Predictive medicine: severe trauma and burns

Severe trauma and burn injury are often associated with a life-threatening systemic inflammatory response, only to be followed by severe infections. Although many parameters of the immune system are depressed or altered, only the innate immune system has been directly correlated with infections in these patients. The innate immune system plays an important role in both the inflammatory response and defense against infections. These types of sequelae suggest that at any particular point in time, depending upon the patient status, either a hyperactive or suppressed polymorphonuclear neutrophil (PMN) response may be detected. In fact, this dichotomy has been shown to occur in numerous published studies.     

CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of CaV1.2, CaV2.2, and CaV2.3 ion channel proteins, the cAMP-mediated activation of motility by HCO3-, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca2+ channel that controls Ca2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.     

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

Bilirubin is the principal end product of heme degradation. Prompted by epidemiologic analyses demonstrating an inverse correlation between serum bilirubin levels and cancer mortality, we examined the effect(s) of bilirubin on the growth and survival of colon adenocarcinoma cells. Adenocarcinoma cell monolayers were treated with bilirubin over a range of bilirubin:BSA molar ratios (0-0.6), and viability was assessed colorimetrically. Apoptosis was characterized by TUNEL assay, annexin V staining and caspase-3 activation. The mechanism(s) by which bilirubin induces apoptosis was investigated by Western blotting for cytochrome c release, assaying for caspase-8 and caspase-9 activation and for mitochondrial depolarization by JC-1 staining. The direct effect of bilirubin on the membrane potential of isolated mitochondria was evaluated using light-scattering and fluorescence techniques. Bilirubin decreased the viability of all colon cancer cell lines tested in a dose-dependent manner. Cells exhibited substantial apoptosis when exposed to bilirubin concentrations ranging 0-50 microM, as demonstrated by an 8- to 10-fold increase in TUNEL and annexin V staining and in caspase-3 activity. Bilirubin treatment evokes specific activation of caspase-9, enhances cytochrome c release into the cytoplasm and triggers the mitochondrial permeability transition in colon cancer monolayers. Additionally, bilirubin directly induces the depolarization of isolated rat liver mitochondria, an effect that is not inhibited by cyclosporin A. Bilirubin stimulates apoptosis of colon adenocarcinoma cells in vitro through activation of the mitochondrial pathway, apparently by directly dissipating mitochondrial membrane potential. As this effect is triggered at concentrations normally present in the intestinal lumen, we postulate a physiologic role for bilirubin in modulating colon tumorigenesis.     

RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties

Tripartite motif protein 32, Trim32, mRNA and protein expression was elevated in independently transformed and tumorigenic keratinocytes of a mouse epidermal carcinogenesis model, in ultraviolet B (UVB)-induced squamous cell carcinomas (SCC), and in approximately 20-25% of chemically induced mouse papillomas and human head and neck SCCs. This suggests that elevated Trim32 expression occurs frequently in experimental epidermal carcinogenesis and is relevant to human cancer. Transduced Trim32 increased colony number in an epidermal in vitro transformation assay and epidermal thickening in vivo when skin-grafted to athymic nu/nu mice. These effects were not associated with proliferation and were not sufficient for tumorigenesis, even with 12-O-tetradecanoylphorbol-13-acetate treatment or defects in the tumor suppressor p53. However, transduced Trim32 inhibited the synergistic effect of tumor necrosis factor alpha (TNFalpha) on UVB-induced apoptosis of keratinocytes in vitro and the apoptotic response of keratinocyte grafts exposed to UVB-light in vivo. Consistent with its RING domain, Trim32 exhibited characteristics of E3-ubiquitin ligases, including being ubiquitylated itself and interacting with ubiquitylated proteins, with increases in these properties following treatment of cultured keratinocytes with TNFalpha/UVB. Interestingly, missense point mutation of human TRIM32 has been reported in Limb-Girdle Muscular Dystrophy Type 2H, an autosomal recessive disease. We propose a model in which Trim32 activities as an E3-ubiquitin ligase favor initiated cell survival in carcinogenesis by blocking UVB-induced TNFalpha apoptotic signaling.     

Blockade of HIV-1 infection of New World monkey cells occurs primarily at the stage of virus entry

HIV-1 naturally infects chimpanzees and humans, but does not infect Old World monkeys because of replication blocks that occur after virus entry into the cell. To understand the species-specific restrictions operating on HIV-1 infection, the ability of HIV-1 to infect the cells of New World monkeys was examined. Primary cells derived from common marmosets and squirrel monkeys support every phase of HIV-1 replication with the exception of virus entry. Efficient HIV-1 entry typically requires binding of the viral envelope glycoproteins and host cell receptors, CD4 and either CCR5 or CXCR4 chemokine receptors. HIV-1 did not detectably bind or utilize squirrel monkey CD4 for entry, and marmoset CD4 was also very inefficient compared with human CD4. A marmoset CD4 variant, in which residues 48 and 59 were altered to the amino acids found in human CD4, supported HIV-1 entry efficiently. The CXCR4 molecules of both marmosets and squirrel monkeys supported HIV-1 infection, but the CCR5 proteins of both species were only marginally functional. These results demonstrate that the CD4 and CCR5 proteins of New World monkeys represent the major restriction against HIV-1 replication in these primates. Directed adaptation of the HIV-1 envelope glycoproteins to common marmoset receptors might allow the development of New World monkey models of HIV-1 infection.     

Early persistent activation of sperm K+ channels by the egg peptide speract.

Transduction by sperm of the instructive signal provided by the egg peptide speract involves rapid, complex changes in internal ion and cyclic nucleotide content. Here, investigations of hypotonically swollen sperm provide insight into the underlying processes and identify K+ channel activation as an initial ionic event in gamete recognition. A sustained hyperpolarization of swollen sperm is promoted by less than 2.5 pM speract and is followed (with greater than 100 pM speract) by transient repolarization and (with greater than 10 nM speract) by depolarization that is dependent on external Ca2+. Monophasic increases in pHi are produced only by greater than 25 pM speract, indicating that hyperpolarization may not directly promote alkalinization. Increased K(+)-selective (K+ greater than Rb+ greater than Cs+ greater than Na+) membrane permeability is found after all speract greater than 2.5 pM, suggesting that hyperpolarization results from persistent activation of K+ channels and that repolarization has a different ionic basis. Supporting this contention, the K+ channel blocker tetraethylammonium (20 mM) inhibits the increased K+ permeability that follows treatment of swollen sperm (and of sperm in seawater) with 2.5 pM speract. Such induced activation of K+ channels is observed in patch-clamped swollen sperm examined in the cell-attached configuration, upon application of 5-50 pM speract to the bath medium. The efficacy of externally applied speract and its potency indicate that activation is indirect and probably involves an as yet unidentified diffusible mediator whose production is promoted by speract at concentrations 0.01-0.001 times those predicted from reported estimates of the Kd for the known speract receptor.     

39K NMR measurement of intracellular potassium during ischemia in the perfused guinea pig heart

The hyperfine shift reagent, TmDOTP^5?, was used to resolve the ³⁹K NMR resonances of intra- (K_i⁺) and extracellular (K_e⁺) potassium in isolated, perfused guinea pig hearts. [K_i⁺] as measured by ³⁹K NMR was 25.9 ± 10.3 mM, compared with 114.4 ± 10.8 mM as measured by atomic absorption spectros-copy (AAS) using TmD0TP^5- as a marker of extracellular space. Thus, only approximately 23% of intracellular potassium was detected by ³⁹K NMR using our experimental conditions. The area of the K_i⁺ signal increased during early ischemia then returned to baseline levels during reperfusion. In an effort to learn more about the K_i⁺ not detected by ³⁹K NMR, hearts were perfused with a Rb⁺-enriched, K⁺ -depleted buffer for an extended period. This resulted in loss of the entire ³⁹K NMR signal, and K_i⁺, as measured by AAS, decreased from ～60 to ～6 to 7 μmol/g wet weight. When K⁺-depleted hearts were subjected to global ischemia, a small ³⁹K NMR signal reappeared, suggesting that at least a portion of the nonexchangeable K_i⁺ becomes detectable by NMR during ischemia. This newly visible K⁺ signal subsequently dissipated during reperfusion of ischemic hearts. We conclude that ischemia induces changes in the NMR visibility of ³⁹K in perfused guinea pig hearts.     

Role of cellular RNA polymerases in virus-induced leukemogenesis.

Friend murine leukemia virus induces splenic enlargement and an increase in RNA polymerase activity of spleen nuclei. Actinomycin D, administered at 60 mug/kg body weight/day prevents the development os splenomegaly and the elevation of polymerase activity following infection, but it has only a slight effect on the production of virus in spleen tissue. Thus, the alteration of RNA synthesis is not a result of virus proliferation, but instead may be a manifestation of leukemic erythropoiesis. Normal erythropoiesis, stimulated by erythropoietin administration, produces a similar but transient increase in RNA polymerase activity in spleen nuclei. Erythropoietin administered before, but not after, Friend virus infection results in an enhancement of RNA polymerase activity, as measured 9 days after inoculation. This effect is most simply explained by assuming that there is a common target cell pool for both erythropoietin and Friend virus, and that this pool becomes refractory to the influence of the hormone as a result of the leukemic process.     

Prolonged survival of reconstituted skin grafts without immunosuppression

Reconstituted skin composed of a cultured allogeneic epithelial sheet (CAES) and a cultured allogeneic dermis (CAD) was evaluated in a rat model to determine whether it could survive for a prolonged period without immunosuppression. Additionally, free CAD grafts were evaluated for their suitability as dermal substitutes. Male Buffalo rats were used as donors and male Lewis rats as recipients. Split-thickness skin obtained from Buffalo rats was separated into epidermis and dermis by means of Dispase II enzyme. The epidermal layers were minced and trypsinized. Then dispersed single keratinocytes were inoculated onto a irradiated 3T3 cell feeder layer. After a suitable period, a confluent cultured keratinocyte layer was detached and provided CAES grafts. Cultured allogeneic dermis grafts were prepared from cultures of the dermal component. Cultured allogeneic dermis grafts, covered by split thickness isografts (STIG) or local skin flaps, became revascularized at a rate of 94.6% and 90.9%, respectively, 7 days after grafting. However, only 25% of CAD grafts covered by synthetic materials became vascularized. Four types of wound coverage were compared including: (1) CAES grafts, (2) CAES over CAD grafts, (3) split-thickness isografts, and (4) STIG over CAD grafts. In groups 2 and 4, CAD grafts were applied 7 days before CAES grafts or STIG. Grafts of groups 1 and 2 were successful in only 36.7% and 31.1% of the animals and resulted in a high rate of wound contracture--72.4%, 66.7%, respectively. On the other hand, in groups 3 and 4, higher average rates of revascularization (92.0% and 88.3%) and lower rates of wound contracture (25.4% and 24.2%) were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)