A real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to reach a sensitivity of 0.0<em>1</em>25 parasites/<em>ml</em> of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the Leishmania DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different L. infantum and Leishmania donovani strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for Leishmania quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of <em>1</em>8 to 42, 0.7 to 42, and 0.<em>1</em>2 to 22.5 parasites/<em>ml</em> for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to <em>1</em>88,700 parasites/<em>ml</em>, with a median of 837 parasites/<em>ml</em>), while in bone marrow, parasite load was more than <em>1</em>00 parasites per <em>1</em>0(6) nucleated human cells. After successful therapy, parasitemia levels remain lower than <em>1</em> parasite/<em>ml</em>. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of Leishmania DNA in the blood of 2<em>1</em>% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.

OBJECTIVE

Little is known about the association of leisure-time physical activity (LTPA) and cardiorespiratory fitness with development of the metabolic syndrome, which predisposes diseases such as diabetes and atherosclerosis. We studied the associations of LTPA and cardiorespiratory fitness with development of the metabolic syndrome (World Health Organization [WHO] and the National Cholesterol Education Program [NCEP] definitions).

METHODS

LTPA over the previous <em>1</em>2 months, VO(2max) (<em>ml</em>. kg(-<em>1</em>). min(-<em>1</em>)), and cardiovascular and metabolic risk factors were assessed in a population-based cohort of 6<em>1</em>2 middle-aged men without the metabolic syndrome.

RESULTS

At the 4-year follow-up, <em>1</em>07 men had metabolic syndrome (WHO definition). Men engaging in >3 h/week of moderate or vigorous LTPA were half as likely as sedentary men to have the metabolic syndrome after adjustment for major confounders (age, BMI, smoking, alcohol, and socioeconomic status) or potentially mediating factors (insulin, glucose, lipids, and blood pressure), especially in high-risk men. Vigorous LTPA had an even stronger inverse association, particularly in unfit men. Men in the upper third of VO(2max) were 75% less likely than unfit men to develop the metabolic syndrome, even after adjustment for major confounders. Adjustment for possible mediating factors attenuated the association. Associations of LTPA and VO(2max) with development of the metabolic syndrome, as defined by the NCEP, were qualitatively similar.

CONCLUSIONS

In particular, high-risk men engaging in currently recommended levels of physical activity were less likely to develop the metabolic syndrome than sedentary men. Cardiorespiratory fitness was also strongly protective, although possibly not independent of mediating factors.

BACKGROUND

In accordance with WHO guidelines, people with HIV infection in Botswana receive daily isoniazid preventive therapy against tuberculosis without obtaining a tuberculin skin test, but duration of prophylaxis is restricted to 6 months. We aimed to assess effectiveness of extended isoniazid therapy.

METHODS

In our randomised, double-blind, placebo-controlled trial we enrolled adults infected with HIV aged <em>1</em>8 years or older at government HIV-care clinics in Botswana. Exclusion criteria included current illness such as cough and an abnormal chest radiograph without antecedent tuberculosis or pneumonia. Eligible individuals were randomly allocated (<em>1</em>:<em>1</em>) to receive 6 months' open-label isoniazid followed by 30 months' masked placebo (control group) or 6 months' open-label isoniazid followed by 30 months' masked isoniazid (continued isoniazid group) on the basis of a computer-generated randomisation list with permuted blocks of ten at each clinic. Antiretroviral therapy was provided if participants had CD4-positive lymphocyte counts of fewer than 200 cells per <em>μL</em>. We used Cox regression analysis and the log-rank test to compare incident tuberculosis in the groups. Cox regression models were used to estimate the effect of antiretroviral therapy. The trial is registered at ClinicalTrials.gov, number NCT00<em>1</em>6428<em>1</em>.

RESULTS

Between Nov 26, 2004, and July 3, 2009, we recorded 34 (3·4%) cases of incident tuberculosis in 989 participants allocated to the control group and 20 (2·0%) in <em>1</em>006 allocated to the continued isoniazid group (incidence <em>1</em>·26% per year vs 0·72%; hazard ratio 0·57, 95% CI 0·33-0·99, p=0·047). Tuberculosis incidence in those individuals receiving placebo escalated approximately 200 days after completion of open-label isoniazid. Participants who were tuberculin skin test positive (ie, ≥5 mm induration) at enrolment received a substantial benefit from continued isoniazid treatment (0·26, 0·09-0·80, p=0·02), whereas participants who were tuberculin skin test-negative received no significant benefit (0·75, 0·38-<em>1</em>·46, p=0·40). By study completion, 946 (47%) of <em>1</em>995 participants had initiated antiretroviral therapy. Tuberculosis incidence was reduced by 50% in those receiving 360 days of antiretroviral therapy compared with participants receiving no antiretroviral therapy (adjusted hazard ratio 0·50, 95% CI 0·26-0·97). Severe adverse events and death were much the same in the control and continued isoniazid groups.

CONCLUSIONS

In a tuberculosis-endemic setting, 36 months' isoniazid prophylaxis was more effective for prevention of tuberculosis than was 6-month prophylaxis in individuals with HIV infection, and chiefly benefited those who were tuberculin skin test positive.

BACKGROUND

US Centers for Disease Control and Prevention and US Agency for International Development.

OBJECTIVE

To report 5-year follow-up results of a randomised clinical trial comparing holmium laser enucleation of the prostate (HoLEP) with open prostatectomy (OP).

METHODS

One hundred twenty patients with prostates greater than <em>1</em>00g in weight according to transrectal ultrasound were randomised to either the HoLEP or the OP group (ie, 60 patients to each group). Preoperative and postoperative assessments included American Urological Association Symptom Score (AUA-SS), maximum urinary flow rates (Qmax), and postvoid residual urine (PVRU) volumes. Measurements were performed at <em>1</em>, 3, 6, <em>1</em>2, <em>1</em>8, 24, 36, 48, and 60 mo. Postoperative outcome data were compared. All complications were recorded.

RESULTS

Five years postoperatively, a total of 46 patients (38.3%) were lost to follow-up or had to be excluded from the study. All the remaining 74 patients (42 HoLEP vs. 32 OP patients, p=0.<em>1</em><em>1</em>) had undergone the 5-yr follow-up assessments. Mean AUA-SS was 3.0 in both groups (p=0.98), mean Qmax was 24.4 ml/s in both groups (p=0.97) and PVRU volume was <em>1</em><em>1</em> ml in the HoLEP and 5 ml in the OP group (p=0.25). Late complications consisted of urethral strictures and bladder-neck contractures; reoperation rates were 5% in the HoLEP and 6.7% in the OP group (p=<em>1</em>.0). No patient developed benign prostatic hyperplasia recurrence.

CONCLUSIONS

Five years after the operation, the improvements in micturition obtained with HoLEP and OP were equally good, and reoperation rates similarly low. HoLEP seems to be a true endourological alternative to OP.

BACKGROUND

Fluorouracil (FU) is an antimetabolite with activity against numerous types of neoplasms, including those of the breast, esophagus, larynx, and gastrointestinal and genitourinary tracts. Systemic toxicity, including neutropenia, stomatitis, and diarrhea, often occur due to cytotoxic nonselectivity. Capecitabine was developed as a prodrug of FU, with the goal of improving tolerability and intratumor drug concentrations through tumor-specific conversion to the active drug.

OBJECTIVE

The purpose of this article is to review the available information on capecitabine with respect to clinical pharmacology, mechanism of action, pharmacokinetic and pharmacodynamic properties, clinical efficacy for breast and colorectal cancer adverse-effect profile, documented drug interactions, dosage and administration, and future directions of ongoing research.

METHODS

Relevant English-language literature was identified through searches of PubMed (1966 to August 2004), International Pharmaceutical Abstracts (1977 to August 2004), and the Proceedings of the American Society of Clinical Oncology (January 1995 to August 2004). Search terms included capecitabine, Xeloda, breast cancer, and colorectal cancer. The references of the identified articles were reviewed for additional sources. In addition, product information was obtained from Roche Pharmaceuticals. Studies from the identified literature that addressed this article's objectives were selected for review, with preference given to Phase II/III trials.

RESULTS

Capecitabine is an oral prodrug that is converted to its only active metabolite, FU, by thymidine phosphorylase. Higher levels of this enzyme are found in several tumors and the liver, compared with normal healthy tissue. In adults, capecitabine has a bioavailability of approximately 100% with a Cmax of 3.9 mg/L, Tmax of 1.5 to 2 hr, and AUC of 5.96 mg.h/L. The predominant route of elimination is renal, and dosage reduction of 75% is recommended in patients with creatinine clearance (CrCl) of 30 to 50 mL/min. The drug is contraindicated if CrCl is < 30 mL/min. Capecitabine has shown varying degrees of efficacy with acceptable tolerability in numerous cancers including prostate, renal cell, ovarian, and pancreatic, with the largest amount of evidence in metastatic breast and colorectal cancer. Single-agent capecitabine was compared with IV FU/leucovorin (LV) using the bolus Mayo Clinic regimen in 2 Phase III trials as first-line treatment for patients with metastatic colorectal cancer. Overall response rate (RR) favored the capecitabine arm (26% vs 17%, P < 0.001); however, this did not translate into a difference in time to progression (TTP) (4.6 months vs 4.7 months) or overall survival (OS) (12.9 months vs 12.8 months). In Phase II noncomparative trials, combinations of capecitabine with oxaliplatin or irinotecan have produced results similar to regimens combining FU/LV with the same agents in patients with colorectal cancer. In metastatic breast cancer patients who had received prior treatment with an anthracycline-based regimen, a Phase III trial comparing the combination of capecitabine with docetaxel versus docetaxel alone demonstrated superior objective tumor RR (42% vs 30%, P = 0.006), median TTP (6.1 months vs 4.2 months, P < 0.001), and median OS (14.5 months vs 11.5 months, P = 0.013) with the combination treatment. Noncomparative Phase II studies have also supported efficacy in patients with metastatic breast cancer pretreated with both anthracyclines and taxanes, yielding an overall RR of 15% to 29% and median OS of 9.4 to 15.2 months. The most common dose-limiting adverse effects associated with capecitabine monotherapy are hyperbilirubinemia, diarrhea, and hand-foot syndrome. Myelosuppression, fatigue and weakness, abdominal pain, and nausea have also been reported. Compared with bolus FU/LV, capecitabine was associated with more hand-foot syndrome but less stomatitis, alopecia, neutropenia requiring medical management, diarrhea, and nausea. Capecitabine has been reported to increase serum phenytoin levels and the international normalized ratio in patients receiving concomitant phenytoin and warfarin, respectively. The dose of capecitabine approved by the US Food and Drug Administration (FDA) for both metastatic colorectal and breast cancer is 1250 Mg/M2 given orally twice per day, usually separated by 12 hours for the first 2 weeks of every 3-week cycle.

CONCLUSIONS

Capecitabine is currently approved by the FDA for use as first-line therapy in patients with metastatic colorectal cancer when single-agent fluoropyrimidine therapy is preferred. The drug is also approved for use as (1) a single agent in metastatic breast cancer patients who are resistant to both anthracycline- and paclitaxel-based regimens or in whom further anthracycline treatment is contra indicated and (2) in combination with docetaxel after failure of prior anthracycline-based chemotherapy. Single-agent and combination regimens have also shown benefits in patients with prostate, pancreatic, renal cell, and ovarian cancers. Improved tolerability and comparable efficacy compared with IV FU/LV in addition to oral administration make capecitabine an attractive option for the treatment of several types of cancers as well as the focus of future trials.

BACKGROUND

Vascular cell adhesion molecule (VCAM)-<em>1</em>, intercellular adhesion molecule (ICAM)-<em>1</em>, and E-selectin mediate adhesion and transmigration of leukocytes to the vascular endothelial wall and may promote plaque growth and instability. In a prospective study, we evaluated the effect of soluble adhesion molecules on the risk of future cardiovascular events among patients with angiographically documented coronary artery disease (CAD). Methods and Results- -We obtained baseline samples from a prospective cohort of <em>1</em>246 patients with CAD. Besides various markers of inflammation, soluble VCAM-<em>1</em> (sVCAM-<em>1</em>), sICAM-<em>1</em>, and sE-selectin were determined. Follow-up information on cardiovascular events was obtained (mean, 2.7; maximum, 4.<em>1</em> years). Independently higher levels of sVCAM-<em>1</em> (<em>1</em>932 versus <em>1</em><em>1</em>28 ng/<em>mL</em>; P<0.000<em>1</em>), sICAM-<em>1</em> (353 versus 287 ng/<em>mL</em>; P=0.0<em>1</em>5), and sE-selectin (8<em>1</em> versus 63 ng/<em>mL</em>; P=0.003) were observed in patients with future death from cardiovascular causes. In a multivariate model, fatal risk was 2.<em>1</em>-fold (<em>1</em>.<em>1</em> to 4.0) higher in patients within the top quartile of baseline sVCAM-<em>1</em> concentrations compared with lower quartiles. This association was present independent of general inflammatory response as reflected by low or high C-reactive protein (hs-CRP) levels. In a model that simultaneously controlled for all inflammatory and soluble adhesion markers determined, only sVCAM-<em>1</em> remained independently significant for future fatal cardiovascular events, with a 2.8-fold increase in risk (P=0.003).

CONCLUSIONS

Soluble adhesion molecules sVCAM-<em>1</em>, sICAM-<em>1</em>, and sE-selectin were significantly related to future death from cardiovascular causes among patients with documented CAD. Especially sVCAM-<em>1</em> added to the predictive value of classic risk factors and hs-CRP in determining the risk of future cardiovascular death.

BACKGROUND

There are several reports that engrafted mesenchymal stem cells (MSCs) stimulate angiogenesis in the ischemic heart, but the mechanism remains controversial. We hypothesize that transplantation of MSCs enhances vascular regeneration through a paracrine action.

METHODS

A transmural myocardial infarction was created by ligation of the left anterior descending coronary artery in rats. Those with an ejection fraction less than 0.70 <em>1</em> week after myocardial infarction were included. Autologous MSCs (<em>1</em> x <em>1</em>0(7); 0.2 <em>mL</em>) or culture medium (0.2 <em>mL</em>) was injected intramyocardially into the periinfarct zone (50 microL/injection at four sites; n = 20/group). At 2 weeks after transplantation, Western blot analysis was used to assay the paracrine factors and proapoptotic proteins. Echocardiography to assess heart function was performed on additional groups at 8 weeks after implantation.

RESULTS

The angiogenic factors basic fibroblast growth factor, vascular endothelial growth factor, and stem cell homing factor (stromal cell-derived factor -<em>1</em>alpha) increased in the MSC-treated hearts compared with medium-treated hearts. This was accompanied by a downregulation of proapoptotic protein Bax in ischemic myocardium. Similarly, capillary density increased about 40% in MSC-treated hearts compared with medium-treated hearts (p = 0.00<em>1</em>). Left ventricular contractility, indicated by fractional shortening, improved in MSC-treated hearts at 2 months after implantation (MSCs: 48.6% +/- <em>1</em>9.9%; medium: <em>1</em>8.7% +/- 6.4%; p = 0.004).

CONCLUSIONS

Autologous MSC transplantation attenuates left ventricular remodeling and improves cardiac performance. The major mechanism appears to be paracrine action of the engrafted cells, increasing angiogenesis and cytoprotection.

We sought to examine mechanisms underlying nitroglycerin (NTG) tolerance and "cross-tolerance" to other nitrovasodilators. Rabbits were treated for 3 d with NTG patches (0.4 mg/h) and their aortic segments studied in organ chambers. Relaxations were examined after preconstriction with phenylephrine. In NTG tolerant rabbit aorta, relaxations to cGMP-dependent vasodilators such as NTG (45 +/- 6%), SIN-<em>1</em> (69 +/- 7%), and acetylcholine (ACh, 64 +/- 5%) were attenuated vs. controls, (90 +/- 2, 94 +/- 3, and 89 +/- 2% respectively, P < 0.05 for all), while responses to the cAMP-dependent vasodilator forskolin remained unchanged. In tolerant aorta, endothelial removal markedly enhanced relaxations to NTG and SIN-<em>1</em> (82 +/- 4 and 95 +/- 3%, respectively). Other studies were performed to determine how the endothelium enhances tolerance. Vascular steady state .-O2 levels (assessed by lucigenin chemiluminescence) was increased twofold in tolerant vs. control vessels with endothelium (0.3<em>1</em> +/- 0.0<em>1</em> vs. 0.6<em>1</em> +/- 0.0<em>1</em> nmol/mg per minute). This difference was less in vessels after denudation of the endothelium. Diphenylene iodonium, an inhibitor of flavoprotein containing oxidases, and Tiron a direct .-O2 scavenger normalized .-O2 levels. In contrast, oxypurinol (<em>1</em> mM) an inhibitor of xanthine oxidase, rotenone (50 microM) an inhibitor of mitochondrial electron transport and NG-nitro-L-arginine (<em>1</em>00 microM) an inhibitor of nitric oxide synthase did not affect the chemiluminescence signals from NTG-tolerant aortas. Pretreatment of tolerant aorta with liposome-entrapped, pH sensitive superoxide dismutase (600 U/<em>ml</em>) significantly enhanced maximal relaxation in response to NTG, SIN-<em>1</em>, and ACh, and effectively reduced chemiluminescence signals. These studies show that continuous NTG treatment is associated with increased vascular .-O2-production and consequent inhibition of NO. mediated vasorelaxation produced by both exogenous and endogenous nitrovasodilators.

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-25<em>1</em>MG, U-<em>1</em>05MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (<em>1</em>-20 ng/<em>ml</em>) stimulation of glioma cells resulted in a 25-<em>1</em>25% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.<em>1</em>, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.<em>1</em> pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.

BACKGROUND

Experimental and initial clinical studies suggest that transplantation of circulating blood- (CPC) or bone marrow-derived (BMC) progenitor cells may beneficially affect postinfarction remodeling processes after acute myocardial infarction (AMI). To relate functional characteristics of the infused cells to quantitative measures of outcome at 4-month follow-up, we performed serial contrast-enhanced MRI and assessed the migratory capacity of the transplanted progenitor cells immediately before intracoronary infusion.

RESULTS

In 28 patients with reperfused AMI receiving either BMCs or CPCs into the infarct artery 4.7+/-<em>1</em>.7 days after AMI, serial contrast-enhanced MRI performed initially and after 4 months revealed a significant increase in global ejection fraction (from 44+/-<em>1</em>0% to 49+/-<em>1</em>0%; P=0.003), a decrease in end-systolic volume (from 69+/-26 to 60+/-28 <em>mL</em>; P=0.003), and unchanged end-diastolic volumes (<em>1</em>22+/-34 versus <em>1</em><em>1</em>7+/-37 <em>mL</em>; P=NS). Infarct size, measured as late enhancement (LE) volume, decreased significantly, from 46+/-32 to 37+/-28 <em>mL</em> (P<0.05). There was a significant correlation between the reduction in LE volume and global ejection fraction improvement. The migratory capacity of transplanted cells as assessed ex vivo toward a gradient of vascular endothelial growth factor for CPCs and stromal cell derived factor-<em>1</em> for BMCs was closely correlated with the reduction of LE volume. By multivariate analysis, migratory capacity remained the most important independent predictor of infarct remodeling.

CONCLUSIONS

Analysis of serial contrast-enhanced MRI suggests that intracoronary infusion of adult progenitor cells in patients with AMI beneficially affects postinfarction remodeling processes. The migratory capacity of the infused cells is a major determinant of infarct remodeling, disclosing a causal effect of progenitor cell therapy on regeneration enhancement.

Chronic elevations in circulating angiotensin II (AngII) levels produce sustained hypertension and increased intrarenal AngII contents through multiple mechanisms, which may include sustained or increased local production of AngII. This study was designed to test the hypothesis that chronic AngII infusion increases renal angiotensinogen mRNA and protein levels, thus contributing to the increase in intrarenal AngII levels. AngII (80 ng/min) was infused subcutaneously for <em>1</em>3 d into Sprague-Dawley rats, using osmotic minipumps. Control rats underwent sham operations. By day <em>1</em>2, systolic arterial BP increased to <em>1</em>84 +/- 3 mmHg in AngII-treated rats, whereas values for sham-treated rats remained at control levels (<em>1</em>25 +/- <em>1</em> mmHg). Plasma renin activity was markedly suppressed (0.2 +/- 0.<em>1</em> versus 5.3 +/- <em>1</em>.2 ng AngI/<em>ml</em> per h); however, renal AngII contents were significantly increased in AngII-treated rats (273 +/- 29 versus 99 +/- <em>1</em>8 fmol/g). Western blot analyses of plasma and liver protein using a polyclonal anti-angiotensinogen antibody demonstrated two specific immunoreactive bands, at 52 and 64 kD, whereas kidney tissue exhibited one band, at 52 kD. Densitometric analyses demonstrated that AngII infusion did not alter plasma (52- or 64-kD), renal (52-kD), or hepatic (52-kD) angiotensinogen protein levels; however, there was a significant increase in hepatic expression of the highly glycosylated 64-kD angiotensinogen protein, of almost fourfold (densitometric value/control value ratios of 3.79 +/- <em>1</em>.<em>1</em>6 versus <em>1</em>.00 +/- 0.35). Renal and hepatic expression of angiotensinogen mRNA, which was examined by semiquantitative reverse transcription-PCR, was significantly increased in AngII-treated rats, compared with shamtreated rats (kidney, densitometric value/glyceraldehyde-3-phosphate dehydrogenase mRNA value ratios of 0.82 +/- 0.<em>1</em><em>1</em> versus 0.58 +/- 0.04; liver, densitometric value/glyceraldehyde-3-phosphate dehydrogenase mRNA value ratios of 2.34 +/- 0.07 versus <em>1</em>.32 +/- 0.<em>1</em>5). These results indicate that increases in circulating AngII levels increase intrarenal angiotensinogen mRNA levels, which may contribute to the sustained renal AngII-generating capacity that paradoxically occurs in AngII-treated hypertensive rats.

Rapidly growing primary cultures of normal human mammary epithelial cells (HMEC) were exposed to <em>1</em> microgram of benzo[a]pyrene (B[a]P) per <em>ml</em> for two or three 24-hr periods. The B[a]P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells. Widespread heterogeneity in morphology and growth patterns was evidenced in these "extended life" (EL) cultures, with multiple sequential changes in these parameters occurring during the course of their life in culture. Two apparently immortal continuous cell lines have thus far emerged from these EL cultures. These lines have been characterized to be of human mammary epithelial origin and derived from the originally treated HMEC specimen. The continuous lines do not appear to be malignantly transformed as they do not cause tumor formation in nude mice and show little or no anchorage-independent growth. Nonetheless, they have acquired several properties characteristic of tumor-derived HMEC, which distinguish them from their normal progenitors. These cell lines, as well as the EL strains, may provide useful substrates for studies to determine what agents can induce further transforming events. Additionally, analysis of the multiple steps occurring in the El cultures, as well as in the emergence of the continuous cell lines, could potentially elucidate the processes occurring during human epithelial cell carcinogenesis and escape from senescence.

Vascular endothelial cells are among the first cells that ventricular zone neuroblasts encounter during early development. The ventricular zone cells promote angiogenesis by the invading vasculature, with the release of endothelial mitogens. Yet the feedback support of young neurons by endothelial cells (ECs) has not hitherto been explored. We therefore asked whether ECs might participate in neuronal recruitment, by providing neurotrophic support to newly generated neurons. We used the neurogenic subependymal zone (SZ) of the adult rat forebrain as a model system, because of its well-characterized and relatively homogeneous population of neuronal precursor cells. We found that explants of the adult rat SZ raised on ECs generated more neurons, which survived longer, than explants raised on astrocytes, fibroblasts, or laminin. This endothelial trophic effect was humoral, in that it was also noted in SZ explants raised in noncontiguous coculture with ECs grown on porous inserts. RT-PCR for neurotrophin family members revealed that cultures of both human brain- and umbilical cord-derived ECs produced brain-derived neurotrophic factor (BDNF) mRNA, but no detectable NGF, NT-3, or NT-4 mRNA. ELISA revealed that BDNF protein was secreted by ECs into the medium at>><em>1</em> ng/<em>ml</em>. The neurotrophic effect of ECs could be replaced by added BDNF, and was blocked by addition of 5 microg/<em>ml</em> trkB-Fc to endothelial-SZ cocultures. Thus, endothelial cells can act as sources of secreted BDNF, through which the capillary microvasculature may act to support neuronal recruitment and survival in the CNS.

OBJECTIVE

To evaluate the frequency of discontinuation of the first highly active antiretroviral regimen (HAART) and the factors predictive of discontinuing for toxicity and failure in a population-based cohort of HIV-positive individuals in Italy, naïve from antiretrovirals at enrolment.

METHODS

The study population consisted of individuals who initiated HAART and had at least one follow-up visit. The primary end-points were discontinuation of any component of HAART for drug toxicity and discontinuation for failure. Survival analyses were performed to identify predictive factors for reaching the two end-points.

RESULTS

Eight hundred and sixty-two individuals initiated HAART; in 727 of them (84.3%) this consisted of two nucleoside reverse transcriptase inhibitors (NRTI) and one protease inhibitor (PI). Over a median follow-up of 45 weeks, 3<em>1</em>2 patients (36.2%) discontinued therapy: <em>1</em>82 (2<em>1</em>.<em>1</em>%) discontinued due to toxicity, 44 (5.<em>1</em>%) due to failure. The probability of discontinuing HAART at <em>1</em> year was 25.5% [95% confidence interval (CI), 2<em>1</em>.9-28.9] due to toxicity and 7.6% (95% CI, 4.9-<em>1</em> 0.3) due to failure. Independent factors associated with discontinuation for toxicity were: gender [relative hazard (RH) = 0.5<em>1</em>; 95% CI, 0.32-0.80 for men versus women], type of treatment (indinavir-containing regimens, RH = <em>1</em>.94; 95% CI, <em>1</em>.<em>1</em>0-3.4<em>1</em> and ritonavir-containing regimens, RH = 3.83; 95% CI, 2.09-7.03 versus hard-gell saquinavir) and time spent on treatment (RH = 0.89; 95% CI, 0.80-0.98 for each additional month). Discontinuation due to failure was independently associated with the most recent HIV-RNA (RH = 3.20; 95% CI, <em>1</em>.74-5.88 for log<em>1</em>0 copies/<em>ml</em> higher), and with type of treatment (indinavir-containing regimens, RH = 0.2<em>1</em>; 95% CI, 0.06-0.78 and ritonavir-containing regimens, RH = 0.23; 95% CI, 0.04-<em>1</em>.26 versus hard-gell saquinavir).

CONCLUSIONS

If the current HAART regimen caused no toxicity, less than <em>1</em>0% of naïve patients discontinue their first HAART regimen because of failure after <em>1</em> year from starting therapy.

BACKGROUND

The safety and immunogenicity of the MRK adenovirus type 5 human immunodeficiency virus type <em>1</em> clade B gag/pol/nef vaccine, a replication-incompetent adenovirus type 5-vectored vaccine designed to elicit cell-mediated immunity against conserved human immunodeficiency virus proteins, was assessed in a phase <em>1</em> trial.

METHODS

Healthy adults not infected with human immunodeficiency virus were enrolled in a multicenter, dose-escalating, blind, placebo-controlled study to evaluate a 3-dose homologous prime-boost regimen of the trivalent MRK adenovirus type 5 human immunodeficiency virus type <em>1</em> vaccine containing from 3 x <em>1</em>0(6) to <em>1</em> x <em>1</em>0(<em>1</em><em>1</em>) viral particles per <em>1</em>-mL dose administered on day <em>1</em>, during week 4 and during week 26. Adverse events were recorded for 29 days after each intradeltoid injection. The primary immunogenicity end point was the proportion of study participants with a positive unfractionated Gag-, Pol-, or Nef-specific interferon-gamma enzyme-linked immunosorbent spot response measured 4 weeks after administration of the last dose.

RESULTS

Of 259 randomized individuals, 257 (99%) received>> or = <em>1</em> dose of vaccine or placebo and were included in the safety analyses. Enzyme-linked immunosorbent spot results were available for 2<em>1</em>7 study participants (84%) at week 30. No serious vaccine-related adverse events occurred. No study participant discontinued participation because of vaccine-related adverse events. The frequency of injection-site reactions was dose dependent. Vaccine doses of>> or = 3 x <em>1</em>0(9) viral particles elicited positive enzyme-linked immunosorbent spot responses to>> or = <em>1</em> vaccine component in>> 60% of recipients. High baseline antibody titers against adenovirus type 5 diminished enzyme-linked immunosorbent spot responses at all doses except the 3 x <em>1</em>0(<em>1</em>0) viral particle dose.

CONCLUSIONS

The vaccine was generally well tolerated and induced cell-mediated immune responses against human immunodeficiency virus type <em>1</em> peptides in most healthy adults. Despite these findings, vaccination in a proof-of-concept trial with use of this vaccine was discontinued because of lack of efficacy.

BACKGROUND

After acute myocardial infarction (AMI), diastolic function assessed by Doppler echocardiography provides important prognostic information that is incremental to systolic function. However, Doppler variables are affected by multiple factors and may change rapidly. In contrast, left atrial (LA) volume is less influenced by acute changes and reflects subacute or chronic diastolic function. This may be of importance when one assesses risk in patients with AMI.

RESULTS

Three hundred fourteen patients with AMI who had a transthoracic echocardiogram with assessment of left ventricular (LV) systolic and diastolic function and measurement of LA volume during admission were identified. The LA volume was corrected for body surface area, and the population was divided according to LA volume index of 32 <em>mL</em>/m2 (2 SDs above normal). LA volume index was >32 <em>mL</em>/m2 in <em>1</em>42 (45%). The primary study end point was all-cause mortality. During follow-up of <em>1</em>5 (range 0 to 33) months, 46 patients (<em>1</em>5%) died. LA volume index was a powerful predictor of mortality and remained an independent predictor (hazard ratio <em>1</em>.05 per <em>1</em>-<em>mL</em>/m2 change, 95% CI <em>1</em>.03 to <em>1</em>.06, P<0.00<em>1</em>) after adjustment for clinical factors, LV systolic function, and Doppler-derived parameters of diastolic function.

CONCLUSIONS

Increased LA volume index is a powerful predictor of mortality after AMI and provides prognostic information incremental to clinical data and conventional measures of LV systolic and diastolic function.

BACKGROUND

Bacterial endotoxin is known to induce interferon gamma and interleukin 12 production, and therefore has the potential to decrease allergen sensitisation. To find out the role of early chronic endotoxin exposure in the development of allergen sensitisation and asthma, we compared concentrations of endotoxin in house dust with allergen sensitisation in infants at high risk for developing asthma.

METHODS

61 infants 9-24 months old with at least three physician-documented episodes of wheezing were studied. Concentrations of house-dust endotoxin and allergens were measured in the infants' homes. Allergen sensitisation was measured by skin-prick testing with a panel of common inhalant and food allergens. In a subset of these infants, proportions of T lymphocytes producing interferon gamma, and interleukins 4, 5, and 13 were calculated by cell-surface and intracellular cytokine staining, with flow cytometry.

RESULTS

House-dust endotoxin concentrations ranged from 104 to 10,000 endotoxin units (EU) per mL (geometric mean 912 EU/mL). Concentrations did not vary significantly over a 6-month interval. Ten infants (16%) were sensitised to at least one allergen. The homes of allergen-sensitised infants contained significantly lower concentrations of house-dust endotoxin than those of non-sensitised infants (mean 468 vs 1035 EU/mL, respectively; p=0.01). Increased house-dust endotoxin concentrations correlated with increased proportions of interferon-gamma-producing CD4 T cells (p=0.01). Such concentrations did not correlate with proportions of cells that produced interleukins 4, 5, or 13.

CONCLUSIONS

This study may provide the first direct in-vivo evidence that indoor endotoxin exposure early in life may protect against allergen sensitisation by enhancing type 1 immunity.

The enormous complexity, wide dynamic range of relative protein abundances of interest (over <em>1</em>0 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-<em>1</em> receptor antagonist protein (approximately 200 pg/<em>mL</em>), cathepsin L (approximately <em>1</em> ng/<em>mL</em>), and transforming growth factor beta <em>1</em> (approximately 2 ng/<em>mL</em>). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF<em>1</em>65 and VEGF<em>1</em>2<em>1</em> were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-<em>1</em> and FLK-<em>1</em>/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-<em>1</em>/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF<em>1</em>65 or VEGF<em>1</em>2<em>1</em> induced a time- and dose-dependent increase in IL-6 secretion (<em>1</em>4- to 27-fold at 50 ng/<em>mL</em> after 24 hours, P <.00<em>1</em>). Conversely, rhIL-6 stimulated VEGF expression and secretion in myeloma cell lines (40%-60%; P <.05) and to a variable degree (up to 5.3-fold; P <.005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6. (Blood. 2000;95:2630-2636)

We investigated regional therapy of recurrent malignant brain tumors with transferrin-CRM<em>1</em>07, a conjugate of human transferrin (Tf) and a genetic mutant of diphtheria toxin (CRM<em>1</em>07) that lacks native toxin binding. Physiological barriers to delivering proteins to tumor and surrounding infiltrated brain were circumvented with high-flow interstitial microinfusion. At least a 50% reduction in tumor volume on magnetic resonance imaging (MRI) occurred in 9 of <em>1</em>5 patients who could be evaluated (60%), including two complete responses. Peritumoral toxicity developed <em>1</em>-4 weeks after treatment in three of three patients at <em>1</em>.0 microg/<em>ml</em>, but in zero of nine patients treated at lower concentrations. No symptomatic systemic toxicity occurred. Regional perfusion with Tf-CRM<em>1</em>07 produces tumor responses without systemic toxicity in patients with malignant brain tumors refractory to conventional therapy. Direct interstitial infusion can be used successfully to distribute a large protein in the tumor and infiltrated brain surrounding the tumor.

Age, ultrasound score, menopausal status, a clinical impression score and serum CA <em>1</em>25 level were assessed to see how they could best distinguish between patients with benign (n = <em>1</em>0<em>1</em>) and malignant (n = 42) pelvic masses. Each criteria used alone provided statistically significant discrimination. The most useful individual criteria were a serum CA <em>1</em>25 level of 30 U/<em>ml</em> (sensitivity 8<em>1</em>%, specificity 75%) and an ultrasound score of 2 (sensitivity 7<em>1</em>%, specificity 83%). Three criteria could be combined in a risk of malignancy index (RMI) which is simply calculated using the product of the serum CA <em>1</em>25 level (U/<em>ml</em>), the ultrasound scan result (expressed as a score of 0, <em>1</em> or 3) and the menopausal status (<em>1</em> if premenopausal and 3 if postmenopausal). This index was statistically virtually as effective a discriminant between cancer and benign lesions as more formal methods. Using an RMI cut-off level of 200, the sensitivity was 85% and the specificity was 97%. Patients with an RMI score of greater than 200 had, on average, 42 times the background risk of cancer and those with a lower value 0.<em>1</em>5 times the background risk.

BACKGROUND

Adiponectin, a novel adipocyte-derived collagen-like protein, is the gene product of the adipose most-abundant gene transcript <em>1</em> (apM<em>1</em>), which has been considered to have anti-inflammatory and anti-atherogenic effects.

OBJECTIVE

To characterize the relationship between adiponectin and leptin, the ob gene product, in normal-weight and obese women.

METHODS

In this cross-sectional study, we measured fasting plasma adiponectin by ELISA, leptin concentrations by RIA, and related parameters such as blood pressure, body mass index (BMI), body fat mass, lipids, fasting blood glucose and insulin in 353 non-diabetic adult women with a wide range of BMI values.

RESULTS

Plasma adiponectin concentrations in women with the highest tertile of BMI (at least 25.0 kg/m(2)) were decreased compared with those in the middle (22.0-25.0 kg/m(2)) or lowest (<or=22.0 kg/m(2)) tertile of BMI (means+/-s.e.m.: 6.7+/-0.3 microg/ml compared with 8.6+/-0.4 microg/ml and 9.2+/-0.3 microg/ml; both P<0.000<em>1</em>). Serum leptin concentrations in women with the highest tertile of BMI were increased compared with those in women in the middle or lowest tertile of BMI (<em>1</em>3.2+/-0.4 ng/ml compared with 8.<em>1</em>+/-0.2 ng/ml and 5.2+/-0.2 ng/ml; both P<0.000<em>1</em>). These relationships were similar after adjustment for BMI or body fat mass. Adiponectin was negatively correlated with serum leptin concentration, fasting immunoreactive insulin, calculated insulin resistance (homeostasis model assessment), BMI and body fat mass. These negative relationships became stronger after adjustment for BMI or body fat mass. In stepwise regression analyses, leptin was the significant independent variable for adiponectin, and adiponectin was also the significant independent variable for leptin before and after adjustment for BMI or body fat mass.

CONCLUSIONS

In this study, we found an inverse correlation between adiponectin and leptin in vivo.

A sensitive assay for detecting double-stranded (ds) DNA in solution is described. This assay employs a new dye, PicoGreen dsDNA quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The brightness of this reagent is due to its high quantum yield (approximately 0.5, bound to ds calf thymus DNA) and large molar extinction coefficient (approximately 70,000 cm-<em>1</em> M-<em>1</em>). The fluorescence enhancement of this dye upon binding dsDNA is>> <em>1</em>000-fold, with excitation and emission maxima near those of fluorescein. Unlike Hoechst 33258, PicoGreen reagent fluorescence intensity was the same upon binding to poly(dA).poly(dT) and poly(dG).poly(dC) homopolymers. The PicoGreen assay allowed the detection of 25 pg/<em>ml</em> dsDNA, surpassing the sensitivity achieved with Hoechst 33258 by 400-fold. The linear concentration range for DNA quantitation extended over four orders of magnitude-25 pg/<em>ml</em> to <em>1</em> microgram/<em>ml</em>-with a single dye concentration. Assay linearity was maintained even in the presence of salts, proteins, poly(ethylene glycol), urea, chloroform, ethanol, and agarose; some ionic detergents and heparin interfered. Linear DNAs yielded slightly brighter signals than supercoiled plasmids. Finally, the assay showed greater dsDNA:RNA selectivity than Hoechst 33258 in low ionic strength buffer and better dsDNA:single-stranded DNA selectivity in <em>1</em> M NaCl.

Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of <em>1</em>0(-2) to <em>1</em>0(-<em>1</em>) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from <em>1</em>0 pg/<em>ml</em> to <em>1</em>00 ng/<em>ml</em>. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT<em>1</em>, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of <em>1</em>0(-2).