W. Al-Ghoul

Effects of burn with and without Escherichia coli infection in rats on intestinal vs. splenic T-cell responses.

Objective To evaluate the effect of burn injury with and without an Escherichia coli septic complication on T-cell proliferation, interleukin-2 production, and Ca2+ signaling responses in intestinal Peyer’s patch and splenic T cells. Design Prospective, randomized, sham-controlled animal study. Setting University medical center research laboratory. Subjects Adult male Sprague-Dawley rats. Interventions Rats were subjected to a 30% total body surface area, full skin thickness burn. Infection in rats was induced via intraperitoneal inoculation of E. coli, 109 colony forming units/kg, with or without a prior burn. Measurements and Main Results Rat Peyer’s patch and splenic T lymphocytes were isolated by using a nylon wool cell purification protocol. T-cell proliferation, interleukin-2 production, and Ca2+ signaling responses were measured after stimulation of cells with the mitogen, concanavalin A. T-cell proliferation was determined by measuring incorporation of 3H-thymidine into T-cell cultures. Interleukin-2 production by T-cell cultures was measured by using enzyme-linked immunosorbent assay. Intracellular T-cell Ca2+ concentration, [Ca2+]i, was measured by the use of Ca2+-specific fluorescent label, fura-2, and its fluorometric quantification. [Ca2+]i was also evaluated by the use of digital video imaging of fura-2 loaded individual T cells. T-cell proliferation and interleukin-2 production were suppressed substantially in both Peyer’s patch and splenic T cells 3 days after either the initial burn alone or burn followed by the E. coli inoculation at 24 hrs after the initial burn. There seemed to be no demonstrable additive effects of E. coli infection on the effects produced by burn injury alone. The T-cell proliferation and interleukin-2 production suppressions with burn or burn-plus-infection insults were correlated with attenuated Ca2+ signaling. E. coli infection alone suppressed T-cell proliferation in Peyer’s patch but not in splenic T cells at 2 days postbacterial inoculation;E. coli infection had no effect on Peyer’s patch or splenic T cells at 1 day postinjury. On the other hand, burn injury alone caused a substantial T-cell proliferative suppression at 2 days postburn in both Peyer’s patch and splenic cells and a significant suppression in T-cell proliferation on day 1 postburn in Peyer’s patch but not in the spleen. Conclusion An initial burn injury suppressed T-cell proliferation at a level that it would not be further affected by a subsequent infection even if the infection by itself has the potential of suppressing T-cell proliferation. An earlier onset of T-cell suppression in Peyer’s patch cells than in the spleen with burn could be attributable to an initial hypoperfusion-related intestinal mucosal tissue injury. Overall, our study supports the concept that burn injury per se can significantly suppress T-cell mediated immunity and that the intestine is an early tissue site of such suppression.

Enteral nutritional supplementation prevents mesenteric lymph node T-cell suppression in burn injury

OBJECTIVE To determine the effects of an immune-enhancing diet supplemented with glutamine, arginine, fish oil, and dietary nucleotides on mesenteric lymph node T-cell functional disturbances encountered after burn injury in rats. DESIGN A prospective animal study. SETTING University medical center research laboratory. SUBJECTS Adult male Sprague-Dawley rats. INTERVENTIONS Rats received a 30%, total body surface, full-thickness burn. Burn-injury rats received the IMPACT diet supplemented with glutamine, arginine, fish oil, and nucleotides or arginine, fish oil, and nucleotides, or an isocaloric/isonitrogenous diet without supplementation with glutamine, arginine, fish oil, or nucleotides. MEASUREMENTS AND MAIN RESULTS Two days after injury, we found a significant decrease in the proliferation and interleukin-2 production by mesenteric lymph node T cells derived from rats fed on conventional chow compared with sham rats. The burn-related suppression of mesenteric lymph node T-cell proliferation and interleukin-2 production was prevented when the rats were fed on a high-protein diet rich in glutamine, arginine, fish oil, and nucleotides. We found that the immunostimulatory effects of the enriched diet are dependent on the presence of glutamine, arginine, fish oil, and nucleotides as feeding of rats on the isocaloric/isonitrogenous diet deficient in glutamine, arginine, fish oil, and nucleotides did not prevent the burn-related suppression of mesenteric lymph node T-cell dysfunction. Finally, our studies suggested that immunostimulatory effects of the diet are mediated by prostaglandin E(2) regulation of T-cell activation signaling molecule P59fyn. CONCLUSION These results suggest that a diet rich in arginine, fish oil, and nucleotides, with and without glutamine, can effectively prevent T-cell dysfunction encountered after burn injury.

Effects of pentoxyfylline on mesenteric lymph node T-cells in a rat model of thermal injury.

Cutaneous burn injury-induced T lymphocyte suppression is a well-known phenomenon. In this study, we evaluated the effect of treatment of burn rats with pentoxifylline (PTX) on the burn-induced suppression of T lymphocytes. Anesthetized rats were subjected to 30% total body surface area burn by exposing skin to 95°C water for 10 s. T lymphocytes were isolated from sham and burn rats with or without PTX treatment (120 mg/kg, ip). T cell proliferation and interleukin (IL)-2 production in response to T cell mitogen concanavalin A was measured using 3 H-thymidine uptake and enzyme-linked immunosorbent assay, respectively. P59 fyn autophosphorylation and its kinase activity was determined using in vitro kinase assay. In addition, T lymphocyte Ca2+ signaling was assessed using Ca2+ imaging technique. Two days after injury, there was a significant decrease in mesenteric lymph node T cell proliferation and IL-2 production in burn injured rats compared with those obtained from sham-injured rats. This decrease in T cell proliferation and IL-2 production in burn-injured rats was accompanied by a significant suppression in both P59 autophophorylation and kinase activity as well as Ca2+ signaling. Treatment of burn-injured rats with PTX produced a near complete recovery of T cell proliferation and IL-2 production. Furthermore, PTX treatment also prevented the burn-mediated suppression in P59fyn and kinase activity as well as restored Ca2+ signaling similar to those observed in sham injured rats. These findings altogether suggested that PTX treatment attenuate T cell suppression in burn-injured rats and that the effects of PTX are mediated via modulating P59 fyn and Ca2+ signaling.

Age-related compaction of lens fibers affects the structure and optical properties of rabbit lenses

BackgroundThe goal of this investigation was to correlate particular age-related structural changes (compaction) to the amount of scatter in rabbit lenses and to determine if significant fiber compaction occurred in the nuclear and inner cortical regions.MethodsNew Zealand White rabbits at 16–20 months old (adult; n = 10) and at 3.5–4 years old (aged; n = 10) were utilized for this study. Immediately after euthanising, scatter was assessed in fresh lenses by low power helium-neon laser scan analysis. Scatter data was analyzed both for whole lenses and regionally, to facilitate correlation with morphometric data. After functional analysis, lenses were fixed and processed for scanning electron microcopy (SEM; right eyes) and light microscopy (LM; left eyes). Morphometric analysis of SEM images was utilized to evaluate compaction of nuclear fibers. Similarly, measurements from LM images were used to assess compaction of inner cortical fibers.ResultsScatter was significantly greater in aged lenses as compared to adult lenses in all regions analyzed, however the difference in the mean was slightly more pronounced in the inner cortical region. The anterior and posterior elliptical angles at 1 mm (inner fetal nucleus) were significantly decreased in aged vs. adult lenses (anterior, p = 0.040; posterior, p = 0.036). However, the average elliptical angles at 2.5 mm (outer fetal nucleus) were not significantly different in adult and aged lenses since all lenses examined had comparable angles to inner fetal fibers of aged lenses, i.e. they were all compacted. In cortical fibers, measures of average cross-sectional fiber area were significantly different at diameters of both 6 and 7 mm as a function of age (p = 0.011 and p = 0.005, respectively). Accordingly, the estimated fiber volume was significantly decreased in aged as compared to adult lenses at both 6 mm diameter (p = 0.016) and 7 mm diameter (p = 0.010).ConclusionMorphometric data indicates that inner cortical fibers undergo a greater degree of age-related compaction than nuclear fibers. Increased scatter appears to be only tentatively correlated with regions of fiber compaction, suggesting that it is simply one of an array of factors that contribute to the overall decreased transparency in aged rabbit lenses.

Enterococcus faecalis exacerbates burn injury-induced host responses in rats.

Pathophysiology of burn injury with complications of gram-positive infections is not well characterized. We have developed an in vivo rat model to study the effects of burn injury along with intra-abdominal inoculation of Enterococcus faecalis. We hypothesized that although burn injury or E. faecalis inoculation by itself may not induce significant pathophysiological responses, the combination of the two can lead to adverse pathophysiological consequences. Sprague-Dawley rats were divided into 4 groups: group 1(C), controls; group 2(B), burn injury on 30% total body surface area; group 3(EF), intra-abdominal implantation of bacterial pellet impregnated with E. faecalis; group 4(B+EF), burn injury plus bacterial pellet implantation. The mortality was 25% and 60% on day 1 and 2 in Group 4(B+EF), respectively; no significant mortality was observed in other groups. In group 4(B+EF), metabolic acidosis, respiratory alkalosis, and a hyperdynamic state developed on day 1, and metabolic and respiratory acidosis and a hypodynamic state on day 2. There were no significant alterations in metabolic or hemodynamic measurements in other groups. Intestinal microvascular permeability to albumin on day 1 and 2 was increased in group 4(B+EF). In group 2(B), microvascular permeability was not increased significantly. Although the permeability was increased on day 1 in group 3(EF), it declined on day 2. The metabolic and hemodynamic alterations were correlated with increased intestinal microvascular permeability to albumin. E. faecalis appeared to be involved in initiating a vicious cycle of burn injury-mediated disruption of intestinal integrity along with metabolic and hemodynamic derangements.

Abstract 2662: Statins modulate MMP-9 and beta catenin in epithelial cells

The effect of statins (Simvastatins) mediated mechanisms that affect inflammatory or non-inflammatory pathways causing formation and progression of cancer need to be investigated. Currently, sufficient data are lacking to support the use of statins for the prevention of cancer and further research is clearly warranted. We studied these antineoplastic mechanisms in rodent inflammatory model, which might be instrumental in drug discovery research for the development of future cancer therapeutics. MMP9 is a member of the family of zinc-containing endopeptidases, which degrade various components of the extracellular matrix, thereby regulating matrix remodeling. Since matrix remodeling plays an important role during growth and progression of cancer and considering the fact that, tumor cells switch to aerobic glycolysis as its major energy source, this study was designed to analyze the effect of Statins on MMP9 and epithelial cell integrity at gastrointestinal gut barrier. We used an established model of thermal injury (TI) to study the effects of simvastatins. We found that the percentage of stained MMP-9 in the control tissue and TI+SMV tissue was lower than the percentage in thermal injury tissue (P Note: This abstract was not presented at the meeting. Citation Format: Nadeem Fazal, Walid Al-Ghoul. Statins modulate MMP-9 and beta catenin in epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2662. doi:10.1158/1538-7445.AM2017-2662