Citations

Background and Purpose— Recombinant-activated factor VII (rFVIIa) is an investigational treatment for intracerebral hemorrhage (ICH). We have evaluated the drug’s treatment effect based on time to treatment. Methods— ICH patients treated up to 4 hours from symptom onset were divided based on time to treatment: ≤3 hours (3H) and 3 to 4 hours (4H). Head CT was done at baseline and 24 hours. Outcome measures included: ICH growth at 24 hours, mortality, favorable outcome and discharge disposition. A cohort of nontreated matched ICH patients was used to asses the clinical efficacy. Results— Forty-six patients were treated with rFVIIa: 24 in the 3H group (range 70 to 180 minutes), 22 in the 4H group (range 181 to 300). One hundred and forty-eight patients formed the control group. Mean baseline ICH volume was 8.8 mL for 3H and 10.1 mL for 4H. Mean 24-hour volume was 9.3 mL for 3H (absolute increase 1.05 mL, relative increase 11.9%) and 11.5 mL for 4H (absolute increase 1.1 mL, relative increase 10.9%); P =0.47 is for the difference in relative increase. Mortality was 12.5% for 3H group, 13.6% for 4H, and 13.1% for the control. In the 3H group, 58.3% were discharged with a poor outcome, compared with 54.5% in 4H and 54.1% in the control. Thrombotic adverse events occurred in 11.1% of patients treated with rFVIIa. Conclusions— In our off-label with rFVIIa, we did not find evidence of a treatment effect based on time to treatment. Other criteria should be sought to identify patients who might benefit clinically from rFVIIa.

We report 2 patients with isolated 3-methylcrotonyl-coenzyme A carboxylase deficiency whose urine was devoid of, or contained only trace, 3-methylcrotonylglycine, the pathognomonic marker for this disorder. The first patient, a girl with trisomy 21, was detected through newborn screening with an elevated 5 carbon hydroxycarnitine species level, and the second patient came to clinical attention at the age of 5 months because of failure to thrive and developmental delay. Investigation of urinary organic acids revealed an elevated 3-hydroxyisovaleric acid level but no demonstrable 3-methylcrotonylglycine in both patients. Enzyme studies in cultured fibroblasts confirmed isolated 3-methylcrotonyl-coenzyme A carboxylase deficiency with residual activities of 5% to 7% and 12% of the median control value, respectively. Incorporation of 14C-isovaleric acid into intact fibroblasts was essentially normal, showing that the overall pathway was at least partially functional and potentially explaining the absence of 3-methylcrotonylglycine in urine. Mutation analysis of the MCCA and MCCB genes revealed that both patients were compound heterozygous for a missense mutation, MCCB-c.1015G→A (p.V339M), and a second mutation that leads to undetectable MCCB messenger (poly A+) RNA. Absent or trace 3-methylcrotonylglycine levels in urine raises the potential for misdiagnosis in the clinical biochemical genetics laboratory based solely on urine organic acid analysis using combined gas chromatography-mass spectrometry.

AbstractPurpose: Passive immunotherapy with antitumor antibodies has the potential to induce active tumor immunity via the opsonic enhancement of immunogenicity of tumor antigen. We have assessed whether immune sensitization to the HER-2/neu tumor antigen occurs during treatment with the anti-HER-2/neu monoclonal antibody trastuzumab.Experimental Design: Twenty-seven patients treated with trastuzumab and chemotherapy were assessed for the induction of HER-2/neu–specific immunity. Sera and peripheral blood mononuclear cells obtained before and after trastuzumab therapy were compared for the presence of anti-HER-2/neu endogenous Igλ antibodies and HER-2/neu–specific CD4 responses by ELISA and enzyme-linked immunospot, respectively.Results: Anti-HER-2/neu antibodies were detectable in 8 of 27 (29%) patients before trastuzumab treatment and in 15 of 27 (56%) patients during trastuzumab treatment. In the overall study population, anti-HER-2/neu humoral responses significantly increased during therapy (P < 0.001) and were not associated with development of an anti-idiotypic response. In 10 evaluable individuals, 6 showed augmented HER-2/neu–specific CD4 T-cell responses during therapy. Of the 22 individuals treated for metastatic disease, those patients showing objective clinical responses exhibited more frequent (P = 0.004) and larger (P = 0.006) treatment-associated anti-HER-2/neu humoral responses.Conclusion: Humoral immune sensitization occurs during treatment with chemotherapy and trastuzumab. Further studies are warranted to investigate whether augmented anti-HER-2/neu humoral and cellular immunity contributes mechanistically to clinical outcome.

Proteolytic processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid β (Aβ) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP C ), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease in humans, remains enigmatic. Because both APP and PrP C are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP C in the proteolytic processing of APP. Cellular overexpression of PrP C inhibited the β-secretase cleavage of APP and reduced Aβ formation. Conversely, depletion of PrP C in mouse N2a cells by siRNA led to an increase in Aβ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Aβ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the β-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP C on the β-secretase cleavage of APP required the localization of PrP C to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP C via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Aβ is regulated by PrP C and may have implications for both Alzheimer's and prion diseases.

Suppression of colon carcinogenesis by peroxisome proliferator-activated receptor (PPAR)-γ is likely due to some effect of PPAR-γ on normal colonic epithelial cells. However, our understanding of the effects of PPAR-γ in such cells is limited. We analyzed the abundance, distribution, and function of PPAR-γ in epithelial cells isolated from the murine proximal and distal colon. Marked differences in PPAR-γ abundance and distribution were observed, suggesting tissue-specific responses. Analysis of PPAR-γ effects on DNA synthesis, formation of preneoplastic lesions, and activation of MAPK signaling in proximal and distal colonic epithelial cells in vivo indicates that PPAR-γ regulates both tissue-specific and common responses within the proximal and distal colon. Three major functional cohorts of PPAR-γ target genes were identified by genomic profiling of isolated colonic epithelial cells: genes that are involved in metabolism, in signaling, and in cellular adhesion and motility. Two subsets of PPAR-γ target genes were differentially expressed in the proximal and distal epithelium. Proximal target genes were primarily involved in metabolic activities, whereas signal transduction, adhesion, and motility targets were more pronounced in the distal colon. Remarkably, those target genes that are differentially expressed in the proximal colon were all induced on activation of PPAR-γ, whereas all target genes that are preferentially expressed in the distal colon were repressed. Our data indicate that PPAR-γ exerts both common and tissue-specific effects in the colon and challenge the general conclusions that PPAR-γ is induced on differentiation of colonic epithelial cells and that this receptor stimulates differentiated function in epithelial cells throughout the colon.

ABSTRACT Influenza A virus was detected at higher rates and for more extended time periods with real-time PCR than with cell cultures. We show here that, using the theranostic approach, rapid viral detection and reporting can provide for early implementation and assessment of available antiviral therapy.

Background— Synthetic vascular grafts cannot be used in small vessels because of graft failure caused by thrombosis and neointima formation. Rapid endothelialization may overcome this limitation. We hypothesized that a magnetic graft would be able to capture and retain endothelial cells labeled with paramagnetic particles. Methods and Results— Porcine blood derived endothelial cells were allowed to endocytose superparamagnetic iron oxide microspheres. Cell survival was assessed by trypan blue exclusion and demonstrated a dose-dependent cell survival of 75% to 95%. A flexible magnetic sheet was annealed to the external surface of a knitted Dacron graft. Labeled cells (10 6 /mL) were placed within the graft for 5 minutes. Confocal and electron microscopy confirmed uniform cell capture at the magnetized surface. The effect of shear forces on the adherent cells was evaluated in a flow chamber. The cells remained attached at rates up to 300 mL/min, with cell loss commencing at 400 mL/min. Prototype magnetic grafts were implanted in porcine carotid arteries. Labeled cells were placed within the graft for 10 minutes at the time of implantation. The grafts were evaluated after one day and uniform cell coverage was noted on the magnetized surface. In comparison, relatively few labeled cells were seen attached to a nonmagnetized surface. Conclusions— Magnetic forces can be used to rapidly cover a vascular graft with paramagnetically labeled cells. This biophysical interaction is sufficient to retain cells in the presence of blood flow. Applications of this technique may include rapid endothelialization of synthetic vascular grafts and dialysis fistulas.

Abstract We recently showed that atypical protein kinase Cι (PKCι) is required for transformed growth of human non–small-cell lung cancer (NSCLC) cells by activating Rac1. Genetic disruption of PKCι signaling blocks Rac1 activity and transformed growth, indicating that PKCι is a viable target for development of novel therapeutics for NSCLC. Here, we designed and implemented a novel fluorescence resonance energy transfer–based assay to identify inhibitors of oncogenic PKCι signaling. This assay was used to identify compounds that disrupt the interaction between PKCι and its downstream effector Par6, which links PKCι to Rac1. We identified aurothioglucose (ATG), a gold compound used clinically to treat rheumatoid arthritis, and the related compound, aurothiomalate (ATM), as potent inhibitors of PKCι-Par6 interactions in vitro (IC50 ∼1 μmol/L). ATG blocks PKCι-dependent signaling to Rac1 and inhibits transformed growth of NSCLC cells. ATG-mediated inhibition of transformation is relieved by expression of constitutively active Rac1, consistent with a mechanism at the level of the interaction between PKCι and Par6. ATG inhibits A549 cell tumor growth in nude mice, showing efficacy against NSCLC in a relevant preclinical model. Our data show the utility of targeting protein-protein interactions involving PKCι for antitumor drug development and provide proof of concept that chemical disruption of PKCι signaling can be an effective treatment for NSCLC. ATG and ATM will be useful reagents for studying PKCι function in transformation and represent promising new agents for the clinical treatment of NSCLC. (Cancer Res 2006; 66(3): 1767-74)