Biomedical subjects

A Ayala

Publications and source records attributed to A Ayala.

At least 73 records · Page 4Linked to original sources

PubMed1999

Hemorrhage decreases macrophage inflammatory protein 2 and interleukin-6 release: a possible mechanism for increased wound infection.

OBJECTIVE: To determine whether alteration in wound exudate cell immune function occurs after trauma-hemorrhage. BACKGROUND: Although clinical and experimental studies indicate that the rate of wound infection is increased after trauma and hemorrhagic shock, the underlying mechanism for this increased susceptibility remains unknown. METHODS: Male C3H/HeN mice were subjected to a midline laparotomy and polyvinyl alcohol sponges were implanted subcutaneously in the abdominal wound before hemorrhage (35+/-5 mm Hg for 90 minutes and resuscitation) or sham operation. The wound exudate cells from the sponges were harvested on the first, third, and fifth postoperative day and cultured for 24 hours in the presence of lipopolysaccharide (10 microg/ml) or heat-killed Staphylococcus aureus. Interleukin (IL)-1beta, IL-6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and nitrite levels were determined in the supernatants. The distribution of macrophages and polymorphonuclear leukocytes was assessed in the sponge with and without in vivo injection of S. aureus. The phagocytic activity of isolated wound exudate cells was determined using fluorescent S. aureus. RESULTS: The composition of exudate cells was unaltered by hemorrhagic shock; however, in vivo injection of S. aureus significantly decreased the percentage of macrophages under such conditions. Wound exudate cell phagocytic activity and the release of IL-1beta, IL-6, monocyte chemotactic protein 1, and macrophage inflammatory protein 2 was decreased on the first postoperative day. The release of IL-1beta and IL-6 was also decreased on the third postoperative day in hemorrhaged mice. On the fifth postoperative day, wound exudate cell cytokine production was comparable to that in shams. CONCLUSIONS: Because most wound infections occur early after severe trauma, these results suggest that the dysfunction of wound exudate cells after hemorrhage might contribute to the increased incidence of wound infections. Therefore, attempts to enhance or restore wound cell immune function might be helpful for decreasing the incidence of wound infections in trauma victims.

Animals↗

PubMed1999

Do different rates of fluid resuscitation adversely or beneficially influence immune responses after trauma-hemorrhage?

BACKGROUND: Although rapid fluid resuscitation continues to be an important component of the initial therapy for trauma patients, it remains unknown whether the rate of fluid administration after trauma-hemorrhage has any deleterious or beneficial effects on immunity. METHODS: Male C3H/HeN mice were subjected to sham operation or soft-tissue trauma (midline laparotomy) and hemorrhagic shock (mean arterial blood pressure of 35+/-5 mm Hg for 90 minutes) followed by resuscitation with four times the volume of shed blood in the form of lactated Ringer's solution over 30 minutes (rapid rate), 60 minutes (moderate rate), or 120 minutes (slow rate). The animals were killed at either 4 hours or 4 days after the end of trauma-hemorrhage. Spleens were harvested and splenocyte interleukin (IL)-3 and interferon-gamma (IFN-gamma) release as well as splenic macrophage IL-1beta and IL-6 release were determined. RESULTS: The results indicate that at 4 hours after trauma-hemorrhage, splenocyte IL-3 and IFN-gamma release were significantly depressed in all animals subjected to trauma-hemorrhage compared with sham-operated animals. At 4 days after trauma-hemorrhage, splenocyte IL-3 and IFN-gamma release were restored in mice resuscitated with the slow rate of resuscitation; however, the release of these cytokines remained significantly depressed in animals resuscitated with the moderate or rapid rates. Splenic macrophage IL-1beta and IL-6 release were significantly depressed at 4 hours after trauma-hemorrhage. At 4 days after trauma-hemorrhage, the release of these proinflammatory cytokines was still depressed in animals resuscitated with the rapid rate. In contrast, splenic macrophage IL-1beta and IL-6 release were restored in mice receiving the slow rate of resuscitation. CONCLUSION: These results suggest that a slower rate of fluid resuscitation after trauma-hemorrhage leads to a faster restoration of the depressed cell-mediated immunity, whereas rapid fluid resuscitation produces a prolonged depression of immune responses. In view of this, we propose that a prospective clinical study of this type must be performed in a select group of trauma patients to determine whether or not a slower rate of fluid resuscitation also improves immune responses in trauma patients.

Animals↗

PubMed1999

L-arginine: a unique amino acid for restoring the depressed macrophage functions after trauma-hemorrhage.

BACKGROUND: Immune responses are markedly depressed very early after the onset of hemorrhage. Furthermore, endothelial cell dysfunction occurs after trauma-hemorrhage and may contribute to alterations in immune function. Recent studies have shown that administration of L-arginine restores the depressed organ blood flow, probably because of the provision of substrate for constitutive nitric oxide synthase. It remains unknown, however, whether administration of L-arginine would have any salutary effect on the depressed macrophage function after trauma-hemorrhage. METHODS: Male rats underwent midline laparotomy (i.e., trauma was induced). After this, the animals were bled to and maintained at a mean blood pressure of 40 mm Hg until 40% of the maximum shed blood volume was returned in the form of lactated Ringer's solution. Sham-operated rats underwent both femoral artery cannulation and ligation, but these animals were neither bled nor resuscitated. Hemorrhaged rats were then resuscitated with lactated Ringer's solution, receiving four times the maximum shed blood volume over 1 hour. During resuscitation, one group received 300 mg/kg L-arginine and the other group received saline (vehicle) intravenously. At 4 hours after resuscitation, splenic and peritoneal macrophage interleukin (IL)-1beta and IL-6 release as well as plasma IL-6 were measured. RESULTS: Splenic and peritoneal macrophage IL-1beta and IL-6 release was significantly decreased in trauma-hemorrhage vehicle-treated rats. Administration of L-arginine after trauma-hemorrhage, however, improved splenic and peritoneal macrophage IL-1beta and IL-6 release. Moreover, the up-regulated plasma levels of IL-6 were attenuated by L-arginine administration. CONCLUSION: L-Arginine administration after trauma-hemorrhage significantly improves the depressed macrophage function, presumably by decreasing the increased plasma IL-6 levels and improving organ blood flow. Early enhancement of the depressed constitutive nitric oxide synthase activity by provision of L-arginine after trauma-hemorrhage, therefore, represents a novel and safe approach for improving the depressed immune function and decreasing plasma IL-6 levels under such conditions.

Animals↗

PubMed1999

Does Fas ligand or endotoxin contribute to thymic apoptosis during polymicrobial sepsis?

Recent studies have shown that with the onset of sepsis there is an increase in apoptosis (Ao) in the thymus, mediated in part by steroids, which may contribute to a loss of T-cell progenitors, thereby, reducing immune functions. However, reports also suggest that these steroid effects could be mediated by Fas ligand (FasL) and/or by endotoxin (ETX). Thus, our study was to determine: 1) if polymicrobial sepsis (cecal ligation and puncture; CLP) alters thymocyte Fas antigen/receptor (Fas+) expression and 2) if the increase in Ao in septic ETX-sensitive C3H/HeN mice is seen in thymocytes from ETX-tolerant, C3H/HeJ, or the FasL-deficient/ETX-tolerant, C3H/HeJ-FasL(gld), male mouse strains subjected to CLP or sham-CLP (Sham) 12 or 24 h before they were killed. The results of flow cytometric analysis indicated that increased %Ao+ seen in thymocytes of CLP C3H/HeN mice was associated with either no change (12 h) or a decrease in %Fas+ expression at 24 h, although the %Bcl-2+ (an antiapoptotic protein) cells was depressed at both times. Additional studies examining C3H/HeJ or C3H/HeJ-FasL(gld) mice subjected to CLP show that as with the ETX-sensitive mouse, thymocyte Fas and Bcl-2 antigen expression as well as Bcl-2/Bcl-X(L/S) mRNA levels decreased although the %Ao+ increased after CLP in both ETX-tolerant and ETX-tolerant/FasL-deficient mice. Furthermore, if ETX-tolerant/FasL-deficient CLP animals were administered the steroid receptor antagonist RU-38486 (s.c., immediately after CLP) the increase in Ao was markedly attenuated, along with restoration of the percentage of cells expressing Bcl-2 and Fas antigen as well as Bcl-2/Bcl-X(L/S) mRNA levels. Thus, we concluded that increased septic thymocyte Ao is not regulated through either Fas mediated pathway or ETX, but is a result of the release of endogenous steroids possibly acting directly or indirectly on Bcl-2 expression.

Animals↗

PubMed1999

Gender dimorphism in trauma-hemorrhage-induced thymocyte apoptosis.

Studies indicate that immune responses after trauma-hemorrhage are significantly depressed in males compared with enhanced immune responses in females under such conditions. Although androgen depletion in male mice by castration before soft tissue trauma and hemorrhagic shock prevents the depression of cell-mediated immunity, the underlying mechanism responsible for this remains unclear. Because the thymus is the primary location of T-cell lymphopoiesis and thymocytes express a large number of androgen receptors, we investigated whether differences in thymic apoptosis might contribute to the divergent immune response in males versus females after trauma-hemorrhage. To study this, male and female C3H/HeN mice were subjected to sham operation or soft tissue trauma (laparotomy) and hemorrhagic shock followed by fluid resuscitation. Animals were killed 72 h thereafter and thymocytes were isolated. Thymocyte interleukin 3 (IL-3) release was significantly suppressed in males, but not females, after trauma-hemorrhage. A parallel increase in thymic apoptosis that was primarily in the CD8+ thymocyte subset was observed in the males. Furthermore, in vitro treatment of thymocytes with 5a-dihydrotestosterone (DHT) increased the rate of apoptosis and decreased IL-3 release in a dose-dependent manner. Thus, the gender-dependent dimorphic immune response after trauma-hemorrhage may be in part due to an androgen-induced increase in thymic apoptosis in males under such conditions.

Animals↗

PubMed1999

IL-1beta stimulation induces paracrine regulation of PMN function and apoptosis.

Polymorphonuclear leukocytes (PMN) play a crucial role in the primary immunological defense against infectious agents. PMN activation and function is influenced in a paracrine manner by cytokines and bacterial products. While cell-cell communication has been demonstrated between PMN and other cell types, little data is available addressing PMN-PMN communication. Therefore, the aim of this study was to determine whether PMN were able to affect PMN function in vitro in a cell-contact independent manner, and whether IL-1beta influenced this effect. Conditioned medias (CM) were prepared by incubating PMN in HBSS +/- IL-1beta for 1-4 h. Incubation of fresh PMN in these conditioned medias had little or no effect on the expression of cell surface FcgammaR expression or oxidative metabolism. However, incubation of PMN in CM-IL1beta, but not control CM, increased phagocytotic activity and suppressed apoptosis. Additionally, CM-IL1beta, but not control CM, slowed the changes in Mac-1 and CR1 cell surface expression that occurred in HBSS within 2 h of incubation. Finally, control CM down-regulated the cell surface expression of PSGL-1; an effect that was not observed with CM-IL1beta. In conclusion, we demonstrate that PMN are able to communicate with and influence the immunological function of other PMN independent of cell-cell contact, and that this influence is regulated by cytokines such as IL-1beta. The major impact of this paracrine regulation is to down-regulate PMN apoptosis with the potential for an upregulated inflammatory response.

Apoptosis↗

PubMed1999

L-Arginine restores splenocyte functions after trauma and hemorrhage potentially by improving splenic blood flow.

Several studies indicate that immune responses are markedly depressed early after onset of hemorrhage. Decreased organ blood flow has been implicated in the pathophysiology of altered immune responses after trauma-hemorrhage. In this regard, administration of L-arginine has been shown to restore depressed intestinal and hepatic blood flow after trauma-hemorrhage, probably due to provision of substrate for constitutive nitric oxide synthase (cNOS). It remains unknown, however, whether administration of L-arginine also ameliorates depressed splenic blood flow and whether this agent has any salutary effects on depressed splenocyte functions after trauma-hemorrhage. Male rats underwent sham operation or laparotomy and were bled to and maintained at a mean arterial blood pressure of 40 mmHg until 40% of maximum shed blood volume (MBV) was returned as Ringer lactate (RL). Hemorrhaged rats were then resuscitated with RL (4 times MBV over 1 h). During resuscitation, rats received 300 mg/kg L-arginine or saline (vehicle) intravenously; 4 h later, splenic blood flow, splenocyte proliferation, and splenocyte interleukin (IL)-2 and IL-3 were determined. Administration of L-arginine improved depressed splenic blood flow and restored depressed splenocyte functions after trauma-hemorrhage. Therefore, provision of L-arginine during resuscitation after trauma-hemorrhage should be considered a novel and safe approach for improving splenic organ blood flow and depressed splenocyte functions under such conditions.

Animals↗

PubMed1999

Hypoxemia in the absence of blood loss upregulates iNOS expression and activity in macrophages.

Regional hypoxia, associated with hemorrhage, is thought to induce a variety of alterations in immune cell function, including upregulation of macrophage-inducible nitric oxide synthase (iNOS) expression and activity (NO production). Furthermore, NO may cause immune cell dysfunction similar to that associated with hemorrhagic shock. However, it remains unknown whether hypoxia per se in the absence of any blood loss is a sufficient stimulus to cause iNOS expression and NO production by macrophages. To study this, male Sprague-Dawley rats (275-325 g) were placed in a plastic box flushed with a gas mixture containing 5% O2-95% N2 for 60 min. Peritoneal and splenic macrophages were isolated 0-5.5 h thereafter, and blood samples were obtained. Nitrite and nitrate (stable degradation products of NO) production by splenic and peritoneal macrophages cultured for 48 h was significantly increased 3 and 5.5 h after hypoxemia. The increase in NO production by macrophages was preceded by elevated expression of iNOS mRNA at 1.5 h after hypoxia. Additionally, interferon-gamma (IFN-gamma) levels in plasma from rats subjected to hypoxemia were significantly elevated soon after the insult (0-1.5 h posthypoxemia), suggesting a causal relationship between IFN-gamma production and upregulation of iNOS activity. We propose that a hypoxemia-induced increase in macrophage iNOS activity following hemorrhage may in part be responsible for the observed immune dysfunction. Thus attempts to suppress macrophage iNOS activity after this form of trauma may be helpful in improving immune function under those conditions.

Animals↗

PubMed1999

Sex steroids regulate pro- and anti-inflammatory cytokine release by macrophages after trauma-hemorrhage.

Studies indicate that macrophage immune responses in males are depressed after trauma-hemorrhage, whereas they are enhanced in females under such conditions. Nonetheless, the involvement of male and female sex steroids in this gender-dependent dimorphic immune response after trauma-hemorrhage remains unclear. To study this, male C3H/HeN mice were castrated and treated with pellets containing either vehicle, 5alpha-dihydrotestosterone (DHT), 17beta-estradiol, or a combination of both steroid hormones for 14 days before soft tissue trauma (i.e., laparotomy) and hemorrhagic shock (35 +/- 5 mmHg for 90 min followed by adequate fluid resuscitation) or a sham operation. Twenty-four hours later the animals were killed, plasma was obtained, and Kupffer cell and splenic and peritoneal macrophage cultures were established. For DHT-treated mice, we observed significantly decreased releases of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-6 by splenic macrophage (-50 and -57%, respectively) and peritoneal macrophage (-51 and -52%, respectively) cultures after trauma-hemorrhage compared with releases by cultures of cells from mice subjected to a sham operation; in contrast, responses of splenic and peritoneal macrophage cultures from other groups subjected to trauma-hemorrhage did not change significantly. In addition, only DHT-treated animals exhibited increased Kupffer cell IL-6 release (+634%). The release of IL-10 in DHT-treated hemorrhaged animals was increased compared with that in sham-operated animals but was decreased in estrogen-treated mice under such conditions. These results suggest that male and female sex steroids exhibit divergent immunomodulatory properties with respect to cell-mediated immune responses after trauma-hemorrhage.

Animals↗

PubMed1999

Early kidney TNF-alpha expression mediates neutrophil infiltration and injury after renal ischemia-reperfusion.

The purpose of this study was to determine whether isolated renal ischemia and reperfusion (I/R) induces renal tumor necrosis factor (TNF) mRNA production, TNF protein expression, or TNF bioactivity and, if so, whether local/early TNF production acts as mediator of ischemia-induced, neutrophil-mediated renal injury. After rats were anesthetized, varying periods of renal ischemia, with or without reperfusion, were induced. Kidney mRNA content (RT-PCR), TNF protein expression (ELISA), TNF bioactivity (WEHI-164 cell clone cytotoxicity assay), and neutrophil infiltration [myeloperoxidase (MPO) assay] were determined. In other animals, renal MPO and serum creatinine were assessed after TNF was neutralized [binding protein (TNF-BP)]. Thirty minutes of ischemia induced renal TNF mRNA. TNF protein expression and bioactivity peaked after 1 h ischemia and 2 h reperfusion, whereas neutrophil infiltration peaked at 4 h reperfusion. TNF-BP neutralized TNF bioactivity, reduced neutrophil infiltration, and protected postischemic function. These results constitute the initial demonstration that 1) early renal tissue TNF expression contributes to neutrophil infiltration and injury after I/R and 2) TNF-BP may offer a new adjunctive therapy in renal preservation prior to planned ischemic insults.

Animals↗

PubMed1999

Trauma-hemorrhage delays wound healing potentially by increasing pro-inflammatory cytokines at the wound site.

BACKGROUND: Studies indicate impaired wound healing after trauma. The underlying mechanism remains unknown. METHODS: Mice were subjected to midline laparotomy, and polyvinyl alcohol sponges were implanted subcutaneously before hemorrhage (35 +/- 5 mmHg for 90 minutes, resuscitated) or sham operation. Wound exudate cells from the sponges were harvested on the first, third, and fifth postoperative day and cultured for 24 hours. Interleukin (IL)-1 beta, IL-6, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and transforming growth factor (TGF)-beta were determined in the supernatants. IL-1 beta and IL-6 were measured in the wound fluid. RESULTS: Hemorrhage decreased collagen deposition in the wound. TGF-beta release was significantly decreased on the first and third postoperative days after hemorrhage, whereas IL-1 beta and IL-6 release was increased at 3 and 5 days after hemorrhage. Similarly, IL-1 beta and IL-6 in the wound fluid were significantly increased at 3 days after hemorrhage. CONCLUSIONS: Because increased levels of pro-inflammatory cytokines and decreased amounts of TGF-beta have been reported to impair the process of wound healing, the increased release of IL-1 beta and IL-6 and the decreased release of TGF-beta after hemorrhage might contribute to the decreased collagen production in those animals. Thus, attempts to locally change the ratio of those cytokines in trauma victims might be useful for improving wound healing in those patients.

Animals↗

PubMed1999

What is the role of interleukin 10 in polymicrobial sepsis: anti-inflammatory agent or immunosuppressant?

BACKGROUND: Controversy exists concerning the role of interleukin 10 (IL-10) in sepsis. When IL-10 is used in models of endotoxemia, it appears to protect (by anti-inflammatory effects), whereas in models of polymicrobial sepsis it seems to be deleterious (by immunosuppression?). However, little direct evidence for such an immunosuppressive role is available for polymicrobial sepsis. Thus the aim of this study was to determine whether IL-10 contributes to lymphocyte immunosuppression in a model of cecal ligation and puncture (CLP) and whether neutralization of IL-10 has any salutary effects on survival after sepsis. METHODS: To assess the former, polymicrobial sepsis was induced in male C57BL/6J wild-type (+/+) and C57BL/6J-IL-10 knockout(-/-) mice by CLP. Splenocytes were harvested 24 hours later and stimulated with concanavalin A to assess their proliferative capacity and their ability to release the Th1 lymphokines interleukin 2 and interferon gamma (by enzyme-linked immunosorbent assay, nanograms/millilter). To further verify the immunosuppressive role of IL-10, splenocytes were obtained from male C3H/HeN mice 24 hours after CLP and then stimulated in the presence or absence of anti-IL-10 monoclonal antibody (Mab, 4 micrograms/mL). To assess the in vivo effects of IL-10 neutralization on survival after CLP, C3H/HeN mice (16 per group) were given 250 micrograms of anti-IL-10 Mab (intraperitoneally) either immediately after CLP (before the initiation of the hyperdynamic phase) or 12 hours after CLP (the beginning of the hypodynamic state). Control mice were given nonspecific rat immunoglobulin G. RESULTS: These data indicate that IL-10 deficiency (-/-) prevents the depression of the proliferative capacity and Th1 lymphokine production after sepsis. Analysis of the interleukin 2-interferon gamma production patterns and proliferative capacity in lymphocytes treated with anti-IL-10 Mab confirmed the role of IL-10 in suppressing lymphocyte responsiveness in CLP. Interestingly, however, only delayed administration (12 hours after CLP) of anti-IL-10 markedly increased survival of mice (Fisher's exact test, P < .05). CONCLUSION: The results not only illustrate IL-10's role in septic immune dysfunction but document that anti-IL-10 administration beyond the initial proinflammatory hyperdynamic state of polymicrobial sepsis improves survival of animals subjected to sepsis.

Animals↗

PubMed1998

Increased mucosal B-lymphocyte apoptosis during polymicrobial sepsis is a Fas ligand but not an endotoxin-mediated process.

Sepsis is reported to induce an increase in the rate of apoptosis (Ao), in immature lymphoid cells residing in hematopoietic tissues such as the thymus and bone marrow. Alternatively, secondary lymphoid tissue, such as the spleen exhibit little innate (unstimulated) Ao. However, it is unknown whether or not polymicrobial sepsis has any effects on the frequency of Ao in mucosal lymphoid tissue and what, if any, are the functional consequences of such a change. To assess this, Peyer's patch cells were harvested from C3H/HeN (endotoxin-sensitive) mice killed 12 or 24 hours after the onset of polymicrobial sepsis (cecal ligation and puncture [CLP]). The results indicate that the percentage of cells that were Ao+ as determined by flow cytometry were markedly increased at 24 hours, but not at 12 hours post-CLP. This correlates well with evidence of increased DNA fragmentation as well as histological changes observed both at a light and transmission electron microscopic level of the Peyer's patch Ao. Phenotypically, these changes were restricted to the B220+ (B-cell) population that also exhibited a marked increase of Fas/Apo-1 antigen expression. The functional consequence of this increased apoptosis appears to be associated with the endogenous stimulation (activation) of IgA production by mucosal B lymphocytes and increased nuclear c-Rel expression. Furthermore, we found that Peyer's patch lymphocytes isolated from C3H/HeJ-Faslgld (endotoxin-tolerant/Fas ligand- [FasL] deficient) as opposed to C3H/HeJ (endotoxin-tolerant) inbred mice did not exhibit increased Ao after CLP. These findings indicate that increased B-cell Ao appears to be a FasL-Fas antigen-mediated process, but is not due to endotoxin sensitivity. In conclusion, we speculate that the increased Fas-associated apoptosis detected in mucosal B cells (as opposed to splenic or bone marrow B cells) may be due to increased luminal antigens other than endotoxin, released due to gut barrier integrity breakdown during sepsis.

Animals↗

PubMed1998

Is Fas ligand or endotoxin responsible for mucosal lymphocyte apoptosis in sepsis?

BACKGROUND: Apoptosis (Ao) is a normal constitutive process that seems to have pathological effects in diseases of immune deficiency and autoimmune disorders, as well as in certain lymphoid tissues during sepsis. Little is known about this process in mucosal lymphoid tissue, such as intestinal intraepithelial lymphocytes (IELs). OBJECTIVES: To determine whether sepsis induces increased Ao in small intestinal IEL, whether this was associated with functional changes in cytokine gene expression in the IEL, and which mediators control this process and their impact on the survival of the mouse with sepsis. DESIGN: Male C3H/HeN (endotoxin-sensitive), C3H/HeJ (endotoxin-tolerant), and C3H/HeJ-FasLgld (endotoxin-tolerant/Fas ligand [FasL]-deficient) mice were subjected to sepsis (cecal ligation and puncture [CLP]) and IELs were harvested at 4 (early) or 24 hours (late sepsis). Alterations in the cell phenotype and Ao (TUNEL [terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling] assay) were determined by 3-color flow cytometry. Cytokine gene expression was assessed by multiprobe RNase protection assay. RESULTS: At 4 hours after CLP, only the frequency of IEL which was CD8+ decreased markedly. By 24 hours after CLP, the number of CD8+ and CD4+ cells decreased while the proportion of double-negative cells showed a marked increase when compared with sham-controls. The percentage of Ao positive in CD8+ and CD4+ double-positive and double-negative cells increased markedly 24 hours after CLP concomitant with a significant (P<.05 vs sham-controls, Mann-Whitney U test) increase in expression of the IL-2, IL-10, and IL-15 gene. These data collectively suggest that sepsis causes lymphocyte activation-induced Ao that may be mediated by FasL. Additional studies were done to determine if the increased Ao was due to either endotoxin or FasL. The results of studies with endotoxin-tolerant C3H/HeJ or FasL-deficient C3H/HeJ-FasLgld mice showed an increase in A,, in CD4+ and CD8+ cells from septic C3H/HeJ but not C3H/HeJ-FasLgld mice. With regard to septic mortality, our results indicated that there was a marked reduction in mortality in C3H/HeJ-FasLgld vs C3H/HeJ mice. CONCLUSIONS: We conclude that the phenotypic changes associated with increased Ao may be a reflection of localized immune cell activation due to a FasL-mediated process and not endotoxin. Thus, FasL directly and/or indirectly contributes to higher septic mortality.

Animals↗

PubMed1998

Dehydroepiandrosterone: an inexpensive steroid hormone that decreases the mortality due to sepsis following trauma-induced hemorrhage.

BACKGROUND: Recent studies suggest that male sex steroids play a role in producing immunodepression following trauma-hemorrhage. This notion is supported by studies showing that castration of male mice before trauma-hemorrhage or the administration of the androgen receptor blocker flutamide following trauma-hemorrhage in noncastrated animals prevents immunodepression and improves the survival rate of animals subjected to subsequent sepsis. However, it remains unknown whether the most abundant steroid hormone, dehydroepiandrosterone (DHEA), protects or depresses immune functions following trauma-hemorrhage. In this regard, DHEA has been reported to have estrogenic and androgenic properties, depending on the hormonal milieu. OBJECTIVE: To determine whether administration of DHEA after trauma-hemorrhage has any salutary or deleterious effects on immune responses, and whether it improves the survival of animals subjected to subsequent sepsis. DESIGN: Male C3H/HeN mice underwent laparotomy (ie, trauma-induced) and hemorrhagic shock (blood pressure, 35+/-5 mm Hg for 90 minutes) followed by fluid resuscitation, or sham operation. The animals then received 100 mg of DHEA (4 mg/kg) or propylene glycol (hereafter referred to as vehicle). At 24 hours after trauma-hemorrhage and resuscitation, the animals were killed and blood, spleens, and peritoneal macrophages were harvested. Splenocyte proliferation and interleukin (IL) 2 release and splenic and peritoneal macrophage IL-1 and IL-6 release were determined. In a separate set of experiments, sepsis was induced by cecal ligation and puncture at 48 hours after trauma-hemorrhage and resuscitation. For those studies, the animals received vehicle, a single 100-microg dose of DHEA, or 100 microg/d DHEA for 3 days following hemorrhage and resuscitation. Survival was monitored for 10 days after the induction of sepsis. RESULTS: Administration of DHEA restored the depressed splenocyte and macrophage functions at 24 hours after trauma-hemorrhage. Moreover, daily administration of DHEA for 3 days significantly increased the survival of animals subjected to subsequent sepsis (P=.01). CONCLUSION: The finding that DHEA markedly improves the depressed immune functions and survival of animals subjected to subsequent sepsis suggests that short-term treatment with DHEA after trauma-hemorrhage is a safe and novel approach for preventing immunodepression and for decreasing the mortality rate due to subsequent sepsis.

Adjuvants, Immunologic↗

PubMed1998

Human myocardial tissue TNFalpha expression following acute global ischemia in vivo.

Although human myocardial TNFalpha levels are increased during the ischemia associated with chonic heart failure, it remains unknown whether an acute global ischemic insult further increases TNFalpha expression in human cardiac myocytes. To study this, biopsies of human myocardium were obtained before and after cardiopulmonary bypass (in vivo acute global ischemia), and myocardial TNFalpha levels were determined by ELISA and cytotoxicity assay (WEHI-164 clone 13 cell line). TNFalpha was immunolocalized by immunohistochemistry. Results indicate that cardiopulmonary bypass induces an increase in human myocardial TNFalpha by both ELISA and cytotoxicity assays. Immunolocalization revealed that prior to cardiopulmonary bypass TNFalpha was located predominantly in the myocardial interstitial cells; however, following bypass, increased TNFalpha was observed in the cardiocytes themselves. Locally-produced myocardial TNFalpha may be an important contributor to myocardial functional depression and injury following acute ischemia. Targeted anti-TNFalpha therapy in the treatment of cardiac ischemic injury may further elucidate its clinical relevance.

Biopsy↗

PubMed1998

Does hepatocellular injury in sepsis involve apoptosis?

Apoptosis (AO) is a process by which cells typically undergo a form of nonnecrotic cellular suicide. AO normally allows the host to selectively delete cells from a given tissue site without producing bystander injury associated with necrosis. However, inappropriate induction of AO has been associated with a variety of acute as well as chronic pathological states and may contribute to the therapeutic nonresponsiveness frequently encountered in the septic animal/patient's organ function. In this respect, while AO has been demonstrated in a variety of immune cell tissues of septic animals it is unclear if it is present in the septic liver. Therefore, it was the aim of our study to determine if AO is evident in hepatocytes of polymicrobial septic animals. To assess this, C3H/HeN male mice were subjected to polymicrobial sepsis (cecal ligation and puncture) or sham- CLP (Sham). Hepatocytes were then harvested at 4 h (early hyperdynamic phase) or 24 h (late hypodynamic state) later, and indices of AO were assessed [cell cycle analysis of Annexin V/propidium iodide (PI) staining for flow cytometric analysis, DNA extracted, and cell death ELISA]. Plasma glutamic pyruvic transaminase (GPT) was also colorimetrically assessed as well as total viable cell yield as an index of hepatocellular necrosis. The results indicate that indices of hepatocellular AO, as determined by cell cycle analysis and cell death ELISA, were markedly increased in polymicrobial septic mice at 24 h. However, while an increase in DNA fragmentation/degradation could be consistently detected, the pattern was typically faint. Similarly, although there was an increase in Annexin V staining it was not dissociated from that of PI (necrotic index). Alternatively, necrosis (as evidenced by increased GPT levels at both 4 and 24 h) preceded the induction of all the indices of AO. Taken together, these data suggest a role for both necrosis and apoptosis in the evolution of hepatocellular injury encountered in the polymicrobial septic animal/patient which may represent a unique pattern of cell death under such conditions.

Animals↗

PubMed1998

Sepsis induces increased apoptosis in lamina propria mononuclear cells which is associated with altered cytokine gene expression.

Studies indicate that lymphoid tissue (e.g., thymus, bone marrow, and Peyer's patches) shows evidence of increase apoptosis (Ao, a form of nonnecrotic cell death) during sepsis. However, it is not known if mucosal lymphoid tissue, such as lamina propria (LP), also shows evidence of increased Ao and if so, is this associated with functional changes, i.e., cytokine gene expression in the LP. To examine this, male C3H/HeN mice were subjected to cecal ligation and puncture (CLP) and lamina propria mononuclear cells (LPMC) were harvested at 4 h (early sepsis) or 24 h (late sepsis). Alterations in the cell phenotype as well as Ao (Tunel assay) were determined by three-color flow cytometry. Cytokine gene expression was assessed by multiprobe RNase protection assay. Sham LPMC preparations were found to be 34.4 +/- 2.4% B220(+) (B-cells), while 12.4 +/- 2.1% were CD8(+) (cytotoxic T-cells), 22.0 +/- 0.8% were CD4(+) (helper T-cells), and 6.4 +/- 0.7% were F4/80(+) (macrophages). The frequency of B220(+) (9%* upward arrow) and CD8 (6%* upward arrow) populations increased markedly at 4 h after CLP; however, this increase was not seen at 24 h. The percentage of Ao+ in CD8(+), B220(+), and F4/80(+) cells increased markedly at both 4 and 24 h. CD4(+) cells showed a marked increase in Ao only at 24 h after CLP. When LPMC mRNA expression was examined, a significant increase in IL-2, -10, and -15 gene expression was observed only at 24 h but not 4 h after CLP. Thus, the early phenotypic changes associated with increased Ao may be a reflection of localized immune cell activation in early sepsis contributing to the increased cytokine gene expression seen in late sepsis. This localized activation may contribute to gastrointestinal inflammation and/or immune dysfunction in sepsis.

Animals↗