Regulation of the expression of lactate dehydrogenase isozymes in human lymphocytes

Mitogen activation of human peripheral lymphocytes leads to a switch in the isozymes of LDH; resting cells contain low activities of only the B₄ and B₃A forms, whereas activated cells contain high activities of the A₄ and A₃B forms. B₄ LDH is not altered in activated cells. In this study we show that the appearance of the A subunits occurs concomitantly with a several fold increase in the steady state levels of LDH-A mRNA. Responses in LDH-A mRNA are observed within 12 hrs of activation, and are, thus, associated with the G0/G1 transition or with early G1 (Marjanovicet al. Exp. Cell Res. (1991) 193: 425–431). Maximal expression of LDH-A mRNA requires both phorbol ester and concanavalin A, implying a complex regulatory pathway involving cascade systems activated through both the antigen receptor (TR) and protein kinase C.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

The physiological scavenger receptor function of hepatic sinusoidal endothelial and Kupffer cells is independent of scavenger receptor class A type I and II

N-cadherin, a cell adhesion molecule normally found in neural cell tissue, has been found recently to be expressed on the surface of malignant T-cells. The function of N-cadherin on these cells remains unclear. Heterotypic assays between Molt-3 T lymphoblastic leukemia cells and Caco-2 epithelial monolayers were examined under different conditions to assess the functional role of N-cadherin. The results indicate that adherence of Molt-3 cells to Caco-2 monolayers was reduced significantly following pretreatment of Molt-3 cells with 100 M of an N-cadherin-derived antagonist decapeptide. In contrast, pretreatment of Molt-3 cells with an anti-N-cadherin antibody raised against the first 20 amino acids of N-cadherin sequence led to a surprisingly marked enhancement of Molt-3 cell adherence to Caco-2 monolayers. In addition, the presence of anti-N-cadherin antibody neutralized the inhibitory effect of anti-ICAM-1 on Molt-3 adhesion to Caco-2 monolayers. This novel finding demonstrates that external stimulus through the N-cadherin amino terminus can modulate adhesion of malignant T-cells to epithelia and may promote their ability to invade or metastasize to inflammatory sites.     

Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing

Cryopreservation of peripheral blood mononuclear cells (PBMC) from animal model studies and clinical trials is utilized as a primary method for long-term storage of PBMC for future in vitro and in vivo applications. The objective of this study was to define the mechanistic pathways involved in cryopreservation-induced apoptosis of CD4+ T-cells in PBMC, and to evaluate a cytokine treatment of the cryopreserved samples to rescue apoptosis for the potential future use of the cryopreserved PBMC. Using cryopreserved PBMC samples isolated from na?ve and Simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, frozen PBMC showed significantly increased levels of apoptosis-induced CD4+ T-cell death compared to fresh PBMC over a 5-day culture period as detected by Annexin V/PI and trypan blue staining. Mechanistic studies using a broad-spectrum caspase inhibitor z-VAD demonstrated a crucial involvement of caspases in cryopreservation-induced apoptosis of CD4+ T-cells. Furthermore, the ability of z-VAD to inhibit both mitochondrial membrane perturbation and apoptotic cell death implicated the involvement of caspase-mediated mitochondrial membrane damage in cryopreservation-induced apoptosis of CD4+ T-cells. Due to their known properties to promote T-cell survival and inhibit apoptosis, we evaluated the ability of IL-2, IL-4, and IL-7 combination cytokine treatment of the cryopreserved cells to rescue apoptosis of the CD4+ T-cells. The cytokine treatment resulted in a significant inhibition (p<0.01) of apoptosis-induced cell death and rescued CD4+ T-cell survival (p<0.01) in the cryopreserved cells. Efficient rescue of cryopreserved CD4+ T-cells has clinical significance in immune function analysis of longitudinal samples and in various long-term protocols requiring cryopreservation, including bone marrow and stem cell transplantation.     

Pathophysiological mechanisms of asthma. Application of cell and molecular biology techniques

Asthma is a common increasing and relapsing disease that is associated with genetic and environmental factors such as respiratory viruses and allergens. It causes significant morbidity and mortality. The changes occurring in the airways consist of a chronic eosinophilic and lymphocytic inflammation, together with epithelial and structural remodeling and proliferation, and altered matrix proteins, which underlie airway wall narrowing and bronchial hyperresponsiveness (BHR). Several inflammatory mediators released from inflammatory cells such as histamine and cysteinyl-leukotrienes induce bronchoconstriction, mucus production, plasma exudation, and BHR. Increased expression of T-helper 2 (Th2)-derived cytokines such as interleukin-4 and 5 (IL-4,5) have been observed in the airway mucosa, and these may cause IgE production and terminal differentiation of eosinophils. Chemoattractant cytokines (chemokines) such as eotaxin may be responsible for the chemoattraction of eosinophils to the airways. The initiating events are unclear but may be genetically determined and may be linked to the development of a Th2-skewed allergen-specific immunological memory. The use of molecular biology techniques on tissues obtained from asthmatics is increasing our understanding of the pathophysiology of asthma. With the application of functional genomics and the ability to transfer or delete genes, important pathyways underlying the cause if asthma will be unraveled. The important outcome of this is that new preventive and curative treatments may ensue.     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.     

Trisomy of the Dscr1 gene suppresses early progression of pancreatic intraepithelial neoplasia driven by oncogenic Kras

Individuals with Down syndrome exhibit remarkably reduced incidence of most solid tumors including pancreatic cancer. Multiple mechanisms arising from the genetic complexity underlying Down syndrome has been suggested to contribute to such a broad cancer protection. In this study, utilizing a genetically engineered mouse model of pancreatic cancer, we demonstrate that trisomy of the Down syndrome critical region-1 (Dscr1), an endogenous calcineurin inhibitor localized on chromosome 21, suppresses the progression of pancreatic intraepithelial neoplasia-1A (PanIN-1A) to PanIN-1B lesions without affecting the initiation of PanIN lesions mediated by oncogenic Kras^G12D. In addition, we show that Dscr1 trisomy attenuates nuclear localization of nuclear factor of activated T-cells (NFAT) accompanied by upregulation of the p15^Ink4b tumor suppressor and reduction of cell proliferation in early PanIN lesions. Our data suggest that attenuation of calcineurin–NFAT signaling in neoplastic pancreatic ductal epithelium by a single extra copy of Dscr1 is sufficient to inhibit the progression of early PanIN lesions driven by oncogenic Kras, and thus may be a potential mechanism underlying reduced incidence of pancreatic cancer in Down syndrome individuals.     

The DEAD box proteins DDX5 (p68) and DDX17 (p72): Multi-tasking transcriptional regulators

Members of the DEAD box family of RNA helicases, which are characterised by the presence of twelve conserved motifs (including the signature D-E-A-D motif) within a structurally conserved ‘helicase’ core, are involved in all aspects of RNA metabolism. Apart from unwinding RNA duplexes, which established these proteins as RNA helicases, DEAD box proteins have been shown to also catalyse RNA annealing and to displace proteins from RNA. DEAD box proteins generally act as components of large multi-protein complexes and it is thought that interactions, via their divergent N- and C-terminal extensions, with other factors in the complexes may be responsible for the many different functions attributed to these proteins.