The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes

Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.     

Targeted Cancer Therapy with a Novel Anti-CD37 Beta-Particle Emitting Radioimmunoconjugate for Treatment of Non-Hodgkin Lymphoma

¹⁷⁷Lu-DOTA-HH1 (¹⁷⁷Lu-HH1) is a novel anti-CD37 radioimmunoconjugate developed to treat non-Hodgkin lymphoma. Mice with subcutaneous Ramos xenografts were treated with different activities of ¹⁷⁷Lu-HH1, ¹⁷⁷Lu-DOTA-rituximab (¹⁷⁷Lu-rituximab) and non-specific ¹⁷⁷Lu-DOTA-IgG₁ (¹⁷⁷Lu-IgG₁) and therapeutic effect and toxicity of the treatment were monitored. Significant tumor growth delay and increased survival of mice were observed in mice treated with 530 MBq/kg ¹⁷⁷Lu-HH1 as compared with mice treated with similar activities of ¹⁷⁷Lu-rituximab or non-specific ¹⁷⁷Lu-IgG1, 0.9% NaCl or unlabeled HH1. All mice injected with 530 MBq/kg of ¹⁷⁷Lu-HH1 tolerated the treatment well. In contrast, 6 out of 10 mice treated with 530 MBq/kg ¹⁷⁷Lu-rituximab experienced severe radiation toxicity. The retention of ¹⁷⁷Lu-rituximab in organs of the mononuclear phagocyte system was longer than for ¹⁷⁷Lu-HH1, which explains the higher toxicity observed in mice treated with ¹⁷⁷Lu-rituximab. In vitro internalization studies showed that ¹⁷⁷Lu-HH1 internalizes faster and to a higher extent than ¹⁷⁷Lu-rituximab which might be the reason for the better therapeutic effect of ¹⁷⁷Lu-HH1.     

Effect of chitosan chain architecture on gene delivery: comparison of self-branched and linear chitosans

Chitosan possesses many characteristics of an ideal gene delivery system. However, the transfection efficiency of conventional chitosans is generally found to be low. In this study, we investigated the self-branching of chitosans as a strategy to improve its gene transfer properties without compromising its safety profile. Self-branched (SB) and self-branched trisaccharide-substituted (SBTCO) chitosans with molecular weights of 11-71 kDa were synthesized, characterized, and compared with their linear counterparts with respect to transfection efficiency, cellular uptake, formulation stability, and cytotoxicity. Our studies show that in contrast with unmodified linear chitosans that were unable to transfect HeLa cells, self-branched chitosans mediated high transfection efficiencies. The most efficient chitosan, SBTCO30, yielded gene expression levels two and five times higher than those of Lipofectamine and Exgen, respectively, and was nontoxic to cells. Nanoparticles formed with SBTCO chitosans exhibited a higher colloidal stability of formulation, efficient internalization without excessive cell surface binding, and low cytotoxicity.     

Perspectives on providing good access to dental services for elderly people: patient selection,dentists’ responsibility and budget management

doi: 10.1111/j.1741‐2358.2012.00646.x Perspectives on providing good access to dental services for elderly people: patient selection, dentists’ responsibility and budget management Objectives: To suggest a model for organizing and financing dental services for elderly people so that they have good access to services. Background: There are few studies on how dental services for elderly people should be organized and financed. This is surprising if we take into consideration the fact that the proportion of elderly people is growing faster than any other group in the population, and that elderly people have more dental diseases and poorer access to dental services than the rest of the adult population. In several countries, dental services are characterized by private providers who often operate in a market with competition and free price‐setting. Private dentists have no community responsibility, and they are free to choose which patients they treat. Material and methods: Literature review and critical reasoning. Results: In order to avoid patient selection, a patient list system for elderly people is recommended, with per capita remuneration for the patients that the dentist is given responsibility for. The patient list system means that the dentist assumes responsibility for a well‐defined list of elderly people. Conclusion: Our model will lead to greater security in the dentist/patient relationship, and patients with great treatment needs will be ensured access to dental services.     

Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.     

Alpha-tocopherol and MRI Outcomes in Multiple Sclerosis – Association and Prediction

Objective

Alpha-tocopherol is the main vitamin E compound in humans, and has important antioxidative and immunomodulatory properties. The aim of this study was to study alpha-tocopherol concentrations and their relationship to disease activity in Norwegian multiple sclerosis (MS) patients.

Methods

Prospective cohort study in 88 relapsing-remitting MS (RRMS) patients, originally included in a randomised placebo-controlled trial of omega-3 fatty acids (the OFAMS study), before and during treatment with interferon beta. The patients were followed for two years with repeated 12 magnetic resonance imaging (MRI) scans and nine serum measurements of alpha-tocopherol.

Results

During interferon beta (IFNB) treatment, each 10 µmol/L increase in alpha-tocopherol reduced the odds (CI 95%) for simultaneous new T2 lesions by 36.8 (0.5–59.8) %, p = 0.048, and for combined unique activity by 35.4 (1.6–57.7) %, p = 0.042, in a hierarchical regression model. These associations were not significant prior to IFNB treatment, and were not noticeably changed by gender, age, body mass index, HLA-DRB1*15, treatment group, compliance, or the concentrations of 25-hydroxyvitamin D, retinol, neutralising antibodies against IFNB, or the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid. The corresponding odds for having new T1 gadolinium enhancing lesions two months later was reduced by 65.4 (16.5–85.7) %, p = 0.019, and for new T2 lesions by 61.0 (12.4–82.6) %, p = 0.023.

Conclusion

During treatment with IFNB, increasing serum concentrations of alpha-tocopherol were associated with reduced odds for simultaneous and subsequent MRI disease activity in RRMS patients.     

Spermine and Citrate as Metabolic Biomarkers for Assessing Prostate Cancer Aggressiveness

Separating indolent from aggressive prostate cancer is an important clinical challenge for identifying patients eligible for active surveillance, thereby reducing the risk of overtreatment. The purpose of this study was to assess prostate cancer aggressiveness by metabolic profiling of prostatectomy tissue and to identify specific metabolites as biomarkers for aggressiveness. Prostate tissue samples (n = 158, 48 patients) with a high cancer content (mean: 61.8%) were obtained using a new harvesting method, and metabolic profiles of samples representing different Gleason scores (GS) were acquired by high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS). Multivariate analysis (PLS, PLS-DA) and absolute quantification (LCModel) were used to examine the ability to predict cancer aggressiveness by comparing low grade (GS = 6, n = 30) and high grade (GS≥7, n = 81) cancer with normal adjacent tissue (n = 47). High grade cancer tissue was distinguished from low grade cancer tissue by decreased concentrations of spermine (p = 0.0044) and citrate (p = 7.73·10⁻⁴), and an increase in the clinically applied (total choline+creatine+polyamines)/citrate (CCP/C) ratio (p = 2.17·10⁻⁴). The metabolic profiles were significantly correlated to the GS obtained from each tissue sample (r = 0.71), and cancer tissue could be distinguished from normal tissue with sensitivity 86.9% and specificity 85.2%. Overall, our findings show that metabolic profiling can separate aggressive from indolent prostate cancer. This holds promise for the benefit of applying in vivo magnetic resonance spectroscopy (MRS) within clinical MR imaging investigations, and HR-MAS analysis of transrectal ultrasound-guided biopsies has a potential as an additional diagnostic tool.     

Metabolic switching of human skeletal muscle cells in vitro

In this review we will focus on external factors that may modify energy metabolism in human skeletal muscle cells (myotubes) and the ability of the myotubes to switch between lipid and glucose oxidation. We describe the metabolic parameters suppressibility, adaptability and substrate-regulated flexibility, and show the influence of nutrients such as fatty acids and glucose (chronic hyperglycemia), and some pharmacological agents modifying nuclear receptors (PPAR and LXR), on these parameters in human myotubes. Possible cellular mechanisms for changes in these parameters will also be highlighted.