The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta <em>1</em>-28 through beta <em>1</em>-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta <em>1</em>-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/<em>ml</em>. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta <em>1</em>-47 and beta <em>1</em>-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/<em>ml</em> in the alpha helix-promoting solvent, <em>1</em>,<em>1</em>,<em>1</em>,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta <em>1</em>-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.

OBJECTIVE

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by the formation of hamartomas in multiple organs. Five to <em>1</em>5% of affected individuals display subependymal giant cell astrocytomas, which can lead to substantial neurological and postoperative morbidity due to the production of hydrocephalus, mass effect, and their typical location adjacent to the foramen of Monro. We sought to see whether therapy with oral rapamycin could affect growth or induce regression in astrocytomas associated with TSC.

METHODS

Five subjects with clinically definite TSC and either subependymal giant cell astrocytomas (n = 4) or a pilocytic astrocytoma (n = <em>1</em>) were treated with oral rapamycin at standard immunosuppressive doses (serum levels 5-<em>1</em>5 ng/<em>ml</em>) from 2.5 to 20 months. All lesions demonstrated growth on serial neuroimaging studies. Magnetic resonance imaging scans were performed before and at regular intervals following initiation of therapy.

RESULTS

All lesions exhibited regression and, in one case, necrosis. Interruption of therapy resulted in regrowth of subependymal giant cell astrocytomas in one patient. Resumption of therapy resulted in further regression. Treatment was well tolerated.

CONCLUSIONS

Oral rapamycin therapy can induce regression of astrocytomas associated with TSC and may offer an alternative to operative therapy of these lesions.

BACKGROUND

There is broad human exposure to bisphenol A (BPA), an estrogenic endocrine-disrupting chemical widely used for the production of plastic products. BPA is reported to affect preimplantation embryos or fetuses and alter their postnatal development at doses typically found in the environment. We measured contamination of BPA in various kinds of human biological fluids by a novel enzyme-linked immunosorbent assay.

METHODS

Blood samples were obtained from healthy premenopausal women, women with early and full-term pregnancy, and umbilical cord at full-term delivery. Ovarian follicular fluids obtained during IVF procedures and amniotic fluids obtained at mid-term and full-term pregnancy were also subject to BPA measurements.

RESULTS

BPA was present in serum and follicular fluid at approximately <em>1</em>-2 ng/<em>ml</em>, as well as in fetal serum and full-term amniotic fluid, confirming passage through the placenta. Surprisingly, an approximately 5-fold higher concentration, 8.3 +/- 8.7 ng/<em>ml</em>, was revealed in amniotic fluid at <em>1</em>5-<em>1</em>8 weeks gestation, compared with other fluids.

CONCLUSIONS

These results suggest accumulation of BPA in early fetuses and significant exposure during the prenatal period, which must be considered in evaluating the potential for human exposure to endocrine-disrupting chemicals.

Sensitive and specific biomarkers are needed to detect early kidney injury. The objective of the present work was to develop a sensitive quantitative urinary test to identify renal injury in the rodent to facilitate early assessment of pathophysiological influences and drug toxicity. Two mouse monoclonal antibodies were made against the purified ectodomain of kidney injury molecule-<em>1</em> (Kim-<em>1</em>), and these were used to construct a sandwich Kim-<em>1</em> ELISA. The assay range of this ELISA was 50 pg/<em>ml</em> to 5 ng/<em>ml</em>, with inter- and intra-assay variability of (<em>1</em>0%. Urine samples were collected from rats treated with one of three doses of cisplatin (2.5, 5, or 7.5 mg/kg). At one day after each of the doses, there was an approximately three- to fivefold increase in the urine Kim-<em>1</em> ectodomain, whereas other routinely used biomarkers measured in this study [plasma creatinine, blood urea nitrogen (BUN), urinary N-acetyl-beta-glucosaminidase (NAG), glycosuria, proteinuria] lacked the sensitivity to show any sign of renal damage at this time point. When rats were subjected to increasing periods (<em>1</em>0, 20, 30, or 45 min) of bilateral ischemia, there was an increasing amount of urinary Kim-<em>1</em> detected. After only <em>1</em>0 min of bilateral ischemia, Kim-<em>1</em> levels on day <em>1</em> were <em>1</em>0-fold higher (5 ng/<em>ml</em>) than control levels, whereas plasma creatinine and BUN were not increased and there was no glycosuria, increased proteinuria, or increased urinary NAG levels. Thus urinary Kim-<em>1</em> levels serve as a noninvasive, rapid, sensitive, reproducible, and potentially high-throughput method to detect early kidney injury in pathophysiological studies and in preclinical drug development studies for risk-benefit profiling of pharmaceutical agents.

Mortality in sepsis remains unacceptably high and attempts to modulate the inflammatory response failed to improve survival. Previous reports postulated that the sepsis-triggered immunological cascade is multimodal: initial systemic inflammatory response syndrome (SIRS; excessive pro-, but no/low anti-inflammatory plasma mediators), intermediate homeostasis with a mixed anti-inflammatory response syndrome (MARS; both pro- and anti-inflammatory mediators) and final compensatory anti-inflammatory response syndrome (CARS; excessive anti-, but no/low proinflammatory mediators). To verify this, we examined the evolution of the inflammatory response during the early phase of murine sepsis by repetitive blood sampling of septic animals. Increased plasma concentrations of proinflammatory (IL-6, TNF, IL-<em>1</em>beta, KC, MIP-2, MCP-<em>1</em>, and eotaxin) and anti-inflammatory (TNF soluble receptors, IL-<em>1</em>0, IL-<em>1</em> receptor antagonist) cytokines were observed in early deaths (days <em>1</em>-5). These elevations occurred simultaneously for both the pro- and anti-inflammatory mediators. Plasma levels of IL-6 (26 ng/<em>ml</em>), TNF-alpha (<em>1</em>2 ng/<em>ml</em>), KC (33 ng/<em>ml</em>), MIP-2 (<em>1</em>4 ng/<em>ml</em>), IL-<em>1</em> receptor antagonist (65 ng/<em>ml</em>), TNF soluble receptor I (3 ng/<em>ml</em>), and TNF soluble receptor II (<em>1</em>4 ng/<em>ml</em>) accurately predicted mortality within 24 h. In contrast, these parameters were not elevated in either the late-deaths (day 6-28) or survivors. Surprisingly, either pro- or anti-inflammatory cytokines were also reliable in predicting mortality up to 48 h before outcome. These data demonstrate that the initial inflammatory response directly correlates to early but not late sepsis mortality. This multifaceted response questions the use of a simple proinflammatory cytokine measurement for classifying the inflammatory status during sepsis.

<em>1</em>. The effects of the IP(3)-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. 2. 2-APB elicited both stimulatory and inhibitory effects on Ca(2+) influx through CRAC channels. At concentrations of <em>1</em>-5 microM, 2-APB enhanced Ca(2+) entry in intact cells and increased I(CRAC) amplitude by up to fivefold. At levels>> or = <em>1</em>0 microM, 2-APB caused a transient enhancement of I(CRAC) followed by inhibition. 3. 2-APB altered the kinetics of fast Ca(2+)-dependent inactivation of I(CRAC). At concentrations of <em>1</em>-5 microM, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations >> or = <em>1</em>0 microM) reduced or completely blocked inactivation. 4. 2-APB inhibited Ca(2+) efflux from mitochondria. 5. 2-APB inhibited I(CRAC) more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. 6. Neither I(CRAC) activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 microg <em>ml</em>(-<em>1</em>) heparin. 7. I(CRAC) is present in wild-type as well as mutant DT40 B cells lacking all three IP(3) receptor isoforms. 2-APB also potentiates and inhibits I(CRAC) in both cell types, indicating that 2-APB exerts its effects independently of IP(3) receptors. 8. Our results show that CRAC channel activation does not require physical interaction with IP(3) receptors as proposed in the conformational coupling model. Potentiation of I(CRAC) by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.

OBJECTIVE

To determine if photodynamic therapy with verteporfin (Visudyne; Novartis AG, Bülach, Switzerland), termed verteporfin therapy, can safely reduce the risk of vision loss compared with a placebo (with sham treatment) in patients with subfoveal choroidal neovascularization caused by age-related macular degeneration who were identified with a lesion composed of occult with no classic choroidal neovascularization, or with presumed early onset classic choroidal neovascularization with good visual acuity letter score.

METHODS

This was a double-masked, placebo-controlled (sham treatment), randomized, multicenter clinical trial involving 28 ophthalmology practices in Europe and North America. The study population was patients with age-related macular degeneration, with subfoveal choroidal neovascularization lesions measuring no greater than 5400 microm in greatest linear dimension with either <em>1</em>) occult with no classic choroidal neovascularization, best-corrected visual acuity score of at least 50 (Snellen equivalent approximately 20/<em>1</em>00), and evidence of hemorrhage or recent disease progression; or 2) evidence of classic choroidal neovascularization with a best-corrected visual acuity score of at least 70 (better than a Snellen equivalent of approximately 20/40); assigned rando<em>ml</em>y (2:<em>1</em>) to verteporfin therapy or placebo therapy. Verteporfin (6 mg per square meter of body surface area) or placebo (5% dextrose in water) was administered by means of intravenous infusion of 30 <em>ml</em> over <em>1</em>0 minutes. Fifteen minutes after the start of the infusion, a laser light at 689 nm delivered 50 J/cm(2) by application of an intensity of 600 mW/cm(2) over 83 seconds using a spot size with a diameter <em>1</em>000 microm larger than the greatest linear dimension of the choroidal neovascularization lesion on the retina. At follow-up examinations every 3 months, retreatment with the same regimen was applied if angiography showed fluorescein leakage. The main outcome measure was at least moderate vision loss, that is, a loss of at least <em>1</em>5 letters (approximately 3 lines), adhering to an intent-to-treat analysis with the last observation carried forward to impute for missing data.

RESULTS

Two hundred ten (93%) and <em>1</em>93 (86%) of the 225 patients in the verteporfin group compared with <em>1</em>04 (9<em>1</em>%) and 99 (87%) of the <em>1</em><em>1</em>4 patients in the placebo group completed the month <em>1</em>2 and 24 examinations, respectively. On average, verteporfin-treated patients received five treatments over the 24 months of follow-up. The primary outcome was similar for the verteporfin-treated and the placebo-treated eyes through the month <em>1</em>2 examination, although a number of secondary visual and angiographic outcomes significantly favored the verteporfin-treated group. Between the month <em>1</em>2 and 24 examinations, the treatment benefit grew so that by the month 24 examination, the verteporfin-treated eyes were less likely to have moderate or severe vision loss. Of the 225 verteporfin-treated patients, <em>1</em>2<em>1</em> (54%) compared with 76 (67%) of <em>1</em><em>1</em>4 placebo-treated patients lost at least <em>1</em>5 letters (P =.023). Likewise, 67 of the verteporfin-treated patients (30%) compared with 54 of the placebo-treated patients (47%) lost at least 30 letters (P =.00<em>1</em>). Statistically significant results favoring verteporfin therapy at the month 24 examination were consistent between the total population and the subgroup of patients with a baseline lesion composition identified as occult choroidal neovascularization with no classic choroidal neovascularization. This subgroup included <em>1</em>66 of the 225 verteporfin-treated patients (74%) and 92 of the <em>1</em><em>1</em>4 placebo-treated patients (8<em>1</em>%). In these patients, 9<em>1</em> of the verteporfin-treated group (55%) compared with 63 of the placebo-treated group (68%) lost at least <em>1</em>5 letters (P =.032), whereas 48 of the verteporfin-treated group (29%) and 43 of the placebo-treated group (47%) lost at least 30 letters (P =.004). Other secondary outcomes, including visual acuity letter score worse than 34 (approximate Snellen equivalent of 20/200 or worse), mean change in visual acuity letter score, development of classic choroidal neovascularization, progression of classic choroidal neovascularization and size of lesion, favored the verteporfin-treated group at both the month <em>1</em>2 and month 24 examination for both the entire study group and the subgroup of cases with occult with no classic choroidal neovascularization at baseline. Subgroup analyses of lesions composed of occult with no classic choroidal neovascularization at baseline suggested that the treatment benefit was greater for patients with either smaller lesions (4 disc areas or less) or lower levels of visual acuity (letter score less than 65, an approximate Snellen equivalent of 20/50(-<em>1</em>) or worse) at baseline. Prospectively planned multivariable analyses confirmed that these two baseline variables affected the magnitude of treatment benefit. (ABSTRACT TRUNCATED)

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is characterized by an accelerated decline in lung function. No drug has been shown conclusively to reduce this decline.

OBJECTIVE

In a post hoc analysis of the Toward a Revolution in COPD Health (TORCH) study, we investigated the effects of combined salmeterol 50 microg plus fluticasone propionate 500 microg, either component alone or placebo, on the rate of post-bronchodilator FEV(<em>1</em>) decline in patients with moderate or severe COPD.

METHODS

A randomized, double-blind, placebo-controlled study was conducted from September 2000 to November 2005 in 42 countries. Of 6,<em>1</em><em>1</em>2 patients from the efficacy population, 5,343 were included in this analysis.

RESULTS

Spirometry was measured every 24 weeks for 3 years. There were 26,539 on-treatment observations. The adjusted rate of decline in FEV(<em>1</em>) was 55 ml/year for placebo, 42 ml/year for salmeterol, 42 ml/year for fluticasone propionate, and 39 ml/year for salmeterol plus fluticasone propionate. Salmeterol plus fluticasone propionate reduced the rate of FEV(<em>1</em>) decline by <em>1</em>6 ml/year compared with placebo (95% confidence interval [CI], 7-25; P < 0.00<em>1</em>). The difference was smaller for fluticasone propionate and salmeterol compared with placebo (<em>1</em>3 ml/year; 95% CI, 5-22; P = 0.003). Rates of decline were similar among the active treatment arms. FEV(<em>1</em>) declined faster in current smokers and patients with a lower body mass index, and varied between world regions. Patients who exacerbated more frequently had a faster FEV(<em>1</em>) decline.

CONCLUSIONS

Pharmacotherapy with salmeterol plus fluticasone propionate, or the components, reduces the rate of decline of FEV(<em>1</em>) in patients with moderate-to-severe COPD, thus slowing disease progression. Clinical trial (GSK Study Code SCO30003) registered with www.clinicaltrials.gov (NCT002682<em>1</em>6).

BACKGROUND

Autologous bone marrow cells (BMCs) transplanted into ventricular scar tissue may differentiate into cardiomyocytes and restore myocardial function. This study evaluated cardiomyogenic differentiation of BMCs, their survival in myocardial scar tissue, and the effect of the implanted cells on heart function.

RESULTS

METHODS

BMCs from adult rats were cultured in cell culture medium (control) and medium with 5-azacytidine (5-aza, <em>1</em>0 micromol/L), TGFbeta<em>1</em> (<em>1</em>0 ng/<em>mL</em>), or insulin (<em>1</em> nmol/L) (n=6, each group). Only BMCs cultured with 5-aza formed myotubules which stained positively for troponin I and myosin heavy chain. In vivo studies: a cryoinjury-derived scar was formed in the left ventricular free wall. At 3 weeks after injury, fresh BMCs (n=9), cultured BMCs (n=9), 5-aza-induced BMCs (n=<em>1</em>2), and medium (control, n=<em>1</em>2) were autologously transplanted into the scar. Heart function was measured at 8 weeks after myocardial injury. Cardiac-like muscle cells which stained positively for myosin heavy chain and troponin I were observed in the scar tissue of the 3 groups of BMC transplanted hearts. Only the 5-aza-treated BMC transplanted hearts had systolic and developed pressures which were higher (P<0.05) than that of the control hearts. All transplanted BMCs induced angiogenesis in the scar.

CONCLUSIONS

Transplantation of BMCs induced angiogenesis. BMCs cultured with 5-aza differentiated into cardiac-like muscle cells in culture and in vivo in ventricular scar tissue and improved myocardial function.

<em>1</em>. Szurszewski (<em>1</em>969) described a cyclic recurring, caudally migrating band of intense action potential activity, the activity fromt, in the small bowel of dogs fasted <em>1</em>8-2<em>1</em> hr. The finding has been confirmed by Carlson, Bedi & Code (<em>1</em>972) and by Grivel & Ruckebusch (<em>1</em>972). The objectives of the present study were to extend these observations first by indentifying the full sequence of myo-electric events in the stomach and small bowel of healthy conscious dogs fasted for 24-48 hr and for longer periods and second by determining the effect of ingestion of mild and of saline solution on the complex and the role of gastric distension in their action. 2. Under surgical anaesthesia, silver-silver chloride electrodes were implanted on the serosal surface of the stomach and small bowel of seven dogs, and recordings of electric activity were started when the dogs had recovered. One hundred and nine interdigestive complexes were studied in detail in five of the dogs during period ranging from 5 to <em>1</em>4 months. All observations were made while the dogs were healthy, conscious, and fasted. 3. The period of intense action potential activity, the activity frot or band, was found to be one phase of a cyclic-recurring sequence of changes in action potential activity. The entire sequence, composed of four phases, occured almost simultaneously in the stomach and duodenun and then migrated distally in sequence over the entire small bowel. As one cycle terminated in the distal ileum, another had started in the stomach and duodenum, and this cyclic recurrence continued during fasts of 4 and 5 days. 4. The cycles of the interdigestive complex tended to recur at the same time each day in three of the dogs. The mean periods of the cycles ranged from 90 to <em>1</em><em>1</em>4 min, and the mean time of their propagation from stomach to terminal ileim ranged from <em>1</em>05 to <em>1</em>34 min. The mean velocity of the activity fronts (phase III of the cycles) was 5-7-<em>1</em><em>1</em>-7 cm/min in the orad portion of the small bowel and 0-9-2-5 cm/min in the distal half. The mean calculated length of the activity front diminished from a range of 42-62 cm in the duodenum to 5-<em>1</em>0 cm in the ileum. 5. Intragastric instillation of 400 <em>ml</em>. milk always interrupted the complex present in the bowel at the time of instillation and usually suppressed the next, whereas 400 <em>ml</em>. saline solution interrupted the complex present in the bowel only at the time of instillation. Distension of the stomach with a ballon always suppressed the interdigestive complex in the stomach and duodenum but sometimes failed to interrupt its migration along the bowe.

Age-related endothelial dysfunction could be caused by an alteration in the L-arginine-NO system and the production of oxidative stress in both normotensive and hypertensive individuals. In 47 normotensive subjects and 49 patients with essential hypertension, we evaluated forearm blood flow (by strain-gauge plethysmography) modifications induced by intrabrachial sodium nitroprusside (<em>1</em>, 2, and 4 microg/<em>1</em>00 <em>mL</em> per minute) and acetylcholine (0.<em>1</em>5, 0.45, <em>1</em>.5, 4.5, and <em>1</em>5 microg/<em>1</em>00 <em>mL</em> per minute), an endothelium-independent vasodilator and an endothelium-dependent vasodilator, respectively. Acetylcholine was repeated in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA, <em>1</em>00 microg/<em>1</em>00 <em>mL</em> per minute), the antioxidant vitamin C (8 mg/<em>1</em>00 <em>mL</em> per minute), or both. Vasodilation to acetylcholine, but not to sodium nitroprusside, was lower (P<0.0<em>1</em>) in hypertensive patients compared with control subjects. Moreover, in both groups, endothelium-dependent vasodilation declined with aging. In normotensive subjects, the inhibiting effect of L-NMMA on response to acetylcholine decreased in parallel with advancing age, whereas vitamin C increased vasodilation to acetylcholine in only the oldest group (age >60 years). In young hypertensive patients (age <30 years), vasodilation to acetylcholine was sensitive to L-NMMA, whereas in hypertensive patients age >30 years, vitamin C enhanced endothelium-dependent vasodilation and restored the inhibiting effect of L-NMMA on response to acetylcholine. In normotensive individuals, an earlier primary dysfunction of the NO system and a later production of oxidative stress cause age-related reduction in endothelium-dependent vasodilation. These alterations are similar but anticipated in hypertensive patients compared with normotensive subjects.

We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for approximately 0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S--NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosothiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at approximately <em>1</em> microM; 60 min after administration of NG-monomethyl-L-arginine at 50 mg/<em>ml</em>, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by approximately 40% (greater than 95% of which were accounted for by S-nitrosoproteins, and approximately 80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone.

PURPOSE To conduct a pilot study to determine the safety, feasibility, and engraftment of haploidentical natural killer (NK) cell infusions after an immunosuppressive regimen in children with acute myeloid leukemia (AML). PATIENTS AND METHODS Ten patients (0.7 to 2<em>1</em> years old) who had completed chemotherapy and were in first complete remission of AML were enrolled on the Pilot Study of Haploidentical Natural Killer Cell Transplantation for Acute Myeloid Leukemia (NKAML) study. They received cyclophosphamide (60 mg/kg on day -7) and fludarabine (25 mg/m(2)/d on days -6 through -2), followed by killer immunoglobulin-like receptor-human leukocyte antigen (KIR-HLA) mismatched NK cells (median, 29 x <em>1</em>0(6)/kg NK cells) and six doses of interleukin-2 (<em>1</em> million U/m(2)). NK cell chimerism, phenotyping, and functional assays were performed on days 2, 7, <em>1</em>4, 2<em>1</em>, and 28 after transplantation. Results All patients had transient engraftment for a median of <em>1</em>0 days (range, 2 to <em>1</em>89 days) and a significant expansion of KIR-mismatched NK cells (median, 5,800/<em>mL</em> of blood on day <em>1</em>4). Nonhematologic toxicity was limited, with no graft-versus-host disease. Median length of hospitalization was 2 days. With a median follow-up time of 964 days (range, 569 to <em>1</em>,<em>1</em>62 days), all patients remain in remission. The 2-year event-free survival estimate was <em>1</em>00% (95% CI, 63.<em>1</em>% to <em>1</em>00%). CONCLUSION Low-dose immunosuppression followed by donor-recipient inhibitory KIR-HLA mismatched NK cells is well tolerated by patients and results in successful engraftment. We propose to further investigate the efficacy of KIR-mismatched NK cells in a phase II trial as consolidation therapy to decrease relapse without increasing mortality in children with AML.

Growth factors released from activated platelets initiate and modulate wound healing in both soft and hard tissues. A recent strategy to promote the wound-healing cascade is to prepare an autologous platelet concentrate suspended in plasma, also known as platelet-rich plasma, that contains growth factors and administer it to wound sites. The purpose of this study was to quantitate platelet number and growth factors released from a prepared platelet concentrate. Whole blood was drawn from <em>1</em>0 healthy patients undergoing cosmetic surgery and concentrated into platelet-rich plasma. Platelet counts on whole blood and platelet-rich plasma were determined using a Cell-Dyn 3200. Platelet-derived growth factor-BB, transforming growth factor-beta<em>1</em>, vascular endothelial growth factor, endothelial growth factor, and insulin-like growth factor-<em>1</em> were measured in the platelet-rich plasma using the enzyme-linked immunosorbent assay method. In addition, platelet activation during the concentration procedure was analyzed by measuring P selectin values in blood serum. An 8-fold increase in platelet concentration was found in the platelet-rich plasma compared with that of whole blood (baseline whole blood, <em>1</em>97 +/- 42 x <em>1</em>0 platelets/microl; platelet concentrate, <em>1</em>600 +/- 330 x <em>1</em>0 platelets/microl). The concentration of growth factors also increased with increasing platelet number. However, growth factor concentration varied from patient to patient. On average for the whole blood as compared with platelet-rich plasma, the platelet-derived growth factor-BB concentration increased from 3.3 +/- 0.9 ng/<em>ml</em> to <em>1</em>7 +/- 8 ng/<em>ml</em>, transforming growth factor-beta<em>1</em> concentration increased from 35 +/- 8 ng/<em>ml</em> to <em>1</em>20 +/- 42 ng/<em>ml</em>, vascular endothelial growth factor concentration increased from <em>1</em>55 +/- <em>1</em><em>1</em>0 pg/<em>ml</em> to 955 +/- <em>1</em>030 pg/<em>ml</em>, and endothelial growth factor concentration increased from <em>1</em>29 +/- 6<em>1</em> pg/<em>ml</em> to 470 +/- 320 pg/<em>ml</em>. No increase was found for insulin-like growth factor-<em>1</em>. In addition, no increase in platelet activation occurred during the concentration procedure as determined by the platelet surface receptor P selectin (45 +/- <em>1</em>6 pg/<em>ml</em> to 52 +/- <em>1</em><em>1</em> pg/<em>ml</em>, p = 0.65). In conclusion, a variety of potentially therapeutic growth factors were detected and released from the platelets in significant levels in platelet-rich plasma preparations. Sufficient concentrates and release of these growth factors through autologous platelet gels may be capable of expediting wound healing in a variety of as yet undetermined specific wound applications.

Proteins potentiated by epidermal growth factor (EGF) to induce a transformed phenotype in non-neoplastic rat kidney fibroblasts in cell culture have been isolated from many non-neoplastic tissues of the adult mouse, including submaxillary gland, kidney, liver, muscle, heart, and brain. They resemble previously described transforming growth factors (TGFs) isolated from neoplastic cells as follows: they are extractable by acid/ethanol and are acid-stable, low molecular weight (6000-<em>1</em>0,000) polypeptides requiring disulfide bonds for activity; and they cause anchorage-independent growth of non-neoplastic indicator cells that will not grow in soft agar in their absence. Partial purification of these TGFs from submaxillary glands of male mice shows that they are distinct from EGF. Unlike previously described extracellular TGFs, but like certain cellular TGFs from neoplastic cells, they are potentiated by EGF in their ability to promote anchorage-independent growth. The isoelectric point of the submaxillary gland TGF protein is near neutrality. Chromatography on Bio-Gel P-30 followed by high-pressure liquid chromatography has resulted in a 22,000-fold overall purification. The most purified protein is active in inducing growth in soft agar at <em>1</em> ng/<em>ml</em> when assayed in the presence of EGF. The data add further evidence to the concept that neoplasia may result from a quantitative, rather than qualitative, alteration in non-neoplastic biochemical processes.

BACKGROUND

Insulin resistance is a risk factor for type 2 diabetes and cardiovascular disease progression. Current diagnostic tests, such as glycemic indicators, have limitations in the early detection of insulin resistant individuals. We searched for novel biomarkers identifying these at-risk subjects.

METHODS

Using mass spectrometry, non-targeted biochemical profiling was conducted in a cohort of 399 nondiabetic subjects representing a broad spectrum of insulin sensitivity and glucose tolerance (based on the hyperinsulinemic euglycemic clamp and oral glucose tolerance testing, respectively).

RESULTS

Random forest statistical analysis selected alpha-hydroxybutyrate (alpha-HB) as the top-ranked biochemical for separating insulin resistant (lower third of the clamp-derived M(FFM) = 33 [<em>1</em>2] micromol x min(-<em>1</em>) x kg(FFM) (-<em>1</em>), median [interquartile range], n = <em>1</em>40) from insulin sensitive subjects (M(FFM) = 66 [23] micromol x min(-<em>1</em>) x kg(FFM) (-<em>1</em>)) with a 76% accuracy. By targeted isotope dilution assay, plasma alpha-HB concentrations were reciprocally related to M(FFM); and by partition analysis, an alpha-HB value of 5 microg/<em>ml</em> was found to best separate insulin resistant from insulin sensitive subjects. alpha-HB also separated subjects with normal glucose tolerance from those with impaired fasting glycemia or impaired glucose tolerance independently of, and in an additive fashion to, insulin resistance. These associations were also independent of sex, age and BMI. Other metabolites from this global analysis that significantly correlated to insulin sensitivity included certain organic acid, amino acid, lysophospholipid, acylcarnitine and fatty acid species. Several metabolites are intermediates related to alpha-HB metabolism and biosynthesis.

CONCLUSIONS

alpha-hydroxybutyrate is an early marker for both insulin resistance and impaired glucose regulation. The underlying biochemical mechanisms may involve increased lipid oxidation and oxidative stress.

Since the initial proposal of the glucose fatty acid cycle, considerable controversy has arisen concerning its physiologic significance in vivo. In the present study, we examined the effect of acute, physiologic elevations of FFA concentrations on glucose production and uptake in normal subjects under three controlled experimental conditions. In group A, plasma insulin levels were raised and maintained at approximately <em>1</em>00 microU/<em>ml</em> above base line by an insulin infusion, while holding plasma glucose at the fasting level by a variable glucose infusion. In group B, plasma glucose concentration was raised by <em>1</em>25 mg/<em>1</em>00 <em>ml</em> and plasma insulin was clamped at approximately 50 microU/<em>ml</em> by a combined infusion of somatostatin and insulin. In group C, plasma glucose was raised by 200 mg/<em>1</em>00 <em>ml</em> above the fasting level, while insulin secretion was inhibited with somatostatin and peripheral glucagon levels were replaced with a glucagon infusion (<em>1</em> ng/min X kg). Each protocol was repeated in the same subject in combination with a lipid-heparin infusion designed to raise plasma FFA levels by <em>1</em>.5-2.0 mumol/<em>ml</em>. With euglycemic hyperinsulinemia (study A), lipid infusion caused a significant inhibition of total glucose uptake (6.3 +/- <em>1</em>.3 vs. 7.4 +/- 0.6 mg/min X kg, P less than 0.02). Endogenous glucose production (estimated by the [3-3H]glucose technique) was completely suppressed both with and without lipid infusion. With hyperglycemic hyperinsulinemia (study B), lipid infusion also induced a marked impairment in glucose utilization (6.2 +/- <em>1</em>.<em>1</em> vs. 9.8 +/- <em>1</em>.9 mg/min X kg, P less than 0.05); endogenous glucose production was again completely inhibited despite the increase in FFA concentrations. Under both conditions (A and B), the percentage inhibition of glucose uptake by FFA was positively correlated with the total rate of glucose uptake (r = 0.69, P less than 0.0<em>1</em>). In contrast, when hyperglycemia was associated with relative insulinopenia and hyperglucagonemia (study C), thus simulating a diabetic state, lipid infusion had no effect on glucose uptake (2.9 +/- 0.2 vs. 2.6 +/- 0.2 mg/min X kg) but markedly stimulated endogenous glucose production (<em>1</em>.4 +/- 0.5 vs. 0.5 +/- 0.4 mg/min X kg, P less than 0.005). Under the same conditions as study C, a glycerol infusion producing plasma glycerol levels similar to those achieved with lipid-heparin, enhanced endogenous glucose production (<em>1</em>.5 +/- 0.5 vs. 0.7 +/- 0.6 mg/min X kg, P less than 0.05). We conclude that, in the well-insulinized state raised FFA levels effectively compete with glucose for uptake by peripheral tissues, regardless of the presence of hyperglycemia. When insulin is deficient, on the other hand, elevated rates of lipolysis may contribute to hyperglycemia not by competition for fuel utilization, but through an enhancement of endogenous glucose output.

BACKGROUND

Racial differences in tobacco-related diseases are not fully explained by cigarette-smoking behavior. Despite smoking fewer cigarettes per day, blacks have higher levels of serum cotinine, the proximate metabolite of nicotine.

OBJECTIVE

To compare the rates of metabolism and the daily intake of nicotine in black smokers and white smokers.

METHODS

Participants received simultaneous infusions of deuterium-labeled nicotine and cotinine. Urine was collected for determination of total clearance of nicotine and cotinine, fractional conversion of nicotine to cotinine, and cotinine elimination rate. Using cotinine levels during ad libitum smoking and clearance data, the daily intake of nicotine from smoking was estimated.

METHODS

Metabolic ward of a university-affiliated public hospital.

METHODS

A total of 40 black and 39 white smokers, average consumption of <em>1</em>4 and <em>1</em>4.7 cigarettes per day, respectively, of similar age (mean, 32.5 and 32.3 years, respectively) and body weight (mean, 73.3 and 68.8 kg, respectively).

METHODS

Clearance (renal and nonrenal), half-life, and volume of distribution of nicotine and cotinine and the calculated daily intake of nicotine.

RESULTS

The total and nonrenal clearances of nicotine were not significantly different, respectively, in blacks (<em>1</em>7.7 and <em>1</em>7.2 <em>mL</em> x min(-<em>1</em>) x kg(-<em>1</em>)) compared with whites (<em>1</em>9.6 and <em>1</em>8.9 <em>mL</em> x min(-<em>1</em>) x kg(-<em>1</em>)) (P=.<em>1</em><em>1</em> and .20). However, the total and nonrenal clearances of cotinine were significantly lower, respectively, in blacks (0.56 and 0.47 <em>mL</em> x min(-<em>1</em>) x kg(-<em>1</em>)) than in whites (0.68 vs 0.6<em>1</em> <em>mL</em> x min(-<em>1</em>) x kg(-<em>1</em>); P=.009 for each comparison). The nicotine intake per cigarette was 30% greater in blacks compared with whites (<em>1</em>.4<em>1</em> vs <em>1</em>.09 mg per cigarette, respectively; P=.02). Volume of distribution did not differ for the 2 groups, but cotinine half-life was higher in blacks than in whites (<em>1</em>064 vs 950 minutes, respectively; P = .07).

CONCLUSIONS

Higher levels of cotinine per cigarette smoked by blacks compared with whites can be explained by both slower clearance of cotinine and higher intake of nicotine per cigarette in blacks. Greater nicotine and therefore greater tobacco smoke intake per cigarette could, in part, explain some of the ethnic differences in smoking-related disease risks.

There is substantial evidence supporting the CD4 molecule as the principal cellular receptor for the human immunodeficiency virus type <em>1</em> (HIV-<em>1</em>). A number of truncated recombinant soluble CD4 (sCD4) molecules have been produced and shown to easily neutralize infection of laboratory strains of HIV-<em>1</em> in vitro, and clinical trials using these sCD4 preparations have begun in patients with AIDS. Infectious HIV-<em>1</em> titers in the plasma and peripheral blood mononuclear cells of five patients receiving sCD4 at 30 mg/day were sequentially monitored. No significant decrease in viral titers was found during therapy. Furthermore, plasma samples from eight patients with AIDS were titrated for HIV-<em>1</em> with and without the addition of sCD4 ex vivo. Despite the addition of sCD4 at up to <em>1</em> mg/<em>ml</em>, there was little change in plasma viral titers. Subsequently, <em>1</em>0 primary HIV-<em>1</em> isolates were tested for their susceptibility to neutralization in vitro by one preparation of sCD4. Neutralization of these clinical isolates required 200-2700 times more sCD4 than was needed to inhibit laboratory strains of HIV-<em>1</em>. Similar results were observed using one other monomeric sCD4 preparation and two multimeric CD4-immunoglobulin hybrid molecules. We conclude that unlike laboratory strains, primary HIV-<em>1</em> isolates require high concentrations of sCD4 for neutralization. This phenomenon may pose a formidable problem for sCD4-based therapeutics in the treatment of HIV-<em>1</em> infection.

BACKGROUND

Tidal volume and plateau pressure limitation decreases mortality in acute respiratory distress syndrome. Computed tomography demonstrated a small, normally aerated compartment on the top of poorly aerated and nonaerated compartments that may be hyperinflated by tidal inflation.

OBJECTIVE

We hypothesized that despite tidal volume and plateau pressure limitation, patients with a larger nonaerated compartment are exposed to tidal hyperinflation of the normally aerated compartment.

RESULTS

Pulmonary computed tomography at end-expiration and end-inspiration was obtained in 30 patients ventilated with a low tidal volume (6 <em>ml</em>/kg predicted body weight). Cluster analysis identified 20 patients in whom tidal inflation occurred largely in the normally aerated compartment (69.9 +/- 6.9%; "more protected"), and <em>1</em>0 patients in whom tidal inflation occurred largely within the hyperinflated compartments (63.0 +/- <em>1</em>2.7%; "less protected"). The nonaerated compartment was smaller and the normally aerated compartment was larger in the more protected patients than in the less protected patients (p = 0.0<em>1</em>). Pulmonary cytokines were lower in the more protected patients than in the less protected patients (p < 0.05). Ventilator-free days were 7 +/- 8 and <em>1</em> +/- 2 d in the more protected and less protected patients, respectively (p = 0.0<em>1</em>). Plateau pressure ranged between 25 and 26 cm H(2)O in the more protected patients and between 28 and 30 cm H(2)O in the less protected patients (p = 0.006).

CONCLUSIONS

Limiting tidal volume to 6 ml/kg predicted body weight and plateau pressure to 30 cm H(2)O may not be sufficient in patients characterized by a larger nonaerated compartment.

A peptide designated DP-<em>1</em>07 was synthesized containing amino acid residues 558-595 of the envelope glycoprotein gp<em>1</em>60 of human immunodeficiency virus type <em>1</em> strain LAI (HIV-<em>1</em>LAI). Algorithms for secondary structure have predicted that this region of the envelope transmembrane protein should form an extended alpha-helix. Consistent with this prediction, analysis by circular dichroism (CD) indicated that, under physiological conditions, DP-<em>1</em>07 is approximately 85% helical. The high degree of stable secondary structure in a synthetic peptide of this size suggests self-association typical of a coiled coil or leucine zipper. In biological assays, the peptide efficiently blocked virus-mediated cell-cell fusion processes as well as infection of peripheral blood mononuclear cells by both prototypic and primary isolates of HIV-<em>1</em>. A single amino acid substitution in the peptide greatly destabilized its solution structure as measured by CD and abrogated its antiviral activity. An analogue containing a terminal cysteine was oxidized to form a dimer, and this modification lowered the dose required for antiviral effect from 5 to about <em>1</em> microgram/<em>ml</em>. These results suggest that both oligomerization and ordered structure are necessary for biological activity. They provide insights also into the role of this region in HIV infection and the potential for development of a new class of antiviral agents.

OBJECTIVE

The established chronic kidney disease (CKD) progression end point of end-stage renal disease (ESRD) or a doubling of serum creatinine concentration (corresponding to a change in estimated glomerular filtration rate [GFR] of −57% or greater) is a late event.

OBJECTIVE

To characterize the association of decline in estimated GFR with subsequent progression to ESRD with implications for using lesser declines in estimated GFR as potential alternative end points for CKD progression. Because most people with CKD die before reaching ESRD, mortality risk also was investigated.

METHODS

Individual meta-analysis of <em>1</em>.7 million participants with <em>1</em>2,344 ESRD events and 223,944 deaths from 35 cohorts in the CKD Prognosis Consortium with a repeated measure of serum creatinine concentration over <em>1</em> to 3 years and outcome data.

METHODS

Transfer of individual participant data or standardized analysis of outputs for random-effects meta-analysis conducted between July 20<em>1</em>2 and September 20<em>1</em>3, with baseline estimated GFR values collected from <em>1</em>975 through 20<em>1</em>2.

METHODS

End-stage renal disease (initiation of dialysis or transplantation) or all-cause mortality risk related to percentage change in estimated GFR over 2 years, adjusted for potential confounders and first estimated GFR.

RESULTS

The adjusted hazard ratios (HRs) of ESRD and mortality were higher with larger estimated GFR decline. Among participants with baseline estimated GFR of less than 60 mL/min/<em>1</em>.73 m2, the adjusted HRs for ESRD were 32.<em>1</em> (95% CI, 22.3-46.3) for changes of −57% in estimated GFR and 5.4 (95% CI, 4.5-6.4) for changes of −30%. However, changes of −30% or greater (6.9% [95% CI, 6.4%-7.4%] of the entire consortium) were more common than changes of −57% (0.79% [95% CI, 0.52%-<em>1</em>.06%]). This association was strong and consistent across the length of the baseline period (<em>1</em> to 3 years), baseline estimated GFR, age, diabetes status, or albuminuria. Average adjusted <em>1</em>0-year risk of ESRD (in patients with a baseline estimated GFR of 35 mL/min/<em>1</em>.73 m2) was 99% (95% CI, 95%-<em>1</em>00%) for estimated GFR change of −57%, was 83% (95% CI, 7<em>1</em>%-93%) for estimated GFR change of −40%, and was 64% (95% CI, 52%-77%) for estimated GFR change of −30% vs <em>1</em>8% (95% CI, <em>1</em>5%-22%) for estimated GFR change of 0%. Corresponding mortality risks were 77% (95% CI, 7<em>1</em>%-82%), 60% (95% CI, 56%-63%), and 50% (95% CI, 47%-52%) vs 32% (95% CI, 3<em>1</em>%-33%), showing a similar but weaker pattern.

CONCLUSIONS

Declines in estimated GFR smaller than a doubling of serum creatinine concentration occurred more commonly and were strongly and consistently associated with the risk of ESRD and mortality, supporting consideration of lesser declines in estimated GFR (such as a 30% reduction over 2 years) as an alternative end point for CKD progression.

BACKGROUND

Autosomal dominant polycystic kidney disease (ADPKD) is a slowly progressive hereditary disorder that usually leads to end-stage renal disease. Although the underlying gene mutations were identified several years ago, efficacious therapy to curtail cyst growth and prevent renal failure is not available. Experimental and observational studies suggest that the mammalian target of rapamycin (mTOR) pathway plays a critical role in cyst growth.

METHODS

In this 2-year, double-blind trial, we rando<em>ml</em>y assigned 433 patients with ADPKD to receive either placebo or the mTOR inhibitor everolimus. The primary outcome was the change in total kidney volume, as measured on magnetic resonance imaging, at <em>1</em>2 and 24 months.

RESULTS

Total kidney volume increased between baseline and <em>1</em> year by <em>1</em>02 <em>ml</em> in the everolimus group, versus <em>1</em>57 <em>ml</em> in the placebo group (P=0.02) and between baseline and 2 years by 230 <em>ml</em> and 30<em>1</em> <em>ml</em>, respectively (P=0.06). Cyst volume increased by 76 <em>ml</em> in the everolimus group and 98 <em>ml</em> in the placebo group after <em>1</em> year (P=0.27) and by <em>1</em>8<em>1</em> <em>ml</em> and 2<em>1</em>5 <em>ml</em>, respectively, after 2 years (P=0.28). Parenchymal volume increased by 26 <em>ml</em> in the everolimus group and 62 <em>ml</em> in the placebo group after <em>1</em> year (P=0.003) and by 56 <em>ml</em> and 93 <em>ml</em>, respectively, after 2 years (P=0.<em>1</em><em>1</em>). The mean decrement in the estimated glomerular filtration rate after 24 months was 8.9 <em>ml</em> per minute per <em>1</em>.73 m2 of body-surface area in the everolimus group versus 7.7 <em>ml</em> per minute in the placebo group (P=0.<em>1</em>5). Drug-specific adverse events were more common in the everolimus group; the rate of infection was similar in the two groups.

CONCLUSIONS

Within the 2-year study period,as compared with placebo, everolimus slowed the increase in total kidney volume of patients with ADPKD but did not slow the progression of renal impairment [corrected]. (Funded by Novartis; EudraCT number, 2006-00<em>1</em>485-<em>1</em>6; ClinicalTrials.gov number, NCT004<em>1</em>4440.)

Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 5<em>1</em> HAdV prototypes. Sensitivity of the assay was <or=<em>1</em>5 copies/run and the linear range of quantitation <em>1</em>.5 x <em>1</em>0(<em>1</em>) to <em>1</em>.5 x <em>1</em>0(8) copies/run. TaqMan PCR gave identical results compared to an established conventional one-step PCR protocol in 2<em>1</em>8 (38 positive and <em>1</em>80 negative) of 234 clinical samples including blood, serum, eye swabs, and feces, and had divergent results in <em>1</em>6 samples (<em>1</em>5 positive only in TaqMan PCR, all with low copy numbers, and one positive only in conventional PCR), indicating a higher sensitivity of TaqMan PCR. Adenovirus viremia was detected by TaqMan PCR in 4 of 27 (<em>1</em>4.8%) paediatric and 8 of 93 (8.6%) adult stem cell transplant recipients but only in 5 of 306 healthy controls (blood donors, <em>1</em>.6%). Virus loads of pediatric patients (median <em>1</em>.7 x <em>1</em>0(5)) were significantly higher than in adult patients (median 2.3 x <em>1</em>0(3)) and than in controls (all samples <or=<em>1</em>.7 x <em>1</em>0(3) copies/<em>ml</em>). A few immunosuppressed children had very high virus loads (up to <em>1</em>.<em>1</em> x <em>1</em>0(<em>1</em>0) copies/<em>ml</em>), which were associated with symptoms of disseminated disease. In conclusion, real-time PCR is a sensitive and quantitative procedure for the detection of adenovirus infections.