NKG2D blockade prevents autoimmune diabetes in NOD mice

NKG2D is an activating receptor on CD8(+) T cells and NK cells that has been implicated in immunity against tumors and microbial pathogens. Here we show that RAE-1 is present in prediabetic pancreas islets of NOD mice and that autoreactive CD8(+) T cells infiltrating the pancreas express NKG2D. Treatment with a nondepleting anti-NKG2D monoclonal antibody (mAb) during the prediabetic stage completely prevented disease by impairing the expansion and function of autoreactive CD8(+) T cells. These findings demonstrate that NKG2D is essential for disease progression and suggest a new therapeutic target for autoimmune type I diabetes.     

Expression of a G-protein {beta} subunit-related gene during lymphocyte activation

Using a subtractlve strategy, we have cloned an activation-relatedgene from a human B cell cDNA library. Sequence analysis revealedthat this gene was identical to H12.3, a gene belonging to anexpanding family of guanlne nucleotlde-blndlng protein ßsubunlts. The expression of H12.3 was induclble in the latephase of mltogen-stlmulated T and B cells. In T cells, IL-2and IL-4 by themselves had no direct effect on the expressionof H12.3, but they could augment the level of steady-state H12.3mRNA stimulated by phytohemagglutlnln. On the other hand, IFN-and IL-6 had no obvious effect on the expression in B cellswith or without Staphytococcus aureus Cowan l-stlmulatlon. CyclosporinA, a strong immunosuppressant, Inhibited the mltogen-stlmulatedexpression of H12.3, but rapamycin, another such agent, didnot. In synchronized Jurkat cells, the expression of H12.3 hadno cell cycle-dependent decrease in S and G₂/M phase, whilecyclin E, which controls the progression of the cell cycle unlate G₁ phase, did show a periodic expression pattern. The resultssuggest that H12.3 might be involved in regulation of lymphocyteactivation.     

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes

We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA) and V4+-OOH, induced a differential translocation of ADP-ribosylation factor (ARF), RhoA, and protein kinase Calpha (PKCalpha), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+-OOH only elicited RhoA translocation and PMA induced ARF and PKCalpha translocation. Three tyrosine kinases inhibitors, ST-638, methyl 2,5-dihydroxycinnamate, and genistein reduced fMLP-stimulated PLD activity by up to 80%. Furthermore, tyrosine kinase inhibitors reduced the fMLP-induced increase of GTPgammaS-stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKCalpha to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKCalpha in the signaling pathway leading to PLD activation by fMLP. RO 31-8220, a specific inhibitor of PKC, reduced PMA-induced PLD activity by 80% in intact HL60 granulocytes but enhanced fMLP-stimulated PLD activity by 60%. Although PMA alone had no effect on RhoA recruitment to the membrane fraction, in the presence of RO 31-8220 the levels of membrane-bound RhoA were increased. The levels of membrane-bound ARF and PKCalpha were unaffected by RO 31-8220 during PMA stimulation. In contrast, fMLP-induced recruitment of ARF and RhoA was insensitive to RO 31-8220 but PKCalpha translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATP-independent manner. Furthermore, increased fMLP-induced PKCalpha translocation in the presence of RO 31-8220 may partially account for the synergistic activation of PLD observed when both fMLP and RO 31-8220 are used together in intact HL60 cells.     

Emergency treatment and long-term follow-up of posterior urethral valves

Posterior urethral valves have a broad spectrum of clinical severity determined by the degree and reversibility of the long stepwise sequence of secondary pathology. Neonatal azotemia and severe bilateral reflux are particularly important negative prognostic factors. In the mild cases, valve ablation with or without delayed reconstruction is good therapy. In the very severe cases, our interpretation of all the clinical and experimental information now available suggests that the time interval and the level of decompression are extremely important. Achievement of consistent low caliceal pressure without stasis and infections should be achieved as soon as possible. We do not agree with the philosophy of "valve ablation and wait and see" for secondary reconstruction as applied to the severe cases. An analogous philosophy would be treating all respiratory infections as upper respiratory infections and applying aggressive appropriate therapy for pneumonia only if the patient does not respond clinically. However, all controversy aside, the management of neonatal infants with posterior urethral valves remains a difficult and challenging problem for us all. The real challenge will be to improve published management results to the point that the family faced with decisions regarding an in utero diagnosis of posterior urethral valves will have enough hope to continue the pregnancy.     

Imaging of cAMP-specific phosphodiesterase-IV: comparison of [11C]rolipram and [11C]Ro 20-1724 in rats

The phosphodiesterase type IV (PDEIV) family of enzymes contributes to the metabolism of cAMP formed by the stimulation of beta-adrenergic, A2-adenosine, and H2-histamine receptors in the brain. Disturbances in cAMP-mediated signaling have been implicated in several neuropsychiatric disorders, and there is evidence that increasing cAMP levels through PDEIV inhibition improves the symptoms of these disorders. In the present study, the selective PDEIV inhibitors rolipram and Ro 20-1724, labeled with C-11, were evaluated in vivo in rats, as potential radioligands for imaging PDEIV enzymes with positron emission tomography (PET). Biodistribution experiments revealed a greater than threefold increased regional brain retention of [11C]rolipram as compared to [11C]Ro 20-1724. [nC]Rolipram uptake was higher in rat brain areas (e.g., cortical regions and olfactory system) showing higher expression of PDEIV enzymes, as determined previously using [3H]rolipram autoradiography or molecular genetic techniques. Binding of [n1C]rolipram and [11C]Ro 20-1724 was specific, since coadministration of high doses of unlabeled rolipram (10 mg/Kg, i.v.) or Ro 20-1724 (30 mg/Kg with [11C]rolipram and 10 mg/Kg with [11C]Ro 20-1724, i.v.) reduced radioactivity uptake in brain regions. Pretreatment with high doses of the PDEI selective inhibitor vinpocetine (10 mg/Kg, i.p., 15 min prior), or the noradrenaline reuptake inhibitor desipramine (10 mg/Kg, i.p., 30 min prior), or coinjection with the adenylyl cyclase activator forskolin (6.5 or 15 mg/Kg, i.v.), did not inhibit [11C]rolipram uptake in brain areas, suggesting binding selectivity for PDEIV enzymes. We conclude that [11C]rolipram, but not [11C]Ro 20-1724, is a promising radioligand for imaging the PDEIV enzymes with PET.     

Amplifications of oncogeneerbB-2 and chromosome 20q in breast cancer determined by differentially competitive polymerase chain reaction

Summary A new method of measuring gene copy number in small samples of DNA was used to measure amplification of theerbB-2 gene and of chromosome 20q in breast cancers. This method, termed differentially competitive polymerase chain reaction (DC-PCR) combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR and competitive PCR. The DC-PCR methodology was evaluated for sensitivity and specificity by comparing amplification oferbB-2 measured by DC-PCR with that obtained by fluorescencein situ hybridization (FISH) for 42 cases or Southern blotting and/or slot blot analysis for 34 cases. There was over 90 percent concordance with both FISH and Southern blotting and/or slot blot analysis.DC-PCR was used to further characterize the newly described amplicon at chromosome 20q. By analyzing DNA from 10 breast cancer cell lines at 7 different loci, we identified a potential common region of amplification of approximately 5 centimorgans at chromosome 20q13 bordered by loci D20S52 and RMC20C001-S1. One hundred and seventeen cases of primary breast cancer were evaluated for amplification at these two loci. Amplification at one or more loci, defined as > 1.5 fold higher copy number than that of normal DNA, was found in 25 cases (21%). Sixteen cases were amplified at only one of the two probes (12 cases for RMC20C001-S1 and 4 cases for D20S52), suggesting that the target gene lies between the two markers or that there are two independent target genes within a small chromosome region.     

Novelty and Familiarity Activations in PET Studies of Memory Encoding and Retrieval

Nine young right-handed men viewed colored pictures of people,scenes, and landscapes. Then, 24 hr later while undergoing PETscanning, they viewed previously studied (OLD) pictures in onetype of scan, and previously not seen (NEW) pictures in another.The OLD-NEW subtraction of PET images indicates familiarity,and the NEW-OLD indicates novelty. Familiarity activations,signalling aspects of retrieval, were observed in the left andright frontal areas, and posterior regions bilaterally. Noveltyactivations were in the right limbic regions, and bilaterallyin temporal and parietal regions, including area 37. These latteractivations were located similarly to novelty activations inprevious PET studies using visual words and auditory sentences,suggesting the existence of brain regions specializing in transmodalnovelty assessment The effects of novelty are seen both be haviorallyand in replicable patterns of cortical and subcortical activation.We propose a "novelty/encoding hypothesis": (1) novelty assessmentrepresents an early stage of long-term memory encoding; (2)elaborate, meaning-based encoding processes operate on the incoming information to the extent of its novelty, and therefore(3) the probability of long-term storage of information vanesdirectly with the novelty of the information.     

Relationship between dopamine D(2) occupancy, clinical response, and side effects: a double-blind PET study of first-episode schizophrenia

OBJECTIVE: Since all antipsychotics block dopamine D(2) receptors, the authors investigated how well D(2) receptor occupancy in vivo predicts clinical response, extrapyramidal side effects, and hyperprolactinemia. METHOD: In a double-blind study, 22 patients with first-episode schizophrenia were randomly assigned to 1.0 or 2. 5 mg/day of haloperidol. After 2 weeks of treatment, D(2) receptor occupancy was determined with [(11)C]raclopride and positron emission tomography, and clinical response, extrapyramidal side effects, and prolactin levels were measured. Patients who showed adequate responses continued taking their initial doses, those who did not respond had their doses increased to 5.0 mg/day, and evaluations were repeated at 4 weeks for all patients. RESULTS: The patients showed a wide range of D(2) occupancy (38%-87%). The degree of receptor occupancy predicted clinical improvement, hyperprolactinemia, and extrapyramidal side effects. The likelihood of clinical response, hyperprolactinemia, and extrapyramidal side effects increased significantly as D(2) occupancy exceeded 65%, 72%, and 78%, respectively. CONCLUSIONS: The study confirms that D(2) occupancy is an important mediator of response and side effects in antipsychotic treatment. The data are consistent with a "target and trigger" hypothesis of antipsychotic action, i.e., that the D(2) receptor specificity of antipsychotics permits them to target discrete neurons and that their antagonist properties trigger within those neurons intracellular changes that ultimately beget antipsychotic response. While limited to haloperidol, the relationship between D(2) occupancy and side effects in this study helps explain many of the observed clinical differences between typical and atypical antipsychotics.