We decided to investigate, in a group of patients from a geographical area with a low incidence of breast cancer, whether HER-2/neu positive tumours are correlated with established or suspected risk factors for breast cancer and thus to identify distinct subgroups of high risk women.Several researchers have claimed that classification of tumours on the basis of HER-2/neu protein, estrogen and progesterone receptor expression were performed using immunochemistry. Odds ratios and 95% confidence intervals were determined by chi-square test and logistic regression analysis. Case-case odds ratios were calculated in order to measure the risk heterogeneity between HER-2/neu+ and HER-2/neu-tumours. Separate analyses were performed for premenopausal and postmenopausal women and according to estrogen receptor status.This study analysed data from patients who attended the Breast Unit at the University Hospital of Heraklion, Crete, Greece between 1996 and 2002. 384 women with primary invasive breast cancer were compared with 566 screened women who were referred to the Unit and had not developed breast neoplasm by the time the data were analysed. Risk factor data were obtained from each subject by personal interviews using a structured questionnaire. The detection and scoring of the HER-2/neu stratification, an increased breast cancer risk was associated with only four of the factors examined: use of oral contraceptives (OR = 4.40, 95%C.I: 1.46–13.28), use of HRT (OR = 7.34, 95%C.I: 2.03–26.53), an age at first full pregnancy more than 23 years (OR = 1.91, 95%C.I: 1.29–2.83) and body mass index more than 29 kg/m2 (OR = 3.13, 95%C.I: 2.02–4.84). Additionally, a history of abortion or miscarriage (OR = 0.56, 95%C.I: 0.38–0.82) was correlated with a decreased risk of breast cancer. In the case to case comparison only BMI >29 kg/m2 revealed a relative connection that was stronger with positive than with negative HER-2/neu tumours (ratio of OR's = 2.23, 95%C.I: 1.20–4.15, p = 0.011). This may indicate evidence of heterogeneity of a rather significant degree for this factor. In the ER negative group an age at first full pregnancy >23 years and a BMI >29 kg/m2 were associated with an increased risk in both HER-2/neu groups, but the association was significantly stronger for the latter factor in the positive HER-2/neu tumours (ratio of OR's = 2.46, 95%CI: 0.97–6.21).In multivariate analysis without HER-2/neu+ versus HER-2/neu-breast tumours, with the exception of increased BMI. Further innovative studies with larger sample sizes are needed to examine how the status of these potentially modifiable breast cancer risk factors interacts with biological markers such as HER-2/neu oncoprotein.Our study did not confirm that the established or putative hormonal breast cancer risk factors differ regarding their relations with HER-2/ Indeed,survival . In addi lesions . As a regenesis. ,7-9.neu overexpression or amplification may define a subset of breast cancer in which the net effect of a risk factor could be rather more obvious and its impact on breast cancer development more clear [On the other hand, the contribution of even well established breast cancer risk factors to the aetiology of carcinogenesis in the breast remains obscure, ill-defined and tenuous, mostly because of the existence of different pathways for the initiation and the evolution of a breast tumour . In ordere clear ,7,8.neu overexpression could indicate either that a HER-2/neu alteration is the way that this risk factor evolves into the carcinogenesis or that there is a parallel interaction between them that leads to breast tumour initiation and development. Since the data in the literature supporting the above hypothesis are few and conflicting, we decided to investigate, in a group of patients from a geographical area with a low incidence of breast cancer, whether HER-2/neu positive tumours are correlated with established or suspected risk factors for breast cancer and thus to identify distinct subgroups of high risk women.Thus, a close correlation of a risk factor with HER-2/This study analysed data derived from the database of the Breast Unit of the Department of Surgical Oncology at the University General Hospital of Heraklion on the island of Crete, Greece. The study considered all women who were consecutively diagnosed with primary, invasive breast cancer in our unit from 1996 to 2002. Subjects of other races, ethnicity, with residence outside Crete or diagnosed with DCIS or LCIS were excluded. Finally, 384 women, all originating from the island of Crete, were eligible for analysis.An age-stratified random sample of 566 women was used as a control group, derived from the Breast Unit's database of screened patients who had not developed breast cancer after a median follow up period of 40 months (range 12–92 months). Personal interviews were conducted with each woman during her first visit (both patients and controls) by a consultant or a senior resident. The interview followed a structured questionnaire, which did not change during the study period. Anthropometric measures were also made during the first visit.Women were classified as postmenopausal if their menstrual cycles had ended naturally at least 12 months before the interview or from surgery or radiation therapy at any age. Those who reported not having menstrual cycles for the last 10 months were considered as perimenopausal and were combined with premenopausal women for the purpose of our analysis.2 vs. BMI>29 kg/m2, median value for the study population) and radiation history of the chest (yes/no).The following variables were analysed for all patients and controls: residence , age at interview (≤ 50 and >50 years), age at menarche (≤ 12 and >12 years old), age at first full birth (<23 vs. ≥ 23 years old), parity (nulliparous, 1 or 2, and >3), lactation (yes/no), use of medications to suppress lactation (yes/no), abortions and miscarriages (yes/no), age at menopause for postmenopausal women (≤ 50 and >50 years old), use of HRT for more than 2 months (yes/no), use of oral contraceptives for more than 2 months (yes/no), family history of breast cancer in a first degree relative (yes/no), history of benign breast disease (yes/no), obesity on the day of the interview (BMI ≤ 29 kg/mneu protein and hormone-receptor expression, respectively.For this study, tumour blocks were successfully retrieved in 378 (98.4%) and in 377 (98.17%) of the 384 interviewed cases for the immunohistochemical detection of HER-2/Immunohistochemistry with the monoclonal antibody CB11 (NCLCB11, Novocastra Laboratories, UK 12 8EW), at a dilution of 1/50 with incubation period of 60 min, was performed using the OPTIMAX automated system with the Super Sensitive Link-Label Detection System RTU Multilink AP/Fast Red, QA200OXE (purchased from Biogenex Laboratories, San Ramon CA 94583 USA), following antigen retrieval by microwave pre-treatment at 500 Watts for 3 × 5 min in citrate buffer (0.01 M, pH 6). Sections from breast cancer of known positivity were used as positive controls. Negative controls were processed by omitting the primary antibody and substituting non-immune serum. Scoring was based on the criteria recommended by DAKO A/S for the HercepTest . Only meThree (3) μm-thick sections taken on negatively-charged (SuperFrostPlus) slides were dewaxed in xylene, and rehydrated through graded alcohols. Following antigen retrieval by microwave pre-treatment at 500 W for 3 × 5 min in citrate buffer (0.01 M, pH 6), estrogen receptor (ERa) and progesterone receptor (PR) expression was detected by immunohistochemistry using the same automated system and detection kit as above, and primary monoclonal antibodies to ERa (DAKO M7047) and PR (Biogenex code # MU 328-UC) at dilutions of 1/50 and 1/20, respectively, with incubation time 60', at room temperature. Positive and negative controls were processed as above. Positive nuclei counting was performed at a final magnification of 400× (Teaching double-headed NICON, ECLIPSE E400 microscope, equipped with CFI 10X/22 oculars). After scanning at a final magnification of 100× for locating the areas with highest density of ER+ or PR+ carcinoma cell nuclei (hot spots), a 40X/¥/0.17 WD 0.65 objective lens was used for cell counting. All carcinoma cells in three hot spots per immunostained slide were evaluated by two pathologists working simultaneously, though independently, and the mean of the two independent counts was considered the final counting value for each field and hot spot. The ratio of the ER+ or PR+ carcinoma cell nuclei was recorded separately for each of the hot spots. The final immunoreactivity index (score) was calculated as the mean percentage of ER+ or PR+ carcinoma cell nuclei in the three hot spots. Specimens were interpreted as positive for ER or PR if at least 10% of the cells demonstrated nuclear staining of any intensity of reactivity, from 1+ to 3+. Staining intensity was graded as negative (0), weak (1+), intermediate (2+) or strong (3+), and reported separately. A mean value of intensity was assigned for specimens in which the staining intensity varied from field to field, and/or from hot spot to hot spot.2) test. A p value <0.05 was defined as significant. The potential association between breast cancers stratified by HER-2/neu status and well known predisposing factors was further investigated by using a stepwise logistic regression analysis (backward LR), testing the independent effect of breast cancer risk factors (independent variables) on breast cancer (dependent variable) for all women and also separately for premenopausal and postmenopausal females. In addition, we undertook further stratification with estrogen receptor status by using the same multivariate logistic regression model. These patient-controls odds ratios helped us to detect the pattern of heterogeneity and to explore plausible aetiological correlations between patient subgroups. Additionally, case-case odds ratios were calculated in order to measure the risk heterogeneity between HER-2/neu+ and HER-2/neu-tumours. It seems that the departure of the OR from unity can reveal the degree of heterogeneity between these subgroups [Odds ratios (OR) and 95% CI , as approximators of relative risk, were calculated to measure the association of the groups of breast cancer and the risk factors, using the chi-square , use of HRT (OR = 7.34, 95%C.I: 2.03–26.53), an age at first full pregnancy more than 23 years (OR = 1.91, 95%C.I: 1.29–2.83) and body mass index more than 29 kg/m2 (OR = 3.13, 95%C.I: 2.02–4.84). Additionally, a history of abortion or miscarriage (OR = 0.56, 95%C.I: 0.38–0.82) was correlated with a decreased risk of breast cancer. However, the number of oral contraceptive and HRT users was too small for reliable estimates of risk.Risk factor distributions in breast cancer patients and matched controls are presented in table neu protein overexpression. HER-2/neu positive tumours were not related with menopausal state, age at interview, tumour size, grade and stage, nodal and estrogen receptor status, but there was a modest positive association between HER-2/neu and progesterone negative tumours.Tumour characteristics of breast cancer patients are shown in table premenopausal group of women, an increased risk for HER-2/neu-tumours was observed for those women who reported an age at first full pregnancy ≥ 23 years (OR = 3.56, 95%C.I: 1.70–7.46), a BMI>29 kg/m2(OR = 6.89, 95%C.I: 2.23–21.25), first degree family history (OR = 3.30, 95%C.I:1.10–9.96) or use of oral contraceptives (OR = 11.19, 95%C.I 3.70–33.84), while an age at menarche less than 12 years was the only factor which slightly increased the risk in premenopausal HER-2/neu+ patients (OR = 2.09, 95%C.I 0.99–4.42). Abortion played a less protective role (p = 0.068) for HER-2/neu-breast cancer in premenopausal than in postmenopausal women (p = 0.038). However, the intercase comparison in the premenopausal subgroup showed an evidence of heterogeneity only for the HER-2/neu+ women who had ever had an abortion (ratio of the OR's = 3.12, 95%C.I:1.18–8.24), while use of oral contraceptives (OR = 0.16, 95%C.I: 0.04–0.60, p = 0.007) and a positive first degree family history (OR = 0.09, 95%C.I: 0.01–0.85, p = 0.035) showed a stronger association for HER-2/neu negative tumours.Subgroups of women stratified by menopausal status were further analysed by a multivariate stepwise logistic regression model adjusted for the remaining variables table . In the all women and the postmenopausal groups of patients due to the large sample size. Patients with an age of menarche ≤ 12 years (OR = 1.72, 95%C.I: 1.07–2.75), first degree family history (OR = 2.23, 95%C.I:1.08–4.63), use of HRT (OR = 10.70, 95%C.I: 2.71–42.31) or OC (OR = 6.47, 95%C.I:1.89–22.16) were at increased risk of developing HER-2/neu-breast cancer only, although the significance of the latter two factors was of little value due to the limited sample size. On the other hand, an age at first full pregnancy ≥ 23 years and a BMI greater than 29 kg/m2 increased breast cancer risk independently of HER-2/neu status, while a history of abortion decreased risk in the same way. In the case to case comparison only BMI >29 kg/m2 revealed a relative stronger connection with positive than with negative HER-2/neu tumours (ratio of OR's = 2.23, 95%C.I: 1.20–4.15, p = 0.011) and this may indicate an evidence of heterogeneity of a rather significant degree for this factor. The stronger association between an age at menarche ≤ 12 years, use of HRT and negative as opposed to positive HER-2/neu status did not reach statistical significance (p = 0.067 and p = 0.062, respectively).The results of logistic regression were identical for neu positivity and obesity already shown in the analysis so far. In more detail, although BMI >29 kg/m2 elevated risk for both ER negative and positive tumours independently of HER-2/neu status, the association was significantly stronger for positive HER-2/neu tumours in the ER negative group (ratio of OR's = 2.46, 95%CI: 0.97–6.21). Additionally, in the same group an age at first full pregnancy >23 years revealed an increase of risk in both HER-2/neu groups, while first degree family history (OR = 2.72, 95%C.I: 1.05–7.07, p = 0.040), age at menopause >50 years (OR = 2.05, 95%C.I: 1.10–3.79, p = 0.023) and birth of 1–2 children (OR = 2.38, 95%C.I: 1.21–4.67, p = 0.012) elevated risk for HER-2/neu negative tumours only. In the ER+ group of women the direct comparison between cases revealed no associations with any factor at all, while abortion showed a protective pattern against breast cancer which expressed estrogen receptors independently of HER-2/neu status.A different stratification pattern of our study's population is presented in table neu oncoprotein. In fact, increased BMI elevated risk in both groups, but the comparison between HER-2/neu+ and HER-2/neu- tumours revealed a much stronger association with HER-2/neu+ breast cancers.This epidemiological study, conducted in a low incidence Mediterranean population,  found thneu status can help discriminate aetiologically distinct subgroups of breast cancer cases, and none of them has identified the effect of increased BMI with HER-2/neu positive tumours [Very few studies have examined the possibility whether HER-2/ tumours ,5,7-9.neu+ tumours with an early age at first full pregnancy, we found a strong elevated risk with a late age regardless of HER-2/neu protein expression [neu+ breast cancers, while we also found this inverse association but independently of HER-2/neu status [neu+ tumours only in the premenopausal group of women. Early contraceptive use has been positively associated with HER-2/neu+ breast cancer in two studies [neu negative tumours. However, because the number of oral contraceptive (and HRT) users in this study was small, this subgroup analysis was hindered by decreased power to detect associations of any magnitude. The slightly protective effect of parity found in the age-adjusted analysis was diminished after logistic regression and did not reveal any association with HER-2/neu status, in contrast with previous findings [neu positive tumours in one study while other investigators reported opposite results [More specifically, in contrast with other investigators who have shown an elevated risk for HER-2/pression . Previouu status . Interes studies ,8, but ofindings . Breastf results ,9. Althoneu status. This inconsistency may reflect differences in study design, populations, and laboratory methodology. In this study we used immunochemistry  to assess the HER-2/neu protein overexpression, which is highly correlated with gene amplification according to previous reports [neu protein overexpression found here was within the limits reported elsewhere [Our findings have similarities and differences with respect to previous reports that examined the associations of breast risk factors with HER-2/ reports . Also, tlsewhere ,5-9.neu protein overexpression and most of the hormone-related breast cancer risk factors does not completely agree with several hypotheses which have maintained that combined estrogen and HER-2/neu activation is closely involved in the same pathway in breast cancer carcinogenesis [This lack of relationship between HER-2/ogenesis ,18.neu positive tumours in our study was high body mass index, which is an established risk factor that has an estrogen-mediated oncogenic effect on the mammary gland. More specifically, obesity is associated with higher breast cancer risk among postmenopausal women through greater lifetime exposure to higher levels of estrogens produced in adipose tissue and lower SHBG production [neu gene [neu receptor, and so promote carcinogenesis through common means [The only hormone-related factor that was found to be related with HER-2/oduction ,20. Highneu gene ,21. Accoon means ,17,18.neu-positivity and lack of hormone receptor expression in breast tumours [neu positive tumours were weakly related with the absence of estrogen receptors, although this was not statistically significant .Numerous epidemiological and experimental studies have shown the strong relationship between HER-2/ tumours ,18,22. IThe interview was conducted during the subjects' first visit to the unit and before clinical examination or any other intervention took place. This constitutes an advantage, because there was no chance that the subjects (both cases and controls) would be influenced by the diagnosis and might therefore falsely inflate the relative risk. Thus, the likelihood of recall bias is not high, improving the comparability of several covariates in both groups, and the selection bias is lessened since all subjects had taken the same route through the Breast Unit's standard routine procedures.neu status subgroup. Some caution regarding our findings is related to the size of the study group. In the analyses stratified by HER-2/neu and menopausal or ER status numbers are quite small and for some risk estimates the confidence intervals are wide and the estimates of risk unstable.Since each case group was compared with the same control group, any selection bias would be expected to have a similar effect on the estimates in the tumour subgroups. Thus, it is extremely unlikely that recall bias issues would apply only to those cases in a specific HER-2/neu+ versus HER-2/neu-breast tumours, with the exception of increased BMI. Further innovative studies with larger sample sizes are needed to examine how the status of these potentially modifiable breast cancer risk factors interacts with biological markers such as HER-2/neu oncoprotein. Their findings will provide us with greater insight into breast cancer aetiology and will help us identify any association that would help discriminate subgroups of women at higher risk.In conclusion, our study did not confirm that the established or putative hormonal breast cancer risk factors differ regarding their relations with HER-2/EGFR: epidermal growth factor receptor, HRT: hormonereplacement therapy, BMI: body mass index, SHBG: sex hormone-binding protein, ER: estrogen receptor, PR: progesterone receptor, OC: oral contraceptives.The author(s) declare that they have no competing interests.NT conceived the study, participated in its design and drafted the manuscript.ES participated in the design of the study, assisted in writing and reviewed the final article.EfS and KM scheduled and performed the laboratory analysis.NA performed the statistical analyses.VG and DDT participated in the design and coordination of the study and reviewed the final article.All authors have read, discussed and approved the manuscript.The pre-publication history for this paper can be accessed here:


A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P). However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line.RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments.We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA) or all-trans-retinoic acid (ATRA), and strongly inhibited by N-(4-hydroxyphenyl) retinamide (HPR). The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that the potency of retinoids on cell growth inhibition may be increased through combination of estrogen and progesterone treatment.Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of  It is well documented that women who experience a full-term pregnancy early in their lives have a significantly reduced risk for developing breast cancer ,2. The mRetinoids are a promising class of chemopreventive agents against breast cancer because of their antiproliferative and proapoptotic properties ,6. RetinThe 76N TERT cells were derived from a reduction mammoplasty specimen ,9. They In this study, we investigated whether E/P induce growth inhibition in 76N TERT cells; and the molecular mechanisms by which E/P inhibited 76N TERT cell growth. For comparison purpose, the anti-growth effect of both natural and synthetic retinoids was examined in parallel in this immortalized mammary epithelial cell line. We also investigated whether we could increase the responsiveness of retinoids by using retinoids in combination with E/P. Our studies demonstrate that 1) inhibition of cell growth by E/P and retinoids could be partially mediated through a p53-dependent mechanism; 2) induction of p21 expression, inhibition of telomerase activity, or up-regulation of estrogen receptor beta (ERβ) by E/P and retinoids may contribute to their anti-growth effects; 3) combination of retinoids with E/P lead to increased inhibitory effects on cell growth.As the ability of estrogen, progesterone and retinoids to influence cell proliferation is mediated by their respective receptors in most cases, we first examined the expression of these receptors in 76N TERT cells. Using Western blot analysis, the proteins of RAR (RARα, RARβ and RARγ), RXR (RXRα, RXRβ and RXRγ), and ER (ERα and ERβ) were observed as suggested by the manufactures Figure . The ant3H]thymidine incorporation assay. The concentrations of E/P and retinoids used in the experiments were chosen based on previous studies and are clinically or physiologically relevant [We then tested the influence of E/P or retinoids on 76N TERT cell growth, using Hthymidinrelevant ,12. TreaRAREs) and regulation of transcriptional activity [RARE gene activation in response to E/P and retinoids was carried out using cells transfected with a RARE-luciferase reporter gene construct. As shown in Figure RARE gene in response to 9-cis RA, ATRA or E/P alone. In contrast, HPR caused no significant change in luciferase activity relative to the control. In the presence of E/P, the effects of 9-cis RA and ATRA on RARE gene activation were essentially the same as without E/P.Many biological responses to retinoids are thought to be mediated through receptors by binding to retinoic acid response elements  the expression of p53 protein. Consistent with the data on p53 gene activation, no additive effects between E/P and retinoids on induction of p53 protein were observed  and dissolved in ethanol. The final concentration was 2 μM for ATRA and HPR, 1 μM for 9-cis RA, 1 ng/ml for β-estradiol and 1 μg/ml for progesterone. These concentrations were chosen based on previous studies and are clinically or physiologically relevant ,4,12.2 and sub-cultured weekly. All experiments were performed on cells with passage numbers from 6 to 15.Cell line was originally supplied and cultured as described by Band et al. . The cul4 cells per well and incubated at 37°C overnight. Cells were then treated in triplicates with indicated retinoids in the presence or absence of E/P for 24 hours. After labelling cells with 1 μCi/ml of [3H]thymidine (Amersham, Arlington Heights, IL, USA) for 24 hours, cells were harvested by washing with PBS and 10% TCA, solubilizing with the mixture of 0.1% SDS and 0.1N NaOH. Aliquots were taken for the quantification of radioactivity by the Tri-Carb 2900 TR Liquid Scintillation Analyzer (Perkin Elmer, Wellesley, MA, USA). Incorporation of [3H]thymidine was expressed as a fold change from vehicle control under the same conditions.Cells were seeded into 24-well plates at 5 × 104 cells per well. Cells were transiently transfected with 0.5 μg of either a RARE-luciferase or a p53-luciferase plasmid along with 0.05 μg of pCMV-Renilla luciferase using the SuperFect transfection reagent , following the manufacturer's recommended procedure. Twenty-four hours after transfection, triplicate cultures were treated with retinoids for 24 h in the presence or absence of E/P. The cells were then washed and lysed. The luciferase activities were measured using the DUAL-luciferase Assay System (Promega, Madison, WI, USA), and normalized by pCMV-Renilla luciferase activity for each sample.Cells were seeded in 24-well plates at 5 × 10g for 20 min at 4°C, and the supernatant was collected. The protein concentration was measured in each extract using the BCA protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Telomerase activity was determined in duplicates by a quantitative real-time PCR telomerase detection Kit (Allied Biotech, Ijamsville, MD, USA) according to the manufacturer's protocol, using Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA).Cells were lysed in CHAPS lysis buffer  and incubated at 4°C for 30 min. The lysate was then centrifuged at 12000 × Cells at subconfluence were treated with retinoids in the presence or absence of E/P for 72 hours. Total RNA was extracted with TRIZOL Reagent from Gibco (Carlsbad, CA, USA) according to the instructions of the manufacture. Single-stranded cDNA was made from 1 μg of total RNA with the Cells-to-cDNA kit (Ambion, Inc., Austin, TX, USA) at 42°C for 15 min. The primers for ERβ were 5'CGA TGC TTT GGT TTG GGT GAT 3' (forward) and 5'GCC CTC TTT GCT TTT ACT GTC 3' (reverse). The primers for GAPDH were 5'CCA TGG AGA AGG CTG GGG 3' (forward) and 5'CAA AGT TGT CAT GGA TGA CC 3' (reverse). All primers were from Integrated DNA Technologies, Inc. . cDNA (1 μl) was amplified in duplicates in Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA) by using Brilliant SYBR Green QPCR Master Mix from Stratagene (La Jolla, CA, USA). The reaction was carried out at 95°C for 10 min to denature, 40 cycles of 95°C for 30 sec, 55°C for 60 sec, 72°C for 60 sec. ERβ gene was quantified and normalized with external standard GAPDH.2, and protease inhibitor cocktail (Sigma, St. Louis, MO, USA), followed by centrifuged at 12,000 rpm for 20 min. Protein content was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Cell lysates (~30 μg protein) were separated on 10% polyacrylamide gels in the presence of 0.1% SDS with prestained low-range molecular-weight standards (Biorad, Richmond, CA, USA). After transfer, the membranes were blocked and then probed with antibodies against interested proteins as suggested by manufactures, followed by incubation with a peroxidase-conjugated secondary antibody. Immunoreactive bands were developed with an ECL reagent from Amersham (Arlington Heights, Il, USA). All blots were probed with anti-actin to normalize for loading differences. Quantification of gels was carried out using ImageJ software (NIH, Bethesda, Maryland, USA).Cells were treated with retinoids in the presence or absence of E/P for 72 hours. Cell lysates were obtained by incubating cells for 30 minutes at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 100 mM NaF, 10 mM MgClThe p53 (DO-1), p21 (F-5) and RARγ (G-1) mouse monoclonal antibodies, and RXRα (D-20) polyclonal antibody were from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Anti-ERβ (Ab-2), ER (Ab-1), PR (Ab-1), RARα and RARβ mouse monoclonal antibodies, and Bax (Ab-1) polyclonal antibody were from Calbiochem (San Diego, CA, USA). Monoclonal anti-actin, RXRβ and RXRγ were from Sigma (St Louis, MO, USA).Statistical differences were analyzed by T-test. Levels of statistical significance were set at p < 0.05.9-cis RA: 9-cis-retinoic acid; ATRA: all-trans-retinoic acid; E/P: ?β-estradiol and progesterone; ERβ: estrogen receptor beta; HPR: N-(4-hydroxyphenyl) retinamide; PR: progesterone receptors; RARs: retinoic acid receptors; RXRs: retinoid X receptors.The author(s) declare that they have no competing interests.JZ designed and carried out most of the assays and drafted the manuscript. YT carried out the real-time PCR experiments. SS conceived of the study and participated in its coordination. All authors read and approved the final manuscript.


The aim of this study is to validate the questionnaire ECOS-16  for the evaluation of health related quality of life (HRQoL) in post-menopausal women with osteoporosis.An observational, prospective and multi-centre study was carried out among post-menopausal women with osteoporosis in primary care centres and hospital outpatient clinics. All patients attended 2 visits: at baseline and at 6 months. In addition, the subgroup of outpatients attended another visit a month after the baseline to assess the test-retest reliability. The psychometric properties of the questionnaire were evaluated in terms of feasibility, validity  and internal consistency in baseline, and in terms of test-retest reliability and responsiveness to change in visit at month and visit at 6 months, respectively. In all visits, ECOS-16, EUROQoL-5D (EQ-5D) and four 7-point items about health status  were administered, whereas only outpatients were given MINI-OQLQ , besides all clinical variables; and sociodemographic variables at baseline.Feasibility: 94.3% of patients answered all items of the questionnaire. The mean administration time was 12.3 minutes. Validity: factor analysis suggested that the questionnaire was unidimensional. In the multivariate analysis, patients with vertebral fractures, co-morbidity and a lower education level showed to have worse HRQoL. Moderate to high correlations were found between the ECOS-16 score and the other health status questionnaires (0.47–0.82). Reliability: internal consistency (Cronbach's α) was 0.92 and test-retest reliability (ICC) was 0.80. Responsiveness to change: ECOS-16 scores increased according to change perceived by the patient, as well as the effect size (ranges between 1.35 to 0.43), the greater the perception of change in patients' general health status, the greater the changes in patients' scores. The Minimal Clinically Important Difference (MCID) suggested a change of 0.5 points in the ECOS-16 score, representing the least improvement in general health status due to their osteoporosis: "slightly better".316 women were consecutively included, 212 from primary care centres and 104 from hospital outpatient clinics. ECOS-16 has been proven preliminarily to have good psychometric properties, so that it can be potentially a useful tool to evaluate HRQoL of post-menopausal women with osteoporosis in research and routine clinical practice. Osteoporosis is characterised by low bone mass and a deterioration in bone tissue micro-architecture, leading to increasing bone weakness and consequently risk of fracture. The most common clinical complications of osteoporosis are hip fracture, vertebral deformity and wrist fracture. According to bone densitometry values, osteoporosis affects approximately 2 million women in Spain ,2. The mTo date, clinical trials on osteoporosis have been based on outcomes measured by imaging tests. But these measurements do not adequately reflect the extent to which the patient is affected in their usual daily activities, and are not appropriate to assess patients' disability and symptoms . NeverthDisease specific questionnaires for osteoporosis are available, such as the Osteoporosis Quality of Life Questionnaire (OQLQ)  or the QThe aim of the present study is to evaluate the psychometric properties of the ECOS-16 in post-menopausal women with osteoporosis.316 post-menopausal women with primary osteoporosis attended in Primary Care Centres or in outpatient clinics were included in the study. Diagnosis was confirmed by a Bone Mineral Density (BMD) using Dual Energy X-Ray Absorptiometry (DEXA). Furthermore, outpatients should have at least one prevalent vertebral fracture confirmed by Genant's radiological criteria due to osteoporosis, a requirement which was not essential in patients from Primary Care Centres. 212 patients from 49 Primary Care Centres and 104 patients attended in outpatient clinics from 14 hospitals were consecutively selected and evaluated from March 2000 to August 2001.All patients attended two visits, a baseline and a follow-up visit 6 months after the inclusion. In order to evaluate test-retest reliability, outpatients were also attended in another follow-up visit one month after the baseline. All patients received the study information and gave their informed consent.An observational, prospective and multi-centre study was carried out for the validation of the ECOS-16 in post-menopausal women with vertebral fractures due to osteoporosis in conditions of clinical practice. At the baseline, feasibility together with the content and construct validity of the ECOS-16 were evaluated. At the visit after 6 months, responsiveness to change with regard to ECOS-16 was evaluated. Outpatients also attended another visit a month after the inclusion so that the test-retest reliability of the ECOS-16 could be evaluated.In the baseline, data on the patients' sociodemographic characteristics (age, education level) and clinical variables  were collected as well as the ECOS-16, the EUROQoL-5D and four 7-point items which refer to general health status, back pain, limitation in daily activities and emotional status. These items were used in a previous study and showed its validity . OutpatiIn the two follow-up visits, any modifications to the specific treatment for osteoporosis prescribed in the baseline, the number of concomitant treatments, and patients' withdrawals causes were registered. Moreover, the ECOS-16, EUROQoL-5D and the four 7-point change items were again administered. Outpatients were again administered the MINI-OQLQ questionnaire.In the present study, the EQ-5D and MINI-OQLQ questionnaires were used in order to assess the validity of the ECOS-16.The ECOS-16  where it was acceptable for the questionnaire to be administered by health care personnel or experienced interviewers.The EUROQoL-5D (EQ-5D) is a generic HRQoL questionnaire. The EQ-5D consists of two parts: a descriptive system and a Visual Analogue Scale (VAS) . The desThe VAS of the EQ-5D is a vertical scale divided into millimetres along a 20 centimetres long thermometer where the two ends are labelled "worst imaginable health state" and "best imaginable health state" with a score of 0 and 100, respectively. The respondent should mark the point on the thermometer which, in their opinion, best describes their actual overall health status.The MINI-OQLQ is a specific HRQoL questionnaire for women with vertebral fractures due to osteoporosis. The MINI-OQLQ is based on a selection of the two highest impact items from each of the five domains of the Osteoporosis Quality of Life Questionnaire (OQLQ). The MINI-OQLQ is, therefore, composed of 10 items grouped into the same five HRQoL dimensions  .Each item has seven response options ranging from 1 (worse HRQoL) to 7 (better HRQoL). The scoring is obtained per dimension, by calculating the mean score of the response items for each dimension, so that the higher the score in each dimension, the better the resulting HRQoL.Changes in four health status items were assessed through four different items regarding the change in the patient's overall health status: the change in the patient's general health status due to osteoporosis, the change in the patient's back pain, the change in the patient's limitation in daily activities and the change in the patient's emotional status, all of them due to osteoporosis, with reference to the baseline. The items have seven possible response options, ranging from "Much better" to "Much worse" and including the category "More or less the same". These items were designed to be self-administered and validated in previous studies ,14.® for Windows version 10.0 (SPSS, Inc., Chicago, Illinois) was used to carry out the entire data analysis.Double data entry was carried out with a subsequent validation to guarantee the quality and consistency of the data. A statistical significance level of p < 0.05 was used in all statistical tests performed. The statistical program SPSSPreviously the statistical analysis, a Kolmogorov-Smirnov test was conducted to assess the distribution of the variables in order to use a parametric or non-parametric tests.With the aim to describe the study sample characteristics and to evaluate differences among patients with and without vertebral fractures a descriptive and comparative analysis was done on patients' sociodemographic characteristics (age, education level) and clinical variables (Body Mass Index (BMI), number of years with menopause, presence or absence of non-vertebral fractures, concomitant diseases and received treatment during the previous year) according to vertebral fracture presence. In order to compare the two groups, the chi-squared test was used, given that the variables were categorical, and the Bonferroni correction for multiple tests.The feasibility of the ECOS-16 was analysed on the basis of missing data, time and method of administration. In order to evaluate the missing data, the number and percentage of missing response items in the whole questionnaire as well as the number and percentage of patients who failed to respond to any of the questionnaire's items were both calculated. The time spent administering the questionnaire was evaluated according to the method of administration (self-administered or administered by an interviewer).The floor (percentage of patients with the lowest score) and ceiling (percentage of patients with the highest score) effect were calculated for each one of the ECOS-16 items and for the overall score.An exploratory factor analysis was used to assess the content validity of the ECOS-16, using the scores obtained during the baseline by all patients for all 16 questionnaire items. Factors were extracted using principal-axis factoring method and varimax rotations. The adequacy of the factor analysis was assessed with the Kaiser-Meyer-Olkin measure and the Bartlett's test of sphericity .To evaluate the construct validity, the correlations between the ECOS-16 scores and the patients' sociodemographic and clinical characteristics  were analysed. Pearson's correlation coefficient was used for continuous variables and the analysis of variance (ANOVA) for categorical variables. A multivariate analysis was also carried out taking the ECOS-16 score as a dependent variable and the sociodemographic and clinical variables that were significant in the bivariant analysis as independent variables, so that any possible confounding factors could be controlled. Secondly, the relationship between the ECOS-16 and the EQ-5D scores, the four 7-point items  and the MINI-OQLQ (only outpatients) were all analysed using Spearman's correlation coefficient, apart from the VAS which was analysed using Pearson's correlation coefficient. Higher correlations were expected between dimensions that measure the same HRQoL aspects.Because the lowest score in ECOS-16, in EQ-5D and in 7-point general health status item represent the best HRQoL, the expected correlations among their dimensions would be positive. The opposite applies to the dimensions of MINI-OQLQ and the other three 7-point items , where the lowest score represent the worst HRQoL and, therefore, the expected correlations between ECOS-16 would be negative.The reliability of the ECOS-16 was evaluated in terms of internal consistency and test-retest reliability. Internal consistency was calculated by Cronbach's α coefficient using the baseline scores of all questionnaire items. Test-retest reliability was evaluated only for outpatients who did not perceive a change in their general health status due to osteoporosis after a month, as shown by the change in 7-point general health status item (response category: 'More or less the same'). The Intraclass Correlation Coefficient (ICC) between the scores for both visits was used for this analysis. The hypothesis that the standard psychometric recommendations for Cronbach's α and ICC were greater than or equal to 0,7 was taken as a starting point for both internal consistency and test-retest reliability [Longitudinal validity of the ECOS-16 was evaluated by analysing the correlations among changes registered in the ECOS-16, and changes in the EQ-5D, changes in the MINI-OQLQ and changes in the four 7-point change items from baseline to visit at 6 months. For this purpose the Spearman's correlation coefficient was used. The expected correlations are the same as in the construct validity.To assess responsiveness to change, first of all, attention has been drawn to whether the questionnaire detects the changes which are perceived by patients between the baseline and the visit at 6 months. In order to do so, the Student's t-test for paired data was used. To assess the magnitude of changes, the effect size was calculated, thus it was establish that the changes in the patients' scores increased at the same time that the changes perceived by patients. In order to calculate the effect size, the change in 7-point general health status item is used .The Minimal Clinically Important Difference (MCID) has been defined as the smallest difference between the scores in a questionnaire that the patient perceives to be beneficial . The MCITable Statistically significant differences (p < 0.01) were found for age, concomitant diseases (patient-reported), and length of time with menopause, it being the case that those patients with vertebral fractures were older and had had menopause for a longer time. In addition, those patients with vertebral fracture were those that had received more treatments during the previous year (p < 0.01) and also had a greater BMI (p < 0.05). However, those patients without a vertebral fracture presented concomitant diseases more frequently (p < 0.01). Only 14.4% of patients showed some type of non vertebral fracture and about 70% had completed at least primary education level.After the Bonferroni correction, the analysis that were still significant (p < 0,007) were age, years with menopause, concomitant disease and previous treatment.At baseline, all osteoporotic patients were prescribed or changed their treatment by the physicians, some having changed his previous treatment but others having received no treatment before.The mean (SD) administration time of the questionnaire was 12.3 (7.8) minutes for all patients, with a median of 10 minutes. In 55.1% of cases, the questionnaire was self-administered, the remainder being administered by health care personnel (due to eyesight difficulties or illiteracy). No statistically significant differences were observed in administration times according to the type of administration.The 99.7% of the patients answered all items of the questionnaire. Only one patient failed to respond to any item.A third of the items had a floor effect greater than or equal to 20%, 38.3% in item "Do you have problems with dressing?". The maximum ceiling effect was observed in 55.7% of the patients in item "How often have you had back pain in the last week?". In the remaining items, the ceiling effect was lower, ranging from 1.3% to 20.3%, the highest being in item "Are you afraid of getting a fracture?". Two patients (0.7%) recorded the highest scores for all the items .In the factor analysis, the Kaiser-Meyer-Olkin measure was 0.916 indicating a good sampling adequacy. The Bartlett's test of sphericity (p < 0.001) made it possible to accept the identity of the matrix correlations for the ECOS-16 items, thus indicating the suitability of the factor analysis.Table A multivariate analysis was carried out to identify patients' characteristics that were related to the ECOS-16 score . All these correlations were also statistically significant.The internal consistency of the ECOS-16 was very high, with a Cronbach's α coefficient of 0.92. Test-retest reliability was analysed for 44 outpatients who declared that their general health status due to osteoporosis had not changed after a month, with an Intraclass Correlation Coefficient of 0.80 and a mean (SD) score change of 0.2 (0.5) points.Figure The measurement of HRQoL has attracted increasing attention as a clinically relevant outcome of research and clinical practice. HRQoL questionnaires reflect the impact of health care interventions on health aspects such as physical, mental and social well-being. However, either in clinical research and in practice, a lengthy questionnaire is problematic for both the health care personnel and the patient. Shortish measure attempt to minimize time and effort as well as to increase patient interest . Thus, sThe ECOS-16 originates from the reduction of two validated and widely used HRQoL questionnaires in osteoporosis patients with vertebral fracture . HoweverAlmost all the patients responded to all the questionnaire items. The presence of a floor effect for one third of the questionnaire items could lead to the conclusion that the questionnaire detects changes only when the severe disease status occurs. However, according to a previous qualitative division , the floAs some previous studies, this study shows that the variables usually used to evaluate patients with osteoporosis, such as Bone Mineral Density (BMD) and the presence of vertebral fractures, have low or no correlation with HRQoL scores. This finding is not new ,20 and sIn this study, the fact that the presence of vertebral fractures does not have a negative effect on HRQoL  deserves special attention. There seems to be some discrepancy regarding this issue in literature. Several studies have shown that HRQoL progressively deteriorates in relation to the presence and number of vertebral fractures ,24. HoweRecently, a significant number of studies have highlighted the importance of the site of the vertebral fracture and its effect on HRQoL. In this regard, it seems that the site of vertebral fracture has a much greater effect on HRQoL than the presence and number of vertebral fractures -30. ThisAlthough in the present study, the bivariate analysis does not discriminate between patients with and without vertebral fracture, the multivariate model shows that the presence of fractures is indeed significant even if the percentage of explained variation is small. This is not surprising given the fact that such analysis is dealing with prevalent fractures in a short term observational study . The incThe results obtained for internal consistency and test-retest reliability showed high levels of homogeneity among questionnaire items and good reproducibility over time. Moreover, the outcomes, have also been shown that the new questionnaire effectively detects changes in patients' perceived health status due to osteoporosis. Expressing responsiveness to change is an important characteristic of the new instrument, one which will doubtless allow its use in clinical research. The high correlation between the ECOS-16 and generic (EQ-5D) and specific (MINI-OQLQ) HRQoL questionnaires corroborates this hypothesis. It also highlights the new questionnaire's validity by demonstrating that it measures concepts which are closely related to already validated HRQoL questionnaires ,13.The mean change in score per question corresponding to the effect size in general and to the MCID in particular is consistent, in terms that the larger the change assessed by the ECOS-16, the larger the effect size. Moreover, MCID is consistent with the results of other HRQoL questionnaires, and it is useful to compare the magnitude of changes detected between them. MCID will also be useful in the planning of new trials, as sample size depends on the magnitude of the difference investigators consider clinically important and are not willing to risk failing to detect .The potential limitations of this study are mainly due to it being unable to rely on certain variables which have shown a clear influence on HRQoL. In particular, the "time spent since the fracture occurred"  was not The low education level of the study sample must also be taken into account. It is consistent with previous studies ,9, and aIn conclusion, the ECOS-16 is a HRQoL questionnaire which is short, easy to administer  and with adequate preliminary psychometric properties. This makes the ECOS-16 potentially very useful during routine clinical practice or/and research for the treatment and follow-up of post-menopausal women with osteoporosis. Nevertheless, its actual potential must be proven in future clinical trials in order to recommend its use in research and clinical practice.ECOS Appendix1.doc, the ECOS-16 questionnaireClick here for file


Intense and long-standing problems in burn centers in Tehran have led nurses to burnout. This phenomenon has provoked serious responses and has put the nurses, patients and the organization under pressure. The challenge for managers and nurse executives is to understand the factors which would reduce or increase the nurses' responses to burnout and develop delivery systems that promote positive adaptation and facilitate quality care. This study, as a part of more extensive research, aims to explore and describe the nurses' perceptions of the factors affecting their responses to burnout.Grounded theory was used as the method. Thirty- eight participants were recruited. Data were generated by unstructured interviews and 21 sessions of participant observations. Constant comparison was used for data analysis.Nurses' and patients' personal characteristics and social support influenced nurses' responses to burnout. Personal characteristics of the nurses and patients, especially when interacting, had a more powerful effect. They altered emotional, attitudinal, behavioral and organizational responses to burnout and determined the kind of caring behavior. Social support had a palliative effect and altered emotional responses and some aspects of attitudinal responses.The powerful effect of positive personal characteristics and its sensitivity to long standing and intense organizational pressures suggests approaches to executing stress reduction programs and refreshing the nurses' morale by giving more importance to ethical aspects of caring. Moreover, regarding palliative effect of social support and its importance for the nurses' wellbeing, nurse executives are responsible for promoting a work environment that supports nurses and motivates them. Working in a burn unit has been described as a stressful occupation . Every nWhile no health-care professional is immune to these pressures, there is evidence that suggests that areas of nursing particularly those areas we think of as critical care environments such as burn units, are often the most vulnerable to stress, and in need of much support ,7. NurseHowever, regarding emotional, attitudinal, psychosomatic, behavioral and organizational responses of these nurses to burnout, it is vital to identify the factors that involve in their perception of burnout. Some authors also referred to these factors in burn centers  and otheThe challenge for managers and nurse executives of burn centers is to understand the intervening factors and their impacts on these burn nurses' responses to burnout. As a result they can develop and promote delivery systems that support positive adaptation to stressors in burn centers, retain nurses and facilitate quality nursing care.In order to understand nurses' perceptions of factors modifying their responses to job stress and burnout, qualitative research adapted from the grounded theory method was chosen .The value of using a qualitative research method such as grounded theory is embedded in the subjective and often emotional nature of care, stress and coping. As a descriptive study, the qualitative paradigm, with its emphasis on understanding factors modifying nurses' responses to job stress and burnout from the view point of practicing nurses themselves seemed logical. Grounded theory is a theory that is derived from data, systematically gathered and analyzed through the research process  The aim In effect, grounded theory is guided by simultaneous analysis. Both analysis and data collection inform each other. The analysis process is systematic and ends when new data no longer generate new insights. This has been also described as ' category saturation' ,14.Five clinical nursing instructors participated in the pilot study. They were faculties of School of Nursing in Iran University of Medical Sciences (IUMS) and had been supervising nursing students in burn centers of Tehran for many years. Their age ranged from 40–48 years and had been working in burn centers for 7–14.5 years. The aim of pilot study was using the experiences of nursing instructors in the original study and reducing the informant and researcher bias in the interviews and participant observations .The results of the study indicated that the staff of burn units felt drained, they haven't had any motivation or desire to care and they had been working purely for their pay. The findings were strongly indicative of the symptoms of burnout. It revealed that their behavior was representative of an indication of their professional dilemma. The pilot study also indicated that social support, patients' cooperation/motivation and the nurses' unique characteristics had been modified to alter the nurses' responses to burnout. Analysis of data from the original study was conducted keeping these findings in mind.The research proposal was approved by the ethics committee of IUMS. Then permission was granted from the managers of two burn centers and their nursing administrators. Further permission and written consent was obtained from all who participated in this study.Following ethical approval, data was collected through tape- recorded, unstructured interviews. Initially data was collected in one center and analyzed. Then data gathering was initiated in the second center. There were 19 informants from the first center and 14 from the second center that participated (except 5 participants in pilot study). From this sample, 25 were nurses in different levels and positions and 8 were other members of burn team. The nurses' sample included 8 staff nurses, 8 licensed practical nurses, 2 nurses' aids, 3 head nurses, 2 supervisors, and 2 nursing administrators. Since nursing staff pointed to some issues concerning the burn team, the researcher interviewed one physician, one social worker, 2 physiotherapists, and 4 patients in the process of theoretical sampling.Criterion for recruiting nursing participants was at least one year of experience in the burn center. Patients were selected according to their desire as well as their physical and psychological stability. Selecting patients occurred by consultation with head nurses.Participants were 11 males and 22 females. The nursing staff participated were 19 females and 6 males. Twelve of the nursing staff had been working 2–3 shifts in burn centers or other hospitals as well as working in other jobs due to financial needs. Other demographic information of the nursing staff is displayed in Table Samples were recruited from all units of both centers. The first purposeful sample included 6 of nursing staff. Theoretical sampling was used after emerging the tentative theory. The basis for theoretical sampling was the questions which emerged during data analysis. At this stage the researcher interviewed nursing administrators and other members of the burn team. Theoretical sampling helped in verifying nursing staff's responses and credibility of categories and resulted in more conceptual density. No new data were emerged in the last two interviews; therefore data gathering by interviews were terminated.Interviews were conducted in a private place with mutual agreement of the interviewer and interviewees. All interviews were completed by the main researcher. The duration of interviews ranged between 30–165 minutes. All the interviews were tape recorded except one. Some notes were taken during dialogues.Unstructured interviews were conducted using a topic guide which has been drawn up by the researchers initial review of the literature related to the concepts of the subject of the study. This topic guide included the structure, process and outcome of care .The following grand tour question guided the study:" please tell me about the nursing care in your unit". Subsequent questions were based on the participants' responses and demands of the emerging theory. Interviews were terminated when data redundancy occurred.In each center after the termination of interviews, participant observations were performed in all wards at morning, evening and night. 14 sessions of observation in the first center and 7 in the second center occurred. For this purpose the researcher informed the nursing administrators of her program. By selecting all the wards in all shifts there was no need for theoretical sampling in this stage (place and time) , but theDescriptive, focused and selective observations were occurred in a non- linear fashion. Theoretical sampling occurred during focused and selective observations. Some of the questions which guided the theoretical sampling were," is there any difference between nursing cares received by different patients?", "is nursing care different in a large or small ward?"Prolonged engagement of the researcher in the field reduced the obtrusiveness. The level of participation varied from complete observation to participation in some activities. Some informal interview also occurred during the observations. Immediately after each session of observation field notes were completed systematically. Analysis of the field notes helped in determining contextual conditions and explaining variations in the nurses' responses in each context. This led to proposition of several hypotheses.Data collection, analysis and interpretation occurred simultaneously, in keeping with grounded theory methodology . After eThe process of integrating and refining the theory occurred in selective coding . In thisThe researchers accepted the perspective of Guba and Lincoln. They translated internal validity into credibility, external validity into transferability, reliability into dependability, and objectivity into confirm ability .Credibility enhanced by the researchers' describing and interpreting her experiences. For this purpose the researcher kept a field journal in which she noted the content and the process of interactions, including reactions to various events. This journal became the record of relationships and provided material for reflection. Prolonged engagement and persistent observation helped to data credibility. In this way the process of data collection and analysis took 8 months. Data triangulation and method triangulation confirmed credibility . MaximumConfirm ability requires one to show the way in which interpretations have been arrived at via the inquiry. In this study, confirm ability was established, because credibility, transferability and dependability were achieved . The sigThe findings related to factors which involved in the nurses' responses to burnout are presented in this article. Analysis and interpretation of data indicated that personal characteristics and social support have involved in the nurses' responses. These factors are presented in Table Data from interviews and participant observations indicated that special personal characteristics and personality traits have involved in the nurses' emotional, attitudinal, behavioral, and organizational responses to burnout. Personal characteristics such as conscience, religious beliefs, personal philosophy, commitment, a sense of responsibility, and altruism facilitated caring behaviors. Nurses with these characteristics were more patient and empathetic. They were more cooperative and rarely justified their faults by fatigue, workload or staff shortage. Conscience, commitment, and religious beliefs such as fear of divine requital were the most prominent traits that modified the responses to burnout. One of participants stated:" God knows. I always feel it's me there on the bed. Sometimes a patient calls and I ignore, but I tell myself, what I expected if I were on this bed? I fear god and say to myself, his authority is great and whatever I do, I will see the reflection of my doings". Some of the participants pointed to interest and love in caring of burn victims. One of participants stated:" these patients are different from other ones. I have worked more than 18 years in general hospitals, I haven't worked more than 7 years in burn centers, but I think that was a blessing in disguise, I am glad because clinical work for a burn patient means love, means everything, believe me. I don't care the managers' behaviors, workload, nursing shortage and other deficiencies, because I love burn victims. Believe me". Many of the nursing staff, distanced from patients, they had immoral beliefs and demonstrated humiliation and reproach in their behaviors. They related these attitudes to fatigue, micro and macro conditions in burn centers, and loss of motivation; but participant observation indicated that, this isn't the case for all the nurses. Nurses, who had been known as good nurses, were very calm and intimate with their patients and focused on the patients' needs. The researcher wrote in one of her field notes:" she is very calm and speaks with compassion. She makes jokes and patients are relaxed with her. She follows the principles and procedures more strictly than others". Nurses in all levels were under pressure of workload, low pay, staff shortage, environmental conditions of burn units and other structural inhibitors, but as the excerpts of interviews indicated, appraisal of these inhibitors was different in the presence of specific personal characteristics.Data indicated that in some instances, when there were a number of inhibitors and they were long stay, even positive personal characteristics couldn't work. This was often the case in infectious dressing rooms and busy wards. The worst kinds of treating patients were seen in these places. It seemed that the inhibitory factors which are persistent and too frequent interact with personal characteristics and finally overcome the positive characteristics. One of participants stated:" patients expect to receive care, expect a friendly encounter which does not happen. To tell the truth, some days I am excessively distressed and tired that I don't have the patience to answer the patient' questions and concerns. I emotionally can not do what I really want to do on a daily basis for the patients. The reason is persistent day and night problems occurring with this job". This process is displayed in Table Data strongly indicated that nurses' appraisal of the patients' characteristics have influenced some aspects of their attitudinal and behavioral responses. When the appraisal was positive, the relationships improved, and when it was negative, relationships deteriorated. Positive appraisal occurred often when the patients were cooperative and motivated for recovery and in cases where they had an advantage of high socio- economic class, cultural and educational levels, or whenever they stimulated the nurses' senses of compassion and pettiness. Negative appraisal occurred often when the patients were from lower socio- cultural levels, addicts or there was a possibility of having acquired immune deficiency syndrome (AIDS) or hepatitis.The first group was treated kindly and more respectfully. The use of humiliating words and reproach towards them diminished and as a result the aggressive behavior and physical withdrawal less occurred. One of participants stated:" ...I am more supportive and compassionate towards children, those who are very alone, who have no one to love and care for them, those who have committed self-inflicted burns, a woman whose husband caused her to burn herself and doesn't have any one to support her. In many occasions I have even paid them to buy juice from outside the hospital. I feel that these patients are needy".The second group was treated very unethical. They encountered a humiliating, reproaching, and aggressive behavior. One of participants stated:" ...he fights when I am dressing him, he pulls his hand and leg, he isn't cooperative, and he has no class. They drain all my energy to the point that I don't want to talk to them. I think they are mentally retarded. They keep still when I shout at them just the way that the children act. I tell them I'll pull your ear, and I'll beat you up. This is the behavior that has worked with them ".Moreover patients with extensive burns, whose survival was an improbable event, not only were badly treated, but also were sometimes ignored and received poor care. In other words, they received only those treatments which had been ordered by physicians to prevent being reprimanded by supervisors. One of the participants justified herself and stated:" if you want the truth, a patient with 90% burns can not benefit from tetracycline ointment, but it's there in his order, I prefer to spend my time with a patient who has a better chance of surviving. Right or wrong I don't apply the ointment, because I can spend that time for a patient who will survive". We can conclude that burnout has made nurses to modify their caring behaviors to fit the different type of patients they care for.As described later, nurse's and patient's characteristics modified the nurse's responses to burnout and altered caring behaviors. More analysis and interpreting of data indicated that interaction between these two variables resulted in a more powerful combination that alters responses to burnout and identifies the kind of caring behavior. This process is displayed in table Table One of participants stated: "I take care of some patients with love and conscience and take care of the other patients only with conscience and some nurses doesn't have love at all, I take care of silent, calm and lonely patients better, I perform routine care for the other". Another participant also stated:" it seems I give positive energy to the patients to whom I am more interested and take care of them with more love. I've seen that they respond better to therapeutic measures". Therefore interaction between nurses and patients' characteristics has a very powerful effect on the nurses' responses to burnout and determines their nature of caring behavior.Data from interviews and participant observations strongly suggested that social support influences the nurses' responses to burnout.Supportive behavior of head nurses, nursing administrators and coworkers modified the nurses' responses. Among these, head nurse's the support was the most effective factor. Nurses believed that they do not have any motivation or desire to perform well when they are not supported well. One of participants stated:" we have a very close loving relationship with the head nurses who are supportive. In that situation I do many things for her. Her supportive attitude and caring/positive attitude helps me a lot. Do you understand?" another participant believed that he couldn't endure if the nursing administrator weren't supportive. He stated:" I have seen that she is doing a good job. I have been so stressed at times that I have thought of quitting. The nursing administrator has changed my mind during those stressful moments by being caring, loving and supportive and I have decided to stay in spite of low pay and benefits." Support from peers also modified the nurses' responses. They could tolerate more with the support of their coworkers. One of participants stated:" by god, I like every single one of them. It seems like we live together seven hours a day (major part of our day). We are so familiar with each other's character and behavior patterns. You may not believe this, these relationships has been very helpful and caused us to have a very strong unit. We care for each other in times of weakness, illness or pressure".It is worthy of mention that the effect of social support on the nurses' responses to burnout was not as powerful as the nurses' and patients' characteristics. Social support influenced the emotional and in some cases the attitudinal responses, but it didn't have enough power to alter the organizational and behavioral responses, therefore it didn't change caring behaviors significantly. Moreover, there were not any data indicative of the modifying effect of the factors proposed in this article on the psycho- somatic responses to burnout.Findings of this study indicated that nurses' and patients' characteristics and social support modified the nurses' responses to burnout. These factors altered the nurses' perceptions of the inhibitory factors; in other words they could have a more positive appraisal and this in turn modified their responses.Lazarus and Folkman 1988) proposed that the initiative of behavioral manifestations is a transactional appraisal which is important for the person's wellbeing and betrays the confrontation as noxious, useful, threatening or requiring struggle 88 propos. In thisIn this study, when inhibitory factors were persistent and frequent, even positive personal characteristics defeated. Selye (1976) concluded that the influence of stressors on a person depends on the number of stressors that must be confronted in a time, the duration of confrontation and existence of previous experience with the same stressors . This haPatients with different characteristics treated differently. Nurses changed their caring behavior with different patients. It seemed that they didn't have enough emotional and physical energy and motivation for caring for all patients; but some patients stimulated their emotions and gave them the needed positive energy for caring. In other words, patients' characteristics could both reduce and intensify the nurses' responses to burnout. When these characteristics appraised as positive, caring behaviors improved, and when it was negative, the worst kind of behaviors occurred. Maslach (1982) in congruence with this conclusion believed that there is little evidence that caring is a uniform state , and BenPatients with extensive burns received the worst kind or care. Nurses stated that caring for these patients is futile. Data implied that caring for these patients have been incongruous with the nurses' values. Meltzer and Huckabay (2004) in their study of the relationship between critical care nurses' perceptions of futile care and its effect on burnout, concluded that feeling of emotional exhaustion in these nurses was highly influenced by the frequency with which nurses were involved in life- sustaining interventions that conflicted with the nurses' values and standards in term of what the nurses thought are ethically appropriate and could result in improvement in a patient's condition and outcome .It was the interaction between nurses' and patients' characteristics that identified the caring behavior. Some authors have found according to their clinical practice that nurses have the ability to adjust their approach and their style of interaction with different patients. These authors proposed that they not only alter the nature of dialogue and the tone of voice to meet each patient's needs, but also adjust their affective response. They pointed that delineation of these behaviors would be a significant contribution, yet to date these styles of care have not been explored . In thisSocial support from head nurses, nursing administrators and peers made nurses to endure and tolerate in the face of problems. Lazarus (1976) proposed that the resources reducing the potential harm could be found in the environment and essentially in others who indicated one can rely on them . Social This study as a part of more extensive research (PhD dissertation) identified the most important factors that intervened in the nurses' responses to burnout in burn centers of Tehran. Nurses had responded to burnout. These responses included emotional, attitudinal, psychosomatic, behavioral and organizational. This part of study indicated that the nurses and patients characteristics and interaction between these two factors had a very powerful effect on the responses and determined the kind of caring behavior. Moreover social support from managers (e.g. head nurses and nursing administrators) and peers modified some of the nurses' responses to burnout.The influence of positive personal characteristics, especially conscience, religious beliefs, philosophy, commitment, a sense of responsibility and altruism on the nurses' responses to burnout, the finding that, long lasting and persistent problems in the work setting can deteriorate even the personal characteristics, and regarding the numerous problems in burn centers of Tehran, there is an urgent need for helping the nurses. We suggest that due to the intense staff shortage in these centers, the managers try to keep these nurses. The only way they can do this is by using stress reduction programs. Data strongly indicated that these nurses need to rest periodically to preserve energy and to refresh their morale. Moreover, giving importance to moral and ethical aspects of care by managers could be helpful and motivating. Changing burn patients' inherent characteristics and their other characteristics such as poverty and socio- cultural level is not a possible alternative. Promoting nurses' morale is possible and must be done promptly if we want our burn survivors receive at least an ethical effective care.Social support made the problems tolerable. Therefore we recommend nurse executives in burn centers of Tehran promote a work environment that help to decrease the perception of pressures and increase perceptions of social support. The best solution is of course eliminating the micro and macro conditions which overshadow the nurses' responses and caring behaviors.This grounded theory created several hypotheses in this stage. Nurses with specific traits were more resistant to burnout and were more caring. It is suggestive of conducting a quantitative research to test the relationship between personality traits, burnout and caring behaviors. Differences in caring behavior for different patients were related to the influence of burnout on the nurses' personality traits and appraisal of patients' characteristics. It was a very new finding that needs to be investigated in more detail.The dramatic effect of social support on the nurse' perceptions of pressures in burn centers of Tehran is suggesting of identifying the relationship between social support and the level of burnout the nurses experience in these burn centers.However the nature of a qualitative research like this, limits it's generalizability; therefore we suggest conducting more qualitative research in other critical care units to support these findings.Application of the findings of this study and conducting the suggested studies would help the managers of burn centers to enhance an environment conductive to morale, promote natural and ethical care, refresh the nurses' positive emotions and facilitate a supportive environment for their nurses.The author(s) declare that they have no competing interests.FR initiated and designed the research, collected and analyzed the data and wrote the paper. FO was the main supervisor, helped in analysis, and revised and edited the drafts. MN was co- supervisor and revised the drafts.The pre-publication history for this paper can be accessed here:


Neutrophil infiltration is one of the critical cellular components of an inflammatory response during peritonitis. The adhesion molecules, P-selectin and intercellular adhesion molecule (ICAM)-1, mediate neutrophil-endothelial cell interactions and the subsequent neutrophil transendothelial migration during the inflammatory response. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy, suggesting that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the objective of this study was to determine the role of P-selectin and ICAM-1 in neutrophil infiltration into the peritoneal cavity during early and late phases of peritonitis.in situ immunohistochemical staining. Comparisons between groups were made by applying ANOVA and student t-test analysis.Peritonitis was induced in both male wild-type and P-selectin/ICAM-1 double deficient (P/I null) mice by cecal ligation-puncture (CLP). Peripheral blood and peritoneal lavage were collected at 6 and 24 hours after CLP. The total leukocyte and neutrophil contents were determined, and neutrophils were identified with the aid of CLP induced a severe inflammatory response associated with a significant leukopenia in both wild-type and P/I null mice. Additionally, CLP caused a significant neutrophil infiltration into the peritoneal cavity that was detected in both groups of mice. However, neutrophil infiltration in the P/I null mice at 6 hours of CLP was significantly lower than the corresponding wild-type mice, which reached a similar magnitude at 24 hours of CLP. In contrast, in peritonitis induced by intraperitoneal inoculation of 2% glycogen, no significant difference in neutrophil infiltration was observed between the P/I null and wild-type mice at 6 hours of peritonitis.The data suggest that alternative adhesion pathway(s) independent of P-selectin and ICAM-1 can participate in neutrophil migration during peritonitis and that the mode of stimuli and duration of the injury modulate the neutrophil infiltration. Sepsis is a common cause of morbidity and mortality following surgery or trauma, and is characterized by activation of a systemic inflammatory response, severe hypotension, and major damage to multiple organs . Althougendothelium (P-selectin and E-selectin) [The selectins mediate neutrophil rolling while the β-2 integrins are important for firm adhesion and transendothelial migration . The selelectin) . ICAM-1 electin) . ICAM-1 electin) .In vivostudies have shown that administration of small molecule ligands, and/or neutralizing antibodies to the selectins, ICAM-1 or β2-integrins can protect tissues from injury following endotoxin exposure, bacterial infection, or ischemia [et al. has shown that soluble P-selectin attenuated skeletal muscle reperfusion injury by inhibition of the classical complement pathway [et al. have demonstrated a significant reduction in neutrophil infiltration into the peritoneal cavity at 4 hours of streptoccocal peritonitis in ICAM-1 mutant mice [et al. has shown that P-selectin and ICAM-1 significantly contributed to liver and lung injury at 4 hours of systemic endotoxemia in ICAM-1 and P-selectin/ICAM-1 double mutant mice [et al. have shown that P-selectin and ICAM-1 double mutant mice exhibited complete loss of neutrophil migration into the peritoneum during S. pneumoniae-induced peritonitis [et al. have shown no differences in survival between wild type and ICAM-1-deficient mice following an intra-peritoneal injection with E. coli, S. auerus, or P. aeruginosa [ischemia -10. Howe pathway . Geneticant mice . Anotherant mice . Furtheritonitis . In contruginosa . More imruginosa . In an aruginosa . Additioruginosa . This moAll chemicals were purchased from Sigma Chemical (St. Louis, MO), unless otherwise noted.Guide for the Care and Use of Laboratory Animals . Experimental protocols were reviewed and approved by the Michigan State University Animal Use and Care Committee.Only adult male mice (i.e., 8–10 wk) were used in this study. All animals received humane care in compliance with the tm1BaySelptm1Bay, backcrossed to C57BL/6, were used in this study. Breeding pairs of double-knockout mice were purchased directly from Jackson Laboratory (Bar Harbor, ME) and bred under the guidance of University Laboratory Animal Resources at Michigan State University. The wild-type (WT) mice were male C57BL/6, which were acclimated to the animal laboratory environment for one week before the start of experimentation. Before and after surgery, all the animals had unlimited access to food and water.Gene-targeted double mutant mice deficient in P-selectin and ICAM-1 (P/I double mutant), C57BL/6-Icam1et al. [Polymicrobial sepsis was induced by CLP as previously described by Chaudry, et al. . SurgicaAdult male mice (i.e., 8–10 wk) were lightly anesthetized by inhalation anesthesia with methoxyflurane. A volume of 2% sterile glycogen solution, equal to 10% of body weight, was injected intraperitoneally (i.p.). Six hours after glycogen inoculation, mice were euthanized and peritoneal lavage and blood samples were collected. The collected samples were identified with ID numbers to assure a blind fashion performance of the tests and data analysis. Total leukocyte and differential counts were performed as described below.Blood samples were obtained from the right ventricle via a left anterior thoracotomy at the time of sacrifice, using a sterile heparinized syringe containing 50 μl of heparin (100 USP Units/ml). Blood smears were prepared and stained with Wright-stain (LeukoStat, Fisher Scientific, Pittsburgh, PA) for differential cell counts. The total number of peripheral blood leukocytes was determined by lysing the red blood cells using 3% acetic acid solution with the aid of a Neubauer hemocytometer. The remaining blood was centrifuged, and plasma was collected and stored at -70°C. A portion of the liver was fixed in buffered 10% formalin and embedded in paraffin, and a second portion was snap frozen in liquid nitrogen and stored at -70°C until used for immunohistochemistry staining.The peritoneal fluids were collected using repetitive (2 times) instillation and withdrawal of 2 and 5 ml respectively of sterile saline solution using a syringe with a 22 G needle. The peritoneal lavage sample was placed on ice, immediately processed for centrifugation at 4°C, and the supernatant and the cell pellet were collected separately. The cell pellet was used for total and differential cell counts. Differential cell counts were determined on cytospin preparations of peritoneal lavage stained with Wright-stain (LeukoStat). The total number of peritoneal leukocytes was determined as described above for the peripheral blood leukocyte count. Additionally, the presence of neutrophils in peritoneal lavage was confirmed by immunohistochemical staining of cytospin preparation using a primary antibody (IgG2a) specific to mouse neutrophil as described below. Further, the neutrophil content was quantified by measuring the myeloperoxidase (MPO) level of peritoneal lavage as described below.The MPO contents of the peritoneal lavage supernatant and the cell pellet were quantified as previously published by our laboratory . Briefly2 clone, PharmMingen, San Diego, CA) and a biotin-conjugated secondary antibody. Tissue sections were counter stained with hematoxylin (Gill's formula, Vector Laboratories) and mounted with DAKO Mounting Media (DAKO Corp, Carpinteria, CA). The samples were examined using a Nikon light microscope interfaced with a spot 24-Bit Digital Color Camera.To confirm the identity of neutrophils in peritoneal lavage, immunohistochemical staining was performed using acetone-fixed cytospin cell preps. The primary antibody (clone 7/4, IgG2a) specific to mouse neutrophil (Cedarlane, Westbury, NY), the biotin-conjugated secondary antibody (PharMingen, San Diego, CA), and a Vectastain avidin-biotin complex reagent and 3,3'-diaminobenzidine chromogen kits (Vector Laboratories, Inc., Burlingame, CA) were used as previously described in detail . The expt-test by way of StatView version 5.0.1 software© for Windows. Comparisons between multiple groups and various time points were analyzed using ANOVA with subsequent Fisher's PLSD test. P ≤ 0.05 was considered significant.All data were expressed as means ± standard error of the mean. Comparisons between two groups were performed using an unpaired The Jackson Laboratory, where the P/I null breeding pairs were purchased, had initially tested the double knockout of the P/I null mice. In addition, the ICAM-1/P-selectin deficiency was confirmed in our laboratory in randomly selected litter mice tissue samples using RT-PCR and immunohistochemical staining of the liver tissue, as previously published by our laboratory (18). The ICAM-1 expression was determined in all the animals used in this study. Figure Clinical signs of sepsis were manifested as quietness, lack of response to stimulus, absence of socializing behavior, ruffled hair coat and lack of appetite. All mice subjected to CLP displayed these signs that became significant at 24 hours of CLP, and no striking differences were observed between the wild-type and P/I null mice.To establish the blood cell parameters under normal physiological conditions in wild-type and P/I null mice, peripheral blood samples were collected from randomly selected mice. In this article these mice are denoted as "Control", which were not subjected to the sham or CLP operation. The leukocyte and differential counts of peripheral blood were measured. As shown in Table i.p. inoculation of 2% glycogen. Further, the inflammatory response to the sham operation, which elicited a trauma-induced peritonitis, caused leukopenia in both wild-type and P/I null mice. As shown in Table The induction of CLP-induced peritonitis resulted in a significant leukopenia in wild-type as well as P/I null mice as compared to their control counterparts Table . It is i6 vs. P/I = 3.6 ± 1.2 × 106). However, it is interesting to note that monocytes/macrophages were the predominant cell type present in the peritoneal cavities of the wild-type mice, whereas in the P/I null mice lymphocytes were the predominant cell type (Table p = 0.08).Similar to peripheral blood, the cell parameters of peritoneal fluid under normal physiological conditions in the wild-type and P/I null mice were determined in randomly selected mice. The mice were not subjected to the sham or CLP operation. As Table Sepsis induced by CLP (i.e., 6 and 24 hr) caused a significant leukocyte infiltration into the peritoneal cavities of both wild-type and P/I null mice when compared to their corresponding sham group Figure . The perPeritonitis was also examined in response to 2% glycogen. A significant leukocyte infiltration into the peritoneal cavity occurred in both wild-type and P/I groups at 6 hours after 2% glycogen injection Figure . SimilarTo further quantify the degree of neutrophil infiltration into the peritoneal cavity, the MPO levels of both the peritoneal lavage cell pellets and supernatants were measured. Although the MPO levels of the cell pellets indicated the presence of a significant number of neutrophils in the peritoneal cavity after CLP, the MPO values did not always correlate with the absolute number of infiltrated neutrophils. The Wright's staining of cytospin preparations of the peritoneal cells demonstrated highly activated phagocytes with vacuolized cytoplasm often containing numerous intracellular bacteria and in most cases with loss of cellular integrity Figure . Thus, iPrevious studies have indicated that tissue MPO activity can be affected by other factors (19, 20). and that MPO values may not indicate the true presence of neutrophils. Additionally, recent studies have identified MPO as the cellular component of macrophages (i.e., Kupffer cells) (21), as well as histiocytes (22). Therefore, an immunohistochemical staining technique was used to confirm the presence of neutrophils in the peritoneal cavity. The cytospin preparations of the peritoneal lavages were immunostained with a specific antibody to mouse neutrophil. Figure Neutrophil infiltration is coordinated by the interplay of the adhesion molecules and the chemoattractants, which plays an important role in inflammatory tissue injury. Recent clinical trials of anti-adhesion therapy did not demonstrate a protective effect in trauma-induce shock despite very strong pre-clinical data (15). Further studies to clear this disparity suggested that the model systems applied and the length of injury are important factors that might modulate the underlying mechanism of neutrophil activation and migration (15). Therefore, this study was undertaken to examine the role of P-selectin and ICAM-1 molecules in the wild-type and P/I null mice subjected to a short and longer periods of peritonitis induce by CLP. The CLP model, chosen to mimic the normal course of sepsis in humans and animals, induced the classic signs of sepsis and a significant systemic inflammatory response. This response was associated with increased leukocyte infiltration into the peritoneal cavities of both the wild-type and P/I null mice. The leukocyte influx into the peritoneal cavity consisted predominantly of neutrophils i.e., 62–80%), which significantly increased in both wild-type and P/I null mice at 6 and 24 hours of CLP. At the early phase of CLP (i.e., 6 h), the total number of neutrophils infiltrated into the peritoneal cavities of P/I null mice were significantly lower than those of the corresponding wild-type mice, which reached to a comparable level at 24 hours of CLP. In contrast to CLP-induced peritonitis, peritonitis induced by 2% glycogen exhibited no significant differences in the number of neutrophils infiltrated into the peritoneal cavities between the wild-type and those of the P/I null mice at 6 hours  or chemically-induced peritonitis (i.e 2% glycogen) and during the late phase of CLP (i.e, 24 hours). The data suggests that there might be a functional role for these adhesion molecules during the initial stages of the inflammatory response, and as the inflammatory process progresses, deficient adhesion mechanisms are bypassed. This notion has also been proposed in leukocyte recruitment in an adhesion .2-integrins (eg., CD11b and CD18) expressed on neutrophils has shown to be a critical event mediating stable neutrophil adhesion and migration across the vascular endothelial barrier [per se, is not a critical factor that results in dysfunctional β2-integrin-mediated migration. Other adhesion molecule(s), ligand(s), and/or yet unknown counter-receptor(s) could mediate neutrophil infiltration. For example, ICAM-2, a ligand for β2-integrins, could be a potential candidate [Interaction of ICAM-1 expressed on the surface of vascular endothelial cells with the β barrier -28. Alth barrier . Furtherandidate . Other sandidate ,31. Thisandidate . Neutropandidate ,34. Addiandidate . Neutropandidate . The imp2-integrin-mediated cell adhesion and motility of neutrophils has been described [2 integrins, such as those produced during coagulation as well as complement activation and tissue factor, which could facilitate neutrophil adhesion and infiltration into the peritoneal cavity [It is possible that neutrophil infiltration utilizes other secondary or tertiary adhesion pathways, and/or is facilitated by proteins that mediate the function(s) of the adhesion molecules. For example, a novel glycosylphosphatidyl inositol-anchored protein (GPI-80) that may regulate βescribed . Additiol cavity ,40. Morel cavity . It has l cavity .i.p. inoculation of Streptococcus pneumonia [et al. study, the inflammatory response and neutrophil infiltration into the peritoneal cavity was studied at 4 hours of the induction of peritonitis, while, in our study neutrophil infiltration was evaluated at 6 and 24 hours of peritonitis. In support of this reasoning is the study reported by Mizgerd et al. who have shown a compromised neutrophil migration into the peritoneal cavity of E-/P-selectin and ICAM-1 mutant mice in response to the injection of Streptococcus pneumoniae at 4 hours after injection, with no impairment present at 24 hours of bacteria injection [et al. demonstrated that ICAM-1 is not necessary for neutrophil migration during glycogen-induced peritonitis, as shown otherwise by other authors [Streptococcus pneumonia organisms. In our study, the data of peritoneal supernatant MPO levels have demonstrated a significant level of MPO released into the peritoneal cavity in mice subjected to CLP (Figure in situ immunohistochemical staining of the peritoneal cells clearly confirms the presence of neutrophils in the cytospin preparations (Figure Contrary to several other studies that have demonstrated the functional importance of P-selectin in models of myocardial infarction and inflammatory lung and liver injury, the data presented in this study indicated that P-selectin is not essential for neutrophil migration into the peritoneal cavity -45. In sneumonia . The difnjection . Additio authors . FurtherP Figure . In Bulls Figure . The datStreptococcus- or LPS-induced peritonitis, the present study employed polymicrobial septic peritonitis as well as chemical and trauma-induced peritonitis. Published studies have shown quantitative and qualitative differences in the inflammatory response induced by gram-positive, gram-negative, polymicrobial sepsis and purified endotoxin [Another important difference between the results of the study presented in this article and those of previous studies may relate to the employment of essentially different models of sepsis and inflammation. While previous studies employed hemorrhagic shock-, ndotoxin -52. Thesndotoxin ,54.The data presented in this study have shown a clear, time-dependent neutrophil migration and infiltration into peritoneal cavity in responses to peritonitis, which is independent of P-selectin and ICAM-1 adhesion molecules. The negative nature of the data presented here and the failure of the anti-adhesion clinical trials, are of great importance. These findings demand innovative alternative approaches to neutrophil transmigration in inflammatory response and suggest that targets other than these adhesion molecules need to be identified. A better understanding of the mechanisms leading to neutrophil migration is critical for the development of new therapeutic strategies for treating inflammatory disease without compromising the host's immune response mechanisms.None declared.CR participated in the technical procedures for CLP, collection of blood and tissue samples, and MPO assay. KH and HA participated in preparation of tissue sample for immunohistochemistry and special staining. KH also assisted in preparation of the manuscript. EC conceived the study, and participated in its design, coordination, and preparation of the manuscript. All authors read and approved the final manuscript.CLP, cecal ligation-puncture; ICAM-1, Intercellular adhesion molecule-1; mAbs, monoclonal antibodies; MPO, Myeloperoxidase; P/I null mice, P-selectin/ICAM-1-deficient miceThe pre-publication history for this paper can be accessed here: