study was undertaken to address the current deficient knowledge of cellular response to nanosized particle exposure. The study evaluated the acute toxic effects of metal/metal oxide nanoparticles proposed for future use in industrial production methods using the in vitro rat liver derived cell line (BRL 3A). Different sizes of nanoparticles such as silver (Ag; <em>1</em>5, <em>1</em>00 nm), molybdenum (MoO(3); 30, <em>1</em>50 nm), aluminum (Al; 30, <em>1</em>03 nm), iron oxide (Fe(3)O(4); 30, 47 nm), and titanium dioxide (TiO(2); 40 nm) were evaluated for their potential toxicity. We also assessed the toxicity of relatively larger particles of cadmium oxide (CdO; <em>1</em> microm), manganese oxide (MnO(2); <em>1</em>-2 microm), and tungsten (W; 27 microm), to compare the cellular toxic responses with respect to the different sizes of nanoparticles with different core chemical compositions. For toxicity evaluations, cellular morphology, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH) levels, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were assessed under control and exposed conditions (24h of exposure). Results showed that mitochondrial function decreased significantly in cells exposed to Ag nanoparticles at 5-50 microg/<em>ml</em>. However, Fe(3)O(4), Al, MoO(3) and TiO(2) had no measurable effect at lower doses (<em>1</em>0-50 microg/<em>ml</em>), while there was a significant effect at higher levels (<em>1</em>00-250 microg/<em>ml</em>). LDH leakage significantly increased in cells exposed to Ag nanoparticles (<em>1</em>0-50 microg/<em>ml</em>), while the other nanoparticles tested displayed LDH leakage only at higher doses (<em>1</em>00-250 microg/<em>ml</em>). In summary the Ag was highly toxic whereas, MoO(3) moderately toxic and Fe(3)O(4), Al, MnO(2) and W displayed less or no toxicity at the doses tested. The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, reduced mitochondrial membrane potential and increase in ROS levels, which suggested that cytotoxicity of Ag (<em>1</em>5, <em>1</em>00 nm) in liver cells is likely to be mediated through oxidative stress.

The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38,000 daltons. Human CobINA was found to be a glycoprotein containing at least <em>1</em>0.7% carbohydrate and to have a normal serum concentration of 34 +/- 7 mug/<em>ml</em> (mean +/- <em>1</em> SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating beta<em>1</em>H were removed from these proteins with anti-beta<em>1</em>H Sepharose. However, in the presence of highly purified beta<em>1</em>H and C3bINA, both C3bIna, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and beta<em>1</em>H had no effect on their cleaved. Incubation of native C3 or C4 with C3bINA and beta<em>1</em>H had no effect on their structure. The action of C3bINA and beta<em>1</em>H on C3b produced two fragments of the alpha<em>1</em>-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40,000 daltons. The action of C3bINA and beta<em>1</em>H on C4b resulted in cleavage of the alpha'-chain giving rise to the <em>1</em>50,000-dalton C4c and the 49,000-dalton C4d fragments which dissociated without reduction. To produce from C3b the immunochemically defined C3c and C3d, fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.

Type 2 diabetes (T2DM) is associated with chronic low-grade inflammation. Adipose tissue (AT) may represent an important site of inflammation. 3T3-L<em>1</em> studies have demonstrated that lipopolysaccharide (LPS) activates toll-like receptors (TLRs) to cause inflammation. For this study, we <em>1</em>) examined activation of TLRs and adipocytokines by LPS in human abdominal subcutaneous (AbdSc) adipocytes, 2) examined blockade of NF-kappaB in human AbdSc adipocytes, 3) examined the innate immune pathway in AbdSc AT from lean, obese, and T2DM subjects, and 4) examined the association of circulating LPS in T2DM subjects. The findings showed that LPS increased TLR-2 protein expression twofold (P<0.05). Treatment of AbdSc adipocytes with LPS caused a significant increase in TNF-alpha and IL-6 secretion (IL-6, CONTROL: 2.7+/-0.5 vs. LPS: 4.8+/-0.3 ng/<em>ml</em>; P<0.00<em>1</em>; TNF-alpha,

METHODS

<em>1</em>.0+/-0.83 vs. LPS: 32.8+/-6.23 pg/<em>ml</em>; P<0.00<em>1</em>). NF-kappaB inhibitor reduced IL-6 in AbdSc adipocytes (

METHODS

2.7+/-0.5 vs. NF-kappaB inhibitor: 2.<em>1</em>+/-0.4 ng/<em>ml</em>; P<0.00<em>1</em>). AbdSc AT protein expression for TLR-2, MyD88, TRAF6, and NF-kappaB was increased in T2DM patients (P<0.05), and TLR-2, TRAF-6, and NF-kappaB were increased in LPS-treated adipocytes (P<0.05). Circulating LPS was 76% higher in T2DM subjects compared with matched controls. LPS correlated with insulin in controls (r=0.678, P<0.000<em>1</em>). Rosiglitazone (RSG) significantly reduced both fasting serum insulin levels (reduced by 5<em>1</em>%, P=0.0395) and serum LPS (reduced by 35%, P=0.0<em>1</em>39) in a subgroup of previously untreated T2DM patients. In summary, our results suggest that T2DM is associated with increased endotoxemia, with AT able to initiate an innate immune response. Thus, increased adiposity may increase proinflammatory cytokines and therefore contribute to the pathogenic risk of T2DM.

The definition and classification of chronic kidney disease (CKD) have evolved over time, but current international guidelines define this condition as decreased kidney function shown by glomerular filtration rate (GFR) of less than 60 <em>mL</em>/min per <em>1</em>·73 m2, or markers of kidney damage, or both, of at least 3 months duration, regardless of the underlying cause. Diabetes and hypertension are the main causes of CKD in all high-income and middle-income countries, and also in many low-income countries. Incidence, prevalence, and progression of CKD also vary within countries by ethnicity and social determinants of health, possibly through epigenetic influence. Many people are asymptomatic or have non-specific symptoms such as lethargy, itch, or loss of appetite. Diagnosis is commonly made after chance findings from screening tests (urinary dipstick or blood tests), or when symptoms become severe. The best available indicator of overall kidney function is GFR, which is measured either via exogenous markers (eg, DTPA, iohexol), or estimated using equations. Presence of proteinuria is associated with increased risk of progression of CKD and death. Kidney biopsy samples can show definitive evidence of CKD, through common changes such as glomerular sclerosis, tubular atrophy, and interstitial fibrosis. Complications include anaemia due to reduced production of erythropoietin by the kidney; reduced red blood cell survival and iron deficiency; and mineral bone disease caused by disturbed vitamin D, calcium, and phosphate metabolism. People with CKD are five to ten times more likely to die prematurely than they are to progress to end stage kidney disease. This increased risk of death rises exponentially as kidney function worsens and is largely attributable to death from cardiovascular disease, although cancer incidence and mortality are also increased. Health-related quality of life is substantially lower for people with CKD than for the general population, and falls as GFR declines. Interventions targeting specific symptoms, or aimed at supporting educational or lifestyle considerations, make a positive difference to people living with CKD. Inequity in access to services for this disease disproportionally affects disadvantaged populations, and health service provision to incentivise early intervention over provision of care only for advanced CKD is still evolving in many countries.

BACKGROUND

Recruitment of circulating leukocytes at sites of atherosclerosis is mediated through a family of adhesion molecules. The function of circulating forms of these adhesion molecules remains unknown, but their levels may serve as molecular markers of subclinical coronary heart disease (CHD).

RESULTS

To determine the ability of circulating vascular cell adhesion molecule-<em>1</em> (VCAM-<em>1</em>), endothelial-leukocyte adhesion molecule-<em>1</em> (E-selectin), and intercellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>) to serve as molecular markers of atherosclerosis and predictors of incident CHD, we studied 204 patients with incident CHD, 272 patients with carotid artery atherosclerosis (CAA), and 3<em>1</em>6 control subjects from the large, biracial Atherosclerosis Risk In Communities (ARIC) study. Levels of VCAM-<em>1</em> were not significantly different among the patients with incident CHD, those with CAA, and control subjects. Higher levels of E-selectin and ICAM-<em>1</em> were observed for the patients with CHD (means [ng/<em>mL</em>]: E-selectin, 38.4; ICAM-<em>1</em>, 288.7) and those with CAA (E-selectin, 4<em>1</em>.5; ICAM-<em>1</em>, 283.6) compared with the control subjects (E-selectin, 32.8; ICAM-<em>1</em>, 244.2), but the distributions were not notably different between the patients with CHD and CAA. Results of logistic regression analyses indicated that the relationship of ICAM-<em>1</em> and E-selectin with CHD and CAA was independent of other known CHD risk factors and was most pronounced in the highest quartile. The odds of CHD and CAA were 5.53 (95% CI, 2.5<em>1</em>-<em>1</em>2.2<em>1</em>) and 2.64 (95% CI, <em>1</em>.40-5.0<em>1</em>), respectively, for those with levels of ICAM-<em>1</em> in the highest quartile compared with those in the lowest quartile. Odds of CAA were 2.03 (95% CI, <em>1</em>.<em>1</em>4-3.62) for those with levels of E-selectin in the highest quartile compared with those in the lowest quartile.

CONCLUSIONS

These data indicate that plasma levels of ICAM-<em>1</em> and E-selectin may serve as molecular markers for atherosclerosis and the development of CHD.

During their lifespan, immature cells normally pass through sequential transitions to a differentiated state and eventually undergo cell death. This progression is aberrant in cancer, although the transition to differentiation can be reestablished in inducible leukemia cell lines. This report describes a gene, MCL<em>1</em>, that we isolated from the <em>ML</em>-<em>1</em> human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Our results demonstrate that expression of MCL<em>1</em> increases early in the induction, or "programming," of differentiation in <em>ML</em>-<em>1</em> (at <em>1</em>-3 hr), before the appearance of differentiation markers and mature morphology (at <em>1</em>-3 days). They further show that MCL<em>1</em> has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(<em>1</em>4;<em>1</em>8) chromosome translocation. MCL<em>1</em> and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the association of MCL<em>1</em> with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-associated genes, expression of MCL<em>1</em> and BCL2 relates to the programming of differentiation and cell viability/death. The discovery of MCL<em>1</em> broadens our perspective on an emerging MCL<em>1</em>/BCL2 gene family and will allow further comparison with oncogene families.