Neo-adjuvant chemotherapy is an integral part of multi-modality approach in the management of locally advanced breast cancer and it is vital to predict the response in order to tailor the regime for a patient. The common final pathway in the tumor cell death is believed to be apoptosis or programmed cell death and chemotherapeutic drugs like other DNA-damaging agents act on rapidly multiplying cells including both the tumor and the normal cells by following the same common final pathway. This could account for both the toxic effects and the response. Absence or decreased apoptosis has been found to be associated with chemo resistance. The change in expression of apoptotic markers (Bcl-2 and Bax proteins) brought about by various chemotherapeutic regimens is being used to identify drug resistance in the tumor cells. A prospective clinical study was conducted to assess whether chemotherapy induced toxic effects could serve as reliable predictors of apoptosis or response to neo-adjuvant chemotherapy in patients with locally advanced breast cancer.50 cases of locally advanced breast cancer after complete routine and metastatic work up were subjected to trucut biopsy and the tissue evaluated immunohistochemically for apoptotic markers (bcl-2/bax ratio). Three cycles of Neoadjuvant Chemotherapy using FAC regime (5-fluorouracil, adriamycin, cyclophosphamide) were given at three weekly intervals and patients assessed for clinical response as well as toxicity after each cycle. Modified radical mastectomy was performed in all patients three weeks after the last cycle and the specimen were re-evaluated for any change in the bcl-2/bax ratio. The clinical response, immunohistochemical response and the drug-induced toxicity were correlated and compared.Descriptive studies were performed with SPSS version 10 and the significance of response was assessed using paired t-test. Significance of correlation between various variables was assessed using chi-square test and coefficient of correlation calculated by Pearson correlation coefficient.There was a statistically significant correlation observed between clinical, immunohistochemical response (bcl-2/bax ratio) and the drug-induced toxicity.Responders also had significant toxicity while non-responders did not show significant toxicity following neoadjuvant chemotherapy. The chemotherapy-induced toxicity was observed to be a cost effective and reliable predictor of response to neo-adjuvant chemotherapy. Neo-adjuvant chemotherapy (NACT) is an integral part of multi-modality approach in the management of locally advanced breast cancer (LABC). It is required both for the local control  and distant or systemic control -5. In thApoptosis is a closely regulated form of active cell death defined by characteristic biochemical and morphological criteria. A large number of anti-cancer agents with widely differing modes of action have been demonstrated to induce apoptosis in vitro, suggesting this as a significant final common pathway for exerting their clinical effects. Mechanisms that suppress apoptosis may be important in the development of intrinsic and acquired resistance to cytotoxic drugs .It was suggested more than 20 years ago that apoptosis might account for much of the spontaneous cell loss (known from kinetic studies) to occur in many tumors and its extent often is enhanced by well-established modalities such as chemotherapy, irradiation and hormone ablation. However, during the past few years, advances in the understanding of the control of apoptosis at the molecular level has extended its potential oncologic significance far beyond the mere provision of a mechanistic explanation for tumor cell deletion. In particular, the discovery that the products of certain proto-oncogenes can regulate apoptosis has opened up exciting avenues for future research .The protean effects of various neoplastic agents on synthesis of DNA, RNA, and inhibition of synthesis which may or may not lead to cell death, requires only that some critical concentrations of active drug or metabolite be present in a cell. Proliferating normal cells may therefore be subject to the same detrimental effects of chemotherapy experienced by neoplastic tissue and successful chemotherapy dictates that recuperative abilities of normal tissues are greater than those of cancer. The two tissues generally most adversely affected by antineoplastics are the hemopoetic cells of the bone marrow and the epithelium of the aero digestive tract as a result of high growth fractions and short cycling times of the cells. The ability of the cancer patients to perform normal activities and function is also recognized both as a determinant of how well the patient may respond to chemotherapy and an index of the general toxicities of the anti cancer agents.A variety of anti-cancer drugs have been shown to induce extensive apoptosis in rapidly proliferating "normal" cell population and the tumors alike. Thus enhanced apoptosis is also likely to be responsible for many of the adverse effects observed following chemotherapy . ApoptosApoptosis is a regulated phenomenon capable of being inhibited and activated. Indeed there is evidence that stimulation of some cells by trophic cytokines or increase in their levels of expression of Bcl-2 proto-ontogeny can greatly increase their resistance to the apoptosis-inducing effects of anticancer drugs. Thus Bcl-2 proto-ontogeny expression may be implicated in the development of resistance of tumors to therapeutic agents and may contribute to tumor growth and perhaps to ontogenesis by allowing the inappropriate survival of cells with DNA abnormalities .Deregulated expression of the Bcl-2 protein represents the best-known example of a potent blocker of apoptosis. Over expression of Bcl-2 has now been shown to protect a wide variety of cell types from induction of apoptosis by many different anticancer agents. Several homologues of Bcl-2 protein have also been shown to act as inhibitors of apoptosis, including Bcl-Xl and others as apoptotic proteins such as Bax. In vitro data suggest that it is the relative ratios of anti-apoptotic and pro-apoptotic proteins that determine the likelihood of cells to undergo apoptosis in response to chemotherapeutic drugs in vivo test bed to further confirm the clinical relevance of these observations. The clinical response or the absence of response along with the toxic effects observed could well help predict the outcome with a particular chemotherapeutic regime and facilitate planning of an alternate regime for better response [The increasing use of pre-operative chemotherapy (PCT) in breast cancer offers an response -8.It is vital to assess the response to NACT in order to tailor the regime for a particular patient to predict the intrinsic or acquired chemo resistance. DNA-damaging agents such as chemotherapeutic drugs can induce apoptosis and increased resistance to chemotherapeutic agents, which has been found to be associated with decreased capacity to undergo apoptosis -9. CentrThe clinical response along with complete pathological response (CPR) is still considered a surrogate marker of response against which all other predictive markers are compared. The need to have a reliable and inexpensive predictor of response in a third world scenario can not be over emphasized since majority of patients present relatively late and the resources are limited and scarce ,3. Since1. To assess the clinical and immunohistochemical response to NACT in patients with LABC.2. To correlate immuno-histochemical (apoptotic markers i.e. Bcl-2/Bax ratio) and clinical response to the drug induced toxicity.3. To ascertain whether the drug induced toxicity could be utilized as a reliable indicator and predictor of response to neoadjuvant chemotherapy.50 FNAC proven cases of locally advanced breast carcinoma according to AJCC (American Joint Committee On Cancer) classification were included in the studyA thorough clinical and ultrasonographic examination (USG) of all the patients including the opposite breast was performed to stage the disease accurately. A core biopsy using a tru-cut needle was performed for immuno-histochemical estimation of the apoptotic markers i.e. base-line Bcl-2/Bax ratio before initiating the chemotherapy. Routine and metastatic work up was done including complete blood examination , chest radiograph, ECG (Echocardiography when ECG had a positive finding), liver function tests, Bone Scan, USG abdomen, KFT (Kidney function tests).2, adriamycin -50 mg/m2, 5-fluorouracil-600 mg/m2) at an interval of three weeks. Before each cycle the patient was clinically and sonologically examined for the breast tumor size, axillary lymph node status & appearance of systemic metastasis. All patients were given the same antitoxicity treatment according to a standardized unit protocol including adequate hydration and inject able antiemetics before initiating the chemotherapy.Patients were subjected to three cycles of FAC regime  solution and incubated with blocking antibodies  at 37°C. Sections were washed with TBS solution. Incubation with avidin-biotin complex (ABC) was done at 37°C for one hour and washed with TBS solution. 3,3 Diaminobenzidine tetra hydrochloride solution applied for 3–5 min. Counter-staining with haemotoxylin solution done for 3–5 min. Sections were washed with distilled water, air dried and mounted using DPX mountant.For Bax, positive controls were taken as germinal centers of the lymphoid follicles and normal breast tissue and negative control was taken as the test slide without primary antibody. For Bcl-2, positive controls were the mantle zone of lymphoid follicles and the negative controls were the test slides without primary antibody.The pattern of positive staining for bcl-2 and bax was cytoplasmic, granular.The primary antibodies for bcl-2 and bax were procured from DAKO.Bax-Rabbit Anti-Human code no. A 3533.Bcl-2 Monoclonal Mouse Anti-Human code no. M 0887.Dilution for both was 1: 40.The results were interpreted on the basis of two criteria:(1) Percentage of cells showing immune bodies; <5%: score = 0, 5–25%: score = 1, 25–75%: score = 2, >75%: score = 3(2) Intensity of staining; mild: score = 1, moderate: score = 2, intense: score = 3.Since there was a strong correlation between the intensity of staining and percentage of cells showing immune bodies, the percentage of cells showing immune antibodies alone was considered for calculating the bcl-2/bax ratio"."Descriptive studies were performed with SPSS version 10. The significance of response assessed using paired t-test. Significance of correlation between various variables assessed using chi-square test and coefficient of correlation was calculated by Pearson correlation coefficient.50 cases of locally advanced breast cancer were included in this study. Mean age of the patients was 45.5 years (range: 28 to 71 years) and 53.3% patients were pre-menopausal. Size of the tumor was measured clinically as well as by ultrasound and the patients were subdivided into four groups: <5 cm(0%), 5–8 cm(56.6%), 8–10 cm(26.6%), >10 cm(16.6%). According to the axillary lymph node status the patients were divided into three groups: N0 (0%), N1 (33%), N2 (67%).Objective clinical response was defined as more than 50% reduction in the tumor size after three cycles of neoadjuvant chemotherapy. Immuno-histochemical response was defined as decrease in the Bcl-2/Bax ratio.Clinical response including the reduction in the tumor size and axillary lymph node status was observed in 70% of patients and was found to be statistically significant p < 0.0001). There were no patients in the No group and 29.4%of the N1 patients were down staged to N0 while70.6% remained N1. In patients with N2 disease 7.7% were down staged to N0 status while 46.2% were downstaged to N1 status and 46.2% did not show any response. Immuno-histochemical response was observed in 60% and was also found to be statistically significant (p = 0.008). Correlation between immuno-histochemical and clinical response was also found to be statistically significant (p < 0.0001) [Table . There wAcute vomiting was observed in 63.3% patients. 81% clinical responders had vomiting (p = 0.002) and 78% immunohistochemical responders also had vomiting which was statistically significant (p = 0.04). Alopecia was observed in 86% clinical responders (p = 0.000) and 94% immuno-histochemical responders (p = 0.000), which was also significant. Leucopoenia was observed in only 14% and 17% of clinical and immuno-histochemical responders respectively and was found to be an insignificant predictor of response in the present study. When multiple toxicities were correlated with the clinical and immuno-histochemical response, 46.7% of patients had both acute vomiting and alopecia. 67% clinical responders (p = 0.001) had both vomiting and alopecia.72% immunohistochemical responders (p = 0.001) had both vomiting and alopecia.A significant positive correlation was observed between the presence of vomiting (r = +0.558), alopeciar = +0.802) and response to neoadjuvant chemotherapy. A significant negative correlation was observed between the absence of side effects and poor response to neoadjuvant chemotherapy in 65% of patients after two courses of FAC . NACT haesection -13.Introduced by Kerr et al (1972) to describe characteristic morphological changes seen during programmed cell death . It is d(1) Chromophin condensation(2) Membrane blebbing(3) Appearance of apoptotic bodies(4) Fragmentation of genomic DNACertain biochemical and genetic events have been identified that are associated with multiple cell types including mammary epithelium. These include the DNA fragmentation via end nuclease activation and cleavage of intracellular proteins, expression of bcl-2 family members, tumor suppressor gene p-53 directed events, proto-oncogene activation and activation of transmembrane receptor signaling pathways such as tumour necrosis factor ,17-22. AThe heterogeneous nature of breast cancer has resulted in overwhelming interest in search for prognostic markers to identify patients who might benefit most from the therapeutic modalities available.Assessment of apoptosis and individual components of apoptotic pathway are therefore relevant in determining prognosis in a particular patient . DNA damBcl-2 is anti-apoptotic in function, whereas bax is proapoptotic and it is the interaction between the two that determines the likelihood of a tumor to undergo cytotoxic drug mediated regression. Therefore any increase in bcl-2 or decrease in bax will push the balance towards chemo resistance and an increase in bax or decrease in bcl-2 will result in increased apoptosis -30.It was observed in a study conducted by Kymionis et al  that incIn the present study the clinical response in terms of reduction of tumor size and immuno-histochemical response in terms of change of bcl-2/bax ratio correlated significantly with the drug induced toxicity following NACT.The time course of various toxic manipulations depends on the drug, its dose and frequency of administration, intrinsic characteristics of the tissue of interest and any local circumstances (e.g. radiation therapy, infection, trauma). There are few general rules however like mucosal toxicities of pain, erythema, ulceration etc. occur 3–10 days after the administration of most offending drugs. Bone marrow effects can be manifestated a few days later averaging 7–14 days. The recovery of normal functioning tissues in both cases is well under way 4–5 days after the zenith of the toxic effects.Alopecia may involve the scalp or the whole body can present within 2 weeks of the drug dose or be a progressive cumulative event. Other cumulative toxicities seen only after administration of a certain quantity of drug over some length of time include cardiomyopathy from anthracyclins.An alkylating agent belonging to the nitrogen mustard subgroup. It is inactive as such produces few acute effects and is not locally damaging. It is transformed in to active metabolites like aldophosphamide and phospharimide mustard in the liver, which then produces the wide variety of antitumour action. The prominent side effects are alopecia and cystitis, which are caused by another metabolite acrolein.It is a pyrimidine antagonist and is converted in the body to the corresponding nucleotide, 5-fluro-2-deoxy-uridine monophosphate, which inhibits thymidine synthetase and blocks the conversion of deoxyuridilic acid to deoxythymidilic acid. Selective failure of DNA synthesis occurs due to non-availability of thymidylate. Flourouracil may itself get incorporated in to nucleic acids and this may contribute to its toxicity. Even resting cells are affected, though multiplying cells are more susceptible like the cells in the gastrointestinal tract (GIT) and bone marrow.It is an anti tumor antibiotic and is capable of causing breaks in the DNA strands by activating topoisomerase II and generating quinone type free radicals. They have mutagenic potential. Maximum action is exerted at S phase, but toxicity is usually exerted in G2 phase. Cardiotoxicity is a unique side effect. Rapidly multiplying cells are more susceptible therefore it also acts on cells of GIT, bone marrow in addition to the tumor cells.Leucopenia has not been observed to be a frequently encountered chemotherapy induced toxicity using commonly used regimen in most of the studies -24. ThisThe rapidly proliferating normal and tumor cells are more susceptible to the action of chemotherapeutic agents, which could explain the significant correlation observed between the effects and the toxicity in the present study. There was a strong correlation observed between the immunohistochemical response (bcl-2/bax ratio), clinical response and drug toxicity. This indirectly indicates a correlation between chemotherapy induced apoptosis and the toxicity and therefore like apoptotic markers, chemotherapy induced toxic effects along with objective clinical response could serve as reliable and cost-effective indicators or predictors of response to NACT in patients with LABC. While many biological markers are in use and many are under trial to tailor the chemotherapy for a particular patient, most of these markers including apoptotic markers or p-glycoprotein etc. are not very frequently available and are expensive for a third world cancer set up. Thus the chemotherapy-induced toxicity along with clinical response may be utilized as a cost effective and reliable predictor of response to NACT in patients with LABC. This would also serve as an intermediate end point in determining drug sensitivity for adjuvant treatment, especially when adjuvant therapy is planned with the same regimen as induction chemotherapy. This can also help in planning an alternative regime in non-responders.None declared.CM, the principal and the corresponding author was the Supervisor and the Chief surgeon who performed and standardized surgery on the patients and designed the study.• VS participated in the designing of the study, performed the statistical analysis and was the first surgical assistant and Senior Postgraduate in charge of the cases in the study.• JP, Postgraduate surgical resident was the second assistant in charge of the cases and participated in the sequence alignment.• AL, Resident surgery participated in the data processing and statistical analysis.• SS was in charge of the molecular genetic studies at the Tumor biology lab ICMR.• AB participated in the genetic studies and data processing.• All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Strictly defined, evolution is a change in the gene pool, or total set of genes, of a given population over time. Genetic changes that increase the fitness of an organism—that is, increase survival or fertility—are more likely to be retained, through natural selection, and passed on to succeeding generations. In the classic case of Darwin's finches, different ecological niches exerted different selective pressures on an original population, and resulted in 14 different species, each sporting a beak uniquely adapted to harvesting particular available food sources.When it comes to microbial evolution, an ecological niche often takes the form of a host. If the microbe is a pathogen, its presence might trigger strong selective pressure from the host's immune system, precipitating an evolutionary two-step between microbe and host. Hosts with strong immune defenses can typically tolerate relatively virulent pests: conversely, ill-defended hosts die, which is bad news for the parasite. When the myxoma virus first infected a population of European rabbits in Australia in 1950, the virus was particularly lethal. Over time, less virulent strains were selected for—killing off your habitat would be an unsustainable fitness cost by most standards—and the rabbits developed resistance.In keeping with evolutionary theory, host immunity should affect the evolution of parasite virulence. Though theory predicts that immunity could potentially heighten virulence, there's no evidence that this is true. Being able to predict how natural selection will act on, and thus shape, virulence is vital for developing effective public health policies—and desperately needed vaccines—to deal with the ever growing roster of rapidly evolving pathogenic threats.Plasmodium in the mouse. Mackinnon and Read first directly injected two groups of mice with infectious parasites: “immunized” mice, which had been exposed to Plasmodium and then treated with the antimalarial drug mefloquine, and “naïve” mice, which had not. Parasites were serially transferred twenty times via a syringe from one mouse host to another. The virulence and infectiousness of the respective strains were evaluated by introducing the strains into another set of immunized and naïve mice.To investigate whether immune system defenses escalate pathogen virulence, Margaret Mackinnon and Andrew Read studied the malarial parasite As theoretically predicted, parasites evolved in the immunized mice were indeed more virulent than parasites evolved in the naïve mice. But what if the parasites were first transmitted through their natural vector, the mosquito, rather than through a syringe? Would they be as virulent? Interestingly, infection was not as severe after mosquito transmission. But parasites evolved in the immunized mice retained a higher level of virulence than those evolved in the naïve mice. This means that immunity accelerates the evolution of virulence in malaria, even after mosquito transmission, making them more dangerous to nonimmunized hosts.How does immune selection create more virulent pathogens? One possibility is that even though many parasites die in immunized hosts, those that “win the race to the syringe”—or the mosquito—are likely genetically equipped to stay ahead of advancing immune system defenses.It's not entirely clear why selection would favor more virulent parasites, but since the virulent strains showed no problems transmitting infection to new hosts, it's likely that such strains would spread throughout an immunized population. While mosquito transmission likely plays a significant role in virulence evolution—it clearly reduced virulence here—the molecular mechanics of this effect are also mostly speculative at this point. Many questions remain, but these results make a strong case that vaccine development aimed at protecting individuals against infectious pathogens would do well to consider the evolutionary implications, or increased pathogen virulence could be an unintended consequence.


Although the effects of resistant starch (RS) on postprandial glycemia and insulinemia have been extensively studied, little is known about the impact of RS on fat metabolism. This study examines the relationship between the RS content of a meal and postprandial/post-absorbative fat oxidation.14C]-triolein with breath samples collected hourly following the meal, and gluteal fat biopsies obtained at 0 and 24 h. RS, regardless of dose, had no effect on fasting or postprandial insulin, glucose, FFA or TAG concentration, nor on meal fat storage. However, data from indirect calorimetry and oxidation of [1-14C]-triolein to 14CO2 showed that addition of 5.4% RS to the diet significantly increased fat oxidation. In fact, postprandial oxidation of [1-14C]-triolein was 23% greater with the 5.4% RS meal than the 0% meal (p = 0.0062).12 subjects consumed meals containing 0%, 2.7%, 5.4%, and 10.7% RS . Blood samples were taken and analyzed for glucose, insulin, triacylglycerol (TAG) and free fatty acid (FFA) concentrations. Respiratory quotient was measured hourly. The 0%, 5.4%, and 10.7% meals contained 50 μCi [1-These data indicate that replacement of 5.4% of total dietary carbohydrate with RS significantly increased post-prandial lipid oxidation and therefore could decrease fat accumulation in the long-term. Resistant starch (RS) is any starch that is not digested in the small intestine but passes to the large bowel for fermentation . RetrogrAcute human studies describe variable postprandial glycemic and/or insulinemic responses to RS ingestion. In general, it is accepted that RS consumption lowers postprandial glucose concentrations marginally and postprandial insulin concentrations markedly. Many groups report a decrease in postprandial glycemic or insulinemic responses to RS ingestion relative to digestible starch (DS) consumption -7, whereMany studies have examined the relationship between RS ingestion and postprandial metabolite and hormone concentrations. Fewer studies have documented the effect of RS on lipid metabolism. In humans, five weeks of RS feeding lowered fasting cholesterol and triglyceride concentrations and postprandial plasma insulin concentrations relative to digestible starch (DS) feeding ,13. It hIt is possible that strong physical association between RS and dietary lipid may slow the absorption, and thereby increase the oxidation, of dietary lipid. Currently, there is no evidence pertaining to the dose-response relationship for RS ingestion  and postprandial glycemia, insulinemia, fat oxidation, or meal fat storage. It is important that these parameters be defined before designing and conducting long-term, prospective RS feeding studies.No difference in fasting or postprandial insulin, glucose, FFA, or triglyceride concentration was observed between any of the RS doses examined Figure .Overall, the dose of RS in the meal had a significant influence on ΔRQ (respiratory quotient) values . Meal fat oxidation at the 5.4% RS dose was significantly higher than both the 0% (p = 0.0062) and 10.7% doses (p < 0.0001). Separate tests at 6 h or 24 h following the test meal gave comparable results  suggest that the inclusion of 5.4% RS in the meal elevated postprandial fat oxidation. Unexpectedly, this effect was lost if the dose was increased to 10.7% RS.Similarly, the oxidation of [s Figure . Taken t14C into gluteal adipose tissue, to be lower for the 5.4% RS meal than all other meals, although this effect did not reach statistical significance . This discovery was verified using two different methods, indirect calorimetry and the oxidation of [12 healthy adults, 7 male and 5 female, participated in the present study. This study was approved by the Colorado Multiple Institution Review Board, in compliance with the Helsinki Declaration, and full written consent was obtained from all subjects. To participate, subjects were required to be between 28 and 45 years of age, have normal glucose tolerance , moderate level of physical activity , and a BMI between 20 and 28. All female subjects were taking oral contraceptive pills or progesterone injections and were tested during the early follicular phase of the menstrual cycle. All subjects underwent dual energy X-ray absorptiometry  for analysis of body composition. As a group, subjects were 33 ± 5 years of age, 1.7 ± 0.07 m tall, weighed 75 ± 11 kg, had a BMI of 24.7 ± 2.4, total fat mass of 18.3 ± 5.0 kg (mean ± SD), and a fasting RQ of 0.750 ± 0.023 (mean ± SEM).Subjects received four meals differing only in resistant starch (RS) content in random order, approximately four weeks apart. Test meals contained either 0%, 2.7%, 5.4%, or 10.7% RS as a percentage of total dietary carbohydrate. All added RS was in the form of high-amylose maize starch, or RS2. High-amylose maize starch was chosen as it has the unique property of a very high gelatinisation temperature which allows it to maintain its granular structure during and after the processing conditions used to manufacture the foods being consumed in this study .All meals were isocaloric, accounting for 30% of the subject's daily energy needs as measured by indirect calorimetry prior to study commencement (RMR × daily activity factor of 1.49). The composition of the test diet was 55% carbohydrate, 15% protein, and 30% fat as a percentage of total energy  on an outpatient basis. Subjects were instructed to eat all of the food/drink provided and not to consume any other foods. Non-caloric beverages could be consumed during the three day lead-in diet.Following an overnight fast (12 h), subjects were admitted to the GCRC and an intravenous catheter was placed for the purposes of drawing blood. The test meal began at 0 min (0800 h) with all food/drink fully consumed within 15 min. Blood samples were taken at 0, 30, 60, 90, 120, 180, 240, 300, and 360 min following meal ingestion and analyzed for glucose, insulin, triacylglycerol (TAG) and free fatty acid (FFA) concentrations. Respiratory quotient (RQ) was measured at hourly intervals after ingestion of the meal via gas collection under a ventilated plexiglass hood for 15 min (Sensormedics 2900 metabolic cart). All urine produced between 0 and 360 min was collected and analyzed for nitrogen content by the GCRC Core Laboratory to facilitate calculation of non-protein RQ.14C]-triolein  suspended in olive oil and the tests were conducted as 24 h inpatient stays at the GCRC. The fat tracer was fed as a triglyceride (glycerol tri [1-14C]oleate) rather than a FFA (eg. [1-14C]oleate) in order to reflect any change in the absorption of triglyceride FFA which might be due to a strong physical association with RS thereby slowing absorption. At hourly intervals following the meal, then at 8, 10, 12, 14 and 24 hours, breath samples were collected via exhalation through a tube with a one-way valve into scintillation vials containing 2 mmol benzethonium hydroxide (to trap 2 mmol CO2), 1 ml methanol, and 1 mg phenolpthalene as a pH indicator. Gluteal fat biopsies were collected by aspiration through a 14 g stainless steel needle at baseline and 24 h after ingestion of the test meal. All breath and fat samples were assayed for the presence of 14C (as described below). For these 24 h tests, subjects received 30% of daily energy needs at each of breakfast, lunch, and dinner, with the remaining 10% of calories received in an evening snack. The timing of meals/snacks was kept constant over all tests. All food was provided by the GCRC on an inpatient basis and the macronutrient content of each meal was the same as that of the test meal. Only the test breakfast contained RS during these 24 h tests, all other meals were composed of standard, commercially available products.In three of the test meals , the bread product in the test meal was spiked with 50 μCi [1-All glucose, FFA, and TAG assays were conducted by the GCRC Core Laboratory using an automated Cobas Mira Plus (Roche Diagnostics, Basel, Switzerland). Serum insulin measurements were also performed by the GCRC Core Laboratory using a human insulin RIA kit (Linco, St. Louis, USA).Fat samples, frozen in liquid nitrogen and stored at -80°C until processing, were incubated in 450 μl Solvable (Packard Bioscience, Groningen, Netherlands) at 50°C for 12 h before the addition of 100 μl 30% (v/v) hydrogen peroxide (for sample bleaching). Fat samples were counted in Aquasol (Packard Bioscience, Groningen, Netherlands) whereas breath samples were counted in Scintisafe 30%  using a Beckman LS6500 scintillation counter (Beckman Instuments, Fullerton, CA). After scintillant was added, all samples were kept in the dark at room temperature for 48 h before being counted to reduce chemiluminescence.et al. .24h/g tissue weight) - (dpmbaseline/g tissue weight) × 1/specific activityμg fat stored/g fat tissue = 2)/vCO2 (min.ml) = (dpmt - dpmbackground) × 1/vCO2 =counts from sample (dpm/mol CO2/ min.mldpm.mol CO2/ min.ml × 0.446 (as 1 ml CO2 = 0.446 mol)dpm/min = dpm.mol COg fat oxidized = AUC(dpm/min) × 1/specific activity2 is the rate of CO2 production as assessed during indirect calorimetry. t is sample time (min). AUC is the incremental area under the curve.where vCOAll statistical analyses were performed using the statistical analysis software SAS, version 8.1 (SAS OnlineDoc, 2000) with a significance level of p = 0.05 and p = 0.01 for interaction terms. All results are presented as mean ± SEM, except for subject characteristics which are described as mean ± SD. To investigate each of the outcomes  we used a mixed model with fixed effect terms for RS DOSE, TIME and the interaction of the two, RS DOSE*TIME. Subjects were included as random effects. The interaction term was not significant for any of the outcomes tested so an additive model was used to test the overall effect of RS DOSE and the differences between doses. To test the effects of RS DOSE at different TIMES, a model that included RS DOSE, TIME and RS DOSE*TIME was used. The repeated measures nature of the study design was taken into account by using the covariance structures available in SAS PROC MIXED. For example, measurements within a subject are assumed to be more highly correlated than between subjects, and within a particular treatment, within a subject, the measurements are assumed to be more correlated. Measurements closer in time to one another were modeled with an autoregressive, or AR(1) covariance structure.RS, resistant starch; DS, digestible starch; TAG, triacylglycerol; FFA, free fatty acid; FFM, fat free mass; SCFA, short-chain fatty acids; GCRC, General Clinical Research Center; RQ, respiratory quotientJanine Higgins and Ian Brown are listed as inventors on RS patents filed by Penford Australia Limited. Both Drs. Higgins and Brown are listed as inventors on these patents as they have intellectual property ownership of some of data used in these but receive no financial benefit.JH conceived of the study design and was responsible for overall study coordination, conducting patient visits, data analysis, and manuscript preparation. DH was responsible for patient scheduling, day-to-day study coordination, conducting patient visits, and data entry. WD contributed to the study design and manuscript preparation, and conducted patient physical examinations and fat biopsies. IB contributed to the study design, selection of RS foods, and assisted with manuscript preparation. MB conducted all statistical analysis. DB contributed to the study design and manuscript preparation, and conducted patient visits, patient physical examinations and fat biopsies. All authors read and approved the final manuscript.Individual meal (a) and total fat oxidation (b) in response to the RS content of a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults.Click here for fileIndividual area under the glucose curve vs. meal (a) and total fat oxidation (b) in response to a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults. Data from all three test meals (0%, 5.4%, and 10.7% RS) is shown. The relationship between area under the glucose curve and fat oxidation remains the same (i.e. no relationship) when represented as individual doses or, as in this plot, for all doses .Click here for fileIndividual area under the glucose curve vs. meal fat oxidation in response to a 0% (a) or 5.4% (b) RS test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in healthy adults. Data from individual test meals is shown.Click here for fileIndividual area under the insulin curve vs. meal (a) and total fat oxidation (b) in response to a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults. Data from all three test meals (0%, 5.4%, and 10.7% RS) is shown. (Document type: Powerpoint, PPT)Click here for file


Familial hypercholesterolemia is a human monogenic disease caused by population-specific mutations in the low density lipoprotein (LDL) receptor gene. Despite thirteen different mutations of the LDL receptor gene were reported from Russia prior to 2003, the whole spectrum of disease-causing gene alterations in this country is poorly known and requires further investigation provided by the current study.Forty-five patients with clinical diagnosis of FH were tested for the apolipoprotein B (apoB) mutation R3500Q by restriction fragment length analysis. After exclusion of R3500Q mutation high-sensitive fluorescent single-strand conformation polymorphism (SSCP) analysis and automatic DNA sequencing were used to search for mutations in the LDL receptor gene.We found twenty one rare sequence variations of the LDL receptor gene. Nineteen were probably pathogenic mutations, and two (P518P, T705I) were considered as neutral ones. Among the mutations likely to be pathogenic, eight were novel (c.670-671insG, C249X, c.936-940del5, c.1291-1331del41, W422X, c.1855-1856insA, D601N, C646S), and eleven (Q12X, IVS3+1G>A, c.651-653del3, E207X, c.925-931del7, C308Y, L380H, c.1302delG, IVS9+1G>A, V776M, V806I) have already been described in other populations. None of the patients had the R3500Q mutation in the apoB gene.Nineteen pathogenic mutations in the LDL receptor gene in 23 probands were identified. Two mutations c.925-931del7 and L380H are shared by St.-Petersburg population with neighbouring Finland and several other mutations with Norway, Sweden or Denmark, i.e. countries from the Baltic Sea region. Only four mutations (c.313+1G>A, c.651-653del3, C308Y and W422X) were recurrent as all those were found in two unrelated families. By this study the number of known mutations in the LDL receptor gene in St.-Petersburg area was increased nearly threefold. Analysis of all 34 low density lipoprotein receptor gene mutations found in St.-Petersburg argues against strong founder effect in Russian familial hypercholesterolemia. Familial hypercholesterolemia (FH) OMIM #143890) is one of the most common monogenic human diseases. It is inherited as an autosomal dominant trait with the prevalence of the heterozygous form conventionally considered to be about 1 of 500 in most populations 43890 is . FurtherThe spectrum of LDL receptor mutations varies between different human populations and more than 900 mutations in the LDL receptor gene have been characterized worldwide -9. A recPatients with FH were recruited from two lipid clinics of St.-Petersburg, namely Institute of Human Brain and Institute of Experimental Medicine. The clinical diagnosis of familial hypercholesterolemia was based on the following criteria: highly elevated plasma total cholesterol and LDL-cholesterol, presence of tendon xanthomata, corneal arcus or both, and positive family history of myocardial infarction and hypercholesterolemia with at least one first-degree relative affected. Forty-five probands fulfilling at least two of three criteria listed above were selected for the study. Full data of patients including their lipid data are given in the Discussion section  in exon 2 that allows a simple detection of this mutation. Restriction enzyme test was performed for the proband and her 4 descendants  that was identified by means of SSCP and verified by sequencing in probands from two unrelated families. SSCP patterns on silver-stained gels differ strikingly in patients with and without the mutation. The presence of the mutation was confirmed in the son of the proband by SSCP.In heterozygotes a deletion of 41 nucleotides at c.1291-1331 in exon 9 (mutation FsV409:S423X) results in occurrence of specific heteroduplexes. Besides, a PCR fragment of smaller size as compared to the normal allele is revealed in silver-stained polyacrylamide gels  were found to be recurrent, i.e. all of those were found in two apparently unrelated families. Together with the data obtained earlier ,38 our rThe author(s) declare that they have no competing interests.FMZ, MYM, VIG, PHN, GGN, AS performed cloning, SSCP, sequencing and familial analysis, BML, VOK, DD, ADD selected patients, VBV and OF participated in the study design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Sign-language comprehension activates the auditory cortex in deaf subjects. It is not known whether this functional plasticity in the temporal cortex is age dependent. We conducted functional magnetic-resonance imaging in six deaf signers who lost their hearing before the age of 2 years, five deaf signers who were >5 years of age at the time of hearing loss and six signers with normal hearing. The task was sentence comprehension in Japanese sign language.The sign-comprehension tasks activated the planum temporale of both early- and late-deaf subjects, but not that of hearing signers. In early-deaf subjects, the middle superior temporal sulcus was more prominently activated than in late-deaf subjects.As the middle superior temporal sulcus is known to respond selectively to human voices, our findings suggest that this subregion of the auditory-association cortex, when deprived of its proper input, might make a functional shift from human voice processing to visual processing in an age-dependent manner. Sign languages involve the use of the hands and face, and are perceived visually -3. Usinget al.  observed [et al.  found thPerformance on the JSL comprehension task was similar across the groups F2, 14) = 1.279, P = 0.309, one-way ANOVA). The patterns of activity evoked during the sign-comprehension task in the hearing signers and the deaf groups are shown in Figure 4 = 1.2794 = 1.279The onset of deafness is related to language acquisition. Prelingual deafness occurs before spoken language is learned. Hearing people generally learn their first language before 5 years of age; hence, prelingual deaf individuals are either deaf at birth or became deaf prior to developing the grammatical basis of their native language, which is usually before the age of 5 years. Postlingual deafness is the loss of acoustic senses, either suddenly due to an accident or as a gradual progression after native-language acquisition . Hence, The posterior STS was activated in all groups during sign comprehension, which is consistent with the proposed neural substrates that subserve human movement perception . The posBoth the early- and late-deaf groups showed activation in the planum temporale, whereas hearing signers did not. Anatomically, the anterior border of the PT is the sulcus behind Heschl's gyrus and the medial border is the point where the PT fades into the insula. The posterior border of the PT involves the ascending and descending rami of the Sylvian fissure . Functioet al. [It has been shown that non-linguistic visual stimuli (moving stimuli) activate the auditory cortex in deaf individuals, but not in hearing subjects ,16. McSwet al.  showed tet al.  revealedet al. ,20 that et al.  indicateet al. .The middle STS, anterior to the Vpc line, was activated more prominently in the early- than the late-deaf subjects. This difference is probably not related to linguistic processes, as both early- and late-deaf subjects are equally capable of learning JSL with the same amount of training. The middle STS region is presumably the area that is selective to human voice processing . This arper se but rather to vocal features that carry nonlinguistic information [Considering that the STS voice-selective area is not sensitive to speech ormation , the funThe results of the present study suggest that in early-deaf subjects, non-auditory processing, such as that involved in the perception and comprehension of sign language, involves the under-utilized area of the cortex that is thought to be selective to the human voice (middle STS). This indicates that the sensitive period for the establishment of human voice processing in the STS might be during the first 2 years of life.The subjects comprised six early-deaf signers (mean age: 22.8 ± 3.1 years), five late-deaf signers (mean age: 34.4 ± 16.2 years) and six hearing signers  and a standard birdcage head coil. Each volume consisted of 11 slices, with a slice thickness of 8 mm and a 1-mm gap, which covered the entire cerebral cortex. The time interval between two successive acquisitions of the same image was 3,000 ms, the echo time was 50 ms and the flip angle was 90 degrees. The field of view was 22 cm. The digital in-plane resolution was 64 × 64 pixels. For anatomical reference, T1-weighted images were also obtained for each subject.x, y and z axes.The first three volumes of each fMRI session were discarded because of unstable magnetization. The remaining 40 volumes per session were used for statistical parametric mapping (SPM99, Wellcome Department of Cognitive Neurology, London, UK) implemented in Matlab (Mathworks, Sherborn, Mass., USA) ,27. Follt statistic (SPM{t}).Statistical analysis was conducted at two levels. First, the individual task-related activation was evaluated. Second, the summary data for each individual were incorporated into the second-level analysis using a random-effects model to make inferences at a population level. The signal was proportionally scaled by setting the whole-brain mean value to 100 arbitrary units. The signal time course for each subject was modeled using a box-car function convolved with a hemodynamic-response function and temporally high-pass filtered. Session effects were also included in the model. The explanatory variables were centered at zero. To test hypotheses about regionally-specific condition effects (that is, sentence comprehension compared with rest), estimates for each model parameter were compared using the linear contrasts. The resulting set of voxel values for each contrast constituted a statistical parametric map (SPM) of the a priori hypothesis that there would be more prominent activation in the early- than late-deaf subjects, we focused on the temporal cortex, which was anatomically defined in standard stereotaxic space [t} was set at P < .001 without a correction for multiple comparisons.The weighted sum of the parameter estimates in the individual analyses constituted 'contrast' images that were used for the group analysis. Contrast images obtained via individual analyses represent the normalized task-related increment of the MR signal of each subject. To examine group differences  in activation due to the sign-comprehension task, a random-effect model was performed with the contrast images (1 per subject) for every voxel. Using the ic space . The thrNS carried out the fMRI studies, data analysis and drafted the manuscript. HY and TO conducted the MR imaging. MY, TH and KM prepared the task materials. YY and HI participated in the task design and coordination. All authors read and approved the final manuscript.


Falls are common among community-dwelling elderly people and can have a considerable impact on quality of life and healthcare costs. People who have sustained a fall are at greater risk of falling again.We replicated a British randomised controlled trial which demonstrated the effectiveness of a multidisciplinary intervention programme to prevent falls.The objective is to describe the design of a replication study evaluating a multidisciplinary intervention programme on recurrent falls and functional decline among elderly persons at risk. The study consists of an effect evaluation, an economic evaluation and a process evaluation.The programme is aimed at community-dwelling elderly people aged 65 years or over who have visited an accident and emergency department (A&E department) or a general practitioners' cooperative (GP cooperative) because of a fall.The design involves a two-group randomised controlled trial. Participants are followed for twelve months after baseline. The intervention programme consists of a detailed medical and occupational therapy assessment with referral to relevant services if indicated. People in the control group receive usual care.The main outcome measures of the effect evaluation are number of falls and daily functioning. The economic evaluation will be performed from a societal perspective. A process evaluation will be carried out to evaluate the feasibility of the intervention programme. This article describes the design of a replication of a randomised controlled trial (RCT) evaluating the effectiveness and cost-effectiveness of a multidisciplinary intervention programme to prevent further falls among elderly people at risk.Publishing the design and protocol of a study before results are available is important for several reasons. A published protocol allows easier comparison between what was originally intended and hypothesised and what was actually done , and it About one-third of people over the age of 65 fall at least once a year . People Since preventing falls has been a matter of interest for years, many programmes aimed at preventing falls have been developed and evaluated. Unfortunately, many of these have turned out to be ineffective . HoweverBecause details of the status of the participants, the context of the intervention and the content and presentation appear to be critical, it has been recommended to re-evaluate effective intervention programmes in different healthcare systems . We therThe main objective of our current study is to evaluate the effects of a multidisciplinary intervention programme on recurrent falls and functional decline among elderly persons who have visit a general practitioners' cooperative (GP cooperative) and/or an accident and emergency department (A&E department) because of a fall. This objective has resulted in the following research questions.• Is a multidisciplinary intervention programme more effective than usual care in preventing new falls and functional decline among community-dwelling elderly people who visit a GP cooperative and/or A&E department at a hospital because of a fall?• Is the multidisciplinary intervention programme cost-effective compared to usual care when assessed from a societal perspective?Besides the effect evaluation and economic evaluation, a process evaluation is being carried out to assess the feasibility and applicability of the intervention programme for those receiving and implementing the intervention.Figure We have taken various measures to ensure blinding in the data collection process. Questionnaires are collected anonymously and sorted by number. Follow-up measurements by phone are contracted out to an independent call centre, whose operators are unaware whether the subjects have been allocated to the intervention or the control group.The study design and protocols were approved by the Medical Ethics Committee of the University Hospital and University of Maastricht.Various studies have been conducted to assess the effectiveness of programmes to prevent falls. Although most studies were aimed at the general population of elderly people, details of the status of the participants appear to be critical ,6. SeverRecruitment of subjects took place at the local GP cooperative and the A&E department of the University Hospital in Maastricht. The Maastricht GP cooperative is a group of GPs from practices in the town of Maastricht and the surrounding area who have founded a non-profit organisation to provide care for their own patients after hours . The MaaThe following inclusion criteria were used: age 65 years or older, community-dwelling, having visited the A&E department or GP cooperative at the University Hospital Maastricht for the consequences of a fall, and living in Maastricht or its surrounding area. People were only allowed to enter the programme after completing and returning an informed consent form. Exclusion criteria were: not able to speak or understand Dutch, not able to complete questionnaires or interviews by telephone, cognitive impairment  ,13, longSample size calculations were based on the expected effects of the intervention on the main outcome measure, the percentage of people sustaining a fall during one year of follow-up. The study by Close et al.  found thTo adapt the programme developed by Close et al.  to the DThe final programme includes a medical and occupational therapy assessment resulting in recommendations. The medical assessment consists of an examination performed by a geriatrician, a geriatric nurse and a rehabilitation physician to identify and address risk factors for falling. The examination includes a comprehensive general examination, but in addition focussed on a more detailed assessment of visual acuity, stereoscopic vision, mobility, balance, cognition, affect, use of medication and examination of feet and footwear. Recommendations or indications for referral resulting from this examination are sent to the patient's GP. After the medical assessments, an occupational therapist visits the patients to identify possible risk factors for falling in the home environment. The therapist makes recommendations regarding adaptations to the home environment, assistive devices, home care and behavioural change. Recommendations by the occupational therapist are sent directly to the subjects themselves and to their GPs. As stated before, the intervention period is scheduled to last for a maximum of 3.5 months after the baseline measurement.An important addition to Close et al.'s protocol  is the cPeople in the control group receive usual care. At present, no guidelines exist for the systematic assessment of the underlying causes of an injurious fall presented at an A&E department or GP cooperative in the Netherlands. In usual care, medical risks and other risk factors such as environmental hazards in the home and patients' risk behaviour are not systematically registered and addressed by hospital physicians, specialists or general practitioners. Moreover, no systematic attention is currently being paid to the specific consequences of an injurious fall for the daily functioning of individual patients in their unique situation. We placed no restrictions on co-interventions.The primary outcome measures of the effect evaluation are number of falls and daily functioning. Number of falls is subdivided into three separate measures: the percentage of elderly people sustaining a fall during the one-year follow-up period, recurrent falls during follow-up , and injurious falls during follow-up . Falls are recorded continuously by means of a fall calendar during the twelve-month follow-up period. Subjects are called monthly to report their falls as recorded on the fall calendar relating to the previous month.We decided to measure daily functioning by means of the Frenchai Activity Index (FAI) , in contOur secondary outcome measures are: recuperation from the fall, health complaints, perceived health measured by means of the first two items of the RAND-36 , ADL andBesides the primary and secondary outcome measures, we assess some background variables which are considered to be predictors, confounders or effect modifiers. The following personal characteristics are assessed: age, sex, marital status, living alone and socio-economic status. In addition, we assess the circumstances and causes of the falls reported at the GP cooperative and/or A&E department, the consequences of the falls , the tyThe economic evaluation in our study is being performed from a societal perspective, which implies that all costs and outcomes are taken into account if possible. The economic evaluation will be a combination of a cost-effectiveness and a cost-utility analysis. The primary outcome measure for the cost-effectiveness analysis will be the percentage of people sustaining a fall during one year of follow-up. As mentioned above, falls are recorded by means of a calendar. Within the cost-utility analysis, the effects are measured in terms of generic health-related quality of life descriptions, measured according to the standard Dutch version of the EuroQol (EQ 5-D)  in self-We will assess programme costs, healthcare costs and patient and family costs. All costs are measured by means of a cost diary , in whicThe process evaluation involves assessing the extent to which the intervention programme is performed according to protocol, the nature of the recommendations made to the participants, participants' compliance with these recommendations and the opinions of participants, physicians and therapists about the intervention programme and recommendations. Data on these topics are collected using the following methods: structured registration forms for the medical and occupational parts of the intervention programme; self-administered evaluation forms filled in by the participants after the medical intervention; interviews by telephone with the participants six weeks or longer after the recommendations are sent and interviews with all participating physicians and therapists at the end of the intervention period.Data will be primarily analysed according to the intention-to-treat principle, i.e., including all participants with valid data, regardless of whether they received or did not receive the intervention. Subsequently, the results of the intention-to-treat analysis will be compared with the results of an on-treatment analysis, to assess whether protocol deviations have caused bias. Participants with documented deviations from the study protocol (i.e., participants in the intervention group who did not receive the entire intervention or participants in either the intervention or the control group with incomplete follow-up data) will be excluded from this on-treatment analysis.Comparability between the intervention and control groups will be assessed at baseline to check for differences between the two groups. Outcome at four and twelve months will be compared between the intervention and control groups by both univariate and multivariate techniques. We will use multivariate analysis to adjust for possible differences in baseline scores and background variables between the intervention and control groups. Dropouts and losses-to-follow up will be described.The economic evaluation will involve calculating cost-effectiveness and cost-utility ratios. The additional costs and additional benefits of the intervention programme compared with usual care will be examined by calculating incremental cost-effectiveness and cost-utility ratios. These incremental ratios represent the difference in mean costs between the intervention and usual care groups in the numerator and the difference in mean effects in the denominator.Since the recruitment period is only 14 months, and the follow-up period is also relatively short (12 months), it is unlikely that there will be substantial differences between costs made by and for patients who started in the first part of the recruitment period and those who started in the last part. Therefore, discounting of costs is not required. Finally, a sensitivity analysis will be done to assess the generalisability of the assumptions made in the costing process. This sensitivity analysis, which involves calculating the upper and lower limits of the confidence interval of cost and effect variables, will allow us to explore and quantify the uncertainty not related to sampling variations.The process evaluation will mainly be analysed by means of descriptive techniques.Recruitment of eligible subjects commenced in December 2002 and ended in February 2004, resulting in a total of 333 eligible subjects being included in the trial. Randomisation resulted in the allocation of 166 participants to the intervention group and 167 to the control group. Of the 333 persons recruited, 105 (32 %) are male and 228 (68 %) are female.The follow-up period is to end in May 2005. Results on the effects of the programme will be available in 2006 and will be published in the relevant journals.Although the intervention has been subject of earlier research, this study will provide new information about the effectiveness in the Dutch situation. Furthermore, the results of the economic evaluation can provide information about the cost-effectiveness of the intervention and the effects on quality of life. In case of shown effectiveness and cost-effectiveness, we aim to implement this intervention into usual healthcare.The author(s) declare that they have no competing interests.All author's read and approved the final version of the manuscript.MH is the investigator and performed most of the writing of this manuscript.JH supervises the planning of the project and wrote the study design.JD supervises the planning and methodological aspects of the project.SA supervises the economic evaluation.HC supervises the clinical part of the project.JE is the principal investigator of the study.The pre-publication history for this paper can be accessed here:


Additional PEEP to 10 cm H2O was added after injury. Animals were randomized to one of the 3 modes of ventilation and followed for 5 hr after injury.We compared gas exchange, respiratory mechanics, and measured bronchoalveolar fluid for inflammatory cytokines, cell counts and surfactant function. Lung injury was scored by light microscopy. Pigs received mechanical ventilation (F2 and respiratory system compliance was significantly greater with biologically variable ventilation compared to the other 2 groups. Mean and mean peak airway pressures were also lower. There were no differences in cell counts in bronchoalveolar fluid by flow cytometry, or interleukin-8 and -10 levels between groups. Lung injury scoring revealed no difference between groups in the regions examined. No differences in surfactant function were seen between groups by capillary surfactometry.PaOIn this porcine model of acute lung injury, various indices to measure injury or inflammation did not differ between the 3 approaches to ventilation. However, when using a low tidal volume strategy with moderate levels of PEEP, sustained improvements in arterial oxygen tension and respiratory system compliance were only seen with BVV when compared to CMV or CMV with a recruitment manoeuvre. T) in patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is alveolar collapse [T) approach the addition of higher PEEP levels offers no further improvement in outcome [IO2, it remains unclear how best to improve and sustain oxygenation, during low VT ventilation strategies for ALI/ARDS management.A negative consequence of mechanical ventilation using lower tidal volumes , a unique computer-controlled version of control mode ventilation (CMV). With BVV, gas exchange and respiratory mechanics improved in animal models, with [T protocols using an ARDSNet algorithm [T ventilation with a recruitment manoeuvre in ALI/ARDS using a low VT approach.Buchman  and othels, with  and withls, with , during lgorithm  and in hlgorithm . After dlgorithm . A recenlgorithm . Other ilgorithm  and a malgorithm . While plgorithm , not a m2O of continuous positive airway pressure for 40 sec performed hourly for 5 hrs. This approach has been shown to improve oxygenation in patients with early ARDS who do not have any chest wall impairment [Therefore, in this study in pigs with oleic acid lung injury, we compared BVV to conventional CMV or CMV with a recruitment manoeuvre (CMV-RM) of 40 cm Hpairment . A multipairment ,20 at en2 of 35–45 mm Hg. Anaesthesia was maintained with 2% isoflurane in 100% oxygen during surgical preparation. Intravenous rocuronium bromide (1 mg/kg/hr) was administered by continuous infusion for muscle relaxation. Lactated Ringer's solution was given intravenously during the surgical preparation and for the duration of the experiment.Pigs (weighing 20–30 kg) received 0.6 mg atropine, 15 mg midazolam, and 300 mg ketamine intramuscularly for sedation. Isoflurane 5% in 100% oxygen was delivered via facemask to induce anaesthesia. When sufficient depth of anaesthesia was achieved, the pigs were intubated orotracheally with a 6.0 mm cuffed endotracheal tube. Mechanical ventilation was instituted with an Ohio 7000 ventilator  with minute ventilation adjusted to maintain a PaCO® ventilator  capable of delivering either CMV or BVV. The ventilator was set to deliver a square wave inspiratory flow pattern with an I:E ratio of 1:2. Isoflurane was discontinued, and a propofol/ketamine infusion at 10/2.5 mg/kg/hr substituted to maintain anaesthesia.A thermodilution pulmonary artery catheter (7.5-Fr) was inserted and advanced into the right external jugular vein until a satisfactory pulmonary capillary wedge tracing was obtained. Temperature was measured from the tip of the pulmonary artery catheter. The right femoral artery was cannulated for continuous pressure transduction and arterial blood gas (ABG) analysis. A 5-Fr single lumen femoral venous catheter was advanced into the inferior vena cava for infusion of oleic acid. A surgical tracheotomy was performed and the animal was switched to an Esprit2O recruitment manoeuvre for 30 sec, animals were ventilated with a VT of 10 ml/kg at an FIO2 of 0.3, with 5 cm H2O of PEEP. Respiratory rate was adjusted to maintain PaCO2 between 35–45 mm Hg.After a 30 cm HAfter 15 min to stabilize, baseline measurements were obtained. Haemodynamic measurements included mean arterial pressure (MAP), heart rate, central venous pressure (CVP), mean pulmonary artery pressure (MPAP), and pulmonary artery occlusion pressure (PAOP). All haemodynamic data were continuously recorded on a Gould 2600 Oscillograph (Gould, Cleveland, OH). Cardiac output (CO) was measured by thermodilution, in triplicate, at stated measurement periods.T intermittently; this data was recorded using an advanced CODAS (Dataq Instruments, Akron, OH) data acquisition system.A pneumotachograph  with the sensor immediately proximal to the tracheotomy was used to measure airway pressures and VArterial and mixed venous gases were analyzed using a Radiometer ABL 500 (Copenhagen NV, Denmark). Arterial and mixed venous oxygen content, oxygen saturation, and haemoglobin concentrations were measured with a Radiometer OSM3 set for porcine blood.T used was that which the animal was receiving at that time.Static respiratory system compliance (Crs) was measured in triplicate by clamping the expiratory limb of the ventilator circuit at the end of inspiration for 1 sec to obtain a plateau pressure. The V2 <60 mm Hg for two consecutive measurements (PaO2/FIO2 <200). Dopamine was started at 5 μg/kg/min and was titrated to keep the MAP >60 mmHg. When the oxygenation target was achieved, the oleic acid infusion was stopped and the PEEP was increased to 10 cm H2O. Ten minutes after the increase in PEEP, an arterial blood gas sample was obtained to determine if PaO2 had increased. PaO2 had to be >75 mm Hg but <90 mm Hg. This was considered to represent adequate lung injury, but indicate that collapsed alveoli could be recruited with the additional PEEP. If the PaO2 did not increase, the experiment was terminated. If the PaO2 increased to >90 mm Hg, additional oleic acid was infused until the PaO2 decreased to <60 mm Hg.Oleic acid was infused via the 5-Fr femoral venous catheter at 0.2 ml/kg/hr until PaOT (7 ml/kg) protocol was initiated and the respiratory rate increased to 30 bpm. After 10–15 min arterial blood gas sampling was done to assess the stability of the PaO2. If the PaO2 remained stable, the animals were then randomized into one of three groups: conventional ventilation with a VT of 7 ml/kg (CMV); conventional ventilation with VT of 7 ml/kg with a 40 sec, 40 cm H2O recruitment manoeuvre performed hourly (CMV-RM). The recruitment manoeuvre was performed at end-expiration with the PEEP level maintained at 10 cm H2O at an FIO2 of 0.3; or biologically variable ventilation (BVV) with a mean VT of 7 ml/kg.A low VFollowing stable oleic acid lung injury, haemodynamic, gas exchange and respiratory system compliance (Crs) measurements were recorded and obtained hourly thereafter. Measurements in the CMV-RM group were obtained 5 min after each recruitment manoeuvre (RM). An additional measure of gas exchange and Crs was made in the CMV-RM group 50 min after the RM, to ascertain the duration of effect of the RM.en bloc. Following removal, the lungs were suspended upside down for 10 min to collect bronchoalveolar (BAL) fluid. Samples of BAL fluid were collected in heparinized saline and then frozen immediately at -80oC. Fluid was then sent for cytokine analysis, measures of surfactant function and flow cytometry. The lungs and previously collected BAL fluid were weighed and the lungs were suspended and aerated overnight. The following day, the lungs were placed in an oven to dry, and following a stable dry measurement, wet:dry weight ratio was calculated.At the end of the experiment, a sternotomy was performed. Animals were sacrificed with a lethal dose of thiopental. The trachea was then clamped at end inspiration and the heart and lungs were removed Surfactant function was assessed on BAL fluid samples using a capillary surfactometer  in the manner of Enhorning and colleagues ,22. Such1. Raw BAL fluid surfactant function analysis – centrifuging at 200 g for 5 min to rid large debris, then the supernatant spun at 10,000 g and pellet resuspended in 100 μL saline and analyzed using the surfactometer.2. Surfactant resuspended in 100 μL of saline after chloroform/methanol extraction. Chloroform/methanol extraction permits the lipid and phospholipid fraction to dissolve in the organic nonpolar solvent (chloroform) and the solvent evaporated to dryness. Volume of the final ratio for chloroform:methanol:water was 1:1:0.9. Following extraction, the pellet was resuspended and analyzed using the surfactometer.The percentage of time that the capillary tube was open for 2 min was determined for each sample, in triplicate, then averaged. Standards were saline; 0% patent for a 2 min time period and bovine surfactant; >98% patent for a 2 min time period. Each sample was measured in a blinded fashion in both conditions.The BAL fluid sample was passed through a cell strainer and 100 μL was transferred into a 5 × 75 mm tissue culture tube. Red cells were lyses by the addition of 500 μL Optilyse C (Beckman Coulter, Mississauga, Canada) and following 15 min incubation at room temperature, the cell suspension was diluted by the addition of 500 μL Isoton II and 100 μL FlowCount Fluorospheres (Beckman Coulter). The cell suspension was immediately analyzed on a Beckman Coulter EPICS Altra cell sorter configured with a high-speed quartz flow cell tip and a water-cooled argon laser emitting 150 mW at 488 nm. Forward and side light scatter signals were employed to derive 2-parameter histograms which clearly defined the fluorescent beads and three populations of cells, subsequently defined as mononuclear cells, neutrophils and eosinophils. At least 20,000 cells were analyzed for each sample. The acquisition software provided with the instrument automatically calculated the concentrations of each cell population based on the number of events in each of the 4 analysis regions.The incubation times and washes were preformed as specified in each respective kit: . Immunoassay kits for swine IL-8 and IL-10 were used. Sample incubation times were kept as constant as possible by pre-plating on a blank 96 well plate before transferring to the coated assay plate. ELISA plates were read at 450 nm by an SLT Rainbow plate reader (Lab Instruments, Research Triangle Park, NC). Standard curves and concentration calculations were performed according to kit directions.For light microscopy, four blocks of tissue from the right lung – upper lobe, middle lobe, nondependent and dependent lower lobe, fixed in buffered formalin, processed and embedded in formalin. Sections were cut and stained with haematoxylin and eosin. Under light microscopy, a lung injury scoring system modified from Rotta et al. ,20 was uParametric data were analyzed by repeated measures ANOVA using least squares means test matrices to identify differences within and between groups from group × time or group effects. Bonferroni's correction was applied where appropriate. Non-parametric comparison of the lung injury scores was by Kruskal-Wallis test. In all circumstances a p-value ≤0.05 corrected for multiple comparisons was considered significant.Twenty-five experiments were undertaken. One animal was discarded because oleic acid injury could not be established after 1.5 hrs of infusion. In the CMV-RM group, one animal died 2 hr into the experiment but data are included in analysis in this experiment up to time of the animal's demise. Complete experiments were done in twenty-three animals; .Temperature and haemodynamic data are shown in Table 1: see 2 over time . In all groups, the PaCO2 increased following oleic acid injury and was essentially stable at these elevated levels for the remainder of the experiment. The group × time for PvO2 was significant at p = 0.002 with significantly lower PvO2 over time in the CMV-RM group. Shunt fraction was lower in the BVV group (group × time interaction p = 0.003).Table 2: see 2 tended to decrease immediately following a recruitment manoeuvre, but increased over time as measured at 50 min. PaO2 significantly decreased in RM4 and 5 (-11.1 ± 5.2 mm Hg and -5.2 ± 4.4 mm Hg respectively). Following recruitment, PaO2 increased slowly early in the experiment at RM2 and RM3 (10.1 ± 5.5 mm Hg and 11.8 ± 9.1 mm Hg respectively), but failed to do so in the later time periods. For respiratory system compliance, no statistically significant differences were seen at any time period over the course of the experiment in the CMV-RM group.PaO2O with BVV; 15.1 ± 0.2 with CMV-RM and 15.1 ± 1.0 with CMV respectively). In all 3 groups, mean Paw increased following oleic acid. A more pronounced effect was seen with peak Paw  with mean peak pressures over time least with BVV . There was no group × time interaction for VT in ml/kg measured over time (p = 0.617).A group × time interaction for mean Paw was seen (p = 0.002) with Paw lowest with BVV  and exponential (R2 = 0.41) curve fits.The ELISA results to assess the cytokine IL-8 concentration in BAL fluid indicated no statistically significant difference for the 3 groups: 564 ± 551 pg/mL with BVV; 653 ± 639 pg/mL with CMV and 300 ± 235 pg/mL with CMV-RM. As in the measurement of neutrophil counts, there was significant variability with IL-8 levels ranging over a 385-fold concentration difference. Such a broad range of data in both neutrophil counts and IL-8 concentration suggested the possibility of power law behaviour and correlation between these variables. A very strong power law relationship was found between IL-8 pg/mL and absolute neutrophil count/mL for pooled data; y = 77609x-6x1.38; n = 13, R2 = 0.83. In this situation the linear and exponential fits were R2 = 0.61 and R2 = 0.69 respectively. In this analysis, IL-8 concentration is presumed to be the dependent variable – being released by the monocytes (counts on the x-axis).The number of mononuclear cells from cell cytometry of BAL fluid was not different between groups: for BVV, 1346 ± 743 cells/μL; for CMV, 1767 ± 905 cells/μL; and for CMV-RM, 1068 ± 579 cells/μL. These cells presumably in large part represent alveolar macrophages. A power law relationship was seen between pooled data for a correlation between IL-8 concentration in pg/mL and monocyte count/mL – 1.0 × 100.15; n = 13, R2 = 0.07.The IL-10 results from BAL fluid, as well, indicate no statistically significant difference for the 3 groups: 119 ± 173 pg/mL for BVV; 124 ± 146 pg/mL for CMV; and 149 ± 168 pg/mL for CMV-RM. Unlike IL-8 concentrations the range of variation for IL-10 concentrations was significantly less – the maximum range differing by only 16-fold. There was no power-law relationship found between IL-10 and absolute monocyte count for pooled data: y = 7.5xT but at lower peak and mean airway pressure and greater respiratory system compliance.The main finding in this study of acute lung injury in a porcine model is that BVV significantly improved oxygenation and respiratory mechanics with no difference in indices of lung injury, inflammation or surfactant function compared to the more conventional ventilation techniques – CMV or CMV with a standard recruitment manoeuvre. The improvements seen with BVV were sustained over the course of the experiment; an effect not seen with recruitment. Such improvement was obtained at similar measured mean VT model of ALI/ARDS; not in healthy lung. As well, we have confirmed the differences between BVV and CMV seen in other work from the laboratory using the same model [These findings are similar to previously published results from our laboratory showing that BVV was superior to both CMV and CMV with sigh breaths. These sigh breaths were of no advantage in a model of healthy lung recruitment – lung reinflation after a controlled collapse for one hr with contralateral one-lung ventilation . In thatme model . No impo2. Thus, lower PvO2 in the CMV-RM group should have minimized shunt but here the shunt fraction is greatest. The lowest shunt with BVV indicates that the numerator of the shunt equation is less in this group, indicating enhanced blood flow in aerated lung units.Shunt fraction was significantly lower with BVV. Lynch et al.  and SandA multimodal approach to assess lung injury, lung inflammation and surfactant function indicated no discernable differences between the 3 approaches to ventilation:1. Light microscopy studies demonstrated no significant differences between groups for lung injury in any region examined – from upper to dependent lower lobes. This finding indicates no difference between groups in lung injury as assessed by histology, with the caveat that only small areas of lung in each region were examined in a condition known to be heterogeneous.2, wet:dry weight ratios, respiratory system compliance, or peak airway pressure. Arold et al. [2. No difference in surfactant function was seen between groups as assessed by capillary surfactometry. Following oleic acid lung injury, surfactant function was markedly depressed when compared to a surfactant standard – see Figure d et al.  have sho3. No difference between groups is seen for cell counts in the BAL fluid. There is considerable variation in this analysis and a full data set is not present. A significant number of studies of cell count could not be undertaken because of the nature of the BAL fluid. When thick with proteinaceous debris the samples often were unable to be prepared for meaningful cytometry analysis. Of the data represented, no difference in the proportion of neutrophils, monocytes or eosinophils is apparent for any of the 3 approaches to ventilation. Allen and Bates  have sho2 = 0.007 in this experiment, n = 24). Failure to reconfirm these findings may, in part relate to the nonlinear relationships highlighted above. Small changes in initial conditions may preclude similar findings at end experiment.4. IL-8 is considered to be the major neutrophil chemoattractant cytokine in lung diseases like ARDS . IL-10 m2 and respiratory system compliance. BVV has been shown to recruit in a pure model of lung collapse – re-expansion of lung following cessation of one lung ventilation [The above observations, collectively, indicate no fundamental differences between the ventilation strategies studied in regards oleic acid or superimposed ventilator associated lung injury. No one technique seems clearly advantageous. However, BVV alone improves oxygenation significantly following acute lung injury over time, an effect that was sustained, suggesting it is the best technique of the 3 studied to recruit atelectatic lung as assessed by greater PaOtilation . With BVtilation .2O to 2 cm H2O above Pflex in an attempt to prevent derecruitment, we maintained PEEP at 10 cm H2O throughout. In a porcine model of lung lavage, lung remained recruited if PEEP was at 10 cm H2O: the value chosen in this study [2 will not increase if the sustained inflation does not restore aeration to the atelectatic regions because a significant fraction of pulmonary blood flow is shunted to nonaerated regions [2O for 60 sec – was not beneficial in another animal model and that a more aggressive recruitment manoeuvre – PEEP 40 cm H2O and pressure control ventilation of 20 cm H2O, with a respiratory rate of 10, and I:E ratio 1:1 for 2 min – was successful only after the second hour. Allen et al. [2O) did not influence the time constants for recovery in elastance both before and after lung lavage. They conclude that for a deep inflation to be beneficial in their model that it may be necessary to apply the inflation several times a minute. Thus, the appropriate PEEP level, with and following recruitment, remains controversial [It could be argued that we have not chosen the optimal recruitment strategy to compare to the two other modes of mechanical ventilation. That said, we have chosen a well recognized approach to recruitment , one assis study . This leis study . Furtheris study . Failure regions . Such ma regions  have shon et al.  have shooversial ,39, but The human variability file for respiratory rate used to program the ventilator is shown in Figure T strategies are used for management of ALI/ARDS. A well accepted recruitment strategy has been compared to BVV in this animal study. It is possible that other approaches for recruitment could be superior to the one chosen. However, all chosen modes of recruitment would increase airway pressures over and above those seen with BVV, potentially increasing the risk of volutrauma.This study shows that BVV with a human variability file was superior to either CMV or CMV with a recruitment manoeuvre (CMV-RM) for sustained improvement in gas exchange and respiratory mechanics in a porcine model of acute lung injury. Prior work has demonstrated an advantage of BVV in a clinical setting of atelectasis . The curD.J.F. was responsible for conduct of the experiments as a fellow in the Anesthesia Laboratory. M.R.G. supervised conduct of the experiments, helped analyze data and helped write the paper. L.G.G. helped with the experiments, data retrieval and collation and table and figure production. J.A.T. did the histological assessments and analysis. B.M.M. supervised the cytokine assays and their interpretation and helped write the paper. E. K-Y. W. conducted the cytokine assays. C.H. did the surfactant assays while a fellow in the Oral Biology Laboratory. J.E.S. supervised and interpreted the results of the surfactant assays. W.A.C.M. conceived the study, analyzed and interpreted data and helped write the paper.Table 1: Temperature and HaemodynamicsClick here for fileTable 2: Respiratory Gas and Derived DataClick here for file


Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. The invasion of trophoblast cells into the endometrium during the implantation process can be blocked by inhibitors of serine proteases, illustrating the role of these enzymes in the invasion process. As in vitro developing embryos resulted in lower implantation rate than those developed in vivo we assume that a reduced PAs activity may lead to it. There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. The purpose of this study was to assess the expression of urokinase type (uPA) and tissue type (tPA) plasminogen activators in in vivo and in vitro preimplantation development in rat embryos using immunofluorescence confocal microscopy and computerized image analysis.Zygotes, 2-cell, 4-cell, 8-cell, morula and blastocyst stages of development were flushed from the reproductive tract (control groups) of Wistar rats. Zygotes were flushed and grown in vitro to the above mentioned developmental stages and comprised the experimental groups. Immunofluorescence microscopy and computerized image analysis were used to evaluate both qualitative  and quantitative expression of plasminogen activators.uPA and tPA were found to be expressed in rat embryos throughout their preimplantation development, both in vivo and in vitro. While uPA was localized mainly in the cell cytoplasm, the tPA was detected mainly on cell surface and in the perivitelline space. In blastocysts, both in vivo and in vitro, uPA and tPA were localized in the trophectoderm cells. Total uPA content per embryo was higher in the in vivo as compared with the in vitro developed embryos at all stages measured. Blastocyst uPA content was significantly low as compared with the four-cell, eight-cell, and morula stages. Total tPA content was higher in embryos developed in vivo than those developed in vitro except for the 4-cell and 8-cell stages.In vitro embryo development leads to lower PAs expression in a stage dependent manner as compared with in vivo developing controls. The enzymes studied vary probably in the ratio of their active and inactive forms as there is no correlation between their content and the activity observed in our previous study. The localization of both PAs in the blastocysts' trophectoderm supports the assumption that PAs plays a role in the implantation process in rats. The inv failure ,13. In t failure .In the implantation process, two major factors participate: the uterus undergoes changes that prepare it for the arrival and implantation of embryos, and the embryos undergo cellular reorganization that enables them to penetrate the endometrium and to form the placenta. We assume that one of the reasons for low implantation rate of embryos developed in vitro involves reduced PAs activity.In a previous study we demonstrated differences in PAs activities between in vivo and in vitro preimplantation developed embryos. In both, uPA activity increased from the zygote towards the blastocyst stage while tPA activity remained relatively unchanged. However, tPA and uPA activities were lower in in vitro developed embryos as compared with in vivo developing ones, at all developmental stages, which may lead to a reduced implantation rate of in vitro developed embryos .There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. Therefore, the purpose of this study was to investigate the PAs expression and localization during embryo development in vivo and in vitro by immunofluorescence confocal microscopy.The following study was approved by the Institutional committee for animal care and ethics at Ben-Gurion University of the Negev, Beer-Sheva, Israel.Mature female Wistar rats 2–3 months old, weighing 180–230 g were used. The animals were kept in a temperature-controlled room maintained at 22–24°C with lighting regimen of 14 hours light 10 hours dark (light on 5:00 AM – 7:00 PM). The rats were allowed free access to rat chow and tap water.Daily vaginal smears were taken at 10:00 AM, and the stage of estrous cycle was determined. Overnight caging of a proestrous female with a male of proven fertility induced pregnancy. The next day, the presence of a vaginal plug or spermatozoa in the vaginal smear was designated as day 1 of pregnancy.Zygotes, two-cell, four-cell, eight-cell embryos and morulae were flushed with rat 1-cell embryo culture medium (R1ECM)  from oviZygotes in their cumulus mass were flushed and the cumulus cells were removed by gentle aspiration through a micropipette (diameter, 150–200 μm) several times in R1ECM containing 80 U/mL of hyaluronidase. Clean zygotes were washed 3 times by transfer into fresh R1ECM to remove traces of hyaluronidase. Flushed embryos were collected with a mouth-controlled micropipette (diameter, 150–200 μm).Blastocysts were flushed from the uterine horns by insertion of a 23-gauge needle attached to a syringe containing R1ECM. Flushed, free-floating blastocysts were collected into a polypropylene tube inserted through the vagina and pushed gently to surround the cervical openings. Tubes were removed, and their contents were poured into Petri dishes. Blastocysts were washed and collected as described for zygotes and embryos. These embryos developed in vivo were the control groups.2 in air. This medium was shown by Miyoshi et al. [As described for the in vivo embryos, clean zygotes were grown in vitro to the same developmental stages as controls; these were the experimental groups. Each group of embryos consisted of 25–35 embryos collected from six pregnant females. This was repeated three times for each developmental stage . Groups of 25–35 zygotes were placed into 35-mm-diameter culture dishes (Nunc, Roskilde, Denmark) containing 50μL of R1ECM medium under a layer of mineral oil  and cultured at 37°C under 5% COi et al.  to enablThe method used was basically that of Dubey et al.  with varThe distribution and concentration of PAs in the embryos were visualized by fluorescent microscopy on a Zeiss laser scanning confocal microscope equipped with an X100 objective. Z-sections and XZ-sections were obtained from 3D scanning by using LSM510 software (Zeiss, Feldbach, Switzerland) The PAs density for each embryo was computed by image analysis based on the same principles as manual counting described elsewhere . The embData are expressed as means ± SEM. Statistical analysis was performed with two-way analysis of variance, followed by the least significant differences test for multiple comparisons using computer software (Statistica 6.0, Statsoft, Inc. Tulsa, OK). P < 0.05 was defined as statistically significant difference.Immunohistochemical staining for the location of tPA and uPA in preimplantation embryos developed in vivo and in vitro are shown in figure 3, respectively, Fig. 3, respectively).Quantitative measurement of total uPA in an embryo at each stage showed significantly lower expression (p < 0.01) in in vitro developed embryos from the 4-cell stage up to the blastocyst stage compared with the in vivo developed corresponding timepoint. The highest expression of uPA was found in in vivo developed embryos from 4-cell to the morula stage (90.58, 78.78 and 79.35 Pixels per embryo × 103, respectively, Fig. Total tPA expression in a whole embryo showed highest expression in in vivo developed embryos at the 2-cell, 8-cell, morula and blastocyst stages (61.59, 52.71, 48.03 and 52.34 Pixels per embryo × 10Our study demonstrates that uPA and tPA are expressed throughout all stages of preimplantation development of rat embryos. Zhang et al.  reportedThe results show localization of immunoreactive uPA in the embryonic cell cytoplasm and plasma membrane in all developmental stages both in vivo and in vitro, while tPA is detected on the cell membrane and in the perivitelline space. In the blastocyst stage, PAs are localized mainly in the trophectoderm. In our previous study  we showePro-uPA is synthesized as an inactive single chain that can be stored or secreted. The secreted pro-uPA can be cleaved to produce the two-chain active molecule, uPA, by the aid of limited proteolytic activity of plasmin . The secHigh tPA expression was detected at the zygote stage which is in accordance with high tPA activity found in this stage . This isThe embryonic extracellular matrix is in a continuous turnover during the embryonic development. The 8-cell stage is characterized by structural changes taking place in the embryo during the compaction process. It is therefore very likely that such changes at the 8-cell stage could be associated with increased tPA expression and activity which is known to participate in tissue remodeling [Lower expression of uPA was observed in in vitro developed embryos as compared with in vivo ones from the 4-cell up to the blastocyst stage while tPA expression was lower only in the morula and blastocyst stages. This could be explained by reduced metabolic activity in the in vitro developed embryos as suggested by Krisher et al. . In addiAdditional studies addressing the regulation of PA/Plasmin system by adding exogenous factors may provide insights into its role in early embryo development and implantation.The purpose of the study was to determine the relative importance of tPA and uPA in preimplantation embryo development. In vitro embryo development leads to lower PAs expression in a stage dependent manner as compared with in vivo developing ones. The localization of both PAs in the blastocysts' trophectoderm supports the assumption that PAs may play a role in the implantation process in rats.EDA participated in the planning of the project, carried out the animal experimentation, immunohistochemistry and the image analysis studies. USM participated in the planning of the project, animal experimentation and participated in preparation of the manuscript. GP participated in preparation of the manuscript. IHV participated in the planning of the project, statistical analysis and in preparation of the manuscript.