Citations

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.

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.

PurposeMantle cell lymphoma (MCL) is characterized by a t(11;14) resulting in overexpression of cyclin D1 messenger RNA. This study tested whether temsirolimus (previously known as CCI-779), an inhibitor of the mammalian target of rapamycin kinase that regulates cyclin D1 translation, could produce tumor responses in patients with MCL.Patients and MethodsPatients with relapsed or refractory MCL were eligible to receive temsirolimus 250 mg intravenously every week as a single agent. Patients with a tumor response after six cycles were eligible to continue drug for a total of 12 cycles or two cycles after complete remission, and were then observed without maintenance.ResultsThirty-five patients were enrolled and were assessable for toxicity; one patient had MCL by histology but was cyclin D1 negative and was ineligible for efficacy. The median age was 70 years (range, 38 to 89 years), 91% were stage 4, and 69% had two or more extranodal sites. Patients had received a median of three prior therapies (range, one to 11), and 54% were refractory to the last treatment. The overall response rate was 38% (13 of 34 patients; 90% CI, 24% to 54%) with one complete response (3%) and 12 partial responses (35%). The median time-to-progression in all patients was 6.5 months (95% CI, 2.9 to 8.3 months), and the duration of response for the 13 responders was 6.9 months (95% CI, 5.2 to 12.4 months). Hematologic toxicities were the most common, with 71% (25 of 35 patients) having grade 3 and 11% (four of 35 patients) having grade 4 toxicities observed. Thrombocytopenia was the most frequent cause of dose reductions but was of short duration, typically resolving within 1 week.ConclusionsSingle-agent temsirolimus has substantial antitumor activity in relapsed MCL. This study demonstrates that agents that selectively target cellular pathways dysregulated in MCL cells can produce therapeutic benefit. Further studies of this agent in MCL and other lymphoid malignancies are warranted.

ABSTRACT Niemann‐Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. In the current study, we used protein transduction to further investigate Rab9‐mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22‐Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 µM. A fraction of the VP22‐Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22‐Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY‐lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL‐receptor levels. In addition, we demonstrated reduction of intracellular cholesterol by VP22‐Rab9 transduction in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease.

Abstract We detected circulating plasma cells (PCs) by flow cytometry in 302 patients with newly diagnosed multiple myeloma (MM) by gating on CD38+CD45- cells. The number of circulating PCs per 50 000 mononuclear cells was reported. In 80 (27%) patients, no circulating PC were seen; 106 (35%) patients had 1 to 10 and 115 (38%) patients had more than 10 circulating PCs. Median overall survival for the 302 patients was 47 months. Patients with 10 or fewer circulating PCs had a median survival of 58.7 months, whereas patients with more than 10 circulating PCs had a median survival of 37.3 months (P = .001). On multivariate analysis, the prognostic value of circulating PCs was independent of β2-microglobulin, albumin, and C-reactive protein. There was only a weak correlation between tumor mass and circulating PCs, suggesting that the appearance of circulating PCs may be a reflection of tumor biology. We conclude that the number of circulating PCs measured by flow cytometry in patients with newly diagnosed MM is an independent predictor of survival.

Patients with Alzheimer's disease (AD) have abnormally low positron emission tomography (PET) measurements of the cerebral metabolic rate for glucose (CMRgl) in regions of the precuneus and the posterior cingulate, parietotemporal, and frontal cortex. Apolipoprotein E (APOE) ε4 gene dose (i.e., the number of ε4 alleles in a person's APOE genotype) is associated with a higher risk of AD and a younger age at dementia onset. We previously found that cognitively normal late-middle-aged APOE ε4 carriers have abnormally low CMRgl in the same brain regions as patients with probable Alzheimer's dementia. In a PET study of 160 cognitively normal subjects 47–68 years of age, including 36 ε4 homozygotes, 46 heterozygotes, and 78 ε4 noncarriers who were individually matched for their gender, age, and educational level, we now find that ε4 gene dose is correlated with lower CMRgl in each of these brain regions. This study raises the possibility of using PET as a quantitative presymptomatic endophenotype to help evaluate the individual and aggregate effects of putative genetic and nongenetic modifiers of AD risk.