Citations

ABSTRACTLatently human immunodeficiency virus (HIV)-infected memory CD4+T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCθ, PKCθA148E, and PKCα, PKCαA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4+T cells and that both PKCθA148E and PKCαA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCα and PKCθ with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCα or PKCθ. Since suppression of PKCα and PKCθ together has no greater inhibitory effect on HIV reactivation than inhibition of PKCα alone, our data confirm that PKCα and PKCθ act in sequence. The requirement for PKCα and PKCθ for prostratin-induced HIV reactivation and the ability of selective PKCα or PKCθ agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.

ABSTRACTThe human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90°; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

AbstractOBJECTIVE:Olfaction is often sacrificed to gain access to the cranial base in anterior craniofacial surgery. We describe the long-term results of olfactory function in patients who underwent anterior craniofacial surgery and a cribriform plate osteotomy to preserve olfaction.METHODS:Between 1992 and 2004, 28 patients underwent 29 cribriform plate osteotomies in an attempt to preserve olfaction during anterior craniofacial surgery performed through modified extended transbasal approaches. Patients' charts and office notes were reviewed retrospectively. Formal olfactory testing was available in 5 patients, but most data were based on patients' subjective reports of olfaction. Olfactory preservation was defined by the subjective ability to detect fumes such as coffee, chocolate, roses, and orange juice regardless of the intensity of the sensation. Follow-up was based on phone calls to patients.RESULTS:Four patients were lost to follow-up and excluded. Therefore, follow-up was available in 24 patients after 25 procedures. On the basis of patients' subjective reports, olfaction was spared in 22 patients after 23 procedures (92%) and was confirmed objectively in the five patients formally tested. After surgery, only two patients were anosmic.CONCLUSION:Olfaction can be preserved in selected patients undergoing anterior craniofacial surgery. At least 1 cm of nasal mucosa should remain attached to the cribriform plate, which can be achieved by including the nasal bone in the osteotomy of the orbital bar. A medial orbital canthopexy is therefore necessary after these procedures.

The protozoan parasite Toxoplasma gondii relies on calcium-mediated exocytosis to secrete adhesins on to its surface where they can engage host cell receptors. Increases in intracellular calcium occur in response to Ins(1,4,5)P3 and caffeine, an agonist of ryanodine-responsive calcium-release channels. We examined lysates and microsomes of T. gondii and detected evidence of cADPR (cyclic ADP ribose) cyclase and hydrolase activities, the two enzymes that control the second messenger cADPR, which causes calcium release from RyR (ryanodine receptor). We also detected endogenous levels of cADPR in extracts of T. gondii. Furthermore, T. gondii microsomes that were loaded with 45Ca2+ released calcium when treated with cADPR, and the RyR antagonists 8-bromo-cADPR and Ruthenium Red blocked this response. Although T. gondii microsomes also responded to Ins(1,4,5)P3, the inhibition profiles of these calcium-release channels were mutually exclusive. The RyR antagonists 8-bromo-cADPR and dantrolene inhibited protein secretion and motility in live parasites. These results indicate that RyR calcium-release channels that respond to the second-messenger cADPR play an important role in regulating intracellular Ca2+, and hence host cell invasion, in protozoan parasites.

Here we examine the use of glycopeptides containing tumour‐associated carbohydrate antigens (TACA) as potential preventive vaccines for carcinomas. Our recent results suggest that CD8+ T cells (CTL) are capable of recognizing TACA in a conventional class I MHC‐restricted fashion. The Thomsen–Friedenreich antigen (TF), a disaccharide, and Tn, its immediate precursor, are TACA largely expressed in carcinomas. TF and Tn can be successfully used as Th‐independent vaccines when conjugated to designer peptides with optimal binding affinity for class I MHC molecules. TF‐ and Tn‐specific CTL generated using this strategy are capable of recognizing TACA‐expressing tumours in vitro, suggesting that glycopeptides are as effectively presented by class I MHC molecules as non‐glycosylated peptides. Because the exact sequences of endogenously synthesized glycopeptides are unknown, the TACA‐specific T cell repertoire elicited by carbohydrate‐based vaccines is assumed to be degenerate. Here we report that mice genetically manipulated to develop TACA‐expressing mammary tumours are not tolerant to glycopeptide vaccination. Moreover, we tested the immunogenicity of designer glycopeptides capable of binding multiple HLA alleles as a novel approach for the development of vaccines potentially useful for vaccination of a large fraction of the general population. Our results have suggested that CTL derived from normal donors respond with high efficiency to glycopeptides in vitro, opening a new avenue for the design of prospective vaccines for cancer prevention.

PurposeAlthough rare overall, marginal zone B-cell lymphoma (MZBCL) is the most common primary low-grade CNS lymphoma reported in the literature. The aim of this study is to elucidate the biology and genetic features of this unusual tumor.Patients and MethodsFifteen CNS MZBCLs were studied clinically, pathologically, and genetically, including fluorescent in situ hybridization analyses with commercially available MALT1 and IgH break-apart and centromere 3, 7, 12, and 18 probes.ResultsCNS MZBCLs preferentially affect middle-aged women (female-to-male ratio, 4:1), with 93% presenting as dural-based masses mimicking meningioma. Ten patients with 1 to 7.6 years of follow-up after diagnosis showed no evidence of disease after radiation and/or chemotherapy. Like MZBCLs outside of the CNS, they consisted of CD20+, CD3−small B lymphocytes with varying degrees of plasmacytic differentiation and predominantly κ light-chain restriction (78%). Lymphoid follicles with follicular colonization were seen in three patients and deposition of amyloid was noted in samples from two patients, one of which was tumefactive. Neither Bcl-6 protein nor Epstein-Barr virus–encoded RNA was expressed. Trisomy 3 was detected in six of 12 patients, with no rearrangements of MALT1 or IgH and no trisomies of 7, 12, or 18 detected.ConclusionOur data suggest that intracranial MZBCL is an indolent primary CNS lymphoma that typically presents as a meningioma-like dural-based mass. Trisomy 3, but not MALT1 or IgH translocation, is a common genetic abnormality that may contribute to the pathogenesis of this CNS lymphoma.

Purpose This phase I study was undertaken to define the toxicity, pharmacokinetics, pharmacodynamics, maximum tolerated dose (MTD), and clinical activity of CI-1040, a small-molecule inhibitor of the dual-specificity kinases MEK(mitogen-activated protein kinase kinase) -1 and MEK2 , in patients with advanced malignancy. Patients and Methods CI-1040 was tested in multiple daily dosing frequencies administered for 21 days repeated every 28 days leading ultimately to continuous administration, and effect of food on absorption was tested. Single dose and steady-state pharmacokinetics were assessed during cycle 1 and phosphorylated extracellular receptor kinase (pERK) levels were assessed in WBCs and also in tumor tissue from selected patients. Results Seventy-seven patients received CI-1040 at dose levels ranging from 100 mg QD to 800 mg tid. Grade 3 asthenia was dose limiting at the highest dose level tested, 800 mg tid administered with food. Ninety-eight percent of all drug-related adverse events were grade 1 or 2 in severity; most common toxicities included diarrhea, asthenia, rash, nausea, and vomiting. Plasma concentrations of CI-1040 and its active metabolite, PD 0184264, increased in a less than dose proportional manner from 100 to 800 mg QD. Administration with a high-fat meal resulted in an increase in drug exposure. The MTD and recommended phase II dose was 800 mg BID administered with food. Sixty-six patients were assessable for response. One partial response was achieved in a patient with pancreatic cancer and 19 patients (28%) achieved stable disease lasting a median of 5.5 months (range, 4 to 17 months). Inhibition of tumor pERK (median, 73%; range, 46% to 100%) was demonstrated in 10 patients. Conclusion CI-1040 was well tolerated at 800 mg BID administered with food. Both target suppression and antitumor activity were demonstrated in this phase I study.

Abstract Context: Cortisol is a potent regulator of carbohydrate, fat, and protein metabolism. Objective: The objective of the study was to determine whether obesity alone or in combination with type 2 diabetes increases splanchnic and/or leg cortisol production. Design: Splanchnic and leg cortisol production were measured using the hepatic and leg catheterization technique combined with infusion of D4-cortisol. Setting: The study was conducted in a General Clinical Research Center. Participants: Nine lean nondiabetic, 10 obese nondiabetic, and 11 obese diabetic subjects were studied. Interventions: Diabetic volunteers were withdrawn from their glucose-lowering medications before study. Main Outcome Measures: Rates of total body, splanchnic and leg cortisol, and D3-cortisol production were measured. Results: Rates of splanchnic cortisol production equaled or exceeded those occurring in extrasplanchnic tissues (e.g. the adrenals) in all three groups. However, because concurrent splanchnic cortisol uptake also occurred, net splanchnic cortisol release was minimal. Splanchnic cortisol production and splanchnic D3-cortisol production (an index of splanchnic 11β-hydroxysteroid dehydrogenase type 1 activity) did not differ among the three groups. In addition, splanchnic cortisol production did not correlate with either visceral fat or endogenous glucose production. On the other hand, splanchnic cortisol uptake was greater in the obese diabetic than lean nondiabetic subjects (25 ± 2.9 vs. 15.3 ± 2.5 μg/min; P < 0.05). Splanchnic, but not leg, D3-cortisol production was correlated with total body D3-cortisol production (r = 0.70; P < 0.001). Conclusions: Although large amounts of cortisol are produced within the splanchnic bed, implying high intrahepatic glucocorticoid concentrations, rates do not differ in lean and obese nondiabetic humans and are not influenced by the presence of type 2 diabetes mellitus. On the other hand, obesity but not diabetes increases splanchnic cortisol uptake.

ABSTRACT A real-time PCR method using the LightCycler (Roche Applied Science, Indianapolis, IN) was compared to a conventional PCR assay for the detection of Tropheryma whipplei in 321 clinical specimens. The LightCycler method had sensitivity and specificity comparable to the conventional method but required considerably less labor and time (3 h versus 3 to 5 days).