Chloroquine attenuates hemorrhagic shock-induced immunosuppression and decreases susceptibility to sepsis.

Hemorrhagic shock causes a severe suppression of cellular immunity and an increased susceptibility to sepsis that may be due to increased release of prostaglandin E2 by macrophages. Since chloroquine inhibits the secretion of prostaglandin E2 by macrophages in vitro, the effects of chloroquine administration in vivo following hemorrhagic shock on macrophage prostaglandin E2 secretion and on depressed cellular immunity were examined. Inbred C3H/HeN male mice, aged 6 to 8 weeks, were bled to a mean blood pressure of 35 mm Hg, which was maintained for 60 minutes, and adequately, resuscitated. Mice then received intramuscular injections of either saline (vehicle) or chloroquine (10 mg/kg of body weight). Prostaglandin E2 in macrophage supernatants (radioimmunoassay) concanavalin A-dependent splenocyte proliferation, and interleukin 2 in splenocyte supernatants (CTLL 20 interleukin 2-dependent proliferation) were determined 2 or 24 hours later. Hemorrhage caused a significant decrease of splenocyte proliferation (47%) and interleukin 2 release (49%) at 24 hours, while prostaglandin E2 secretion from macrophages was elevated at 2 hours. Chloroquine treatment attenuated depression of splenocyte functions and reduced prostaglandin E2 release. Furthermore, chloroquine treatment decreased the mortality of septic mice after hemorrhage to levels comparable with those of sham-operated mice. Thus, chloroquine may be a useful adjunct in the clinical setting for the treatment of shock-induced immunodepression and increased susceptibility to sepsis following hemorrhage.     

Ibuprofen restores cellular immunity and decreases susceptibility to sepsis following hemorrhage.

Although hemorrhage depresses splenocyte (SPL) functions and increases susceptibility to sepsis, it is not known whether increased tumor necrosis factor (TNF) or prostaglandin (PG) production are responsible for it. To study this, mice (C3H/HeN) were bled to a mean blood pressure of 35 mm Hg, maintained at that pressure for 60 min, resuscitated, and treated with ibuprofen (1.0 mg/kg body weight) or vehicle (saline). Hemorrhage reduced (P less than 0.05) SPL proliferation by 60%, SPL release of interleukin-2 (IL-2) by 47%, interferon-gamma (IFN-gamma) by 67%, TNF by 54%, and interleukin-6 (IL-6) by 46% compared to sham. In addition, splenic macrophage (sM phi) release of interleukin-1 (IL-1) and TNF was decreased by 58 and 67% (P less than 0.05), respectively. However, ibuprofen treatment increased (P less than 0.05) SPL proliferation, lymphokine (IL-2, IFN-gamma, and IL-6) synthesis, and IL-1 release by sM phi compared to hemorrhage alone. Furthermore, ibuprofen enhanced the release of TNF by SPL (+175%, P less than 0.05) and sM phi (+68%) compared to the vehicle group. Ibuprofen also decreased (P = 0.011) the susceptibility to sepsis following hemorrhage. These results indicate that PGs are involved in hemorrhage-induced suppression of cellular immunity and in the increased mortality of such animals following a septic challenge.     

γ-干扰素对前列腺癌细胞粘附和侵袭能力影响的初步研究

目的：探讨γ-干扰素对前列腺癌细胞粘附和侵袭行为的影响。方法：用纤维粘连蛋白和层粘连蛋白处理细胞培养板，检测γ-干扰素对前列腺癌细胞系LNCaP和PC-3粘附作用的影响；用Transwell小室，以Matrigel和纤维粘连蛋白构建基底膜，检测γ-干扰素对前列腺癌细胞侵袭人工基底膜能力的影响；应用Western-blot法检测γ-干扰素对前列腺癌细胞膜粘连蛋白-2（annexin-2）表达的影响。结果：γ-干扰素未处理的两种人前列腺癌细胞系细胞LNCaP、PC-3粘附率分别为46％和40％，γ-干扰素处理的两种细胞系细胞LNCaP、PC-3粘附率分别为21％和23％，同种细胞系γ-干扰素处理组与未处理组间差异有显著性（P均〈0．05）。在相同细胞系γ-干扰素处理组较未处理组前列腺癌细胞24h侵袭能力明显降低（P均〈0．05）。Western印迹结果表明γ-干扰素可显著抑制annexin-2蛋白的表达（P〈0．05）。结论：γ-干扰素可能通过下调annexin-2的表达来抑制前列腺癌细胞的粘附和侵袭。     

Diltiazem restores IL-2, IL-3, IL-6, and IFN-gamma synthesis and decreases host susceptibility to sepsis following hemorrhage

Various beneficial effects of calcium channel blockers on cell and organ function following endotoxic shock, organ ischemia, and reperfusion have been reported; however, it is not known whether these agents have any salutary or deleterious effects on immune responses after low-flow conditions. Therefore, the aim of this study was to determine (a) the effect of hemorrhage on lymphocyte IL-2, IL-3, IL-6, and IFN-gamma synthesis, and (b) whether diltiazem has any salutary or adverse effects on these parameters when administered following hemorrhage and resuscitation. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mm Hg, maintained at that level for 60 min, and resuscitated with shed blood plus twice that volume of Ringer's lactate. Immediately following resuscitation mice received either diltiazem (2400, 800, or 400 micrograms/kg body wt), or an equivalent volume of saline. The mice were sacrificed 24 hr later, splenic lymphocytes were obtained, and their capacity to produce lymphokines was assessed. The results indicated that in the vehicle-treated animals, hemorrhage significantly decreased (P less than 0.05) IL-2, IL-3, IL-6, and IFN-gamma synthesis by 82 +/- 19%, 64 +/- 28%, 71 +/- 11%, and 86 +/- 14%, respectively. However, diltiazem (400 but not 2400 micrograms/kg) treatment after hemorrhage restored lymphocyte capacity to produce IL-2, IL-3, IL-6, and IFN-gamma (P less than 0.05). Additional groups of animals were subjected to sepsis by cecal ligation and puncture 3 days following hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interferon-gamma gene polymorphisms and the development of sepsis in patients with trauma

BACKGROUND: The outcome of patients with trauma does not always correlate with injury severity or premorbid health status. This study evaluates the relationship between polymorphisms in the first intron of the interferon-gamma gene and the development of sepsis after trauma. METHODS: DNA was extracted from peripheral leukocytes of patients with trauma and an injury severity score of 16 or greater. Data collected included demographics, injury mechanism, injuries sustained, development of sepsis, and outcome. A previously identified cytosine/adenine repeated polymorphism was amplified, alleles/genotypes identified, and the results correlated with patient outcome. RESULTS: Sixty-one patients were evaluated. Thirty patients (49%) became septic. The injury severity score, race, age, and gender distribution was similar for both the septic and nonseptic groups. Six alleles and 10 genotypes were identified. Alleles C (34%) and D (52%) were the most common. Patients who were septic had a 62% chance of having a D allele (P =.06), whereas they had only a 29% chance of having a C allele. Homozygotes for allele D (DD) were the most likely to become septic (65%). CONCLUSIONS: Homozygotes for the D allele (DD) of the interferon-gamma gene have an increased chance of developing sepsis after traumatic injury compared with other allelic combinations. This supports the hypothesis that genetic composition plays a role in patient outcome.     

Hypothyroidism abolishes the hyperdynamic phase and increases susceptibility to sepsis

To evaluate the role of thyroid hormones in sepsis, 250-400 g rats were surgically thyroidectomized and 2-6 weeks later sepsis was produced by cecal ligation and puncture (CLP). In normal rats, total body O2 consumption (VO2) increased by 12.8% (P less than 0.05) in early sepsis (6 hr after CLP) and decreased slightly in late sepsis (16 hr after CLP). In hypothyroid (HT) rats, VO2 was depressed by 19.8% (P less than 0.05) in early sepsis and further decreased to 46.7% (P less than 0.001) of preoperative levels in late sepsis. Hepatic blood flow increased in early sepsis in normal rats but was unchanged in HT rats. The normal hyperglycemic response to early sepsis was also absent in HT rats. The respiratory control ratio (RCR) of isolated mitochondria with succinate was not increased in HT rats in early sepsis. In late sepsis, hypothyroid animals showed further decreases in VO2 and mitochondrial RCR, and, in contrast to normal rats, showed no change in blood glucose levels. Survival (5 days) following late sepsis in normal, HT, and HT rats given daily ip injections of thyroxine (30 micrograms/kg) were 65.2% (15/23), 30% (6/20) (P less than 0.025), and 77.1% (14/18), respectively. Thus, absence of thyroid hormone abolishes the hyperdynamic phase of sepsis and significantly increases mortality in sepsis, and thyroxine replacement following thyroidectomy prevents the increased mortality from sepsis.     

Determinants of susceptibility to sepsis and mortality: Malnutrition vs anergy

This study investigated whether increased susceptibility to sepsis and mortality is best determined by the nutritional state or by depressed host resistance, as indicated by anergy. One hundred and fifty Sprague-Dawley rats, 120–130 g, were presensitized with keyhole limpet hemocyanin (KLH) and fed a normal diet. At 3 weeks all rats had a reactive delayed-hypersensitivity skin test (DTH) response to KLH. Thirty rats were continued on this diet (Group I) and the rest were fed a 0.2% protein deficient diet. After 5 weeks of protein depletion 30 rats that maintained reactive DTH responses formed Group II and 30 rats that were anergic, as defined by a DTH response to KLH of < 5 mm induration at 24 hr, formed Group III. Carcass nitrogen studies confirmed that malnourished Groups II and III were comparably malnourished. All three groups were then challenged, via intraperitoneal injection, with a suspension of bacteriae and 10% BaSO₄ (total 2.5 cc/100 g) at three doses. There was no significant difference in survival between nourished reactive (78%) and malnourished reactive (60%) rats. However, malnourished anergic rats had a significantly higher mortality (80%) compared to nourished reactive rats (22%, χ² = 6.34, P < 0.025). Furthermore, a 40% difference in survival was observed between severely malnourished anergic and malnourished reactive DTH populations. We conclude that, while protein deprivation produces the state of anergy, survival from peritonitis challenge is determined, not by the malnutritional status per se, but by the adversely affected host immune state.     

Organ interactions in sepsis. Host defense and the hepatic-pulmonary macrophage axis

Endotoxin (lipopolysaccharide [LPS]) and tumor necrosis factor (TNF-alpha) have been implicated in the pathogenesis of sepsis-induced adult respiratory distress syndrome. To evaluate the possible interaction of the hepatic-pulmonary macrophage axis in the adult respiratory distress syndrome, we compared the kinetics of immunosuppressive prostaglandin E2, TNF-alpha, and interleukin 6 production in LPS-stimulated Kupffer cells and alveolar macrophages (AMs). Interleukin 6 production by Kupffer cells was significantly higher than for equal numbers of AMs. Kupffer cell TNF-alpha levels peaked early before decreasing as regulatory prostaglandin E2 levels rose. In contrast, AM TNF-alpha levels rose sharply and remained significantly higher than for Kupffer cells throughout culture coincident with negligible prostaglandin E2 production. Kupffer cell sequestration of LPS may normally invoke a coordinated cytokine response able to locally induce acute-phase hepatocytes. In hepatic failure, however, LPS spillover to the lung may promote adult respiratory distress syndrome by inducing unregulated AM TNF-alpha production within the pulmonary microenvironment.     

Hemorrhage without tissue trauma produces immunosuppression and enhances susceptibility to sepsis

To determine whether hemorrhage without major tissue trauma can itself produce immunosuppression, the effect of hemorrhage on the lymphocyte response to T-cell mitogen in endotoxin-resistant C3H/HEJ mice was measured. The mice were bled to achieve a mean blood pressure of 35 mm Hg, maintained at that level for one hour, and then adequately resuscitated. On days 1 through 10 thereafter, the proliferative responses of the splenocytes to concanavalin A were measured and allogeneic mixed lymphocyte reaction was performed. The proliferative responses to mitogen stimulation as well as the results of mixed lymphocyte reaction studies indicated that marked immunosuppression occurred at day 1. Immunosuppression persisted for at least five days following hemorrhage, as evidenced by mitogen stimulation assay. Another group of mice was subjected to sepsis three days after hemorrhage and resuscitation. The mortalities in the sham-hemorrhage and hemorrhage groups following sepsis were 58% and 100%, respectively. Thus, a significant depression of cellular immunity occurred following simple hemorrhage despite adequate resuscitation, and this immunosuppression enhanced the susceptibility to sepsis.     

Anti-CD18 antibody attenuates neutropenia and alveolar capillary-membrane injury during gram-negative sepsis.

Activated polymorphonuclear leukocytes (PMNs) are implicated in the pathogenesis of acute lung injury (ALI) associated with sepsis. Adhesion of activated PMNs to endothelial monolayers is mediated by the CD18 adhesion-receptor complex on the PMN cell surface. Monoclonal antibody 60.3 (MoAb 60.3) blocks CD18-dependent PMN-endothelial adhesion in vitro and in vivo. This study was designed to determine the role of CD18-dependent PMN adhesion in ALI associated with gram-negative sepsis. Anesthetized, ventilated (FiO2 0.5, positive end-expiratory pressure 5 cm H2O) pigs received sterile saline (control, n = 8) or live Pseudomonas aeruginosa, 5 x 10(8) colony-forming units/ml at 0.3 ml/20 kg/min (septic, n = 9) for 1 hour. A third group (n = 7) received MoAb 60.3, 2 mg/kg intravenously, 15 minutes before Pseudomonas infusion. Animals were studied for 300 minutes. MoAb 60.3 significantly (p less than 0.05) attenuated the neutropenia seen in sepsis (15 +/- 1 vs 6 +/- 1 x 10(3) PMNs/mm3 at 300 min). Alveolar-capillary membrane injury was assessed by bronchoalveolar-lavage protein content and extravascular lung water determination. MoAb 60.3 significantly (p less than 0.05) reduced BAL protein at 300 minutes (388 +/- 75 vs 1059 +/- 216 micrograms/ml in septic animals) and attenuated the increase in extravascular lung water to 240 minutes (7.1 +/- 2 vs 14.2 +/- 1.2 ml/kg in septic animals). Systemic hypotension, decreased cardiac index, pulmonary hypertension, and relative hypoxemia, all characteristic of this model, were not altered by MoAb 60.3. These data suggest that, in this model of septic ALI, neutropenia is, in part, CD18 dependent and that blocking CD18-dependent PMN adhesion protects the alveolar-capillary membrane independently of altered hemodynamic status.     

Enhanced susceptibility to sepsis after simple hemorrhage. Depression of Fc and C3b receptor-mediated phagocytosis

To determine whether phagocytosis mediated by Fc receptors and/or receptors for the third component of complement (C3b) are altered after hemorrhage, C3H/HeN mice were subjected to nonlethal hemorrhage and then adequately resuscitated. Twelve hours after the hemorrhagic episode, a significant decrease in both Fc (-55.2%) and C3b (-46.6%) receptor-positive peritoneal macrophages was observed compared with controls. At 24 hours the extent of the depression, while still marked, was only -22.5% and -17.4% for Fc and C3b receptors, respectively. By day 3 after hemorrhage, no differences could be observed for either of these receptors. The capacity of macrophages from mice after hemorrhage to elaborate interleukin 1 or tumor necrosis factor-alpha showed no increase over that of the sham controls, and serum levels of endotoxin were not elevated 2 or 24 hours after hemorrhage. Moreover, endotoxin-tolerant C3H/HeJ mice also exhibited depression of both receptors after hemorrhage. Thus, the inability of the host macrophages to clear opsonized infectious agents after hemorrhage may be due in part to the loss of Fc and C3b receptors on macrophages.     

TIMP-1 overexpression in pancreatic cancer attenuates tumor growth, decreases implantation and metastasis, and inhibits angiogenesis.

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in pancreatic cancer progression. This study was undertaken to determine the effects of overexpression of a natural tissue inhibitor of MMP (TIMP-1) on pancreatic cancer cell growth, metastasis, and angiogenesis. METHODS: A poorly differentiated human pancreatic cancer cell line (PANC-1) underwent gene transfection to overexpress TIMP-1 (CD-1 cells). One million PANC-1 or CD-1 cells were injected into 40 nude mice subcutaneously (N = 20) or orthotopically (N = 20). Mice were sacrificed at 120 days. TIMP-1 overexpression by CD-1 cells was confirmed prior to injection and after necropsy. Immunohistochemical staining was undertaken after necropsy to evaluate tumors for TIMP-1, MMP-2, apoptosis (TUNEL), and angiogenesis (CD-31). RESULTS: Tumors of CD-1 cells were less likely to implant (35% vs 70%, P = 0.05) and grew to smaller size (0.5 g +/- 0.03 vs 1.5 g +/- 0.20, P < 0.001) than tumors of PANC-1 cells. As well, subcutaneous CD-1 tumors appeared later than PANC-1 tumors (45 days +/- 2.0 vs 27 days +/- 2.2, P < 0.001). Orthotopic CD-1 tumors metastasized less often than PANC-1 tumors (20% vs 60%, P < 0.05). MMP-2 expression was similar in PANC-1 and CD-1 tumors, while CD-1 tumors showed increased TIMP-1 expression, increased apoptosis, and decreased angiogenesis relative to PANC-1 tumors. CONCLUSIONS: TIMP-1 overexpression reduces pancreatic cancer cell implantation, growth, metastasis, and angiogenesis, while increasing tumor apoptosis, all without altering MMP-2 production. This study demonstrates the potential role of TIMP-1 as a target in gene therapy for pancreatic cancer.     

Hypocalcemia during sepsis. Relationship to resuscitation and hemodynamics.

The ionized calcium (IC) and parathyroid hormone response to polymicrobial intra-abdominal sepsis and the relationship between IC and hemodynamic alterations with and without crystalloid resuscitation were investigated. Thirty swine underwent cecal ligation and incision (n = 19) or sham laparotomy (n = 11), with seven animals that had cecal ligation and incision administered Ringer's solution (50 mL/kg) after each set of measurements recorded on days 0, 1, 2, 4, and 8. An early decrease in mean arterial pressure and cardiac index in animals that had cecal ligation and incision reversed with resuscitation. The IC also fell early and parathyroid hormone level increased in both the unresuscitated and resuscitated septic groups. However, correlation coefficients of mean arterial pressure and cardiac index with IC ranged from .034 to .287 in the septic animals and were lower in the group that had sham laparotomy. We conclude that polymicrobial intra-abdominal sepsis results in decreased IC and an elevated parathyroid hormone level. Hemodynamics do not correlate with IC levels, and resuscitation can be achieved without calcium administration.