Effects of Alginate Hydrogels and TGF-β1 on Human Dental Pulp Repair In Vitro

The objective of this study was to investigate the use of alginate hydrogels to present either exogenous or endogenous transforming growth factor (TGF)- &#103 1 to the dentin-pulp complex to signal reparative processes. Hydrogels were prepared, applied to cultured human tooth slices and the effects on tertiary dentinogenesis examined histologically. Both TGF- &#103 1-containing and acid-treated alginate hydrogels, but not untreated hydrogels, upregulated dentin matrix secretion and induced odontoblast-like cell differentiation with subsequent secretion of regular tubular dentin matrix on cut pulpal surfaces. It is concluded that TGF- &#103 1 can signal both induction of odontoblast-like cell differentiation and upregulation of their matrix secretion in the human dentin-pulp complex. Alginate hydrogels provide an appropriate matrix in which dental regeneration can take place and may also be useful for delivery of growth factors, including TGF- &#103 s, to enhance the natural regenerative capacity of the dental pulp.     

In Vitro and In Vivo Effects of Genistein on Murine Alveolar Macrophage TNFα Production

The present study was performed to determine whether genistein could inhibit in vivo LPS-induced alveolar macrophage TNF production and thus reduce the alveolar neutrophil influx following LPS. In vitro incubation with genistein completely inhibited LPS-induced TNF production by alveolar macrophages (AM) from BALB/c mice. Subsequently mice were pretreated with intraperitoneal genistein or vehicle, then received nasal LPS to induce an alveolitis. Genistein was then administered every eight hours for five days following LPS. At 24 hours after LPS, the bronchoalveolar lavage (BAL) TNF and ex vivo TNF production from AM, were lower in the genistein treated animals. As well, total BAL white blood cell (WBC) count was reduced in the genistein as compared to the vehicle-only group. The percent neutrophils and the resolution of neutrophils were similar between genistein and vehicle groups. Therefore, genistein was able to decrease AM TNF production, and was associated with a decrease in BAL WBC count post-LPS.     

The Effects of Cyclic Stretching on the Proliferation of Lung Adenocarcinoma Cells in Vitro

Using FX-4000 strain unit, the prolieration in human lung adenocarcinoma A549 cells that underwent mechanical strain of different waveform、frequency and duration were studied. Image analysis revealed that cellular proliferation rate(PR) reduced significantly after cells were subjected to square wave with 0～20% elongation at frequency 30,40,50 and 60 cycles/min for 2h. The PR had no distinct difference at heart wave , triangle wave and sine wave group compared with control. It is concluded that square wave and higher frequency play an important role in inhibiting A549 cells proliferation.     

Macrophage Inflammatory Protein-1α Expression in Vivo and in Vitro: The Role of Lipoteichoic Acid

Lipoteichoic acid (LTA), a component of the cell wall of most gram-positive bacteria, has been shown to play a significant role in the initiation and progression of bacterial infection. However, little is known of its position in the cytokine network involved in the induction and perpetuation of inflammation. In this study, we assessed whether the macrophage activating and chemotactic cytokine macrophage inflammatory protein-1α (MIP-1α) was expressed in the setting of localized gram-positive infection. Furthermore, we determined whether LTA purified from either Staphylococcus aureus or Streptococcus pyogenes could induce the expression of MIP-1α mRNA and protein from human blood monocytes. Immunohistochemical staining of human endocardial samples obtained from patients with acute S. aureus endocarditis revealed cell-associated MIP-1α expression by neutrophils, macrophages, and fibroblasts. Treatment of human peripheral blood monocytes in vitro with LTA isolated from either S. aureus or S. pyogenes resulted in both the time- and dose-dependent expression of MIP-1α mRNA. Similarly, staphylococcal and streptococcal LTA induced the dose-dependent production of MIP-1α protein after 24 h in culture. These studies suggest that LTA may play an important role in triggering the recruitment and activation of leukocytes that characterizes the host response to gram-positive bacterial invasion.     

Hepatitis C virus-induced furin and thrombospondin-1 activate TGF-β1: role of TGF-β1 in HCV replication

In this study, we demonstrated the molecular mechanisms of TGF-β1 induction as well as proteolytic activation in HCV (JFH-1)-infected cells. Our studies showed the synthesis and secretion of TGF-β1 in HCV-infected cells which was reduced in the presence of Ca²⁺ chelators, an inhibitor of mitochondrial Ca²⁺ uptake, and antioxidants. We also showed that the expression of HCV NS proteins NS3/4A, and NS5A can induce TGF-β1 by cell-based luciferase assay. Furthermore, mutational analysis revealed that the functionally active protease domain of NS3 and N-terminus domain of NS5A are required for TGF-β1 activity. Using siRNA approach we demonstrated that HCV-induced furin and thrombospondin-1 (TSP-1) are involved in the proteolytic activation of TGF-β1. Our results also suggest that TGF-β1 positively regulates HCV RNA replication. Collectively, these observations provide insight into the mechanism of TGF-β1 activation, which likely manifest in liver fibrosis associated with hepatitis C infection.     

Immunomodulatory Activities of HERBSnSENSES™ Cordyceps — in Vitro and in Vivo Studies

The commercially available HERBSnSENSES? Cordyceps (HSCS) belongs to a cultivated strain of Cordyceps sinensis whose immunomodulatory activities has been renowned in traditional Chinese medicine (TCM) for centuries. The present report is the first that describes its immunomodulatory features through a series of in vitro and in vivo experiments. We measured, in peripheral blood mononuclear cells the in vitro effects of HSCS on the gene expression of cytokines and cytokine receptors, cytokine release, and surface expression of cytokine receptors using cDNA expression array, cytometric bead array (CBA), and immunoflorescence staining, respectively, as well as macrophage phagocytosis and monocyte production of H₂O₂ using flow cytometry. Sixty female BALB/c mice were fed with either HSCS (40 mg/kg/day) or water consecutively for 14 days. Proliferation, cytokine liberation, and CD3/4/8 expression of splenic cells were measured using 5-bromo-2′-deoxyuridine proliferation ELISA, CBA, and cytometry immunoflorescence staining, respectively. In vitro results demonstrated that HSCS induced the production of interleukin(IL)-1β, IL-6, IL-10 and tumor necrosis factor alphaα from PBMC, augmented surface expression of CD25 on lymphocytes, and elevated macrophage phagocytosis and monocyte production of H₂O₂. In vivo results showed that HSCS did not induce splenomegaly and cytokine overliberation. Our results possibly provide the biochemical basis for future clinical trials.     

Tuning of CD40–CD154 Interactions in Human B-Lymphocyte Activation: A Broad Array of In Vitro Models for a Complex In Vivo Situation

Naive and memory B-lymphocyte populations can be activated through the binding of CD154 to CD40, a receptor that is constitutively expressed on the surface of these cells. Models based on the in vitro stimulation of human B lymphocytes through CD40 have greatly contributed to our understanding of the human immune response in healthy individuals and patients suffering from immune disorders. The nature of the engineered CD40 ligands is as diverse as the in vitro models used in studies of CD40-activated B lymphocytes. Monoclonal anti-CD40 antibodies, recombinant CD154 proteins, soluble CD154⁺ membranes as well as CD154⁺ cell lines have turned out to be very useful tools, and are still in use today. As for any receptor–ligand interaction, parameters such as duration and strength of contact, timing, affinity, and receptor density are major determinants of CD40 binding by CD154 or anti-CD40. Furthermore, variation in the intensity of CD40 stimulation has been shown to influence proliferation, differentiation and immunoglobulin secretion of human hybridomas, B-cell lines, tonsil and blood B lymphocytes. The objective of this review is to present an overview of the great diversity of CD40 agonists used in in vitro models of B-lymphocyte activation, with a particular emphasis on variations in the resulting strength of CD40 signaling generated by these models. A better understanding of these models could open up new avenues for the rational use of human B lymphocytes as antigen-presenting cells in cellular therapies.     

β-Endorphin Effects on Antibody Production, Proliferation, and Secretion of Th1/Th2 Cytokines In Vivo

Intraperitoneal injection of β-endorphin in doses of 1, 0.01, and 0.0005 μg/kg under conditions of systemic immunization increased the count of antibody-producing cells in the spleen and the titer of anti-erythrocyte antibodies in the plasma of experimental animals. Intraperitoneal β-endorphin stimulated proliferative activity of splenocytes in mice in the presence of both B- and T-cell mitogen, did not change the production of IFN-γ, reduced the level of IL-2, and stimulated the secretion of IL-4, the main Th2-polarizing factor.     

Rat Macrophage Inflammatory Protein-1α, a CC Chemokine, Acts as a Neutrophil Chemoattractant in Vitro and in Vivo

Recombinant rat macrophage inflammatory protein-1 (rMIP-1) at a concentration of 3 × 10^–8 M had strong neutrophil chemotactic activity, though the potency of rMIP-1 was less than that of cytokine-induced neutrophil chemoattractant (CINC)-1 at lower concentrations. In addition, rMIP-1 induced neutrophil chemotaxis in vivo when rMIP-1 was injected into the preformed air-pouch on the back of rats. The adhesion of rMIP-1-treated neutrophils to fibrinogen significantly increased, reaching a maximum adhesion at 10^–8 M. Stimulation of neutrophils with rMIP-1 induced a transient increase in intracellular free [Ca²⁺] dose-dependently. rMIP-1 still induced an increase in the intracellular [Ca²⁺] of rat neutrophils stimulated first with CINC-1, CINC-3 or C5a, suggesting that rat neutrophils have a specific receptor for rMIP-1. Supporting these findings, an additive increase in chemotactic potency was found when both rMIP-1 and CINC-1 were added to the lower wells of Boyden chamber in vitro. In addition, high levels of rMIP-1 were detected in the inflammatory site of air-pouch/carrageenan-induced inflammation in rats. Our results suggest that rMIP-1 acts as a neutrophil chemoattractant and, together with CINCs, plays an important role in infiltration of neutrophils into inflammatory sites in rats.     

In Vitro Effects of Folic Acid on γ-Glutamyltransferase and Glutathione Reductase Activities in Malignant Lung and Thymus Tumors

In vitro effects of folic acid (10^-5, 10^-4, and 10^-3 M) on activities of -glutamyltransferase and glutathione reductase, the enzymes involved in glutathione metabolism, were studied in tissue samples obtained after surgical treatment of the lungs and thymus. Folic acid did not change -glutamyltransferase activity in lung cancer tissue, but in thymoma tissue this substance in a concentration of 10^-3 M inhibited it by 16%. Folic acid had no effects on glutathione reductase activity in benign tumors and normal lung and thymus tissues, but increased this activity in thymoma and lung cancer tissues. Activation of glutathione reductase was probably related to binding of folic acid in the allosteric center of the enzyme, which probably induced conformational changes in the catalytic center, acceleration of electron transport from NADPH₂ to oxidized glutathione via flavin adenine nucleotide, and intense production of reduced glutathione.