René Zellweger

Females in proestrus state maintain splenic immune functions and tolerate sepsis better than males.

OBJECTIVES To determine: a) whether the cell-mediated immune response during sepsis differs in females vs. males; and b) whether the survival rate in females is different than in males after a septic insult. DESIGN A prospective, randomized animal study. SETTING University research laboratory. SUBJECTS Male and female proestrus C3H/HeN mice. INTERVENTIONS After anesthesia, male and proestrus female mice underwent cecal ligation puncture to induce sepsis. The mice were killed at 24 hrs after the onset of sepsis. MEASUREMENTS AND MAIN RESULTS Splenocyte proliferation, as well as splenocyte interleukin (IL)-2 and IL-3 release, was determined by bioassay. In additional studies, survival rate after septic challenge was measured over 10 days. Splenocyte proliferative capacity and splenocyte IL-2 and IL-3 release were markedly decreased in male, but not in female, septic mice. Furthermore, the survival rate of septic female proestrus mice was significantly higher than in comparable male mice. CONCLUSIONS These results support the concept that the immune response of females differs from males, and that females are immunologically better positioned to meet the challenge of sepsis.

Trauma-hemorrhage causes prolonged depression in cellular immunity

ABSTRACT A number of clinical studies have shown that multiple and severe trauma causes immunosuppression and increases the susceptibility to sepsis. However, because there is a close temporal relationship between trauma and hemorrhage in humans, it is difficult to dissociate the effects of tissue trauma versus hemorrhage on immunity in the clinical setting. Studies in mice have shown that simple hemorrhage per se as well as laparotomy alone produces a marked depression in cellular immunity and no difference was seen in the extent of depression at 2 h if these two insults were combined. Nonetheless, it remains unknown whether the combined model of trauma-hemorrhage produces a more protracted depression in immune function. To study this, 5 days after either sham operation, laparotomy (i.e.trauma), hemorrhage alone (35 mmHg for 1 h, followed by resuscitation), or the combination of laparotomy and hemorrhage, mice (C3H/HeN) were sacrificed, after which splenocyte and peritoneal macrophage cultures were established. The proliferative capacity of the splenocytes, as well as their ability to release IL-2 and IL-3, was markedly decreased in the trauma-hemorrhage animals but was normal in the other groups. Furthermore, the release of IL-6 by peritoneal macrophages from animals that underwent trauma-hemorrhage was also significantly depressed. These results support the concept that traumatic injury in the form of a midline laparotomy combined with hemorrhage produces a more protracted impairment in cell-mediated immunity than laparotomy or hemorrhage alone.

Effect of surgical trauma on splenocyte and peritoneal macrophage immune function.

Although previous studies have shown that simple laparotomy produces a depression in peritoneal macrophage (Mphi) antigen presentation capacity, it remains unknown whether the adverse effects of laparotomy are limited to peritoneal Mphi or whether such an insult also affects splenocyte immune function. To study this, mice were anesthetized and a 1-inch midline abdominal incision was made, followed by abdominal closure. At 2 and 24 hours after the surgical procedure, the animals were killed, splenocyte cultures established and stimulated for 48 hours with concanavalin A (2.5 micrograms/mL), while peritoneal macrophage cultures were stimulated with LPS (10 micrograms/mL). The proliferative capacity of the splenocytes, as well as their ability to release interleukin-2 and interleukin-3, was markedly decreased at 2 as well as 24 hours after laparotomy. Furthermore, the release of interleukin-6 by splenic and peritoneal macrophages from animals that underwent laparotomy were also significantly depressed at both 2 and 24 hours. These results support the concept that surgical stress in the form of midline laparotomy per se is sufficient to produce a significant impairment in cell-mediated immunity, thus setting the stage for increased incidence of postoperative complications.

Minimal invasive treatment of proximal femur fractures with the long gamma nail: indication, technique, results.

BACKGROUND The surgical treatment of complex unstable proximal femur fractures from the trochanteric region to the middle shaft area is difficult and often highly invasive, especially in older patients with osteoporotic bones. METHODS In 1993, we began to treat all unstable proximal femur fractures from the trochanteric region to the middle third of the shaft with the long gamma nail (LGN) and allowed the patients immediate full weightbearing after surgery. Perioperative, postoperative, and follow-up data were analyzed. RESULTS During a 3-year period, we treated 37 patients with unstable proximal femur fractures with the LGN. Five of the patients had sustained pathological fractures, and four patients had sustained multiple trauma. The 37 operations were performed by 22 different surgeons with varying degrees of experience. The LGN implantations through maximal 40-mm skin incisions were performed in a mean skin-to-skin operation time of 125 minutes (range, 65-200 minutes) without any further complications. Thirty-one of the 37 patients were allowed immediate full weightbearing after the second postoperative day. The six patients who were not allowed full weightbearing were the ones who had multiple trauma (4) and those who were unable to walk before surgery (2). Three postoperative complications were as follows: one deep infection 4 months after surgery, one superficial infection 3 weeks after surgery, and one deep vein thrombosis 2 weeks after discharge. All three complications occurred in the group of patients who had sustained only one trauma. All traumatic fractures (n = 32) healed without any bone grafting, which means a union rate of 100%. Although immediate full weightbearing was allowed, there were no implant dislocations, implant failures, or other problems. The 30-day mortality was 0%. The follow-up examinations after a mean time of 6 months were very satisfying. All traumatic fractures were healed. The patients had symmetrical functional findings to the opposite side or before surgery, respectively, except for two patients with a leg shortening of 2 cm, two patients with an external malrotation of 10 degrees, and one patient with a hip extension deficit of 10 degrees after a pathological fracture. CONCLUSION The LGN is, after appropriate introduction and training, a safe and easy implant for the treatment of complex proximal femur fractures from the trochanteric region to the middle shaft area. The minimal invasive technique with low risks and minimal complications and the possibility of immediate full weightbearing sets a new standard, especially for older patients with osteoporotic bones.

Metoclopramide: a novel and safe immunomodulating agent for restoring the depressed macrophage immune function after hemorrhage.

BACKGROUND AND OBJECTIVE Recent studies have shown that administration of the anterior pituitary hormone, prolactin, after hemorrhage restored the depressed immune responses that are observed under those conditions. Because metoclopramide (MCP) is known to increase prolactin secretion and ultimately plasma prolactin levels, we attempted to determine whether administration of metoclopramide after hemorrhage produces any beneficial effects on the depressed splenocyte and peritoneal macrophage immune function after severe hemorrhage. DESIGN, MATERIALS AND METHODS Mice were bled to and maintained at a mean arterial pressure of 35 mm Hg for 60 minutes, then adequately resuscitated and segregated into two groups. One group received saline vehicle; animals in the other group were treated with metoclopramide (100 microg/100 g body weight, subcutaneously) before resuscitation. Two hours after saline or MCP injection, the animals were killed and macrophage as well as splenocyte cultures established. Plasma corticosterone levels were also measured. RESULTS The proliferative capacity of the splenocytes as well as their ability to release interleukin (IL)-2 and IL-3 in response to mitogen was markedly improved in animals that had hemorrhaged and that were treated with MCP compared with saline-injected mice. Moreover, the depressed splenic and peritoneal macrophage IL-1 and IL-6 release after hemorrhage was restored with MCP treatment. Furthermore, treatment with MCP prevented the increase in blood corticosterone levels seen after severe hemorrhage. CONCLUSION These results support the concept that the immunosuppression after hemorrhage may be mediated by hormones from the hypothalamic-pituitary-adrenal axis. Furthermore, MCP may be a useful adjuvant in the treatment of the trauma-hemorrhagic shock-induced immunosuppression.

Effect of naloxone on immune responses after hemorrhagic shock.

Objective: To determine whether naloxone administration after hemorrhagic shock has any beneficial or deleterious effect on immune responses. Background Data: Hemorrhagic shock is known to produce immunodepression in both humans and experimental animals. Although studies suggest that endogenous opioids play a role in immune regulation in adverse circulatory conditions, it remains controversial whether these opioids exert beneficial or detrimental effects on immunity after shock. Moreover, little information is available concerning the effects of opioid receptor blockade using naloxone on cell‐mediated immunity and endocrine responses after shock. Methods: Male C3H/HeN mice (25 g) were bled to and maintained at a mean arterial blood pressure of 35 ± 5 mm Hg for 1 hr. The shed blood was then returned along with lactated Ringers solution (two times the shed blood volume) to provide fluid resuscitation. The animals were randomized to receive either naloxone (1 mg/kg iv) or an equal volume of vehicle (saline) after the shed blood was returned, i.e., immediately before crystalloid resuscitation, and were killed at 2 hrs after resuscitation to obtain splenocytes, macrophages (peritoneal and splenic), and blood. Measurements and Main Results: Bioassays revealed significantly decreased release of all studied interleukins (interleukins‐1, ‐2, ‐3, and ‐6) by peritoneal and splenic macrophages as well as significantly decreased splenocyte proliferative capacity after shock in vehicle‐treated mice. Naloxone administration after hemorrhage resulted in either similar or even more decreased levels of interleukin release compared with vehicle‐treated hemorrhaged mice. Significantly increased plasma corticosterone concentrations were observed in vehicle‐treated animals compared with control animals. Naloxone administration did not have any significant effects on corticosterone plasma concentrations after hemorrhage. Conclusions: These findings indicate the importance of the endogenous opioid system for the maintenance of immunity in adverse circulatory conditions, i.e., hemorrhage. Although additional studies involving different doses and/or times of naloxone administration may provide different results, the present findings raise the concern that naloxone administration in the traumatized host may have deleterious effects because it decreases peritoneal macrophage and splenic immune functions.

Increased melatonin levels after hemorrhagic shock in male and female C3H/HeN mice

Although hemorrhagic shock leads to significant alterations of several hormones, e.g. ACTH, corticosterone and β-endorphin, it is not known whether plasma melatonin levels are affected under this condition and if so, whether the effects are comparable in males and females. Using a radioimmunoassay, it was found that plasma melatonin levels were significantly increased in male and proestrus female C3H/HeN mice immediately after hemorrhagic shock. However, in male mice, by two hours after hemorrhage and resuscitation, plasma melatonin returned to levels comparable to those seen in control and sham-operated animals. Proestrus female mice, on the other hand, showed significantly increased plasma melatonin levels at two hours after surgery when compared to unoperated control animals. Although the significance and biological role of the transient increased plasma melatonin levels after hemorrhagic shock remain to be determined, it appears that the pineal gland and/or an extrapineal source of melatonin, of both male and proestrus female mice responds to severe hypotension by increased release of melatonin.

Predisposing factors: effect of sex, nutritional factors and age on immunity following shock and sepsis

Research from numerous laboratories throughout the world now indicates that many, if not all, of the physiological, metabolic, and immunological responses to trauma, sepsis and cancer are not mediated directly by bacteria or their toxins, or by tumor cells, but rather by groups of host-derived polypeptide molecules which are produced in response to these stimuli and have collectively been called cytokines [1–4]. These molecules work together with classic stress hormones and with other humoral mediators to orchestrate and coordinate the cellular response to critical illnesses. The central role occupied by these polypeptide signals has challenged past teachings and prompted a rethinking of traditional approaches for treating the host with severe infections. New knowledge delineating how these key polypeptide molecules control the catabolic response to stress states has been generated at a staggering pace. From an evolutionary standpoint, these biological responses are the result of a process that favors survival of the fittest in the struggle to survive and preserve the species. Ironically, these polypeptide mediators, which clearly orchestrate many of the appropriate and beneficial responses to these catabolic diseases (i.e. fever, tachycardia, and acute-phase protein synthesis), can also initiate detrimental physiological responses, such as hypotension, organ failure, cachexia, and death [5–7].

Peritoneal macrophages show increased cytokine gene expression following haemorrhagic shock

Tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-1 and transforming growth factor-beta (TGF-beta) have been recognized as important mediators of pathophysiological and immunological events associated with shock. Previous studies have indicated that although peritoneal macrophage (PM phi) antigen presentation was depressed following haemorrhage, the cytokine release capacity in response to lipopolysaccharide (LPS) was not affected in vitro. To determine the effect of haemorrhagic shock on PM phi cytokine mRNA transcription, C3H/HeN male mice were bled to and maintained at a mean arterial blood pressure of 35 mmHg for 60 min, and then adequately resuscitated. PM phi were isolated at 1 or 24 hr after haemorrhage and were incubated without or with 10 micrograms LPS/ml for 1 hr. Total RNA was then extracted followed by Northern blot analysis, as well as semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR). The results of Northern blot analysis indicated that haemorrhage markedly increased LPS-induced IL-1 beta, IL-6, and TNF-alpha mRNA accumulation in PM phi at both 1 and 24 hr after haemorrhage and resuscitation. Furthermore, competitive RT-PCR demonstrated that mRNA of IL-1 beta, IL-6, TNF-alpha, as well as TGF-beta, was increased in PM phi obtained 1 hr after haemorrhage either with or without LPS stimulation. The data from Northern blot analysis and semi-quantitative RT-PCR also revealed that LPS enhanced the effect of haemorrhage on PM phi cytokine gene expression. Thus, following haemorrhage, PM phi showed elevated cytokine mRNA accumulation which was not followed by an increased ability to release cytokines in response to LPS in vitro. These results, therefore, suggest that different mechanisms regulate gene expression and subsequent cytokine secretion by PM phi following haemorrhage.

Immunomodulation following shock and sepsis

Despite the introduction of broad-spectrum antibiotics, sepsis is one of the most important causes of multiple organ failure (MOF) which accounts for 70,000 deaths in the USA each year [1]. Infection is also an important cause of mortality in the severely traumatized patient and is responsible for 50–70% of all burn-related deaths [2–3].