Leukocyte apoptosis and its significance in sepsis and shock

Sepsis and multiple organ failure continue to be significant problems among trauma, burn, and the critically ill patient population. Thus, a number of laboratories have focused on understanding the role of altered apoptotic cell death in contributing to immune and organ dysfunction seen in sepsis and shock. Immune cells that undergo altered apoptotic changes include neutrophils, macrophages, dendritic cells, as well as various lymphocyte populations. Evidence of epithelial as well as endothelial cell apoptotic changes has also been reported. Although mediators such as steroids, tumor necrosis factor, nitric oxide, C5a, and Fas ligand (FasL) appear to contribute to the apoptotic changes, their effects are tissue- and cell population-selective. As inhibiting Fas-FasL signaling (e.g., gene deficiency, Fas fusion protein, or Fas short interfering RNA administration), caspase inhibition (caspase mimetic peptides), and/or the overexpression of downstream antiapoptotic molecules (e.g., Bcl-2, Akt) improve survival of septic mice, it not only demonstrates the pathological significance of this process but points to novel targets for the treatment of sepsis.     

Use of Monoclonal Antibodies That Recognize p60 for Identification of Listeria monocytogenes

Listeria monocytogenes causes major food-borne outbreaks of disease worldwide. Specific identification of this microorganism is of utmost importance to public health and industry. Listeria species are known to secrete a 60-kDa protein collectively termed p60, which is encoded by the iap (invasion-associated protein) gene and secreted in large quantities into the growth media. p60 is a highly immunogenic murein hydrolase that is essential for cell division. Due to these properties, p60 is an ideal diagnostic target for the development of immunological detection systems for L. monocytogenes. We report here two independent lines of monoclonal antibody (MAb): p6007, which specifically recognizes L. monocytogenes p60, and p6017, which reacts with a wide range of Listeria p60 proteins. By combining these antibodies with a polyclonal antibody, we developed efficient sandwich enzyme-linked immunosorbent assay (ELISA) systems which can specifically identify L. monocytogenes or generally detect Listeria species. Since an excess amount of the peptide corresponding to PepA or PepD did not interfere with the ELISA, and direct ELISAs were unable to detect both peptides, we concluded that the epitope presumed to be recognized by p6007 or p6017 could be distinguished from PepA and PepD as described by Bubert et al. (Appl. Environ. Microbiol. 60:3120-3127, 1997). To our best knowledge, this is the first example of an immunological identification system that uses p60-recognizing MAbs.     

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

The effect of alphaMSH analogues on rat bones

Melanocortin is the downstream mediator of leptin signaling and absence of leptin signaling in ob/ob and db/db mice revealed the enhancement of bone formation through the central regulation. While alpha-melanocyte-stimulating hormone (alphaMSH) inhibits the secretion of interleukin-1alpha and tumor necrosis factor-alpha from the inflammatory cells, alphaMSH can also enhance clonal expansion of pro B cells linked to stimulation of osteoclastogenesis. Therefore, we tested the effect of melanocortin on bones. alphaMSH analogues [(6)His]alphaMSH-ND and [(6)Asn]alphaMSH-ND were synthesized and the radio-ligand receptor binding- and cyclic AMP generating activity were analyzed in China Hamster Ovary cell line over- expressing melanocortin receptors. The EC(50) of [(6)His]alphaMSH-ND measured from melanocortin-1, 3, 4 and 5 receptors were 0.008 +/- 0.0045, 1.523 +/- 0.707, 0.780 +/- 0.405, and 250.320 +/- 42.234 nM, respectively, and the EC(50) of [(6)Asn]alphaMSH-ND were 16.8 +/- 6.94, 271.8 +/- 21.95, 8.0 +/- 1.21, and 1132.5 +/- 635.46 nM, respectively. Four weeks after the subcutaneous injection of the analogues, the body weights in the [(6)His]alphaMSH-ND and the [(6)Asn]alphaMSH-ND treated groups (346.0 +/- 20.63 g vs. 350.0 +/- 13.57 g) were lower than that of the vehicle treated group (375.8 +/- 17.31 g, p < 0.05). There was no difference in the total femoral BMD measured by dual x-ray absorptiometry among the three groups. Among the three groups, there were no differences in the total numbers of crystal violet positive- or alkaline phosphatase positive colonies, in the expression of Receptor Activator of Nuclear Factor Kappa-B ligand on the tibia and the total number of multinucleated osteoclast-like cells differentiated from primary cultured bone marrow cells. From the above results, no evidence of bone gain or loss was found after treatment of the alphaMSH analogues peripherally.     

Increased inducible apoptosis in CD4+ T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin

Recent studies suggest that increased lymphocyte apoptosis (Ao) detected in peripheral blood T cells from burn patients appears to contribute to decreased lymphocyte immunoresponsiveness. However, while it is known that sepsis induces a marked depression in the splenocyte immune response (i.e. decreased interleukin-2, interferon-gamma production and proliferation) in response to the T-cell mitogen concanavalin A (Con A), it is unknown whether this depression is associated with an increase in inducible Ao and if so, which mediators control this process. To assess this, splenocytes were harvested from mice at 24 hr (a period associated with decreased Con A response) after the onset of polymicrobial sepsis [caecal ligation and puncture (CLP)] or sham-CLP (Sham) and then stimulated with 2.5 microg Con A/ml (24 hr). Septic mouse splenocytes stimulated with Con A, while not showing a change in their phenotypic make-up, did exhibit a marked increase in the percentage of splenocyte that were Ao+ which was associated with altered cytokine release. This appears to be due to an increase in the percentage of Ao+ cells in the CD4+ CD8- population and was associated with enhanced Fas antigen expression as well as an increase in mRNA for the Fas-FasL gene family. To determine if the changes in Ao are due to either endotoxin (a product of Gram-negative bacteria seen in CLP mice) or the expression of Fas ligand (FasL; a mediator of activation-induced lymphocyte Ao), a second set of studies examining Con A-inducible Ao was performed with splenocytes harvested from septic endotoxin-tolerant C3H/HeJ and the FasL-deficient C3H/HeJ-Fasl gld mice. The results show that increased splenocyte Ao detected following CLP is due to a FasL-mediated process and not to endotoxin. Thus the inadvertent up-regulation of FasL-mediated splenocyte Ao may contribute to the depression of splenocyte immune responses seen during polymicrobial sepsis.     

Preventive and therapeutic effects of oral tolerance in a murine model of asthma

Allergic asthma is an inflammatory disease of the airways, and Th2 cells secreting IL-4 and IL-5 play a pivotal role in its pathogenesis. We have previously demonstrated that oral tolerance can be induced and maintained more profoundly in a Th2-related immune response, and that an ongoing immune response can be suppressed by the oral administration of antigen combined with an appropriate feeding regimen. In the present study, we examined the preventive and therapeutic effects of the oral administration of allergen on a Th2-mediated immune disorder using a murine model of asthma. Our results show that the development of asthma can be blocked completely by orally administering allergen. Airway hyperreactivity, allergen-specific IgE production, Th2-derived cytokines, allergen-induced T cell proliferation and the infiltration of inflammatory effector cells into the lung were prevented by such oral administration. To assess the therapeutic effects of oral administration on the progression of asthma, we tested the effects of oral tolerance in an established asthma model, and found that a multiple high dose-feeding regimen was effective at suppressing the progression of mild asthma. In the high dose-feeding group, the number of eosinophils in bronchoalveolar lavage fluid was reduced and airway reactivity also decreased. However, this was insufficient to reduce airway reactivity and eosinophilia in bronchoalveolar lavage fluid in cases of severe asthma. These results demonstrate that allergic asthma may be ameliorated by feeding allergen; there is hope that these results will provide a new immunotherapeutic strategy for allergic asthma.     

Transient expression of osteopontin mRNA and protein in amoeboid microglia in developing rat brain

To investigate a potential role of osteopontin (OPN) in developing rat brain, the expression of OPN mRNA and protein in the developing rat brain relative to the distribution of brain macrophages was investigated using in situ hybridization and immunohistochemistry, and the phagocytic capability of OPN-expressing cells was accessed using rhodamine isothiocyanate (RhIc) as a tracer. OPN-expressing cells appeared from embryonic day 16. During the first week of postnatal life, OPN-labeled cells increased markedly, and peaked around P7, then declined and had completely disappeared by the end of the second postnatal week. The spatiotemporal distribution pattern of OPN mRNA closely matched that of OPN protein. Their morphology and localization were compared with those of cells expressing the established microglial marker OX-42 in adjacent sections, and double-labeling studies demonstrated that OPN was localized to the amoeboid microglia which stain with the lectin GSI-B₄, another marker for microglia. Furthermore, OPN-labeled cells were confirmed to be active phagocytes emitting RhIc fluorescence indicating that the tracer into the brain tissues was engulfed by phagocytosis. Therefore, these results provide the first evidence that OPN is transiently expressed in active brain macrophages in the embryonic and early postnatal brain, and suggest that OPN may contribute to the migration and phagocytic function of brain macrophages in the developing brain.This work was supported by a Korea Research Foundation grant (KRF-2002-015-EP0106)     

Regulation of phosphatidylinositol kinases by arachidonic acid in rat submandibular gland cells

Phosphoinositide kinases were characterized in membrane extracts of rat submandibular gland cells. Both phosphatidylinositol (PI) 4-kinase and phosphatidylinositol-4-phosphate (PI(4)P) 5-kinase phosphorylated endogenous substrates in reactions that were linear for up to 5 min, were activated by Mg²⁺ and showed maximal activity around neutral pH. PI 4-kinase was stimulated by Triton X-100 at an optimal concentration of 0.22%, but the detergent had an inhibitory effect on PI(4)P 5-kinase. Arachidonic acid (AA), at concentrations greater than 100 M, inhibited the activity of both enzymes in a dose-dependent manner. The inhibitory effect was replicated by other unsaturated fatty acids, but not by a saturated fatty acid of the sn-20 series. The nature of AA inhibition of the kinases was examined in enzyme kinetic studies with exogenous phosphoinositide and adenosine 5-triphosphate (ATP) substrates. Lineweaver-Burk plots of PI 4-kinase activity showed that AA had no effect on the apparent K _m for either PI or ATP, but that the fatty acid significantly reduced V _max (PI) from 331 to 177 pmol.mg^–1.min^–1 and V _max (ATP) from 173 to 59 pmol.mg^–1.min^–1. This inhibitory action was consistent for PI(4)P 5-kinase kinetics, where again, AA did not alter apparent K _m values, but lowered V _max for both PI(4)P and ATP by around 50%. Since the combination of a reduced V _max and an unchanged K _m value indicates noncompetitive enzyme inhibition, it is proposed that AA regulates phosphoinositide cycle activity in submandibular gland cells by acting as a noncompetitive inhibitor of PI 4-kinase and PI(4)P 5-kinase.     

Brain tumor in the first year of life: a single institute study.

Brain tumors in infants present special diagnostic and therapeutic challenges. To figure out the clinical features, pathological classification of the tumors and the treatment outcome of infantile brain tumors, 458 children (age<16) with brain tumors were reviewed retrospectively. Among them 21 cases (4.6%) were diagnosed during the first 12 months of life. Two tumors were definitely of congenital origin. The majority of infants with brain tumors presented with increased intracranial pressure. Fourteen tumors were located at the supratentorial area. Sixteen cases had neuroepithelial tumors; astrocytoma (optic pathway), supratentorial primitive neuroectodermal tumor (PNET) and medulloblastoma were found in three cases each. There were two treatment-related mortalities. Compared with the outcomes in older children, the treatment outcome was poorer in medulloblastoma and the optic pathway glioma which showed a higher growth potential. Because of the limited application of postoperative adjuvant therapy, radical surgical removal played a more important role in this age group. The prognosis of patients in whom the tumors could not be totally removed, largely depended on the pathological malignancy of the tumors. Though the treatment outcome was not always dismal, immaturity of the brain, higher growth potential, perioperative risks, limitations in adjuvant therapy, and pessimistic attitude on the part of parents made management more challenging.