HDAC6 selective inhibition of melanoma patient T-cells augments anti-tumor characteristics.

BACKGROUND: Therapies targeting anti-tumor T-cell responses have proven successful in the treatment of a variety of malignancies. However, as most patients still fail to respond, approaches to augment immunotherapeutic efficacy are needed. Here, we investigated the ability of histone deacetylase 6 (HDAC6)-selective inhibitors to decrease immunosuppression and enhance immune function of melanoma patient T-cells in ex vivo cultures. METHODS: T-cells were harvested from peripheral blood or tumor biopsies of metastatic melanoma patients and cultured in the presence of pan-, class-specific or class-selective histone deacetylase (HDAC) inhibitors. Changes in cytokine production were evaluated by Luminex and intracellular flow cytometry staining. Expression of surface markers, transcription factors, protein phosphorylation, and cell viability were assessed by flow cytometry. Changes in chromatin structure were determined by ATAC-seq. RESULTS: T-cell viability was impaired with low doses of pan-HDAC inhibitors but not with specific or selective HDAC inhibitors. The HDAC6-selective inhibitors ACY-1215 (ricolinostat) and ACY-241 (citarinostat) decreased Th2 cytokine production (i.e. IL-4, IL-5, IL-6, IL-10 and IL-13). Expansion of peripheral blood T-cells from melanoma patients in the presence of these inhibitors resulted in downregulation of the Th2 transcription factor GATA3, upregulation of the Th1 transcription factor T-BET, accumulation of central memory phenotype T-cells (CD45RA-CD45RO + CD62L + CCR7+), reduced exhaustion-associated phenotypes (i.e. TIM3 + LAG3 + PD1+ and EOMES+PD1+), and enhanced killing in mixed lymphocyte reactions. The frequency, FOXP3 expression, and suppressive function of T regulatory cells (Tregs) were decreased after exposure to ACY-1215 or ACY-241. Higher frequencies of T-cells expressing CD107a + IFNγ+ and central memory markers were observed in melanoma tumor-infiltrating lymphocytes (TIL), which persisted after drug removal and further expansion. After ACY-1215 treatment, increased chromatin accessibility was observed in regions associated with T-cell effector function and memory phenotypes, while condensed chromatin was found in regions encoding the mTOR downstream molecules AKT, SGK1 and S6K. Decreased phosphorylation of these proteins was observed in ACY-1215 and ACY-241-treated T-cells. AKT- and SGK1-specific inhibition recapitulated the increase in central memory frequency and decrease in IL-4 production, respectively, similar to the observed effects of HDAC6-selective inhibition. CONCLUSIONS: HDAC6-selective inhibitors augmented melanoma patient T-cell immune properties, providing a rationale for translational investigation assessing their potential clinical efficacy.

Targeting histone deacetylase 6 mediates a dual anti-melanoma effect: Enhanced antitumor immunity and impaired cell proliferation.

The median survival for metastatic melanoma is in the realm of 8-16 months and there are few therapies that offer significant improvement in overall survival. One of the recent advances in cancer treatment focuses on epigenetic modifiers to alter the survivability and immunogenicity of cancer cells. Our group and others have previously demonstrated that pan-HDAC inhibitors induce apoptosis, cell cycle arrest and changes in the immunogenicity of melanoma cells. Here we interrogated specific HDACs which may be responsible for this effect. We found that both genetic abrogation and pharmacologic inhibition of HDAC6 decreases in vitro proliferation and induces G1 arrest of melanoma cell lines without inducing apoptosis. Moreover, targeting this molecule led to an important upregulation in the expression of tumor associated antigens and MHC class I, suggesting a potential improvement in the immunogenicity of these cells. Of note, this anti-melanoma activity was operative regardless of mutational status of the cells. These effects translated into a pronounced delay of in vivo melanoma tumor growth which was, at least in part, dependent on intact immunity as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Given our findings, we provide the initial rationale for the further development of selective HDAC6 inhibitors as potential therapeutic anti-melanoma agents.

Short-chain fatty acids and encephalopathy of Reye's syndrome.

Plasma levels of six short-chain fatty acids (SCFA) were measured in 23 Reye's syndrome patients. In sequential measurements, only propionic acid correlated closely with neurologic severity. Although admission SCFA levels were slightly elevated, there were no significant differences between patients grouped by severity of encephalopathy. Admission SCFA did not predict neurologic outcome; also, they correlated poorly with admission blood ammonia, amino acid nitrogen, and lactate.

The metabolic course of Reye's syndrome: distinction between survivors and nonsurvivors.

We compared the levels of hormones and metabolites in the plasma of 37 survivors of Reye's syndrome with the levels in 8 fatal cases, at four time periods within 72 hours of admission. The most prominent differences were found for norepinephrine (NE), which was significantly elevated in fatal cases compared with survivors at all periods. Lactate and dopamine were elevated in the earlier periods. Epinephrine and alpha-amino acid nitrogen were also elevated in fatal cases, but the differences usually were not significant. NE elevation may reflect an increased sympathoadrenal medullary output associated with brain edema, compounded by impaired hepatic clearance of monoamines. Skeletal muscle ischemia from NE-induced vasoconstriction may explain the association between lactic acidemia and the severity of encephalopathy.

Changes in plasma fibronectin in children after elective repair of congenital heart defects.

Plasma fibronectin is an attachment protein important for maintaining capillary integrity and host defense mechanisms. Depletion of plasma fibronectin has been shown to occur in adults after septic shock, major trauma, and burns. Limited laboratory and clinical studies suggest a correlation between decreased plasma fibronectin levels and increased pulmonary capillary permeability and tissue perfusion. Mild and transient plasma fibronectin depletion has been observed in adults after cardiovascular operations. We measured plasma fibronectin by immunoturbidometric assay in 20 children (age 6 months to 12 years) undergoing repair of congenital heart defects. Plasma fibronectin levels immediately after operations and daily thereafter were compared with the preoperative values. Plasma fibronectin declined on postoperative days 1, 2, 3, 4, and 5 (p < 0.05). A nadir was reached on day 3 with a tendency toward recovery thereafter. Patients with a therapeutic intervention score of more than 35 had greater magnitude of plasma fibronectin decline than those with a score of less than 35 at 24 hours after the operation (p < 0.005). We conclude that (1) significant and prolonged plasma fibronectin depletion occurs after cardiovascular operations in children; and (2) postoperative plasma fibronectin depletion is associated with increasingly complex surgical intervention. Reduced plasma fibronectin synthesis and more extensive operations for congenital heart defects are likely reasons for children being more susceptible than adults to plasma fibronectin depletion after cardiovascular operations.

Idiopathic pulmonary hemorrhage in infancy. Clinical features and management with high frequency ventilation.

STUDY OBJECTIVES: To describe the clinical characteristics of infants with severe acute pulmonary hemorrhage and the effects of mechanical ventilation on gas exchange. SETTING: Tertiary care pediatric ICU in a university hospital. PATIENTS AND DESIGN: Case records of patients with severe acute pulmonary hemorrhage from January 1992 to July 1995 were reviewed. Acute pulmonary hemorrhage was defined as hemoptysis and/or epistaxis or blood obtained from endotracheal tube which could not be attributed to cardiac or vascular malformation, infectious process, or known trauma. INTERVENTIONS: Patients were initially managed with conventional ventilation. High frequency ventilation (HFV) was utilized when hypoxemia (PaO2/PAO2 < 0.2) and/or respiratory acidosis (PaCO2 > or = 60 mm Hg with pH < 7.25) persisted. MEASUREMENTS AND RESULTS: Six African-American male infants from Detroit, with a median age 2.3 months, presented with severe acute pulmonary hemorrhage. Chest radiographs showed diffuse bilateral infiltrates or opacification with a normal sized heart. All infants were managed with HFV, four by oscillation and two by jet. The indications for HFV were persistent hypoxemia (2), respiratory acidosis (1), and a combination of both (3). There was an improvement in pH and PaCO2, and a decreased need for oxygen 6 and 24 h after initiating HFV. PaO2/PAO2 and oxygenation index showed a tendency toward improvement. All infants survived, and there were no complications. No cause for pulmonary hemorrhage was found in any of the infants. CONCLUSIONS: Idiopathic acute pulmonary hemorrhage is a potentially life-threatening disorder encountered among inner-city infants. HFV is highly effective and safe in rapidly reversing the severe oxygenation and ventilation deficits in this setting.

Effects of insulin and metformin on glucose metabolism in rat vascular smooth muscle.

Glucose metabolism in vascular smooth muscle cells (VSMCs) is characterized by substantial lactate production even in fully oxygenated conditions. Insulin and metformin, an insulin-sensitizing agent, have direct effects on the vascular tissue metabolism. We investigated whether insulin or metformin can induce a switch in VSMC glucose metabolism from lactate production to pyruvate oxidation, by measuring lactate oxidation as determined by the conversion of [1-14C]-D,L-lactate to [1-14C]-pyruvate and subsequent oxidation to acetyl coenzyme A and 14CO2 by pyruvate dehydrogenase (PDH). Lactate oxidation was measured in control rat aortic cultured VSMCs incubated for 30 minutes in media with and without additional glucose compared with VSMCs cultured in the presence of insulin or metformin. The addition of glucose to VSMCs decreased lactate oxidation (4.6+/-1.7 v 9.6+/-2.4 pmol/cell/min, P < .001). In the absence of additional glucose, metformin decreased lactate oxidation in VSMCs compared with controls (4.9+/-1.4 v 9.6+/-2.4 pmol/cell/min, P < .01). Metformin in the presence of glucose caused the greatest decline in lactate oxidation (2.5+/-0.4 pmol/cell/min, P < .001). In contrast to the effects of metformin, insulin increased lactate oxidation both with (12.9+/-1.5 pmol/cell/min, P < .001) and without (17.9+/-4.4, P < .01) additional glucose. This suggests that insulin facilitates VSMC utilization of lactate as a source of pyruvate and energy production even during noncontractile periods.

Protective effects of a thromboxane synthetase inhibitor and continuous arteriovenous hemofiltration in rat endotoxic shock.

We determined the efficacy of continuous arteriovenous hemofiltration (CAVH) and a thromboxane synthetase inhibitor (TSI) on survival and their effect on TXA2, PGI2, TNF alpha, and IL-1beta production in rat endotoxemia. Thirty-six endotoxemic rats were randomized to one of 4 groups: (A) no TSI, sham CAVH; (B) no TSI, CAVH; (C) TSI, sham CAVH; and (D) TSI, CAVH. Either CAVH (Group B) or pretreatment with TSI (Group C) resulted in increased survival time. CAVH did not prevent the rise in TX (Group B). TNF alpha levels at 2 h after LPS infusion were higher in Group D compared to Group B (26.1 +/- 3.7 vs 13.2 +/- 4.3 ng/mL, P < 0.05) respectively. IL-1beta was detected earlier in Groups C,D when compared to Groups A,B (P < 0.02). TNF alpha and IL-1beta were not ultrafiltered. CAVH and the inhibition of TX synthesis independently improved survival in endotoxemia, however, their beneficial effects were not additive. While TSI may improve survival by blocking TXA2 production, the salutary effects of CAVH appear to be from removal of an undetermined TXA2 dependent mediator. TNF alpha and IL-1beta concentrations do not appear to influence survival times in this model.

Ibuprofen attenuates cardiopulmonary dysfunction by modifying vascular tone in endotoxemia.

Ibuprofen, a cyclooxygenase inhibitor, improves pulmonary and cardiovascular injury in endotoxemia. We studied the mechanism of the beneficial effects of ibuprofen in relation to production of inflammatory mediators which influence vascular tone in endotoxemia. Rats were randomly assigned to one of three groups: (1) control, (2) endotoxemia alone; and (3) ibuprofen pretreatment and endotoxemia. Plasma and lung lavage concentrations of tumor necrosis factor, thromboxane B2 (TXB2), leukotriene (LT) C4,D4,E4 and nitric oxide (NO) were determined over a 2 h period. Pretreatment with ibuprofen resulted in increased survival, and attenuation of pulmonary and cardiovascular dysfunction when compared to the rats receiving endotoxin alone. The marked elevation in plasma TXB2 concentration in endotoxemic rats was prevented by pretreatment with ibuprofen. Similarly, pretreatment with ibuprofen prevented the decrease in lung lavage NO levels in endotoxemic rats. The improved survival and cardiopulmonary protection in endotoxemic rats pretreated with ibuprofen appears to be related to decreased thromboxane production and preservation of endothelial production of nitric oxide.

Clinical characteristics of respiratory syncytial virus infections in healthy versus previously compromised host.

In an effort to delineate the clinical characteristics of respiratory syncytial virus (RSV) infection in the compromised host, we compared children with bronchopulmonary dysplasia (BPD), congenital heart disease (CHD), premature birth, failure to thrive, and gastroesophageal reflux to previously healthy children. During a four-year period, 262 patients were admitted to the hospital with RSV infection diagnosed by a rapid RSV antigen detection test. Children with BPD or CHD had more hospital days and supplemental oxygen days than the previously healthy group (P less than 0.05). Patients with BPD also had more ICU days, ventilator days, and NPO days, as well as a higher physiologic stability index and therapeutic intervention score than the previously healthy group (P less than 0.05). Premature infants were more likely to present with apnea from RSV (P less than 0.001). Patients with underlying illness tended to be older, although significant difference was demonstrated only for the BPD group (7.0 +/- 5.3 vs. 3.5 +/- 3.3, P less than 0.05). Patients with BPD and CHD had more nosocomial infections than the previously healthy group (P less than 0.0001) and death occurred only in patients with underlying illness. We conclude that previously compromised patients are at risk for more severe and prolonged RSV disease. Earlier diagnosis and therapeutic intervention may be necessary in such patients to improve outcome.

Macrophage inflammatory protein-2 predicts acute lung injury in endotoxemia.

BACKGROUND: Proinflammatory mediators that include tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) and anti-inflammatory mediators such as interleukin-10 (IL-10) modulate the immune response to endotoxemia. IL-10 downregulates the production of TNF-alpha and MIP-2. Acute lung injury may occur secondary to neutrophil chemotaxis mediated by chemokine MIP-2. We studied the temporal relationship of TNF-alpha, MIP-2, and IL-10 in rat endotoxemia and correlation of MIP-2 concentrations with acute lung injury. METHODS: Ten ventilated rats were randomized to receive an intravenous infusion of 2 mg/kg Escherichia coli lipopolysaccharide (n = 6) or saline placebo (n = 4). Blood pressure was continuously monitored and arterial blood was obtained for lactate, blood gas, TNF-alpha, IL-10, and MIP-2 measurements at baseline, 2, 4, and 5.5 hours after LPS or saline infusion. RESULTS: Endotoxemia resulted in hypotension, lactic acidemia, and increased alveolar-arterial oxygen gradient (A-a O2 gradient) compared with the placebo group. TNF-alpha, MIP-2, and IL-10 levels were increased 2 hours after endotoxemia. Subsequently, TNF-alpha levels declined while IL-10 and MIP-2 levels remained elevated. Control rats had no significant increase in cytokine production at any time point. MIP-2 concentrations correlated with A-a O2 gradient, an indicator of lung injury (r = 0.56, p < 0.001). CONCLUSIONS: MIP-2, possibly released by TNF-alpha stimulation of macrophages, is associated with acute lung injury possibly by inducing neutrophil chemotaxis. IL-10 may exert its counter-inflammatory response by inhibiting the release of TNF-alpha in endotoxemia.

Diagnosis and management of acute epiglottitis -- report of 90 consecutive cases.

Ninety children with acute epiglottitis were admitted from 1971 - 1977. Lateral extended neck radiograph was found to be a quick, safe and reliable way to establish the diagnosis. Out of 79, 77 (97%) had blood cultures yielding Hemophilus influenzae type b. Immediate institution of airway and antibiotics were the mainstay of treatment. All patients in the series survived. For short-term airway management, as in acute epiglottitis, nasotracheal intubation under general anesthesia was found to be the airway of choice.

Efficacy of erythromycin for postpyloric placement of feeding tubes in critically ill children: a randomized, double-blind, placebo controlled study.

BACKGROUND: Erythromycin enhances gastric emptying and has been suggested to facilitate nasoenteric feeding tube placement in adults. Our primary objective was to evaluate the effect of erythromycin on the transpyloric passage of feeding tubes in critically ill children, and second, to evaluate the effect of erythromycin on the distal migration of duodenal feeding tubes. METHODS: Seventy-four children were randomly assigned to receive erythromycin lactobionate (10 mg/kg) IV or equal volume of saline placebo 60 minutes before passage of a flexible weighted tip feeding tube. Abdominal radiographs were obtained 4 hours later to assess tube placement. If the tube was proximal to the third part of the duodenum, two additional doses of erythromycin/placebo were administered 6 hours apart. Those receiving additional doses had repeat radiographs 14 to 18 hours after tube placement. RESULTS: The number of postpyloric feeding tubes was similar in the erythromycin and placebo treated groups 4 hours after tube insertion (23/37 vs 27/37, p = .5). Of those with prepyloric tubes at 4 hours, none in the erythromycin group and 3 in the placebo group had the tube migrate to the postpyloric position by 14 to 18 hours (p < .05). Of those with postpyloric tubes proximal to the third part of the duodenum at 4 hours, additional doses of erythromycin did not cause more tubes to advance further into the intestine than did placebo (p = .6). CONCLUSIONS: Erythromycin does not facilitate transpyloric passage of feeding tubes in critically ill children. The distal migration of duodenal tubes further into the small bowel is also not enhanced by erythromycin.

The role of intensive care in managing childhood cancer.

To study the value of intensive care in childhood cancer, we evaluated the clinical course and outcome of all such children admitted to our intensive care unit (ICU) (n = 183) during the five-year period from 1984-1988. Excluding those admitted for postoperative observation, there were a total of 63 admissions for complications of malignancy. Of these, admissions for sepsis, pulmonary parenchymal disease, or coma were associated with poor outcome. Thirty-six percent of patients requiring mechanical ventilation for respiratory failure and 27% requiring inotropic support survived longer than six months. Physiologic Stability Index and Therapeutic Intervention Scores were significantly greater in nonsurvivors than survivors. Of those who survived their ICU stay, 50% went home functioning at their premorbid state. The duration of ICU stay was not different in survivors and nonsurvivors, suggesting that intensive care does not excessively prolong the dying process. We conclude that many life-threatening complications of cancer are potentially reversible. The extent of functional recovery of survivors warrants aggressive intensive support in this setting.

End-of-life decision-making and satisfaction with care: parental perspectives.

OBJECTIVES: To evaluate parents' perceptions of the process by which decisions are made to limit or withdraw life support from critically ill children, and to evaluate parents' perceptions of their child's death in the pediatric intensive care unit (ICU) and their satisfaction with the care provided. DESIGN: Survey. SETTING: University teaching hospital. PARTICIPANTS: Seventy-eight parents who experienced the death of a child in the ICU between January 1, 1995 and June 30, 1998. INTERVENTIONS: Structured telephone interviews. MEASUREMENTS AND MAIN RESULTS: Forty-one parents recalled discussing the limitation or withdrawal of life support from their child with a physician. Of these, 31 (76%) felt they had just the right amount of authority to make decisions for their child, 8 (20%) felt they had too little, and 1 (2%) felt they had too much. Those satisfied with their decision-making authority had more trust in their physician than those who were dissatisfied (5 vs. 1, p <.001 by Mann-Whitney U test, where 1 = no trust and 5 = complete trust). Factors identified by parents as being extremely important in the decision-making process included physician recommendations, diagnosis, expected neurologic recovery, and degree of pain and suffering. A total of 51 parents were with their child at the time of death. Although none regretted being present, 17 parents who were not present later wished they had been (p <.001, Fisher's exact test). The quality of care provided to parents by the ICU staff was graded (1 = poor; 5 = excellent). Eleven parents (14%) scored quality of care

Adult respiratory distress syndrome in children.

In spite of modern technological advances, ARDS continues to be an important cause of morbidity and mortality from a diverse group of disorders such as sepsis, trauma, and aspiration. ARDS represents a target organ injury resulting from activation of the host's inflammatory cells and uncontrolled liberation of inflammatory mediators. In most instances, therefore, ARDS is a localized manifestation of a widespread onslaught characteristic of SIRS. At this time, there are no proven interventions to prevent ARDS, and the management is mainly supportive. Modulation of the host's inflammatory response seems to hold the most promise for prevention and treatment of ARDS. Such strategies need to be explored with well-controlled clinical trials.

Transporting the neurologically compromised child.

The initial evaluation, stabilization, and subsequent transport of the neurologically compromised child should take into account the pathophysiologic response of the CNS to a variety of injurious factors. Little can be done to avoid neuronal damage from the primary event. Secondary insults resulting from hypoxemia, ischemia, intracranial hypertension, and fluid shifts can and must be prevented to ensure maximum neuronal salvage, however. Maintenance of an adequate airway, breathing, and circulation assume an immediate and ongoing priority. Neuroresuscitation should be directed toward reversing alterations in cerebral metabolism, autoregulation, brain water, and ICP associated with individual pathologic states.

The role of tumor necrosis factor in endotoxic shock.

Tumor necrosis factor (TNF) has been implicated in hemodynamic changes of endotoxic shock. The temporal relationship of hypotension and TNF release in endotoxemia was studied. Carotid arteries of five intubated rats were cannulated and Escherichia coli 0127:B8 lipopolysaccharide (LPS) was infused over 10 seconds. Arterial blood pressure (ABP), heart rate, and plasma TNF concentrations were measured at 0, 5, 15, 30, and 60 mins. Five to 15 mins after LPS, there was a marked decline in ABP (146 +/- 23 vs 57 +/- 5 mm Hg, p < 0.005), without a significant rise in TNF. The heart rate did not change. From 15 to 60 mins, there was a rise in TNF concentrations (523 +/- 333 vs 5783 +/- 629 pg/ml, p < 0.005) while the same degree of hypotension persisted. It is concluded that early hypotension after acute endotoxemia is not dependent on TNF alone. However, TNF may play a role in sustaining hypotension after endotoxemia.

Expression of stress proteins HSP 72 & HSP 32 in response to endotoxemia.

Pretreatment with heat decreases mortality and acute lung injury in the rat septic shock model, presumably by the production of heat shock proteins (HSP). However, endotoxin, a severe cell stresser, has not been shown to induce HSP 70. We investigated the effects of severe endotoxemia on the expression of specific protective stress proteins, including HSP 72 (inducible HSP 70), HSP 32 (heme oxygenase-1), and HSP 90. Fifteen rats received intravenously either 3 mg/kg of endotoxin (E. coli O127:B8 lipopolysaccharide, LPS) (n=9) or saline (n=6). Two hr later the spleen was removed and splenocytes were separated into three groups and analyzed for specific HSP by Western blot. In Group 1, both endotoxin-treated and saline-treated splenocytes were incubated for 3 hr at 37 degrees C. In Group 2, the splenocytes were washed twice, then heat shocked for 30 min at 42 degrees C and subsequently incubated for 2.5 hr at 37 degrees C. In Group 3, splenocytes were washed twice, then incubated for 3.0 hr at 37 degrees C. HSP 90 & HSP 70c (constitutive) were present in all groups. Consistent with observations by others, HSP 72 was not induced in Group 1. HSP 72 was induced in both the saline-treated and endotoxin-treated splenocytes after heating (Group 2). However, in the absence of heat stress, HSP 72 was present in endotoxin-treated but not in saline-treated splenocytes after incubation (Group 3). Conversely, HSP 32, while present in Group 1 splenocytes, was not detected in the endotoxin-treated splenocytes of Group 2 and Group 3, but was present in the saline-treated cells. In conclusion, endotoxemic shock results in induction of HSP 72 and depletion of HSP 32, but only after the cells have been washed and further incubated.

Altered T cell cytokine production following mechanical trauma.

BACKGROUND: Cell mediated immunity is suppressed following traumatic injury. The Objective is to determine whether there is a shift from T helper type 1 (TH1) to TH2 cell cytokine production following mechanical trauma in a rat model. METHODS: Male Sprague-Dawley rats were anesthetized and subjected to bilateral femur fractures or sham injury. Spleens were removed 3 days later. T cell proliferation and cytokine production were stimulated by culturing spleen cells with the T cell mitogen concanavalin A (con A). Interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-10 and IL-4 concentrations were measured in spleen cell supernatants using enzyme linked immunosorbent assays. RESULTS: Con A-induced spleen cell proliferation was decreased in traumatized rats compared to controls (p < 0.05). Spleen cell supernatant concentrations of the TH1 cytokines IL-2 and IFN-gamma were decreased in the trauma group (p < 0.05). Supernatant concentrations of the TH2 cytokine IL-10 were also decreased in traumatized rats (p < 0.01). The IL-4 concentrations were below the detection limit (< 15 pg/mL) in all cell supernatants. CONCLUSIONS: Mechanical tissue injury leads to generalized suppression of T helper cell cytokine production rather than a shift from TH1 to TH2 cell activity. Post-trauma cellular immunosuppression is not mediated via excess IL-10 production by TH2 cells.