Anti-AIDS agents. Part 47: Synthesis and anti-HIV activity of 3-substituted 3',4'-Di-O-(S)-camphanoyl-(3'R,4'R)-(+)-cis-khellactone derivatives

Six 3-substituted 3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone derivatives (3-8) were synthesized from 3-methyl DCK (2). 3-Hydroxymethyl DCK (6) exhibited potent anti-HIV activity in H9 lymphocytes with EC(50) and TI values of 1.87 x 10(-4) microM and 1.89 x 10(5), respectively. These values are similar to those of DCK and better than those of AZT in the same assay.     

CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.     

Growth behaviour of human mononuclear cells derived from bone marrow and cord blood on a collagen carrier for osteogenic regeneration]

We investigated the biocompatibility and osteogenetic potency of a porcine collagen I/III carrier in a human bone marrow and cord blood cell culture system. METHODS: Human mesenchymal mononuclear cells were isolated from cord blood and iliac crest bone marrow and cultivated in various cell densities on a semipermeable porcine collagen I/III carrier. After 14 days of in vitro cultivation both cultures were subjected to osteogenic stimulation by dexamethasone, ascorbic acid and beta-glycerol phosphate (DAG) until day 40. Semiquantitative immunochemical evaluation based on osteoblastic and progenitor cell markers was then done. RESULTS: With regard to the minimal local cell density required for growth and osteogenic differentiation, cord blood derived progenitor cells showed lower tolerance in comparison with bone marrow derived cells. For both cell culture systems three-dimensional growth and calcification within the collagen fibres were seen after osteogenic stimulation. CONCLUSION: Human cord blood and bone marrow derived mesenchymal stem cell are capable of differentiating into osteoblasts after incubation with a collagen I/III biomaterial.     

Basal Ganglia Neuronal Activity during Scanning Eye Movements in Parkinson’s Disease

The oculomotor role of the basal ganglia has been supported by extensive evidence, although their role in scanning eye movements is poorly understood. Nineteen Parkinsońs disease patients, which underwent implantation of deep brain stimulation electrodes, were investigated with simultaneous intraoperative microelectrode recordings and single channel electrooculography in a scanning eye movement task by viewing a series of colored pictures selected from the International Affective Picture System. Four patients additionally underwent a visually guided saccade task. Microelectrode recordings were analyzed selectively from the subthalamic nucleus, substantia nigra pars reticulata and from the globus pallidus by the WaveClus program which allowed for detection and sorting of individual neurons. The relationship between neuronal firing rate and eye movements was studied by crosscorrelation analysis. Out of 183 neurons that were detected, 130 were found in the subthalamic nucleus, 30 in the substantia nigra and 23 in the globus pallidus. Twenty percent of the neurons in each of these structures showed eye movement-related activity. Neurons related to scanning eye movements were mostly unrelated to the visually guided saccades. We conclude that a relatively large number of basal ganglia neurons are involved in eye motion control. Surprisingly, neurons related to scanning eye movements differed from neurons activated during saccades suggesting functional specialization and segregation of both systems for eye movement control.     

The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4-phosphate

Signal transduction pathways that co-regulate a given biological process often are organized into networks by molecules that act as coincidence detectors. Phosphoinositides and the Rho-type GTPase Cdc42 regulate overlapping processes in all eukaryotic cells. However, the coincidence detectors that link these pathways into networks remain unknown. Here we show that the p21-activated protein kinase-related kinase Cla4 of yeast integrates signaling by Cdc42 and phosphatidylinositol 4-phosphate (PI4P). We found that the Cla4 pleckstrin homology (PH) domain binds in vitro to several phosphoinositide species. To determine which phosphoinositides regulate Cla4 in vivo, we analyzed phosphatidylinositol kinase mutants (stt4, mss4, and pik1). This indicated that the plasma membrane pool of PI4P, but not phosphatidylinositol 4,5-bisphosphate or the Golgi pool of PI4P, is required for localization of Cla4 to sites of polarized growth. A combination of the Cdc42-binding and PH domains of Cla4 was necessary and sufficient for localization to sites of polarized growth. Point mutations affecting either domain impaired the ability of Cla4 to regulate cell morphogenesis and the mitotic exit network (localization of Lte1). Therefore, Cla4 must retain the ability to bind both Cdc42 and phosphoinositides, the hallmark of a coincidence detector. PI4P may recruit Cla4 to the plasma membrane where Cdc42 activates its kinase activity and refines its localization to cortical sites of polarized growth. In mammalian cells, the myotonic dystrophy-related Cdc42-binding kinase possesses p21-binding and PH domains, suggesting that this kinase may be a coincidence detector of signaling by Cdc42 and phosphoinositides.     

Referral patterns to an ophthalmic outpatient clinic by general practitioners and ophthalmic opticians and the role of these professionals in screening for ocular disease.

Case notes of 1113 consecutive new patients referred to a consultant ophthalmologist at a district general hospital were reviewed to determine the source and efficacy of referrals and the current screening practices of general practitioners and ophthalmic opticians. General practitioners initiated referral in 546 cases (49%) and ophthalmic opticians referral in 439 (39%). Visual loss or visual disturbance was the most important single reason for referral (345 cases; 31%), followed by suspected glaucoma (145 cases; 13%), abnormalities of binocular vision (140; 12.5%), disorders of eyelids or ocular adnexa (127; 11%), and red eye (86; 8%). General practitioners referred many more patients with disorders of the eyelids and adnexa and ophthalmic opticians many more patients with suspected glaucoma. Ophthalmic opticians were far more likely than general practitioners to refer patients with suspected glaucoma correctly. A total of 180 patients (16%) were referred from ocular screening, in 149 cases by ophthalmic opticians and in 10 by general practitioners. Seventy patients had glaucoma or incomplete features of glaucoma, all of them referred by ophthalmic opticians. Of eight diabetic patients referred by ophthalmic opticians, three had asymptomatic disease and in two diabetes was diagnosed as a result of ocular screening. No patient was referred for asymptomatic diabetic retinopathy from screening by general practitioners. Ophthalmic opticians were more likely than general practitioners to diagnose retinopathy requiring photocoagulation. Use of a community based service to screen for glaucoma could save unnecessary consultant outpatient appointments. A similar service could facilitate detection of diabetic retinopathy at a stage when treatment is most effective.     

MAGE-C2/CT10 protein expression is an independent predictor of recurrence in prostate cancer

The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms. In most cases, no CT antigen is found in normal tissues, except in testis, making them ideal targets for cancer immunotherapy. A comprehensive analysis of CT antigen expression has not yet been reported in prostate cancer. MAGE-C2/CT-10 is a novel CT antigen. The objective of this study was to analyze extent and prognostic significance of MAGE-C2/CT10 protein expression in prostate cancer. 348 prostate carcinomas from consecutive radical prostatectomies, 29 castration-refractory prostate cancer, 46 metastases, and 45 benign hyperplasias were immunohistochemically analyzed for MAGE-C2/CT10 expression using tissue microarrays. Nuclear MAGE-C2/CT10 expression was identified in only 3.3% primary prostate carcinomas. MAGE-C2/CT10 protein expression was significantly more frequent in metastatic (16.3% positivity) and castration-resistant prostate cancer (17% positivity; p<0.001). Nuclear MAGE-C2/CT10 expression was identified as predictor of biochemical recurrence after radical prostatectomy (p = 0.015), which was independent of preoperative PSA, Gleason score, tumor stage, and surgical margin status in multivariate analysis (p<0.05). MAGE-C2/CT10 expression in prostate cancer correlates with the degree of malignancy and indicates a higher risk for biochemical recurrence after radical prostatectomy. Further, the results suggest MAGE-C2/CT10 as a potential target for adjuvant and palliative immunotherapy in patients with prostate cancer.     

Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a dimeric, bacterial enzyme that detoxifies many organophosphorus neurotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 are located near the bimetallic active site present in each monomer. It has been proposed that these residues influence catalysis by interacting with active site residues and the substrate in the binding pocket. We replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single-site-modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site (four per dimer), all three of these altered enzymes possessed only two metals per dimer while retaining considerable enzymatic activity for the preferred phosphotriester (P-O bond) substrate, paraoxon (5-100% kcat). The three altered enzymes achieved a 2-30-fold increase in substrate specificity (kcat/Km) for demeton S (P-S bond), an analogue for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (P-F bond) was substantially decreased for each of these enzymes. In addition, H257L and H254R/H257L showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analogue for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and it is suggested that changes in metal requirements may affect these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site, while simultaneously decreasing the catalytic efficiency for smaller substrates.     

Altering an artificial Gagpolnef polyprotein and mode of ENV co-administration affects the immunogenicity of a clade C HIV DNA vaccine

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.