Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-<em>1</em>-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-<em>1</em> infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from <em>1</em>3 of <em>1</em>3 patients receiving HAART with an average treatment time of <em>1</em>0 months and with undetectable (<500 copies HIV RNA/<em>ml</em>) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-<em>1</em> produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-<em>1</em> DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

A potent retrovirus packaging cell line named Platinum-E (Plat-E) was generated based on the 293T cell line. Plat-E is superior to existing packaging cell lines regarding efficiency, stability and safety. The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins. Conventional packaging constructs made use of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env, while our packaging constructs utilized the EF<em>1</em>alpha promoter, which is <em>1</em>00-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozak's consensus sequence upstream of the initiation codon resulting in high expression of virus structural proteins in Plat-E cells. To maintain the high titers of retroviruses under drug selection pressure, we inserted the IRES (internal ribosome entry site) sequence between the gene encoding gag-pol or env, and the gene encoding a selectable marker in the packaging constructs. Plat-E cells can stably produce retroviruses with an average titer of <em>1</em> x <em>1</em>07/<em>ml</em> for at least 4 months. In addition, as we used only the coding sequences of viral structural genes to avoid inclusion of unnecessary retrovirus sequences in the packaging constructs, the probability of generating the replication competent retroviruses (RCR) by recombination can virtually be ruled out.

OBJECTIVE

This report documents that the gastric bypass operation provides long-term control for obesity and diabetes.

BACKGROUND

Obesity and diabetes, both notoriously resistant to medical therapy, continue to be two of our most common and serious diseases.

METHODS

Over the last <em>1</em>4 years, 608 morbidly obese patients underwent gastric bypass, an operation that restricts caloric intake by (<em>1</em>) reducing the functional stomach to approximately 30 <em>mL</em>, (2) delaying gastric emptying with a c. 0.8 to <em>1</em>.0 cm gastric outlet, and (3) excluding foregut with a 40 to 60 cm Roux-en-Y gastrojejunostomy. Even though many of the patients were seriously ill, the operation was performed with a perioperative mortality and complication rate of <em>1</em>.5% and 8.5%, respectively. Seventeen of the 608 patients (< 3%) were lost to follow-up.

RESULTS

Gastric bypass provides durable weight control. Weights fell from a preoperative mean of 304.4 lb (range, <em>1</em>98 to 6<em>1</em>5 lb) to <em>1</em>92.2 lb (range, <em>1</em>04 to 466) by <em>1</em> year and were maintained at 205.4 lb (range, <em>1</em>07 to 5<em>1</em>2 lb) at 5 years, 206.5 lb (<em>1</em>30 to 388 lb) at <em>1</em>0 years, and 204.7 lb (<em>1</em>58 to 270 lb) at <em>1</em>4 years. The operation provides long-term control of non-insulin-dependent diabetes mellitus (NIDDM). In those patients with adequate follow-up, <em>1</em>2<em>1</em> of <em>1</em>46 patients (82.9%) with NIDDM and <em>1</em>50 of <em>1</em>52 patients (98.7%) with glucose impairment maintained normal levels of plasma glucose, glycosylated hemoglobin, and insulin. These antidiabetic effects appear to be due primarily to a reduction in caloric intake, suggesting that insulin resistance is a secondary protective effect rather than the initial lesion. In addition to the control of weight and NIDDM, gastric bypass also corrected or alleviated a number of other comorbidities of obesity, including hypertension, sleep apnea, cardiopulmonary failure, arthritis, and infertility. Gastric bypass is now established as an effective and safe therapy for morbid obesity and its associated morbidities. No other therapy has produced such durable and complete control of diabetes mellitus.

Antibody-secreting hybrid cells have been derived from a fusion between mouse myeloma cells and spleen cells from a mouse immunized with membrane from human tonsil lymphocyte preparations. Hybrids secreting antibodies to cell surface antigens were detected by assaying culture supernatants for antibody binding to human tonsil cells. Six different antibodies (called W6/<em>1</em>, /28, /32, /34, /45 and /46 were analyzed. These were either against antigens of wide tissue distribution (W6/32, /34, and /46) or mainly on erythrocytes (W6/<em>1</em> and W6/28). One of the anti-erythrocyte antibodies (W6/<em>1</em>) detected a polymorphic antigen, since blood group A<em>1</em> and A2 erythrocytes were labeled while B and O were not. Antibodies W6/34, /45 and /46 were all against antigens which were mapped to the short arm of chromosome <em>1</em><em>1</em> by segregation analysis of mouse-human hybrids. Immunoprecipitation studies suggest that W6/45 antigen may be a protein of <em>1</em>6,000 dalton, apparent molecular weight, while W6/34 and /46 antigens could not be detected by this technique. Antibody W6/32 is against a determinant common to most, if not all, of the 43,000 dalton molecular weight chains of HLA-A, B and C antigens. This was established by somatic cell genetic techniques and by immunoprecipitation analysis. Tonsil leucocytes bound 370,000 W6/32 antibody molecules per cell at saturation. The hybrid myelomas W6/32 and W6/34 have been cloned, and both secrete an IgG2 antibody. W6/32 cells were grown in mice, and the serum of the tumor-bearing animals contained greater than <em>1</em>0 mg/<em>ml</em> of monoclonal antibody. The experiments established the usefulness of the bybrid myeloma technique in preparing monospecific antibodies against human cell surface antigens. In particular, this study highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymorphisms.

One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU<em>1</em><em>1</em>248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU<em>1</em><em>1</em>248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU<em>1</em><em>1</em>248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU<em>1</em><em>1</em>248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU<em>1</em><em>1</em>248 selectively inhibited Flk-<em>1</em>/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-<em>1</em>00 ng/<em>ml</em>. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for <em>1</em>2 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU<em>1</em><em>1</em>248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.

OBJECTIVE

As treatment options expand for metastatic colorectal cancer (mCRC), a blood marker with a prognostic and predictive role could guide treatment. We tested the hypothesis that circulating tumor cells (CTCs) could predict clinical outcome in patients with mCRC.

METHODS

In a prospective multicenter study, CTCs were enumerated in the peripheral blood of 430 patients with mCRC at baseline and after starting first-, second-, or third-line therapy. CTCs were measured using an immunomagnetic separation technique.

RESULTS

Patients were stratified into unfavorable and favorable prognostic groups based on CTC levels of three or more or less than three CTCs/7.5 <em>mL</em>, respectively. Patients with unfavorable compared with favorable baseline CTCs had shorter median progression-free survival (PFS; 4.5 v 7.9 months; P = .0002) and overall survival (OS; 9.4 v <em>1</em>8.5 months; P < .000<em>1</em>). Differences persisted at <em>1</em> to 2, 3 to 5, 6 to <em>1</em>2, and <em>1</em>3 to 20 weeks after therapy. Conversion of baseline unfavorable CTCs to favorable at 3 to 5 weeks was associated with significantly longer PFS and OS compared with patients with unfavorable CTCs at both time points (PFS, 6.2 v <em>1</em>.6 months; P = .02; OS, <em>1</em><em>1</em>.0 v 3.7 months; P = .0002). Among nonprogressing patients, favorable compared with unfavorable CTCs within <em>1</em> month of imaging was associated with longer survival (<em>1</em>8.8 v 7.<em>1</em> months; P < .000<em>1</em>). Baseline and follow-up CTC levels remained strong predictors of PFS and OS after adjustment for clinically significant factors.

CONCLUSIONS

The number of CTCs before and during treatment is an independent predictor of PFS and OS in patients with metastatic colorectal cancer. CTCs provide prognostic information in addition to that of imaging studies.

BACKGROUND

Highly active antiretroviral therapy (HAART) is being scaled up in developing countries. We compared baseline characteristics and outcomes during the first year of HAART between HIV-<em>1</em>-infected patients in low-income and high-income settings.

METHODS

<em>1</em>8 HAART programmes in Africa, Asia, and South America (low-income settings) and <em>1</em>2 HIV cohort studies from Europe and North America (high-income settings) provided data for 48<em>1</em>0 and 22,2<em>1</em>7, respectively, treatment-naïve adult patients starting HAART. All patients from high-income settings and 2725 (57%) patients from low-income settings were actively followed-up and included in survival analyses.

RESULTS

Compared with high-income countries, patients starting HAART in low-income settings had lower CD4 cell counts (median <em>1</em>08 cells per muL vs 234 cells per muL), were more likely to be female (5<em>1</em>%vs 25%), and more likely to start treatment with a non-nucleoside reverse transcriptase inhibitor (NNRTI) (70%vs 23%). At 6 months, the median number of CD4 cells gained (<em>1</em>06 cells per muL vs <em>1</em>03 cells per muL) and the percentage of patients reaching HIV-<em>1</em> RNA levels lower than 500 copies/mL (76%vs 77%) were similar. Mortality was higher in low-income settings (<em>1</em>24 deaths during 2236 person-years of follow-up) than in high-income settings (4<em>1</em>4 deaths during 20,532 person-years). The adjusted hazard ratio (HR) of mortality comparing low-income with high-income settings fell from 4.3 (95% CI <em>1</em>.6-<em>1</em><em>1</em>.8) during the first month to <em>1</em>.5 (0.7-3.0) during months 7-<em>1</em>2. The provision of treatment free of charge in low-income settings was associated with lower mortality (adjusted HR 0.23; 95% CI 0.08-0.6<em>1</em>).

CONCLUSIONS

Patients starting HAART in resource-poor settings have increased mortality rates in the first months on therapy, compared with those in developed countries. Timely diagnosis and assessment of treatment eligibility, coupled with free provision of HAART, might reduce this excess mortality.

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H<em>1</em>N<em>1</em>) or A/PR/8/34 (H<em>1</em>N<em>1</em>) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB<em>1</em>, and PA viral polymerases. This strategy yielded>><em>1</em> x <em>1</em>0(3) plaque-forming units (pfu) of virus per <em>ml</em> of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x <em>1</em>0(4)-5 x <em>1</em>0(7) pfu/<em>ml</em>. We also used reverse genetics to generate a reassortant virus containing the PB<em>1</em> gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

BACKGROUND

Previous studies showing that tiotropium improves multiple end points in patients with chronic obstructive pulmonary disease (COPD) led us to examine the long-term effects of tiotropium therapy.

METHODS

In this randomized, double-blind trial, we compared 4 years of therapy with either tiotropium or placebo in patients with COPD who were permitted to use all respiratory medications except inhaled anticholinergic drugs. The patients were at least 40 years of age, with a forced expiratory volume in <em>1</em> second (FEV(<em>1</em>)) of 70% or less after bronchodilation and a ratio of FEV(<em>1</em>) to forced vital capacity (FVC) of 70% or less. Coprimary end points were the rate of decline in the mean FEV(<em>1</em>) before and after bronchodilation beginning on day 30. Secondary end points included measures of FVC, changes in response on St. George's Respiratory Questionnaire (SGRQ), exacerbations of COPD, and mortality.

RESULTS

Of a total of 5993 patients (mean age, 65+/-8 years) with a mean FEV(<em>1</em>) of <em>1</em>.32+/-0.44 liters after bronchodilation (48% of predicted value), we randomly assigned 2987 to the tiotropium group and 3006 to the placebo group. Mean absolute improvements in FEV(<em>1</em>) in the tiotropium group were maintained throughout the trial (ranging from 87 to <em>1</em>03 ml before bronchodilation and from 47 to 65 ml after bronchodilation), as compared with the placebo group (P<0.00<em>1</em>). After day 30, the differences between the two groups in the rate of decline in the mean FEV(<em>1</em>) before and after bronchodilation were not significant. The mean absolute total score on the SGRQ was improved (lower) in the tiotropium group, as compared with the placebo group, at each time point throughout the 4-year period (ranging from 2.3 to 3.3 units, P<0.00<em>1</em>). At 4 years and 30 days, tiotropium was associated with a reduction in the risks of exacerbations, related hospitalizations, and respiratory failure.

CONCLUSIONS

In patients with COPD, therapy with tiotropium was associated with improvements in lung function, quality of life, and exacerbations during a 4-year period but did not significantly reduce the rate of decline in FEV(<em>1</em>). (ClinicalTrials.gov number, NCT00<em>1</em>44339.)

BACKGROUND

The rate of disease progression among persons infected with human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>) varies widely, and the relative prognostic value of markers of disease activity has not been defined.

OBJECTIVE

To compare clinical, serologic, cellular, and virologic markers for their ability to predict progression to the acquired immunodeficiency syndrome (AIDS) and death during a <em>1</em>0-year period.

METHODS

Prospective, multicenter cohort study.

METHODS

Four university-based clinical centers participating in the Multicenter AIDS Cohort Study.

METHODS

<em>1</em>604 men infected with HIV-<em>1</em>.

METHODS

The markers compared were oral candidiasis (thrush) or fever; serum neopterin levels; serum beta 2-microglobulin levels; number and percentage of CD3+, CD4+, and CD8+ lymphocytes; and plasma viral load, which was measured as the concentration of HIV-<em>1</em> RNA found using a sensitive branched-DNA signal-amplification assay.

RESULTS

Plasma viral load was the single best predictor of progression to AIDS and death, followed (in order of predictive strength) by CD4+ lymphocyte count and serum neopterin levels, serum beta 2-microglobulin levels, and thrush or fever. Plasma viral load discriminated risk at all levels of CD4+ lymphocyte counts and predicted their subsequent rate of decline. Five risk categories were defined by plasma HIV-<em>1</em> RNA concentrations: 500 copies/mL or less, 50<em>1</em> to 3000 copies/mL, 300<em>1</em> to <em>1</em>0000 copies/mL, <em>1</em>000<em>1</em> to 30000 copies/mL, and more than 30000 copies/mL. Highly significant (P < 0.00<em>1</em>) differences in the percentages of participants who progressed to AIDS within 6 years were seen in the five risk categories: 5.4%, <em>1</em>6.6%, 3<em>1</em>.7%, 55.2%, and 80.0%, respectively. Highly significant (P < 0.00<em>1</em>) differences in the percentages of participants who died of AIDS within 6 years were also seen in the five risk categories: 0.9%, 6.3%, <em>1</em>8.<em>1</em>%, 34.9%, and 69.5%, respectively. A regression tree incorporating both HIV-<em>1</em> RNA measurements and CD4+ lymphocyte counts provided better discrimination of outcome than did either marker alone; use of both variables defined categories of risk for AIDS within 6 years that ranged from less than 2% to 98%.

CONCLUSIONS

Plasma viral load strongly predicts the rate of decrease in CD4+ lymphocyte count and progression to AIDS and death, but the prognosis of HIV-infected persons is more accurately defined by combined measurement of plasma HIV-<em>1</em> RNA and CD4+ lymphocytes.

Human blood mononuclear leukocytes stimulated with toxoplasma antigen, concanavalin A, mezerein plus lentil lectin, or staphylococcal enterotoxin A secreted a factor (macrophage-activating factor, or MAF) that enhanced the capacity of human macrophages to release H2O2 and to kill toxoplasmas. The same lymphoid supernatants contained IFN gamma but not IFN alpha or IFN beta. The MAF activity of six of seven unfractionated supernatants was completely eliminated by a monoclonal antibody that neutralizes IFN gamma, and MAF in the remaining supernatant was almost completely neutralized. Native IFN gamma partially purified by two independent protocols to specific activities of <em>1</em> X <em>1</em>0(6) and <em>1</em>0(7) U/mg protein was enriched in MAF activity at least as much as in antiviral activity. The capacity of macrophages to secrete H2O2 after incubation in partially purified native IFN gamma (mean peak stimulation, 8.8-fold) was greater than with unpurified lymphokines (3.8-fold) and sometimes equaled or exceeded the capacity of freshly harvested monocytes. The MAF activity of the partially purified native IFN gamma preparations was abolished by monoclonal anti-IFN gamma. Finally, IFN gamma of greater than 99% estimated purity was isolated (at Genentech, Inc.) from bacteria transformed with the cloned human gene for this lymphokine. Recombinant IFN gamma had potent MAF activity, stimulating the peroxide-releasing capacity of macrophages an average of <em>1</em>9.8-fold at peak response and enhancing their ability to kill toxoplasmas from 2.6 +/- <em>1</em>.3% for untreated cells to 54 +/- 0.4% for treated cells. Attainment of 50% of the maximal elevation in peroxide-releasing capacity required a geometric mean concentration of 0.<em>1</em> antiviral U/<em>ml</em> of recombinant IFN gamma, which is estimated to be approximately 6 picomolar for this preparation. Peroxide secretory capacity and toxoplasmacidal activity of macrophages peaked 2-4 d after exposure to IFN gamma. Peroxide-secretory capacity remained elevated during at least 6 d of continuous exposure, but the effect of IFN gamma was reversed within about 3 d of its removal. Activation was usually but not invariably accompanied by characteristic changes in cell morphology. Thus, IFN gamma activates human macrophage oxidative metabolism and antimicrobial activity, and appeared to be the only factor consistently capable of doing so in the diverse LK preparations tested.

Most humans depend on sun exposure to satisfy their requirements for vitamin D. Solar ultraviolet B photons are absorbed by 7-dehydrocholesterol in the skin, leading to its transformation to previtamin D3, which is rapidly converted to vitamin D3. Season, latitude, time of day, skin pigmentation, aging, sunscreen use, and glass all influence the cutaneous production of vitamin D3. Once formed, vitamin D3 is metabolized in the liver to 25-hydroxyvitamin D3 and then in the kidney to its biologically active form, <em>1</em>,25-dihydroxyvitamin D3. Vitamin D deficiency is an unrecognized epidemic among both children and adults in the United States. Vitamin D deficiency not only causes rickets among children but also precipitates and exacerbates osteoporosis among adults and causes the painful bone disease osteomalacia. Vitamin D deficiency has been associated with increased risks of deadly cancers, cardiovascular disease, multiple sclerosis, rheumatoid arthritis, and type <em>1</em> diabetes mellitus. Maintaining blood concentrations of 25-hydroxyvitamin D above 80 nmol/L (approximately 30 ng/<em>mL</em>) not only is important for maximizing intestinal calcium absorption but also may be important for providing the extrarenal <em>1</em>alpha-hydroxylase that is present in most tissues to produce <em>1</em>,25-dihydroxyvitamin D3. Although chronic excessive exposure to sunlight increases the risk of nonmelanoma skin cancer, the avoidance of all direct sun exposure increases the risk of vitamin D deficiency, which can have serious consequences. Monitoring serum 25-hydroxyvitamin D concentrations yearly should help reveal vitamin D deficiencies. Sensible sun exposure (usually 5-<em>1</em>0 min of exposure of the arms and legs or the hands, arms, and face, 2 or 3 times per week) and increased dietary and supplemental vitamin D intakes are reasonable approaches to guarantee vitamin D sufficiency.

ICAM-<em>1</em> is a cell surface glycoprotein originally defined by a monoclonal antibody (MAb) that inhibits phorbol ester-stimulated leukocyte aggregation. Staining of frozen sections and immunofluorescence flow cytometry showed intercellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>) is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-<em>1</em> staining on vascular endothelial cells is most intense in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-<em>1</em> is expressed in low amounts on peripheral blood leukocytes. Phorbol ester-stimulated differentiation of myelomonocytic cell lines greatly increases ICAM-<em>1</em> expression. ICAM-<em>1</em> expression on dermal fibroblasts is increased threefold to fivefold by either interleukin <em>1</em> (IL <em>1</em>) or interferon-gamma at <em>1</em>0 U/<em>ml</em> over a period of 4 or <em>1</em>0 hr, respectively. The induction is dependent on protein and mRNA synthesis and is reversible. ICAM-<em>1</em> displays Mr heterogeneity in different cell types with a Mr of 97,000 on fibroblasts, <em>1</em><em>1</em>4,000 on the myelomonocytic cell line U937, and 90,000 on the B lymphoblastoid cell JY. ICAM-<em>1</em> biosynthesis involves a Mr approximately 73,000 intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment has a Mr of 55,000. ICAM-<em>1</em> isolated from phorbol myristic acetate (PMA) stimulated U937 and from fibroblasts yields an identical major product of Mr = 60,000 after chemical deglycosylation. ICAM-<em>1</em> MAb interferes with the adhesion of phytohemagglutinin blasts, and the adhesion of the cell line SKW3 to human dermal fibroblast cell layers. Pretreatment of fibroblasts but not lymphocytes with ICAM-<em>1</em> MAb, and of lymphocytes but not fibroblasts with lymphocyte function-associated antigen <em>1</em> MAb inhibits adhesion. Intercellular adhesion is increased by prior exposure of fibroblasts to IL <em>1</em>, and correlates with induction of ICAM-<em>1</em>.

In <em>1</em>996 the American Society for Therapeutic Radiology and Oncology (ASTRO) sponsored a Consensus Conference to establish a definition of biochemical failure after external beam radiotherapy (EBRT). The ASTRO definition defined prostate specific antigen (PSA) failure as occurring after three consecutive PSA rises after a nadir with the date of failure as the point halfway between the nadir date and the first rise or any rise great enough to provoke initiation of therapy. This definition was not linked to clinical progression or survival; it performed poorly in patients undergoing hormonal therapy (HT), and backdating biased the Kaplan-Meier estimates of event-free survival. A second Consensus Conference was sponsored by ASTRO and the Radiation Therapy Oncology Group in Phoenix, Arizona, on January 2<em>1</em>, 2005, to revise the ASTRO definition. The panel recommended: (<em>1</em>) a rise by 2 ng/<em>mL</em> or more above the nadir PSA be considered the standard definition for biochemical failure after EBRT with or without HT; (2) the date of failure be determined "at call" (not backdated). They recommended that investigators be allowed to use the ASTRO Consensus Definition after EBRT alone (no hormonal therapy) with strict adherence to guidelines as to "adequate follow-up." To avoid the artifacts resulting from short follow-up, the reported date of control should be listed as 2 years short of the median follow-up. For example, if the median follow-up is 5 years, control rates at 3 years should be cited. Retaining a strict version of the ASTRO definition would allow comparisons with a large existing body of literature.

BACKGROUND

For patients with advanced hepatocellular carcinoma, sorafenib is the only approved drug worldwide, and outcomes remain poor. We aimed to assess the safety and efficacy of nivolumab, a programmed cell death protein-<em>1</em> (PD-<em>1</em>) immune checkpoint inhibitor, in patients with advanced hepatocellular carcinoma with or without chronic viral hepatitis.

METHODS

We did a phase <em>1</em>/2, open-label, non-comparative, dose escalation and expansion trial (CheckMate 040) of nivolumab in adults (≥<em>1</em>8 years) with histologically confirmed advanced hepatocellular carcinoma with or without hepatitis C or B (HCV or HBV) infection. Previous sorafenib treatment was allowed. A dose-escalation phase was conducted at seven hospitals or academic centres in four countries or territories (USA, Spain, Hong Kong, and Singapore) and a dose-expansion phase was conducted at an additional 39 sites in <em>1</em><em>1</em> countries (Canada, UK, Germany, Italy, Japan, South Korea, Taiwan). At screening, eligible patients had Child-Pugh scores of 7 or less (Child-Pugh A or B7) for the dose-escalation phase and 6 or less (Child-Pugh A) for the dose-expansion phase, and an Eastern Cooperative Oncology Group performance status of <em>1</em> or less. Patients with HBV infection had to be receiving effective antiviral therapy (viral load (<em>1</em>00 IU/mL); antiviral therapy was not required for patients with HCV infection. We excluded patients previously treated with an agent targeting T-cell costimulation or checkpoint pathways. Patients received intravenous nivolumab 0·<em>1</em>-<em>1</em>0 mg/kg every 2 weeks in the dose-escalation phase (3+3 design). Nivolumab 3 mg/kg was given every 2 weeks in the dose-expansion phase to patients in four cohorts: sorafenib untreated or intolerant without viral hepatitis, sorafenib progressor without viral hepatitis, HCV infected, and HBV infected. Primary endpoints were safety and tolerability for the escalation phase and objective response rate (Response Evaluation Criteria In Solid Tumors version <em>1</em>.<em>1</em>) for the expansion phase. This study is registered with ClinicalTrials.gov, number NCT0<em>1</em>658878.

RESULTS

Between Nov 26, 20<em>1</em>2, and Aug 8, 20<em>1</em>6, 262 eligible patients were treated (48 patients in the dose-escalation phase and 2<em>1</em>4 in the dose-expansion phase). 202 (77%) of 262 patients have completed treatment and follow-up is ongoing. During dose escalation, nivolumab showed a manageable safety profile, including acceptable tolerability. In this phase, 46 (96%) of 48 patients discontinued treatment, 42 (88%) due to disease progression. Incidence of treatment-related adverse events did not seem to be associated with dose and no maximum tolerated dose was reached. <em>1</em>2 (25%) of 48 patients had grade 3/4 treatment-related adverse events. Three (6%) patients had treatment-related serious adverse events (pemphigoid, adrenal insufficiency, liver disorder). 30 (63%) of 48 patients in the dose-escalation phase died (not determined to be related to nivolumab therapy). Nivolumab 3 mg/kg was chosen for dose expansion. The objective response rate was 20% (95% CI <em>1</em>5-26) in patients treated with nivolumab 3 mg/kg in the dose-expansion phase and <em>1</em>5% (95% CI 6-28) in the dose-escalation phase.

CONCLUSIONS

Nivolumab had a manageable safety profile and no new signals were observed in patients with advanced hepatocellular carcinoma. Durable objective responses show the potential of nivolumab for treatment of advanced hepatocellular carcinoma.

BACKGROUND

Bristol-Myers Squibb.

BACKGROUND

Interleukin-6 (IL-6) plays a central role in inflammation and tissue injury. However, epidemiological data evaluating the role of IL-6 in atherogenesis are sparse.

RESULTS

In a prospective study involving <em>1</em>4 9<em>1</em>6 apparently healthy men, we measured baseline plasma concentration of IL-6 in 202 participants who subsequently developed myocardial infarction (MI) and in 202 study participants matched for age and smoking status who did not report vascular disease during a 6-year follow-up. Median concentrations of IL-6 at baseline were higher among men who subsequently had an MI than among those who did not (<em>1</em>.8<em>1</em> versus <em>1</em>. 46 pg/<em>mL</em>; P=0.002). The risk of future MI increased with increasing quartiles of baseline IL-6 concentration (P for trend <0.00<em>1</em>) such that men in the highest quartile at entry had a relative risk 2.3 times higher than those in the lowest quartile (95% CI <em>1</em>.3 to 4.3, P=0.005); for each quartile increase in IL-6, there was a 38% increase in risk (P=0.00<em>1</em>).This relationship remained significant after adjustment for other cardiovascular risk factors, was stable over long periods of follow-up, and was present in all low-risk subgroups, including nonsmokers. Although the strongest correlate of IL-6 in these data was C-reactive protein (r=0.43, P<0.00<em>1</em>), the relationship of IL-6 with subsequent risk remained after control for this factor (P<0.00<em>1</em>).

CONCLUSIONS

In apparently healthy men, elevated levels of IL-6 are associated with increased risk of future MI. These data thus support a role for cytokine-mediated inflammation in the early stages of atherogenesis.

BACKGROUND

Most patients requiring mechanical ventilation for acute lung injury and the acute respiratory distress syndrome (ARDS) receive positive end-expiratory pressure (PEEP) of 5 to <em>1</em>2 cm of water. Higher PEEP levels may improve oxygenation and reduce ventilator-induced lung injury but may also cause circulatory depression and lung injury from overdistention. We conducted this trial to compare the effects of higher and lower PEEP levels on clinical outcomes in these patients.

METHODS

We randomly assigned 549 patients with acute lung injury and ARDS to receive mechanical ventilation with either lower or higher PEEP levels, which were set according to different tables of predetermined combinations of PEEP and fraction of inspired oxygen.

RESULTS

Mean (+/-SD) PEEP values on days <em>1</em> through 4 were 8.3+/-3.2 cm of water in the lower-PEEP group and <em>1</em>3.2+/-3.5 cm of water in the higher-PEEP group (P<0.00<em>1</em>). The rates of death before hospital discharge were 24.9 percent and 27.5 percent, respectively (P=0.48; 95 percent confidence interval for the difference between groups, -<em>1</em>0.0 to 4.7 percent). From day <em>1</em> to day 28, breathing was unassisted for a mean of <em>1</em>4.5+/-<em>1</em>0.4 days in the lower-PEEP group and <em>1</em>3.8+/-<em>1</em>0.6 days in the higher-PEEP group (P=0.50).

CONCLUSIONS

These results suggest that in patients with acute lung injury and ARDS who receive mechanical ventilation with a tidal-volume goal of 6 ml per kilogram of predicted body weight and an end-inspiratory plateau-pressure limit of 30 cm of water, clinical outcomes are similar whether lower or higher PEEP levels are used.

<AbstractText>Coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9) causes severe community and nosocomial outbreaks. Comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not yet available. Nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. We aimed to ascertain the serial respiratory viral load of SARS-CoV-2 in posterior oropharyngeal (deep throat) saliva samples from patients with COVID-<em>1</em>9, and serum antibody responses.</AbstractText><AbstractText>We did a cohort study at two hospitals in Hong Kong. We included patients with laboratory-confirmed COVID-<em>1</em>9. We obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. Serial viral load was ascertained by reverse transcriptase quantitative PCR (RT-qPCR). Antibody levels against the SARS-CoV-2 internal nucleoprotein (NP) and surface spike protein receptor binding domain (RBD) were measured using EIA. Whole-genome sequencing was done to identify possible mutations arising during infection.</AbstractText><p><div><b>FINDINGS</b></div>Between Jan 22, 2020, and Feb <em>1</em>2, 2020, 30 patients were screened for inclusion, of whom 23 were included (median age 62 years [range 37-75]). The median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5·2 log_<em>1</em>0 copies per <em>mL</em> (IQR 4·<em>1</em>-7·0). Salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope -0·<em>1</em>5, 95% CI -0·<em>1</em>9 to -0·<em>1</em><em>1</em>; R²=0·7<em>1</em>). In one patient, viral RNA was detected 25 days after symptom onset. Older age was correlated with higher viral load (Spearman's ρ=0·48, 95% CI 0·074-0·75; p=0·020). For <em>1</em>6 patients with serum samples available <em>1</em>4 days or longer after symptom onset, rates of seropositivity were 94% for anti-NP IgG (n=<em>1</em>5), 88% for anti-NP IgM (n=<em>1</em>4), <em>1</em>00% for anti-RBD IgG (n=<em>1</em>6), and 94% for anti-RBD IgM (n=<em>1</em>5). Anti-SARS-CoV-2-NP or anti-SARS-CoV-2-RBD IgG levels correlated with virus neutralisation titre (R²>0·9). No genome mutations were detected on serial samples.</p><AbstractText>Posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. Unlike severe acute respiratory syndrome, patients with COVID-<em>1</em>9 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. This finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. Serological assay can complement RT-qPCR for diagnosis.</AbstractText><AbstractText>Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, and Sanming Project of Medicine.</AbstractText>

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is characterised by both an accelerated decline in lung function and periods of acute deterioration in symptoms termed exacerbations. The aim of this study was to investigate whether these are related.

METHODS

Over 4 years, peak expiratory flow (PEF) and symptoms were measured at home daily by <em>1</em>09 patients with COPD (8<em>1</em> men; median (IQR) age 68.<em>1</em> (63-74) years; arterial oxygen tension (PaO(2)) 9.00 (8.3-9.5) kPa, forced expiratory volume in <em>1</em> second (FEV(<em>1</em>)) <em>1</em>.00 (0.7-<em>1</em>.3) l, forced vital capacity (FVC) 2.5<em>1</em> (<em>1</em>.9-3.0) l); of these, 32 (29 men) recorded daily FEV(<em>1</em>). Exacerbations were identified from symptoms and the effect of frequent or infrequent exacerbations >> or < 2.92 per year) on lung function decline was examined using cross sectional, random effects models.

RESULTS

The <em>1</em>09 patients experienced 757 exacerbations. Patients with frequent exacerbations had a significantly faster decline in FEV(<em>1</em>) and peak expiratory flow (PEF) of -40.<em>1</em> ml/year (n=<em>1</em>6) and -2.9 l/min/year (n=46) than infrequent exacerbators in whom FEV(<em>1</em>) changed by -32.<em>1</em> ml/year (n=<em>1</em>6) and PEF by -0.7 l/min/year (n=63). Frequent exacerbators also had a greater decline in FEV(<em>1</em>) if allowance was made for smoking status. Patients with frequent exacerbations were more often admitted to hospital with longer length of stay. Frequent exacerbations were a consistent feature within a patient, with their number positively correlated (between years <em>1</em> and 2, 2 and 3, 3 and 4).

CONCLUSIONS

These results suggest that the frequency of exacerbations contributes to long term decline in lung function of patients with moderate to severe COPD.

OBJECTIVE

We sought to reexpress the 4-variable Modification of Diet in Renal Disease (MDRD) Study equation for estimation of glomerular filtration rate (GFR) using serum creatinine (S(cr)) standardized to reference methods.

METHODS

Serum specimens included creatinine reference materials prepared by the College of American Pathologists (CAP), traceable to primary reference material at the NIST, with assigned values traceable to isotope dilution mass spectrometry (IDMS), a calibration panel prepared by the Cleveland Clinic Research Laboratory (CCRL), and frozen samples from the MDRD Study. Split specimens were measured at the CCRL using the Roche enzymatic and Beckman CX3 kinetic alkaline picrate assays.

RESULTS

Roche enzymatic assay results on CAP samples were comparable to IDMS-assigned values. Beckman CX3 assay results in 2004-2005 were significantly higher than but highly correlated with simultaneous Roche enzymatic assay results (r(2) = 0.9994 on 40 CCRL samples) and showed minimal but significant upward drift from Beckman CX3 assay results during the MDRD Study in <em>1</em>989-<em>1</em>99<em>1</em> (r(2) = 0.9987 in 253 samples). Combining these factors, standardized S(cr) = 0.95 x original MDRD Study S(cr). The reexpressed 4-variable MDRD Study equation for S(cr) (mg/dL) is GFR = <em>1</em>75 x standardized S(cr)(-<em>1</em>.<em>1</em>54) x age(-0.203) x <em>1</em>.2<em>1</em>2 (if black) x 0.742 (if female), and for S(cr) (micromol/L) is GFR = 30849 x standardized S(cr)(-<em>1</em>.<em>1</em>54) x age(-0.203) x <em>1</em>.2<em>1</em>2 (if black) x 0.742 (if female) [GFR in <em>mL</em> x min(-<em>1</em>) x (<em>1</em>.73 m(2))(-<em>1</em>)].

CONCLUSIONS

When the calibration of S(cr) methods is traceable to the S(cr) reference system, GFR should be estimated using the MDRD Study equation that has been reexpressed for standardized S(cr).

Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective intercellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hematopoietic progenitor cells (HPC) as a model. We found that ES-MV (<em>1</em>0 microg/<em>ml</em>) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-<em>1</em>) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA.

BACKGROUND

Renal sympathetic hyperactivity is associated with hypertension and its progression, chronic kidney disease, and heart failure. We did a proof-of-principle trial of therapeutic renal sympathetic denervation in patients with resistant hypertension (ie, systolic blood pressure>>/=<em>1</em>60 mm Hg on three or more antihypertensive medications, including a diuretic) to assess safety and blood-pressure reduction effectiveness.

METHODS

We enrolled 50 patients at five Australian and European centres; 5 patients were excluded for anatomical reasons (mainly on the basis of dual renal artery systems). Patients received percutaneous radiofrequency catheter-based treatment between June, 2007, and November, 2008, with subsequent follow-up to <em>1</em> year. We assessed the effectiveness of renal sympathetic denervation with renal noradrenaline spillover in a subgroup of patients. Primary endpoints were office blood pressure and safety data before and at <em>1</em>, 3, 6, 9, and <em>1</em>2 months after procedure. Renal angiography was done before, immediately after, and <em>1</em>4-30 days after procedure, and magnetic resonance angiogram 6 months after procedure. We assessed blood-pressure lowering effectiveness by repeated measures ANOVA. This study is registered in Australia and Europe with ClinicalTrials.gov, numbers NCT 00483808 and NCT 00664638.

RESULTS

In treated patients, baseline mean office blood pressure was <em>1</em>77/<em>1</em>0<em>1</em> mm Hg (SD 20/<em>1</em>5), (mean 4.7 antihypertensive medications); estimated glomerular filtration rate was 8<em>1</em> mL/min/<em>1</em>.73m(2) (SD 23); and mean reduction in renal noradrenaline spillover was 47% (95% CI 28-65%). Office blood pressures after procedure were reduced by -<em>1</em>4/-<em>1</em>0, -2<em>1</em>/-<em>1</em>0, -22/-<em>1</em><em>1</em>, -24/-<em>1</em><em>1</em>, and -27/-<em>1</em>7 mm Hg at <em>1</em>, 3, 6, 9, and <em>1</em>2 months, respectively. In the five non-treated patients, mean rise in office blood pressure was +3/-2, +2/+3, +<em>1</em>4/+9, and +26/+<em>1</em>7 mm Hg at <em>1</em>, 3, 6, and 9 months, respectively. One intraprocedural renal artery dissection occurred before radiofrequency energy delivery, without further sequelae. There were no other renovascular complications.

CONCLUSIONS

Catheter-based renal denervation causes substantial and sustained blood-pressure reduction, without serious adverse events, in patients with resistant hypertension. Prospective randomised clinical trials are needed to investigate the usefulness of this procedure in the management of this condition.

The incidence of tuberculosis has been increasing substantially on a worldwide basis over the past decade, but no tuberculosis-specific drugs have been discovered in 40 years. We identified a diarylquinoline, R2079<em>1</em>0, that potently inhibits both drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro (minimum inhibitory concentration 0.06 mug/<em>ml</em>). In mice, R2079<em>1</em>0 exceeded the bactericidal activities of isoniazid and rifampin by at least <em>1</em> log unit. Substitution of drugs included in the World Health Organization's first-line tuberculosis treatment regimen (rifampin, isoniazid, and pyrazinamide) with R2079<em>1</em>0 accelerated bactericidal activity, leading to complete culture conversion after 2 months of treatment in some combinations. A single dose of R2079<em>1</em>0 inhibited mycobacterial growth for <em>1</em> week. Plasma levels associated with efficacy in mice were well tolerated in healthy human volunteers. Mutants selected in vitro suggest that the drug targets the proton pump of adenosine triphosphate (ATP) synthase.

BACKGROUND

Among the many adipocyte-derived endocrine factors, we recently found an adipocyte-specific secretory protein, adiponectin, which was decreased in obesity. Although obesity is associated with increased cardiovascular mortality and morbidity, the molecular basis for the link between obesity and vascular disease has not been fully clarified. The present study investigated whether adiponectin could modulate endothelial function and relate to coronary disease.

RESULTS

For the in vitro study, human aortic endothelial cells (HAECs) were preincubated for <em>1</em>8 hours with the indicated amount of adiponectin, then exposed to tumor necrosis factor-alpha (TNF-alpha) (<em>1</em>0 U/<em>mL</em>) or vehicle for the times indicated. The adhesion of human monocytic cell line THP-<em>1</em> cells to HAECs was determined by adhesion assay. The surface expression of vascular cell adhesion molecule-<em>1</em> (VCAM-<em>1</em>), endothelial-leukocyte adhesion molecule-<em>1</em> (E-selectin), and intracellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>) was measured by cell ELISA. Physiological concentrations of adiponectin dose-dependently inhibited TNF-alpha-induced THP-<em>1</em> adhesion and expression of VCAM-<em>1</em>, E-selectin, and ICAM-<em>1</em> on HAECs. For the in vivo study, the concentrations of adiponectin in human plasma were determined by a sandwich ELISA system that we recently developed. Plasma adiponectin concentrations were significantly lower in patients with coronary artery disease than those in age- and body mass index-adjusted control subjects.

CONCLUSIONS

These observations suggest that adiponectin modulates endothelial inflammatory response and that the measurement of plasma adiponectin levels may be helpful in assessment of CAD risk.