Elimination of pesticides in olive oil by refining using bleaching and deodorization.

The effects of the processing steps on some pesticides during physical refining of olive oil were determined. Oil samples were spiked with four types of pesticides, i.e. endosulfan (alpha-endosulfan, beta-endosulfan and endosulfan sulphate), simazine, oxifluorfen and diflufenican, and subjected to physical refining (bleaching and deodorization). The removal of contaminants by bleaching earths was compared using five types of commercial earths. Deodorization assays under laboratory (10 kg) and discontinuous pilot plant (200 kg) conditions at different temperatures and treatment times were carried out. Bleaching seemed effective only for the elimination of simazine. For removal of the other pesticides tested, a physical refining treatment was required at 240 degrees C in the deodorizing stage for different periods (1 h for diflufenican, oxifluorfen and alpha-endosulfan, 2 h for beta-endosulfan and 3 h for endosulfan sulphate).

Phenolic compounds in olive oils intended for refining: formation of 4-ethylphenol during olive paste storage.

The phenolic composition of "lampante olive oil", "crude olive pomace oil", and "second centrifugation olive oil" was characterized by high-performance liquid chromatography with UV, fluorescence, and mass spectrometry detection. The phenolic profile of these olive oils intended for refining was rather similar to that previously reported for virgin olive oil. However, a new compound was found in these oils, which is mainly responsible of their foul odor. It was identified as 4-ethylphenol by comparison of its UV and mass spectra with those of a commercial standard. Although 4-ethylphenol was discovered in all oils intended for refining, its presence was particularly significant in "second centrifugation olive oils", its concentration increasing with time of olive paste storage. Similar trends were observed for hydroxytyrosol, hydroxytyrosol acetate, tyrosol, and catechol, the concentration of these substances reaching values of up to 600 mg/kg of oil, which makes their recovery for food, cosmetic, or pharmaceutical purposes attractive.

Changes in cellular motility and cytoskeletal actin in fibroblasts from patients with chronic venous insufficiency and in neonatal fibroblasts in the presence of chronic wound fluid.

PURPOSE: Fibroblasts (fb) play an important role in wound healing involving motility, contraction, fibrosis, and expression of the cytoskeletal protein alpha-smooth muscle actin (alpha-sma). Patients with chronic venous insufficiency (CVI) are known to have dermal changes and impaired venous ulcer healing. To investigate whether these dermal-fb have an altered ability to migrate and whether chronic wound fluid from venous ulcers alters neonatal fb motility, we examined cell migration and alpha-sma. METHODS: Fibroblasts were cultured from the margin of venous ulcers (du-fb, n = 4, CEAP 6), from patients with venous reflux without ulcer (dr-fb, n = 5, CEAP 2), and from the ipsilateral thigh of the same patients with (pu-fb) and without (pr-fb) ulcer, respectively. The abbreviations used are p and d, which represent proximal and distal, respectively; u and r represent ulcer and reflux, respectively. Neonatal foreskin fibroblasts (nf-fb) were exposed to chronic venous ulcer wound fluid (CVUWF, 300 microg protein/mL, n = 3) or bovine serum albumin (BSA, control). Fibroblast motility was determined by means of time-lapse photo-images, and the rate (micrometer per hour) was calculated. Immunohistochemistry for alpha-sma was analyzed with confocal laser microscopy. RESULTS: The rate of motility (micrometer per hour +/- SEM) was decreased for both du-fb (11.4 +/- 0.7) and dr-fb (13.8 +/- 0.6), when compared with pu-fb (21.9 +/- 0.9) and pr-fb (24.7 +/- 1.1), respectively. The motility rate for nf-fb was lower in CVUWF (24.7 +/- 2.0) than in BSA (37.1 +/- 6.7). An elevated level of microfilament bundles of alpha-sma for both du-fb and dr-fb, compared with those of pu-fb and pr-fb, and also in nf-fb treated with CVUWF was demonstrated by means of immunohistochemistry. CONCLUSION: These data demonstrate a reduced motility in the dermal fb of patients with CVI. Patients with reflux disease without ulcer are predisposed to these changes. Furthermore, it appears that CVUWF causes changes in motility and alpha-sma expression in nf-fb as demonstrated in du-fb. These findings suggest that reduced motility and CVUWF, representing the microenvironment of venous ulcers, play a significant role in impaired wound healing.

Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates.

We studied microorganisms associated with infant diarrhea in a group of 256 children admitted to a public pediatric hospital in Montevideo, Uruguay. Diagnostic procedures were updated to optimize detection of potential pathogens, which were found in 63.8% of cases, and to be able to define their characteristics down to molecular or antigenic type. Coinfection with two or more agents was detected in more than one-third of positive studies. Escherichia coli enteric virotypes, especially enteropathogenic E. coli (EPEC), were shown to be prevalent. Rotavirus, Cryptosporidium, Campylobacter (mainly Campylobacter jejuni), and Shigella flexneri were also often identified. Enterotoxigenic E. coli, Salmonella, and Giardia lamblia were sporadically recognized. Unusual findings included two enteroinvasive E. coli strains, one Shigella dysenteriae 2 isolate, and a non-O:1 Vibrio cholerae culture. EPEC bacteria and S. flexneri (but not Salmonella) showed unusually frequent antimicrobial resistance, especially towards beta-lactam antibiotics, which is the subject of ongoing work.

Storage time and deodorization temperature influence the formation of aniline-derived compounds in denatured rapeseed oils.

In 1981 an epidemic, named Toxic Oil Syndrome, occurred in Spain as a result of ingestion of rapeseed oil denatured with 2% aniline, which had been imported for industrial use but was fraudulently diverted and processed for human consumption. Two groups of chemical compounds have been identified in the ingested toxic oil: fatty acid anilides and amino-propanediol derivatives. The objective of this work was to assess the effect of several refining process variables on the formation of 3-(N-phenylamino)-1,2-propanediol (PAP) esters. The amount of PAP esters in aniline-denatured oil increased dramatically when oil was heated from 250 degrees C to 300 degrees C. However, the ones formed when 300 degrees C was reached were lost during processing at that temperature. The level maintained during the operation time at 300 degrees C was higher in denatured samples stored for 3 weeks before refining than in denatured samples stored only for 1 week. Anilides were also analyzed. We found that anilides decreased very little with distillation time. In this paper we discuss the influence of storage time prior to refining and of elevated refining temperature, such as temperatures that might occur in close proximity to a deodorizer coil.

Pathology of "toxic oils" and selected metals in the MRL/lpr mouse.

The Toxic Oil Syndrome epidemic that occurred in Spain in 1981 and affected nearly 20,000 people was caused by ingestion of oil mixtures that contained analine-denatured rapeseed oil. To date, an animal model in which to identify the actual etiologic agents(s) and to investigate the pathogenesis of the disease has not been discovered. In this study, the MRL/lpr was used to assess the histopathological response of 3 "toxic oils" and 3 metals. The oils tested were a denatured rapeseed oil collected from a family who were affected by the Toxic Oil Syndrome epidemic in Spain (CO756) plus two synthesized oils (RSD and RSA). Female mice, 7 weeks of age, received an undiluted (neat) or a 1:10 diluted dose of each oil; mercury (50 ppm), cadmium (100 ppm), or lead (50 ppm). Half of each group was killed after 5 weeks of exposure and the remaining mice after 10 weeks of exposure. Body and organ weights (liver, kidney, thymus, and spleen) were recorded and selected organs were collected for histopathology. Ten weeks after treatment, body weights (BW) of the cadmium and lead groups were significantly suppressed, and the body weight of the C0756-neat group was significantly increased compared to their respective controls. Kidney/BW were decreased in the RSA-neat and RSA 1:10 groups after 10 weeks of exposure, and the kidney/BW in the mercury and cadmium groups were increased. Spontaneous development (12 weeks of age) and progression (17 weeks of age) of histopathological lesions are described for selected organs examined in the naïve mice as are changes that resulted from exposure to the "toxic oils" and metals. C0756-neat, mercury, and lead suppressed progression of the glomerulonephritis that normally occurs in the MRL/lpr mouse. Also of interest were lesions that included mononuclear cuffing of hepatic bile ducts, progression of the granulomas that formed in the renal glomeruli, vessels in the lymphoid organs that contained tightly packed lymphocytes, and the presence of plasma cells in the thymus. All 3 oils stimulated early development of the lymphoproliferative syndrome characteristic of the MRL/lpr mouse as demonstrated by an increase in the thymus/BW and spleen/BW ratios after 5 weeks of treatment. These data contribute to our knowledge of spontaneous disease progression in the thymus, spleen, lymph nodes, and kidneys in the MRL/lpr mouse and the effects of 3 different "toxic oils" and metals on the development and progression of those lesions.

Initial evaluation of carotid angioplasty and stenting with three different cerebral protection devices.

OBJECTIVE: The purpose of the study was to assess the effectiveness of cerebral protection devices during carotid artery angioplasty and stent placement. METHODS: Between September 1998 and September 1999, carotid angioplasty and stenting were performed in 46 patients with symptomatic (39.1%) or asymptomatic (60.9%) severe carotid artery stenosis. Wallstents were used in all patients with selective predilatation. Cerebral protection devices were used in 25 of these patients. Primary end points were perioperative neurologic complications and mortality. Data were collected prospectively. RESULTS: The overall combined end point of all neurologic deficits and death rate was 4.34%. Two neurologic events (one transient ischemic attack and one minor stroke) occurred in the unprotected group (9.53%) versus none in the group with cerebral protection. This difference is not statistically significant. The mortality rate was 0% for both groups. On an intention to treat basis, the overall technical success rate for carotid angioplasty was 97.8%, and for placement of cerebral protection devices it was 100%. An important number of particles of different sizes were captured in all cases in which cerebral protection devices were used. CONCLUSION: Experience has shown that cerebral protection during carotid angioplasty and stenting is technically feasible and appears to be effective in preventing procedure-related neurologic complications. Further investigation is warranted.

The proliferative capacity of neonatal skin fibroblasts is reduced after exposure to venous ulcer wound fluid: A potential mechanism for senescence in venous ulcers.

PURPOSE: We have previously shown that fibroblasts cultured from venous ulcers display characteristics of senescence and have reduced growth rates. Susceptibility of young fibroblasts to the microcirculatory changes associated with venous ulcers, such as macrophage trapping and activation, could explain the prevalence of senescent fibroblasts in these wounds. METHODS: We tested the in vitro effect of venous ulcer wound fluid (VUWF), as well as pro-inflammatory cytokines known to be present in VUWF (TNF-alpha, IL-1beta, and TGF-beta1), on neonatal foreskin fibroblasts (NFFs). NFF growth rates, cellular morphology, and senescence-associated beta-galactosidase (SA-beta-Gal) activity were determined in the presence or absence of VUWF and the above cytokines. VUWF TNF-alpha concentration and the effect of anti-TNF-alpha antibody on VUWF inhibitory activity were determined in samples obtained from four patients with venous ulcers. RESULTS: NFF growth rates were significantly reduced by VUWF (42,727 +/- 6301 vs 3902 +/- 2191 P =. 006). TNF-alpha also significantly reduced NFF growth rates in a dose-dependent manner (P =.01). No significant growth-inhibitory activity was seen for IL-1alpha or TGF-beta. Incubation with VUWF significantly increased the percentage of SA-beta-Gal-positive fibroblasts in vitro on culture day 12 (P =.02). TNF-alpha and TGF-beta1 had similar effects. TNF-alpha was detected in all VUWF tested, with a mean of 254 +/- 19 pg/mL. CONCLUSION: These data suggest that the venous ulcer microenvironment adversely affects young, rapidly proliferating fibroblasts such as NFFs and induces fibroblast senescence. Pro-inflammatory cytokines such as TNF-alpha and TGF-beta1 might be involved in this process. The role of other unknown inhibitory mediators, as well as pro-inflammatory cytokines, in venous ulcer development and impaired healing must be considered.

The effect of passage number on fibroblast cellular senescence in patients with chronic venous insufficiency with and without ulcer.

BACKGROUND: Fibroblasts (fb) cultured from venous ulcer patients and patients with venous reflux disease without ulcer demonstrate characteristics of cellular senescence, such as increased fibronectin level and senescence-associated beta-galactosidase (SA beta-gal) positive cells. Cellular senescence is an in vitro event characterized by the progressive loss of proliferative capacity with increased passage number, and has been associated with impaired healing in vivo. This report examines progressive stages of cellular senescence in fb from the distal area (du-fb) and proximal fb (pu-fb) of patients with venous ulcer, as well as in distal fb (dr-fb) and proximal fb (pr-fb) from patients with venous reflux without ulcer, by comparing the population doubling time (T) and percent SA beta-gal expression. RESULTS: The mean value of T over 6 passages for fb in the ulcer group was 132.5 +/- 29.0 hours for pu-fb and 492.9 +/- 146.2 hours for du-fb (P = 0.0009). For fb in the reflux group the mean value of T over 5 passages was 79.3 +/- 12.8 hours for pr-fb and 94.2 +/- 16.8 hours for dr-fb (P = 0.8). Comparing ulcer and reflux fb, no difference in T was observed between pu-fb and pr-fb (P = 0.6), but a difference was noted between du-fb and dr-fb (P = 0.0004). The mean percent SA beta-gal activity for fb in the ulcer group was 11.2% +/- 3.1% for pu-fb and 63.8% +/- 8.9% for du-fb (P = 0.0001). Individual passages demonstrated significant difference (P <0.05) in SA beta-gal activity between pu-fb and du-fb at early and late passages. No difference was noted in SA beta-gal activity for fb in the reflux group or between pu-fb and pr-fb, but comparison between du-fb and dr-fb was significant (63.8% +/- 8.9% versus 7.8% +/- 2.9%; P = 0.0001). CONCLUSIONS: The in vitro passage of du-fb and pu-fb in chronic venous ulcer patients has an effect on T and cellular senescence as measured by SA beta-gal activity. Our data further suggest that du-fb are at a more progressive stage of cellular senescence when compared with pu-fb, and more importantly with fb cultured from patients with venous reflux without ulcer. These findings are consistent with impaired wound healing of venous stasis ulcer. The accumulation of senescent fb and a more advanced stage of cellular senescence of du-fb may explain why repeated episodes of venous ulceration are resistant to conservative treatment and require more aggressive measures of therapy.

Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence.

PURPOSE: Venous reflux precedes the development of venous ulcers. Our earlier work showed that the fibroblasts that are cultured from these wounds display more characteristics of senescence. We evaluated fibroblast senescence in patients with venous reflux but without ulcers to further investigate the role of venous reflux in the predisposition to venous ulcers. METHODS: Fibroblasts that were isolated from skin biopsy specimens of the "gaiter" area (distal) and of the ipsilateral thigh of the same patient (proximal) were compared. Twelve patients with venous reflux (9 patients in clinical, etiologic, anatomic, and pathologic classification 4; 3 patients in classification 5) with an average venous filling index of 5.45 mL/s and 4 patients without venous reflux were enrolled in the study. The growth rates, the response to basic fibroblast growth factor (b-FGF), and the senescence markers (beta-galactosidase activity at a pH level of 6, unstimulated fibroblasts fibronectin protein, and messenger RNA levels) were determined for each cell population. RESULTS: The number of senescence-associated beta-galactosidase positive cells (8.3% +/- 1.9% vs 2.2% +/- 0.8%; P =.008) and the level of cellular fibronectin protein (455.7 +/- 80 vs 210 +/- 51; P =.04) and messenger RNA (16.8 +/- 6.8 vs 13.5 +/- 5.7; P =.042) were significantly higher in the distal fibroblasts as compared with the proximal fibroblast cultures. The growth rates of the distal fibroblasts were lower when compared with the proximal fibroblasts (15,746 +/- 4287 cells/day vs 29,550 +/- 5035 cells/day; P <.002) but were not different in the presence of b-FGF (41,717 +/- 9542 cells/day vs 47,030 +/- 6133 cells/day; P =.53). In the patients without venous reflux, no site differences were noted in the growth rates or the senescence markers between the proximal and distal fibroblasts. CONCLUSION: Distal fibroblasts that are isolated from patients with venous reflux display more senescence characteristics than do proximal fibroblasts and have significantly lower growth rates. Despite senescence, b-FGF restored the distal-fibroblasts growth rate to that of the stimulated proximal fibroblasts, which proposes a therapeutic role for b-FGF. These changes precede ulcer formation and suggest a mechanism that is focal and intrinsically related to venous reflux.

Carotid endarterectomy in a patient with anterior ischemic neuropathy.

A case of anterior ischemic optic neuropathy (AION) associated with 90% stenosis of the ipsilateral internal carotid artery (ICA) and diminished blood flow through the ophthalmic artery was studied. After carotid endarterectomy (CEA), the patient showed subjective and measurable improvement in vision and increased ophthalmic artery flows. This observation supports the role for screening carotid duplex scanning and transcranial Doppler in patients with AION. Early CEA in patients with associated ipsilateral ophthalmic artery hypoperfusion may improve visual outcome.

Fibroblasts cultured from venous ulcers display cellular characteristics of senescence.

PURPOSE: A well-recognized characteristic of venous ulcers is impaired healing. Fibroblasts cultured from venous ulcers (wound-fb) have been shown to have reduced growth rates and are larger than normal fibroblasts (normal-fb) from the ipsilateral limb. Reduced growth capacity and morphologic changes are 2 well-known traits of cellular senescence. Other molecular changes are overexpression of matrix proteins, such as cellular fibronectin (cFN), and enhanced activity of beta-galactosidase at pH of 6.0 (senescence associated beta-Gal, or SA-beta-Gal). Senescence, an irreversible arrest of cell proliferation with maintenance of metabolic functions, may represent in vivo aging and thus may be related to impaired healing. METHODS: Cultured normal-fb and wound-fb from 7 venous ulcer patients (average age, 51 years) were obtained by taking punch biopsies of the perimeter of the ulcer and from the ipsilateral thigh of the same patient. Growth rates, SA-beta-Gal activity, and level of cFN protein (immunoblot) and message (Northern blot) were measured. RESULTS: In all patients, wound-fb growth rates were significantly lower than those of normal-fb (P =.006). A higher percentage of SA-beta-Gal positive cells were found in all wound-fb (average, 6.3% vs. 0.21%; P =.016). The level of cFN, was consistently higher in all wound-fb tested. Also, in 4 patients, the level of cFN messenger RNA (mRNA) was increased. CONCLUSION: Fibroblasts cultured from venous ulcers exhibited characteristics associated with senescent cells. Accumulation of senescent cell in ulcer environment may be associated with impaired healing.

An association between periodontal disease and peripheral vascular disease.

BACKGROUND: Periodontal disease has been shown to be associated with increased risk of coronary heart disease. Because coronary heart disease and peripheral vascular disease (PVD) have similar pathophysiologies, we hypothesized that periodontal disease might be a risk factor for PVD. METHODS: Using the combined data from the Normative Aging Study and Dental Longitudinal Study of the US Department of Veterans Affairs, we examined the relationship between PVD and periodontal disease. Multivariate logistic regression analysis was used. RESULTS: Over the 25 to 30 years of follow-up, 80 of these initially healthy subjects developed PVD. Compared with controls (n = 1,030), subjects with clinically significant periodontal disease at baseline had a 2.27 increment in the risk of developing PVD (95% confidence interval 1.32 to 3.9, P value = 0.003). CONCLUSIONS: Periodontal disease emerged as a significant independent risk factor for PVD in a multivariate analysis that adjusted for other established risk factors.

[Acoustic masking and Feldmann curves in tinnitus. Clinical study]. / Enmascaradores acústicos y curvas de Feldman en acúfenos. Estudio clínico.

Eight-seven patients with severe tinnitus were studied. All patients underwent a complete audiological examination and tinnitus evaluation (pitch-matching, loudness, post-masking effect, masking curves, etc.). Most of the patients with a positive residual inhibition test had Feldman type I and III masking curves.

[Functional nasal tests for the evaluation of the mucociliary transport]. / Test funcional nasal para la determinación del transporte mucociliar.

A study was made of a noninvasive functional test of mucociliary clearance using micronized indigo blue colorant, which is insoluble in airway secretions. Transport time to the nasopharynx was measured optically with an endoscope. This method was more objective and less irritating than others tested by other authors. Measurements were made in each subject using different endoscopes. The results were highly suggestive of a difference in nasal mucociliary clearance time between healthy and unhealthy subjects. This may mean that chronic inflammation, atrophic rhitis, dyskinesia, and other diseases lead to malfunction of the nasal mucociliary system.

Multiple system organ failure may be influenced by macrophage hypoactivation as well as hyperactivation--importance of the double challenge.

OBJECTIVE: To find out if an infective challenge caused by a burn followed by caecal ligation and puncture in mice caused more abnormalities of the immune response than burn alone or caecal ligation and puncture alone. DESIGN: Laboratory study. SETTING: University hospital, USA. MATERIAL: 80 male 7-8 week old A/J mice. INTERVENTIONS: Burn followed 10 days later by caecal ligation and puncture (n = 18), caecal ligation and puncture alone (n = 24), burn alone (n = 20), and controls (n = 18). The mice had their spleens removed on day 11 (n = 28; 6, 8, 8, and 6 in the respective groups), day 12 (n = 26; 6, 8, 6, and 6), and day 13 (n = 26; 6, 8, 6, and 6), and splenocytes and adherent cells were harvested for measurement of prostaglandin E2 (PGE2), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF-alpha). MAIN OUTCOME MEASURES: Alterations in the production of the cytokines. RESULTS: After the double challenge (burn followed by caecal ligation and puncture) there were significant reductions in production of TNF-alpha and IL-6 compared with caecal ligation and puncture alone (p < 0.05), burn alone (p < 0.05), and controls (p < 0.05). These findings indicate that activation of macrophages was reduced after infection; production of TNF-alpha, IL-1, and IL-6 by splenocytes stimulated by lipopolysaccharide was reduced. CONCLUSIONS: The differences do not seem big enough to indicate that mortality would be increased after caecal ligation and puncture alone. Only when there has been a previous injury (which resulted in hyperactivation of macrophages followed by a more pronounced hypoactivation) would mortality increase. In view of clinical trials with antiendotoxin and antiTNF antibodies that failed to improve survival in infected patients, we suggest that the mechanisms of the cellular immune response need further clarification.

Long-term immunotherapeutic intervention with pentoxifylline in a mouse model of thermal injury and infection.

Major thermal or traumatic injury often results in abnormalities of immune function, and these abnormalities contribute to the increased susceptibility to infection observed in these patients. Abnormalities of T-cell function, including decreased proliferation and secretion of cytokines are observed following major injury and, conversely, there is markedly increased monokine production. Thus, therapy of this syndrome might logically be aimed at modulating the immune system to upregulate T-cell function and downregulate monocyte hyperactivation. Pentoxifylline (PTX), a methylxanthine derivative, has been shown to be therapeutically effective in several animal models. The purpose of this study was to evaluate PTX and its effect on cytokine production in a mouse model of thermal injury and to study its effect on survival after septic challenge. The results show that PTX therapy after injury can restore T-cell production of IL-2 and downregulate the hyperactive macrophage secretion of proinflammatory cytokines. However, improvement in survival resulting from this therapy following thermal injury and septic challenge depends on timing of dosage.

Interleukin-2 receptor expression and function following thermal injury.

BACKGROUND/OBJECTIVE: Serious traumatic or thermal injury is associated with depression of cellular immunity, including the failure of T-lymphocyte proliferation in response to stimulation that depends both on production of interleukin-2 (IL-2) and on expression of functional IL-2 receptors (IL-2R). While decreased IL-2 production following thermal injury is undisputed, the status of IL-2R expression and function in this setting is controversial; therefore, we sought to investigate this issue. DESIGN: A total of 220 male A/J mice (n = 22 per group) were subjected to a 20% scald burn injury or sham burn, killed 4, 7, 10, 14, or 21 days later, and splenocytes harvested. In vitro parameters of both IL-2R expression and function were measured. RESULTS: On day 7, splenic lymphocyte proliferation and IL-2 production in response to mitogenic stimulation were both suppressed following burn injury to 50% and 60% of controls, respectively. Northern blot analysis revealed normal IL-2R p55 messenger RNA expression in response to mitogenic stimulation on days 7, 10, and 14 in thermally injured animals. Phenotypic IL-2R p55 expression in concanavalin A-stimulated CD3+ cells was unchanged following burn injury. Binding of fluorescein-labeled IL-2 to cell membranes was increased in burned animals at days 10 and 14. The addition of IL-2 to cultures of spleen cells from burned mice consistently restored the mitogenic response to that of the controls. CONCLUSIONS: Thermal injury in this model does not result in either quantitative or functional suppression of IL-2R. Suppression of T-cell activation and proliferation, seen following thermal injury, appears primarily related to abnormal IL-2 production.

Altered gene transcription after burn injury results in depressed T-lymphocyte activation.

OBJECTIVE: Patients with major burns and an animal model of burn injury were studied to determine the mechanism of depressed interleukin-2 (IL-2) production after thermal injury and to determine the effect of such injury on IL-2 receptor (IL-2R) expression and function. SUMMARY BACKGROUND DATA: Major burn injury is known to diminish resistance to infection by altering cytokine production and prostanoic secretion and by inhibiting T-lymphocyte activation. T-cell activation requires production of regulatory cytokines, principally IL-2, and expression of the appropriate cytokine receptors. Depressed IL-2 production after major burn injury is undisputed, although the molecular mechanisms remain undefined; the effect of burn injury on IL-2R expression and function currently is controversial. METHODS: The authors studied serial samples of peripheral blood mononuclear cells (PBMC) from 11 patients with 25% to 95% surface area burns and 7 age-matched volunteer control subjects. Peripheral blood mononuclear cells were stimulated by the T-cell mitogen phytohemagglutinin (PHA), and IL-2 production and mRNA expression by Northern blot were determined. Expression and function of IL-2R were determined by monoclonal antibodies to the p55 and p75 chains of the IL-2R, binding of fluorescein-labeled IL-2, and response to exogenous recombinant IL-2. We also studied a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from mice with burns (20-22 per group) were studied for IL-2 production and IL-2 mRNA expression after stimulation with the T-cell mitogen concanavalin A (ConA) and compared with sham burn control subjects. Kinetics of mRNA expression after ConA stimulation also were determined and a nuclear run-on assay performed to determine IL-2 gene transcription. The mRNA expression was determined for the proto-oncogenes c-jun and c-fos, whose protein products join to form transcription factor AP1, which is necessary for activation of the IL-2 promoter. Splenocytes from mice with burns after ConA stimulation also were studied for expression and function of the IL-2R. RESULTS: Peripheral blood mononuclear cells from burn patients compared with healthy control subjects showed diminished (p < 0.05) IL-2 production and mRNA expression 4 to 10 days after burn injury. Burn PBMC demonstrated normal expression of IL-2R, p55, and p75 chains 0 to 7, 8 to 20, and 21 to 37 days after burn injury, normal IL-2R binding of fluorescein-labeled IL-2, and a normal proliferative response to PHA in the presence of exogenous recombinant IL-2. Splenocytes from mice 7 days after burn injury showed diminished production (p < 0.05) of IL-2 and IL-2 mRNA expression after ConA stimulation as compared with sham burn control subjects. Kinetics of mRNA expression after ConA stimulation were the same for burn and control mice, indicating that reduced IL-2 mRNA expression was not caused by altered mRNA degradation. A nuclear run-on assay confirmed decreased IL-2 gene transcription in burn splenocytes. Burn splenocytes showed normal expression of mRNA for c-jun but diminished expression of mRNA for c-fos. Finally, splenocytes from mice with burns after ConA stimulation showed normal expression and function of the IL-2R 7, 10, 14, and 21 days after burn injury. CONCLUSIONS: These human and animal studies indicate that major burn injury depresses T-cell activation at the level of IL-2 gene transcription at least in part by inhibiting c-fos expression, whereas IL-2R expression and function remain normal and T-cell proliferation can be restored to normal levels by exogenous IL-2.