Mashkoor A. Choudhry

Cecal ligation and puncture.

The model of cecal ligation and puncture (CLP) in rodents has been used extensively to investigate the clinical settings of sepsis and septic shock. This model produces a hyperdynamic, hypermetabolic state that can lead to a hypodynamic, hypometabolic stage, and eventual death. Blood cultures are positive for enteric organisms very early after CLP. The model has been widely used over the past 26 years and is highly versatile in adapting to a range of severity and testing objectives. It is inexpensive to prepare and technically straightforward. Aspects of sepsis research investigated using CLP include energetics, metabolism, resuscitation, antibiotic therapy, microbial factors, cardiovascular responses, immune function, mediator release, and cytokine expression patterns. The challenge of the small circulating blood volume in rodents can be overcome by using micromethods that enable analysis of small volumes, or alternatively, by using a large number of animals to obtain serial samples.

Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical consequences: summary of a symposium.

This report is a summary of the symposium on Alcohol, Intestinal Bacterial Growth, Intestinal Permeability to Endotoxin, and Medical Consequences, organized by National Institute on Alcohol Abuse and Alcoholism, Office of Dietary Supplements, and National Institute of Diabetes and Digestive and Kidney Diseases of National Institutes of Health in Rockville, Maryland, October 11, 2006. Alcohol exposure can promote the growth of Gram-negative bacteria in the intestine, which may result in accumulation of endotoxin. In addition, alcohol metabolism by Gram-negative bacteria and intestinal epithelial cells can result in accumulation of acetaldehyde, which in turn can increase intestinal permeability to endotoxin by increasing tyrosine phosphorylation of tight junction and adherens junction proteins. Alcohol-induced generation of nitric oxide may also contribute to increased permeability to endotoxin by reacting with tubulin, which may cause damage to microtubule cytoskeleton and subsequent disruption of intestinal barrier function. Increased intestinal permeability can lead to increased transfer of endotoxin from the intestine to the liver and general circulation where endotoxin may trigger inflammatory changes in the liver and other organs. Alcohol may also increase intestinal permeability to peptidoglycan, which can initiate inflammatory response in liver and other organs. In addition, acute alcohol exposure may potentiate the effect of burn injury on intestinal bacterial growth and permeability. Decreasing the number of Gram-negative bacteria in the intestine can result in decreased production of endotoxin as well as acetaldehyde which is expected to decrease intestinal permeability to endotoxin. In addition, intestinal permeability may be preserved by administering epidermal growth factor, l-glutamine, oats supplementation, or zinc, thereby preventing the transfer of endotoxin to the general circulation. Thus reducing the number of intestinal Gram-negative bacteria and preserving intestinal permeability to endotoxin may attenuate alcoholic liver and other organ injuries.

High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this “divide-and-conquer” strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 different proteins with 1494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 “classic” cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

Gender differences in acute response to trauma-hemorrhage.

To understand the pathogenesis of a disease, experimental models are needed. A good experimental model is the one that simulates responses observed in the clinical setting. In recent years, clinical studies have indicated that gender might be a factor that plays a significant role in the outcome of patients with shock, trauma, and sepsis. These observations are now being evaluated in experimental setting. Studies performed in a rodent model of trauma-hemorrhage have concluded that alterations in immune and cardiac functions after trauma-hemorrhage are more markedly depressed in adult males, and ovariectomized and aged females. However, both are maintained in castrated males and in proestrus females. Moreover, the survival rate of proestrus females subjected to sepsis after trauma-hemorrhage is significantly higher than age-matched males or ovariectomized females. Although these observations suggest gender-specific response after trauma-hemorrhage, the mechanisms responsible for gender specificity remain largely unknown. Furthermore, in other injuries such as burn, experimental studies dealing with sexual dimorphism are limited. Therefore, more studies in clinical and experimental settings are required to determine whether gender-specific responses are global across the injuries or are observed in specific injury situations. Studies are also needed to delineate underlying mechanisms responsible for differences between males and females after trauma-hemorrhage. The information gained from the experimental studies will help in designing innovative therapeutic approaches for the treatment of trauma patients.

Cutaneous Expression of CRH and CRH-R: Is There a “Skin Stress Response System?”

ABSTRACT: The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH‐R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic‐pituitary‐adrenal axis functions in mammalian skin, in response to local stress (see Reference 1 ). To further define such system we used immunocytochemistry, RP‐HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH‐R1 was documented in mouse and human skin using RT‐PCR and Northern blot techniques; CRH binding sites and CRH‐R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose‐dependent increases in intracellular Ca2+. The latter were inhibited by the CRH antagonist α‐helical‐CRH(9–41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH‐R1, supports the existence of a local CRH/CRH‐R neuroendocrine pathway that may be activated within the context of a skin stress response system.

Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways.

Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Rα). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

Tissue-specific expression of estrogen receptors and their role in the regulation of neutrophil infiltration in various organs following trauma-hemorrhage

Although 17β‐estradiol (E2) administration after trauma‐hemorrhage (T‐H) reduces tissue neutrophil sequestration in male rodents, it remains unknown which of the estrogen receptor (ER) subtypes mediates this effect and whether the same ER subtype is involved in all the tissues. We hypothesized that the salutary effects of E2 on attenuation of neutrophil accumulation following T‐H are tissue and receptor subtype‐specific. Male Sprague‐Dawley rats underwent sham operation or T‐H (mean blood pressure, 40 mmHg for 90 min and then resuscitation). E2 (50 μg/kg), ER‐α agonist propyl pyrazole triol (PPT; 5 μg/kg), ER‐β agonist diarylpropiolnitrile (DPN; 5 μg/kg), or vehicle (10% dimethyl sulfoxide) was administered subcutaneously during resuscitation. Twenty‐four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine‐induced neutrophil chemoattractant (CINC)‐1, CINC‐3, and intercellular adhesion molecule (ICAM)‐1 levels in the liver, intestine, and lung were measured (n=6 rats/group). ER‐α and ER‐β mRNA levels in sham‐operated rats were also determined. T‐H increased MPO activity, CINC‐1, CINC‐3, and ICAM‐1 levels in the liver, intestine, and lung. These parameters were improved significantly in rats receiving E2 after T‐H. Administration of the ER‐α agonist PPT but not the ER‐β agonist DPN improved the measured parameters in the liver. In contrast, DPN but not PPT significantly improved these parameters in the lung. In the intestine, ER subtype specificity was not observed. ER‐α mRNA expression was highest in the liver, whereas ER‐β mRNA expression was greatest in the lung. Thus, the salutary effects of E2 administration on tissue neutrophil sequestration following T‐H are receptor subtype and tissue‐specific.

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll‐like receptors (TLR). Although mitogen‐activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP ∼35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF‐α, interleukin‐6 (IL‐6), monocyte chemoattractant protein‐1 (MCP‐1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF‐α, MCP‐1, and KC production by Kupffer cells were significantly increased following trauma‐hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG‐stimulated TLR9 signaling increased TNF‐α and IL‐6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, ‐4, and ‐9 signaling. In contrast, TNF‐α and IL‐6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma‐hemorrhage. J. Cell. Physiol. 210: 667–675, 2007.

Gender and susceptibility to sepsis following trauma.

An analysis of current literature on sexual dimorphism in response to trauma-hemorrhage revealed conflicting reports on the role of gender in outcomes of trauma patients. In contrast, results obtained from experimental studies clearly support the suggestion that gender plays a significant role in post injury pathogenesis. As discussed in this review, experimental studies suggest that the suppression of immune and cardiac function is severe in males and ovariectomized females; however, both immune and cardiac functions are maintained in proestrus females. Furthermore, findings from a number of studies have shown that the depletion of male sex hormones by castration or by blocking the interaction between male sex steroids and their receptors in males prevented the suppression of both immune and cardiac functions following trauma-hemorrhage. Moreover, administration of estrogen in males and ovariectomized females also prevented the suppression of immune and cardiac functions following trauma-hemorrhage. Thus, these experimental findings collectively suggest that female sex hormones (i.e., estrogen) produce salutary effects following trauma-hemorrhage whereas male sex steroids (i.e. 5alpha-dihydrotestosterone, 5alpha-DHT) are suppressive to immune and cardiac functions under those conditions. Such dramatic differences in the outcome of trauma-hemorrhage in proestrus females and males clearly suggest that the prevailing sex hormonal levels at the time of injury play a critical role in shaping the host response to trauma-hemorrhage. While a definitive cause for the conflicting data obtained in the clinical setting remains to be established, the discrepancy could be due to the differences in the hormonal levels at the time of injury. Since there is no information on hormonal status in the clinical studies, it is difficult to ascertain the role of sex hormones in post trauma pathogenesis. Therefore, in order to establish the role of gender in the outcome of trauma patients, more planned studies are needed in which the levels of sex hormones should be measured at the time of hospital admission. Furthermore, more studies, both in the clinical and experimental settings, should be performed to determine the mechanism by which the sex hormones improve immune and organ functions following trauma-hemorrhage. The findings obtained from these studies will help in designing innovative therapeutic approaches for the treatment of trauma patients.

Acute ethanol exposure inhibits macrophage IL-6 production: role of p38 and ERK1/2 MAPK.

Acute ethanol consumption has been linked to an increase in infectious complications in trauma and burn patients. Ethanol modifies production of a variety of macrophage‐derived immunoregulatory mediators. Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in macrophages, activates several intracellular signaling pathways, including mitogen‐activated protein kinases (MAPK). In the current study, we investigated the effect of acute ethanol exposure on in vivo activation of p38 and extracellularly regulated kinases 1 and 2 (ERK1/2) MAPK in murine macrophages and the corresponding, LPS‐stimulated interleukin (IL)‐6 production. We demonstrated that a single dose of ethanol transiently down‐regulated p38 and ERK1/2 activation levels (3–24 h after treatment) and impaired IL‐6 synthesis. Ethanol‐related reduction in IL‐6 production was not further affected by the presence of inhibitors of p38 and ERK1/2 (SB 202190 and PD 98059, respectively). These results demonstrate that acute ethanol exposure can impair macrophage IL‐6 production and indicate that this effect may result from ethanol‐induced alterations in intracellular signaling through p38 and ERK1/2.