Since the early 1900s, the scope of burn resuscitation has evolved dramatically. Due to various advances in pre-hospital care and training, under-resuscitation of patients with severe burns is now relatively uncommon. Over-resuscitation, otherwise known as “fluid creep”, has emerged as one of the most important problems during the initial phases of burn care over the past decade. To avoid the complications of over-resuscitation, careful hourly titration of fluid rates based on compilation of various clinical end points by a bedside provider is vital. The aim of this review is to provide a practical approach to the resuscitation of severely burned patients. Since the early 1900s, advances in burn resuscitation have significantly reduced morbidity and mortality.–5 It appIn this review, a concise practical guide for clinicians involved in leading a resuscitation of the thermally burned will be provided. In addition, we review the current controversies and the application of potential adjunctive therapies when faced with a difficult resuscitation in an effort to avoid “fluid creep”.Typically, patients are at risk for developing burn shock if they have greater than 20% of their total body surface area (TBSA) burned. Factors such as the depth and extent of the burn, pre-existing illness, and the presence of inhalational injury greatly influence the magnitude and duration of shock. Burn shoThe initial phase of burn shock, also known as the emergent or ebb phase, is at maximum 12 h into the postburn and usually lasts for up to 72 h. This phase of injury is characterized by a combination of increased capillary permeability and cellular changes. Capillary permeability is a direct result of histamine, prostaglandins, and other vasoactive substances released into circulation. Overall,The primary goal of fluid resuscitation after severe burn is to maintain adequate tissue perfusion to the end-organs with intravenous crystalloid in an effort to avoid ischemic injury at the lowest physiologic cost. Extreme Delayed or inadequate replacement of intravascular volume results in suboptimal tissues perfusion. Suboptimal tissue perfusion results in end-organ damage that may become irreversible and ultimately contributes to death. Fortunately, under resuscitation is relatively uncommon since the adoption of weight and injury-based resuscitation formulas. On occasion, however, the patient can develop a concurrent distributive shock which necessitates additional volume to maintain adequate perfusion.Over-resuscitation often results in “resuscitation morbidity”. Resuscitation morbidity is a term used to describe complications of fluid overload which includes orbital compartment syndrome, extremity compartment syndrome, pulmonary edema, and abdominal compartment syndrome.13 Of theP < 0.0001). A greater percentage of patients in the Parkland group exceeded the Ivy Index, defined as 24 h volumes exceeding 250 mL/kg .It is important to emphasize that this method of deriving the initial fluid rate should not be applied to pediatric burn patients. In a recent guideline, the American Burn Association recommended that pediatric patients with thermal burns are resuscitated with initial fluid rates derived by the Parkland formula. In additOnce the fluid resuscitation is initiated at a given rate, emphasis must be placed on the ongoing resuscitation by attentive bedside care providers with close monitoring of hourly urine outputs, base deficit, mean arterial pressure and if a central venous line is available, central venous pressure. Fluids rates should be titrated, increased or decreased, hourly based on a compilation of various clinical endpoints centered on a target urine output of 0.5–1 mL/kg/h or approximately 30–50 mL/h. In children, the target urine output should be closer to 1 mL/kg/h. In general, titration of fluid rates up or down at each point should not exceed 30% of the current rate in an effort to avoid wide fluctuations. Within the US military, providers are asked to carefully track fluid resuscitation on a burn-specific resuscitation flow-sheet during global evacuation back to our burn center in Texas . Impleme12Despite timely and adequate fluid resuscitation, many patients with severe burns will develop refractory burn shock leading to a “runaway” resuscitation. As such the following adjunctive therapies may be required.et al. demonstrates a decreased likelihood of death.[et al. demonstrated that the addition of colloid to crystalloid resuscitation rapidly reduced hourly fluid requirements and ameliorated fluid creep.[et al. demonstrated decreased overall fluid volumes and lower intra-abdominal pressures when comparing a crystalloid resuscitation versus one supplemented with fresh frozen plasma.[Despite some reservation with the use of albumin in the early phases of burn resuscitation, recent work by Cochran of death. In a recid creep. We routiid creep.. In the id creep. In selecn plasma.et al. were able to exhibit in burned mice, burn-mediated changes in the liver increase perioxidation and decrease antioxidant capacity.[et al. were able to demonstrate in a study analyzing the oxidative stress response after severe burn injury that the type of fluid resuscitation has only a moderate effect on the pro-oxidant state and does not influence the changes of endogenous antioxidants in burned patients.[The development of oxidative stress is a major element in burn pathophysiology. Burn injury induces the formation of reactive oxygen species, which distorts the balance between radical formation and elimination. As Pham capacity. Foldi etpatients.et al. found that adjuvant ascorbic acid administered during the first 24 h after thermal injury significantly decreased fluid administered compared to control .[Using high dose ascorbic acid, 66 mg/kg/h for 24 h, Tanaka  < 0.01). High doset al. when therapeutic plasma exchange was utilized as a salvage maneuver, significant physiologic improvement and cessation of increasing fluid rates occured. Also, both MAP and UOP greatly increase while lactate levels decrease.[Originally described almost 30 years ago, the plasma exchange is thought to be effective in removing the circulating mediators responsible for the phenomenon of burn shock. Accordindecrease. Prospectvia convection as delivered by hemofiltration.[n = 29) aggressively treated with high volume continuous venovenous hemofiltration (CVVH) when compared to a closely matched historical cohort (n = 28).[n = 21), a majority of them with septic shock, we observed a dramatic reduction in the vasopressor requirement at 24 and 48 h that did not exist in the historical cohort (*P < 0.05 both to baseline and between groups). Due to potential effects in reversing shock, we believe this mode of therapy may have a potential role as a salvage maneuver in patients with burn shock undergoing a “runaway” resuscitation.Another method of extracorporeal blood purification is hemofiltration. Clearance of molecules of middle molecular weight (up to 50 kDa) is possible ltration. Our grou(n = 28). In a subOptimal care for the seriously burned patient should start with prompt recognition and evaluation of the burn. Once that has been established, resuscitation should be initiated via a simple formula right away. The initial fluid rate is not as important as the actual resuscitation itself guided by an attentive bedside clinician. Fluids should be titrated based on a compilation of various end points with the main goal of maintaining urine output 0.5–1 cc/kg/h, or roughly 30–50 cc/h. Best results are obtained by tracking the resuscitation on a separate flow-sheet. The need to define better endpoints of resuscitation to avoid excessive volume administration represents a high priority for future investigations. Additionally, the role for various adjunctive and salvage therapies when anticipating a “runaway” resuscitation must be carefully considered weight risk and benefit. Many of these adjuncts will need to be defined more clearly by future clinical trials.


Primary urethral squamous cell carcinoma is rare. Its management is particularly challenging owing to the paucity of evidence from randomised trials to inform practice. We report two male and female cases of squamous cell carcinoma of the urethra, which were treated with concomitant cisplatin and radiotherapy. These cases add to the body of case reports that have shown benefit for concomitant chemoradiotherapy in urethral squamous cell carcinoma. They also illustrate that single agent chemotherapy, namely, cisplatin, may be used successfully with limited toxicities. Primary urethral squamous cell carcinoma (SCC) is a rare entity accounting for less than 1% of all cancers [Historically, surgery and radiotherapy have been the main treatment modalities; however, significant morbidity may be conferred if an “R0” resection  is to be achieved . ConcomiWe report two male and female cases of SCC of the urethra, which were treated successfully with concomitant cisplatin and radiotherapy.A 55-year-old lady presented with a history of intermittent per vaginal bleeding and haematuria in January 2011. Cystoscopy demonstrated a mobile solid, fleshy tumour at the external urethral meatus. Wedge biopsy confirmed a moderate to poorly differentiated distal urethral SCC, which invaded into the muscle. Magnetic resonance imaging (MRI) demonstrated malignant local invasion and possible extension into the perineum and vaginal orifice; see −2 for five weeks. Conformal radiotherapy using 50.4 Gray in 28 fractions was given in 2 phases over 5.5 weeks. Phase 1: 45 Gray in 25 fractions to the primary tumour and the regional lymph nodes and phase 2: 5.4 Gray in 3 fractions to the primary tumour. The patient underwent concomitant chemoradiotherapy with weekly cisplatin 40 mg mDuring the treatment, the patient experienced a grade 3 pelvic skin reaction and a grade 2 diarrhoea (common terminology criteria version (CTC) 3.0) . ToxicitPosttreatment MRI demonstrated a complete response; see A 43-year-old man presented with a 2-month history of difficulty voiding urine and terminal dribbling. Cystourethroscopy demonstrated a polypoid growth in the bulbar urethra that prevented navigation to the bladder. A further cystourethroscopy under anaesthetic revealed only the polypoid tumour. There were no other abnormalities in the lower urinary tract. Subsequent biopsies confirmed a poorly to moderately differentiated polypoid SCC of the bulb of the urethra, which invaded into the stroma, at least stage pT2. Subsequent MRI scan revealed a 3.5 × 1.7 cm tumour invading into the proximal corpus spongiosum with some associated small inguinal nodes at 8 mm, which were not thought to be malignant. See −2 for 6 weeks with 64 Gray in 32 fractions of conformal radiotherapy was given in 2 phases. Phase 1: 44 Gray in 22 fractions to the primary tumour with a margin of 2.5 cm craniocaudally and 1.5 cm circumferentially. Phase 2: 20 Gray in 10 fractions to the primary tumour with a geometric margin of 1.5 cm. Radical cystoprostatectomy was recommended, but the patient declined given the morbidity associated. Chemoradiotherapy treatment was therefore offered. A suprapubic catheter was sited due to the risk of obstruction from inflammation in the urethra during radiotherapy. Chemoradiotherapy with weekly cisplatin 40 mg mCTC grade 3 pelvic skin toxicity was noted, this responded well to supportive measures. The suprapubic catheter was removed two months following completion of chemoradiotherapy. MRI at two months after treatment confirmed a complete response with low signal changes of the tumour sized at 2.2 × 0.8 cm, which was felt to be postradiotherapy change. The inguinal lymph nodes previously noted remained unchanged at 8 mm, supporting their benign nature. Urethroscopy was undertaken which confirmed scar tissue in the bulbar urethra. Biopsies were taken which did not show any evidence of residual tumour. See Both cases were discussed at the Urology Multi-Disciplinary Meeting where it was agreed that they represented primary urethral cancers rather than metastases from other pelvic structures.Urethral SCC is rare and can be challenging to manage due to the lack of randomised evidence to inform practice. Most patients present with locally advanced disease as urethral tumours tend to invade local structures such as bladder, prostate, or vagina. The above cases illustrate that urethral SCC can be successfully treated with concomitant cisplatin and radiotherapy in selected patients. In case one, a conventional conformal pelvic radiotherapy schedule of 50.4 Gray in 28 fractions was given due to the location of the tumour at the terminal urethra. Although testing for human papilloma virus (HPV) DNA or p16 was not undertaken, it was anticipated that this tumour would behave in a similar way to other anogenital SCCs that have an aetiological link to HPV such as anal cancer, and cancer of the uterine cervix –9. PerinIn the last few decades, there has been a drive to establish genital preserving strategies for urethral squamous cell cancer. Intensity modulated radiotherapy (IMRT) is now standard practice for some tumour sites in most UK hospitals, for example, for head and neck cancer. IMRT allows a more conformal dose distribution and may allow for dose escalation to the primary tumour with better sparing of organs at risk such as bladder and rectum. Human papilloma virus (HPV) has been implicated in many types of SCC, conferring a better prognosis in head and neck in particular . HPV hasThese two cases add to the body of case reports that have shown benefit for chemoradiotherapy in urethral SCC. They also illustrate that single agent chemotherapy, namely, cisplatin, may be used successfully with limited toxicities. Randomised clinical trials remain the ultimate goal; however, with rare cancers, amalgamating experience is vital to inform practice.


Many varieties of working memory have been linked to fluid intelligence. In Duncan et al. (Journal of Experimental Psychology:General 137:131–148,  Tests of “fluid intelligence,” such as Raven’s Progressive Matrices .For optimal correlational data, we sought a broad distribution of fluid-intelligence scores, approximately representative of variability in the normal population. On the basis of prior testing with the Culture Fair test, Scale 2, Form A . The net rule working memory score was the mean of these values across the total of ten rules employed.On each trial, the experimenter read a list of numbers, which the participant was immediately to repeat back in the same order. The test used the materials and procedures from the WAIS-III  followed by either an addition or a subtraction. All of the values used on the left side of each equation were single-digit numbers from 1 to 9. The equations were given with incorrect answers (deviating from the correct answer by either 1 or 2) on half of the trials. The words were monosyllabic concrete nouns of length 4–6 characters, with Kučera–Francis  written In the VSTM task  between all measures are shown in Table Descriptive statistics for each measure are shown in Table t(83) = 1.69, p < .05], operation span , or VSTM ; for spatial span, the difference was not significant .  From the published reliability of the Culture Fair  with Culture Fair IQ. As is shown in Fig. To define a “general intelligence” factor, a well-established alternative to fluid-intelligence tests is principal-components analysis, where the first principal component accounts for much of the general tendency to positive correlations in a diverse battery of tests  for rule working memory, and for the other working memory tests, it changed the Culture Fair correlation by a maximum of .02.These results confirm a strong link between fluid intelligence and working memory for task rules. At .57, the correlation between Culture Fair and rule working memory was significantly higher than the equivalent correlations for digit span, operation span, and VSTM, and it was numerically higher than the correlation for spatial span. We propose that, in all tasks, a mental control program must implement an integrated series of attentional episodes defining the separate parts or stages that the task contains Duncan, . If fluiGiven this proposal, an open question is how best to interpret the correlation of fluid intelligence with other working memory tests, including those examined in the present study. Many previous studies have reported positive correlations between fluid intelligence and different varieties of working memory; on average these have been quite modest (Ackerman et al., In this context, an intriguing finding is the comparatively strong correlation between fluid intelligence and spatial span. In many kinds of task, frontoparietal activity—resembling that linked to fluid-reasoning tasks—increases with task difficulty (Duncan & Owen, Though any task will show some positive correlation with fluid intelligence Cattell, , the magWhen behavior is simple and familiar, the quality of the control program may contribute only modestly to between-person variability. With increased complexity and novelty, however, programs may vary substantially in their effectiveness, increasingly contributing to individual differences. In this respect, the present measures of rule working memory may form a bridge between traditional, relatively simple working memory tasks, with their relatively modest fluid-intelligence correlations, and the full complexity of novel reasoning.


In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) ‘colonizers’ if microorganisms were detected within the follicular fluid, but not within the vaginal swab ; or (2) ‘contaminants’ if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners.Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the  The presence of opportunistic pathogens in the lower female reproductive tract has been associated with adverse pregnancy outcomes after both natural and IVF conceptions et alIn their study, Cottell The aim of this current study was to screen follicular fluid collected from IVF patients at the time of trans-vaginal oocyte retrieval for the presence of microorganisms. We hypothesized that follicular fluid microorganisms cause adverse IVF outcomes. Therefore, we identified the microorganisms in follicular fluids and vaginal swabs and correlated these with IVF outcomes.From September 2007 to November 2008, 263 consenting couples commencing fully stimulated IVF cycles at Wesley-Monash IVF  were enrolled in this study. Pregnancy was classified by the World Health Organization definition of a clinical pregnancy with fetal heartbeat: that is ultrasonographic evidence of a gestational sac containing one or more fetuses with a heart beat Chlamydia trachomatis in either partner, pelvic inflammatory disease, sexually transmitted infection, or laparoscopic diagnosis of tubal disease; or (4) idiopathic infertility if the female and male partners had undergone screening for infertility and no abnormalities were detected. Couples were enrolled in this study in the order in which they consented and began IVF treatment. No patients were prescribed antimicrobial therapy during the course of their IVF cycle. No efforts were made to control the distribution of group sizes based on the etiology of infertility.Women from couples where the only cause of infertility was male factor infertility were classified as the ‘fertile’ control group. The remaining women were grouped according to an etiology of infertility due to: (1) endometriosis, which was laparoscopically diagnosed by identification of endometrial explants within the pelvic cavity; (2) polycystic ovary syndrome, diagnosed by the Rotterdam criteria Specimen collection and storage was performed as previously described Ethical approval was obtained from the review boards of Uniting Care Health, Human Research Ethics Committee and Queensland University of Technology Human Ethics Committee. All patients provided informed written consent for their follicular fluids to be used in this study and gave permission for researchers to access medical records to obtain their reproductive history and IVF outcomes. All patients gave permission for researchers to access medical records to obtain their reproductive history and IVF outcomes.3 for a 1 µL loop or 102 for a 10 µL loop) to give the colony forming units (CFU)/mL. The limit of detection using this method is 103 CFU/mL.Culture and identification of clinical isolates, was performed as previously described Statistical analyses were performed on the microbiological data and IVF outcomes using SPSS version 17 for Windows XP and R . Proportions were compared using a normal approximation or Fisher's exact test. Continuous variables were compared across groups using Wilcoxon's rank sum or the Kruskal-Wallis test. Multinomial logistic regression and Fisher's exact test were used to model the relationship between categorical outcomes  and explanatory variables (colonizing or contaminating species). A p-value of p<0.05 was considered statistically significant.Clinical specimens were collected from 263 women for microbiological analyses. One woman had no microorganisms detected in her follicular fluid by either traditional culture or molecular microbiology techniques, and her IVF outcome results were excluded from further analysis. For the remaining 262 women, the microorganisms identified in follicular fluid were analyzed to determine if there was a relationship between the microbial flora and IVF outcomes.The mean age of women in this study was 37 years ±4 years. There were no statistical differences in the mean age of women from ‘fertile’ women with male factor infertility (37±4 years) compared to infertile women . There was no relationship between the age of the women who had a live birth (34±4 years) compared to those women who did not . For fertile and infertile women there was no difference in the number of prior IVF treatment cycles. Fertile women participated in an average of 1 (range 0–4±1 cycle) previous trans-vaginal oocyte retrieval procedure as did infertile women . There was no difference in the number of oocytes collected at the time of retrieval for fertile (11±6) or infertile women .Lactobacillus spp., Bifidobacterium spp. and Staphylococcus spp. were the most prevalent species detected in the lower genital tract specimens of all women. The culture analyses revealed that cultivable bacterial species (1–5 species) were present in 99% of follicular fluids tested. A range of microorganisms was isolated from the follicular fluid. The follicular fluids were classified as: (1) ‘colonized’  if microorganisms were detected within the follicular fluid, but not in the vagina ; or (2) ‘contaminated’  if microorganisms detected in the vagina at the time of trans-vaginal oocyte retrieval were also detected within the follicular fluid. Both fertile women  and infertile women  demonstrated both colonization and contamination of follicular fluid specimens  (Actinomyces spp. and Propionibacterium spp. were the only species isolated significantly more often from the colonized fluid than from contaminated follicular fluids (p<0.05). Also, Peptostreptococccus spp. was isolated more frequently from right contaminated follicular fluid than from colonized follicular fluid (p<0.05).The Lactobacillus spp., Bifidobacterium spp., Staphylococcus spp., and S. agalactiae were the most prevalent species identified as vaginal flora  . In the al flora  A. C. allonizers . None ofLogistic regression analysis was used to test for the degree of dependence between colonization of follicular fluid (either left or right) and etiology of infertility. Endometriosis, polycystic ovary syndrome, genital tract infection, male factor and idiopathic etiologies were considered. Colonization of left follicular fluid and endometriosis were found to be associated (p<0.05). There were no other statistically significant results.Overall, for women with microbial colonization of follicular fluid there was no significant difference between fertilization rates for infertile women with a diagnosis of endometriosis, polycystic ovary syndrome or a history of upper genital tract infection when normalized against the control group of fertile women .Adverse IVF outcomes were associated with microbial colonization of follicular fluid. Embryo discard rates were higher for women with endometriosis with colonized follicular fluid  but not for women with contaminated follicular fluid (34%) . DecreasLactobacillus spp. in both right and left follicles was associated with higher rates of embryo transfer (p<0.05). By contrast, the presence of Propionibacterium spp. and Streptococcus spp. in right follicles was associated with poor embryo transfer rates (p<0.05 and p<0.01 respectively). Propionibacterium spp. were more frequently isolated from colonized right ovarian follicular fluids, whilst S. agalactiae was isolated more frequently from the left ovarian follicular fluids.No single species was associated with decreased fertilization rates; however, the presence of Actinomyces spp., Bifidobacterium spp., Propionibacterium spp. and Streptococcus spp. within the left ovarian follicular fluid had a significant association with negative pregnancy outcomes (p<0.01). Lactobacillus spp. as a contaminant within the left ovary was associated with a positive pregnancy outcome (p<0.001). For the right ovary, negative pregnancy outcomes were associated with Actinomyces spp., Bifidobacterium spp., and Streptococcus intermedius (p<0.01), and to a lesser extent with Propionibacterium spp. and Staphylococcus spp. (p<0.05). To demonstrate the relationship between individual or combination microbial species and IVF outcomes in this study, combinations of microbial species were color coordinated based on known genital tract pathogenicity for presentation as a heat map  and negative . The heat map demonstrates that the lactobacilli are more frequently isolated alone or in combination with microaerophiles or anaerobes when a positive IVF outcome was achieved. In contrast, there is an absence of lactobacilli when poor IVF outcomes were reported. As the map is viewed from top (positive IVF outcomes) to bottom (negative IVF outcomes) there is a transition from green through to red, demonstrating the particularly negative impact of isolating anaerobes in the absence of Propionibacterium spp., Streptococcus spp. Actinomyces spp., Staphylococcus spp. and Bifidobacterium spp.) correlated with adverse IVF outcomes whereas improved embryo transfer rates are associated with the presence of Lactobacillus spp. in both the left and right ovarian follicular fluids. Our data demonstrates that not only is follicular fluid not sterile but that bacterial species in follicular fluid can have both positive and negative effects on IVF outcomes.This study found that human follicular fluid contains microorganisms. The presence of some microorganisms (E. coli and Streptococcus spp.) in porcine follicular fluid might inhibit follicle-stimulating hormone (FSH) from binding to its receptor on granulosa cells Previous studies have also reported the presence of bacteria within porcine follicular fluid. These studies concluded that some bacteria  . However, the presence of bacteria other than Lactobacillus spp. , reported as cervicovaginal flora, were associated with a reduced number of successful IVF pregnancies and an increase in early miscarriage rates Improved embryo transfer rates are associated with the presence of t ovary) . Each spBacteroides spp. and Streptococcus spp. have been recovered from peritoneal fluid, but not from the vagina or cervix for 25% of women tested Lactobacillus spp.), the gastrointestinal tract , the skin (Staphylococcus spp.) and the oral mucosa (Streptococcus spp.)  . This fuLactobacillus spp. were the most prevalent species isolated from these specimens in women with colonized and contaminated follicular fluids. This is in agreement with previous studies investigating the vaginal microflora in women undergoing IVF cycles, which also concluded that the lactobacilli are the most prevalent isolates We employed a culture-dependent method for the primary isolation and identification of the microorganisms present within follicular fluids and vaginal swabs. We confirmed that the et alThe species of microorganisms we isolated from follicular fluid were consistent with previous reports documenting asymptomatic contamination of follicular fluid collected as part of an IVF cycle et alThe follicular fluid microorganisms were discordant for fluids collected from the left and the right ovaries. This study identified a significant difference in the median number of microorganisms isolated from the left compared to the right follicular fluids. This study confirms the results we reported in our smaller microbiological study  in vivo follicular environment. Poor quality embryos will have been discarded following developmental arrest prior to transfer. Inequality in function between the left and right ovaries has also been reported in other mammals It has been proposed that ovulation occurs more often in the right ovary than the left ovary throughout the reproductive lifetime because of the separate, independent vasculature of each ovary Propionibacterium spp., Streptococcus spp. Actinomyces spp., Staphylococcus spp. and Bifidobacterium spp.) and are related to adverse IVF outcomes including decreased embryo transfer rates, which may reflect poor quality embryos due to damage by microorganisms or their metabolites and decreased pregnancy rates or no pregnancy (failed implantation); (2) Lactobacillus spp. within follicular fluid correlates with positive IVF outcomes; (3) follicular fluid is not sterile, but in fact contains a diverse range of microorganisms at varying concentrations; (4) follicular fluid can be colonized or contaminated by microorganisms; (5) follicular fluid colonization or contamination does not correlate with any one cause of infertility; and (6) the microflora of the follicular fluid is asymmetrical with differences observed between the microflora collected from the left and from the right ovary and in the IVF outcomes for each side.The data from this study demonstrate several new findings: (1) some species isolated from follicular fluid appear to be opportunistic pathogens Click here for additional data file.


The design of gp120 monomeric probes with modified antigenic profiles that are specific for a target epitope has been successfully used for the isolation of broadly neutralizing HIV-1 antibodies. Existing probes, however, do not possess sufficient specificity and can bind antibodies with undesired properties . To achieve improved epitope specificity, positive and negative design stages can be incorporated into the probe design process.Here, we apply a combination of structure- and sequence-based methods for improving the epitope specificity of gp120 monomeric probes. Specifically, structure-based redesign using the OSPREY protein design software suite was used to predict gp120 knock-out mutations for selected antibodies. Additionally, using a sequence-based mutual information approach, knock-out mutations were designed by identifying gp120 residues that are predicted to associate with neutralization resistance for a given antibody.Using stabilized (Ds12F123) and resurfaced stabilized (RSC3) HXB2 gp120 cores as templates, we designed a set of mutants with improved epitope specificity. In particular, RSC3 (a prototypic probe previously used for the isolation of VRC01 and other CD4-binding-site antibodies) was redesigned to specifically bind CD4-binding-site antibodies that are broadly neutralizing  but not moderately/weakly neutralizing . Additionally, CD4i-specific probes were designed by introducing mutations that destabilize binding to the entire class of CD4-binding-site antibodies (negative design), while retaining binding to CD4i antibodies (positive design). The desired epitope specificity of the redesigned probes was confirmed by ELISA binding. The probe design approach was further validated with knock-out mutations for a diverse set of antibodies, including PG9 and 2F5.Probes with enhanced epitope specificity can select more precisely for antibodies with desired properties. A set of our redesigned probes are currently being utilized for the isolation of antibodies targeting different epitopes of interest on the HIV-1 Envelope.


The Research Grant number in Acknowledgments section in page 9 stating that “This work was supported in part by Research Grant 20590847” was wrong and the corrected one is “23591070.”


P ≤ 0.001). Mean tumour thickness was also reduced from diagnosis to treatment. At 3-month followup, complete tumour response was found in 93% and the cosmetic outcome was rated excellent or good in 100% of cases. In conclusion, deep curettage significantly reduces BCC thickness and may with topical PDT provide a favourable clinical and cosmetic short-term outcome.Topical photodynamic therapy (PDT) has limitations in the treatment of thick skin tumours. The aim of the study was to evaluate the effect of pre-PDT deep curettage on tumour thickness in thick (≥2 mm) basal cell carcinoma (BCC). Additionally, 3-month treatment outcome and change of tumour thickness from diagnosis to treatment were investigated. At diagnosis, mean tumour thickness was 2.3 mm (range 2.0–4.0). Pre- and post-curettage biopsies were taken from each tumour prior to PDT. Of 32 verified BCCs, tumour thickness was reduced by 50% after deep curettage ( Basal cell carcinoma (BCC) is the most common cancer in the white population, and its incidence is still increasing , 2. ThisAmong several therapeutic options available for the treatment of this tumour excision surgery is regarded as the most effective . HoweverTopical PDT, with beneficial cosmesis, may in such cases be an attractive treatment option , 8. ThisFive-year clearance rates in BCC from 64 to 81% are reported –13.Evidence-based guidelines support the use of topical PDT in the treatment of BCC, particularly low risk, superficial lesions , 15. A cAmong several strategies to increase PDT effect, pre-treatment curettage has been shown to improve treatment efficacy in nodular tumours . The comIt is recommended to perform a pre-treatment biopsy to obtain an indication of tumour thickness . HoweverConsequently, it is of interest to examine to what extent tumour thickness may be reduced by deep curettage and examine to which degree this may affect treatment outcome.The main objective of this study was to evaluate the effect of deep curettage on tumour thickness in thick BCC. Additionally, short-term treatment efficacy and cosmesis as well as changes in tumour thickness from diagnosis to treatment were investigated.The study was conducted at the Department of Dermatology, St. Olav's Hospital HF, Trondheim over a two-year period. Patients with histological verified BCC ≥2.0 mm thick, selected for PDT were eligible. The study was approved by the Regional Ethics Committee, and informed consent was obtained from all patients before study entry.The size was defined as the mean of the length and width of the lesion. Pre- and post-curettage biopsies were taken from the central tumour area by one investigator (EC) using a 2.0–3.0 mm disposable punch biopsy . The centre was defined as the midpoint of the line following the greatest tumour length and was marked using a skin marker. The biopsies were taken approximately 0.5 mm from either side of the midpoint. In lesions with a central ulceration, the biopsies were taken outside of the ulcerated area, along the line following the greatest tumour length, approximately 1.0 mm apart. Bleeding after deep curettage was dried with gauze prior to the taking of post-curettage biopsy.μm, and stained with haematoxylin, eosin, and saffron (HES). The biopsy tissue was fixed in 10% formaldehyde, routinely processed, embedded in paraffin, cut perpendicular to the skin surface at three places in sections of 4 The histological prepared slides were examined by pathologists at St. Olav's University Hospital. The tumour thickness was measured from the stratum corneum to the bottom of the tumour nest. The pre-curettage biopsies were classified histologically as of nonaggressive (nodular) or aggressive (micronodular and morpheform/infiltrative) by one pathologist. To ensure as little variation as possible deep curettage was performed by one investigator (EC). The procedure was comprised of an intratumoural debulking within clinical margins of the lesion followed by multiple passes of curettage in various directions across the tumour base. A small surgical curette was used to remove soft, friable tissue and a disposable 4 mm ring curette  used to remove hyperkeratosis and crusts and to scrape clinically firm tumour areas. In addition, a 4 mm broad brim of normal appearing skin surrounding the tumour was superficially scraped using a ring curette only to remove stratum corneum.2 and light dose of 37 J/cm2.The area was then treated with PDT using methyl aminolevulinate (MAL) as a precursor of photosensitive porphyrins. MAL cream  was applied onto the treatment site in a 1 mm thick layer and occluded with a light-shielding dressing. Any residual cream was wiped off after 3 hours and the area exposed to red light (570–670 nm). A noncoherent LED light source  was used with a fluence rate of 70–100 mW/cmEfficacy was evaluated by dermatologists through inspection and palpation three months after treatment and classified as either in complete response (complete disappearance of tumour) or as noncomplete response.The cosmetic outcome in the clinical complete response areas was rated on a 4-point ordinal scale as (1) excellent (absence of any stigmata of treatment), (2) good , (3) poor , or (4) fair .All statistics were calculated using SPSS version 15 . t-test was used to analyse the differences in tumour thickness. Simple linear regression was used to analyse the association between treatment waiting time  and tumour thickness reduction . One-way ANOVA was used to analyse possible differences in tumour reduction and cosmetic result. P < 0.05 was considered statistically significant.Data distributions were examined by Q-Q plots. Two-tailed, one-sample Student A total of 36 patients with each one BCC tumour were included. Twenty-one patients were men, mean age 72 years (range 39–92), and 15 were women, mean age 77 years (range 62–85).Thirty-four of the tumours were located to scalp or face and two located to the back. Mean lesion size was 11 mm (range 3–38). The mean diagnostic biopsy tumour thickness was 2.3 mm (range 2.0–4.0). The average time from diagnosis to treatment was 91 days (range 13–339). In three cases BCC was not demonstrated in either the pre- or post-curettage biopsies. One post-curettage biopsy was lost after fixation. Thirty-two lesions were included in the analyses. Histologically, 20 tumours were of nonaggressive and 12 were of aggressive  growth type. All data were found to be normally distributed.P = 0.001 (Mean tumour thickness before curettage was 2.0 mm (range 0.7–4.0) and after curettage was 1.0 mm (range 0.0–3.1). The differences between measurements was statistically significant . The dif = 0.001 . In threP = 0.04). For nonaggressive tumours only, the difference was 0.34 mm (SD 0.6) (P = 0.02) and for the aggressive subtype it was 0.13 mm (SD 0.9) (P = 0.60). A difference between the diagnostic and pre-curettage mean biopsy measurement of 0.3 mm (SD 0.7) was found to be statistically significant  (P = 0.11).A weak but statistical significant linear regression was found between the treatment waiting time and tumour reduction of the same period (re 0.13) . One patFive of the initial 36 included lesions were missed for 3-month PDT follow-up. Two lesions in two patients were excised after a reassessment of treatment shortly after the first PDT session. This decision was based on the post-curettage biopsy tumour thickness measurement, combined with factors as lesion size and location. Further, two patients discontinued the scheduled PDT sessions because of the experience of severe pain during light exposure. They were referred to treatment by excision surgery. One patient with one lesion died from a non-PDT-related cause.n = 31) for the 3-month follow-up. When disregarding the three pre- and post-curettage non-BCC-verified cases, noncomplete response was observed in 2 out of 28 (7%) treated tumours. One was a nodular tumour with a thickness reduction of 0.4 mm and the other was an aggressive subtype showing a 0.1 mm increase after curettage. Data from the five dropout patients were excluded from the data analyses using the per-protocol population (The cosmetic outcome was rated excellent in 11 of 26 (42%) and good in 15 of 26 (58%) of cases, of which two examples are demonstrated in P = 0.36).No difference between tumour thickness reduction after curettage and the cosmetic outcome was found (The main finding of this study was a mean 50% reduction of BCC tumour thickness measurement after deep curettage. The reduction was significant for both the aggressive (nodular) and nonaggressive (micronodular and morpheaform/infiltrative) histological subtypes. These findings support the practice of pre-treatment curettage as an effective intervention for tumour thickness reduction before the use of PDT in thick BCC.There are a number of BCCs that are currently difficult to treat by traditional invasive therapies because of size, site, or multiple lesion presentation, particularly if trying to avoid complications such as scar formation. Although treatment by topical PDT has been shown to be less effective compared with excision surgery in nodular BCC , 23, cosDespite the use of deep curettage ahead of PDT, we still achieved a favourable cosmetic result assessed as good or excellent in all the evaluated cases. The 3-month complete response rate of 93% is comparable to published short-term treatment results after MAL-PDT following curettage showing clearance from 91 to 97% for superficial and from 82 to 91% for nodular BCC , 24, 25.Most BCCs appear in the face and neck area  of whichThere are, however, various factors that may have affected the thickness measurements reported in this study. The pre- and post-curettage punch biopsies were, as described, not taken from identical tumour areas within the tumour. BCC can have an irregular growth with infiltrating extensions . The disBCCs grow slowly and may take years to double in size . HoweverSimilar mechanisms may explain why BCCs in three cases were not detected in either pre- or post-curettage biopsies and apparently underwent spontaneous clearance. However, we cannot exclude sampling error. In the treatment of BCC, curettage is frequently combined with other modalities such as surgery, electrodessication, cryosurgery, and topical PDT. It is commonly performed to delineate tumour margins and/or to reduce tumour thickness. To what extent curettage exerts an independent effect on treatment outcome is unclear. A few studies report on the treatment of small (≤15 mm) tumours and/or selected groups of BCC with curettage alone , 33. ThiThe belief that curettage alone is not sufficient to erase all parts of tumour is, on the other hand, supported by a finding in the study by Jih et al.  evaluatiPDT as a topical monotherapy still appears to be a less attractive option for the treatment of thicker BCC. The combination of PDT and pre-treatment curettage has proved more effective compared to treatment by placebo cream and pre-treatment curettage alone. In a randomized, double blinded study of nodular BCC the complete response rates were 73% versus 27%, respectively . In a study by Fantini et al. , a low rFurther, prior debulking curettage to PDT achieved a complete response rate for nodular BCC of 92% compared to non complete response in the control groups . The conThese results imply that pre-treatment curettage contribute to make nodular BCC more responsive to topical PDT.Within the PDT regime, pre-treatment curettage is a practical, technique-dependant supportive method practised with great variability. In trying to obtain uniformity of this procedure, the same investigator performed the curettage in all cases in the present study. However, it was difficult to achieve a standardization of the practical exercise, which again may have influenced the study results. The main purpose of deep curettage was to erase the main bulk of the tumour within its clinical margins. The purpose was not to remove all parts of the tumour in an attempt to prevent damage afflicted to surrounding tissue, as reflected by the favourable post-treatment cosmetic results obtained.The study showed a significant reduction of tumour thickness in thick BCC after deep curettage. A favourable short-term efficacy was found, and cosmesis was maintained following deep curettage and PDT. Topical PDT combined with deep curettage may be considered as a treatment option of selected thick BCCs in cases where surgery or other invasive treatment methods are regarded as suboptimal.Out-patient clinic at the Department of Dermatology, St. Olav's University Hospital HF, Trondheim, Norway.The authors declare no conflict of interests.


Motivation: The Illumina Infinium 450 k DNA Methylation Beadchip is a prime candidate technology for Epigenome-Wide Association Studies (EWAS). However, a difficulty associated with these beadarrays is that probes come in two different designs, characterized by widely different DNA methylation distributions and dynamic range, which may bias downstream analyses. A key statistical issue is therefore how best to adjust for the two different probe designs.Results: Here we propose a novel model-based intra-array normalization strategy for 450 k data, called BMIQ (Beta MIxture Quantile dilation), to adjust the beta-values of type2 design probes into a statistical distribution characteristic of type1 probes. The strategy involves application of a three-state beta-mixture model to assign probes to methylation states, subsequent transformation of probabilities into quantiles and finally a methylation-dependent dilation transformation to preserve the monotonicity and continuity of the data. We validate our method on cell-line data, fresh frozen and paraffin-embedded tumour tissue samples and demonstrate that BMIQ compares favourably with two competing methods. Specifically, we show that BMIQ improves the robustness of the normalization procedure, reduces the technical variation and bias of type2 probe values and successfully eliminates the type1 enrichment bias caused by the lower dynamic range of type2 probes. BMIQ will be useful as a preprocessing step for any study using the Illumina Infinium 450 k platform.Availability: BMIQ is freely available from http://code.google.com/p/bmiq/.Contact:a.teschendorff@ucl.ac.ukSupplementary information:Supplementary data are available at Bioinformatics online In the past few years, the field of epigenomics has risen to prominence . EpigenoA key statistical issue with the Illumina 450 k beadchip is that probes come in two different designs, which causes the methylation values derived from these two designs to exhibit widely different distributions  head and neck cancers (HNCs), of which 18 were HPV+ and 14 HPV−, as well as five fresh frozen HNCs (FF), of which 2 were HPV+ and 3 HPV−. The data are available from GEO under accession number GSE38271.This 450 k dataset consists of 81 glioblastoma multiformes (GBMs)  consists of 10 samples as provided in the Bioconductor data package TCGAmethylation 450 k, Dataset7 (LIV) consists of nine normal liver tissue samples from Batch203 in the TCGA data portal, Dataset8 (LC) consists of 22 lung cancer samples from Batch196, Dataset9 (BLDC) consists of 12 bladder cancer samples from Batch86 and Dataset10 (HCC) consists of 10 hepatocellular carcinoma samples from Batch153.Fitting of a three-state  beta mixture model to the type1 and type2 probes separately. For sake of convenience we refer to intermediate allelic methylation as hemimethylation even though hemimethylation is most often used in the context of strand-specific methylation. Let For those type2 probes assigned to the U-state, transform their probabilities of belonging to the U-state to quantiles using the inverse of the cumulative beta distribution with beta parameters For those type2 probes assigned to the M-state, transform their probabilities of belonging to the M-state to quantiles using the inverse of the cumulative beta distribution with beta parameters For the type2 probes assigned to the H-state, we perform a dilation  transformation to ‘fit’ the data into the ‘gap’ with endpoints defined by The normalization of type2 probe values into type1 must satisfy the following criteria. (i) It must allow for the different biological characteristics of type1 and type2 probes, i.e type1 probes are significantly more likely to map to CpG islands than type2 probes, and hence the relative proportion of methylated and unmethylated probes will vary between the two designs. In the case of the type2 probes, this means that these proportions must be invariant under the normalization transformation. (ii) The transformation of the type2 probe values should reduce the bias, which amounts to matching of the density distributions of the two design types, specially at the unmethylated and methylated extremes. (iii) The transformation must be monotonic, that is, the relative ranking of beta values of the type2 probes must be invariant under the transformation. Next, we propose a normalization strategy for the type2 probes satisfying the above properties and which is based on three steps:We next describe each of the above steps in detail. We first model each beta value Further, let Supplementary Fig. S1). Specifically, we first identify the minima and maxima of the type2 Finally, it remains to normalize the type2 We want the new normalized maximum and minimum values of Supplementary Fig. S1), presumably as a result of the dye bias, which is specific to the type2 distribution.This algorithm is flexible in that the dilation can be performed also including the Supplementary Fig. S1) to then obtain type2-specific thresholds using a simple correction reflecting the difference in the modes between the type1 and type2 distributions. Specifically, if Supplementary Fig. S1). In this regard we have also verified that using a beta mixture model with more than three states does not improve the overall type2 fit. Fortunately however, as explained above, the goodness of fit problem associated with the empirical distribution. Hence, the probe values that are not well described by a beta distribution are not normalized using estimated beta parameters, which means that their normalization is insensitive to the goodness of fit.There are a number of other important points to note about BMIQ: (i) First, it is important to choose reasonable initial weight parameters To validate BMIQ, we first applied it to data where the PBC method has been shown to work reasonably well. Thus, we applied it to a fresh frozen breast tumour sample from Dataset1 A. We canTo further test BMIQ we applied it to Dataset2 (CL) consisting of three replicates of a given cell-line, to investigate if reproducibility is improved. First, we computed for each of the probes its standard deviation across the three replicates and for each of the three scenarios: no normalization, PBC and BMIQ. As seen, BMIQ performed similarly to PBC and led to a significant reduction in inter-replicate variability A. To cheUsing replicates to evaluate normalization methods assesses the method in terms of reducing technical variability but does not evaluate whether the actual values of the replicates are closer to the true estimate. This requires comparison with a gold-standard, which is provided by matched BPS data , althougTo further test BMIQ, we considered the supervised context, in which a ranked list of probes correlating with a phenotype of interest is derived. Given the higher dynamic range of type1 probes, one expects that this would favour type1 probes and that therefore there would be a relative over enrichment of type1 over type2 probes in a top ranked list of probes. However, one key difficulty when assessing whether there is a bias towards type1 probes is that type1 and type2 probes differ significantly in terms of their biological characteristics, in particular in terms of CpG density. Hence, in order to avoid confounding by CpG density, we only selected probes that mapped to CpG islands and to 200 bp upstream of the TSS, thus allowing a sensible comparison between type1 and type2 probes. We considered three different datasets and derived for each a ranked list of probes associated with a phenotype of interest: breast cancer versus normal breast [Dataset1 (BT)], HPV + versus HPV−HNC [Dataset3 (FFPE)], and CIMP+ versus CIMP− (GBM) (Dataset5). The ranking was performed using the magnitude of differential methylation. Although this ranking does not take the within-phenotype variability into account, it remains a popular method . Using PSupplementary Table S1).To further assess BMIQ, we devised an evaluation framework which exploits the well known spatial correlation of DNA methylation at scales <500 bp . In factFinally, it must be verified that the reduction in technical variance obtained with BMIQ is not at the expense of a reduced biological signal. Since it is difficult to establish what constitutes a true positive, we used a training test set strategy, to identify features in a training set and calling them true positives if validated in a test set. This strategy thus allows for a comparison of sensitivity and positive predictive value (PPV) between the different normalization methods. To perform this analysis, we used Dataset4(FF) consisting of 2 HPV+ and 3 HPV− fresh frozen head and neck cancers to derive features associated with HPV status. As test set we used Dataset3(FFPE) consisting of 18 HPV+ and 14 HPV− head and neck cancers (FFPE tissue). Using limma  and an FIn this work we have presented a novel mixture-model-based algorithm (BMIQ) for correcting the bias associated with type2 probe values in 450 k studies. Confirming the observations made in minfi package  and matched BPS data . Using these criteria, we have seen that BMIQ leads to significant improvements, similar to the improvements noted for PBC  and 3. I package . Of note package .In summary, using a number of different evaluation criteria and numerous datasets, we have seen that BMIQ compares favourably with both PBC and SWAN. Although we did not compare BMIQ to SQN , this laWe have presented a mixture model assumption-free normalization algorithm, BMIQ, which will be useful for correcting the bias associated with the type2 assay in DNA methylation studies using the Illumina Infinium 450 k platform.Funding: A.E.T. is supported by a Heller Research Fellowship. M.L. was supported by a Wellcome Trust Research Training Fellowship (093855). S.B. was supported by a Royal Society Wolfson Research Merit Award (WM100023) and grants from the Wellcome Trust (084071) and EU-FP7 BLUEPRINT (282510). We also thank FP7 SYNERGY-COPD , BILS (D.G.) and Stockholm County (J.T.).Conflict of Interest: none declared.


DK of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with  Ricin is a 60–65 kDa glycoprotein derived from castor beans There are potentially two major groups of antidotes against toxins, antibodies and chemical compounds. The history of using antibodies as effective antidotes against toxins can be traced back to 1890 Macaca fascicularis) and this ScFv antibody demonstrated a high sequence similarity (90%) to human counterparts. However, the rest of these antibodies were all derived from mice. While structurally similar, antibody sequence difference between mice and humans is sufficient to invoke an immune response in humans when murine antibodies were injected into humans. The immune response would result in a rapid removal of murine antibodies from the human blood, systemic inflammatory effects, and possible anaphylaxis, which can sometimes be fatal Much work has been done on developing antibodies, both polyclonal and monoclonal, as antidotes against the toxin in vivo. Intraperitoneal (i.p.) administration of D9, at a low dose of 5 µg per mouse, 6 hours after or 6 weeks before the i.p. challenge with 5×LD50 ricin rescued 100% of the mice (manuscript submitted). These results indicate that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin.In our previous study, a murine anti-ricin monoclonal antibody (mAb), D9 was found to be exceptionally effective in both pre- and post-exposure efficacy assays in vivo. It has the potential for both prophylactic and therapeutic uses.In this study, in order to humanize D9, the CDRs from the heavy and light chain (HC and LC) variable regions (VH and VL) forming antigen binding site of the murine mAb D9 were grafted onto antibody FRs from the selected donor human germline V and J genes of HC and LC. The humanized D9 (hD9) VH and VL were further grafted onto human HC and LC constant regions (CH and CL) to assemble the whole antibody gene. After expression in mammalian cells and purification, the recombinant hD9 (>95% human sequences) was demonstrated to retain antigen-binding specificity and therapeutic efficacy comparable to its parental murine D9. To the best of our knowledge, this is the first humanized anti-ricin mAb with demonstrated therapeutic efficacy All mouse experiments were performed in strict accordance with the guidelines set out by the Canadian Council on Animal Care (CCAC). The animal care protocol was reviewed and approved by the Committee on the Ethics of Animal Experiments of Defence Research and Development Canada – Suffield (DRDC Suffield) (protocol number: J1C-10-1-1-0).D9 VH and VL DNA sequences were determined as follows. Briefly, total RNA was isolated using a Qiagen RNeasy Plus Mini kit  from a D9 hybridoma cell line, made from female Balb/c mice in our laboratory (manuscript submitted). This was reverse-transcribed with Superscript II RNase H  and an oligo dT primer (12–18 mer) to produce cDNA. Platinum Taq DNA Polymerase High Fidelity (Invitrogen) was used to amplify the VH and VL genes with the degenerate primers specific for mouse antibodies  by polymerase chain reaction (PCR) in an Eppendorf Mastercycler . Distinct bands of ∼340 bp for VH and ∼325 bp for VL were detected on a 1.5% agarose gel after PCR. The bands were purified using a Qiagen Gel Extraction kit (Qiagen) and cloned into a Zero Blunt TOPO PCR cloning vector (Invitrogen) for sequencing.http://www.biocomputing.it/pigs), a web server for the automatic modeling of antibody Fv. When VH and VL protein sequences of D9 were entered, the sequences were analyzed by the PIGS server to search over a database of known antibody structures. The three-dimensional (3D) structure of D9 Fv was then constructed by homology modeling based on the structures of the VH (PDB No. 2NR6) of a mouse mAb against cockroach allergen Bla g 2 and the VL structure of a mAb against chicken egg-white lysozyme (PDB No. 1MLB). The final 3D structure of D9 was then visualized using a PDB molecular visualisation program, Deepview, for identification of the key FR residues, including Vernier Zone residues located within 5? of the CDRs, supporting CDR conformation and interchain packing residues located within 5? of VH-VL interface, contributing to VH-VL interaction. Meanwhile, the residue accessibility was calculated to determine their positions. The residue was defined as on the surface of antibody when it was greater than 30%.The homology modeling of D9 Fv was performed using PIGS (http://www.bioinf.org.uk/abs/seqtest.html). In order to select human germline V genes as FR donors (FRs 1 to 3) for humanization of D9, the VH and VL amino acid sequences of D9 were separately subjected to IgBlast and IMGT searches against the entire human antibody germline V genes. Then human germline V genes with the same canonical structure as D9 were shortlisted. The human donor V genes were selected from the shortlist based on their highest CDR similarities  to those of D9 VH and VL with consideration of key FR residue similarity. Human donor J genes to provide FR 4 for humanization of D9 were selected respectively based on their highest similarities to D9 VH and VL CDR3 and key FR residues in FR3. Finally, CDRs of D9 VH and VL were grafted onto the FRs of the selected human germline donor V and J genes respectively, resulting in humanized D9 (hD9) Fv. Furthermore, the hD9 VH and VL were grafted onto human gamma 1 HC CHs and kappa 1 LC CL, respectively, to assemble the complete humanized antibody gene, resulting in the full-length humanized antibody, hD9.The humanization of D9 Fv was done by CDR-grafting as described previously with minor modification The hD9 full-length DNA sequence (2 kb) including a LC leader sequence, the humanized LC (VL+CL), foot-and-mouth-disease virus (FMDV)-derived 2A self-cleavage linker encoding APVKQTLNFDLLKLAGDVESNPGP, a human antibody gamma 1 HC leader sequence, and humanized HC (VH+CH1 to 3) was synthesized by GenScript Corporation  and cloned into pUC57, resulting in pUC57-hD9. A recombinant adenovirus vector, pAdhD9 expressing hD9 was constructed and the recombinant hD9 was expressed in HEK 293 mammalian cells using the AdEasy system  Antibodies were separated by 10% NuPAGE Bis-Tris SDS-PAGE gels using an XCell SureLock Novex Mini-Cell System (Invitrogen). The bands were visualized by SimplyBlue safestain staining (Invitrogen). The molecular weights of the samples were estimated by comparison to the relative mobility values of standards of known molecular weights.koff) and association (kon) rate constants were obtained with the SensiQ Qdat software, corrected by subtraction of the zero antibody concentration flow cell as well as zero ricin flow cell; values for the apparent equilibrium dissociation constant (DK) were calculated from the ratio of koff and kon.The affinities for antibodies binding to ricin were determined using a Surface Plasmon Resonance (SPR) biosensor, SensiQ Pioneer . Briefly, ricin (10 µg/mL) diluted in 10 mM acetate buffer pH 4.5 was first immobilized onto the COOH1 chip following the standard 1-ethyl-3-(3-dimethylpropyl)-carbodiimide (EDC) plus N-hydroxysuccinimide (NHS) (Sigma-Aldrich) coupling chemistry, resulting in 250 response units (RU) of ricin being immobilized. The system was operated at 25°C. Kinetic measurements were carried out by 2 min injection at a flow rate of 25 µl/min of serial dilutions of each mAb from 0 to 500 nM in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered saline containing 3 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl and 0.05% Tween-20 and dissociation for 6 min. The ricin immobilized chip surface was regenerated by injection of 10 mM phosphoric acid for 120 seconds after each cycle. The data of dissociation  were obtained from the pathogen-free mouse-breeding colony at DRDC Suffield, with the original breeding pairs purchased from Charles River Canada . For post-exposure therapeutic efficacy study, groups of 8 mice were given 5×LD50 of ricin per mouse and then 5 µg of hD9 per mouse both by the i.p. route to mice at 2, 4, or 6 hours post-ricin poisoning. The mice were observed for morbidity and mortality over one week.To evaluate the half-life of hD9 or D9 in serum, groups of 4 mice were injected by the i.p. route with 5 µg/mouse of antibody in 100 µl phosphate buffered saline (PBS), and were bled from a superficial tail vein at 1, 7, 14, and 23 days post injection. hD9 or D9 concentrations in sera over time were then measured by the anti-ricin immunoassay. Briefly, 96-well Nunc Maxisorp immunoassay plates  were coated with 100 µl per well of 2.5 µg/ml ricin in carbonate bicarbonate buffer, pH 9.6, then incubated overnight at 4°C. After blocking with SuperBlock blocking buffer , the plates were incubated with 100 µl of serum dilutions for 2 hours at room temperature. Anti-ricin mAbs were detected by incubation with 1∶3,000 diluted horseradish peroxidase (HRP)-goat anti-mouse IgG  for D9 and 1∶5,000 diluted HRP-rabbit anti-human IgG (Jackson ImmunoResearch Laboratories) for hD9 followed by the addition of tetramethylbenzidine (TMB) . Absorbance was measured at 615 nm by a microplate autoreader . The data were expressed as percentages of the hD9 or D9 concentration in sera on day 1, and then plotted against time in days post treatment.http://www.biocomputing.it/pigs) based on the most homologous antibody VH (2NR6), sharing 86% identity and VL (1MLB), sharing 70% identity with the corresponding VH and VL of D9 in the database of known antibody structure. 3D structure of D9 was then visualized using a protein data bank (PDB) molecular visualisation program, Deepview, as shown in D9 VH and VL mRNA was extracted from D9 hybridoma cells, amplified by Reverse Transcription-PCR and then sequenced. The deduced amino acid sequences are shown in In order to select human germline antibody V genes as FR donors for D9 humanization, a shortlist of human germline antibody HC or LC V gene candidates with the same CDR canonical structure as the D9 counterpart was then formed to ensure that the human antibody donor FRs support the mouse CDR canonical structures  or 2. WiTo select human germline antibody J genes as FR4 donors, CDR3 and FR4 sequences from D9 VH or VL were aligned with human germline JH or JK genes  or 4. AsConsequently, human HC germline genes 1–18 and JH6 were selected as FR donors for humanization of D9 VH; human LC germline genes O12 and JK4 were selected as FR donors for humanization of D9 VL shown in The VH of hD9 was further grafted onto the human gamma 1 HC CHs to form a complete HC, while the VL was grafted onto the human kappa 1 LC CL to form a whole LC.In order to obtain an equi-molar expression of antibody HC and LC in a single open reading frame (ORF) driven by one single promoter, a 2A self-cleavage linker encoding a 24-residue-oligopeptide, was introduced between HC and LC DNA sequences to express a full-length antibody from a ORF driven by a single promoter in an adenoviral vector The recombinant hD9 was expressed in HEK-293 cells and purified using an ImmunoPure Protein (L) agarose column. The purified product was subjected to 10% SDS-PAGE, and one prominent band of ∼150 kDa in non-reducing conditions, and two clear bands of ∼50 kDa and ∼25 kDa in reducing conditions (cleavage of disulfide bridges) were observed .DK) for antibodies binding to ricin were performed by SPR. Ricin was captured on a biosensor chip, various concentrations of hD9 or D9 were passed through the chip. The kinetics of association and dissociation were recorded in SPR sensorgram .In order to compare the antigen-binding affinity between hD9 and parental murine D9, measurements of affinity constant  to RTA To date, most of anti-ricin neutralizing antibodies have been developed from mice. Only one was developed from macaques that share a high degree of antibody sequence similarity with humans There are two sources of human antibody sequences: mature  and germline . Mature sequences result from the recombination of germline genes V, D, and J for VH or V and J for VL CDR-grafted antibodies tend to lose the parental binding affinity In order to have equimolar expression of HC and LC in mammalian cells, an FMDV-derived 2A self-cleavage linker was introduced between the two chain genes. The 2A oligopeptide sequence was expected to undergo self-cleavage to generate separate HC and LC after translation. The exact mechanism of 2A self-cleavage is still unknown. It has been hypothesized that the 2A sequence impairs peptide bond formation between 2A G and 2B P through a ribosomal skip mechanism After the recombinant hD9 was expressed in mammalian cells, HC and LC appeared completely cleaved without detectable unpaired chains as shown in DK of 1.63 nM, comparable to D9 (2.55 nM), indicating humanization of D9 was successful.The specific binding activity of purified hD9 to ricin was evaluated. As shown in hD9 appeared less efficacious than its parental murine D9. The difference is understandable, since hD9 had a much shorter half-life than D9 in mice. The half-life of hD9 was estimated to be around 3 days, compared to 19 days of D9 in mice. hD9 decayed in mice around 6 times faster than D9. There was only 25% of the hD9 left while still 85% of the D9 left in mice at day 7 post administration of antibodies, when 50% of the mice were dead in hD9-administrated group compared to no death in D9 group. Therefore, hD9 short half-life might be attributed to its slight lower efficacy than D9 in mice.In conclusion, D9 was humanized by CDR-grafting to minimize murine antibody immunogenicity in humans. The resultant hD9 was expressed in mammalian cells and the recombinant hD9 was demonstrated to retain ricin-binding specificity and anti-ricin efficacy comparable to its parental murine D9. This hD9 has the potential utility for prophylactic or therapeutic purposes against ricin poisoning.


SMARCB1/INI1 tumour suppressor gene in familial schwannomatosis. SMARCB1 is located centromeric to NF2 on 22q and loss of function of SMARCB1 is also a hallmark of malignant rhabdoid tumour (MRT), a highly aggressive tumour of infancy. Both familial and sporadic schwannoma tumours show a mosaic pattern of SMARCB1 protein expression, suggestive of tumour cells either haploinsufficient, or null for SMARCB1 protein. Familial schwannomas linked to constitutional SMARCB1 mutation can also have somatic mutation of NF2, and conversely, schwannoma tumours associated with constitutional NF2 mutation show mosaic loss of SMARCB1, suggesting the involvement of a four-hit mechanism. Molecular analysis for evidence of constitutional SMARCB1 mutation is important in both familial and sporadically occurring schwannomatosis because of the transmission risk for a mutation predisposing to the incurable MRT, in early childhood. However as for NF2, recent evidence suggests that constitutional SMARCB1 mutations have variable penetrance and exhibit mosaicism, highlighting the importance of examining multiple tumour tissue samples as well as blood in affected individuals to ascertain germline predisposition and to provide accurate counseling for transmission risk. Further studies are needed to define the SMARCB1 mutation spectrum in schwannomatosis and to dissect the strikingly different biology between schwannoma and MRT.Schwannomatosis is a form of Neurofibromatosis type 2 (NF2) characterized by multiple schwannomas without vestibular involvement, affecting the cranium, spine and periphery. Several recent genetic studies have implicated the


In the STa (80 ng/mL) challenged jejunal loop, the fluid absorption rate of 28.6 ± 5.8 (14) μL/cm/hr was significantly below (P < .001) the normal rate of 98.8 ± 6.2 (17) μL/cm/hr. Intraluminal (300 uM) glibenclamide added to STa perfused loops failed to improve the inhibited fluid absorption rate, which was 7.4 ± 3.2 (6) μL/cm/hr on coperfusion with STa. Similarly, on coperfusion with 30 uM clotrimazole, the fluid absorption rate with STa present remained inhibited at 11.4 ± 7.0 (4) μL/cm/hr. On coperfusion with intraluminal 1 uM cromakalim, STa reduced fluid absorption significantly (P < .02) to 24.7 ± 8.0 (10) μL/cm/hr, no different from STa challenge in the absence of cromakalim. Infusion i.v. with these agents also failed to restore fluid absorption after STa challenge. These observations do not support the proposed potassium ion permeability event as a necessary corollary of enterotoxin-mediated secretion. This makes it unlikely that modulators of such permeability prevent enterocyte secretion in diarrhoeal disease.Enhanced potassium ion permeability at the enterocyte basolateral membrane is assumed to facilitate sustained chloride ion and fluid secretion into the intestinal lumen during episodes of secretory diarrhoeal disease. To examine this concept  Fluid and electrolyte absorption is an essential function of the enterocytes of the small intestine. In addition, enterocytes are assumed also to be capable of fluid secretion into the intestine resulting from the transport of chloride ion into the lumen although this enterocyte origin of secretion has been questioned , 2. Net in vitro studies of electrical events in cell systems after STa and cholera toxin challenge apparently confirm this aspect of the secretion model [in vivo studies on potassium channel openers and blockers in the enterotoxin challenged small intestine, and the most detailed account seems to contradict the above model of the role of potassium ion channel opening. Reduced fluid absorption in vivo from the rat jejunum after STa challenge was partially restored by glibenclamide and also, less explicably, by cromakalim, the potassium channel opener [in vivo study, a brief report on the pig intestine, indicated that the potassium channel blocker clotrimazole did not overcome the effect of STa challenge [Many on model –5. In col opener . The othhallenge .in vivo on the effect of clotrimazole, cromakalim, and glibenclamide on jejunal fluid absorption in vivo in the rat compromised by perfusion with E. coli STa enterotoxin. These experiments intended to determine whether or not K+-channel modulators administered in vivo could restore or even further reduce the effects of STa on fluid absorption, as has been hypothesised should occur on the basis of in vitro findings. Jejunum was chosen rather than ileum, as the jejunum has higher rates of fluid absorption that are susceptible to STa derangement. Ileum can be used, but absorption rates are about one half those in the jejunum. When tested in vivo, these potassium channel modulators had no effect on STa-inhibited fluid absorption, making it unlikely that they would be effective antidiarrhoeal compounds. In addition, the failure of potassium channel modulators to alter STa action adds to the available in vivo evidence against the enterocyte secretory model of diarrhoeal disease.We report here on similar findings in the rat jejunum in vivo rat jejunal loops was measured by a recirculation procedure [i.p.) were tracheotomised to allow unimpeded breathing of room air. Anaesthesia was sustained by periodic i.p. dosage sufficient to maintain abolition of the hind limb flexor withdrawal reflex. Body temperature was kept at 37°C by a heating table controlled by rectal thermistor. Twenty five cm jejunal loops, starting ten cms below the ligament of Treitz, were perfused via PVC tubing at 1.8 mL/min by peristaltic pump  from a reservoir containing an initial volume of 25 mLs of perfusate maintained at 37°C. After three hours, the experiment was ended by pumping air into the loop and collecting all remaining perfusate in the reservoir. In addition, the loop was excised, and any fluid remaining in the loop was expelled by air with a 20-mL syringe into the reservoir under gravity drainage. Final recovered volume in the reservoir was measured to the nearest 0.1 mL. Loop length was measured by ruler after gently laying out the excised loop on the bench without stretching it. Fluid absorption as measured by this fluid recovery [All experiments complied with current UK legislation and were approved after internal ethical review. Fluid absorption from perfused rocedure  describerocedure . Adult Srecovery  method iIn preliminary experiments to confirm the activity of the potassium ion channel agents, arterial blood pressure was measured via a carotid cannula  using a Statham 23 B pressure transducer coupled via a 1401 CED ac/dc board to a Dell Pentium and recorded by the “Chart” data capture programme . Where these drugs were given as part of the perfusion protocol for assessing their effect on absorption, blood pressure was measured with a Harvard pressure transducer, coupled to a Toshiba lap-top computer and recorded using Spike 2 for data capture via the CED 1401 interface . Heart rate and respiration frequency (from the Traube-Hering waves) were also measured from the blood pressure record.Cromakalim and clotrimazole were dispensed from stock solutions of 5 mM in ethanol. Experiments were done using ethanol vehicle to confirm that this was without effect on the reduced fluid absorption caused by STa. Glibenclamide was dispensed from a 300 mM stock solution in DMSO (dimethyl sulphoxide). Similar control experiments with DMSO confirmed that vehicle alone had no effect on STa action.Cromakalim and glibenclamide had been previously identified as being antisecretory , using ain vitro experiments by measuring changes in longitudinal tension in the rabbit ileum mounted in a Burn-Dale apparatus and maintained in aerated Krebs-Ringer at 37°C. Tension was measured by a Harvard instruments tension transducer via an ac/dc converter board recorded by a Dell Pentium PC using the “Chart” data capture programme. Rabbit ileal tissue became available during the time of this study but was not purposefully selected as a test assay for motility changes.The ability of the potassium channel active agents to alter intestinal smooth muscle function was also tested in separate E. coli STa and other chemicals were purchased from Sigma Chemical Co . For some of the later experiments, STa from a P16 strain was used at an equivalent dose after the synthetic peptide became unavailable. There were no significant differences in the effect of STa on fluid absorption, since the reduced fluid absorption of 23.5 ± 8.5 (8) μL/cm/hr after P16 STa exposure did not differ significantly from the rate of 33.2 ± 8.6 (9) μL/cm/hr after Sigma STa exposure. Sagatal (sodium pentobarbitone) was obtained from Rhone Merieux Ltd .t values after Dunnett's [Results are given as the mean and standard error of the mean, followed by the number of experiments, equal to the number of animals, in parenthesis. Implementation of all statistical analysis was done using BMDP . Where runnett's  correctiin vivo and on smooth muscle tension in vitro. This was intended to confirm, by using the same stock solutions, that the drugs introduced into the small intestine had demonstrable effects on other organ systems. In a representative group of untreated animals, systolic/diastolic pressure was 128.3 ± 11.7 (6) over 112.0 ± 13.8 (6) mm Hg, giving a mean arterial blood pressure of 117.4 ± 13.6 (6) mm Hg. Intravenous glibenclamide (P < .01) increased systolic by 21.7 ± 5.8 (7), diastolic by 20.3 ± 4.7 (7) and mean arterial blood pressure by 20.8 ± 4.8 (7) mm Hg. Clotrimazole as a bolus dose of 1 mg/kg depressed mean systolic pressure significantly (P < .01) to 87.2 ± 7.6 (6) mm Hg, the diastolic pressure to 66.3 ± 5.6 (6) mm Hg and mean arterial blood pressure to 73.3 ± 13.1 (6) mm Hg. Clotrimazole administration caused marked bradycardia in some animals for 20 seconds or longer, coupled with a compensatory increase in pulse pressure, as predicted by the Frank-Starling law of the heart. Cromakalim at a dose of 24 ug/kg depressed mean arterial blood pressure to 58 ± 5 (3) which was significantly less (P < .02) by 38 ± 5 (3) mm Hg pressure than control values. A higher dose of 60 ug/kg depressed mean arterial blood pressure even further to 42 ± 10 (3) mm Hg. In contrast to clotrimazole, there were no episodes of bradycardia with cromakalim or late pressor effects as with glibenclamide. However, it was evident that all three drugs prepared from the same stock solutions used to perfuse the small intestine showed demonstrable effects on the cardiovascular system.The potassium channel active agents used to attempt to prevent the effect of STa on fluid absorption were tested for their known pressor effects on mean arterial blood pressure nclamide  as a bolin vitro in experiments on rabbit ileum incubated in Krebs-Ringer solution. Confirming the effects on vascular smooth muscle tone in vivo, 7 uM cromakalim and 100 uM clotrimazole reduced intestinal smooth muscle longitudinal tension (P < .001) to 4.6 ± 0.6 (4) grams and reduced average tension and tension amplitude, without affecting the rate of contraction. Cromokalim similarly reduced (P < .05) tension amplitude from 5.1 ± 1.3 (3) to 2.8 ± 1.3 (3) grams without altering frequency of contraction. In contrast, 50 uM glibenclamide significantly (P < .001) increased the basal tension from 2.0 ± 0.5 (3) to 3.1 ± 0.6 (3) grams and also had no effect on frequency of contraction. These results are not novel and served only to confirm the blood pressure results in that clotrimazole and cromakalim reduced smooth muscle tone and glibenclamide showed a pressor action, as is well known and as others have found [The ability of these compounds to affect smooth muscle tension was also determined  tension . Basal tve found , 14, 15. E. coli STa.In a group of animals perfused luminally with STa, mean arterial blood pressure was 108 ± 11.7 (3) mm Hg at the onset of perfusion. After three hours perfusion, the mean arterial blood pressure in the STa perfused animals rose to 125 ± 5 (3) mm Hg, indicating that pressure was maintained and unaffected by intraluminal STa perfusion. In a similarly representative group perfused with STa, heart rate was 375 ± 10 (5) beats per minute, and the respiratory rate was 53 ± 5 (5) breaths per minute, with neither variable significantly changing over the perfusion period. Hence, three or more hours of perfusion under anaesthesia did not lead to respiratory or cardiovascular depression even when the lumen was perfused withμL/cm/hr. In the STa challenged loop, there was a significantly lesser (P < .001) rate of 28.6 ± 5.8 (17) μL/cm/hr absorption that was also significantly higher (P < .001) than zero net absorption. When glibenclamide was added to E. coli STa perfused loops at a concentration of 100 uM, known to normalise the increased membrane currents found in Caco2 cells after STa exposure [μL/cm/hr which did not differ from absorption in the presence of STa without glibenclamide. This value of fluid absorption was still highly significantly (P < .001) below control rates of absorption, indicating that simultaneous glibenclamide perfusion had not improved the poor fluid absorption rates caused by STa enterotoxin. Glibenclamide at a concentration of 300 uM, known to reduce the short-circuit current increases in tracheal fibroblasts [μL/cm/hr, which was significantly (P < .05) below the rate in the presence of STa but without glibenclamide.When perfused without STa, the unchallenged jejunum  absorbedexposure , fluid a.6 ± 5.8  μL/cm/hrμL/cm/hr, not significantly different from the value of  28.6 ± 5.8 (17) μL/cm/hr for luminal perfusion with STa without glibenclamide. Glibenclamide perfusion caused a noticeable pressor effect of approximately 16.7 ± 5.1 (5) mm Hg which indicated that while there was no effect on fluid uptake, the familiar effect of glibenclamide on mean arterial blood pressure was found.When glibenclamide was given intravenously , the meain vitro) of clotrimazole in the lumen as well as STa, fluid absorption was 11.4 ± 7.0 (4) μL/cm/hr, indicating that fluid absorption hindered by STa was not improved by inclusion of the potassium channel blocker. The i.v. perfusion of clotrimazole  μL/cm/hr for fluid absorption in the presence of STa without simultaneous perfusion i.v. with clotrimazole. Clotrimazole was confirmed to have the anticipated depressor action on mean arterial blood pressure.With a 30 uM concentration (known to inhibit completely the increment in short-circuit current after STa exposure rimazole  during SP < .01) to 24.7 ± 8.0 (10) μL/cm/hr, which did not differ from the value for fluid absorption after STa challenge alone of 28.6 ± 5.8 (17) μL/cm/hr. When cromakalim was administered intravenously (μL/cm/hr, indicating that fluid absorption after STa exposure was not restored to normal values but was worsened by cromakalim.In the presence of 1 uM luminal cromokalim , STa redvenously , during A prevalent theory regarding secretion by the small intestine during diarrhoeal disease proposes that the enterocytes secrete chloride ion into the lumen. This is a necessary aspect of any hypothesis concerning an enterocyte origin for fluid secretion, because water movement occurs normally only in response to osmotic gradients. The mucosally secreted chloride and associated sodium ions purportedly increase osmolarity at the mucosal surface, causing fluid flow into the small intestinal lumen. To achieve chloride secretion from the enterocyte interior against the disadvantageous chemical gradient, this model incorporates enhanced potassium ion permeability to maintain a favourable electrical gradient. According to the enterocyte secretion concept, potassium ion exits across the basolateral membrane and maintains the mucosal membrane potential difference. As sustained secretion requires increased basolateral potassium ion permeability, fluid secretion should be inhibited by potassium channel blockers and possibly should also be accelerated by potassium channel openers.in vitro short-circuit current and ion efflux studies in tumour cells in Ussing chambers although neither parameter measures secretion as the mass transport of fluid. Prostaglandin E 1 or E. coli STa increases a barium inhibitable rubidium flux across the serosal membrane, increases short circuit currents, and alters sodium and chloride fluxes across T84 cells [E. coli STa enterotoxin [in vitro evidence from electrical events and ion efflux studies points to potassium channel blockers preventing secretion.Evidence for this particular aspect of the enterocyte secretion model derives largely from 84 cells , 19. Pot84 cells  and shou84 cells . Clotrimerotoxin . Hence, in vivo pharmacological tests of the role of enhanced enterocyte potassium ion permeability during enterotoxin challenge are rare, and the results are contradictory. Since clotrimazole [in vivo by measuring loop weight, STa was modestly secretory, as was glibenclamide alone without toxin [ E. coli STa-mediated secretion despite being a potassium channel opener that should worsen or at least not improve secretion. Hence, neither agent affected fluid movement in the STa challenged jejunum in a manner predicted by the potassium ion permeability concept. The ability of cromakalim, the potassium channel opener, to prevent STa action and the ability of glibenclamide to cause secretion despite being a potassium channel blocker do not accord with the enterocyte potassium permeability model at all.In contrast, rimazole  and glibrimazole  reduce tut toxin . In contE. coli STa enterotoxin. STa reduces fluid absorption severely, but if the potassium ion permeability model is valid, clotrimazole and glibenclamide should improve the poor fluid absorption, and cromakalim should perhaps worsen it. Although the theory predicts this, the observations of Shirgi-Degen and Beubler are at variance both with the present observations and with secretion theory. A three-hour perfusion time as used here allows large changes in volume to arise, since a reduction of 60 μL/cm/hr in the rate of fluid absorption after STa challenge in a 25 cm loop translates into 4.5 millitres of fluid additionally present in the fluid reservoir at the end of experiment. Any net secretion and also any ability to inhibit secretion will be detected in this comparatively robust system and, in fact, was not found. Over a three-hour perfusion experiment, mean arterial blood pressure rose from 100 mm Hg to 120 mm Hg at the end of the experiment, indicating that mean arterial blood pressure is not compromised. Using the recovered volume technique, glibenclamide, cromakalim, and clotrimazole were shown not to have any ameliorating effect on STa induced reductions in fluid absorption.In the present experiments, neither luminal nor intravenous administration of clotrimazole, glibenclamide, or cromakalim affected the reduced fluid absorption occurring after exposure to in vivo in the rat, and glibenclamide had a moderate pressor effect, as others have found [To establish that the potassium channel modulators were active prior to intravenous administration or luminal perfusion, aliquots from the stock solutions were tested in separate, unrelated experiments. Intravenous injections of cromakalim and clotrimazole reduced mean arterial blood pressure ve found , 20. Theve found . Confirmweight also measures additionally fluid entering and leaving the vasculature, causing changes in the interstitial volume. Since measurement of loop weight also includes interstitial volume, which the luminal fluid recovery method does not, differing findings might result from vasomotor changes. This might explain why potassium channel active agents do not alter fluid absorption after STa challenge when assessed by luminal volume recovery in the present experiments but seem to reduce secretion when this is defined and measured as an increase in loop weight. This could also explain why cromakalim is antagonized by glibenclamide in the Beubler study, as a depressor and a pressor substance compete to alter peripheral resistance and hence intestinal interstitial volume. If these changes are not explicable by local vascular changes within the intestine, then any explanation defaults to the level of the enterocyte. The cardiovascular actions of the selected agents may explain differences between this study and that of Shirgi-Degen and Beubler . Luminalin vitro evidence indicating that it should be relevant. One explanation for the in vitro results is that rubidium efflux and short-circuit-current changes relate to the sodium: potassium pump rate and not to chloride secretion. The known inhibition by clotrimazole of the sodium pump may explain the fall in short circuit current in isolated cells [Since these compounds do not inhibit fluid movement in the STa challenged intestine when the recovered volume technique is used, then the potassium channel activation model does not apply despite the availability of ed cells . Similarin vivo the reduced net fluid movement caused by E. coli STa enterotoxin, widely regarded as causing secretion. Enhanced potassium ion permeability to maintain the electrical gradient for adequate luminal exit of chloride ion is a sub-hypothesis of the chloride secretory model [E. coli STa enterotoxin is not prevented by potassium channel modulators despite promising data from in vitro techniques that are assumed to but may not measure secretion. The lack of effect of potassium channel modulators is further evidence that STa is not a secretory toxin but acts solely by inhibiting sodium ion and fluid absorption.In conclusion, potassium ion active agents do not restore ry model  rather t