Platelet transfusion is universally employed in acute leukemia. Platelet concentrate supernatants contain high concentrations of biologic mediators that might impair immunity. We investigated whether washed platelet and red cell transfusions could improve clinical outcomes in adult patients with acute leukemia.A pilot randomized trial of washed, leukoreduced ABO identical transfusions versus leukoreduced ABO identical transfusions was conducted in 43 adult patients with acute myeloid or lymphoid leukemia during 1991–94. Primary endpoints to be evaluated were platelet transfusion refractoriness, infectious and bleeding complications and overall survival.There were no significant differences in infectious or major bleeding complications and only one patient required HLA matched platelet transfusions. Minor bleeding was more frequent in the washed, leukoreduced arm of the study. Confirmed transfusion reactions were more frequent in the leukoreduced arm of the study. Overall survival was superior in the washed arm of the study (40% versus 22% at 5 years), but this difference was not statistically significant (p = 0.36). A planned subset analysis of those ≤50 years of age found that those in the washed, leukoreduced arm (n = 12) had a 75% survival at five years compared with 30% in the leukoreduced arm (n = 10) (p = 0.037)This study provides the first evidence concerning the safety and efficacy of washed platelets, and also raises the possibility of improved survival. We speculate that transfusion of stored red cell and platelet supernatant may compromise treatment, particularly in younger patients with curable disease. Larger trials will be needed to assess this hypothesis. In recent years, data have accumulated that platelet transfusion refractoriness and transfusion reactions in patients with hematologic malignancies can be reduced by use of leukoreduced -3 and/orOver the last two decades it has become apparent that allogeneic blood transfusions can modify host immunity and clinical outcomes . EpidemiPlatelet transfusions are almost universally used in the supportive care of acute leukemia in adults. The original study design hypothesized a potential benefit from removing soluble immunomodulatory mediators in red cell and platelet concentrates derived from plasma and white cells. White cells and their secreted products are now largely removed prior to storage through filtration. However, there is now reason to be concerned about soluble platelet derived substances that would not be removed by pre-storage leukoreduction, as well as immunomodulatory mediators from plasma itself, such as IgG and soluble HLA antigens. More recent data document that stored platelet concentrate supernatants accumulate striking levels of biologic response modifiers during storage, including vascular endothelial growth factor (VEGF), soluble CD40L, histamine and transforming growth factor (TGF-β1) -18. TherPlatelet transfusions are given to patients undergoing myelotoxic chemotherapy for acute leukemia over a two to three week period when the peripheral blood immune system is regenerating. We speculated that platelet transfusions, in addition to causing platelet refractoriness and transfusion reactions, might impair anti-leukemic immunity because allogeneic transfusions have been shown to favor type 2 immunity (e.g., characterized by cytokines such as IL4 and IL10) and suppress type 1 cellular immunity (e.g., IL2, γ-interferon and TNF-α) ,21. TherTo investigate the possible efficacy of washed platelet transfusions in preventing platelet refractoriness, transfusion reactions, bleeding and improving long term survival in adult acute leukemia, we performed a randomized trial in patients receiving either washed, leukoreduced, ABO identical platelet and red cell transfusions compared with our standard protocol of leukoreduced, ABO identical transfusions.The diagnosis of acute leukemia was based upon laboratory results from our hematopathology laboratory. Only patients receiving chemotherapy with curative intent were included. Three patients died before receiving a full course of induction therapy but are included in the data. Data retrieval of clinical information was done in a blinded fashion. Patients and clinicians were not blinded as to study allocation due to the obvious difference in packaging of washed versus unwashed transfusions.For acute myeloid or undifferentiated leukemia treatment invariably involved initial remission induction attempts with seven days of cytosine arabinoside and three days of an anthracycline. Patients with acute lymphoid leukemia received vincristine and prednisone, as well as other drugs. Patients with high risk features or those who failed to achieve remission received additional courses of induction therapy and/or additional agents depending on attending physician preference. Cytogenetic results, when available, were classified retrospectively according to a currently used scheme . All stePatients entering remission also typically underwent consolidation therapy and bone marrow transplant depending on age, performance status, availability of a sibling HLA matched allogeneic donor and other factors. Our institutional review board for studies involving humans approved the study protocols and informed consent documents. Once placed on a particular transfusion protocol, as described below, a patient received only transfusions of that type throughout their treatment. This included consolidation, transplant and relapses, continuing until they were cured or died of their leukemia.In 1991–94, 43 patients participated in a randomized trial of ABO identical, leukoreduced versus ABO identical, leukoreduced, plasma reduced (washed) transfusions. Study endpoints were platelet transfusion refractoriness, total platelet transfusion requirements during induction therapy, infections during induction therapy and overall survival. Randomization was arranged by our Department of Biostatistics employing computer generated random assignments and a blocked design with separate, sequential, sealed, opaque envelopes for patients with diagnoses of acute myeloid (AML) or acute lymphoid (ALL) leukemias. Leukoreduction for all patients was by post-storage, bedside filtration with Pall filters (RC50 and PL100), and plasma reduction of both red cell and platelet transfusions was by saline washing using the Cobe 2991 by a previously published method . Neither9/l and usually prior to invasive procedures at <50 × 109/l. Protocol violations in which patients received a transfusion of the incorrect type were <0.5% of all transfusions. All transfusions were treated with 2500 centigray gamma irradiation. We did not collect data on storage time of the red cells or platelets transfused in the study.Transfusion practice was consistent during the period of the study, employing almost exclusively whole blood derived random platelet concentrates, either ABO identical, leukoreduced, or washed, ABO identical, leukoreduced, according to protocol. Prophylactic transfusions were consistently given at morning platelet counts of <20 × 10Patients were to be excluded from the analysis if they did not have acute leukemia or died prior to randomization. Since randomization occurred upon admission to the hospital with the diagnosis of suspected acute leukemia, some patients were found to have other diseases after randomization. These exclusions involved two patients who were randomized to the unwashed group but found not to have acute leukemia upon further investigation. Subgroup analysis was planned for patients ≤50 years of age because they are known to have substantially better prognosis, and for patients with acute myeloid leukemia, who may have a greater risk of HLA alloimmunization. Fifty years of age was also the maximal age for allogeneic bone marrow transplantation in patients with acute leukemia at the time of the study.Data were collected by a blinded coauthor (JMH) from medical record review of the admission for initial induction, and follow-up data on survival obtained from the local tumor registry. The clinical data for blood component use and morbidity were collected only for the initial admission to achieve treatment time comparability. Bleeding was evaluated for the initial admission for remission induction, and defined as minor (1–2 days not requiring any therapeutic intervention such as wound site hematoma, guaiac positive stools, mild epistaxis not requiring transfusion nor packing, etc.) or major . Refractoriness was evaluated for the entire duration of a patient's transfusion therapy until cessation of transfusions or death, and defined as the need for HLA matched platelet transfusions, as determined by the attending hematologist. Transfusion reactions were reported at the discretion of the nursing staff and evaluated by Transfusion Service residents who were not blinded as to the component received. A new fever or new rigors occurring during or shortly after a transfusion was considered a reaction whereas a fever or rigor that occurred shortly before the transfusion started was considered unrelated.Statview 5.0 (SAS Institute, Cary, NC) was employed to calculate survival curves by the Kaplan-Meier method and Cox proportional hazards regressions. For continuous variables, the Mann-Whitney test was used for bivariate comparisons, and for categorical variables, Fisher's exact test or Chi square with continuity correction were employed as appropriate. No corrections were made for multiple comparisons, because except for the major outcome variables of survival, platelet refractoriness, infectious complications and platelet utilization, the data comparisons are considered exploratory.The demographic pre-treatment and initial hospitalization clinical outcomes data for patients in the study are shown in Tables Clinically evident bleeding was uncommon (8 of 43 patients, 19%) and major bleeding occurred in only 2 of 43 patients (5%). Minor bleeding was more common in those receiving washed platelets (6 of 25, 24%) than unwashed (0 of 18, 0%) (p = 0.03 by Fisher's exact test) but major bleeding occurred in only one patient in each arm of the study . Bleeding was in all instances related to specific anatomic lesions, such as minor epistaxis, hematomas at the site of trauma or invasive procedures, or guaiac positive stools. For six patients these mild bleeding episodes were only seen on one or two days during their admission, without generalized petechiae or purpura. One patient in the washed arm had melena for five days, which resolved. The only life threatening bleeding was non-fatal and occurred in one patient in the unwashed arm of the study who experienced eight days of hemoptysis and received 416 units of platelets and 52 units of red cells during induction therapy. Bleeding did not influence overall survival at last follow-up, which was 38% in those who had bleeding and 35% in those patients with no bleeding (p = 0.94 by logrank test). Patients with bleeding required more platelet (mean of 141 ± 118) (1 SD) and red cell transfusions (21 ± 13) than patients with no bleeding (61 ± 44 platelets and 15 ± 7 red cells), but this was almost exclusively due to the one patient in the unwashed arm of the study with eight days of hemoptysis. When this patient was removed from the analysis, the patients with bleeding received no more transfusions than those without bleeding.Reported numbers of transfusion reactions were similar in both arms of the study (a mean of 0.4 per patient). In the washed arm of the study there were eight transfusion reactions reported due to intercurrent fever and two allergic reactions (rash and/or urticaria) to unwashed platelet transfusions given in violation of protocol due to clinical urgency. In the unwashed arm of the study there were three transfusions with intercurrent fever and four allergic reactions. Reactions were reported in 7 of 25 (28%) of patients in the washed study arm and 7 of 18 (39%) of the patients in the unwashed arm (p = 0.52). When evaluated by the transfusion medicine resident, seven of the reactions in the washed arm were considered to be pre-existing fever and the reported reaction likely unrelated to transfusion. When only "on protocol" transfusions judged causally related by a blood bank physician are considered, the patients in the washed arm were less likely to experience reactions  than patients in the unwashed arm  (p = 0.0058 by Fisher's exact test). During the course of the treatment, three of 18 patients randomized and analyzed in the unwashed arm had severe or repeated transfusion reactions and were placed on a washed protocol for future transfusions.As determined by a Cox proportional hazards regression, survival was not associated significantly with type of leukemia (p = 0.37), cytogenetic results (p = 0.63), or receipt of washed blood transfusions (p = 0.62) but was significantly associated with age (p = 0.0002), with younger patients surviving longer.Because long-term survival in acute leukemia is uncommon in those over the age of 50–60 years, we also performed a planned subset comparison of the 22 patients in the trial ≤50 years of age. We recognized that this subset would be small, rendering statistical analysis more difficult. These results are shown in figure There were more patients with ALL (p = 0.32) and more patients with poor risk cytogenetics in the washed arm of the study (p = 0.09). Otherwise there were few differences between the patients ≤50 years of age in the two cohorts. Patients in the washed arm had significantly better overall survival and required fewer platelets per day or per red cell transfused than patients in the unwashed arm. There were no significant differences in bleeding, use of growth factors or white cell transfusions between the two arms of the study, in either the entire or younger cohorts (data not shown but available from the authors on request).Keeping in mind the extremely small number of patients involved, a Cox proportional hazards regression was performed on these younger patients in the randomized trial. Receipt of washed transfusions was a significant predictor of longer survival (p = 0.011), as was cytogenetics  but age and type of leukemia were not statistically significant predictors of survival time.The survival of all patients ≤50 restricted to those with AML is shown in Figure In the subset of all patients ≤50 years of age with either type of leukemia who both achieved a complete remission and also received definitive post-remission therapy in the form of bone marrow transplantation, the survival in patients receiving washed transfusions (n = 12) was significantly better than those receiving unwashed transfusions (n = 8) (p = 0.031 by log rank test) (graph not shown).To our knowledge, these data represent the first randomized trial of washed platelet transfusions in any setting. The issues of the efficacy and safety of washed platelet transfusions are of importance because (1) some patients require plasma-reduced transfusions to treat allergic or febrile reactions, (2) new methods of viral and bacterial pathogen-inactivation for transfusions may require washing prior to transfusion, and (3) a growing body of evidence suggests that immunologically important molecules are present in the stored supernatant of blood transfusions that might, speculatively, affect clinical outcomes. Our study design was less likely to detect a benefit of platelet washing because, unlike many centers, we routinely use ABO identical platelet transfusions for patients with leukemia. There is evidence from two small randomized trials in leukemia that this reduces refractoriness ,5 and inThere was no apparent benefit to washed transfusions in terms of reduced platelet transfusion refractoriness, reduced bacterial infections, reduction in reported febrile or allergic transfusion reactions or reduced length of stay. Confirmed transfusion reactions were less frequent in the washed arm of the study, and 15% of the patients in the leukoreduced arm of the study had severe or repeated reactions that led to their receiving washed transfusions, which abrogated those reactions. Our data do provide evidence, for the first time, that washed platelet transfusions are probably as safe and efficacious as standard leukoreduced transfusions. The mean number of days with bleeding was marginally but not significantly reduced in the patients in the washed group, as were the number of platelet transfusions needed per day in younger patients, and the ratio of platelet transfusions to red cell transfusions. However, there was more minor bleeding in the washed group raising the possibility that washed platelets are slightly less effective. However, the prevalence of major bleeding requiring treatment was the same in each group and very low (<5%). The only life threatening hemorrhage in these 43 patients occurred in a patient in the leukoreduced arm.This study is also the first attempt to investigate whether transfusion practices during initial induction therapy are associated with changes in overall survival in acute leukemia in adults. The underlying rationale for studying this issue are observations demonstrating that allogeneic transfusions alter host T cell and natural killer cell immune function in surgical patients and experimental animals . The majLarge numbers of red cell and platelet transfusions are given to patients with acute leukemia during the period of recovery from aplasia that is caused by cytotoxic chemotherapy given during remission induction. Additional transfusions are given during consolidation and bone marrow or peripheral blood stem cell transplantation. Many of these transfusions occur during the period when at least the bone marrow and peripheral blood compartments of the immune system are reconstituting and might be susceptible to the immunomodulatory effects of transfusions. Both white cell and platelet-derived mediators are present in the transfused red cells as well as platelets. Prestorage leukoreduction (not employed in this study) removes the vast majority of the white cells and platelets from stored red cells, and red cell concentrates would be expected to have much lesser concentrations of biologic mediators, even without washing. Platelet derived mediators would, of course, still be present in prestorage leukoreduced platelet concentrates.Transfusions are known to cause suppression of type 1 cellular immunity and upregulation of type 2 humoral immunity ,21. SuchLeukoreduction filters remove white cells from the transfused blood components. In the case of pre-storage leukoreduction, filtration also removes some of the biologic response modifiers secreted by white cells during storage. Platelet concentrate supernatant contains large amounts of mediators such as soluble CD40L (CD154), VEGF, TGF-β1, histamine and other biologic response modifiers that might impair cellular immunity -18. WashThere are distinct limitations to what can be concluded from our data. The number of patients studied is very small and almost all of them had AML. The statistically significant improvement in survival in patients ≤50 years of age with AML who had unusually good survival represents a subset analysis, not a primary outcome group and thus may represent the play of chance. The survival of 75% of any cohort of adult patients with acute leukemia for 4–5 years is a distinctly unusual circumstance . This co9/μl.These data, however promising, raise hypotheses for additional testing rather than proving a principle. If transfusion practice impacts anti-leukemic immunity, and/or survival as our data suggest, a moderate sized randomized clinical trial should be able to confirm this in relatively few years. This is a propitious time for such trials as the need for prophylactic platelet transfusion therapy is being revisited. The question is being raised as to whether platelet transfusions in non-bleeding patients are truly necessary or need be as frequent as currently employed in this disease . One strPlatelet washing is time consuming, delaying transfusion by about 2.5–3 hours, may present an additional opportunity for bacterial contamination, and involves some loss of platelets (about 20%). Platelet washing may not be feasible in clinically urgent situations. Direct costs are modest, at less than $40 per transfusion. Washing adds about $500–2000 to the total cost of caring for patients with acute leukemia in our center from diagnosis to cure or death. This is less than 0.5–1% of the total costs of treating AML or ALL with curative intent. There are few or no proven side effects of platelet washing other than reduced dose of platelets transfused. There are some additional potential clinical benefits of washing including a reduced likelihood of transfusion complications such as febrile non-hemolytic transfusion reactions, transfusion-related acute lung injury, and allergic reactions. These preliminary data, albeit from a very small number of patients, raise the possibility that some patients with acute leukemia might benefit from washed transfusions. Larger studies are indicated to explore this possibility.Gambro BCT provided partial support for these studies. Dr. Blumberg has received lecture honoraria and a previous research grant from Pall Biomedical Corp. and lecture honoraria from Baxter.NB and JMH had the original idea for the study. All authors contributed to design of the study, drafting of the manuscript and all revisions. NB supervised the laboratory preparation of blood components for the study. JMR provided overall clinical supervision of the study and care for many of the patients. JMH collected the data and JMH and NB analyzed the data.The pre-publication history for this paper can be accessed here:


The high fat, low carbohydrate ketogenic diet (KD) was developed as an alternative to fasting for seizure management. While the mechanisms by which fasting and the KD inhibit seizures remain speculative, alterations in brain energy metabolism are likely involved. We previously showed that caloric restriction (CR) inhibits seizure susceptibility by reducing blood glucose in the epileptic EL mouse, a natural model for human multifactorial idiopathic epilepsy. In this study, we compared the antiepileptic and anticonvulsant efficacy of the KD with that of CR in adult EL mice with active epilepsy. EL mice that experienced at least 15 recurrent complex partial seizures were fed either a standard diet unrestricted (SD-UR) or restricted (SD-R), and either a KD unrestricted (KD-UR) or restricted (KD-R). All mice were fasted for 14 hrs prior to diet initiation. A new experimental design was used where each mouse in the diet-restricted groups served as its own control to achieve a 20–23% body weight reduction. Seizure susceptibility, body weights, and the levels of plasma glucose and β-hydroxybutyrate were measured once/week over a nine-week treatment period.Body weights and blood glucose levels remained high over the testing period in the SD-UR and the KD-UR groups, but were significantly (p < 0.001) reduced in the SD-R and KD-R groups. Plasma β-hydroxybutyrate levels were significantly (p < 0.001) increased in the SD-R and KD-R groups compared to their respective UR groups. Seizure susceptibility remained high in both UR-fed groups throughout the study, but was significantly reduced after three weeks in both R-fed groups.The results indicate that seizure susceptibility in EL mice is dependent on plasma glucose levels and that seizure control is more associated with the amount than with the origin of dietary calories. Also, CR underlies the antiepileptic and anticonvulsant action of the KD in EL mice. A transition from glucose to ketone bodies for energy is predicted to manage EL epileptic seizures through multiple integrated changes of inhibitory and excitatory neural systems. Epilepsy is a neurological disorder involving recurrent abnormal discharges of neurons that produce epileptic seizures . With thEpilepsy animal models are used widely to test the influence of environmental and genetic factors on seizure mechanisms. The epileptic EL mouse is a natural model for human multifactorial idiopathic epilepsy and was first discovered in 1954 in an outbred DDY mouse colony ,8-10. ELDespite intensive antiepileptic drug (AED) research and development, seizures remain unmanageable or refractory in many persons with epilepsy -26. As aCR is a natural dietary therapy that improves health, extends longevity, and reduces the effects of neuroinflammatory diseases in rodents and humans ,29,39,40Glucose uptake and metabolism increases more during epileptic seizures than during most other brain activities -44. BlooIn this study, we compared the antiepileptic and anticonvulsant effects of both the KD and CR in adult EL mice that experienced at least 15 recurrent complex partial seizures. The results show that seizure control in EL mice is more associated with the amount than with the origin of dietary calories, and that CR underlies the antiepileptic and anticonvulsant action of the KD in EL mice. A preliminary report of these findings was recently presented .The composition of each diet is shown in Table All mice were matched for age (approximately 210 days) and body weight (approximately 31.0 ± 1.5 g) before the start of the dietary treatment  mice were originally obtained from J. Suzuki (Tokyo Institute of Psychiatry). The mice were maintained in the Boston College Animal Care Facility as an inbred strain by brother × sister mating. The mice were group housed (prior to initiation of study) in plastic cages with Sani-chip bedding (P.J. Murphy Forest Products Corp., Montville, N.J.) and kept on a 12-hr light/dark cycle at approximately 22°C. Cotton nesting pads were provided for warmth when animals were individually housed. All cages and water bottles were changed once per week. Only females were used for these studies as adult males die sporadically with age from acute uremia poisoning due to urinary retention . The proSeizure onset in EL mice is generally between 60–70 days of age as previously described . BrieflyMice were designated seizure susceptible if they experienced a generalized seizure during seizure testing. Generalized seizures in EL mice involve loss of postural equilibrium and consciousness, together with excessive salivation, head, limb, and chewing/swallowing automatisms. An erect forward-arching Straub tail, indicative of spinal cord activation, was also seen in most mice having generalized seizures. Mice that displayed only vocalization and twitching without progression to generalized seizure were not considered seizure susceptible ,21. SeizAll mice received PROLAB RMH3000 chow diet (LabDiet, Richmond, IN, USA) prior to the experiment. This is the standard food pellet diet (SD) and contained a balance of mouse nutritional ingredients. According to the manufacturer's specification, this diet delivers 4.4 Kcal/g gross energy, where fat, carbohydrate, protein, and fiber comprised 55 g, 520 g, 225 g, and 45 g/Kg of the diet, respectively. The ketogenic diet (KD) was obtained from the Zeigler Bros., Inc. (Gardners, PA, USA) in butter-like form and also contained a balance of mouse nutritional ingredients. According to the manufacturer's specification, the KD delivers 7.8 Kcal/g gross energy, where fat, carbohydrate, protein, and fiber comprised 700 g, 0 g, 128 g, and 109 g/Kg of the diet, respectively. The fat in this diet was derived from lard and the diet had a ketogenic ratio (fats: proteins + carbohydrates) of 5.48:1. The individual % composition of each dietary energy component for the SD and KD diet is shown on Table Seizure susceptibility, body weight, and food intake was measured four times over a three-week period in 24 singly caged female EL mice (approximately 210 days of age). All mice received the SD during the pre-trial period and food intake was determined by subtracting the weight of food pellets remaining in the food hopper after one week from the initial amount given (200 g). The difference was then divided by seven to estimate the average daily food intake. Thus, all mice were highly seizure susceptible at the initiation of the diet therapy.ad libitum or unrestricted (SD-UR), 2) the KD fed ad libitum or unrestricted (KD-UR), 3) the SD restricted to achieve a 20–23% body weight reduction from the pre-trial weight (SD-R), and 4) the KD restricted to achieve a 20–23% body weight reduction from the pre-trial weight (KD-R). Each mouse in the two R groups served as its own control for body weight reduction. Based on food intake and body weight during the pre-trial period, food in the R-fed mouse groups was reduced until each mouse achieved the target weight reduction of a 20–23%. In other words, the daily amount of food given to each R mouse was reduced gradually until it reached 77–80% of its initial  body weight.After the three-week pre-trial period, the mice were placed into four groups (n = 6 mice/group) where the average body weight of each group was similar (about 31.0 ± 1.5 g) . The dish edges were shaved to reduce the height from 15 mm to about 6 mm. After placing about 5 g of KD in the dish for the KD-UR mice, the dish with the weighed KD was inverted and placed on the top of the food hopper. An empty water bottle was placed on top of the dish to prevent dish movement during animal feeding. The butter-like consistency adhered the KD to the inverted dish. This feeding apparatus allowed the mice easy access to the KD and prevented KD contact with bedding material. After about 24 hr, the amount of KD consumed was determined and another 5 grams of fresh KD were added to the dish. The KD was therefore given fresh every day without moving or disturbing the mice. The total amount of KD consumed per day was summed each week and divided by 7 to obtain the average weekly food intake of each mouse. For the KD-R mice, a calculated restricted amount of KD was placed directly on top of the food hopper bars for easy access. The R-fed mice licked the bars clean of the KD.® procedure (Stanbio, Boerne, TX, USA), or a modification of the Williamson et al procedure [Blood was collected approximately 1 h after seizure testing except for the pre-trial period where blood was not collected. Blood was first collected from all mice about 24 hr prior to the initiation of the 14 hr fast  was used to determine the relationship between body weight, food intake, plasma glucose levels, plasma β-hydroxybutyrate levels, and seizure susceptibility. Binary logistic regression (SPSS) was used to determine the relationship between seizure susceptibility, plasma glucose, and β-hydroxybutyrate levels on mice fed either the SD or the KD. Differences were considered significant at p < 0.01. All values are expressed as mean ± SEM. All statistical data were presented according to the recommendations of Lang et al., [Both ANOVA and a two-tailed  et al., .AED, antiepileptic drug; CR, caloric restriction; KD, ketogenic diet; R, restricted; SD, standard diet; UR, unrestricted.The authors declare that they have no competing interests.JGM carried out all described methods and drafted the manuscript. NAC helped with the blood assays, participated in the feeding of the mice and carried out the incorporation of the data in Excel spreadsheets. MTT helped with the design of the study. RM participated in the data discussion and the statistical analysis. TNS conceived the study, participated in its design and coordination and helped prepare the manuscript. All authors read and approved the final manuscript.


The hedgehog pathway plays a critical role in the development of prostate. However, the role of the hedgehog pathway in prostate cancer is not clear. Prostate cancer is the second most prevalent cause of cancer death in American men. Therefore, identification of novel therapeutic targets for prostate cancer has significant clinical implications.Su(Fu). Furthermore, expression of sonic hedgehog protein is detected in majority of PTCH1 positive tumors (24 out of 27). High levels of hedgehog target genes are also detected in four prostate cancer cell lines (TSU, DU145, LN-Cap and PC3). We demonstrate that inhibition of hedgehog signaling by smoothened antagonist, cyclopamine, suppresses hedgehog signaling, down-regulates cell invasiveness and induces apoptosis. In addition, cancer cells expressing Gli1 under the CMV promoter are resistant to cyclopamine-mediated apoptosis. All these data suggest a significant role of the hedgehog pathway for cellular functions of prostate cancer cells.Here we report that activation of the hedgehog pathway occurs frequently in advanced human prostate cancer. We find that high levels of hedgehog target genes, PTCH1 and hedgehog-interacting protein (HIP), are detected in over 70% of prostate tumors with Gleason scores 8–10, but in only 22% of tumors with Gleason scores 3–6. Furthermore, four available metastatic tumors all have high expression of PTCH1 and HIP. To identify the mechanism of the hedgehog signaling activation, we examine expression of Su(Fu) protein, a negative regulator of the hedgehog pathway. We find that Su(Fu) protein is undetectable in 11 of 27 PTCH1 positive tumors, two of them contain somatic loss-of-function mutations of Our data indicate that activation of the hedgehog pathway, through loss of Su(Fu) or overexpression of sonic hedgehog, may involve tumor progression and metastases of prostate cancer. Thus, targeted inhibition of hedgehog signaling may have significant implications of prostate cancer therapeutics. SMO signaling triggers a cascade of intracellular events, leading to activation of the pathway through GLI-dependent transcription [The polarity . Secretecription -6. More cription -10.Development of prostate requires hedgehog signaling. Although the initial formation of prostate buds does not require sonic hedgehog signaling (shh), shh is critical for maintaining appropriate prostate growth, proliferation and tissue polarity -14. In tAs an important regulator of tissue polarity, active hedgehog signaling is required for ductual morphogenesis and proliferation during prostate development -14. The Ptch1 null MEF cells have no active Ptch1 gene, thus should not have positive staining with PTCH1 antibodies. Indeed, no staining was seen in Ptch1 null MEF cells  or over-expression of hedgehog -10. The Su(Fu), which are predicted to create STOP codons in the coding sequence  specimens in which tumor portion can be as high as 70% of the tissue mass. Prostatectomy specimens (most of our tumors), however, often contain a small percentage (5–10%) of tumor tissue and are therefore not suitable for Western blotting or real-time PCR analyses. As shown in Fig. nce Fig. . In PC48nce Fig. . In PC51nce Fig. . These mnce Fig. , confirmFor tumors with high level of PTCH1 expression, but no changes in Su(Fu) protein expression, we examined expression of sonic hedgehog. It is reported that expression of hedgehog may be responsible for hedgehog signaling activation in lung cancer and GI cancers. Immunohistostaining with sonic hedgehog antibodies indicate that sonic hedgehog is highly expressed in 24 of 27 advanced prostate tumors with elevated expression of PTCH1 and HIP see Fig. . Thus, aTo demonstrate the role of hedgehog pathway in prostate cancer, we screen five available cell lines for the expression of Gli1, PTCH1 and HIP. TSU, LNCap, Du145 and PC3 are prostate cancer cell lines whereas RWPE-1 is a prostate epithelial cell line. We found that the hedgehog target genes were significantly elevated in all cancer cell lines Fig. . Thus, wFollowing treatment with 5 μM cycloapmine in PC3 cells, expression of hedgehog target genes were dramatically inhibited (data not shown here), which was accompanied with a significant reduction of BrdU positive cells see Fig.  and 6C. Prostate cancer progression is accompanied by increased cell invasiveness. Because the hedgehog signaling activation occurs frequently in advanced prostate cancer, we examined if inhibition of the hedgehog signaling can reduce cell invasiveness. Using BD Bio-coat cell invasion chambers, we found that treatment of cyclopamine in PC3 cells reduced the percentage of invasive cells by 70% Fig. . SimilarIt has been shown that cyclopamine induced apoptosis in cancer cells with activated hedgehog signaling . We haveAll these data indicate that the hedgehog pathway is activated in advanced prostate cancers, as indicated by high expression of PTCH1 and HIP. Our results also indicate that hedgehog signaling is required for cell proliferation and cell invasion of prostate cancer cells. Thus, targeted inhibition of the hedgehog pathway may be effective in future prostate cancer therapeutics.Hedgehog signaling pathway regulates cell proliferation, tissue polarity and cell differentiation during normal development. Abnormal signaling of this pathway has been reported in a variety of human cancers, including basal cell carcinomas, medulloblastomas, small cell lung cancer and GI cancers -10,22,23Su(Fu) in two prostate cancers. In addition to inactivated mutations in the coding region, Su(Fu) may be inactivated through promoter methylation. The heterogeneous nature of prostate cancer makes it difficult to screen prostate cancer specimens for Su(Fu) mutations since the tumor content is often less than 5% of the specimens. Future improvement can be achieved using microdissection techniques for collecting pure population of tumor cells in gene mutation analysis.In our studies, we found that some prostate tumors had no detectable Su(Fu) protein expression while others contained high levels of Shh protein expression. We further identified inactivated mutations of Since all available prostate cancer cell lines express Su(Fu) at a high level, the role of Su(Fu) on cellular functions of prostate cancer cannot be investigated in these cells. It appears that over-expression of sonic hedgehog may be responsible for hedgehog signaling activation in these cell lines [our unpublished data and -26]. AftSu(Fu) is reported to affect β-catenin signaling ,28. We aIn addition to Su(Fu) inactivation, over-expression of Shh is another mechanism by which the hedgehog pathway is activated in cancer -10. We nOnce hedgehog pathway is activated, the target gene expression will be up-regulated. Thus, analysis of target gene expression using immunohistochemistry will be an effective way to detect hedgehog pathway activation in prostate cancer. Currently, PTCH1, Gli1 and HIP are good markers for the hedgehog pathway.Our findings not only provide novel basic understanding of prostate cancer, but also allow us to design new ways to treat prostate cancer. With a specific SMO antagonist, cyclopamine, it will be possible in the future to treat prostate cancers, which have over-expressed sonic hedgehog. However, as a downstream molecule, tumors with Su(Fu) inactivation may not respond to cyclopamine treatment. Therefore, additional small molecule inhibitors appear to be necessary to treat Su(Fu) inactivated prostate cancer. One possibility is to use Gli1 SiRNA since we have indicated that down-regulation of Gli1 may be an important mechanism by which inhibition of the hedgehog pathway by cyclopamine induces apoptosis Fig. . SanchezTaken together, our findings suggest that activation of the hedgehog pathway involves prostate cancer progression. There might be several mechanisms by which the hedgehog pathway is activated in advanced prostate cancers, including loss of Su(Fu) protein expression, over-expression of sonic hedgehog or other alterations. We demonstrate that activation of the hedgehog pathway is associated with DNA synthesis and cell invasiveness in prostate cancer cells. Inhibition of the hedgehog pathway, on the other hand, causes apoptosis possibly through down-regulation of Gli1. Our studies predict that targeted inhibition of the hedgehog pathway may be an effective way to prevent prostate cancer progression.® according to manufacturer's instruction .A total of 55 paraffin-embedded tissue blocks from patients with prostate cancer were obtained from UTMB Surgical pathology with approval from the Institutional Review Board (IRB). Pathological reports, H#E staining of each specimen were reviewed to determine the nature of the disease and the Gleason scores. Of 55 specimens, 18 were from tumors with Gleason scores 3–6, 15 with Gleason score 7 and 22 with Gleason scores 8–10. The tumor area was first identified before tissue microarray (1.5 mm in diameter for specimens) was assembled with Beecher's Tissue arrayer-IA standard avidin-biotin immunostaining technique was performed using a kit from Vector laboratories using specific antibodies to Su(Fu) (Santa Cruz Biotechnology Cat# 10933), PTCH1 (Santa Cruz Biotechnology Cat# 6149), HIP (R&D systems Cat# AF1568) and Shh (Santa Cruz Biotechnology Cat# 9024) and PSA (Vector laboratories). Positive staining was in red or brown. The specificity of antibodies was tested using the very peptide used for raising the antibodies, which abolished the specific staining. Hematoxylin was used for counterstaining (in blue). Protein was analyzed by Western analysis with appropriate antibodies [Su(Fu) antibodies were from Santa Cruz, beta-actin antibody was purchased from Sigma). The signals were visualized with the enhanced chemiluminescence detection system (Amersham).Cell lines (RWPE-1, Du145, PC3, LN-CAP were purchase from ATCC and cultured according to the suggested conditions. TSU was kindly provided by Dr. Allen Gao. Cell invasion assay was performed with BD Bio-coat cell invasion chambers according to manufacturer's instruction (BD Bioscience, Inc., Franklin Lakes, NJ), with triplicates for each sample and the experiment was repeated three times with the similar results. Cell were treated with 5 μM cyclopamine (or tomatidine) before (for 12 h) and during cell invasion assay (for 24 h). The rate of cell invasion was calculated by dividing cell numbers penetrated the matrigels by the number of cell in the control chambers (without matrigels).® reagent (Invitrogen), and RT-PCR was performed using Promega's RT-PCR system according to the manufacturer's protocol. Two pairs of Su(Fu) primers were used . PCR products were first purified using Qiagen's Gel Extraction Kit. Due to existence of possible Su(Fu) splicing isoforms in humans, Su(Fu) genetic mutations were screened after the PCR products were cloned into TOPO® TA cloning vectors (Invitrogen). Several independent clones (from three experiments) of each PCR product were selected for sequencing analysis in UTMB sequencing facility. All mutations were confirmed by at least six independent clones.Total RNA was isolated using Trizol® assay reagent (P/N 4319413E) for an internal control. The primers are designed to span exon-exon junctions so as not to detect genomic DNA and the primers and probe sequences were searched against the Celera database to confirm specificity. To obtain the relative quantitation of gene expression, a validation experiment was performed to test the efficiency of the target amplification and the efficiency of the reference amplification. All absolute values of the slope of log input amount vs. ΔCT were <0.1. Separate tubes (singleplex) one-step RT-PCR was performed with 20 ng RNA for both target genes and endogenous control. The reagent we used was TaqMan one-step RT-PCR master mix reagent kit (P/N 4309169). The cycling parameters for one-step RT-PCR was: reverse transcription 48°C for 30 min, AmpliTaq activation 95°C for 10 min, denaturation 95°C for 15 sec and annealing/extension 60°C for 1 min (repeat 40 times) on ABI7000. Triplicate CT values were analyzed in Microsoft Excel using the comparative CT(ΔΔCT) method as described by the manufacturer(Applied Biosystems, Foster City, CA). The amount of target (2-ΔΔCT) was obtained by normalization to an endogenous reference (18sRNA) and relative to a calibrator.Real-time PCR We used Applied Biosystems' assays-by-demand 20× assay mix of primers and TaqMan probes (FAM™ dye-labeled) for the target genes (human Gli and PTCH1, the sequences have been patented by Applied Biosystems, Foster City, CA) and pre-developed 18S rRNA (VIC™-dye labled probe) TaqManin situ cell proliferation kit  [in situ cell death kit  [BrdU labeling was performed using an emicals) . Cells wemicals) ,29. Cellpatched 1; Shh – sonic hedgehog; SMO – smoothened, BCC – basal cell carcinoma.PSA – prostate specific antigen; HIP – hedgehog-interacting protein; Su(Fu) – suppressor of fused; PTCH1 – human homologue of Tao Sheng contributed to Figures Table 1 Prostate cancer specimens and protein expression. Prostate cancer specimens and expression of several hedgehog signaling proteins are summarized in this table (A). A total of 55 specimens were used in this study. The Gleason scores and protein expression of Shh, PTCH1 and Su(Fu) are shown (B).Click here for file


To determine whether previously reported loci predisposing to nonsyndromic high myopia show linkage to common myopia in pedigrees from two ethnic groups: Ashkenazi Jewish and Amish. We hypothesized that these high myopia loci might exhibit allelic heterogeneity and be responsible for moderate /mild or common myopia.Cycloplegic and manifest refraction were performed on 38 Jewish and 40 Amish families. Individuals with at least -1.00 D in each meridian of both eyes were classified as myopic. Genomic DNA was genotyped with 12 markers on chromosomes 12q21-23 and 18p11.3. Parametric and nonparametric linkage analyses were conducted to determine whether susceptibility alleles at these loci are important in families with less severe, clinical forms of myopia.There was no strong evidence of linkage of common myopia to these candidate regions: all two-point and multipoint heterogeneity LOD scores were < 1.0 and non-parametric linkage p-values were > 0.01. However, one Amish family showed slight evidence of linkage (LOD>1.0) on 12q; another 3 Amish families each gave LOD >1.0 on 18p; and 3 Jewish families each gave LOD >1.0 on 12q.Significant evidence of linkage (LOD> 3) of myopia was not found on chromosome 18p or 12q loci in these families. These results suggest that these loci do not play a major role in the causation of common myopia in our families studied. Myopia is one of the leading causes of vision loss around the world. In the Despite many decades of research, little is known about the precise molecular defects and abnormal biochemical pathways that result in myopia. Compelling data from familial aggregation and twin studies indicate that susceptibility to myopia is inherited. Several familial aggregation studies have reported a greater prevalence of myopia in children of myopic parents compared to children of nonmyopic parents -12. SeveThe combination of genetic and environmental influences on the development of myopia suggests that myopia is a complex disorder and should not be classified as a simple Mendelian trait. Further evidence is shown by studies that have reported correlation coefficients for myopia between offspring and parents and between pairs of siblings to lie between 0.07–0.36 -32. Due To avoid some of the complexity in mapping genes for myopia we have collected refractive measurements and DNA samples from Amish and orthodox Jewish families with myopia. The Old Order Amish are mostly rural farmers and craftsmen. They lead a culturally and technologically distinct lifestyle. They are a genetically well-defined founder population with large families and well-documented genealogies ,35. FamiThe orthodox Jewish families in this study are all of Ashkenazi descent, a population with known founder effects in other common diseases. This poThe study protocol adhered to the tenet of the Declaration of Helsinki and was approved by the University of Pennsylvania and the National Human Genome Research Institute, National Institutes of Health institutional review boards. Informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. The collection of orthodox Jewish individuals was begun by a mass mailing of 3900 letters to all the known orthodox Jewish families living in Lakewood, New Jersey. Questionnaires were sent with letters explaining the study. If willing to participate, individuals completed and returned questionnaires that included their contact and physician information. Second and third mailings went out to individuals who did not respond – either positively or negatively – to the first mailing. The total number of questionnaires returned was 1,310. All Jewish individuals included in the study were of Ashkenazi heritage. Collection of Amish families was done by an advertisement in an Amish newspaper, referrals from local eye doctors in the Lancaster County community and word of mouth. Criteria for entry into the study included the following: 1) Negative history of systemic or ocular disease which may predispose to myopia, 2) negative history of a premature birth, 3) proband must be affected and must have a family history of myopia in either their parents or children, 4) only one parent (as opposed to both parents) of the proband can be myopic. For the Orthodox Jewish population, an individual's myopic status was obtained from the most recent (within 2 years) measurement of refractive error. If not recent, an individual was given a repeat exam by their local eye doctor or one of the study investigators (D.S.). For the Amish subjects, all participants were examined by a study investigator (D.S.) at the Amish Eye Clinic in Strasburg, PA. Amish participants were brought to the study site because they do not have phone access making it difficult to obtain a past history and records. Cycloplegic refractions were done on all individuals less than 40 years of age with one drop each of 1% cyclogyl, 1% mydriacyl and 2.5% phenylephrine. A manifest refraction was performed if an individual was older than 40 years of age. Classification as myopic required at least -1.00D in each meridian of both eyes. Individuals were classified as nonmyopes if they were over 21 years of age and did not meet the above criteria for myopia. Other individuals were classified as nonmyopic if they were 5–10 years old and had ≥ +3.00D in each meridian, 10–18 years old with ≥ +2.00D in each meridian or 18–21 years old with ≥ +0.50D in each meridian. All other individuals were classified as unknown for the trait.This ascertainment protocol resulted in the collection of 40 Amish families and 38 orthodox Jewish families. Of the 40 Amish families, phenotype data were available on 340 persons  but only 323 DNA samples were available to be genotyped. In the 38 Jewish families, phenotype data were available for 313 persons (177 affected, 122 unaffected and 14 of indeterminate phenotype) and DNA samples were available and genotyped for 290 of these family members.2; 10 mM Tris/HCl (pH8.3); 50 mM KCl; 0.6 uM of each primer; and 7.6 units/ul of Taq polymerase. Standard thermocycling was as follows: 94°C for 30 sec., 55°C for 30 sec. and 72°C extension time for 30 sec. Markers used included D12S85, D12S1706, D12S346, D12S78, D12S79, D12S86, D18S59, D18S481, D18S63, D18S452, D18S53, and D18S474 located in the 18p and 12q regions implicated in high myopia [Peripheral blood was collected from family members. High molecular weight genomic DNA extraction from the blood samples was performed with a kit . Polymerase chain reactions were performed in a 17.05 ul volume containing 12–320 ng/ul of DNA; 880 uM each of dATP, dCTP, dGTP, and dTTP; 3 mM MgClh myopia ,17.A simulation study was conducted on the first 44 Ashkenazi Jewish families collected in this study, using the computer program SIMLINK ,39, to c: D12S85-42.78-D12S1706-0.53-D12S346-7.22-D12S78-13.44-D12S79-9.23-D12S86; D18S59-6.94-D18S481-1.36-D18S63-10.4-D18S452-22.54-D18S53-30.08-D18S474. To carefully explore the possibility of linkage of common myopia to these high myopia candidate regions, we utilized 12 different parametric models  divided by the square root of the total number of families (N = 78)[The data on 40 Amish and 38 Ashkenazi Jewish families were checked for misspecification of family structures, data entry errors and genotyping errors using the program SIBPAIR,42 and tAs expected, the estimated average maximum LOD score decreases with the distance between the linked marker locus and the trait locus, and with increasing heterogeneity. However, only minimal changes in projected power for our Ashkenazi families were observed as penetrance, phenocopy rate and disease allele were varied. Projected power was always higher when an allele frequency of 0.01 was used for the susceptibility allele at the trait locus than when an allele frequency of 0.05 was used; however, these differences in power were very small. Table Parametric and nonparametric LOD scores were calculated for 40 Amish pedigrees and 38 Jewish pedigrees. The six markers on chromosome 12q21-q23 spanned 73 cM, and the six markers on chromosome 18p11.2-p11.32 spanned 70 cM. Markers D12S1706, D12S346, D18S59, D18S481 and D18S63 were previously reported by Young et al. ,17 as shResults of two-point parametric linkage analysis of myopia assuming linkage heterogeneity to the chromosome 12 and 18 markers in 40 Amish families are presented in Table The same markers on chromosome 12q21-q23 and chromosome 18p11.2-p11.32 were analyzed using 38 Ashkenazi Jewish families Table . StatistHeterogeneity testing using HOMOG in the combined Jewish and Amish families also did not yield any significant evidence of linkage in these two regions, with the maximum HLOD's being 0.39 and 0.95 on chromosomes 12 and 18 respectively.p = 0.09 but was not close to the significance level of at least p = 0.01 needed to provide confirmation of a prior linkage[Furthermore, nonparametric two-point NPL scores did not show any significant evidence for linkage in either the Amish or Jewish populations. The observed combined NPL score of 1.37 for D12S1706 approached nominal significance at r linkage.Multipoint parametric linkage analyses assuming homogeneity were consistently negative in both the Amish and Jewish datasets. A maximum multipoint parametric HLOD of 0.92 was observed at D18S474 in the Amish population. However, multipoint parametric HLOD scores were essentially zero for the chromosome 12 region in the Amish and for both the chromosome 12 and 18 regions in the Jewish families.p = 0.05)  showed marginal evidence for linkage (LOD > 1.0) to the region previously reported on chromosome 12 (D12S1706 and D12S346) for both two-point and multipoint parametric analyses Table . Three AThe overall results of these preliminary studies do not indicate any strong evidence for linkage of myopia in these families to the candidate regions on chromosomes 12 or 18. Although some families show marginal evidence of linkage to one of these regions, the results could be due to chance. Our negative results for these candidate regions have several possible explanations. First, the diagnostic criteria used in the previous studies ,10 that Previous studies attempting to confirm the high myopia loci on chromosomes 18 and 12 have yielded inconsistent results. Naiglin et al.collectedThese studies suggest that myopia is complex and probably caused by the interaction of multiple genes with the environment. Therefore, to understand myopia it is necessary to apply the equation: Genes + Environment=Outcome. The difficulty here is the uncertainty surrounding both terms in the equation; ideally, one set of genetic factors will interact with one set of environmental influences to produce identical outcomes, but it is unknown whether this is always going to be the case. Therefore, to lessen the problem of multiple gene interaction as well as gene-environment interaction confounding the results, strategies to limit this problem should be utilized in the genetic mapping of myopia. The use of isolated populations is one approach to limiting the heterogeneity across populations and is the approach we are using for a genome wide scan in these families. Furthermore, the definition of myopia needs to be standardized so comparisons across studies can be made accurately. Previous studies have utilized different requirements with regard to affection status making cross comparisons difficult. In conclusion, we find little evidence implicating previously described susceptibility loci for high myopia on chromosomes 12 and 18 as being important in the etiology of common, moderate/mild myopia in our two population samples.None declared.Dwight Stambolian, Lauren Reider, Debra Dana, Robert Owens, and Elise Ciner recruited patients for the study. Melissa Schlifka carried out the genotyping in chromosomes 18 and 12. Dwight Stambolian and Joan E. Bailey-Wilson performed the study design and wrote part of the manuscript. Joan Bailey-Wilson oversaw all statistical analyses; Grace Ibay performed statistical analyses and wrote part of the manuscript; Taura Holmes, Betty Doan and Jennifer O'Neill assisted with analyses of the data. All authors read and approved of the final manuscript.The pre-publication history for this paper can be accessed here:


Chlamydia pneumoniae and stroke, the possibility exists that previous exposure to C. pneumoniae may play a role in vascular dementia. The objective of this study was to determine if there was an association between serological evidence of C. pneumoniae infection or inflammatory markers with vascular dementia.Chronic inflammation appears to play a role in the pathogenesis of vascular dementia. Given the association between C. pneumoniae IgG and IgA antibodies. The association between vascular dementia and C. pneumoniae titres as well as inflammatory markers was estimated by using both conditional logistic regression and stratified logistic regression.28 case-patients with vascular dementia at a geriatric clinic and 24 caregiver-controls were tested for C. pneumoniae specific IgG antibodies (titre ≥ 1:32), odds ratio [OR] 1.3 , p = 0.71, or in elevated C. pneumoniae specific IgA antibodies (titre ≥ 1:16), OR 2.0 (95%CI 0.5 to 8.0), p = 0.33 indicative of past or persistent C. pneumoniae infection. Similarly, no difference in high IgG or IgA antibody levels (IgG titre ≥ 1:512 or IgA titre ≥ 1:64) between the two groups, indicative of recent C. pneumoniae infection, was found, OR 0.4 (95%CI 0.1 to 2.1), p = 0.27. For C-reactive protein (CRP), the mean difference between 18 matched pairs (case – control) was – 3.33 mg/L. There was no significant difference between cases and controls when comparing log transformed values, OR 0.03 (95%CI 0.00 to 2.89), p = 0.13 or comparing CRP values above or below the median, OR 0.8 (95%CI 0.2 to 3.4), p = 0.71. For fibrinogen, the mean difference between pairs (case – control) was -0.07 g/L. There was no statistical difference between cases and controls when comparing log transformed values, OR 0.6 (95%CI 0.0 to 31.2), p = 0.79 or between fibrinogen values above and below the median, OR = 0.5 (95%CI 0.1 to 2.0), p = 0.50.When matched cases were compared to controls, there was no significant difference in elevated C. pneumoniae infection, inflammatory markers such as CRP and fibrinogen, and vascular dementia.We found no evidence for a significant association between  Vascular dementia is characterized by a loss of cognitive function and social adaptive functions in individuals with cerebrovascular disease ,2. VascuChlamydia pneumoniae and cerebrovascular disease. C. pneumoniae has been associated with stroke, transient cerebral ischemia, and atherosclerosis in the middle cerebral artery in both prospective and case-control studies [C. pneumoniae infection may also be a risk factor for vascular dementia. To our knowledge, this potential relationship has not previously been assessed. We conducted a pilot case-control study to determine an association between serological evidence of C. pneumoniae infection and vascular dementia. We also sought to determine if the inflammatory markers, C-reactive protein (CRP) and fibrinogen were associated with this illness.Recent data from serological and PCR studies support an association between  studies -12. SincC. pneumoniae (erythromycin, clarithromycin, azithromycin, levofloxacin, trovafloxacin, doxycycline, or tetracycline).Patients with vascular dementia were enrolled from the Geriatric Clinic at Henderson Hospital, an outpatient clinic affiliated with a tertiary hospital in Hamilton, Ontario. The diagnosis of vascular dementia for participants enrolled was determined in accordance with criteria established by the Neuroepidemiology Branch of the National Institute of Neurological Disorders and Stroke and Association Internationale pour la Recherche et l'Enseignement en Neurosciences (NINDS-AIREN) International workshop . This inC. pneumoniae IgG and IgA antibodies, CRP, and fibrinogen.The controls for this study were chosen from a list of all caregivers who attended the geriatric clinic at the time of the study, regardless of the diagnosis of their spouse or family member. For each case, one caregiver matched for age (within five years) and sex was selected. Caregivers were excluded if they had a diagnosis that included any of the following: dementia, stroke, or cognitive impairment as determined by a Standardized Mini-Mental Status Examination score of < 27 . EnrolmeThis study was approved by the research ethics board at McMaster University. Signed consent was obtained for all participants .C. pneumoniae IgG and IgA antibody detection, all sera were titrated at two-fold dilutions from 1:16 to endpoint. Samples were analyzed by microimmunofluorescence (MIF), using a 16 hour incubation of serum and substrate at 4–8°C with the same batch of C. pneumoniae IgG/IgM MIF slides (LabSystems OY, Helsinki) (23). To prevent IgG interference, sera used for IgA detection were first treated with goat anti-human IgG antibodies . CRP was measured using a high sensitivity automated rate nephelometric immunoassay (Dade Behring high-sensitivity CRP, BNII Nephelometer System, Marburc, DE). Fibrinogen was assayed using an automated STA fibrinogen assay (von Clauss method) on a Roche/Stago (Diagnostica Stago SA).For C. pneumoniae infection, was defined as an IgG titre of 1:32 or greater and IgA of 1:16 or greater [C. pneumoniae, suggesting a more recent infection, was defined by IgG titres of 1:512 or greater or IgA titres of 1:64 or greater [The presence of elevated antibody levels, indicative of past or persistent  greater ,16. HighC. pneumoniae titres and inflammatory markers was estimated using a matched analysis. Conditional logistic regression analyses were performed for antibody levels, dichotomized and log transformed CRP, as well as dichotomized and log transformed fibrinogen.The association between vascular dementia and C. pneumoniae titres and inflammatory markers (both log transformed and dichotomized at the median), stratifying by age and sex (the following age strata were used: ≤ 70 years, 71–80 years, 81–90 years). All analyses for C. pneumoniae titres and inflammatory markers were also performed with adjustment for current smoking status. Data analyses were performed with SPSS version 10 or Egret for Windows version 2.0.3.A stratified logistic regression analysis was also conducted for C. pneumoniae titres, for an alpha of 0.05 and 80% power, matching 30 cases to 60 controls would allow for detection of an odds ratio of 3.8 or higher. As the study proceeded, it became apparent that enrolling the Alzheimer's patients was not feasible. We decided then to limit the analysis to a 1:1 case:control ratio focusing on 30 patients with vascular dementia.The original protocol involved two concurrent case-control studies: one including 30 vascular dementia patients and 30 controls and the other with 30 Alzheimer disease patients and 30 controls. The analysis was to include the additional 30 Alzheimer disease controls in the vascular dementia analysis (giving a 1:2 case:control ratio). Assuming that one third of controls would have elevated A total of 28 case-patients were enrolled: mean age 76.2 years (minimum to maximum: 56 to 90 years); 18 (64%) were male. Nine of the 28 cases had at least one comorbidity ; 1 case had 3 or more comorbidities. Thirteen of the cases were current smokers.Twenty of the 28 cases could be matched to caregiver-controls, for a total of 20 case-control pairs. Of these 20 caregiver-controls, 16 were unrelated to a case, and four were spouses of a case. However, none of these four were matched to their spouse. Four additional caregiver-controls were selected for the unmatched analyses, so that data on a total of 24 caregiver-controls was obtained.Where there was incomplete data on antibody or inflammatory marker levels, those pairs were excluded from matched analyses. Incomplete information on cases and controls occurred when individuals consented to participate in the study and provided medical information but did not attend the out-patient clinic for the required blood collection. All individuals with complete data were included in the unmatched analyses.C. pneumoniae specific IgG antibodies showed no statistically significant difference in elevated antibody levels between matched case-patients and controls, odds ratio [OR] = 1.3 , p = 0.71. When pairs were broken and stratified analyses were performed the difference was not statistically significant, OR = 1.8 (95%CI 0.4 to 8.3), p = 0.46. The analysis was also performed with adjustment for participants' current smoking status, OR = 1.8 (95%CI 0.2 to 12.2), p = 0.57 , p = 0.33. The stratified analysis produced slightly higher odds ratio estimates, although not statistically significant. For the unadjusted analysis, OR = 2.8 (95%CI 0.7 to 10.4), p = 0.13 and for the adjusted analysis, OR = 2.7 (95%CI 0.5 to 14.2), p = 0.24 , p = 0.27 or in the stratified analysis, OR = 0.6 (95%CI 0.2 to 2.0), p = 0.40 for the unadjusted analysis and OR = 0.5 (95%CI 0.1 to 2.5), p = 0.41 for the adjusted analysis  revealed no significant difference between log-transformed values, OR of 0.03 (95%CI 0.00 to 2.89), p = 0.13. Similarly, there was no difference comparing matched cases and controls with CRP values above or below the median, OR = 0.8 (95%CI 0.2 to 3.4), p = 0.71. In the stratified analysis , p = 0.79. Similarly, there was no difference when comparing the pairs on fibrinogen values above and below the median, OR = 0.5 (95%CI 0.1 to 2.0), p = 0.33. In the stratified analysis  are similar to the recent meta-analysis odds ratio estimates for coronary heart disease of 1.25 (95% CI 1.03 to 1.53) and 1.15 (95% CI 0.97 to 1.36), respectively ,22. In bnfection ,15,10.C. pneumoniae infection. In 1998 Balin et al [C. pneumoniae in post-mortem brain samples and late-onset Alzheimer's disease. However, more recent studies have not repeated these findings [C. pneumoniae infection contributes to Alzheimer's disease, found an improved long-term cognitive state in patients with mild to moderate Alzheimer's disease who had been treated with doxycycline and rifampin. However, the serological data did not suggest that this clinical effect was due to treatment of chronic C. pneumoniae infection. One study has looked for C. pneumoniae in brain samples of vascular dementia patients. This study, like the later AD studies, did not identify C. pneumoniae in any of the brain samples [C. pneumoniae in the brains is not strongly associated with late-onset Alzheimer's disease or vascular dementia.Vascular dementia is the second most common cause of dementia, second only to Alzheimer's disease. It was previously believed that most cases of dementia were the outcome of one of these two distinct diseases. However, the clear division between them has recently been challenged. It is now widely believed that vascular risk factors are also associated with Alzheimer's disease and Alzheimer's and vascular dementia may share many common clinical and pathological characteristics ,24-26. Ain et al  found anfindings -31. A refindings , based o samples . These rInflammatory responses are also known to be associated with cardiovascular disease and have recently been implicated in dementia . ElevateWe found no significant association between increased fibrinogen levels and vascular dementia. Abnormalities of haemostasis are thought to be important in the pathogenesis of cardiovascular disease, ischaemic stroke, and vascular dementia. Within the pathways of coagulation and fibrinolysis, fibrinogen represents an important marker. Elevated levels of fibrinogen are associated with increased risks of cardiovascular disease and ischaemic stroke ,40 but tC. pneumoniae titres; and between smoking and vascular dementia [C. pneumoniae serology is an imperfect test of C. pneumoniae exposure and chronic infection. First, the high prevalence of C. pneumoniae exposure makes it difficult to detect true serological differences between cases and controls. Second, it is unclear what the appropriate serological cut-offs should be for identifying exposure versus chronic infection or recent infections. As a result, different groups have used different criteria making comparisons across studies more difficult. However, the importance of this inconsistency is unclear. In the meta-analysis reported by Danesh et al [C. pneumoniae DNA in peripheral blood mononuclear cells [C. pneumoniae is infectious an increased exposure in the caregivers could potentially mask a statistically significant association between the patients and controls. There is also evidence that caregivers, because of stress, may have altered immune systems [We acknowledge several limitations of this study. Because of the relatively small sample size, the analyses were adjusted for only a small number of potentially important covariates and the analysis of CRP and fibrinogen was restricted to above and below the median (while quartiles would have been more sensitive). To adjust for variables that were not used as matching criteria and to maximize the data collected a stratified analysis was also done. The additional stratified analysis adjusted for current smoking status. We adjusted for smoking status because there is a known strong association between smoking and dementia . Howeversh et al  no signiar cells . Another systems  which co systems -49.C. pneumoniae antibodies and vascular dementia. We found no evidence for a significant association between systemic inflammatory markers and vascular dementia. While this study can rule out a strong association, larger studies are necessary to determine if a weak association exists.In summary, a case-control study of vascular dementia patients suggests that there is no significant association between The authors declare that they have no competing interests.ML, MS, WM, CG, JM, and MC conceived and designed the original study. SCC conducted the analysis of data and drafted the manuscript. SS and TS coordinated the study and collected data. JG conducted the serological testing. All authors offered critical input into the manuscript and all have read and approved the final version.The pre-publication history for this paper can be accessed here:


Journal of Experimental & Clinical Assisted Reproduction is an open access, online, peer-review journal publishing papers on all aspects of research into reproductive endocrinology, infertility, bioethics and the advanced reproductive technologies. The journal reports on important developments impacting the field of human reproductive medicine and surgery. The field exists as a sub-specialty of obstetrics & gynecology, focusing on the diagnosis and treatment of complex human reproductive problems. The continued growth of this relatively new field depends on quality research by proven scientists as well as junior investigators who, together, make contributions to this area of medical and surgical practice. The publishing revolution made possible by internet technology presages a bright future for continued interdisciplinary collaboration among researchers. Against this background, Journal of Experimental & Clinical Assisted Reproduction exists for the scientific community to facilitate this scholarly dialogue. Journal of Experimental & Clinical Assisted Reproduction began in Atlanta after discussions with London-based publisher BioMed Central to develop a new electronic peer-reviewed journal reporting on the rapidly growing field of reproductive endocrinology and infertility. Working with colleagues who shared this vision, an exploratory organizational group was established to design a journal that would not only meet the current demands of the specialty, but also anticipate its challenges and needs for the future.In early 2002, planning for Journal of Experimental & Clinical Assisted Reproduction already had received submissions and inquiries from authors seeking to publish manuscripts in the categories of original research, review, case report, opinion/debate, medical history, and letters. Although column space does not constrain electronic publishing in the same way as traditional print journals, our peer-review process determined that not every submission was appropriate for publication. Hence, from the first instance the journal has pledged to observe a peer-review policy that ensures only the best quality manuscripts are published.By the time of its formal launch in July 2004, Journal of Experimental and Clinical Assisted Reproduction is fully committed to the philosophy of Open Access as articulated in the following policies:Our editorial board membership reflects a depth of expertise required to guide editorial policies of a journal that aspires to join the top tier of selective scientific journals. Journal of Experimental & Clinical Assisted Reproduction (ISSN 1743-1050) are also accessioned in PubMed Central, maintained by the U.S. National Library of Medicine – the world's largest medical library. Accordingly, all journal publications are available free and without password requirements to anyone with internet access. Furthermore, the platform provided by our publisher, BioMed Central (NLMID b101153627), allows author(s) to track how frequently their manuscript has been accessed and viewed by the public at no charge.While the availability of published articles on the journal's homepage provides high and unrestricted visibility for accepted articles, contents of The journal reports on important developments impacting the field of human reproductive medicine and surgery. We accept manuscripts describing research in reproductive endocrinology, infertility, bioethics and the advanced reproductive technologies. 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Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown.To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential.Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed.We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms. Integral to both normal and pathological tissue remodeling, the matrix metalloproteinase (MMP) family of proteolytic enzymes collectively degrades laminin, collagen, gelatin, and other protein components of the extracellular matrix ,2. MMP eLittle is known about the mechanisms underlying MT1-MMP and MMP-11 induction. Previous studies have revealed induction of MT1-MMP in human fibroblasts grown on interstitial collagen (collagen I), which is abundant around tumours and during wound healing . Furtherin vivo. Type IV collagen has been shown to mediate pro-MMP-2 activation in HT1080 cells, a human fibrosarcoma cell line, without inducing either a transcriptional modulation of MMP-2 or MT1-MMP expression nor any alteration of MT1-MMP protein synthesis or processing [As collagen I has been reported to induce MT1-MMP expression, detection of such induction by quantitative RT-PCR would provide an ideal basis for testing further regulatory agents. Collagen IV and V appeared to be ideal candidates for further analysis of MMP induction as they are present in the basement membrane and would therefore be released specifically during early tumour invasion and metastasis, which coincides with MT1-MMP and MMP-11 expression ocessing . Fibroneocessing -15.in situ interaction of MMPs, growth factors and other potentially regulatory agents between the fibroblasts and HBC cells.We investigated the potential role of the following cytokines, interleukin (IL)-1β, IL-2, IL-6, IL-8 and transforming growth factor (TGF)-β in MT1-MMP and MMP-11 regulation. All of these cytokines are reportedly released by neoplastic cells and are capable of affecting transcription in fibroblasts -19. IL-22 tissue culture flasks (Costar) DMEM (Dulbecco's modified Eagles medium) with 10% FCS (Invitrogen) and allowed to attach for 6–12 hrs at 37°C, 5% CO2 prior to use. HBC cell lines were originally obtained from the ATCC and maintained by The Lombardi Cancer Center. ML-20 cells were derived from The Lombardi Cancer Center MCF-7 cells by clonal selection after transfection with a CMV-driven expression plasmid encoding bacterial β-galactosidase.NIH3T3 culture was obtained from the ATCC, while MEF cultures were constructed at the John Curtin School of Medical Research, Australian National University. Briefly, embryos were removed from 13-day pregnant mice. Following removal of the liver and head, the remaining tissue was placed in PBS (1× pH 7.4, Sigma-Aldrich). Tissue was homogenised using an 18 guage syringe (Terumo) in PBS, plated in 25 cm2 of flask area were added to each well in 2 ml 24 well plates (Costar). The wells were then incubated at 37°C for 1 hour to allow the collagen coats and fibronectin to set in the wells. Excess solution was aspirated from all wells before seeding with 105 cells from either NIH3T3, or MEF cultures in 1 ml DMEM - 10% fetal calf serum (Invitrogen). Treated cultures were then incubated at two time-points, 24 hours and 48 hours at 37°C, 5% CO2, before undergoing RNA extraction.Collagen coats and fibronectin treatments were prepared on non-coated tissue culture plates as follows: prior to seeding, triplicate sets of 2 ml 24 well plates (Costar) were overlaid with 150 μl of collagen I, IV and V (Sigma, 1 mg/ml) solutions. For the fibronectin treatments, 15 μg of fibronectin (Promega), equivalent to 5 μg/cm5 cells in 1 mL Dulbecco's modified Eagles medium (DMEM) - 10% fetal calf serum (Invitrogen). Treatments consisted of 25 μg Con-A added to the 1 ml of DMEM in each well. Treated cultures were then incubated at two time-points, 24 hours and 48 hours at 37°C, 5% CO2, before undergoing RNA extraction.Non-coated 2 ml 24 well tissue culture plates (Costar) were seeded with either NIH3T3 or MEF at 105 cells from either NIH3T3 or primary mouse fibroblast cultures (passage 5) in 1 mL Dulbecco's modified Eagles medium (DMEM) - 10% fetal calf serum (Invitrogen). Cultures were incubated at 37°C, 5% CO2 for 24 hours prior to cytokine treatment, thus allowing complete attachment. All treatments were separate and consisted of final solutions of 100 units/ml IL-1β, IL-2, IL-6, 0.1 units/ml of IL-8 and 1 ng/ml pan-TGF-β (Sigma-Aldrich) in cultures grown in triplicate.2 ml 24 well plates (Costar) were seeded with 106 cells of either NIH3T3 or MEFs (passage 5), which were each allowed to attach overnight in 1 mL Dulbecco's modified Eagles medium (DMEM) - 10% fetal calf serum (Invitrogen). The DMEM was then aspirated and 24 mm Transwell Porous Cell Culture Inserts (Costar) were seeded with 105 cells of either MDA-MB-231, MDA-MB-435, MDA-MB-436, MCF-7s or ML-20 epithelial tumour cultures in 2 ml of RPMI medium (Trace Biosystems) - 10% fetal calf serum. Use of the porous inserts meant that co-cultures were not in direct physical contact, but shared growth media at all times. These HBC cultures exhibit different levels of invasive and metastatic potential (see Table 2, before RNA extraction.30 mm 6 well plates (Costar) were seeded with 10260 using a QuantaGene spectrophotometer .Total RNA was extracted from the fibroblasts using an RNeasy Mini Kit (QIAGEN) as previously described . Total RAll 3' primers were labeled with the TET dye phosphoramidite and were commercially obtained . Oligonucleotides for the target genes, MT1-MMP and MMP-11, were generated using Amplify 2.1 analysis software in conjunction with published cDNA sequences ,25. The cDNA competitive templates for the target genes, MT1-MMP and MMP-11 were made by PCR amplification using purpose built 47-mer oligonucleotides as described . The 47-2O and stored at -70°C. To ensure that amplification would not occur as a result of residual DNA contamination, control samples were run in triplicate without the reverse transcriptase thermal cycle. No amplification products were visualised on resultant agarose gels.The cDNA competitive templates generated by PCR with the 47-mers described above were each ligated into pGem-T Easy Vectors . JM109 (Promega) competent cells were then transformed and grown overnight. Plasmid DNA was extracted using a Magic Miniprep kit (Promega) and linearised by nuclease digest. Mimic RNA was generated from the "mimic" plasmids by transcription of the insert with T7 RNA polymerase (Promega). The post-transcription mixture then underwent RQ1 DNase (Promega) digestion; the remaining RNA was then purified using QIAGEN RNeasy Minikit, RNA competitive template was then resuspended in DEPC-treated H2], 0.4 μM each primer, 1 unit Taq polymerase (Perkin Elmer ABI), 1.2 units AMV reverse transcriptase (Promega) and 900 ng tRNA (Boehringer Mannheim)]. Thermal cycling was as follows: 30 mins 50°C (reverse transcription), then 2 mins 95°C, followed by 35 cycles of 30 sec 95°C, 30 sec 65°C, 45 sec 72°C, followed by a final incubation of 5 mins at 72°C.Single tube, one step RT-PCR was performed as previously described  for all 3' primers for MT1-MMP, MMP-11 and 18S rRNA were labeled with TET fluorescent dye, thus allowing RT-PCR samples to be analysed by capillary electrophoresis using a 310 Genetic Analyzer with GeneScan software (ABI) as described previously . Using lThe GeneScan values for fluorescence peak area of the target gene transcripts and the control transcripts produced the target:control ratio. All graphed values represent averages from triplicate samples and were adjusted to produce a control value of 1.00. These target:control ratio values were used to plot time series graphs of all combined data for each gene. Data on the time series graphs was then normalised to 18S rRNA expression, as measured in the same respective cultures. The 0.5× control and 2.0× control are the resultant target:control ratios produced using half and double the amount of total RNA used in the control sample, respectively.Competitive quantitative RT-PCR using capillary electrophoresis and GeneScan analysis revealed no substantial changes in expression of MT1-MMP in either NIH3T3, or MEF cultures under any treatment conditions carried out over 3, 6, 12, and 24 hours  remained consistently normal for this treatment, effectively acting as an additional unregulated control, lends further credit to the likelihood of specific regulation of MMP-11 after 48 hours of treatment. There are also mild trends toward up regulation for the IL-2 and IL-6 treated samples.Initial results demonstrate no induction or inhibition of either MT1-MMP or MMP-11 in mouse fibroblasts by co-culture with any of the HBC cell lines Table . ResultsThis study investigated the effects of a number of possible regulatory agents on the expression of two members of the MMP gene family for their role in human breast cancer metastases. NIH3T3 and MEF cultures were treated with cytokines, collagens, fibronectin, Con-A or matrigel, and co-cultured with various HBC cell lines. The expression of MT1-MMP and MMP-11 was then determined by competitive RT-PCR using capillary electrophoresis and GeneScan technology. Only MMP-11 expression at 48 hours was affected by treatment with IL-1β, TGF-β, fibronectin and collagen V, in the tested fibroblasts cultures.During tumour progression, the release of exogenous cytokines by neoplastic cells and immunological cells may cause a stimulatory effect on adjacent stromal fibroblasts, resulting in matrix metalloproteinase (MMP) induction. Past studies indicate that some MMPs undergo regulatory changes under the influence of cytokines ,27. AnotOur study did however, find evidence for an inhibitory effect from both collagen I and collagen IV on MT1-MMP expression. Curiously, there is evidence suggesting that collagen I actually stimulates MT1-MMP mRNA production in fibroblasts and activates MMP-2 in a variety of cells over a longer time period ,29. Howein vivo [Despite reports of MDA-MB-231 conditioned media inducing MT1-MMP expression [in vivo .in vitro expansion to an another isolated cell population). Another possible explanation is that the basal level of expression of MT1-MMP and MMP-11 in cultured murine fibroblasts is elevated, thus rendering them refractory to exogenous regulatory agents. We are currently investigating this possibility.Although the co-cultures carried out here did not allow cell-cell contact, the previous study also involved no cell-cell contact, but rather treatment with pre-conditioned media (application of isolated medium from one cell population following This study examined the effects of a number of regulatory agents and treatments on the induction of MMP-11 and MT1-MMP gene expression, factors that may influence breast cancer tumour progression. Stimulation of MMP-11 was demonstrated after treatments by both cytokines (IL-1β, TGF-β) and by matrix constituents (collagen IV and fibronectin), whilst MT1-MMP was inhibited by matrix constituents (collagen I and fibronectin). The results presented suggest that several mechanisms may be involved in MMP-11 and MT1-MMP regulation as part of the complex epithelial-stromal interactions that occur within human breast carcinomas.None declared.in vitro studies, designed PCR primers and drafted the manuscript. L.M.H. contributed to the manuscript design and finalisation. E.W.T. contributed toward the design of the study. K.I.M. contributed toward the design and performance of competitive PCRs. M.G.I. participated in the conception and design of the study. L.R.G. participated in the conception and design of the study and its coordination. All authors read and approved the final manuscript.S.S. performed the molecular genetic and The pre-publication history for this paper can be accessed here: