The regulation of Sema4D exodomain shedding by protein kinase A in platelets.

We have previously shown that Sema4D expressed on the platelet plasma membrane can be cleaved by the metalloprotease ADAM17, producing a 120-kDa exodomain fragment that retains biological activity and remnant fragments of 24-28 kDa that remain associated with the platelet membrane. This process is modulated by calmodulin. Here we investigated the potential role of protein kinase A (PKA) in these events. Using a pharmacological approach, we now show that inhibition of PKA by H89 is sufficient to induce Sema4D exodomain shedding, while activation of PKA inhibits agonist-initiated shedding. Studies on the regulatory mechanism show that the shedding induced by PKA inhibition is mediated by ADAM17, but, unlike agonist-induced shedding, does not involve the dissociation of calmodulin from the Sema4D cytoplasmic domain. In attempt to identify the cleavage sites for shedding, we found that ADAM17 mediates variable cleavages in the juxtamembrane region. Therefore, our data reveal a potential regulatory mechanism for the shedding of Sema4D in platelets.

Tumor-ratio-metastasis staging system as an alternative to the 7th edition UICC TNM system in gastric cancer after D2 resection--results of a single-institution study of 1343 Chinese patients.

BACKGROUND: In this study, we assessed the prognostic value of the lymph node ratio (LNR), established a hypothetical tumor-ratio-metastasis (TRM) staging system and compared it with the 7th edition International Union Against Cancer pathological N (pN) and tumor-node-metastasis (TNM) system. PATIENTS AND METHODS: A total of 1343 gastric cancer patients undergoing D2 resection were staged using the TRM staging system and the 7th edition TNM system. Optimal cut points of LNR were calculated using X-tile software and validated by bootstrapping. Homogeneity, discriminatory ability, and monotonicity of gradients of the TRM and TNM systems were compared using linear trend χ(2), likelihood ratio χ(2) statistics, and Akaike information criterion (AIC) calculations. RESULTS: Optimal cut points classified patients into LNR0 (0%), LNR1 (1%-30%), LNR2 (31%-60%), and LNR3 (61%-100%) groups. In univariate, multivariate and stratified analyses, the LNR staging showed superiority to the 7th edition pN staging. The TRM staging system had higher linear trend and likelihood ratio χ(2) scores and smaller AIC values compared with those for the TNM system, which represented the optimum prognostic stratification. CONCLUSIONS: The novel TRM staging system predicts survival of gastric cancer more accurately than the 7th edition TNM system. It may be considered as an alternative to TNM system.

Virological, serological and biochemical outcomes through 3 years of entecavir treatment in nucleoside-naive Chinese chronic hepatitis B patients.

Hepatitis B virus (HBV) infection has a high prevalence in China. Entecavir has shown superior efficacy over lamivudine in Chinese nucleoside-naive chronic hepatitis B (CHB) patients over 48 weeks, with continued clinical benefit to 96 weeks. The present study evaluates the long-term efficacy of entecavir in Chinese CHB patients who continued entecavir treatment for 144 weeks. Patients receiving either entecavir 0.5 mg/day (n = 258) or lamivudine 100 mg/day (n = 261) entered the initial 96-week randomized, double-blind, controlled efficacy study. Patients who did not achieve a consolidated response [HBV DNA <0.7 MEq/mL; alanine aminotransferase (ALT) <1.25 x upper limit of normal; and if hepatitis B e antigen (HBeAg) positive at baseline, loss of HBeAg for ≥ 24 weeks] or who experienced viral breakthrough or relapse entered a 48-week entecavir rollover study. A total of 160 patients received continuous entecavir for 144 weeks; of these, 89% had undetectable serum HBV DNA, 86% showed ALT normalization, 20% reported HBeAg loss and 8% experienced HBeAg seroconversion. The cumulative rates of HBeAg loss and seroconversion were 36% and 27% at Week 144, respectively. The development of resistance was low, with three patients up to Week 96 and an additional two patients in Weeks 96-144 showing evidence of associated genotypic mutations. Entecavir was well tolerated. Adverse event rates were similar to those in lamivudine-treated patients, but patients receiving entecavir experienced fewer ALT flares. This study demonstrates that entecavir provides durable, long-term suppression of HBV DNA and ALT normalization in Chinese CHB patients, and is associated with low rates of emerging resistance. The results are consistent with the findings using entecavir globally and in Japan.

Dimerization-assisted energy transport in light-harvesting complexes.

We study the role of the dimer structure of light-harvesting complex II (LH2) in excitation transfer from the LH2 [without a reaction center (RC)] to the LH1 (surrounding the RC) or from the LH2 to another LH2. The excited and unexcited states of a bacteriochlorophyll (BChl) are modeled by a quasispin. In the framework of quantum open system theory, we represent the excitation transfer as the total leakage of the LH2 system and then calculate the transfer efficiency and average transfer time. For different initial states with various quantum superposition properties, we study how the dimerization of the B850 BChl ring can enhance the transfer efficiency and shorten the average transfer time.

Prediction of the prognosis of patients with acute-on-chronic hepatitis B liver failure using the model for end-stage liver disease scoring system and a novel logistic regression model.

The objective of this study was to determine the predictive value of the model for end-stage liver disease (MELD) scoring system in patients with acute-on-chronic hepatitis B liver failure (ACLF-HBV), and to establish a new model for predicting the prognosis of ACLF-HBV. A total of 204 adult patients with ACLF-HBV were retrospectively recruited between July 1, 2002 and December 31, 2004. The MELD scores were calculated according to the widely accepted formula. The 3-month mortality was calculated. The validity of the MELD model was determined by means of the concordance (c) statistic. Clinical data and biochemical values were included in the multivariate logistic regression analysis based on which the regression model for predicting prognosis was established. The receiver-operating characteristic curves were drawn for the MELD scoring system and the new regression model and the areas under the curves (AUC) were compared by the z-test. The 3-month mortality rate was 57.8%. The mean MELD score for the patients who died was significantly greater than those who survived beyond 3 months (28.7 vs 22.4, P = 0.003). The concordance (c) statistic (equivalent to the AUC) for the MELD scoring system predicting 3-month mortality was 0.709 (SE = 0.036, P < 0.001, 95% confidence interval 0.638-0.780). The independent factors predicting prognosis were hepatorenal syndrome (P < 0.001), liver cirrhosis (P = 0.009), HBeAg (P = 0.013), albumin (P = 0.028) and prothrombin activity (P = 0.011) as identified in multivariate logistic regression analysis. The regression model that was constructed by the logistic regression analysis produced a greater prognostic value (c = 0.891) than the MELD scoring system (z = 4.333, P < 0.001). The MELD scoring system is a promising and useful predictor for 3-month mortality of ACLF-HBV patients. Hepatorenal syndrome, liver cirrhosis, HBeAg, albumin and prothrombin activity are independent factors affecting the 3-month mortality. The newly established logistic regression model appears to be superior to the MELD scoring system in predicting 3-month mortality in patients with ACLF-HBV.

Kimura disease: review of the literature.

Kimura disease (KD) is a rare, chronic inflammatory disease of unknown cause and is characterized by painless s.c. swellings and lymphadenopathy commonly affecting the head and neck region. Much therapeutics has been used to treat KD, but is not satisfactory because of frequent relapse. Imatinib has been reported previously to be useful for treatment of hypereosinophilic syndrome and may work by selectively blocking protein-tyrosine kinases, such as platelet-derived growth factor receptor, and c-Kit. We carried out immunohistochemical examination of platelet-derived growth factor receptor-alpha and c-Kit in tissues from patients with KD. The results were positive and suggested that Imatinib might be an effective drug for the treatment of the disease. We have also briefly reviewed the epidemiology, aetiology, clinical manifestations, laboratory and pathological examinations, differential diagnoses, treatment and prognosis of KD in this manuscript.

Mesenteric lymph from rats subjected to trauma-hemorrhagic shock are injurious to rat pulmonary microvascular endothelial cells as well as human umbilical vein endothelial cells.

Previously, we have documented that gut-derived lymph from rats subjected to trauma/hemorrhagic shock (T/HS) is injurious to human umbilical vein endothelial cells (HUVEC). To verify these findings in an all rat systems, the ability of T/HS lymph to increase rat pulmonary microvascular endothelial cell (RPMVEC) monolayer permeability and kill RPMVEC was compared with that observed with HUVECs. RPMVEcs isolated from male rats or HUVECs were grown in 24-well plates for the cytotoxicity assays or on permeable filters in a two-chamber system for permeability assays. Mesenteric lymph was collected from male rats subjected to trauma (laparotomy) plus hemorrhagic shock (T/HS group) or to a laparotomy plus sham-shock (T/SS group). The T/HS group had their mean arterial pressure decreased to 30 mmHg and kept there for 90 min. Lymph samples centrifuged to remove the cellular component were incubated with the RPMVECs or HUVECs at a 10% concentration. Neither T/SS lymph nor post-T/HS portal vein plasma was toxic to or increased the permeability of the RPMVECs or HUVECs. The pattern of cytotoxicity observed in the HUVECs incubated with T/HS mesenteric lymph was similar to that observed in the RPMVECs, as reflected by trypan blue dye exclusion, with more than 95% of the HUVECs and RPMVECs being killed after a 16-h incubation with T/HS mesenteric lymph. However, at earlier time points the amount of LDH released from the HUVEC cells incubated with T/HS lymph was greater than that observed with the PRMVEC, although trypan blue dye exclusion was similar. Similarly, incubation with 10% T/HS lymph increased the permeability of both HUVEC and RPMVEC monolayers more than 2-fold, even with an incubation period as short as 1 h. In conclusion, these results provide further evidence that T/HS lymph, but not T/SS lymph or post-T/HS portal vein plasma, is injurious to endothelial cells and that RPMVECs are as susceptible to injury as HUVECs. Additionally, these studies support the emerging concept that gut-induced distant organ injury is mediated by factors contained in mesenteric lymph.

Hypoxia combined with Escherichia coli produces irreversible gut mucosal injury characterized by increased intestinal cytokine production and DNA degradation.

The objective of the present study was to determine whether hypoxia/reoxygenation in the absence or presence of intestinal bacteria would affect the integrity of the gut mucosal epithelium (as evidenced by histologic changes) and increase the local production of cytokines (interleukin 6 [IL-6] and tumor necrosis factor [TNF]). Rat ileal mucosal membranes were harvested and their electrophysiologic properties and barrier function were measured ex vivo in the Ussing chamber system. Membranes were exposed to normoxia, normoxia + Escherichia coli, hypoxia for 40 min followed by normoxia, or hypoxia for 40 min + E. coli followed by normoxia for 3 h. IL-6 and TNF levels were measured using cytokine-dependent cellular assays. Morphological changes and the degree of DNA fragmentation were used as quantitative markers of gut mucosal injury. Mucosal integrity was maintained in the normoxia group. The addition of bacteria increased the IL-6 response and reduced mucosal integrity. During the hypoxic period, a transient decline in resistance (R) occurred and cytokine production was reduced. In the hypoxic ileal membranes not exposed to E coli, reoxygenation reversed the change in R and increased IL-6 production. The combination of hypoxia/reoxygenation plus E. coli bacterial challenge resulted in the greatest extent of gut mucosal injury and increase in TNF production. The results of this study support the hypothesis that the combination of increased intestinal bacterial levels superimposed on an ischemia/reperfusion injury increases the magnitude of gut mucosal injury and the production and subsequent release of proinflammatory cytokines.

Endotoxin-induced mesenteric microvascular changes involve iNOS-derived nitric oxide: results from a study using iNOS knock out mice.

The administration of endotoxin alters intestinal blood flow, increases nitric oxide (NO) production, and induces gut barrier dysfunction. Thus, we investigated the hypothesis that microvascular reactivity and permeability of the mesenteric vascular bed are altered to a lesser degree in iNOS knock out (iNOS -/-) mice than their wild-type (iNOS +/+) litter mates after an endotoxin challenge. To test this hypothesis, we compared the microvascular response of iNOS knockout (iNOS -/-) mice after a topical or systemic endotoxin challenge against that of their wild-type litter mates (iNOS +/+). Intravital microscopy was used to measure arteriolar diameter and postcapillary venular permeability in the mouse ileum. Both parameters were determined by computer-assisted image analysis. Diameter was measured in A1, A2, and A3 arterioles (1, 2, 3 = rank of deployment). Changes in microvascular permeability were measured from changes in interstitial fluorescence caused by extravasation of fluorescein isothiocyanate (FITC)-dextran 150 (molecular weight = 150 kDa) and expressed as changes in integrated optical intensity (IOI). In the first series of experiments, endotoxin (100 microg/mL) was applied topically to the ileal segment. In the second series, endotoxin (10 mg/kg) was administered intraperitoneally (i.p.). Administration of topical or i.p.. endotoxin caused vasoconstriction and was associated with an early increase in permeability in both iNOS +/+ and -/- mice, although over time the responses of the iNOS -/- and iNOS +/+ mice diverged. Twenty minutes after topical endotoxin, the increase in permeability in iNOS -/- mice had reached a plateau whereas it continued to increase in the iNOS +/+ mice, such that at 80 min post-topical endotoxin, IOI was 27+/-7 in iNOS -/- vs. 39+/-5 in iNOS +/+ (P < 0.05). A similar permeability response was observed after i.p.. endotoxin, where the increase in post-capillary venular permeability was greater in the iNOS +/+ mice (P < 0.05). Both iNOS -/- and iNOS +/+ mice had a similar transient vasoconstrictive response after topical endotoxin challenge (reduction in A2 arteriolar diameters by -17+/-4% vs. -24+/-7%), with return to baseline values by 60-80 min post-endotoxin challenge. The iNOS +/+ but not the iNOS -/- mice manifested a secondary vasodilatory response that persisted throughout the experimental period. The arteriolar vasoreactive response of the iNOS -/- and iNOS +/+ mice to i.p.. endotoxin was similar to that of topical endotoxin, but of a lesser magnitude. In conclusion, the similarity in effects between topical and systemic endotoxin indicates that endotoxin causes microvascular dysfunction in the gut by directly on the microcirculation. In addition, our data suggest that NO production by iNOS is involved in the microvascular alterations associated with gut barrier dysfunction.

Mesenteric lymph duct ligation provides long term protection against hemorrhagic shock-induced lung injury.

Recently we have shown that ligation of the main mesenteric lymph (MLN) duct prior to an episode of hemorrhagic shock (HS) prevents shock-induced lung injury. Yet, ligation or diversion of intestinal lymph immediately prior to injury is not clinically feasible. Diversion of intestinally derived lymph after injury to protect against secondary insults is possible, but it is not known how long the protective effects of lymph ligation would last. Thus, we tested whether ligation of the MLN duct seven days prior to HS would still be protective. Male Sprague-Dawley rats were subjected to laparotomy with or without MLN duct ligation. Seven days later, half of the sham and actual MLN duct ligated animals randomly were selected to undergo HS (30 mmHG for 90 min). The other half of the animals was subjected to sham shock. Lung permeability, pulmonary myeloperoxidase (MPO) activity, and bronchoalveolar fluid (BALF) protein content were used to determine lung injury. Lymphatic division 7 days prior to HS continued to prevent shock induced lung injury as assessed by a lower Evans Blue dye concentration, BALF protein and MPO activity. In addition, there was no evidence of Patent Blue dye in the previously ligated MLN duct. Since ligation of the main mesenteric lymphatic duct continues to protect against shock-induced lung injury 1 week after duct ligation, it is feasible that lymphatic ligation performed after an injury remains protective against certain secondary insults for at least 1 week.

Injury-enhanced calcium mobilization in circulating rat neutrophils models human PMN responses.

G-protein coupled (GPC) chemoattractants are important neutrophil (PMN) activators in human shock and sepsis, acting in part by increasing cytosolic calcium ([Ca2+]i). Rats are widely used as laboratory models of shock and sepsis, but reports of [Ca2+]i flux in circulating rat PMN are rare. Moreover, the [Ca2+]i values reported often differ markedly from human systems. We developed study methods where basal [Ca2+]i values in circulating rat PMN were comparable to human PMN, but rat PMN still mobilized calcium poorly after stimulation. Trauma (laparotomy) did not change rat PMN basal [Ca2+]i, but induced brisk [Ca2+]i responses to chemokine and lipid mediators that approximated human PMN responses. This was associated with marked loading of microsomal calcium stores. Formyl peptides still mobilized calcium less well in rat than human PMN. Normal rat PMN appear to circulate in a less mature or primed form than human PMN. A very limited injury rapidly converts rat PMN to a more activated phenotype. PMN thus activated act quite similar to human PMN in terms of GPC receptor-mediated calcium mobilization. Trauma enhances rat PMN responses to GPC agonists at least in part by loading cell calcium stores.

Bacterial translocation from the gut impairs systemic immunity.

The goal of this study was to determine the influence of bacterial translocation on systemic immunity, since bacteria and their products play a major role in the development and maintenance of the host's immune system. To test this hypothesis, we measured the blastogenic response of mononuclear cells harvested from the blood, spleen, Peyer's patches, and mesenteric lymph nodes of control and Escherichia coli C25 monoassociated mice to a battery of mitogens. The E. coli C25 monoassociation model was used because this bacterial translocation model is not associated with experimental manipulations that are likely to affect the systemic immune system. The mitogenic response of lymphocytes isolated from the E. coli C25 monoassociated mice was significantly depressed compared to the control groups (p less than 0.01). Since the biologic significance of depressed in vitro mitogen responsiveness is difficult to determine, we assessed the ability of the mice to control a bacterial challenge using an in vivo Staphylococcus aureus abscess model. It appears that the observed changes in mitogen responsiveness may be of biologic significance, since the ability of the E. coli C25 monoassociated mice to control the injected S. aureus was impaired (p less than 0.01). These results suggest that an association exists between bacterial translocation and decreased systemic immune responsiveness.

Factors larger than 100 kd in post-hemorrhagic shock mesenteric lymph are toxic for endothelial cells.

BACKGROUND: Post-shock mesenteric lymph kills and injures endothelial cells (ECs), but neither the mechanism nor the mediators of lymph's toxic effect are known. Thus, in these studies we investigated and characterized potential factors that may be involved in lymph's toxic effect on ECs. METHODS: Lymph was collected hourly from rats before shock, during the shock period, and for 6 hours post-shock and processed in several ways-including removal of cellular elements, freezing, heating, or separation by molecular weight-after which they were tested for toxicity (lactate dehydrogenase as a marker of cell injury and trypan blue as a marker of cell viability). RESULTS: Controls consisting of medium, pre-shock lymph, and post-shock portal vein plasma had no EC toxicity. Lymph collected 1 to 3 hours post-shock resulted in the death of 90% to 95% of ECs and caused an 8- to 10-fold increase in lactate dehydrogenase release; however, this toxic effect waned by 4 hours post-shock. Endotoxin neutralization and immune cell removal did not decrease lymph cytotoxicity but complement inactivation did. By fractionating the toxic lymph samples by size, it appears that the putative EC cytotoxic mediator(s) is larger than 100,000 d. CONCLUSIONS: Mesenteric lymph collected 1 to 3 hours after hemorrhagic shock is toxic to ECs, but this effect is lost by 4- to 5-hours post-shock and is not dependent on the presence of immune cells or endotoxin but does involve complement and other putative mediators of greater than 100,000 d.

A time course study of the protective effect of mesenteric lymph duct ligation on hemorrhagic shock-induced pulmonary injury and the toxic effects of lymph from shocked rats on endothelial cell monolayer permeability.

BACKGROUND: We have previously documented that lymphatic duct division protects against shock-induced lung injury when tested 3 hours post-shock and that lymph collected at 3 hours post-shock increases endothelial cell monolayer permeability. However, whether lymph collected at other time points post-shock also increases endothelial cell permeability is not known. We tested the protective effects of lymphatic division on lung permeability at 6, 12, and 24 hours post-shock and the ability of lymph collected before, during, and hourly (up to 6 hours) after shock to increase endothelial cell monolayer permeability. METHODS: At 3, 6, 12, or 24 hours after sham or actual shock (30 mm Hg for 90 min), lung permeability was measured by using Evans blue dye in rats subjected to sham or actual mesenteric duct ligation. In separate experiments, the ability of lymph collected from rats subjected to shock or sham shock to increase human umbilical vein endothelial cell (HUVEC) monolayer permeability to a 40 kd dextran rhodamine permeability probe. Lymph was tested at 10% and 1% concentrations. RESULTS: Hemorrhagic shock induced a 3- to 4-fold increase in lung permeability compared with sham-shock rats when tested at 3, 6, 12, or 24 hours post-shock. Lymphatic division prevented this increase in lung permeability at each of these time points. Sham shock lymph did not increase HUVEC permeability, while lymph from the shocked rats did, whether tested at 1% or 10%. Lymph samples collected during the shock period and hourly for 6 hours post-shock all increased HUVEC permeability; however, the greatest relative increase in HUVEC permeability was observed in the 3- and 6- hour post-shock samples. CONCLUSIONS: Lung injury after hemorrhagic shock appears to be caused by toxic factors carried in the mesenteric lymph, and factors capable of increasing HUVEC permeability initially appear in the lymph during the shock period and increase over time.

Oral-TPN-induced bacterial translocation and impaired immune defenses are reversed by refeeding.

Although certain defined diets have been shown to promote bacterial translocation (BT), the ability to reverse diet-induced BT has not previously been investigated. Furthermore, little is known about the effects of defined diets on host immune defenses. To address these questions, we measured BT and immune reactivity in rats fed a normal diet or enteral elemental (ORAL-TPN) diet. After 7 days on the elemental or normal diet, the rats were killed, and BT and mitogen responsiveness to concanavalin A and phytohemagglutinin were measured. In separate experiments, the effects of these diets on in vivo host defenses was measured with a Staphylococcus aureus abscess model. Additional experiments were performed to determine the time required to reverse ORAL-TPN-induced BT and impairment of host immune defenses by reinstituting normal feedings. Administration of the ORAL-TPN diet for 7 days was associated with BT to the mesenteric lymph node complex of all animals, decreased blastogenic response of blood and splenic lymphocytes to mitogens, and decreased ability to control an in vivo infectious challenge with S. aureus. Each of the derangements was reversed by the reinstitution of normal feedings. In summary, the enteral administration of an elemental diet for 7 days is associated with disruption of the gut microflora, BT, and the development of an immunocompromised state, all of which can be reversed by refeeding the animals a normal diet.

Gut-derived mesenteric lymph: a link between burn and lung injury.

BACKGROUND: Previously, we showed that mesenteric lymph generated following hemorrhagic shock increases endothelial cell permeability and contributes to lung injury. It has also been shown that lymph produced at the site of burn injury plays a role in altering pulmonary vascular hemodynamics. In addition, previous experimental work has suggested that organs and tissues distant from the injury site may contribute to pulmonary dysfunction. One explanation would be that gut-derived inflammatory factors (in addition to those produced locally at the site of injury) are reaching the pulmonary circulation, where they exert their effects via the gut lymphatics. HYPOTHESES: The 2 hypotheses herein were that (1) gut-derived factors carried in the mesenteric lymph of rats generated following thermal injury will contribute to lung injury and (2) intestinal bacterial overgrowth will potentiate the degree of burn-induced lung injury. These hypotheses were tested by examining the effect of mesenteric lymph flow interruption prior to thermal injury on burn-induced lung injury in rats with a normal intestinal bacterial flora and in rats with intestinal Escherichia coli overgrowth. These rats were termed E. coli-monoassociated rats. METHODS: Normal intestinal bacterial flora and monoassociated male Sprague-Dawley rats were subjected to sham burn, 40% total body surface area burn, or lymphatic division plus burn. After 3 hours, 10 mg of Evans blue was injected to measure lung permeability. After the rats were killed, a bronchoalveolar lavage was performed and the fluid analyzed spectrophotometrically. Bronchoalveolar lavage fluid protein content, pulmonary myeloperoxidase activity, and alveolar apoptosis served to further quantitate lung injury. RESULTS: Both normal intestinal bacterial flora and monoassociated-burned rats exhibited significant increases in lung permeability, bronchoalveolar lavage fluid protein content, myeloperoxidase activity, and alveolar apoptosis. The combination of monoassociation and thermal injury resulted in even further increases in lung injury over thermal injury alone. Lymphatic division prior to thermal injury ameliorated burn-induced increases in lung permeability, bronchoalveolar lavage fluid protein content, pulmonary myeloperoxidase accumulation, and alveolar apoptosis in both normal intestinal bacterial flora and monoassociated rats. CONCLUSIONS: The results of this study support the hypothesis that gut-derived factors carried in the mesenteric lymph contribute to burn-induced lung injury and may therefore play a role in postburn respiratory failure and suggest that intestinal bacterial overgrowth primes the host such that when animals are exposed to a second stimulus (such as thermal injury) an exaggerated response occurs.

Effect of heat shock and endotoxin stress on enterocyte viability apoptosis and function varies based on whether the cells are exposed to heat shock or endotoxin first.

BACKGROUND: Stress-gene responses, including the heat shock (HS) response and the acute phase response, are protective mechanisms for cells after exposure to stress. Both responses cannot occur simultaneously, and, in endothelial cells, the sequence of stress-gene expression seems to be a critical factor in whether cellular protection or injury occurs. OBJECTIVE: To determine if the sequence of stress-gene expression affects cellular protection or injury in epithelial cells. DESIGN: Randomized controlled in vitro study. SETTING: University research laboratory. SUBJECTS: Rat intestinal epithelial cell-6 (IEC-6) cells were grown on 35-mm culture dishes, chamber slides, or in a bicameral system to confluence or until tight junction integrity was established. INTERVENTIONS: Rat IEC-6 cells were examined for viability, apoptosis, and bacterial translocation (BT) after exposure to 25-micrograms/mL lipopolysaccharide (LPS) for 18 hours to HS (43 degrees C) for 90 minutes, to LPS followed by HS, or to HS followed by LPS. MAIN OUTCOME MEASURES: The IEC-6 cells were stained for viability and apoptosis using trypan blue and a direct immunoperoxidase detection of digoxigenin-labeled genomic DNA (Apop Tag Plus In Situ Apoptosis Detection Kit, Oncor, Gaithersburg, Md), respectively. Bacterial translocation was measured by culturing the bacteria (ie, Escherichia coli) that crossed the IEC-6 cell monolayer in the bicameral system. RESULTS: Control cells (medium only) and cells exposed to LPS alone, HS alone, or HS followed by LPS had a viability from 92% to 98%, and the percentage of apoptotic cells ranged from 2.2% to 5.7%. In contrast, IEC-6 cells exposed to LPS followed by HS had a significantly lower viability (83%, P < .05 vs all other groups) and a higher percentage of apoptotic cells (12.2%, P < .01). At 3 hours after challenge with E coli, the LPS-exposed IEC-6 cell monolayers had significantly increased BT vs control monolayers (P < .05), while the IEC-6 cell monolayers exposed to HS followed by LPS had decreased BT (P < .05). Conversely, cells exposed to LPS followed by HS had the highest magnitude of BT (P < .01 vs all other groups). CONCLUSIONS: These results indicate that preinduction of HS response can diminish LPS-induced cell injury, while induction of HS response after the LPS challenge (ie, the acute phase response) may lead to decreased enterocyte viability, increased apoptosis, and cellular dysfunction as manifested by BT.

Expression of a fusion protein of scFv-biotin mimetic peptide for immunoassay.

We constructed two fusion proteins of scFv linked to biotin mimetic sequence (BMS) via different linkers, and expressed them in the Pichia pastoris expression/secretion system. We found that both bi-functional scFv proteins exhibited their intrinsic binding activities to antigen CA125 determined in competitive radioimmunoassay experiments, but the fusion protein with a spacer between the scFv and BMS (scFv-spacer-BMS) showed higher binding activity of streptavidin than the one with c-Myc peptide as a linker.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes.

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.