Pronounced cancer resistance in a subterranean rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence.

BACKGROUND: Subterranean blind mole rats (Spalax) are hypoxia tolerant (down to 3% O2), long lived (>20 years) rodents showing no clear signs of aging or aging related disorders. In 50 years of Spalax research, spontaneous tumors have never been recorded among thousands of individuals. Here we addressed the questions of (1) whether Spalax is resistant to chemically-induced tumorigenesis, and (2) whether normal fibroblasts isolated from Spalax possess tumor-suppressive activity. RESULTS: Treating animals with 3-Methylcholantrene (3MCA) and 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), two potent carcinogens, confirmed Spalax high resistance to chemically induced cancers. While all mice and rats developed the expected tumors following treatment with both carcinogens, among Spalax no tumors were observed after DMBA/TPA treatment, while 3MCA induced benign fibroblastic proliferation in 2 Spalax individuals out of12, and only a single animal from the advanced age group developed malignancy 18 months post-treatment. The remaining animals are still healthy 30 months post-treatment. In vitro experiments showed an extraordinary ability of normal Spalax cultured fibroblasts to restrict malignant behavior in a broad spectrum of human-derived and in newly isolated Spalax 3MCA-induced cancer cell lines. Growth of cancer cells was inhibited by either direct interaction with Spalax fibroblasts or with soluble factors released into culture media and soft agar. This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation. Cells from another cancer resistant subterranean mammal, the naked mole rat, were also tested for direct effect on cancer cells and, similar to Spalax, demonstrated anti-cancer activity. No effect on cancer cells was observed using fibroblasts from mouse, rat or Acomys. Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells. CONCLUSIONS: This report provides pioneering evidence that Spalax is not only resistant to spontaneous cancer but also to experimentally induced cancer, and shows the unique ability of Spalax normal fibroblasts to inhibit growth and kill cancer cells, but not normal cells, either through direct fibroblast-cancer cell interaction or via soluble factors. Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated. Exploring the molecular mechanisms allowing Spalax to survive in extreme environments and to escape cancer as well as to kill homologous and heterologous cancer cells may hold the key for understanding the molecular nature of host resistance to cancer and identify new anti-cancer strategies for treating humans.

ATP release and autocrine signaling through P2X4 receptors regulate γδ T cell activation.

Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional αß T cells release ATP upon TCR cross-linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the "unconventional" γδ T lymphocytes. We observed that γδ T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or IPP. Pretreatment of γδ T cells with (10)panx-1, CBX, or Bf A reversed the stimulation-induced increase in extracellular ATP concentration, indicating that panx-1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with (10)panx-1 inhibited Ca(2+) signaling in response to TCR stimulation. qPCR revealed that γδ T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP-ATP inhibited transcriptional up-regulation of TNF-α and IFN-γ in γδ T cells stimulated with anti-CD3/CD28-coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human γδ T cells.

A3 adenosine receptor inhibition improves the efficacy of hypertonic saline resuscitation.

We reported previously that hypertonic saline (HS) treatment can prevent or upregulate the function of polymorphonuclear neutrophils (PMNs) via A2a-type adenosine receptors or A3-type adenosine receptors (A3R), respectively. A3R translocate to the cell surface upon PMN stimulation, and thus, HS promotes PMN responses under conditions of delayed HS treatment. Here we investigated if inhibition of A3R improves the protective effects of HS resuscitation in a mouse sepsis model. We found that HS nearly triples extracellular adenosine concentrations in whole blood and that inhibition of A3R with the selective antagonist MRS-1191 dose dependently improves the inhibitory effect of HS. MRS-1191 at a concentration of 1 nM enhanced the inhibitory effect of HS and reduced stimulatory effects of delayed HS treatment. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found that MRS-1191 reduces acute lung injury and PMN accumulation in lung tissue. Whereas delayed HS treatment (4 mL/kg of 7.5% NaCl) of mice 1 h after CLP aggravated PMN accumulation, lung tissue damage, and mortality 24 h after CLP, infusion of MRS-1191 (2 ng/kg body weight) combined with HS reduced these detrimental effects of delayed HS treatment. Our data thus show that A3 receptor antagonists can strengthen the beneficial effects of HS resuscitation by avoiding stimulatory adverse effects that result from delayed HS administration.

Autocrine regulation of T-cell activation by ATP release and P2X7 receptors.

T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X(7) receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4(+) T cells express abundant P2X(7) receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 microM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X(7) receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X(7) receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X(7) receptors, compared to control BALB/c mice, which express fully functional P2X(7) receptors. We conclude that ATP release and autocrine, positive feedback through P2X(7) receptors is required for the effective activation of T cells.

Hypertonic saline increases gammadeltaT cell-mediated killing of activated neutrophils.

OBJECTIVE: Hypertonic saline fluids used to resuscitate trauma patients can prevent neutrophil-mediated lung tissue damage, making them attractive alternatives to conventional resuscitation fluids. We have previously shown that gammadeltaT cells, a small T lymphocyte subset, reduce acute inflammatory lung damage by eliminating activated neutrophils that express heat shock protein 72 on the cell surface. Here, we studied whether these protective effects of hypertonic saline are related to improved gammadeltaT cell-mediated neutrophil killing. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Human peripheral blood from healthy subjects--isolated gammadeltaT lymphocytes and neutrophils. INTERVENTIONS: Isolated blood cells were treated with different concentrations of hypertonic saline and endotoxin of Escherichia coli O111:B4 (lipopolysaccharide). In some experiments, gammadeltaT cells were activated by CD3 cross-linking or by phorbol-myristate acetate and ionomycin, or by phytohemagglutinin. MEASUREMENTS AND MAIN RESULTS: Clinically relevant concentrations of hypertonic saline (20 mM) significantly augmented CD69 expression of gammadeltaT cells that were stimulated with 100 ng/mL lipopolysaccharide. Additionally, lipopolysaccharide induced a three- to five-fold increase in tumor necrosis factor-alpha and interleukin-10 expression by gammadeltaT cells. This response was completely abrogated by hypertonic saline. These data indicate that hypertonic saline can modulate gammadeltaT cell functions. Stimulation of neutrophils with 1-1,000 ng/mL lipopolysaccharide caused a greater than 3-fold increase in heat shock protein-72 expression on the cell surface, which was significantly augmented by hypertonic saline. In cocultures of gammadeltaT cells with autologous neutrophils, 15.6 +/- 3.4% of all neutrophils were killed within 120 min. In the presence of lipopolysaccharide (1 microg/mL), this percentage increased to 23.7 +/- 2.1%, and it was further increased to 31.8 +/- 3.1% when 20 mM hypertonic saline was added with lipopolysaccharide. CONCLUSIONS: Our findings suggest that hypertonic saline enhances the elimination of inflammatory neutrophils by gammadeltaT cells by augmenting heat shock protein-72 expression on the cell surface of neutrophils. Hypertonic saline resuscitation may therefore protect host tissues by enhancing neutrophil clearance from the lungs.

Hypertonic saline up-regulates A3 adenosine receptor expression of activated neutrophils and increases acute lung injury after sepsis.

OBJECTIVE: Hypertonic saline resuscitation reduces tissue damage by inhibiting polymorphonuclear neutrophils. Hypertonic saline triggers polymorphonuclear neutrophils to release adenosine triphosphate that is converted to adenosine, inhibiting polymorphonuclear neutrophils through A2a adenosine receptors. Polymorphonuclear neutrophils also express A3 adenosine receptors that enhance polymorphonuclear neutrophil functions. Here we investigated whether A3 receptors may diminish the efficacy of hypertonic saline in a mouse model of acute lung injury. DESIGN: Randomized animal study and laboratory investigation. SETTING: University research laboratory. INTERVENTIONS: The effect of A3 receptors on the efficacy of hypertonic saline resuscitation was assessed in A3 receptor knockout and wild-type mice. Animals were treated with hypertonic saline (7.5% NaCl, 4 mL/kg) before or after cecal ligation and puncture, and acute lung injury and mortality were determined. The effect of timing of hypertonic saline exposure on A3 receptor expression and degranulation was studied in vitro with isolated human polymorphonuclear neutrophils. MEASUREMENTS AND MAIN RESULTS: Treatment of human polymorphonuclear neutrophils with hypertonic saline before stimulation with formyl methionyl-leucyl-phenylalanine inhibited A3 receptor expression and degranulation, whereas hypertonic saline-treatment after formyl methionyl-leucyl-phenylalanine-stimulation augmented A3 receptor expression and degranulation. Acute lung injury in wild-type mice treated with hypertonic saline after cecal ligation and puncture was significantly greater than in wild-type mice pretreated with hypertonic saline. This aggravating effect of delayed hypertonic saline-treatment was absent in A3 receptor knockout mice. Similarly, mortality in wild-type mice with delayed hypertonic saline-treatment was significantly higher (88%) than in animals treated with hypertonic saline before cecal ligation and puncture (50%). Mortality in A3 receptor knockout mice remained only 50% regardless of timing of hypertonic saline administration. CONCLUSIONS: Polymorphonuclear neutrophil A3 receptors expression determines whether hypertonic saline resuscitation inhibits or aggravates polymorphonuclear neutrophil-induced acute lung injury. These findings suggest that A3 antagonists could improve the efficacy of hypertonic saline resuscitation by reducing side effects in patients whose polymorphonuclear neutrophils are activated before hypertonic saline treatment.

Roles of heat shock proteins and gamma delta T cells in inflammation.

Elimination of activated inflammatory cells that infiltrate and damage host organs can reduce morbidity and mortality. A better understanding of the mechanisms by which these processes occur may lead to new approaches to prevent tissue damage. The lungs, gastrointestinal tract, and skin are particularly prone to infection and collateral damage by inflammatory cells. Specialized lymphocytes protect these organs from collateral tissue damage by eliminating neutrophils and macrophages from inflamed tissues. These lymphocytes recognize signals produced by inflammatory cells. One such signal is heat shock protein (Hsp) expressed on the cell surface of inflamed phagocytes. Mammalian Hsp molecules closely resemble their microbial equivalents, and therefore phagocytes decorated with these molecules are recognized as target cells. T lymphocytes bearing the gammadelta T cell receptor (TCR) elicit cytotoxic activity toward macrophages and neutrophils that express Hsp60 and Hsp70, respectively, protecting host organs from collateral tissue damage by phagocytes.

A3 and P2Y2 receptors control the recruitment of neutrophils to the lungs in a mouse model of sepsis.

We have recently shown that A3 adenosine receptors and P2Y2 purinergic receptors play an important role in neutrophil chemotaxis. Chemotaxis of neutrophils to sites of infections is critical for immune defense. However, excessive accumulation of neutrophils in the lungs can cause acute lung tissue damage. Here we assessed the role of A3 and P2Y2 receptors in neutrophil sequestration to the lungs in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) using adult male C57BL/6J mice (wild type [WT]), homozygous A3 receptor knockout (A3KO) mice, and P2Y2 receptor knockout (P2Y2KO) mice. Animals were killed 2, 4, 6, or 8 h after CLP, and peritoneal lavage fluid and blood were collected. Lungs were removed, and neutrophil infiltration was evaluated using elastase as a marker. Leukocyte and bacterial counts in peritoneal lavage fluid and blood samples were determined. Survival after sepsis was determined in a separate group. Leukocyte counts in the peritoneum were lower in A3KO and P2Y2KO mice than in WT mice. Conversely, initial leukocyte counts in the peripheral blood were higher in KO mice than in WT mice. Neutrophil sequestration to the lungs reached a maximum 2 h after CLP and remained significantly higher in WT mice compared with A3KO and P2Y2KO mice (P < 0.001). Survival after 24 h was significantly lower in WT mice (37.5%) than in A3KO or P2Y2KO mice (82.5%; P < 0.05). These data suggest that A3 and P2Y2 receptors are involved in the influx of neutrophils into the lungs after sepsis. Thus, pharmaceutical approaches that target these receptors might be useful to control acute lung tissue injury in sepsis.

Use of the hemostatic agent QuikClot for the treatment of massive splenic injury in a rat model.

BACKGROUND: In the present study, QuikClot (QC) was used to treat intra-abdominal bleeding induced by massive splenic injury (MSI) in rats. STUDY DESIGN: 40 animals were divided into five groups: (1) sham operated; (2) MSI untreated; (3) MSI treated with 41.5 ml/kg lactated Ringer's solution (RL); (4) MSI treated with QC, and (5) MSI treated with QC and RL. RESULTS: Untreated MSI was followed by mortality of 60%, total blood loss (TBL) of 33.69% and mean survival time (MST) of 153.9 min. MSI treatment with RL resulted in mortality of 100%, TBL of 61.8% (p < 0.001), and MST of 92.2 min (p < 0.05). MSI treated with QC was followed by TBL of 14.1% (p < 0.005) and MST of 237.5 min (p < 0.05) with no mortality. MSI treated by QC and RL led TBL of 27.4% (p < 0.001 vs. group 3), and MST of 233.3 min (p < 0.05) and no mortality. CONCLUSIONS: QC significantly reduced blood loss from the injured spleen with improved survival. Combination of RL and QC prevented the increase in blood loss and improved survival compared to RL alone.

Heparanase pretreatment attenuates endotoxin-induced acute lung injury in rats.

A central event of systemic inflammation and septic organ injury is infiltration of tissues with polymorphonuclear neutrophils, likely modulated by the integrity of the extracellular matrix underlying the vascular endothelium. In the present study, the effect of matrix-modifying endoglycosidase (heparanase) on endotoxin (LPS)-induced inflammatory lung injury was investigated in rats. Animals were treated with heparanase or LPS or pretreated with heparanase before LPS injection, and acute lung injury was verified histologically and characterized by analysis of bronchoalveolar lavage fluids. Pretreatment with heparanase attenuated the mortality of animals and preserved the histological structure of the lungs. Furthermore, polymorphonuclear neutrophil accumulation and activation, analyzed by myeloperoxidase release and reactive oxygen species production associated with lung injury, were significantly reduced upon heparanase pretreatment. In addition, heparanase pretreatment elevated the IL-10 levels in the pulmonary compartment. Moreover, results from in vitro experiments have identified monocyte-derived IL-10 as an important mediator used by heparanase to suppress inflammatory reactions. The protective effect of heparanase may indicate a novel therapeutic strategy for sepsis.

Ultrastructural features of lymphocyte suppression induced by anthrax lethal toxin and treated with chloroquine.

Antibacterial therapy does not fully protect against anthrax because of severe systemic intoxication. Lysosomal processing of anthrax lethal toxin (LTX) is a key event in the disease pathogenesis, and agents interfering with this process, like chloroquine (CQ), may have practical applications. Although LTX is known to induce T-cell suppression, precise mechanisms of this phenomenon are not completely characterized. In the present study, we investigated alterations of lymphocyte ultrastructure caused by LTX and associated with favorable effect of CQ on the LTX-related dysfunction. Peripheral blood lymphocytes were activated via CD3 crosslinking in the presence or absence of LTX and CQ, and examined by transmission electron microscopy, flow cytometry and immunoblotting. Crosslinking of CD3 induced ultrastructural signs of lymphocyte activation, mostly disappeared after LTX treatment. The cell ultrastructure was well preserved in LTX-treated cells, despite dose- and time-dependent inhibition of T-cell function associated with impaired activation of mitogen-activated protein kinase. Regardless of intracellular signaling abnormalities, LTX did not decrease T-cell viability. CQ restored expression of CD69 (P<0.001) and improved phosphorylation of p38 (P=0.022) in LTX-exposed T lymphocytes. The exposure of cells to CQ, with or without LTX, led to appearance of many phagolysosomes with heterogeneous content, possibly representing unprocessed internalized material. In conclusion, LTX suppressed T-cell functions, but did not affect the viability and caused no ultrastructural damage. Ultrastructural observations indicated that CQ reduced harmful effects of LTX, possibly by interfering with lysosomal activity.

Adverse effects of increased intra-abdominal pressure on small bowel structure and bacterial translocation in the rat.

BACKGROUND: The purpose of this study was to evaluate the effects of elevated intra-abdominal pressure (IAP) on intestinal structures and bacterial translocation in the rat. MATERIALS AND METHODS: Forty-two male Sprague-Dawley rats were randomly divided into three experimental groups of 14 rats each: the sham group underwent insertion of a balloon-tipped catheter; the IAP-15 group was subjected to a 15 mm Hg pneumoperitoneum for 60 minutes; and the IAP-25 group was subjected to a 25 mm Hg pneumoperitoneum for 60 minutes. Intestinal structural changes (bowel circumference, overall bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth) and bacterial translocation to mesenteric lymph nodes, liver, spleen, portal blood, and peripheral blood were determined 24 hours following pneumoperitoneum. RESULTS: IAP-15 and IAP-25 rats demonstrated a significant decrease in: bowel and mucosal weight in the duodenum, jejunum, and ileum; mucosal DNA and protein in the jejunum and ileum; villus height in the jejunum: and crypt depth in the jejunum and ileum compared to the sham rats. Bacterial translocation was demonstrated in 60% of IAP-15 rats and in 80% of IAP-25 rats. CONCLUSION: Elevated IAP results in mucosal injury of the gut, causing mucosal hypoplasia, and increases bacterial translocation.

Chloroquine prevents T lymphocyte suppression induced by anthrax lethal toxin.

Lysosomal processing of lethal toxin (LTX) is a key event in the pathogenesis of anthrax. This study investigated the ability of chloroquine (CQ) to interfere with this processing and thereby to reduce suppression of T lymphocytes. T lymphocytes isolated from blood were activated, by cross-linking of CD3, in both the absence and presence of LTX and CQ and then were assayed by flow cytometry and immunoblotting. LTX was found to disrupt intracellular signaling, and it down-regulated T lymphocyte function. CQ significantly reduced the harmful effects of LTX and protected the activation and cytokine production of T lymphocytes. This effect may indicate a promising strategy in the treatment of anthrax.

Hemodynamic effects of combined treatment with oxygen and hypertonic saline in hemorrhagic shock.

OBJECTIVE: In hemorrhagic shock, small volume resuscitation with hypertonic saline transiently increases mean arterial blood pressure (MABP) and cardiac output and augments organ perfusion. Inhalation of 100% oxygen after hemorrhage also increases MABP and redistributes blood flow to the splanchnic and renal vascular beds. We evaluated hemodynamic effects of combined resuscitation with hypertonic saline and oxygen in shock induced by controlled bleeding in rats. DESIGN: Animal study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Animals were assigned to four hemorrhage groups that received posttreatment with a) normal saline; b) normal saline + 100% oxygen; c) hypertonic saline; d) hypertonic saline + oxygen, and a fifth sham-shock group that received hypertonic saline + oxygen. MEASUREMENTS AND MAIN RESULTS: Bolus infusion of small volume hypertonic saline markedly increased MABP (p < .001), hindquarter vascular resistance (p < .05), and distal aorta blood flow (p < .01). Hypertonic saline transiently increased superior (cranial) mesenteric artery (SMA) blood flow (p < .001) and small bowel perfusion (p < .01). Inhalation of oxygen after normal saline rapidly increased MABP (p < .01) and hindquarter vascular resistance (p < .02) and decreased distal aorta blood flow (p < .02) and perfusion of the gracilis muscle (p < .05). When given after normal saline, oxygen did not change SMA resistance and increased SMA flow (p < .05). The supplementation of oxygen after hypertonic saline did not exert additional effects on vascular resistance and blood flows in the two vascular beds. However, the combined treatment prevented the oxygen-induced decrease in distal aorta blood flow and gracilis muscle perfusion and maintained MABP at slightly higher values and SMA flow at significantly higher values than hypertonic saline alone until the end of the protocol (p < .01). The two hemorrhaged groups treated with oxygen exhibited the lowest final plasma lactate concentrations (p < .05 from normal saline and hypertonic saline groups). CONCLUSIONS: We suggest that early combined use of hypertonic saline and oxygen exerts a favorable extended profile of hemodynamic effects that amends shortcomings of each treatment alone in hemorrhagic shock.

Involvement of the multidrug resistance P-glycoprotein in acetaminophen-induced toxicity in hepatoma-derived HepG2 and Hep3B cells.

Acetaminophen overdose causes severe hepatic failure. Although the mechanisms of acetaminophen hepatotoxicity have been well investigated, little is known about the involvement of the P-glycoprotein in acetaminophen transport and toxicity. P-Glycoprotein is a membrane efflux pump, playing a significant role in regulating absorption, excretion, and tissue distribution of many drugs. To evaluate the contribution of P-glycoprotein transporter in the course of acetaminophen-induced toxicity, HepG2 and Hep3B cells with different P-glycoprotein expression and activity, were treated by acetaminophen (1-10 mM) for different time periods, with or without the P-glycoprotein inhibitor verapamil. P-Glycoprotein activity was determined by rhodamine 123 efflux assay and western blot analysis. To assess the acetaminophen-induced toxicity and effect of verapamil, we investigated cellular redox status, phosphatidylserine externalization, nuclear fragmentation and ultrastructural changes. Verapamil markedly enhanced acetaminophen-induced oxidative damage and cell death. Moreover, verapamil revealed acetaminophen toxicity even at subtoxic levels. High acetaminophen concentrations increased P-glycoprotein activity and content in both HepG2 and Hep3B cells. These observations suggest the involvement of P-glycoprotein in acetaminophen transport. Notwithstanding the differences of the investigated hepatoma cell lines in P-glycoprotein function, acetaminophen-induced toxicity was similar, possibly due to different functions of drug-metabolizing systems. We conclude that acetaminophen is a P-glycoprotein substrate and P-glycoprotein is involved in acetaminophen transport and toxicity in HepG2 and Hep3B cells. This study establishes the fact that acetaminophen can modulate P-glycoprotein in tumour cells, suggesting that its routine use in cancer patients in combination with anticancer drugs, may influence the result of chemotherapy.

Surface expression of HSP72 by LPS-stimulated neutrophils facilitates gammadeltaT cell-mediated killing.

During inflammation and sepsis, accumulation of activated neutrophils causes lung tissue damage and organ failure. Effective clearance of neutrophils reduces the risk of organ failure; however, its mechanisms are poorly understood. Because lungs are rich in gammadeltaT cells, we investigated the physiological role of these cells in the protection of lung tissue from infiltrating neutrophils. In a mouse model of sepsis, we found that the lungs of survivors contained significantly higher numbers of gammadeltaT cells than those of mice that died from sepsis. The number of gammadeltaT cells correlated inversely with the number of neutrophils in the lungs and with the degree of lung tissue damage. LPS rapidly elicited the expression of heat shock protein (HSP) 72 on the surface of human neutrophils. Inhibitors of transcription, protein synthesis, and intracellular protein transport blocked HSP72 expression, indicating that de novo synthesis is required. gammadeltaT cells targeted and rapidly killed LPS-treated neutrophils through direct cell-to-cell contact. Pre-treatment with neutralizing antibodies to HSP72 diminished neutrophil killing. Our data indicate that HSP72 expression on the cell surface predisposes inflamed neutrophils to killing by gammadeltaT cells. This intercellular exchange may allow gammadeltaT cells to resolve inflammation and limit host tissue damage during sepsis.

Lactated Ringer's solution and hypertonic saline improve survival in uncontrolled hemorrhagic shock in female rats in metestrus.

BACKGROUND: In the present investigation the effect of fluid treatment in uncontrolled hemorrhagic shock after massive splenic injury (MSI) was comparatively studied in male and female rats. MATERIALS AND METHODS: The anesthetized animals were randomly divided into three groups: in group 1 MSI was induced in males, in group 2 MSI was induced in females in proestrus, in group 3 MSI was induced in females in metestrus. Each group was divided into four subgroups: a) Sham-operated, b) MSI untreated (UT), c) MSI treated with 40 ml/kg lactated Ringer's solution (RL), and d) MSI treated with 5 ml/kg NaCl 7.5% (HTS). RESULTS: Total blood loss (TBL) in groups 1b, 2b, and 3b was 31.7 +/- 3.6%, 33.1 +/- 2.6%, and 36.7 +/- 2.6%, respectively, and mean survival time (MST) was 143.7 +/- 25.3 min, 174.8 +/- 10.4 min, and 67.8 +/- 11.4 min (P < 0.01 versus group 2b), respectively. TBL in groups 1c, 2c, and 3c increased to 52.4 +/- 5.5% (P < 0.02 versus UT), 48.6 +/- 1.6% (P < 0.02 versus UT), and 48.8 +/- 4.1% (P < 0.02 versus UT), respectively, and MST decreased to 126 +/- 19.4 min, (P < 0.05 versus UT), and 136.8 +/- 13.0 min (P < 0.05 versus UT) in groups 1c and 2c, respectively, and increased in group 3c to 120.4 +/- 23.3 min (P < 0.05 versus UT). TBL in groups 1d, 2d, and 3d was 31.3 +/- 4.8%, 38.0 +/- 4.2%, and 40.6 +/- 3.7%, respectively, and MST increased to 198.5 +/- 13.9 min (P < 0.05 versus UT) in group 1d, decreased to 128.4 +/- 17.2 min (P < 0.01) in group 2d, and increased to 102.6 +/- 19.0 min (P < 0.002 versus group 1d) in group 3d. CONCLUSIONS: RL infusion significantly increased blood loss in all three groups, reduced survival time in males and female rats in proestrus, but significantly improved survival in females in metestrus. HTS treatment did not alter blood loss in all three groups, but significantly improved survival in females in metestrus and males.

Effect of subcutaneous insulin on intestinal adaptation in a rat model of short bowel syndrome.

Insulin has been shown to influence intestinal structure and absorptive function. The purpose of the present study was to evaluate the effects of parenteral insulin on structural intestinal adaptation, cell proliferation, and apoptosis in a rat model of short bowel syndrome (SBS). Male Sprague-Dawley rats were divided into three experimental groups: sham rats underwent bowel transection and reanastomosis, SBS rats underwent a 75% small bowel resection, and SBS-INS rats underwent a 75% small bowel resection and were treated with insulin given subcutaneously at a dose of 1 U/kg, twice daily, from day 3 through day 14. Parameters of intestinal adaptation, enterocyte proliferation, and enterocyte apoptosis were determined on day 15 following operation. SBS rats demonstrated a significant increase in jejunal and ileal bowel and mucosal weight, villus height and crypt depth, and cell proliferation index compared with the sham group. SBS-INS animals demonstrated higher jejunal and ileal bowel and mucosal weights, jejunal and ileal mucosal DNA and protein, and jejunal and ileal crypt depth compared with SBS animals. SBS-INS rats also had a greater cell proliferation index in both jejunum and ileum and a trend toward a decrease in enterocyte apoptotic index in jejunum and ileum compared with the SBS untreated group. In conclusion, parenteral insulin stimulates structural intestinal adaptation in a rat model of SBS. Increased cell proliferation is the main mechanism responsible for increased cell mass.

Effect of sex and sex hormones on structural intestinal adaptation after massive small bowel resection in rats.

PURPOSE: The gonadal steroids play a major role in the regulation of many functions. The purpose of the current study was to evaluate the effect of sex and sex hormones on intestinal adaptation in a rat model of short bowel syndrome (SBS). METHODS: In the first experiment, male and female Sprague-Dawley rats underwent bowel transection and re-anastomosis (sham group) or 75% small bowel resection and anastomosis (SBS group). Relative changes in parameters of intestinal adaptation (overall bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth) were measured on day 15 and were compared with respect to sex. In the second experiment, male rats were divided into 4 experimental groups: SBS rats, SBS castrated rats, SBS castrated rats treated with testosterone, and SBS castrated rats treated with estradiol. Parameters of intestinal adaptation were compared with respect to hormonal treatment. Statistical significance was determined by Student's t test and analysis of variance with P < .05 considered significant. RESULTS: Sex had minimal effects on intestinal adaptation. Both male and female rats showed a comparable increase in all parameters of intestinal adaptation. In the second experiment, castration led to significant decrease in bowel and mucosal weight, mucosal DNA and protein in both jejunum and ileum compared with SBS animals. Castrated rats also had lower jejunal villus height and crypt depth compared with SBS animals. Testosterone attenuated this negative effect of castration on bowel regrowth. Rats treated with testosterone showed a significant increase in bowel and mucosal weight, mucosal protein in both jejunum and ileum, mucosal DNA, villus height, and crypt depth in jejunum compared with castrated nontreated animals. Treatment with estradiol after resection and castration had minimal effect on bowel regrowth. CONCLUSIONS: Bowel regrowth after massive small bowel resection is not sex-related. Depletion of androgens by castration inhibited intestinal adaptation. Testosterone has shown a strong stimulating effect on bowel regrowth.

Oral arginine reduces gut mucosal injury caused by lipopolysaccharide endotoxemia in rat.

BACKGROUND: The objective of this study was to evaluate the effects of lipopolysaccharide (LPS) endotoxemia and enteral arginine (ARG) supplementation on intestinal structural changes, enterocyte proliferation, and apoptosis in rat. METHODS: Male Sprague-Dawley rats, weighing 250-280 g, were divided into three experimental groups: control rats, LPS rats treated with lipopolysaccharide given ip at a dose of 10 mg/kg every 24 h (two injections), and LPS-ARG rats treated with enteral arginine given in drinking water (2%) 72 h before and following injection of LPS. Intestinal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined on day 3 following the first LPS injection. RESULTS: LPS rats demonstrated a significant decrease in bowel weight in duodenum, mucosal weight in duodenum, jejunum, and ileum, mucosal DNA and protein in jejunum and ileum, and villus height in jejunum and ileum compared to control animals. LPS rats also had a significantly lower cell proliferation index in jejunum and ileum and a higher apoptotic index in jejunum and ileum compared to control rats. LPS-ARG animals demonstrated greater duodenal bowel weight, duodenal and ileal mucosal weight, ileal mucosal DNA and protein, ileal villus height, and jejunal and ileal cell proliferation index compared to LPS animals. CONCLUSIONS: LPS endotoxemia impairs the integrity of the gastrointestinal mucosa in rat. Decreased cell proliferation and increased apoptosis may be considered the main mechanisms responsible for the decreased cell mass. Enteral arginine administration decreases the mucosal injury caused by lipopolysaccharide.