Effects of exposure of mice to hindlimb unloading on leukocyte subsets and sympathetic nervous system activity.

The hindlimb unloading (HU) rodent model was developed to simulate some of the aspects of spaceflight conditions. Our previous studies showed that exposure to HU for 48 h (h) followed by bacterial challenge, reduces the ability of mice to resist infection. The purpose of this study was to investigate the physiological changes in mice during the 48 h of exposure to HU to understand the mechanisms involved in the increased susceptibility to infection observed in mice subjected to these conditions. Female Swiss Webster mice were hindlimb-unloaded during 48 h. Blood samples, spleen and peritoneal cells were removed before and after 18 or 48 h of HU-exposure. Leukocyte subset analysis was performed in spleen and peritoneal cells by flow cytometry, and catecholamine levels were measured in plasma and whole spleen by a catecholamine enzyme immunoassay. Catecholamine levels measured in plasma and spleen were significantly greater in mice exposed to HU compared to control. This increase coincided with significant reductions in spleen size in the HU group. Flow cytometric analyses showed a significant reduction of splenic CD19 + B-cells and NK1.1+ cells in mice exposed to HU with a concomitant increase in T-cells. These results suggest that exposure to HU increases the activity of the sympathetic nervous system (SNS) and induces lymphocyte sub-population changes that may contribute to the deregulation of immunity seen in mice exposed to HU and, more importantly may predispose the otherwise healthy host to the subsequent reduced ability to resist infections.

Active hexose correlated compound activates immune function to decrease bacterial load in a murine model of intramuscular infection.

BACKGROUND: Infection is a serious, costly, and common complication of surgery and constitutes the principal cause of late death in patients undergoing surgery. The objective of this study was to clarify the mechanisms by which active hexose correlated compound (AHCC) increases survival in a murine model of intramuscular infection. METHODS: Food-deprived mice receiving either AHCC or excipient were infected with bacteria. Kinetics of bacterial load, white blood cell counts, cytokine levels, and antibody levels were compared between groups. RESULTS: AHCC-treated mice had reduced bacterial load at day 5 and cleared bacteria entirely at day 6. Levels of interleukin-12, tumor necrosis factor-alpha, and interleukin-6 peaked earlier in this group (day 3) compared with controls (day 5). Increased percentages of peripheral lymphocytes and monocytes and decreased numbers of polymorphonuclear cells were detected in the AHCC group. CONCLUSIONS: AHCC appears to induce an early activation of the immune response, leading to an effective clearance of bacteria and rapid recovery.

Kinetics of systemic cytokine and brain chemokine gene expression in murine toxoplasma infection.

Toxoplasma gondii often migrates to the central nervous system in immunocompromised patients, where it induces a severe inflammation referred to as Toxoplasma encephalitis. The mechanisms involved in control of parasite multiplication and prevention of Toxoplasma encephalitis remain unclear. The objective of the present study was to characterize the inflammatory response in the brains of mice during acute T. gondii infection, with emphasis on the expression of chemokine receptors. Susceptible C57BL/6 mice were orally infected with 10 cysts of the low-virulent ME49 strain of T. gondii. Levels of cytokines (TNF-alpha, IFN-gamma, IL-10, IL-6, and IL-12p70) and chemokines (CCL/2MCP-1) were measured in plasma at 5, 10, 15, 20, and 30 days after infection. In addition, the mRNA expression of chemokines (CCL5/RANTES, CCL2/MCP-1, CCL4/MIP-1beta) and chemokine receptors (CCR1, CCR2, CCR5, CCR7, CCR8, CXCR4, and CXR5) were measured in brain tissues at the same time points. Plasma levels of IFN-gamma and CCL2/MCP-1 were highly expressed at day 5, whereas TNF-alpha had a moderate increase at day 5, peaked at day 10, and returned to normal levels by day 30. Plasma levels of IL-10, IL-6, and IL-12p70 were not detected throughout the study. Analyses of mRNA expression of chemokines and chemokine receptors in the brain showed that CCL5/ RANTES, CCR7, CXCR4, and CXCR5 were upregulated, peaking after 10 days of T. gondii infection. IgM-specific antibody levels increased at day 5 and peaked at days 10 and 30, whereas IgG levels increased at day 10 and continued to increase thereafter, reaching maximum levels at day 30 postinfection (PI). Our results suggest that T. gondii infection is controlled at local and systemic levels, and that proinflammatory proteins and their receptors may be acting coordinately to induce stage conversion and prevent parasite multiplication and development of Toxoplasma encephalitis. The early production of IFN-gamma and the delayed expression of CXCR4 and CXCR5 indicate that T. gondii induces an early robust cellular immune response, followed by a strong and sustained antibody-mediated immunity.

Effect of the alpha3beta1 integrin on the IL-1 stimulated activation of c-Jun N-terminal kinase (JNK) in CACO-2 cells.

Intestinal epithelial cells (IEC) are capable of responding to IL-1 stimulation by producing a variety of pro-inflammatory cytokines. Recently, we have found that binding of the alpha3beta1 integrin may have a regulatory effect on IL-1 responses and intracellular signaling by suppressing cytokine secretion, mRNA expression and the downstream intracellular signaling events from IKK to NF-kappaB activation. In this study, we extend these findings by showing that treatment of the Caco-2 epithelial cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression in the levels of IL-1 induced AP-1 binding activity in nuclear extracts. Furthermore, suppressed levels of IL-1 induced c-Jun N-terminal kinase (JNK) phosphorylation and kinase activity were seen with the antibody treated cells. Cells cultured on purified laminin-5, the ligand for the alpha3beta1 integrin, did not show significantly elevated levels of JNK phosphorylation after IL-1 stimulation while cells cultured on fibronectin yielded significantly elevated levels of IL-1 induced JNK phosphorylation. These results indicate that binding of the alpha3beta1 integrin results in a suppression in the activation of the IL-1 induced intracellular signaling pathway from JNK to AP-1. This novel regulatory effect may be a potentially important mechanism to regulate IL-1 mediated responses by IEC.

Enhancement of in vitro growth of pathogenic bacteria by norepinephrine: importance of inoculum density and role of transferrin.

Norepinephrine is a stress hormone that enhances bacterial growth. We examined the effects of a small inoculum on the norepinephrine-induced growth of species previously reported to be unaffected by norepinephrine. The results indicated that a reduced inoculum density is essential for observing norepinephrine-induced effects. Additional studies using serum-free media suggested that transferrin plays a role in norepinephrine-induced growth.

Active hexose correlated compound (AHCC) enhances resistance to infection in a mouse model of surgical wound infection.

BACKGROUND: Infection is the most common postoperative complication within the surgical wound and during severe trauma. In spite of the use of modern sterile techniques and prophylaxis, infection continues to be a leading cause of death in these patients. Therefore, it has become crucial to develop new alternatives to prevent the effects of trauma and other complications on the immune system and improve resistance to infection. The objective of this study was to test the prophylactic effects of oral administration of active hexose correlated compound (AHCC), a natural immunoenhancer, on survival in a mouse model of surgical soft tissue infection. METHODS: The model involves the intramuscular administration of a 50% lethal dose (LD50) of K. pneumoniae to mice that have restricted food intake for 24 hours prior to and six hours after infection and simulates local infection and food deprivation that often occur during trauma or surgical procedures. In the present study, AHCC was administrated orally to Swiss Webster mice for eight days prior to and during the infection period. Survival, time of death, LD50, and clearance of bacteria of this group were compared with those control mice receiving the excipient alone. RESULTS: Survival and mean time to death were increased significantly in the AHCC-treated group; the LD50 was greater in mice receiving AHCC than in mice receiving the excipient. Mice receiving AHCC were better able to clear bacteria from their systems than were control animals. CONCLUSIONS: The results suggest that AHCC protects mice in this model by restoring the immune and other systems negatively affected by trauma, infection, and food deprivation. More studies are necessary to determine the intrinsic mechanisms involved in this model and whether AHCC can prevent infection or improve survival in human beings with severe trauma or undergoing surgical procedures.

Regulation of connexin43 protein complexes by intracellular acidification.

Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K+ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury.

Kinetics of protein-protein interactions of connexins: use of enzyme linked sorbent assays.

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.

pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains.

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate "receptor" domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119-144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced alpha-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains.

The proximal promoter region is essential for lipopolysaccharide induction and cyclic AMP inhibition of mouse tumor necrosis factor-alpha.

Bacterial lipopolysaccharide (LPS) induces a rapid and transient increase in transcription of the tumor necrosis factor-alpha (TNF-alpha) gene in cells of monocyte/macrophage lineage. This study examines the role of potential regulatory elements within the proximal promoter region of the mouse TNF-alpha gene in LPS induction and cyclic AMP (cAMP)-mediated inhibition of TNF-alpha in RAW 264.7 murine macrophage-like cells. Transfection of proximal promoter chloramphenicol acetyltransferase (CAT) reporter constructs demonstrated that this region is LPS inducible in murine RAW 264.7 cells, with a 5.9-fold increase over nonstimulated transfectants. Site-specific mutations of the ETS, activated protein-1 (AP-1)/cAMP-responsive element (CRE)-like, or NF-kappaB-like motifs within this region caused a reduction in the LPS response by 52%, 46%, and 51%, respectively. LPS induction of the proximal promoter-CAT reporter construct was reduced by >40% by the addition of 8-bromo-cAMP (8-Br-cAMP). To determine the role of the proximal promoter region in the context of the entire TNF-alpha gene, we produced a hemagglutinin (HA)-tagged genomic TNF-alpha construct that contains a deletion of the proximal promoter region. Transfection of this construct into RAW 264.7 cells demonstrated a decrease in LPS-induced transcripts as well as a lack of response to cAMP. This suggested an essential role for this regulatory region in LPS-induced activation and cAMP inhibition of mouse TNF-alpha gene transcription in murine macrophages.