Neuregulins (i.e. neuregulin-<em>1</em> (NRG<em>1</em>), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG<em>1</em> or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG<em>1</em> (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/<em>ml</em>) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal alpha-actin. Moreover, NRG<em>1</em> mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG<em>1</em> expression in these cells was increased by endothelin-<em>1</em>, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.

Serum immunoreactive parathyroid hormone (PTH) is increased in obese as compared with nonobese subjects and declines with weight loss. To determine whether alteration of the vitamin D-endocrine system occurs in obesity and whether ensuing secondary hyperparathyroidism is associated with a reduction in urinary calcium, a study was performed in <em>1</em>2 obese white individuals, five men and seven women, and <em>1</em>4 nonobese white subjects, eight men and six women, ranging in age from 20 to 35 yr. Body weight averaged <em>1</em>06 +/- 6 kg in the obese and 68 +/- 2 kg in the nonobese subjects (P less than 0.0<em>1</em>). Each of them were hospitalized on a metabolic ward and were given a constant daily diet containing 400 mg of calcium and 900 mg of phosphorus. Whereas mean serum calcium, serum ionized calcium, and serum phosphorus were the same in the two groups, mean serum immunoreactive PTH (5<em>1</em>8 +/- 48 vs. 243 +/- 33 pg/<em>ml</em>, P less than 0.00<em>1</em>), mean serum <em>1</em>,25-dihydroxyvitamin D [<em>1</em>,25(OH)2D] (37 +/- 2 vs. 29 +/- 2, P less than 0.0<em>1</em>), and mean serum Gla protein (33 +/- 2 vs. 24 +/- 2 ng/<em>ml</em>, P less than 0.02) were significantly higher, and mean serum 25-hydroxyvitamin D (25-OHD) (8 +/- <em>1</em> vs. 20 +/- 2 ng/<em>ml</em>, P less than 0.00<em>1</em>) was significantly lower in the obese than in the nonobese men and women. Mean urinary phosphorus was the same in the two groups, whereas mean urinary calcium (<em>1</em><em>1</em>5 +/- <em>1</em>0 vs. <em>1</em>66 +/- <em>1</em>3 mg/d, P less than 0.0<em>1</em>) was significantly lower, and mean urinary cyclic AMP (3.<em>1</em>8 +/- 0.43 vs. <em>1</em>.84 +/- 0.25 nM/dl GF, P less than 0.0<em>1</em>) and creatinine clearance (2<em>1</em>6 +/- <em>1</em>3 vs. <em>1</em>73 +/- 6 liter/d, P less than 0.0<em>1</em>) were significantly higher in the obese than in the nonobese individuals. There was a significant positive correlation between percentage of ideal body weight and urinary cyclic AMP (r = 0.524, P less than 0.0<em>1</em>) and between percentage of ideal body weight and serum immunoreactive PTH (r = 0.7<em>1</em>7, P less than 0.0<em>1</em>) in the two groups. The results provide evidence that alteration of the vitamin D-endocrine system in obese subjects is characterized by secondary hyperparathyroidism which is associated with enhanced renal tubular reabsorption of calcium and increased circulating <em>1</em>,25(OH)2D. The reduction of serum 25-OHD in them is attributed to feedback inhibition of hepatic synthesis of the precursor by the increased serum <em>1</em>,25(OH)2D.

Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G<em>1</em> that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G<em>1</em> supernatants diluted to 8.3 IRU IFN-gamma/<em>ml</em> with 6 X <em>1</em>0(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G<em>1</em> supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G<em>1</em> MAF activity, two murine macrophage cell lines, P388D<em>1</em> and J774, were identified which absorbed significantly reduced amounts of natural IFN-gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D<em>1</em>. Normal macrophages but not P388D<em>1</em> bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G<em>1</em> supernatants. Scatchard plot analysis showed that <em>1</em>2,000 molecules of soluble <em>1</em>25I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X <em>1</em>0(8) M-<em>1</em>. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN-beta competed only weakly. Monoclonal antibodies against IFN-gamma either inhibited or enhanced MAF activity by blocking or increasing IFN-gamma binding to macrophages, respectively. These results indicate that IFN-gamma reacts with a receptor on macrophage in a specific and saturable manner and this interaction initiates macrophage activation.

OBJECTIVE

Although models exist that place patients into discrete groups at various risks for disease recurrence after surgery for prostate cancer, we know of no published work that combines pathologic factors to predict an individual's probability of disease recurrence. Because clinical stage and biopsy Gleason grade only approximate pathologic stage and Gleason grade in the prostatectomy specimen, prediction of prognosis should be more accurate when postoperative information is added to preoperative variables. Therefore, we developed a postoperative nomogram that allows more accurate prediction of probability for disease recurrence for patients who have received radical prostatectomy as treatment for prostate cancer, compared with the preoperative nomogram we previously published.

METHODS

By Cox proportional hazards regression analysis, we modeled the clinical and pathologic data and disease follow-up for 996 men with clinical stage T1a-T3c NXM0 prostate cancer who were treated with radical prostatectomy by a single surgeon at our institution. Prognostic variables included pretreatment serum prostate-specific antigen level, specimen Gleason sum, prostatic capsular invasion, surgical margin status, seminal vesicle invasion, and lymph node status. Treatment failure was recorded when there was either clinical evidence of disease recurrence, a rising serum prostate-specific antigen level (two measurements of 0.4 ng/mL or greater and rising), or initiation of adjuvant therapy. Validation was performed on this set of men and a separate sample of 322 men from five other surgeons' practices from our institution.

RESULTS

Cancer recurrence was noted in 189 of the 996 men, and the recurrence-free group had a median follow-up period of 37 months (range, 1 to 168 months). The 7-year recurrence-free probability for the cohort was 73% (95% confidence interval, 68% to 76%). The predictions from the nomogram appeared to be accurate and discriminating, with a validation sample area under the receiver operating characteristic curve (ie, a comparison of the predicted probability with the actual outcome) of 0.89.

CONCLUSIONS

A postoperative nomogram has been developed that can be used to predict the 7-year probability of disease recurrence among men treated with radical prostatectomy.

Cytokines are recognized as critical early mediators of organ injury. We attempted to determine whether or not severe hepatic ischemia/reperfusion injury results in tumor necrosis factor-alpha (TNF-alpha) release with subsequent local and systemic tissue injury. After 90 min of lobar hepatic ischemia, TNF was measurable during the reperfusion period in the plasma of all <em>1</em>4 experimental animals, with levels peaking between 9 and 352 pg/<em>ml</em>. Endotoxin was undetectable in the plasma of these animals. Pulmonary injury, as evidenced by a neutrophilic infiltrate, edema and intra-alveolar hemorrhage developed after hepatic reperfusion. The neutrophilic infiltrate was quantitated using a myeloperoxidase (MPO) assay; this demonstrated a significant increase in MPO after only <em>1</em> h of reperfusion. Anti-TNF antiserum pretreatment significantly reduced the pulmonary MPO after hepatic reperfusion. After a <em>1</em>2-h reperfusion period, there was histologic evidence of intra-alveolar hemorrhage and pulmonary edema. Morphometric assessment showed that pretreatment with anti-TNF antiserum was able to completely inhibit the development of pulmonary edema. Liver injury was quantitated by measuring serum glutamic pyruvic transaminase which showed peaks at 3 and 24 h. Anti-TNF antiserum pretreatment was able to significantly reduce both of these peak elevations. These data show that hepatic ischemia/reperfusion results in TNF production, and that this TNF is intimately associated with pulmonary and hepatic injury.

BACKGROUND

An endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is elevated in patients with type 2 diabetes mellitus (DM). This study explored the mechanisms by which ADMA becomes elevated in DM.

RESULTS

Male Sprague-Dawley rats were fed normal chow or high-fat diet (n=5 in each) with moderate streptozotocin injection to induce type 2 DM. Plasma ADMA was elevated in diabetic rats (<em>1</em>.33+/-0.3<em>1</em> versus 0.48+/-0.08 micromol/L; P<0.05). The activity, but not the expression, of dimethylarginine dimethylaminohydrolase (DDAH) was reduced in diabetic rats and negatively correlated with their plasma ADMA levels (P<0.05). DDAH activity was significantly reduced in vascular smooth muscle cells and human endothelial cells (HMEC-<em>1</em>) exposed to high glucose (25.5 mmol/L). The impairment of DDAH activity in vascular cells was associated with an accumulation of ADMA and a reduction in generation of cGMP. In human endothelial cells, coincubation with the antioxidant polyethylene glycol-conjugated superoxide dismutase (22 U/<em>mL</em>) reversed the effects of the high-glucose condition on DDAH activity, ADMA accumulation, and cGMP synthesis.

CONCLUSIONS

A glucose-induced impairment of DDAH causes ADMA accumulation and may contribute to endothelial vasodilator dysfunction in DM.

BACKGROUND

Inflammatory immune activation is an important feature in chronic heart failure (CHF). Little is known about the prognostic importance of tumor necrosis factor-alpha (TNF-alpha), soluble TNF-receptor <em>1</em> and 2 (sTNF-R<em>1</em>/sTNF-R2), interleukin-6 (IL-6), and soluble CD<em>1</em>4 receptors (sCD<em>1</em>4) in CHF patients.

RESULTS

In <em>1</em>52 CHF patients (age 6<em>1</em>+/-<em>1</em> years, New York Heart Association [NYHA] class 2.6+/-0.<em>1</em>, peak VO(2) <em>1</em>7.3+/-0.6 mL. kg(-<em>1</em>). min(-<em>1</em>), mean+/-SEM) plasma concentrations of immune variables were prospectively assessed. During a mean follow-up of 34 months >><em>1</em>2 months in all patients), 62 patients (4<em>1</em>%) died. Cumulative mortality was 28% at 24 months. In univariate analyses, increased total and trimeric TNF-alpha, sTNF-R<em>1</em>, and sTNF-R2 (all P</=0.000<em>1</em>), sCD<em>1</em>4 (P=0.0007), and IL-6 (P=0.005) predicted 24-month mortality. With multivariate analysis and receiver operating characteristics, sTNF-R<em>1</em> emerged among all cytokine parameters as the strongest and most accurate prognosticator in this CHF population, regardless of follow-up duration and independently of NYHA class, peak VO(2), VE/VCO(2) slope, left ventricular ejection fraction, and wasting (P<0.00<em>1</em>). The receiver operating characteristic area under the curve for sTNF-R<em>1</em> was greater than for sTNF-R2 at 6, <em>1</em>2, and <em>1</em>8 months (all P<0.05).

CONCLUSIONS

sTNF-R<em>1</em> was the strongest and most accurate prognosticator, independent of established markers of CHF severity. Assessment of sTNF-R<em>1</em> may be useful in identifying patients who are at high risk of death and in monitoring patients undergoing anti-TNF-alpha treatment.

The rates of protein synthesis and degradation and of amino acid transport were determined in the leg muscle of untrained postabsorptive normal volunteers at rest and approximately 3 h after a resistance exercise routine. The methodology involved use of stable isotopic tracers of amino acids, arteriovenous catheterization of the femoral vessels, and biopsy of the vastus lateralis muscle. During postexercise recovery, the rate of intramuscular phenylalanine utilization for protein synthesis increased above the basal value by <em>1</em>08 +/- <em>1</em>8%, whereas the rate of release from proteolysis increased by 5<em>1</em> +/- <em>1</em>7%. Muscle protein balance improved (P < 0.05) after exercise but did not become positive (from -<em>1</em>5 +/- <em>1</em>2 to -6 +/- 3 nmol phenylalanine.min-<em>1</em>.<em>1</em>00 <em>ml</em> leg volume-<em>1</em>). After exercise, rates of inward transport of leucine, lysine, and alanine increased (P < 0.05) above the basal state from <em>1</em>32 +/- <em>1</em>6 to 208 +/- 29, from <em>1</em>22 +/- 8 to 260 +/- 8, and from 384 +/- 7<em>1</em> to 602 +/- 89 nmol.min-<em>1</em>.<em>1</em>00 <em>ml</em> leg-<em>1</em>, respectively. Transport of phenylalanine did not change significantly. These results indicate that, during recovery after resistance exercise, muscle protein turnover is increased because of an acceleration of synthesis and degradation. A postexercise acceleration of amino acid transport may contribute to the relatively greater stimulation of protein synthesis.

<strong class="sub-title"> Background: </strong> An important feature of severe acute respiratory syndrome coronavirus 2 pathogenesis is COVID-<em>1</em>9-associated coagulopathy, characterised by increased thrombotic and microvascular complications. Previous studies have suggested a role for endothelial cell injury in COVID-<em>1</em>9-associated coagulopathy. To determine whether endotheliopathy is involved in COVID-<em>1</em>9-associated coagulopathy pathogenesis, we assessed markers of endothelial cell and platelet activation in critically and non-critically ill patients admitted to the hospital with COVID-<em>1</em>9.

<strong class="sub-title"> Methods: </strong> In this single-centre cross-sectional study, hospitalised adult (≥<em>1</em>8 years) patients with laboratory-confirmed COVID-<em>1</em>9 were identified in the medical intensive care unit (ICU) or a specialised non-ICU COVID-<em>1</em>9 floor in our hospital. Asymptomatic, non-hospitalised controls were recruited as a comparator group for biomarkers that did not have a reference range. We assessed markers of endothelial cell and platelet activation, including von Willebrand Factor (VWF) antigen, soluble thrombomodulin, soluble P-selectin, and soluble CD40 ligand, as well as coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes. We compared the level of each marker in ICU patients, non-ICU patients, and controls, where applicable. We assessed correlations between these laboratory results with clinical outcomes, including hospital discharge and mortality. Kaplan-Meier analysis was used to further explore the association between biochemical markers and survival.

<strong class="sub-title"> Findings: </strong> 68 patients with COVID-<em>1</em>9 were included in the study from April <em>1</em>3 to April 24, 2020, including 48 ICU and 20 non-ICU patients, as well as <em>1</em>3 non-hospitalised, asymptomatic controls. Markers of endothelial cell and platelet activation were significantly elevated in ICU patients compared with non-ICU patients, including VWF antigen (mean 565% [SD <em>1</em>99] in ICU patients vs 278% [<em>1</em>33] in non-ICU patients; p<0·000<em>1</em>) and soluble P-selectin (<em>1</em>5·9 ng/<em>mL</em> [4·8] vs <em>1</em><em>1</em>·2 ng/<em>mL</em> [3·<em>1</em>]; p=0·00<em>1</em>4). VWF antigen concentrations were also elevated above the normal range in <em>1</em>6 (80%) of 20 non-ICU patients. We found mortality to be significantly correlated with VWF antigen (r = 0·38; p=0·0022) and soluble thrombomodulin (r = 0·38; p=0·0078) among all patients. In all patients, soluble thrombomodulin concentrations greater than 3·26 ng/<em>mL</em> were associated with lower rates of hospital discharge (22 [88%] of 25 patients with low concentrations vs <em>1</em>3 [52%] of 25 patients with high concentrations; p=0·0050) and lower likelihood of survival on Kaplan-Meier analysis (hazard ratio 5·9, 95% CI <em>1</em>·9-<em>1</em>8·4; p=0·0087).

<strong class="sub-title"> Interpretation: </strong> Our findings show that endotheliopathy is present in COVID-<em>1</em>9 and is likely to be associated with critical illness and death. Early identification of endotheliopathy and strategies to mitigate its progression might improve outcomes in COVID-<em>1</em>9.

Funding: This work was supported by a gift donation from Jack Levin to the Benign Hematology programme at Yale, and the National Institutes of Health.

Six new permanent cell lines were established from human gliomas and compared to nine other cell lines from human gliomas. All fifteen lines had individually distinct HLA phenotypes and all but two, which were from a black patient, had type B glucose-6-phosphate-de;hydrogenase isoenzymes. Morphologically, the lines could be classified into four patterns descriptively designated as fibroblastic, fascicular, epithelial, or glial. Four of the lines grew progressively and could be serially transplanted when injected into athymic mice; two others grew initially and then regressed. From none to <em>1</em>00% of cells developed elongated tapering processes and showed reduction in nuclear-cytoplasmic ratio in the presence of <em>1</em> mM cyclic AMP and theophylline. Levels of 2'-3' cyclic nucleotide 3'-phosphohydrolase activity ranged from nondetectable to <em>1</em>2.78 +/- <em>1</em>.49 micromoles 2' AMP formed per hr mgm total protein. None of the lines had detectable S-<em>1</em>00 protein, but two had readily demonstrable glial fibrillary acidic protein in indirect immunofluorescence. Fibronectin levels in spent culture supernatants ranged from undetectable levels to 2<em>1</em>.4 micrograms/<em>ml</em>/<em>1</em>0(5) cells. All but one line shared surface antigens with normal human adult or fetal brain, as detected in absorption analyses with nonhuman primate antiserum raised against glioblastoma multiforme tissue or cell line U-25<em>1</em> MG. Although there were many common properties of the lines, each line had a unique profile of the parameters evaluated. This heterogeneity most likely reflects the individuality of the tumors of origin and individual genotypes and capacity for a range of phenotypic expression of cells.

By using the highly sensitive and specific technique of enzyme-linked immunosorbent assay, we investigated the presence and amount of transforming growth factor-beta 2 (TGF-beta 2) in samples of aqueous humor obtained from <em>1</em>5 patients who had a clinically established diagnosis of advanced primary open-angle glaucoma (POAG), as well as from ten age-matched normal human subjects undergoing cataract surgery. The total amount of TGF-beta 2 in the samples of normal aqueous humor ranged from 0.4<em>1</em> to 2.24 ng <em>ml</em>-<em>1</em> (mean +/- S.D.: <em>1</em>.48 +/- 0.68 ng <em>ml</em>-<em>1</em>) of which 4.88 to 37.05% (<em>1</em><em>1</em>.99 +/- 9.95%) was intrinsically active. Compared with normal subjects, the aqueous humor from POAG patients had a statistically significantly greater amount of total TGF-beta 2 (2.70 +/- 0.76 ng <em>ml</em>-<em>1</em>, P < 0.0<em>1</em>), as well as a higher level of intrinsically active TGF-beta 2 (0.45 +/- 0.28 ng <em>ml</em>-<em>1</em>, P < 0.05) which corresponded to <em>1</em>.09 to 60.84% (<em>1</em>8.33 +/- <em>1</em>5.50%) of the total amount. No linear correlation was found between the age of the subjects and the protein concentration of the aqueous humor from either normal or glaucomatous eyes, nor between the age of the patient and the total amount of TGF-beta 2. The negligible amount of TGF-beta 2 present in serum argues against its influx into the aqueous humor after breakdown of the blood-aqueous barrier that is known to occur in glaucomatous eyes; rather, our present findings support the concept of the intraocular derivation of this cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)

We performed the first studies of analytic sensitivity, analytic specificity, and dynamic range for the new Xpert MTB/RIF assay, a nucleic acid amplification-based diagnostic system that detects Mycobacterium tuberculosis and rifampin (RIF) resistance in under 2 h. The sensitivity of the assay was tested with 79 phylogenetically and geographically diverse M. tuberculosis isolates, including 42 drug-susceptible isolates and 37 RIF-resistant isolates containing <em>1</em>3 different rpoB mutations or mutation combinations. The specificity of the assay was tested with 89 nontuberculosis bacteria, fungi, and viruses. The Xpert MTB/RIF assay correctly identified all 79 M. tuberculosis isolates and correctly excluded all 89 nontuberculosis isolates. RIF resistance was correctly identified in all 37 resistant isolates and in none of the 42 susceptible isolates. Dynamic range was assessed by adding <em>1</em>0(2) to <em>1</em>0(7) CFU of M. tuberculosis into M. tuberculosis-negative sputum samples. The assay showed a log-linear relationship between cycle threshold and input CFU over the entire concentration range. Resistance detection in the presence of different mixtures of RIF-resistant and RIF-susceptible DNA was assessed. Resistance detection was dependent on the particular mutation and required between 65% and <em>1</em>00% mutant DNA to be present in the sample for 95% certainty of resistance detection. Finally, we studied whether assay specificity could be affected by cross-contaminating amplicons generated by the GenoType MTBDRplus assay. M. tuberculosis was not detected until at least <em>1</em>0(8) copies of an MTBDRplus amplicon were spiked into <em>1</em> <em>ml</em> of sputum, suggesting that false-positive results would be unlikely to occur.

To determine whether cytokines are generated in vivo in subjects with asthma, we have measured cytokine levels (tumor necrosis factor [TNF], granulocyte-macrophage-colony-stimulating factor [GM-CSF], interleukin [IL]-<em>1</em> alpha, IL-<em>1</em> beta, IL-2, IL-4, and IL-6) in the airways of subjects with symptomatic (N = 24) and asymptomatic (N = 9) asthma with immunoassays (GM-CSF, IL-<em>1</em> alpha, IL-<em>1</em> beta, IL-2, and IL-4) or bioassays (TNF and IL-6) and the polymerase chain reaction (IL-<em>1</em> beta and TNF). Significant levels of TNF (578 +/- 9<em>1</em>7 pg/<em>ml</em> versus 24 +/- 29 pg/<em>ml</em>) (p = 0.0<em>1</em>), GM-CSF (24 +/- 4<em>1</em> pg/<em>ml</em> versus less than 8 pg/<em>ml</em>) (p = 0.02), and IL-6 (225 +/- 327 pg/<em>ml</em> versus 7 +/- <em>1</em>2 pg/<em>ml</em>) (p = 0.0<em>1</em>), but not IL-<em>1</em> alpha or IL-4, were detected in the bronchoalveolar lavage fluid (BALF) of patients with symptomatic compared with BALF of patients with asymptomatic asthma. Levels of IL-<em>1</em> beta (266 +/- 270 pg/<em>ml</em> versus less than 20 pg/<em>ml</em>) (p = 0.00<em>1</em>) and IL-2 (<em>1</em>.4 +/- 2.8 ng/<em>ml</em> versus less than 0.3 ng/<em>ml</em>) (p = 0.05) in BALF in patients with symptomatic compared with that in BALF levels in patients with asymptomatic asthma suggested activation of alveolar macrophages and T cells. Thus, in episodes of asthma, several cytokines, including TNF, GM-CSF, IL-<em>1</em> beta, IL-2, and IL-6 are detectable in BALF.

International variations in cancer rates have been attributed, at least in part, to differences in dietary intake. Recently, it has been suggested that consumption of soyfoods may contribute to the relatively low rates of breast, colon, and prostate cancers in countries such as China and Japan. Soybeans contain a number of anticarcinogens, and a recent National Cancer Institute workshop recommended that the role of soyfoods in cancer prevention be investigated. In this review, the hypothesis that soy intake reduces cancer risk is considered by examining relevant in vitro, animal, and epidemiological data. Soybeans are a unique dietary source of the isoflavone genistein, which possesses weak estrogenic activity and has been shown to act in animal models as an antiestrogen. Genistein is also a specific inhibitor of protein tyrosine kinases; it also inhibits DNA topoisomerases and other critical enzymes involved in signal transduction. In vitro, genistein suppresses the growth of a wide range of cancer cells, with IC50 values ranging from 5 to 40 microM (<em>1</em>-<em>1</em>0 micrograms/<em>ml</em>). Of the 26 animal studies of experimental carcinogenesis in which diets containing soy or soybean isoflavones were employed, <em>1</em>7 (65%) reported protective effects. No studies reported soy intake increased tumor development. The epidemiological data are also inconsistent, although consumption of nonfermented soy products, such as soymilk and tofu, tended to be either protective or not associated with cancer risk; however, no consistent pattern was evident with the fermented soy products, such as miso. Protective effects were observed for both hormone- and nonhormone-related cancers. While a definitive statement that soy reduces cancer risk cannot be made at this time, there is sufficient evidence of a protective effect to warrant continued investigation.

BACKGROUND

Adherence of 95% or more to unboosted protease regimens is required for optimal virologic suppression in HIV-<em>1</em>-infected patients. Whether the same is true for nonnucleoside reverse transcriptase inhibitor (NNRTI)-based therapy is unclear.

OBJECTIVE

To assess the relationship between adherence to NNRTI-based therapy and viral load in treatment-naive patients.

METHODS

Observational cohort study.

METHODS

Private-sector HIV and AIDS disease management program in South Africa.

METHODS

282<em>1</em> adults infected with HIV who began NNRTI-based therapy between January <em>1</em>998 and March 2003 (2764 patients [98%] were enrolled after December 2000).

METHODS

Adherence was assessed by monthly pharmacy claims. The primary end point was sustained viral load suppression (<400 copies/mL) in <em>1</em>00% of recorded viral load measurements throughout follow-up. Secondary end points included time to initial viral load suppression and time to subsequent virologic failure (>400 copies/mL).

RESULTS

The median follow-up period was 2.2 years (interquartile range, <em>1</em>.7 to 2.7 years). The proportion of patients with sustained viral load suppression ranged from <em>1</em>3% (4<em>1</em> of 325 patients) in patients who filled less than 50% of antiretroviral drug prescriptions to 73% (725 of 997 patients) in those who filled <em>1</em>00% of antiretroviral drug prescriptions. Each <em>1</em>0% increase in pharmacy claim adherence greater than 50% was associated with a mean absolute increase of 0.<em>1</em>0 in the proportion of patients with sustained virologic suppression (P < 0.00<em>1</em>). Predictors for shorter time to virologic failure after initial suppression in multivariable Cox regression included CD4+ T-cell counts of 0.50 x <em>1</em>0(9) cells/L or less (hazard ratio, <em>1</em>.60 [95% CI, <em>1</em>.22 to 2.<em>1</em>0] vs. CD4+ T-cell counts >0.20 x <em>1</em>0(9) cells/L), baseline viral load greater than <em>1</em>0(5) copies/mL (hazard ratio, <em>1</em>.39 [CI, <em>1</em>.<em>1</em>4 to <em>1</em>.70]), nevirapine-based regimen (hazard ratio, <em>1</em>.43 [CI, <em>1</em>.<em>1</em>6 to <em>1</em>.75]), and low pharmacy claim adherence (hazard ratio, <em>1</em>.58 [CI, <em>1</em>.48 to <em>1</em>.69], per <em>1</em>0% decrease in adherence to 50%).

CONCLUSIONS

Observational study with adherence stratification at study end and lack of standardized timing for outcome measurement.

CONCLUSIONS

Virologic outcomes improve in a linear dose-response manner as adherence to NNRTI-based regimens increases beyond 50%.

<em>1</em>. Neurotransmitter-receptors in the membrane of Xenopus oocytes have been studied using electrophysiological techniques. Neurotransmitters and related agents were applied while recording either membrane potential or membrane current. The majority of ovarian oocytes used were at stages IV and V.2. Three types of oocytes were examined: inner ovarian epithelium covered (e.c.) oocytes; epithelium manually removed (e.r.) oocytes; and collagenase treated (c.t.) ooctyes.3. Ovarian oocytes are sensitive to some cholinergic and catecholaminergic agents. Responses to serotonin were seldom observed and when present were much weaker than responses to other agents. No responses were observed to the amino acids: aspartate, glutamate, gamma-aminobutyric acid, and glycine; or to octopamine and histamine.4. Acetylcholine (ACh) usually depolarized the membrane, in a dose-dependent manner, with threshold concentrations as low as <em>1</em>0(-9)m. The ACh-potential was due to an increase in Cl permeability and had a reversal potential around - <em>1</em>9 mV. The intracellular Cl ion activity, measured with a Cl-ion sensitive micro-electrode, was about 65 mm and the estimated Cl-ion equilibrium potential, E(Cl), agreed with the reversal potential of the ACh-potential.5. Curare (<em>1</em>0(-4)m), tetrodotoxin (<em>1</em>0(-6)m), or alpha-bungarotoxin (<em>1</em>0(-6) g/<em>ml</em>.) did not block the response to <em>1</em>0(-6)m-ACh; whereas atropine (<em>1</em>0(-7)m) blocked it. No response to nicotinic agents (e.g. nicotine, <em>1</em>,<em>1</em>-dimethyl-4-phenylpiperazinium) was observed. These results suggest that the ACh receptors in the oocyte membrane are muscarinic in nature.6. The apparent latency of the ACh potential, examined by ionophoretic application of ACh to e.r. oocytes and c.t. oocytes, ranged from 0.5 sec to over 20 sec. Intracellular injection of ACh was without effect.7. Responses to catecholamines were observed mostly in e.c. oocytes; while in e.r. and c.t. oocytes they were rare and of very small amplitudes.8. The usual response to both dopamine and (-)-epinephrine was a transient hyperpolarization manifested by an initial increase in K-permeability followed by a decrease. The latency of these responses ranged from <em>1</em>0 sec to over 30 sec and their reversal potential was nearly - <em>1</em>00 mV, which coincided with E(K).9. Oocytes responded to the beta-adrenergic receptor agonist, isoproterenol, as well as (-)-epinephrine. Pre-treatment with the beta-adrenergic receptor blocker, propranolol, abolished the response to both (-)-epinephrine and (-)-isoproterenol. The dopamine potential was also reduced considerably. Both the alpha-adrenergic receptor agonist, phenylephrine, and the alpha-adrenergic receptor blocker, phentolamine, were without effect.<em>1</em>0. Maturation of the oocytes, induced in vivo by gonadotropin or in vitro by progesterone, led to loss of responsiveness to both cholinergic and catecholaminergic agents.

BACKGROUND

Alzheimer's disease (AD) is characterized by extensive loss of neurons in the brain of AD patients. Intracellular accumulation of beta-amyloid peptide (Abeta) has also shown to occur in AD. Neuro-inflammation has been known to play a role in the pathogenesis of AD.

METHODS

In this study, we investigated neuro-inflammation and amyloidogenesis and memory impairment following the systemic inflammation generated by lipopolysaccharide (LPS) using immunohistochemistry, ELISA, behavioral tests and Western blotting.

RESULTS

Intraperitoneal injection of LPS, (250 microg/kg) induced memory impairment determined by passive avoidance and water maze tests in mice. Repeated injection of LPS (250 microg/kg, 3 or 7 times) resulted in an accumulation of Abeta<em>1</em>-42 in the hippocampus and cerebralcortex of mice brains through increased beta- and gamma-secretase activities accompanied with the increased expression of amyloid precursor protein (APP), 99-residue carboxy-terminal fragment of APP (C99) and generation of Abeta<em>1</em>-42 as well as activation of astrocytes in vivo. 3 weeks of pretreatment of sulindac sulfide (3.75 and 7.5 mg/kg, orally), an anti-inflammatory agent, suppressed the LPS-induced amyloidogenesis, memory dysfunction as well as neuronal cell death in vivo. Sulindac sulfide (<em>1</em>2.5-50 microM) also suppressed LPS (<em>1</em> microg/<em>ml</em>)-induced amyloidogenesis in cultured neurons and astrocytes in vitro.

CONCLUSIONS

This study suggests that neuro-inflammatory reaction could contribute to AD pathology, and anti-inflammatory agent could be useful for the prevention of AD.

BACKGROUND

Use of antiretroviral treatment for HIV-<em>1</em> infection has decreased AIDS-related morbidity and mortality and prevents sexual transmission of HIV-<em>1</em>. However, the best time to initiate antiretroviral treatment to reduce progression of HIV-<em>1</em> infection or non-AIDS clinical events is unknown. We reported previously that early antiretroviral treatment reduced HIV-<em>1</em> transmission by 96%. We aimed to compare the effects of early and delayed initiation of antiretroviral treatment on clinical outcomes.

METHODS

The HPTN 052 trial is a randomised controlled trial done at <em>1</em>3 sites in nine countries. We enrolled HIV-<em>1</em>-serodiscordant couples to the study and randomly allocated them to either early or delayed antiretroviral treatment by use of permuted block randomisation, stratified by site. Random assignment was unblinded. The HIV-<em>1</em>-infected member of every couple initiated antiretroviral treatment either on entry into the study (early treatment group) or after a decline in CD4 count or with onset of an AIDS-related illness (delayed treatment group). Primary events were AIDS clinical events (WHO stage 4 HIV-<em>1</em> disease, tuberculosis, and severe bacterial infections) and the following serious medical conditions unrelated to AIDS: serious cardiovascular or vascular disease, serious liver disease, end-stage renal disease, new-onset diabetes mellitus, and non-AIDS malignant disease. Analysis was by intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT0007458<em>1</em>.

RESULTS

<em>1</em>763 people with HIV-<em>1</em> infection and a serodiscordant partner were enrolled in the study; 886 were assigned early antiretroviral treatment and 877 to the delayed treatment group (two individuals were excluded from this group after randomisation). Median CD4 counts at randomisation were 442 (IQR 373-522) cells per μL in patients assigned to the early treatment group and 428 (357-522) cells per μL in those allocated delayed antiretroviral treatment. In the delayed group, antiretroviral treatment was initiated at a median CD4 count of 230 (IQR <em>1</em>97-249) cells per μL. Primary clinical events were reported in 57 individuals assigned to early treatment initiation versus 77 people allocated to delayed antiretroviral treatment (hazard ratio 0·73, 95% CI 0·52-<em>1</em>·03; p=0·074). New-onset AIDS events were recorded in 40 participants assigned to early antiretroviral treatment versus 6<em>1</em> allocated delayed initiation (0·64, 0·43-0·96; p=0·03<em>1</em>), tuberculosis developed in <em>1</em>7 versus 34 patients, respectively (0·49, 0·28-0·89, p=0·0<em>1</em>8), and primary non-AIDS events were rare (<em>1</em>2 in the early group vs nine with delayed treatment). In total, 498 primary and secondary outcomes occurred in the early treatment group (incidence 24·9 per <em>1</em>00 person-years, 95% CI 22·5-27·5) versus 585 in the delayed treatment group (29·2 per <em>1</em>00 person-years, 26·5-32·<em>1</em>; p=0·025). 26 people died, <em>1</em><em>1</em> who were allocated to early antiretroviral treatment and <em>1</em>5 who were assigned to the delayed treatment group.

CONCLUSIONS

Early initiation of antiretroviral treatment delayed the time to AIDS events and decreased the incidence of primary and secondary outcomes. The clinical benefits recorded, combined with the striking reduction in HIV-<em>1</em> transmission risk previously reported, provides strong support for earlier initiation of antiretroviral treatment.

BACKGROUND

US National Institute of Allergy and Infectious Diseases.

<em>1</em>. A new smooth muscle preparation, the rat anococcygeus muscle, is described. The muscle is paired, thin, consists of smooth muscle only and the muscle cells are organized in parallel bundles. It has a dense adrenergic innervation distributed throughout the muscle but apparently no cholinergic innervation. The muscles are easily isolated.2. The muscle contracts to noradrenaline, acetylcholine, furmethide, 5-hydroxytryptamine, but not to histamine. Isoprenaline produces contraction at high concentrations. The effects of noradrenaline and acetylcholine are blocked by phentolamine and atropine respectively. The response to isoprenaline is little affected by propranolol.3. The muscle contracts in response to field stimulation or stimulation of extrinsic nerves. This response is completely blocked by phentolamine but unaffected by hexamethonium or atropine.4. Guanethidine <em>1</em>0(-6)-5 x <em>1</em>0(-6)M blocks the motor response to nerve stimulation and potentiates that to noradrenaline. Higher concentrations of guanethidine raise tone. In the presence of raised tone, field stimulation produces an inhibitory response insensitive to hexamethonium but abolished by tetrodotoxin 2 x <em>1</em>0(-7) g/<em>ml</em>. This inhibitory response to stimulation can also be shown after other drugs which raise tone.5. The inhibitory response to nerve stimulation is not mimicked by acetylcholine, isoprenaline or ATP, nor blocked by atropine, phentolamine, phenoxybenzamine, propranolol, hexamethonium or lysergic acid diethylamide.

Escherichia coli K-<em>1</em>2 strains tested so far (approximately 20) can be separated into three groups on the basis of their abilities to form colonies on nutrient agar supplemented with nalidixic acid (NAL): (i) Nal(s) or wild type (no growth at <em>1</em> to 2 mug/<em>ml</em>); (ii) NalA(r) (growth at 40 mug/<em>ml</em> or higher); and (iii) NalB(r) (growth at 4 mug/<em>ml</em>, but no growth at <em>1</em>0 mug/<em>ml</em>). The NalA(r) group has a spectrum of sensitivity ranging from 60 to over <em>1</em>00 mug/<em>ml</em>. All Hfr strains of the NalA(r) and NalB(r) groups transfer NAL resistance to recipient cells at genetic loci which are at 42.5 +/- 0.5 and 5<em>1</em> +/- <em>1</em> min, respectively, on the Taylor-Trotter map. Some members of the NalA(r) group also have the genetic locus for NalB(r). The nalA(s) allele is completely dominant to nalA(r) in a partial diploid configuration. In haploids, nalA(r)-nalB(r) is phenotypically NalA(r); nalA(r)-nalB(s) is NalA(r); and nalA(s)-nalB(r) is NalB(r). The map location of nalA and the easy differentiation between NalA(r) and NalA(s) allow this marker to be used as a counterselector in bacterial conjugation experiments.

BACKGROUND

Breastfeeding is essential for child health and development in low-resource settings but carries a significant risk of transmission of HIV-<em>1</em>, especially in late stages of maternal disease. We aimed to assess the efficacy and safety of triple antiretroviral compared with zidovudine and single-dose nevirapine prophylaxis in pregnant women infected with HIV.

METHODS

Pregnant women with WHO stage <em>1</em>, 2, or 3 HIV-<em>1</em> infection who had CD4 cell counts of 200-500 cells per μL were enrolled at five study sites in Burkina Faso, Kenya, and South Africa to start study treatment at 28-36 weeks' gestation. Women were randomly assigned (<em>1</em>:<em>1</em>) by a computer generated random sequence to either triple antiretroviral prophylaxis (a combination of 300 mg zidovudine, <em>1</em>50 mg lamivudine, and 400 mg lopinavir plus <em>1</em>00 mg ritonavir twice daily until cessation of breastfeeding to a maximum of 6·5 months post partum) or zidovudine and single-dose nevirapine (300 mg zidovudine twice daily until delivery and a dose of 600 mg zidovudine plus 200 mg nevirapine at the onset of labour and, after a protocol amendment in December, 2006, <em>1</em> week post-partum zidovudine 300 mg twice daily and lamivudine <em>1</em>50 mg twice daily). All infants received a 0·6 mL dose of nevirapine at birth and, from December, 2006, 4 mg/kg twice daily of zidovudine for <em>1</em> week after birth. Patients and investigators were not masked to treatment. The primary endpoints were HIV-free infant survival at 6 weeks and <em>1</em>2 months; HIV-free survival at <em>1</em>2 months in infants who were ever breastfed; AIDS-free survival in mothers at <em>1</em>8 months; and serious adverse events in mothers and babies. Analysis was by intention to treat. This trial is registered with Current Controlled Trials, ISRCTN7<em>1</em>46840<em>1</em>.

RESULTS

From June, 2005, to August, 2008, 882 women were enrolled, 824 of whom were randomised and gave birth to 805 singleton or first, liveborn infants. The cumulative rate of HIV transmission at 6 weeks was 3·3% (95% CI <em>1</em>·9-5·6%) in the triple antiretroviral group compared with 5·0% (3·3-7·7%) in the zidovudine and single-dose nevirapine group, and at <em>1</em>2 months was 5·4% (3·6-8·<em>1</em>%) in the triple antiretroviral group compared with 9·5% (7·0-<em>1</em>2·9%) in the zidovudine and single-dose nevirapine group (p=0·029). The cumulative rate of HIV transmission or death at <em>1</em>2 months was <em>1</em>0·2% (95% CI 7·6-<em>1</em>3·6%) in the triple antiretroviral group compared with <em>1</em>6·0% (<em>1</em>2·7-20·0%) in the zidovudine and single-dose nevirapine group (p=0·0<em>1</em>7). In infants whose mothers declared they intended to breastfeed, the cumulative rate of HIV transmission at <em>1</em>2 months was 5·6% (95% CI 3·4-8·9%) in the triple antiretroviral group compared with <em>1</em>0·7% (7·6-<em>1</em>4·8%) in the zidovudine and single-dose nevirapine group (p=0·02). AIDS-free survival in mothers at <em>1</em>8 months will be reported in a different publication. The incidence of laboratory and clinical serious adverse events in both mothers and their babies was similar between groups.

CONCLUSIONS

Triple antiretroviral prophylaxis during pregnancy and breastfeeding is safe and reduces the risk of HIV transmission to infants. Revised WHO guidelines now recommend antiretroviral prophylaxis (either to the mother or to the baby) during breastfeeding if the mother is not already receiving antiretroviral treatment for her own health.

BACKGROUND

Agence nationale de recherches sur le sida et les hépatites virales, Department for International Development, European and Developing Countries Clinical Trials Partnership, Thrasher Research Fund, Belgian Directorate General for International Cooperation, Centers for Disease Control and Prevention, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and UNDP/UNFPA/World Bank/WHO Special Programme of Research, Development and Research Training in Human Reproduction.

Muscle mass declines with aging. Amino acids alone stimulate muscle protein synthesis in the elderly. However, mixed nutritional supplementation failed to improve muscle mass. We hypothesized that the failure of nutritional supplements is due to altered responsiveness of muscle protein anabolism to increased amino acid availability associated with endogenous hyperinsulinemia. We measured muscle protein synthesis and breakdown, and amino acid transport in healthy young (30 +/- 3 yr) and elderly (72 +/- <em>1</em> yr) volunteers in the basal postabsorptive state and during the administration of an amino acid-glucose mixture, using L-[ring-(2)H(5)]phenylalanine infusion, femoral artery and vein catheterization, and muscle biopsies. Basal muscle amino acid turnover was similar in young and elderly subjects. The mixture increased phenylalanine leg delivery and transport into the muscle in both groups. Phenylalanine net balance increased in both groups (young, -27 +/- 8 to 64 +/- <em>1</em>7; elderly, -<em>1</em>6 +/- 4 to 29 +/- 7 nmol/(min.<em>1</em>00 <em>mL</em>); P: < 0.000<em>1</em>, basal vs. mixture), but the increase was significantly blunted in the elderly (P: = 0.030 vs. young). Muscle protein synthesis increased in the young, but remained unchanged in the elderly [young, 6<em>1</em> +/- <em>1</em>7 to <em>1</em>33 +/- 30 (P: = 0. 005); elderly, 62 +/- 9 to 70 +/- <em>1</em>4 nmol/(min.<em>1</em>00 <em>mL</em>) (P: = NS)]. In both groups, protein breakdown decreased (P: = 0.0<em>1</em>2) and leg glucose uptake increased (P: = 0.0258) with the mixture. We conclude that the response of muscle protein anabolism to hyperaminoacidemia with endogenous hyperinsulinemia is impaired in healthy elderly due to the unresponsiveness of protein synthesis.

OBJECTIVE

The primary goal of this study was to evaluate whether pathologic response to chemotherapy predicts patient survival after preoperative chemotherapy and resection of colorectal liver metastases (CLM). The secondary goal of the study was to identify the clinical predictors of pathologic response.

METHODS

A retrospective review was performed of 305 patients who underwent preoperative irinotecan- or oxaliplatin-based chemotherapy, followed by resection of CLM. Pathologic response was systematically evaluated and reported as the mean of the percentage of cancer cells remaining within each tumor. Univariate and multivariate analyses were performed to identify the predictors of pathologic response and survival.

RESULTS

Cumulative 5-year overall survival rates by pathologic response status were as follows: 75% complete response (no residual cancer cells), 56% major response (<em>1</em>% to 49% residual cancer cells), and 33% minor response >> or = 50% residual cancer cells; complete v major response, P = .037; major v minor response, P = .028). Multivariate analysis revealed that only surgical margin status (P = .050; hazard ratio [HR], <em>1</em>.77) and pathologic response (major response: P = .034; HR, 4.80; minor response: P = .007; HR, 6.93) were independent predictors of survival. Multivariate analysis of the predictors of pathologic response revealed that carcinoembryonic antigen level < or = 5 ng/<em>mL</em>, tumor size < or = 3 cm, and chemotherapy with fluoropyrimidine plus oxaliplatin and bevacizumab were independent predictors of pathologic response.

CONCLUSIONS

Pathologic response predicts survival after preoperative chemotherapy and resection of CLM. Degree of pathologic response represents a new outcome end point for prognosis after resection of CLM.

Transduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only <em>1</em> x <em>1</em>0(<em>1</em>0) scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/<em>mL</em>) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.