Few studies report on the effect of organizational factors facilitating transfer between primary and tertiary care hospitals either within an integrated health care system or outside it. In this paper, we report on the relationship between degree of clinical integration of cardiology services and transfer rates of acute coronary syndrome (ACS) patients from primary to tertiary hospitals within and outside the Veterans Health Administration (VHA) system.Prospective cohort study. Transfer rates were obtained for all patients with ACS diagnoses admitted to 12 primary VHA hospitals between 1998 and 1999. Binary variables measuring clinical integration were constructed for each primary VHA hospital reflecting: presence of on-site VHA cardiologist; referral coordinator at the associated tertiary VHA hospital; and/or referral coordinator at the primary VHA hospital. We assessed the association between the integration variables and overall transfer from primary to tertiary hospitals, using random effects logistic regression, controlling for clustering at two levels and adjusting for patient characteristics.Three of twelve hospitals had a VHA cardiologist on site, six had a referral coordinator at the tertiary VHA hospital, and four had a referral coordinator at the primary hospital. Presence of a VHA staff cardiologist on site and a referral coordinator at the tertiary VHA hospital decreased the likelihood of any transfer (OR 0.45, 95% CI 0.27–0.77, and 0.46, p = 0.002, CI 0.27–0.78). Conversely, having a referral coordinator at the primary VHA hospital increased the likelihood of transfer (OR 6.28, CI 2.92–13.48).Elements of clinical integration are associated with transfer, an important process in the care of ACS patients. In promoting optimal patient care, clinical integration factors should be considered in addition to patient characteristics. Coronary artery disease is the leading cause of death among Americans . HospitaThe coordination and integration between primary and tertiary hospitals has important implications for integrated health care delivery systems. The Veterans Health Administration (VHA) is one of the largest vertically integrated health care delivery systems in the United States . The VHATreatment guidelines for acute coronary syndromes -10 suggeThe objective of this study was to evaluate the association between structural components of clinical integration and patient transfer rates from VHA primary hospitals to tertiary hospitals, both within and outside the VHA system for patients with ACS. We hypothesized that primary VHA hospitals with structural components of clinical integration present would have a higher rate of within-system transfer of ACS patients than primary VHA hospitals lacking these components.The VHA Access to Cardiology study was a prospective cohort study of 2,733 patients with a primary discharge diagnosis of either acute myocardial infarction (ICD9-CM 410.xx) or unstable angina (ICD9-CM 411.xx) discharged over a one year period (March 1, 1998 through February 28, 1999) from 24 VHA hospitals in five regions, including Minnesota and the Dakotas, the Southwest, the Rocky Mountains, the Pacific Northwest, and Southern California. Patient demographics, clinical characteristics, and specific processes of care including hospital transfer were obtained as part of the Access to Cardiology Study.All patients admitted to one of the 12 primary VHA hospitals in the study were eligible for this analysis (n = 862 out of the 2,733 in the larger Access to Cardiology study). The remaining 12 VHA Medical Centers were tertiary hospitals with cardiology services and cardiac catheterization laboratories on site. These were not the focus of the analysis reported in this paper. We excluded 107 patients because they were initially admitted to a private hospital and transferred into a primary VHA hospital. In addition, we excluded 3 patients who were transferred from one primary VHA hospital to another. Finally, 27 patients had missing data in the variable indicating prior history of congestive heart failure, which was included in the final analysis. As a result, a total of 725 patients from 12 primary VHA hospitals were included in these analyses. The study protocol was approved by the Human Subjects Committee at the University of Washington, and by Institutional Review Boards and Research and Development Committees at each participating VHA hospital.Patient transfer from a primary VHA hospital to a tertiary hospital (either VHA or private) was the primary outcome for this study. Secondary outcomes included both transfer from a primary VHA hospital to a tertiary VHA hospital, and transfer from a primary VHA hospital to a private (non-VHA) tertiary hospital.Transfers to a tertiary VHA hospital were considered transfers within the system, while transfers to a private hospital were considered transfers outside the system. Transfer data were available for all 725 patients in the study cohort. We constructed two binary variables for the analyses: transfer to any tertiary care hospital (yes/no), and transfer to a tertiary VHA hospital versus transfer to a private (non-VHA) hospital.The key independent variable for this study was clinical integration of cardiac services. We defined clinical integration ,13 as thThe data used to construct these measures came from on-site interviews conducted with Chiefs of Cardiology at each of the tertiary VHA hospitals associated with the primary VHA hospitals included in this study. During on-site interviews, Chiefs of Cardiology were asked to describe all of the primary VHA hospitals that refer ACS patients to them on a regular basis, and to identify the presence or absence of each of the structural elements of clinical integration. Interviews followed a structured protocol, ensuring uniform data collection. In all cases, the Chiefs of Cardiology were able to provide detailed information about the services available at both the tertiary and primary VHA hospitals. We also asked the Chief of Cardiology about the degree of competitiveness for cardiac services in the local markets for each of the primary VHA hospitals. This was an ordinal variable, with three levels: non-competitive; moderately competitive; or highly competitive market. In all cases, the Chief of Cardiology was able to answer the questions about market competition in the primary hospital market without difficulty, indicating considerable awareness of market conditions and the impact these had on their referral base.In addition, we constructed two separate variables to control for patient distance from the primary VHA hospital to which they were initially admitted, and to control for the distance between primary and tertiary VHA hospitals. The patient distance variable was measured as the distance from the patient's home zip code centroid to the primary VHA hospital. The distance between the primary and tertiary referral VHA hospitals was measured in miles using VHA national databases. We tested different specifications of the distance variables, concluding that it was best to enter the distance between primary and tertiary VHA hospital as a continuous variable, whereas it made no difference in the results of the estimation what form we used for patient distance to primary VHA hospital. In the final analyses, it was dichotomized at greater than or equal to 100 miles – approximately two hours driving time. The patient distance variable is measured at the patient level, while the hospital distance variable is measured at the hospital level.We included several measures of patient clinical characteristics, including age 65 or over; prior history of chronic obstructive pulmonary disease, bleeding disorder (such as hemophilia or anticoagulation therapy), smoking, prior percutaneous coronary intervention (PCI), or chronic heart failure; having a "Do Not Resuscitate" order, and several measures of seriousness or urgency of condition during the index admission in the primary VHA hospital: ST segment elevation on electrocardiogram or elevated cardiac enzymes at presentation; and a composite variable indicating the presence of a serious event during admission. Presence of a serious event during admission was a binary variable taking the value "1" if at least one of the following conditions was present: angina persisting more than 24 hours after admission; hypotensive episode; heart failure during admission; cardiac arrest; or positive stress test during admission. All of these variables were abstracted from the medical record.We explored the bivariate association between clinical integration variables, distance variables, patient characteristic variables, and patient transfer using one-way analysis of variance with Scheffe correction for multiple comparisons.To construct the most parsimonious models using the full set of candidate independent variables , we used backward stepwise logistic regression, beginning with all available patient clinical characteristics that have been shown to be significant in predicting mortality outcomes for ACS patients in prior studies. We eliminated variables from the model if the p-value for the variable was greater than 0.1. A number of the candidate variables, including many of the history and co-morbidity variables, were found to be insignificant, and we created a summary variable described above which included many of the highly significant variables from the index hospital admission (details available from authors). C-statistics for each of the final models ranged from 0.77 to 0.85. We used Stata SE version 8.2 for all analyses.We then investigated the relationship between clinical integration of cardiac services and transfer rates using random effects logistic regression , correctAmong the 12 primary VHA hospitals included in the sample, the mean rate of transfer was 42% (319 of 725). Mean rate of transfer to a tertiary VHA hospital was 31% (237 of 725), and to a private hospital was 11% (82 of 725). Most patients were transferred in order to receive cardiac catheterization or coronary revascularization. In addition, 37% of patients were treated in primary VHA hospitals that were over 250 miles from their tertiary referral VHA hospital, and 18% of patients lived over 100 miles from the primary VHA hospital to which they were admitted.Three of the 12 primary care VHA hospitals had a VHA cardiologist available at least episodically on site; six had a referral coordinator at the associated tertiary center; and four had a referral coordinator at the primary VHA hospital. The distribution of these components is shown in Figure The bivariate associations between the patient characteristic variables, clinical integration variables, and type of transfer are shown in Table Results of the random effects logistic regressions for transfer to any tertiary care hospital are shown in Table Patient factors that decreased the likelihood of transfer to any tertiary hospital included history of chronic obstructive pulmonary disease, or bleeding disorder; and having a do not resuscitate (DNR) order during the hospital admission. In addition, the further the distance between primary and tertiary VHA, the less likely patients were to be transferred at all, and the more competitive the market for cardiac care, the less likely that the patient was transferred to a tertiary care hospital.All three components of integration were significantly associated with transfer to tertiary care, although in different directions. After adjustment for patient and other characteristics, the presence of a VHA staff cardiologist and having a referral coordinator at the tertiary VHA hospital decreased the likelihood of transfer to any tertiary care hospital. In contrast, the presence of a referral coordinator at the primary VHA hospital increased the probability of transfer to a tertiary hospital.The results of this analysis are shown in Table The degree of market competition was not significantly associated with transfer to VHA versus private tertiary hospital. Neither of the distance variables were associated with transfer either to VHA or non-VHA tertiary hospitals. Furthermore, only one of the individual integration variables entered separately were significantly associated with likelihood of transfer to tertiary VHA versus private hospital, and although the parameter estimate for the variable indicating presence of a referral coordinator at the tertiary hospital was large and significant, it was very imprecise (i.e. large standard error). This is probably due to the relatively small number of patients included in the estimation (N = 319) and uneven splits among hospitals, clustered by VISN.The goal of this study was to investigate the association between measures of clinical integration of care and transfer of patients with acute coronary syndromes in the VHA. In particular, we evaluated whether structural components of clinical integration, such as the presence of referral coordinators and on-site cardiologists, were associated with patient transfer within and/or outside of the VHA healthcare system. In multivariate analysis, the presence of referral coordinators located at primary care VHA hospitals increased the overall likelihood of transfer of ACS patients. In contrast, having a VHA staff cardiologist available or a referral coordinator at a tertiary VHA hospital significantly decreased the likelihood of any transfer to a tertiary care hospital. Finally, we found that only one of the three integration components, presence of a referral coordinator at the tertiary VHA hospital, was significantly associated with transfer to a tertiary VHA hospital compared to a non-VHA tertiary hospital.Our finding that referral coordinators at primary care hospitals increase the likelihood of transfer to tertiary care hospitals is consistent with prior studies demonstrating that referral coordinators increase the ease of referral and frequency of transfer ,15-19. PHowever, our finding that the presence of a referral coordinator at a tertiary VHA hospital was negatively associated with transfer appears contradictory. It is possible that referral coordinators at the tertiary centers may facilitate consultation, which may, at least for lower risk patients, appropriately reduce the need for transfer. However, it is of some concern that these referral coordinators may be serving in a gatekeeper role with regard to transfer decisions. Future research should focus on the role and decision-making associated with these referral coordinators. Of note, when transfer did occur, the presence of a referral coordinator at the tertiary VHA hospital was positively associated with transfer to VHA facilities rather than non-VHA facilities. This suggests that referral coordinators may function differently with different kinds of patients, decreasing overall transfer rates but facilitating within-system transfer when transfer occurred.In general, we found that transfers to tertiary care were largely associated with patient characteristics appropriate to transfer: sicker and more urgent patients, except for those for whom more intensive care may not be indicated (e.g. DNR status), were significantly more likely to be transferred. In particular, patients with ST-segment elevation on their presenting electrocardiogram and abnormal cardiac enzymes were significantly more likely to be transferred, most likely for coronary revascularization. These patients are most likely to benefit from revascularization ,9, and tDistance between the patient's home and primary VHA hospital was significantly associated with increased likelihood of subsequent transfer to a tertiary care hospital. This may indicate that patients who live further from the hospital take longer to present and are therefore sicker on arrival, leading to the requirement for higher levels of care. Also of interest, distance between primary and tertiary VHA hospitals was significantly associated with a decreased likelihood of transfer, indicating that in situations where primary and tertiary VHA hospitals are further apart, primary VHA hospitals may elect to keep more ACS patients rather than transfer them at all. Future research is needed on the appropriateness of transfer of ACS patients, as it is not clear that variation in transfer based on distance between hospitals represents appropriate variation in care.The finding that cardiologist availability at the primary VHA hospitals was associated with less transfer to tertiary care hospitals may reflect that local or distant cardiology consultation was sufficient in some cases (e.g. lower risk patients) to avoid transfer. Similarly, the availability of a transfer coordinator at the tertiary VHA hospital may have provided an avenue for consultation and avoidance of transfer in some cases. Future studies are needed to define the mechanisms of association between reduced transfers and both on-site cardiology availability and tertiary hospital transfer coordinators.The findings of this study, that referral coordination is associated with transfer from primary to tertiary hospitals, but may operate differently for different types of patients, and may have one mechanism of operation within a health care system and another outside that system, have potential application outside VHA. Previous studies  have fouFirst, we were not able to conduct full-scale validation and reliability testing of the clinical integration measures, which would have required a larger sample of hospitals participating in the study to conduct split-sample validation. Second, we used structural, rather than process, elements of integration in this analysis. We focus on structural elements both because they are relatively easier to measure (present or not), and because in Donabedian's widely accepted model of quality in health care, structure precedes process and outcome ,23. ThirFourth, because transfer is closely related to patient outcomes, especially for ACS patients , carefulWe found that referral coordinators located at primary care VHA hospitals increase the overall likelihood of transfer of ACS patients. Referral coordinators at tertiary VHA hospitals and the presence of on-site cardiologists appeared to decrease the likelihood of transfer. Only one component of integration, presence of a referral coordinator at the tertiary hospital, was associated with within-system compared to out-of-system transfer. These findings have significant potential implications for the VHA. One of the goals of an integrated health care system is to maintain optimal coordination between its component parts . This stThe author(s) declare that they have no competing interests.AES participated in the design and conduct of the study, conducted the analyses and wrote the manuscript. SLP participated in conducting the project, and assisted in writing the manuscript. DJM participated in writing the manuscript. NRE participated in the design and conduct of the study. NDS participated in writing the manuscript. JSR participated in the statistical analyses and co-wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To determine the frequency, risk factors and mortality of nosocomial pneumonia a prospective study was conducted in the intensive care units. In the study period, 2402 patients were included. The nosocomial pneumonia was defined according to the Centers for Disease Control Criteria. Overall, 163 (6.8%) of the patients developed nosocomial pneumonia and 75.5% (n = 123) of all patients with nosocomial pneumonia were ventilator-associated pneumonia. 163 patients who were admitted to the intensive care unit during the same period but had no bacteriologic or histologic evidence of pneumonia were used as a control group. The APACHE II score, coma, hypoalbuminemia, mechanical ventilation, tracheotomy, presence of nasogastric tube were found as independent risk factors. Crude and attributable mortality were 65% and 52.6%, respectively. The mortality rate was five times greater in the cases . The mean length of stay in the intensive care unit and hospital in the cases were longer than controls (p < 0.0001). Patients requiring mechanical ventilation have a high frequency of nosocomial pneumonia. Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia) are associated with higher mortality [Nosocomial pneumonia (NP) is the most frequent nosocomial infection in the intensive care units (ICU). The reported frequency varies with the definition, the type of hospital or ICU, the population of patients, and the type of rate calculated. In the recent studies, the incidence was reported as 6.8–27% -4. In anortality ,11,12. Portality .Studies on NP are mainly reported from the United States and European countries, whereas studies from around the world are missing. The aims of this study were to assess incidence, risk factors and mortality of NP in Eurasian intensive care units.2) blocker therapy, clinical outcome, length of stay in ICU and in the hospital.Between February 2001 and February 2002 a prospective study was conducted among intensive care units (ICU) patients of the Erciyes University Hospital. This university hospital is a teaching hospital and full time intensivists care the patients in ICUs. Patients from the surgical ICU (SICU) (24 beds), medical ICU (MICU) (9 beds) and burn unit (7 beds) were included. The SICU consist of 8 neurosurgical (NICU), 8 general surgery (GICU) and 8 cardiac surgery (CICU) beds. Patients older than 16 yr of age were included. The same infection control doctor collected data and intensivist reviewed the diagnosis of pneumonia. Data collection included physical examination findings, APACHE II scores on admission, consciousness, risk factors , prior surgery, immunosuppression, prior antimicrobial and antacid or histamine type 2  purulent endotracheal secretions with a Gram stain showing one or more types of bacteria [NP was considered when new and persistent (more than 48 h) pulmonary infiltrates not otherwise explained appeared on chest radiographs. Moreover, at least two of the following criteria were also required: 1) fever >38°C; 2) peripheral leukocyte count >10 000/mmbacteria . VAP wasbacteria . AdmissiExtra length of stay was calculated comparing the extra stay after onset of pneumonia in the cases and after a reference date  in the control group.Giemsa stains of sputum samples were performed for all patients. Sputum samples, containing more than 25 polimorphonuclear leukocyte (pnl) and less than 10 (×100) epithel were classified as purulent. If necessary, samples were obtained by nasotracheal aspiration. In that case, samples containing more than 10 pnl (×1000) were defined as purulent. Quantitative cultures of all purulent samples were performed using standard methods. Susceptibility testing was performed by disc diffusion method. In the absence of an alternative diagnosis a bronchoalveolar lavage was performed. In some case, pleural fluid was obtained by thoracentesis and examined for cell count, smear, Gram- and Giemsa-staining and microbiological culture.All data were evaluated using SPSS. Parameters were compared using univariate and multivariate logistic regression and chi-square tests. Student t test was used to compare the extra length of stay. Data were given as mean ± SD and a p-value of <0.05 was accepted as significant.2 = 19.7, p < 0.0001). Length of stay in the MICU was significantly higher than in the SICU, 21.3 ± 21.4 versus 16.2 ± 8.8 days, respectively (p < 0.05). Characteristics of patients who developed NP are shown in table During the study period, 2402 patients were admitted to the ICUs. Distribution of patients by ICU and length of stay in the ICU are shown in table 2 = 6.6, p < 0.01). Consequently, the incidence of VAP was higher for MICU- than SICU-patients (X2 = 29.2, p < 0.0001). Furthermore, the length of ventilation was higher for patients admitted to MICU (6.3 ± 4.0) than SICU (5.1 ± 3.7), but the difference was not statistically significant.Overall, 17% of all patients requiring MV (n-724) developed VAP. Thereby, VAP accounts for 75.5% (n = 123) of all patients with NP (n = 163) during the study period  Table . MechaniDuring the study period patients received 3128 ventilation days, with an average duration of 11.3 ± 10.0 days per ventilated patient. The device-related incidence rate for VAP was 39.3/1000 ventilation days. The incidence per 1000 ventilation days was 41.9 in SICU, 36.6 in MICU, 66.0 in NICU, 38.0 in GICU, and 9.1 in CICU patients. The mean onset day of NP after MV was 4.2 ± 3.9 days.Univariate analysis suggested the following risk factors for the development of NP: the APACHE II score, coma, COPD, uremia, hypoalbuminemia, MV, tracheotomy, enteral feeding, presence of nasogastric tube and previous treatment with broad-spectrum antibiotic (Table A. baumannii (29.6%), P. aeruginosa (20.6%), Klebsiella pneumoniae (14.4%) were the most common.187 pneumonia episodes were observed during the study period, resulting in the isolation of 257 microorganisms. The most commonly isolated pathogens were Gram-negative bacteria (85.6%). Among these pathogens, Empiric antibiotic therapy was based on previous surveillance cultures and the Gram stain results. Therapy was adjusted according to the reports of susceptibility testing.Crude and attributable mortality were 65% and 52.6%, respectively. The mortality in patients without NP was 26.4% Table . The risThe mean length of stay in the ICU and hospital for the patients with NP were 18.04 ± 14.74 days and 23.49 ± 16.44 days, respectively. The mean length of stay in the ICU and hospital for the control group 3.10 ± 3.03 and 9.64 ± 5.08 days, respectively. This difference was statistically significant (p < 0.0001). The extra stay in the control group was 4.36 ± 3.87 and 17.04 ± 14.17 in the patients (p < 0.001).The incidence of NP was reported different in different studies, which may be justified by the presence of different populations with variable ages, underlying diseases, and other associated risk factors. Incidence ranges from 6.8 to 27% -4 and alMV increases the risk of NP by 3- to 10-fold -23, resuComa was described as another important risk factor for NP. In these patients, local defense mechanisms of the respiratory airway are altered, allowing microorganisms to better attach to and colonize the mucosal surface. Furthermore, depression of the level of consciousness significantly increases the chance of aspiration, and as a result development of NP ,28. In o2 antagonists did not increase the risk of NP.The causative agents of NP differ by the study population and diagnostic techniques but generally Gram-negative bacteria are the most common ones ,4,28-33.In the literature, tracheotomy is described as a significant risk factor for NP. Bronchial colonization during the procedure and (prolonged) continuation of sedation after the procedure will furthermore increase the occurrence of NP , a fact The role of advanced age and high APACHE II scores as risk factors of NP are still under discussion. While Kollef et al.  report tPatients with NP have a significantly higher morbidity and mortality ,35,38. HAs a result of the increased morbidity, patients with VAP remain hospitalized for 4–17 days longer than controls -38. ThisIn conclusion, NP is a major cause of morbidity and mortality in ICU patients. Especially patients on mechanical ventilation are at high risk. Studies determining the impact of "old" and "new" risk factors of NP should repeatedly be performed in order to effectively guide the implementation of preventive measures methods.


Defending against attack is one of the most important challenges facing any organism. But while sticks and stones may break the bones of a lion, microscopic threats such as bacteria require different weapons. And it's not just we humans who have this problem—insects are prey to bacterial infections too. Their immune systems, however, rely on a far simpler set of defenses than those found in mammals. Exactly how one insect immune system recognizes bacteria, and how it fights off the invader, is the subject of a new study in this issue by Johann Deisenhofer and colleagues.Drosophila, has long been known to use a set of molecular sentries called “peptidoglycan recognition proteins,” or PGRPs, that circulate in the fly's bloodstream. When a PGRP recognizes a bacterial invader, it triggers a cascade of events whose ultimate product is a group of antimicrobial compounds that attack and kill the bacteria.The fruitfly, While the family of PGRPs has been extensively studied, exactly how they recognize their target bacteria has been less clear. At the cellular level, recognition requires contact, and the part of the bacterium the PGRP recognizes is, as its name implies, the peptidoglycan. A peptidoglycan is a special sort of molecular polymer found primarily on bacterial cell walls. Peptidoglycan forms when chains of sugar molecules (the glycans) are cross-linked by amino acids (the peptides) to form a meshwork that helps keep the bacterium from bursting under the osmotic strain of its contents.Drosophila defensive reaction. Deisenhofer and colleagues set out to determine whether this difference in triggering ability of particular peptidoglycans was linked to differences in the PGRPs that recognize them. To do this, they determined the three-dimensional structure of one PGRP, called PGRP-SA. They worked out not only the overall shape of PGRP-SA, but also which amino acids sat where on the convoluted surface of the protein.There are several types of peptidoglycans that differ in their precise sugar and amino acid constituents and in their ability to trigger the What they found on that surface was an extended groove down one entire side of the protein. To test whether this groove was indeed the recognition site for peptidoglycan, the group introduced a series of mutations to critical amino acids along the groove, testing each new form for its ability to bind peptidoglycan. Indeed, the binding and defense-triggering ability was worse for almost every mutant, demonstrating conclusively that the normal protein uses the groove to bind and recognize peptidoglycan.Then the team made a surprising discovery. They found that when PGRP-SA comes in contact with bacterial peptidoglycan, it begins to cleave the links between amino acids in the peptide portion of the peptidoglycan. This in itself is not so amazing—animals make plenty of peptide-cleaving proteins. But this protein has a difference, one which makes it unique in the animal kingdom.To understand this difference, consider your two hands. They are mirror images of each other, alike yet not the same. No amount of twisting and turning will allow you to superimpose one exactly on the other—if you align the fingers, the knuckles will point in opposite directions, and if the knuckles point the same way, the fingers are all mismatched. This type of relationship between mirror images, called chirality (from the Greek for “hand”), is found in amino acids as well, a result of the three-dimensional geometry that radiates from their central atom.Drosophila PGRP-SA was able to break apart this “L,D” (levo-dextro) linkage, making it, in their words, “the first eukaryotic protein exhibiting such an activity specific for peptide bonds existing only in prokaryotes.”All the amino acids used by all known animal species are exclusively of the “left-handed” form, and the protein-digesting enzymes we make are designed specifically for these L-amino acids. Bacteria, however, link left-handed and right-handed amino acids together to form peptidoglycan. What Deisenhofer's team discovered was that unlike any other known animal enzyme, the What does it all mean? Deisenhofer and colleages' results are yet another demonstration that at the molecular level, understanding structure is the key to understanding function. They also show that when it comes to defense, it helps to be able to fight with both hands.


Pro and Raz are bisdioxopiperazine compounds. In this work, we evaluated the anti-tumor and anti-metastatic effects of Pro and Raz 5. Pro and Raz were active against primary tumor growth and significantly inhibited pulmonary metastasis of LLC at same dose-ranges (inhibitory rates > 90 %). Both Raz and Pro were effective in 1, 5, and 9 day administration schedules. Three different schedules of Raz and Pro were effective against the primary tumor growth of LLC (35–50 %). The synergistic anticancer effect of Raz with bleomycin (Ble) (from 41.3 % to 73.3 %) was more obvious than those with daunorubicin (Dau) (from 33.1 % to 56.3 %) in the LLC tumor model. Pro was also seen to have synergistic anti-cancer effects with Ble in the LLC model. Both Raz and Pro inhibited the growth of LAX 83 in a statistically significant manner.After determining the lethal dosage of Pro and Raz, we assessed and compared the inhibitory effects of Pro and Raz against primary tumor growth and metastatic occurrences of LLC at the dosage of LDThese data suggest that both Raz and Pro may have anti-tumor potentiality and Raz and Pro have combinative effects with Ble or Dau. The potential targets of bisdioxopiperazines may include lung cancers, especially on tumor metastasis. The anti-cancer effects of Raz and Pro can be increased with the help of other anticancer drugs. Raz), first developed in UK, was the earliest agent against spontaneous metastasis for the murine model (Lewis lung carcinoma) in 1969 [Raz for anticancer actions, like assisting radiotherapy, [Raz, as a cardioprotectant of anthrocyclines, has been licensed in 28 countries in 4 continents. Since morpholine groups in some structures were reported to be responsible for cytotoxic or modulative actions on tumors, an anticancer agent, probimane [1,2-bis (N4-morpholine-3, 5-dioxopeprazine-1-yl) propane; AT-2153, Pro] was synthesized by introducing two morpholine groups into Raz in China.[Raz and Pro belong to bisdiopiperazines. Like Raz, Pro also exhibits anti-tumor activity both in vivo and in vitro against experimental tumor models in a small scale investigation [Pro could inhibit human malignant lymphoma even for those resistant to other anticancer drugs [Raz, like detoxication of Adriamycin (ADR) induced cardiotoxicities, and synergism with ADR against tumors [Pro, like inhibiting the activity of calmodulin (CaM), a cell-signal regulator, which can explain anticancer actions and the combined cytotoxic effect of Pro and ADR [LPO) of erythrocytes [Razoxane ICRF-159) (59 (Raz),therapy,  overcomitherapy, , inhibittherapy,  and so otigation  and limit tumors . We have and ADR . Pro washrocytes , influenhrocytes  and inhihrocytes .MDR) and by high metastatic occurrence in clinics [Pro, like Raz may possess useful therapeutic potentialities, we evaluated in vivo the chemotherapeutical parameters of Pro and Raz for lung cancer of both murine and human origins.Lung cancer is the No 1 killer among all categories of cancers in urban areas in China and many Western countries. The high mortality rate of lung cancer can easily be caused by inducing multi-drug resistance  and Raz (20 mg/kg ip × 7) as equitoxic dosages for further treatment studies.The lethal dosage of Pro and Raz on LLC are tabulated in Table Pro and Raz at equitoxic dosages (LD5) showed a noticeable anticancer effect on primary tumor growth (inhibitory rates, approximately 30–45 %), and significantly inhibited the formation of tumor metastases (inhibitory rates on pulmonary metastasis > 90 %, P < 0.001). Primary tumor growth of LLC was inhibited more by Pro (48 %) than by Raz (40.3%) in a 20 day trial, whereas the inhibition of Pro (35.7%) was slightly less than that of Raz (40 %) on an 11 day trial. Pro seems to be more persistent than Raz in inhibiting primary tumor growth of LLC.Antitumor and antimetastatic effects of Raz and Pro on LLC are included in Table Raz and Pro were effective in a statistically significant manner with the 3 injection schedule of the 1, 5 and 9 day administrations on LLC. If we administered Raz to tumor-bearing mice once on day 1, 5 and 9, there was no difference between treatment and vehicle control. Antitumor effects of Raz in combination with Ble on LLC (73.3 %) were better than those in combination with Dau (56.3 %)  and Pro (80–100 mgKg-1 ip × 5) with inhibitory rates of 25–32 % and 55–60 % respectively (P < 0.01 vs control). CTX, as a positive anticancer drug (40 mgKg-1 ip × 5), exhibited antitumor activities against the growth of LAX-83 with an inhibitory rate of 84 %. Obvious necrosis in tumor tissues was observed by histological evaluation of CTX and Pro treatment groups, but Pro showed larger vacuoles than CTX. Drug inhibition on tumor volumes were calculated and outlined in Table Pro was the same or better than those of MTX, DDP and as well as 5-Fu against LAX-83 tumor growth.The experiments showed that LAX-83 was sensitive to bisdioxopiperazines are now inconclusive. The present explanation for the anticancer mechanisms of Raz has been attributed to antiangiogenesis and topoisomerase II inhibition.[Raz and Pro were much stronger than those actions against primary tumor growth, this special targeting on metastasis ought to be more useful in clinical cancer treatment. Raz and Pro show typical characteristics of antiangiogenesis agents, which target small nodule of tumors. Meanwhile, recent reports on drugs targeting angiogenesis indicate that most anti-vascular drugs have low or even no effects on most cancers when they are used alone in clinics, but they show synergistic effects in combination with other anticancer drugs. [Raz and Pro with Ble or Dau basing on this theory. Previous work showed that Pro and Raz could reduce the cardiotoxicity of anthrocycline,[anthrocyclines. The data in our study suggests that the synergistic effects of Raz with anthrocyclines are present, but not as potent as those with Ble.Explanations of anticancer and antimetastatic mechanisms of hibition. Since thr drugs. ,18 Our socycline,,10 so wePro and Raz, we suggest that anticancer effects of Pro and Raz are within the effective anticancer ranges of commonly available anticancer drugs.Since we have tested the antitumor activity of clinically available anticancer drugs (CTX, 5-Fu, MTX, DDP and VCR) against LAX-83, CTX being the best one, two bisdioxopiperazines studied on this work show overall similar anticancer effective as commonly used drugs. Although the anticancer effects of CTX and VCR are better than those of Pro, for other commonly used drugs, such as DDP, MTX and 5-Fu, the antitumor effects are no better than those of Pro. Since the antitumor effects of MTX and DDP are even less effective than those of Pro is that it is the most water-soluble among the bisdioxopiperazines. Most bisdioxopiperazines are less water-soluble and given orally in clinics. Although oral administration is easy for patients, bioavailability varies from patient to patient. For some patients who have a poor absorption of bisdioxopiperazines in oral administration, Pro can be injected iv to maintain stable drug levels. Our previous work showed that Pro could strongly accumulate in tumor tissue while Pro levels in other tissues decrease rapidly [Raz, (dexrazoxane, ICRF-187), a water-soluble Raz, is being reinvestigated and has aroused the interests of clinical oncologists. Phase III clinical studies are currently underway in the US. More importantly, ICRF-187 was licensed in 28 countries in 4 continents. This work shows a noticeable inhibition of Pro and Raz on lung cancers and suggests possible usage of Raz and Pro on lung cancer in clinics.The other useful property of  rapidly . Presentbisdioxopiperazines in clinical treatment of lung cancers are as follows: (i) Pro and Raz can inhibit the growth of lung cancers, with and without the help of other anticancer drugs, like Dau and Ble; (ii) like Raz, Pro strongly inhibits spontaneous pulmonary metastasis of LLC; (iii) since Pro can inhibit CaM [MDR and metastatic phenotypes, synergistic anticancer effects of Pro and Raz can be expected in combination with other anti-cancer drugs, like Dau or Ble. Now, new concepts of the relationship between tumor metastasis and MDR in cancers have been stated,[bisdioxopiperazines can inhibit both tumor metastasis and MDR. As a counterpart of Raz, Pro might be of interest and have chemotherapeutic potential in clinics.The advantages of ibit CaM , a calcin stated, whereas CTX), daunorubicin (Dau) and bleomycin (Ble), 5-fluororacil (5-Fu), vincristine (VCR), cisplatin (DDP), methotrexate (MTX) were purchased from Shanghai Pharmaceutical Company. Pro and Raz were prepared by Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. C57BL/6J and Kun-Min strain mice were purchased from Shanghai Center of Laboratory Animal Breeding, Chinese Academy of Sciences. Nude mice (Swiss-DF), taken from Roswell Park Memorial Institute, USA, were bred in Shanghai Institute of Materia Medica, Chinese Academy of Sciences under a specific pathogen free condition. Human pulmonary adenocarcinoma xenograft (LAX-83)[LLC) were serially transplanted in this laboratory. All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals, NIH, established by Washington University's Animal Studies Committee. Bouin's solution consists of water saturated with picric acid: formaldehyde: glacial acetic acid (75: 20: 5, v/v/v).Cyclophosphomide ( (LAX-83) and Lewiip injected with Pro and Raz daily for 10 successive days. The deaths of mice were counted after 1 month. Lethal dosage of agents was calculated by Random Probity tests.Mice of Kun-Min strain  were sc with LLC (2 × 106 cells) from donor mice. The mice were injected intraperitoneally with drugs daily or every two days for 7 injections. On day 11 or day 20, mice were sacrificed, and locally growing tumors were separated from skin and muscles and weighed, and lungs of host mice were placed into a Bouin's solution for 24 h, and then the lung samples were submerged into a solution of 95 % alcohol for 24 h. Finally, the numbers of extruding metastatic foci in lungs were counted.C57BL/6J mice were implanted sc with LLC (2 × 106 cells) from donor mice. Mice were injected intraperitoneally with drugs on day 1, 5, 9. Single injection or 3 injections every 3 hours were used. Tumors were separated and weighed on day 11.C57BL/6J mice were implanted LAX-83. Then nude mice were sacrificed, and their kidneys were taken out for measurement of tumor sizes using a stereomicroscope a week after transplantation. Tumor volume was calculated as 1/2(ab2) where a and b are their major and minor axes of the lump. Kidneys with tumors were paraffin-embedded, sliced and hematoxylin dyed. The tumor tissues were then observed from a light microscope.Nude mice were inoculated with LAX-83 under the renal capsule (SRC method). Nude micStudent's t-test was used to assess the differences between control and drug treatment groups of above methods.Pro, probimane; Raz, razoxane; CaM, calmodulin; LPO, lipoperoxidation; Dau, daunorubicin; Ble, bleomycin; LLC, Lewis lung carcinoma, LAX-83; a lung adenocarcinoma xenograft; ADR, adriamycin;The experimental design was made by Bin Xu and Da-Yong Lu.Experiments were performed by Da-Yong Lu (anticancer activity tests)The manuscript was written by Da-Yong Lu, and Jian Ding.


Peritoneal carcinomatosis from gastrointestinal cancer has new treatment options for surgical management. The approach uses cytoreductive surgery which combines peritonectomy and visceral resection in an effort to remove all visible cancer within the abdomen and pelvis. Then the peritoneal cavity is flooded with chemotherapy solution in an attempt to eradicate residual disease. In order to select patients for this approach the quantitative prognostic indicators for carcinomatosis were reviewed, compared and contrasted. Prognostic indicators to be used to select patients for this aggressive approach at the initiation of surgery and after completion of cytoreduction were studied. Four quantitative assessments to be used at the time of abdominal exploration were the Gilly staging, Japanese gastric cancer P score, peritoneal cancer index (PCI), and the simplified peritoneal cancer index (SPCI). All have value with the PCI being the most validated and most precise. Preoperative assessments include the tumor histopathology and the prior surgical score. The completeness of cytoreduction score is an assessment of residual disease after a maximal surgical effort. An opportunity for long-term survival following treatment for carcinomatosis requires a complete cytoreduction in all reports for gastrointestinal cancer. Quantitative prognostic indicators need to be knowledgeably employed when patients with carcinomatosis are being treated. Improved patient selection with greater benefit and reduced morbidity and mortality should result. Peritoneal carcinomatosis has always been regarded as a terminal condition. It is present in 10 to 30% of patients with gastrointestinal cancer at the time of their initial surgery and is a frequent finding in patients who develop recurrent cancer. Important natural history studies establish a 6-month median survival in this group of patients ,5. PatieQuantitative prognostic indicators are to serve as guidelines in the selection of treatments to maximize benefits of therapy and to exclude patients who have little or no chance to improve. They are of greatest utility in high risk and costly management protocols. Requirements of a useful quantitative prognostic indicator include reproducibility, prediction of survivorship, and assessment of morbidity and mortality. The goal is to establish management protocols that standardize the decision making process for multiple caregivers.General surgery has used quantitative prognostic indicators in the past with established benefit to patient care. Examples of quantitative prognostic indicators currently in use include Ranson's criteria, which estimates the risk of life threatening complication or death in patients with acute pancreatitis; and, the Child-Pugh score for liver cirrhosis, which evaluates the severity of liver disease correlating grades with one- and two-year survival. Currently, there are several clinical assessments at many different institutions in use for the evaluation of carcinomatosis  and peritoneal mesothelioma. In these diseases a non-invasive process may be widely disseminated on the peritoneal surfaces. The biological aggressiveness of the malignancy can be estimated by the pathologist in a knowledgeable histologic review of multiple specimens. For pseudomyxoma peritonei syndrome, the histologic classification described by Ronnett and colleagues has been most widely utilized .Histopathologic examination categorizes the disease process into disseminated peritoneal adenomucinosis (DPAM), peritoneal mucinous carcinoma (PMCA) or a hybrid type.This is a minimally invasive disease, and therefore more likely to be completely removed by cytoreduction using peritonectomy. The histology of DPAM shows a bland single layer of epithelium that surrounds lobules of mucin. There are no signet rings and there is minimal atypia. Invasion of the structures upon which tumor accumulates does not occur. The primary site for DPAM is an appendiceal adenoma which has minimally invaded the wall of the appendix. Usually, the widespread intraperitoneal dissemination of mucinous tumor is caused by a rupture of the lumen of the appendix from pressure built up by the malignant mucocele. Figure This is an invasive disease in which the mucinous cancer cells show invasion into surrounding tissues. Sometimes, the signet ring morphology or lymph node metastases are present. The cancer cells will be found in multiple layers surrounding the mucinous tumor globules. There is loss of nuclear polarity and atypia is common. The quantity of mucus may be variable from one patient to the other. However, the PMCA histology may be associated with very large amounts of mucoid ascites fluid. Therefore, it is categorized as pseudomyxoma peritonei syndrome but with an aggressive tumor histology. Figure In the hybrid type of mucinous carcinomatosis the field of view for the pathologist shows 95% or more DPAM. PMCA is present but in 5% or less of the total field of view figure . If therNot surprisingly, the observation has been made by numerous groups that the non-invasive mucinous tumors (DPAM and hybrid type) are amenable to complete cytoreduction. Therefore more definitive treatment and improved survival using the combined approach is expected with DPAM and hybrid type . The hisThe quantitation of tumor found at the time of surgical exploration of the abdomen has proven to be of value in assessment of prognosis and treatment planning. Four different assessments have been published. They are listed in Table The Gilly peritoneal carcinomatosis staging format was first described in Lyon in 1994 . This prAlthough the Gilly system has been used for almost a decade with acceptable prognostic value, there are some criticisms regarding this system. First, it should not be designated a "staging system" because patients can only be staged once in the course of their disease at the time of diagnosis of the primary malignancy. Usually, a TNM staging system is appropriate. The system might better be called the Gilly prognostic index for carcinomatosis.A second weakness of the Gilly prognostic index concerns a failure to quantitate distribution of peritoneal surface implants in the stage 3 and 4 categories. Carcinomatosis confined to one portion of the abdomen may carry an excellent prognosis even if the localized tumor implants are of large size. If group III and group IV nodules by size are diffuse throughout the whole abdomen, certainly a much different prognosis would occur. A definitive assessment of not only the size of the nodules but also the distribution of carcinomatosis is necessary for the most accurate assessment of prognosis.The Japanese have proposed a quantitation of carcinomatosis that is very simple, has been frequently applied, and has been validated for gastric malignancy. For the original staging a "P factor" is indicated for gastric cancer patients. P-0 means that no carcinomatosis was seen by the surgeon or could be established at the time of surgery. It would currently include patients who are cytology positive for gastric cancer cells. P-1 indicates implants immediately adjacent to the stomach and above the transverse colon. P-2 indicates scattered implants within the abdomen but not of great number. P-3 indicates numerous implants throughout the abdomen and pelvis.This staging system can also be applied to patients who have carcinomatosis with recurrent gastric cancer. A major deficit of this staging system is its inability to accurately locate the carcinomatosis. Also, it has no size assessment of the cancerous implants. Although the P factor has been of great value historically in the management of primary gastric cancer as peritonectomy and intraperitoneal chemotherapy are used for treatment of carcinomatosis, a more precise prognostic assessment is needed to manage gastric cancer peritoneal seeding.The Peritoneal Cancer Index (PCI), like the other carcinomatosis assessments, is determined at the time of surgical exploration of the abdomen and pelvis. With invasive cancer it serves as an estimate of probability of complete cytoreduction and has been found to be an accurate assessment of survival when cytoreductive surgery and perioperative intraperitoneal chemotherapy are used as treatment .The PCI quantitatively combines the distribution of tumor throughout 13 abdominopelvic regions with a lesion size score. Two transverse and two sagittal planes divide the abdomen into 9 regions. The upper transverse plane is located at the lowest aspect of the costal margin, and the lower transverse plane is placed at the anterior superior iliac spine. The sagittal planes divide the abdomen into three equal sectors. The lines define 9 regions, which are numbered in a clockwise direction with 0 at the umbilicus and 1 defining the space beneath the right hemidiaphragm. Regions 9 through 12 divide the small bowel into upper and lower jejunum and upper and lower ileum Figure . To makeThe lesion size (LS) score is determined after complete lysis of all adhesions and complete inspection of all parietal and visceral peritonea surfaces within the abdominopelvic regions. LS-0 indicates no implants seen. LS-1 indicates implants less than 0.25 cm. LS-2 indicates implants between 0.25 and 2.5 cm. LS-3 indicates implants greater than 2.5 cm. It refers to the greatest diameter of tumor implants that are distributed on the peritoneal surfaces. Primary tumors or localized recurrences at the primary site that can be removed definitively are excluded from the assessment. If there is a confluence of disease matting abdominal or pelvic structures together, this is automatically scored as LS-3 even if it is a thin layer of cancerous implants.The lesion sizes are then summated for all abdominopelvic regions. The extent of the disease within all regions of the abdomen and pelvis is indicated by a numerical score from 0 to 39.In 1995, Sugarbaker and Jablonski published that the PCI was a meaningful assessment for colon cancer but not for mucinous appendiceal tumors . Elias eThis quantitative prognostic indicator for colon carcinomatosis established that for patients scoring greater than 20, palliation is the goal of treatment. Currently, a PCI of greater than 20 is regarded as a relative contraindication to an elective intervention for carcinomatosis from colon cancer. It is associated with a low median survival, approximately the same as median survival without surgical intervention. In patients who have a PCI greater than 20, palliative surgery is indicated in order to alleviate symptoms or to prevent symptoms that may occur in the near future. In an asymptomatic patient with colon carcinomatosis cytoreductive surgery with intraperitoneal chemotherapy with cure as a goal of treatment is probably not indicated.An exception to the utility of the PCI is found in treating patients with pseudomyxoma peritonei and minimally aggressive mesothelioma. Because the disease is non-invasive, a PCI of 39 can be converted to 0 by cytoreductive surgery. There is a low probability of recurrence after complete cytoreduction with perioperative intraperitoneal chemotherapy and therefore the PCI has no prognostic implication .Another caveat that must be observed when using the PCI occurs in cases in which a low PCI score is recorded in the presence of invasive cancer at a crucial anatomic site. For example, at exploration one may find invasive tumor in and around the common bile duct with little disease elsewhere. Even thought the PCI is low, a complete cytoreduction may not be possible. In these cases, invasive cancer at a crucial anatomic site places the patient into the same category as would systemic metastasis in the lungs or bone. Only palliative surgery is indicated if residual disease post-cytoreduction will be present.The Simplified Peritoneal Cancer Index (SPCI) was established at the Netherlands Cancer Institute and has been used for colorectal and appendieal cancer staging Table . This toVerwaal and colleagues have provided important information regarding the relationship of the Simplified Peritoneal Cancer Index and the incidence of complications in patients who receive combined treatment . In theiThere are marked similarities between the SPCI and the PCI. Both the anatomic distribution of the tumor masses and the size of the tumor masses within each abdominal region are indicated. In the PCI, there are 13 anatomic sites designated by a diagram; in the Dutch SPCI, there are 7 anatomic regions designated by anatomic site. In both systems the volume of tumor in each region is to be scored quantitatively. Some shortcomings of the SPCI could be formulated. First, the epigastric region, very important in determining the completeness of cytoreduction in some diseases is not designated separately. Disease above the stomach in the lesser omental region may cause the cytoreduction to be incomplete .A second major criticism of the Dutch SPCI concerns their misuse of their own tool. In their recent publications they perform a survival analysis by SPCI and a toxicity assessment by the SPCI. However, only the involvement of regions 0–7 was indicated. No tumor size in the regions was indicated ,17.An accepted fact regarding cancer treatment is that the optimal treatment with the highest cure rate, the greatest preservation of function, and the lowest morbidity and mortality is the initial treatment. In the management of carcinomatosis the extent of prior resection before definitive cytoreduction with intraperitoneal chemotherapy has a negative impact on the survival. This occurs because of the cancer cell entrapment phenomenon. Surgery opens tissue planes whose raw surface is a favored site for cancer cell adherence, vascularization and progression. In the use of combined treatment for carcinomatosis, the non-traumatized peritoneal surface is the body's first line of defense against carcinomatosis. Cancer progression deep to peritoneal surfaces, especially disease imbedded in scar, is difficult or impossible to remove by peritonectomy or to eradicate by intraperitoneal chemotherapy.The prior surgical score (PSS) quantitates the extent of surgery prior to definitive combined treatment. It shows that the greater the surgery the poorer the results of carcinomatosis treatment. The assessment uses a diagram similar to that for PCI but excludes abdominopelvic regions 9–12. For a PSS of 0 no prior surgery or only a biopsy was performed; PSS of 1 indicates one region with prior surgery; PSS-2 indicates 2 to 5 regions previously dissected; PSS-3 indicates more than 5 regions previously dissected. This is equivalent to a prior attempt at complete cytoreduction but in the absence of perioperative intraperitoneal chemotherapy. In appendiceal cancer patients with a prior surgical score of 0–2, the survival using combined treatment was 70% at 5 years; with a prior surgical score of 3, the 5-year survival was 51% (p = 0.001) .The Completeness of Cytoreduction Score functions as a major prognostic indicator for the survival in peritoneal mesothelima, colon cancer with carcinomatosis, gastric cancer with carcinomatosis and sarcomatosis . It is tSugarbaker and colleagues found that prognosis can be estimated by completeness of cytoreduction. For colon cancer as shown in Figure Although no formal statement in the literature is available, it is thought that the definition of complete vs. incomplete cytoreduction varies with the histologic type of the malignancy. For example, mucinous tumors by diffusion are well penetrated with intraperitoneal chemotherapy solutions. With minimally invasive mucinous tumors such as pseudomyxoma peritonei, complete cytoreduction may occur in the combined treatment plan with tumor nodules up to a full centimeter in size. In contrast, hard fibrotic non-mucinous colon cancer is poorly penetrated by chemotherapy solution. Only cytoreduction down to no visible evidence of disease would be expected to result in long-term survival with a sclerotic malignant process. Also, some cancers may be remarkably more responsive to chemotherapy than others. This is likely the case with a majority of ovarian cancers. Their complete response to systemic chemotherapy is also frequently seen with intraperitoneal chemotherapy solutions or a bidirectional  approach. In both these situations the definition of a complete cytoreduction scored by a CC-1 designation would vary with the clinical situation.The preoperative CT is an excellent tool in locating and quantifying mucinous adenocarcinoma within the peritoneal cavity . UnfortuThere are some special demands on CT scanning if the radiologic examination is to be optimized. Bowel loops cut in cross section are often indistinguishable from cancer nodules. Only if maximal oral contrast using a barium sulfate compound is utilized to prepare the patient for this examination can the greatest accuracy and the greatest prognostic implications of the examination be realized.Another technical requirement is the imaging of solid tumor layered out on the peritoneal surfaces. Unless there is maximal intravenous contrast with a 60 to 120 second delay after contrast infusion will the confluence of malignancy as a thin layer on the peritoneum be imaged. In some patients, the solid tumor, or semisolid tumor may be distributed to appear as ascites on abdominal and pelvic CT. Much to the surgeon's dismay, upon opening the abdomen, a solid tumor mass filling the abdomen and pelvis and causing adherence of small bowel and small bowel mesentery will be revealed. In this situation, not even palliative surgery can be safely performed. In patients who clinically have a firm abdomen and in whom the surgeon suspects large volume of solid tumor, an ultrasound examination may be required in order to confirm an ascitic versus a solid component of the abdominal and pelvic malignancy. If ultrasound shows that there is only minimal or no ascites and that the large volume of tumor is solid or semisolid, surgical interventions are not beneficial. It is better to determine the nature of the carcinomatosis radiologically than at the time of a major surgical exploration.Quantitative prognostic indicators are of value in management of peritoneal surface malignancy from gastrointestinal cancer. Preoperative CT PCI, intraoperative PCI and postresection CC score have all been reported valuable. As one knowledgeable applies these tests, proper selection of patients for combined treatment may increase benefit and decrease morbidity and mortality.


Periodic epidemics of malaria are a major public health problem for many sub-Saharan African countries. Populations in epidemic prone areas have a poorly developed immunity to malaria and the disease remains life threatening to all age groups. The impact of epidemics could be minimized by prediction and improved prevention through timely vector control and deployment of appropriate drugs. Malaria Early Warning Systems are advocated as a means of improving the opportunity for preparedness and timely response.Rainfall is one of the major factors triggering epidemics in warm semi-arid and desert-fringe areas. Explosive epidemics often occur in these regions after excessive rains and, where these follow periods of drought and poor food security, can be especially severe. Consequently, rainfall monitoring forms one of the essential elements for the development of integrated Malaria Early Warning Systems for sub-Saharan Africa, as outlined by the World Health Organization.The Roll Back Malaria Technical Resource Network on Prevention and Control of Epidemics recommended that a simple indicator of changes in epidemic risk in regions of marginal transmission, consisting primarily of rainfall anomaly maps, could provide immediate benefit to early warning efforts. In response to these recommendations, the Famine Early Warning Systems Network produced maps that combine information about dekadal rainfall anomalies, and epidemic malaria risk, available via their Africa Data Dissemination Service. These maps were later made available in a format that is directly compatible with HealthMapper, the mapping and surveillance software developed by the WHO's Communicable Disease Surveillance and Response Department. A new monitoring interface has recently been developed at the International Research Institute for Climate Prediction (IRI) that enables the user to gain a more contextual perspective of the current rainfall estimates by comparing them to previous seasons and climatological averages. These resources are available at no cost to the user and are updated on a routine basis. It is estimated that more than 110 million Africans live in areas prone to epidemics of malaria. Populations in these areas are infrequently challenged by malaria and, therefore, do not fully develop acquired immunity. As a result, the disease remains life threatening to all age groups. The impact of malaria epidemics could be greatly reduced by timely detection or, ideally, by prediction and prevention through vector control and deployment of appropriate drugs [Rainfall is recognized as one of the major factors influencing variability in malaria transmission in warm semi-arid and desert-fringe areas of the Sahel, the Greater Horn and Southern Africa. Explosive epidemics may occur in these regions after excessive rains, usually with a lag-time of several weeks during which time mosquito vector populations and malaria infections increase rapidly. Epidemics can be especially severe among communities stressed by recent periods of drought and poor food security. Recent research has found that rainfall estimates would be able to provide useful epidemic early warning information, even in highland-fringe settings, such as those in Kenya and Ethiopia, where temperature is also an important limiting factor for the development of the malaria parasite -4. ConseDuring a meeting of the Roll Back Malaria Technical Resource Network on Prevention and Control of Epidemics (RBM-TSN) it was decided that immediate benefit could be realized from the routine availability of a simple indicator of changes in epidemic risk in these regions of marginal transmission . The indThe following article provides an update on existing online rainfall monitoring resources and a description of a new tool that has been developed to address remaining needs of the malaria control communityFollowing discussion among members of the RBM-TSN a consensus map of epidemic risk zones was produced . The map under "RFE Anomaly – Malaria", where the anomaly map for the most recent dekad is displayed along with documentation on how the map is produced. The existence of this online monitoring resource was publicized and their use and validation by control services and researchers was encouraged [The epidemic risk map was then combined with rainfall anomaly data  to provide a simple indicator of changes in risk in epidemic prone areas. Figure couraged . The raicouraged .In the year following the launch of the ADDS dekadal rainfall anomaly maps, WHO commissioned field visits to a number of epidemic prone countries to evaluate whether the National Malaria Control Programmes were aware of this resource and how useful they considered it may be for their efforts. Sudan, Uganda, Niger, Mali and Burkina Faso received field visits. In general, all of the control programmes had been aware of the rainfall anomaly maps, but only those in Uganda and Sudan had monitored them regularly during the previous year. The control programmes in the Sahelian countries did not agree with the epidemic risk zone used in the mask because their recent experience was that epidemic outbreaks had occurred beyond the northern boundary of the epidemic risk zone. This was also partly true in Sudan where epidemic outbreaks have been known to occur along the Nile River margins in the northern half of the country. Uganda's malaria control programme, however, had found the maps to be reasonably accurate and a useful monitoring resource. Further dialogue with malaria control programmes in West Africa and Southern Africa also raised the point that a single dekadal rainfall anomaly map could raise an alert; when in fact the rainfall levels were not abnormally high – but just 10 days earlier than 'normal'. This suggested that additional information about the temporal distribution of rainfall was necessary.. In addition to the most recent map, it is possible to download the dekadal maps for the previous six months and begin to construct a seasonal time series. The integration of the rainfall anomalies maps within HealthMapper also allowed the users to improve their analyses by combining ancillary data related to malaria directly on top of the rainfall anomalies maps for their country. Figure In order to respond to these issues, USGS and WHO-HealthMapper agreed to collaborate on the development of the dekadal anomaly maps in a format which could be downloaded, viewed and archived by surveillance staff directly in HealthMapper, a basic mapping and surveillance software developed by WHO's Communicable Disease Surveillance and Response Department . It displays the most recent dekadal rainfall map  have since developed a web-based Malaria Early Warning System (MEWS) interface that enables the user to gain a broader contextual perspective of the current rainfall season by comparing it to previous seasons and climatological averages. The interface is in the IRI Data Library and takes the form of an online 'clickable map' of Africa: p Figure  over whip Figure . These tAccess to frequently-updated rainfall information is an important requirement for the development of integrated early warning systems for malaria and other climate sensitive diseases ,10. ThesWhile it is recognized that much of Africa does not (yet) have easy access to the internet, email is becoming more prevalent and it is now relatively easy for regional support centres, such as the WHO-Intercountry Programme for Malaria Control in Southern Africa, to prepare bulletins with malaria relevant climate data for distribution by email and courier to district health teams in epidemic prone areas as part of an overall MEWS process .


Actinomycosis is a rare inflammatory disease caused by an anaerobic bacterium that can rarely affect the large intestine.We present a rare case of a cecum and ascending colon actinomycosis in a 72 years old woman, mimicking clinically a malignant inflammatory tumor of the right colon. The patient complained of right lower quadrant pain. Although our first thought was a peri-appendiceal abscess, CT scan suggested a right colon tumor. The patient underwent a right colectomy and the histological examination of the specimen revealed colon actinomycosis.Preoperative diagnosis in colon actinomycosis is difficult to achieve. Treatment of choice is antibiotics administration. A review of the possible pathogenesis and therapeutic modalities is also presented. Actinomycosis is an uncommon inflammatory entity caused by the universally distributed anaerobic bacterium, Actinomyces Israeli. This Actinomyces is a gram-positive, rather microaerophilic bacterium, which consists a component of the normal human flora. Actinomyces requires the presence of many other bacteria, which destroy the over-vascularized regions and convert aerobic microenvironment to an anaerobic one. Then it's easy for Actinomyces to migrate, infect and proliferate in already injured tissue. Primary bowel involvement is rare, although it has been increased in frequency over the last years. The most common sites of the disease are the transverse colon and the cecum with the appendix ,2.Actinomycosis can mimic other abdominal diseases as diverticulitis, abscesses, inflammatory bowel disease and malignant tumors, presenting a diagnostic challenge, and identified post-operatively in most of the cases .The treatment of choice is antibiotic administration, whenever it is possible due to diagnostic difficulties, although in most cases surgical intervention is performed Diagnosis can be achieved with endoscopy and imaging techniques, as computed tomography (CT scan) and magnetic resonance imaging (MRI). We present a rare case of right colon actinomycosis mimicking malignant tumor, causing bowel obstruction. The diagnosis was achieved only postoperatively.A 72 years old woman proceeded to the Emergency Department complaining of acute lower right abdominal pain, mild fever, mild weight loss and constipation. The past medical history of the patient was free.The patient was presented with severe lower right abdominal pain, with signs of local peritonitis and a palpable mass in the same region. The body temperature was 37.2–37.4°C, while arterial blood pressure and cardiac rate 150/90 mmHg and 90/min respectively. The signs of local peritonitis combined with the palpable mass of the lower right abdominal area suggested perforation of the appendix and abscess formation. The laboratory examinations of the patient showed leucocytosis with white blood cell count (WBC) 19000 . Colonoscopy revealed obstruction of the right colon. Biopsies were not acquired because the colon lumen was obstructed and the endoscope could not approach the lesion. Computerized tomography (CT) scans of the abdomen  with focal areas of attenuation invading the adjacent tissues and suggesting the diagnosis ,9.Most common findings in CT scan and/or barium study include mural invasion with stricture formation, mass effect with tapered narrowing of the lumen, and thickened mucosal folds. In many cases the radiologic findings are similar to those of Crohn's disease, intestinal tuberculosis, and excavated malignant tumors ,11. The Goldwag et al suggest that CT guided fine needle aspiration can be both diagnostic and therapeutic. Microbiological analysis of material acquired by FNA may reveal sulfur granules, which are suggesting actinomycosis and nocardiosis. In most of the cases the sample receive is difficult especially when intestinal and colon are involved. We believe that in cases where the CT findings are non-specific, surgical exploration is necessary not only for diagnostic but also for therapeutic reasons .High dose intravenous penicillin injection followed by orally administered penicillin for at least 6–12 months is the treatment of choice. Penicillin administration decreases morbidity and the patient may avoid an unnecessary operation ,16.Correct diagnosis is difficult and can be achieved preoperatively in only 10% of the cases, but it is of great importance because the appropriate treatment includes primarily penicillin administration. Surgical intervention is indicated only in cases with obscure diagnosis and for necrotic debridement removal. Although diagnosis only with imaging techniques and laboratory tests is difficult, abdominal actinomycosis should always be included in the differential diagnosis in patients with abdominal masses [In conclusion, colon actinomycosis should always be included in the differential diagnosis of abdominal masses with tumor of inflammatory characteristics. It should be especially suspected when the appearance on CT scan is of a solid mass with focal areas of attenuation or a cystic mass with a thickened wall showing inhomogeneous contrast enhancement that tends to invade adjacent tissues or structures. Immediate and accurate diagnosis, usually by FNA and cytology examination can prevent unnecessary surgical treatment.The author(s) declare that they have no competing interests.All authors contributed equally to this work. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Kip1 (p27) proteolysis, which is triggered by its phosphorylation on threonine (Thr) 187. Since its levels are abundant in quiescent and scarce in cycling cells, p27 is an approved marker for quiescent cells, extensively used in histopathology and cancer research.G1/S cell cycle progression requires p27However here we showed that by using a specific phosphorylation site (pThr187) antibody, p27 is detectable also in proliferative compartments of normal, dysplastic and neoplastic tissues.In fact, whereas un-phosphorylated p27 and MIB-1 showed a significant inverse correlation , pThr187-p27 was positively and significantly correlated with MIB-1 expression . Thus proliferating cells only stain for pThr187-p27, whereas they are un-reactive with the regular p27 antibodies. However increasing the sensitivity of the immunocytochemistry (ICH) by the use of an ultra sensitive detection system based on tiramide signal amplification, simultaneous expression and colocalisation of both forms of p27 was shown in proliferating compartments nuclei by double immunofluorescence and laser scanning confocal microscopy studies.Overall, our data suggest that p27 expression also occurs in proliferating cells compartments and the combined use of both regular and phospho- p27 antibodies is suggested. Kip1 (p27) is a key inhibitor of cell division that protects tissues from excessive cell proliferation [in vivo regulation. p27 cellular levels, copious in quiescent cells undergoing terminal differentiation, are scanty in cycling cells [Immunocytochemistry (ICH) is an important method for identification of proteins in cells and in tissues. Since the biological activity of many proteins is dependent on their phosphorylation status, a challenge for immunocytochemistry is to characterize the protein form and not just the total amount . p27Kip1feration . Since pferation . Howeverng cells . This evng cells .et al raised an antibody (Ab), specific for pThr187-p27 that was reactive in immunoprecipitates from proliferating cells and negative in quiescent cells [Therefore un-phosphorylated ("plain") p27 is representative of the total protein amounts only in quiescent cells, whereas in cycling cells a fraction of p27 is transiently present in the pThr187 form before degradation . Recentlnt cells . Even mopThr187-p27 was detected by the 71–7100 polyclonal antibody (PcAb) (Zymed Laboratories, San Francisco, CA, USA) and by the sc-16324 PcAb (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). These Ab's were raised against a short peptide corresponding to the portion of human p27 containing phosphorylated Thr-187, in order to detect only phospho-p27 and to be unreactive with "plain"-p27. The 71–7100 Ab was previously employed in immuno-precipitation experiments by Western blot, ,10 and t"Plain" p27 protein levels were detected with the K10125 monoclonal Ab (McAb) from (Transduction Laboratories, Lexington, Ky, USA), and with the rabbit PcAb (C-19) (Santa Cruz Biotechnology). These antibodies were previously shown to share the same staining pattern . MIB-1 MA wide range of normal, dysplastic and neoplastic tissues was obtained from surgical specimens. At least five samples from each type of normal tissue were processed Table . DysplasXylene dewaxed and alcohol rehydrated paraffin sections were placed in Coplin jars filled with a 0.01 M tri-sodium citrate solution, and heated for 3 minutes in a conventional pressure cooker . After h3VO4, and 1 mmol/L phenyl methyl sulfonyl fluoride and protease inhibitors) and 20 to 30 μg of proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred onto nitrocellulose membranes. The membranes were first blocked and then incubated with the primary antibody as it follows: anti-pThr187-p27 (Zymeed), 1:2000 and anti "plain" p27 (Transduction), 1:3000 for 1 hour at room temperature. To confirm equal loading, membranes were immunoblotted with monoclonal anti b-tubulin antibody (1:1000, Santa Cruz). After three washes, filters were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies  for 1 hour at room temperature. Detection of immunocomplexes was performed with an enhanced chemiluminescence system (ECL, Amersham).pThr187-p27 antibody specificity was controlled by Western blot analysis of MDA MB 468 (breast cancer) and NPA (thyroid papillary cancer) cell lines. These were lysed (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.1% Triton X-100, 5 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L Na-1).pThr187-p27 immunostaining specificity was assessed by several control experiments performed in parallel, in which the primary Ab was either replaced by a similarly diluted normal rabbit serum, or adsorbed with increasing concentrations of its phospho and dephosphopeptides  was used to score the nuclei of individual cells for expression of these proteins. As already described, nuclear boundary optical density and Ab threshold were adjusted for each case examined . A minim2O2 for 1 hour. The primary mouse McAb anti p27 (1/4000) was detected by overnight incubation at 4°C, followed by goat anti mouse HRP (1/200, DAKO). After washing in TBS, sections were incubated with fluorescein isotiocyanate (FITC) tyramide amplification (Perkin Elmer Life Sciences) and mounted with Vectashield-DAPI mounting medium (Vector). Control sections in which the second primary Ab was omitted were included to ascertain destruction of peroxidase activity. Single IF stained sections as well as colorimetric immunostaining were used as controls of signal specificity. Slides were examined with a Leica TCS SP2 UltraSpectral LaserScan Confocal microscope. FITC was excited at 488 and detected with a bandpass 500 to 550 nm. Cy3 was excited at 514 nm and detected with a bandpass 580 to 655 nm. Series of images were processed with the Leica confocal soft package. Confocal images were captured and imported into Adobe Photoshop 7 (Adobe Systems, Mountain View, CA) and processed with an eMac personal computer.Tissue sections from both normal and neoplastic specimens were also stained using double IF labelling for pThr187-p27 and "plain" p27, according to previous studies with minor modifications . The priSimilar results were obtained with both anti pThr187-p27 Ab's, with only proliferating tissue compartments being stained. This pattern overlapped to that of Ki67/MIB-1, whereas it was different to that shown by both Mc and Pc Ab's directed to "plain" p27 Table ; on the In other normal tissues, comprising breast, lung, kidney, pancreas, prostate, thyroid and parathyroid, "plain" p27 was expressed by the vast majority of cells, whereas pospho-p27 single labelled cells could be made out by meticulous scrutiny; in these tissues also MIB-1 staining was sporadic.As examples of dysplastic lesions, the expression of the pThr187-p27 was assessed in low-and in high grade squamous intraepithelial lesions (SIL) of the uterine cervix and in colonic adenoma. According to the relationship between phospho-p27 expression and proliferation, in low grade SIL the dysplastic basal and parabasal cells were in most instances positive for the expression of both pThr187-p27 Figure , and Ki-A wide range of different tumours was examined, in order to assess whether staining for phospho-p27 may yield diagnostic information in addition to those provided by "plain p27" Table  whereas Western blot analysis of MDA MB 468 (breast cancer) and NPA (thyroid papillary cancer) cell lines revealed a single anti-pThr187-p27 band, whose molecular weight (27 kDa) corresponded to that showed by "plain"-p27 Fig. . pThr187Tissue sections from both normal and neoplastic specimens were also stained using double IF labelling for pThr187-p27 and "plain" p27 and analyzed by LSCM. The use of an ultra sensitive detection system based on tiramide signal amplification for IF staining, revealed a more precise pattern of protein expression and distribution, slightly different to results above shown of regular colorimetric IHC. In proliferative compartments of normal tissues the cells frequently showed simultaneous expression of the two forms of p27; in these cells, pThr187-p27 was more localized as an inner rim along the nuclei membrane, whereas "plain" p27 was more centrally located in the same nuclei with an intermediate area of colocalization Fig. . Similarin vitro by Montagnoli et al, who also detected p27 expression in proliferating cells by the pThr187-p27 Ab [p27 is an approved marker for quiescent cells, extensively used in histopathology and cancer research. However here we showed that an unusual p27 expression pattern may be obtained when only its portion phosphorylated on threonine 187 is stained. By using a specific phosphorylation site antibody, p27 expression may be detected in the proliferative compartments of squamous and intestinal epithelia, of lymph node germinal centres, of trophoblastic villi, of cervical and colonic pre-invasive lesions and of several carcinomas types. These results are similar to those reported 7-p27 Ab . The samIn this study we showed that p27 expression does not feature only one pattern, but that two different staining may be obtained by the use of either regular or pThr187 antibodies; in fact, when the overall data relative to the expression of p27 by both antibodies in tumours were analysed, a significant inverse correlation between the two forms was found . This data reflects the different relationship between each form of p27 and tissue differentiation:aggressive tumours were strongly labelled by phospho-p27 Ab, whereas plain-p27 was prevalent in well differentiated tumours. Thus the ratio between the two forms of p27, more than the total protein amounts, could yield information on tumour differentiation and behaviour.et al, who also confirmed antibody specificity by biochemical assays, showed that p27 expression may be better detected by using both antibodies than either alone and that there is a portion of p27 that is only recognizable by the anti-phospho Ab [Since cdk2 activity is low in differentiating cells and p27 is only present in its un-phosphorylated form, it is reasonable that squamous superficial layers, upper half of the intestinal crypts, lymphoid mantle cells and syncytiotrophoblastic cells only react with the regular antibodies. More intriguing is the search for the reasons explaining why in proliferating cells p27 is detected by the pThr187-Ab and missed by the regular antibodies. The possibility of an aberrant cross-reactivity by the pThr187-Ab was excluded both by Western blot analysis and by phosphopeptide adsorbtion. Montagnoli ospho Ab . Thus, iIn this study we have reported the following observations: (1) pThr187-p27 Ab selectively stains proliferating cells; (2) this staining does not identify the "plain" p27 protein, expressed by quiescent cells. Therefore, the pThr187-p27 antibody is a useful tool to study p27 in vivo regulation. Indeed, the pattern of expression observed in this study, strongly suggest that the anti-pThr187-p27 Ab identifies proliferating cells. Considering the importance of the different biological functions of p27: regulator of cell growth, cell differentiation, contact inhibition, apoptosis, protection against immunological aggression, and protection against environmental stress, among others, the combined use of both "plain" and pThr187 p27 antibodies will be a useful tool to better characterize the biology of these conditions.The author(s) declare that they have no competing interests.GT conceived the study and wrote the manuscript; JCM carried out the LSCM study; AC scored the immunostaining; AI coordinated the study; MR performed the peptide experiments; PZ evaluated the tumour pathological features; IM participated in its design and coordination and helped to draft the manuscript; MLM carried out the double immunostainings; DC carried out the Western blot analysis; EAP performed the statistical analysis. LP participated in the design of the study and evaluated the results.All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


Several family structure-related factors, such as birth order, family size, parental age, and age differences to siblings, have been suggested as risk factors for schizophrenia. We examined how family-structure-related variables modified the risk of schizophrenia in Finnish families with at least one child with schizophrenia born from 1950 to 1976.We used case-sibling design, a variant of the matched case-control design in the analysis. Patients hospitalized for schizophrenia between 1969 and 1996 were identified from the Finnish Hospital Discharge Register, and their families from the Population Register Center. Only families with at least two children  were included in the analysis. Conditional logistic regression with sex, birth cohort, maternal schizophrenia status, and several family-related variables as explanatory variables was used in the case-sibling design. The effect of variables with the same value in each sibship was analyzed using ordinary logistic regression.Having a sibling who was less than five years older (OR 1.46, 95% CI 1.29–1.66), or being the firstborn (first born vs. second born 1.62, 1.87–1.4) predicted an elevated risk, but having siblings who were more than ten years older predicted a lower risk (0.66, 0.56–0.79).Several family-structure-related variables were identified as risk factors for schizophrenia. The underlying causative mechanisms are likely to be variable. Several factors relating to family structure have been suggested as risk factors for schizophrenia. They include sibship size, number of older siblings, birth order in the sibship, age difference to older siblings, and age-at-onset, and sex of the affected siblings. These family-related risk factors may provide useful hints about the nature of environmental and genetic risk factors for schizophrenia .In several studies, siblings of female probands have had a higher risk of developing schizophrenia than siblings of male probands in several studies, with the number of cases varying from 123 to 500 . The effThere are mixed results about the effect of birth order on risk. Two studies reported lowest risk for the firstborns ,10, and The number and age of older siblings at the time of birth is hypothesized to be connected to the risk of schizophrenia through infection pressure. In a Swedish register-based study (270 cases), having siblings who were 3–4 years older was associated with increased risk (relative was 2.02) . The DanAll risk factors concerning birth order, family size, number of older or younger siblings, and paternal and maternal age are correlated. For example, the number of older siblings is dependent on both the individual's birth order and family size, which are connected to maternal age. If all these correlated variables are not in the model simultaneously, spurious associations may be found. Therefore, a large, population-based data set that contains a sufficient number of sibships with variable structures is needed to analyze family-related variables. Our aim was to investigate which of these variables were risk factors for schizophrenia in Finland among those who were born between 1950 and 1976.Using the Finnish Hospital Discharge Register of Finland, we obtained information on all hospitalizations with a diagnosis of schizophrenia (ICD-8 295*, ICD-9 295*, ICD-10 F20*). The information was sought for all Finns born between 1950 and 1976 for the period between 1969 and 1996. Sibship was defined as a group of individuals having the same mother. Since many of the variables we examined are related to maternal characteristics, we considered it essential that each sibship has the same mother. It should be noticed that generally the mother has a stronger influence on the child than the father especially in early childhood: e.g. mother's womb provides the fetal environment, and after birth the mother has more of a close physical relationship to the child through breast feeding. Siblings and parents of the probands were identified using the data from the Population Register Centre, and their information was then linked back to the Hospital Discharge Register to obtain data on relatives' hospitalizations. Only those who were born between 1950 and 1976 were qualified as probands, but their younger siblings were included in analyses as family members.We assumed our ascertainment was complete, because the two registers used, the Population Register and the Finnish Hospital Discharge Register, are nationwide and accurate . The relAltogether, we identified 17549 sibships with 35481 siblings, of whom 31004 were born between 1950 and 1976. There were 12 siblings in the largest family, and the highest number of cases in one family was six  women. In the 1966 and 1986 Northern Finland birth cohorts, primiparity was a risk factor for having a low birth weight infant and for perinatal mortality . In an eOur results concerning the effect of paternal and maternal age are not comparable to previous studies -18, becaConsistently with a recent meta-analysis , the risOur results showed that the higher was the age-at-onset of the first proband in the family, the lower was the siblings' risk Tables  and 2, wA case-sibling design has some limitations. It can only provide approximate estimates of relative risk. In our analyses only families with two or more siblings were informative, and therefore our results are not applicable to one child families. However, whether matching was taken into account or not did not largely affect the results.Our study was based on a nationwide, representative sample of patients with schizophrenia and their siblings, and our sample size was considerably larger than in any of the previous studies. Register-based information is not as accurate as information based on face-to-face interview, but Finnish registers are quite reliable. The two registers we used, the National Population Register and the National Hospital Discharge Register, are nationwide. The National Hospital Discharge Register covers all public and private hospitals in Finland, and the National Population Register registers all Finnish citizens. However, as information on parents enabling the identification of sibships first appeared in the Population Register only in 1974, sibship data may be more accurate in the younger cohorts. Information in both registers is accurate. Some proportion of siblings of the study population are really half-siblings, but even in this case, the case-sibling design is applicable, because it does not assume that familial aggregation is due to a genetic mechanism. When entries in the National Hospital Discharge Register were compared with data on hospital case notes, the primary diagnosis in the register and hospital case notes was identical in 99 percent of cases for schizophrenia and in 98 percent of cases for all mental disorders -22,37.We investigated concurrently several family-related factors which independently have been suggested to be risk factors for schizophrenia. We were able to verify some of the previous associations, while others were refuted. The risk factors we identified probably have different underlying causative mechanisms. For example, the risk associated with being the firstborn might be explained by increased risk to obstetric complications, while the combination of decreased risk among females but increased risk among siblings of female probands might be explained by genetic factors. The risk factors we identified should be studied further in samples that include more detailed information on possible causative factors, such as infections and obstetric complications.The author(s) declare that they have no competing interests.JH participated in the design of the study, performed the data-analysis and drafted the manuscript. JS participated in the design of the study, and the drafting of the manuscript. JL participated in the design of the study and its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: