Multivariate analysis of complex gene expression and clinical phenotypes with genetic marker data

This paper summarizes contributions to group 12 of the 15th Genetic Analysis Workshop. The papers in this group focused on multivariate methods and applications for the analysis of molecular data including genotypic data as well as gene expression microarray measurements and clinical phenotypes. A range of multivariate techniques have been employed to extract signals from the multi-feature data sets that were provided by the workshop organizers. The methods included data reduction techniques such as principal component analysis and cluster analysis; latent variable models including structural equations and item response modeling; joint multivariate modeling techniques as well as multivariate visualization tools. This summary paper categorizes and discusses individual contributions with regard to multiple classifications of multivariate methods. Given the wide variety in the data considered, the objectives of the analysis and the methods applied, direct comparison of the results of the various papers is difficult. However, the group was able to make many interesting comparisons and parallels between the various approaches. In summary, there was a consensus among authors in group 12 that the genetic research community should continue to draw experiences from other fields such as statistics, econometrics, chemometrics, computer science and linear systems theory.     

Lentiviral vector-mediated autonomous differentiation of mouse bone marrow cells into immunologically potent dendritic cell vaccines.

Approaches facilitating generation of dendritic cell (DC) vaccines for clinical trials and enhancing their viability, bio-distribution, and capacity to stimulate antigen-specific immune responses are critical for immunotherapy. We programmed mouse bone marrow (BM) cells with lentiviral vectors (LV-GI4) so that they produced granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in an autonomous manner. DC/LV-GI4 cells underwent autonomous trans-differentiation to yield typical phenotypic characteristics of DCs. DC/LV-GI4 cells that self-differentiated either ex vivo or in vivo showed persistent and robust viability and stimulated high influx of DCs into draining lymph nodes (LNs). The immunostimulatory efficacy of DC/LV-GI4 cells was evaluated using MART1 and TRP2 as co-expressed melanoma antigens. Mice vaccinated with DC/LV-GI4 cells that self-differentiated in vitro or in vivo produced potent antigen-specific responses against melanoma, which correlated with protective and long-term therapeutic anti-tumor effects. Thus, DC precursors can be genetically engineered after a single ex vivo manipulation, resulting in DC vaccines with improved activity.     

Interferon γ (IFN-γ) Is Necessary for the Genesis of Acetylcholine Receptor–induced Clinical Experimental Autoimmune Myasthenia gravis in Mice

Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-γ, plays a role in the development of EAMG, we immunized IFN-γ knockout (IFN-gko) (−/−) mice and wild-type (WT) (+/+) mice of H-2^b haplotype with AChR in CFA. We observed that AChR-primed lymph node cells from IFN-gko mice proliferated normally to AChR and to its dominant pathogenic α146–162 sequence when compared with these cells from the WT mice. However, the IFN-gko mice had no signs of muscle weakness and remained resistant to clinical EAMG at a time when the WT mice exhibited severe muscle weakness and some died. The resistance of IFN-gko mice was associated with greatly reduced levels of circulating anti-AChR antibody levels compared with those in the WT mice. Comparatively, immune sera from IFN-gko mice showed a dramatic reduction in mouse AChR-specific IgG1 and IgG2a antibodies. However, keyhole limpet hemocyanin (KLH)–priming of IFN-gko mice readily elicited both T cell and antibody responses, suggesting that IFN-γ regulates the humoral immune response distinctly to self (AChR) versus foreign (KLH) antigens. We conclude that IFN-γ is required for the generation of a pathogenic anti-AChR humoral immune response and for conferring susceptibility of mice to clinical EAMG.     

Calcimimetics and the treatment of primary and secondary hyperparathyroidism

OBJECTIVE: To review the mechanism of action, development, and clinical application of the calcimimetic compounds being investigated for the treatment of primary and secondary hyperparathyroidism (HPT). DATA SOURCES: A MEDLINE search (1990-April 2004) was performed to identify all published articles related to calcimimetics. Published abstracts over the previous 5 years from various scientific meetings (American Society of Nephrology, American Society for Clinical Pharmacology and Therapeutics, American Society of Bone and Mineral Research) were also searched for reports regarding investigational calcimimetic agents. Data on cinacalcet HCl were provided by Amgen, Inc. STUDY SELECTION AND DATA EXTRACTION: Studies were selected based on number of patients included, relevance to the approved indications, and inclusion of pharmacokinetic and drug interaction information. DATA SYNTHESIS: The investigational calcimimetic compounds directly modulate the calcium-sensing receptor and can produce at least a 30% reduction in parathyroid hormone (PTH) secretion in secondary HPT. Cinacalcet appears to have more predictable pharmacokinetics and a lower risk of symptomatic hypocalcemia than the older agent, R-568. The safety profile and effective reduction in PTH and simultaneous reductions in calcium and phosphorus (and the calcium-phosphorus product) induced by cinacalcet make this agent advantageous over previously used therapies, such as vitamin D and phosphate-binding agents. More studies are required to determine additional uses of cinacalcet. CONCLUSIONS: Cinacalcet has a unique mechanism for reducing PTH concentration and appears to be a safe and effective oral therapy for both primary and secondary HPT.     

Juvenile idiopathic arthritis: parent-child discrepancy on reports of pain and disability

OBJECTIVE: To examine the incidence and nature of disagreements about pain and functional disability between parents and their children with juvenile idiopathic arthritis (JIA) and to identify demographic and psychosocial predictors of parent-child disagreement about pain and functional disability. METHODS: Participants comprised 63 children 8-16 years of age (mean 12.36 +/- 2.61) and their parents, followed as part of a longitudinal study of pain in children. During routine rheumatology clinic visits, children and their parents completed validated measures of pain, depressive symptoms, and functional disability. RESULTS: Parents and children often disagreed as to the frequency and intensity of pain and to the degree of disability caused by arthritis. Child depressive symptoms (p < 0.01) and parental perceptions of child limitations (p < 0.02) predicted parent-child disagreement about the frequency of the child's pain. Parental perceptions of child limitations also predicted parent-child disagreement about the child's level of functional disability (p < 0.04). Those children who estimated their level of disability to be different than their parents' rating also were more depressed compared to children who agreed with their parents about their level of disability (p < 0.01). CONCLUSION: Discrepancy between parent and child reports of pain and disability in children with JIA is common. Findings suggest that such disagreements in reporting of pain and functional disability by parents and their children with JIA are associated with underlying depressive symptoms in children.     

Hepatic Met-enkephalin immunoreactivity is enhanced in primary biliary cirrhosis

BACKGROUND/AIMS: In contrast to the normal adult liver, the fetal human and rat livers, and the liver of rats with cholestasis secondary to bile duct resection (BDR) express the preproenkephalin (ppENK) mRNA, which codes for the endogenous opioid peptide Met-enkephalin. In addition, Met-enkephalin immunoreactivity (MEIR) is detected in hepatocytes and in proliferating bile ductules in the cholestatic rat liver. These data suggest that cholestasis is associated with the resurgence of cells that produce Met-enkephalin. To explore further the status of opioids in cholestasis, we studied the expression of MEIR in liver tissue. METHODS: The MEIR was sought in paraffin-preserved liver tissues from patients with primary biliary cirrhosis (PBC) (n = 10). RESULTS: The MEIR was detected in all the PBC livers. Its intensity varied from weak to strong on hepatocytes and bile ducts and the strongest expression appeared as coarse granules. The MEIR was either absent or only faintly expressed by some hepatocytes from disease and nondisease control biopsies, but absent from bile ducts. CONCLUSION: These results suggest that the human liver in cholestasis may be a source of endogenous opioids.     

Pilot Study of Low Dose Oral Methotrexate Treatment for Primary Biliary Cirrhosis

Objective: To assess the efficacy of low doses of oral methotrexate as therapy for primary biliary cirrhosis. Methods: Ten symptomatic patients with this disease were treated with methotrexate at a dose of 15 mg/wk in an open label trial. Results: Eight patients completed 1 yr of treatment and six completed 2. Pruritus and fatigue decreased in all patients treated for at least 1 yr. Mean levels of serum alkaline phosphatase, ALT, and IgM were less at 1 and 2 yr than corresponding baseline means. Total serum bilirubin increased in three patients during treatment. Serum aminotransferases and alkaline phosphatase became normal in one patient with stage I disease. Although liver biopsies at 1 and 2 yr revealed a decrease in the intensity of the Inflammatory infiltrate, they also showed an increase in fibrosis suggestive of disease progression. Methotrexate was discontinued in five patients: for disease progression in four (one at 4 months, one at 1 yr, and two at 2 yr) and for intractable pruritus in one (at 4 months). All patients experienced transient mucositis and intermittent dyspepsia. Conclusions: These findings suggest that methotrexate treatment in patients with primary biliary cirrhosis is not beneficial in patients with advanced disease; in patients with early disease, methotrexate may be associated with amelioration of symptoms, reduction in serum biochemical indices of liver disease, and reduction in hepatic inflammation. However, prospective, randomized, controlled trials will he necessary for definitive evaluation of the effects of methotrexate on the quality of life and survival of patients with primary biliary cirrhosis.     

Spatial memory performances of aged rats in the water maze predict levels of hippocampal neurogenesis

Neurogenesis occurs within the adult dentate gyrus of the hippocampal formation and it has been proposed that the newly born neurons, recruited into the preexistent neuronal circuits, might be involved in hippocampal-dependent learning processes. Age-dependent spatial memory impairments have been related to an alteration in hippocampal plasticity. The aim of the current study was to examine whether cognitive functions in aged rats are quantitatively correlated with hippocampal neurogenesis. To this end, we took advantage of the existence of spontaneous individual differences observed in aged subjects in a hippocampal-dependent task, the water maze. We expected that the spatial memory capabilities of aged rats would be related to the levels of hippocampal neurogenesis. Old rats were trained in the water maze, and, 3 weeks after training, rats were injected with 5-bromo-2'-deoxyuridine (BrdUrd, 50 or 150 mg/kg) to label dividing cells. Cell proliferation was examined one day after the last BrdUrd injection, whereas cell survival and differentiation were determined 3 weeks later. It is shown that a quantitative relationship exists between learning and the number of newly generated neurons. Animals with preserved spatial memory, i.e., the aged-unimpaired rats, exhibited a higher level of cell proliferation and a higher number of new neurons in comparison with rats with spatial memory impairments, i.e., the aged-impaired rats. In conclusion, the extent of memory dysfunction in aged rats is quantitatively related to the hippocampal neurogenesis. These data reinforce the assumption that neurogenesis is involved in memory processes and aged-related cognitive alterations.