Brain-derived neurotrophic factor (BDNF) is an activity-dependent secreted protein that is critical to organization of neuronal networks and synaptic plasticity, especially in the hippocampus. We tested hypothesis that reduced CSF BDNF is associated with age-related cognitive decline.42 and total tau were measured in 128 cognitively normal adults , 21 patients with Alzheimer's disease (AD), and nine patients with Mild Cognitive Impairment. Apolipoprotein E and BDNF SNP rs6265 genotype were determined. Neuropsychological tests were performed at baseline for all subjects and at follow-up visits in 50 Normals. CSF BDNF level was lower in AD patients compared to age-matched Normals (p = 0.02). CSF BDNF concentration decreased with age among Normals and was higher in women than men (both p<0.001). After adjusting for age, gender, education, CSF Aβ42 and total tau, and APOE and BDNF genotypes, lower CSF BDNF concentration was associated poorer immediate and delayed recall at baseline (both p<0.05) and in follow up of approximately 3 years duration (both p<0.01).CSF concentration of BDNF, AβReduced CSF BDNF was associated with age-related cognitive decline, suggesting a potential mechanism that may contribute in part to cognitive decline in older individuals. Val66Met or Met-BDNF), inheritance of which has been associated with poorer cognitive performance in healthy older adults Met-BDNF are not clear; however, one study has shown diminished depolarization-induced secretion of Met-BDNF compared to Val-BDNF, and failure of Met-BDNF to localize to secretory granules or synapses in transfected neurons Age-related cognitive decline is a complex convergent phenotype that likely derives in part from brain senescence and in part from prodromal dementing illnesses, most commonly Alzheimer's disease (AD). Brain-derived neurotrophic factor (BDNF) is an activity-dependent secreted protein that, along with its receptors, is expressed widely in the central nervous system and is critical to organization of neuronal networks and synaptic plasticity, especially in the hippocampus, in a variety of animal models and apparently in humans All procedures were approved by the institutional review boards of the University of Washington; all subjects were recruited from University of Washington Alzheimer's Disease Research Center and provided written informed consent. Subjects underwent detailed clinical and laboratory evaluation and were classified as no cognitive impairment , amnestic mild cognitive impairment (MCI) 42 was measured using an X-MAP-based assay APOE genotype was determined by a restriction digest method BDNF SNP rs6265 was genotyped using TaqMan allelic discrimination detection, as previously described CSF concentration of BDNF, total tau and AβNeuropsychological tests included: (i) Paragraph Recall - a test of declarative memory post hoc Bonferroni multiple pair-wise comparisons for continuous variables and Fisher's exact test for categorical variables to assess differences between diagnostic and genetic groups. Linear regression models were used for associations of demographic characteristics with CSF BDNF concentration, and cross-sectional relationships between CSF BDNF concentration and coincident cognitive test performance. Raw scores were used for each test except log-transformed times for Trails B to remove skewness. We used two-stage regression  to assess association of baseline CSF BDNF concentration with subsequent longitudinal changes of cognitive test performance One-way analysis of variance (ANOVA) was used with post-hoc Bonferroni test, p = 0.02). CSF BDNF level for the nine MCI patients was not different from either group (both p>0.05). The frequencies of Met-BDNF genotype (G/G vs. G/A or A/A) were not different between ON, MCI, and AD subjects (Fisher's Exact Test p = 0.36). Moreover, CSF BDNF concentration in AD subjects was not different among Met-BDNF genotype G/G , G/A , or A/A  .42, and total tau, and who underwent neuropsychological evaluation , G/A , or A/A  .Our focus was on 128 cognitively normal volunteers  in whom we measured CSF BDNF, Aβaluation Table 1.APOE ε4 allele, and Met-BDNF (G/G vs G/A or A/A) as independent predictors, both age and gender were significantly associated with CSF BDNF level (p<0.001). Neither presence of APOE ε4 allele (p = 0.33) nor Met-BDNF (p = 0.09) was related to CSF BDNF levels. In addition, there was no interaction between APOE ε4 allele and Met-BDNF genotype (p = 0.25), nor between age and gender (p = 0.45). Removing the three lowest values of BDNF for males did not change the results. Finally, after adjusting for age and gender, CSF BDNF levels were related inversely (slope = −0.03) to CSF levels of Aβ42 (p = 0.05) but not to CSF total tau (p = 0.34).CSF BDNF concentration decreased with age among Normals and was higher in women than men . There was an association between higher CSF BDNF concentrations and better performance on Paragraph Recall - Delayed that was independent of CSF Aβ42 and total tau concentrations (Model 2) as well as presence of APOE ε4 and Met-BDNF (Model 3); correlation with the Paragraph Recall - Immediate was significant when all predictor variables were in the model (Model 3). An association between lower CSF BDNF and poor performance on Trail Making Test Part B also was observed. There was no significant correlation between CSF BDNF concentration and performance on the Category Fluency test.Most (n = 121) Normals underwent extensive neuropsychological testing at baseline Table 1.42 and total tau concentrations (Model 5), suggesting that prediction by baseline CSF BDNF concentration for subsequent cognitive changes is independent of preclinical AD. These relationships also were unchanged after further adjusting for presence of APOE ε4 and Met-BDNF (Model 6). All results were unaffected by removing the subject who had the largest annual decline for Immediate Recall, Delayed Recall, and Category Fluency , aged 40–100 years (mean±SD = 71.5±8.9) had at least one clinical follow-up visit  with an average length of follow-up of 3.3 years  Table 1.Met-BDNF genotype , even after adjusting for age, gender and education in the multiple regression model . Similarly, the longitudinal changes in these cognitive performances (n = 50) were not associated with Met-BDNF genotype .Among all Normals , baseline performance on each of the four cognitive tests  did not differ by Met-BDNF or the APOE ε4 allele. (ii) Women had higher average CSF BDNF concentrations than men. (iii) Lower CSF BDNF concentration was associated strongly with poorer memory and less so with diminished executive function; importantly, these associations were independent of CSF biomarkers of preclinical AD, suggesting that the mechanisms that contribute to early AD and to age-related decline in CSF BDNF might be independent. We must acknowledge that this study has a relatively small sample size and short duration of follow-up and further studies are needed to validate our results.We made three novel observations in cognitively normal individuals. (i) CSF BDNF decreased across the human life-span in the absence of dementia or MCI and was independent of inheritance of While ours is the first study of which we are aware to investigate age-related changes in CSF BDNF, other studies have investigated disease-associated changes in CSF BDNF in patients with idiopathic Parkinson disease The gender difference in CSF BDNF observed in this study is especially interesting, and we speculate that this may be due to hormonal effects. Animal studies have shown that estrogen receptors colocalize to cells that express BDNF and its receptor trkB, and estrogen regulates the expression of BDNF Met-BDNF or APOE ε4. Further reduction in CSF BDNF occurred in AD. Our data suggest that reduced secretion of BDNF in the central nervous system is one mechanism that may contribute to age-related cognitive decline.Our data showed that progressive decline in CSF BDNF concentration was a feature of advancing age independent of preclinical AD or dementia, and was associated strongly with reduced performance in declarative memory tests and less strongly with performance on tests of executive function, perhaps a reflection of the special role of BDNF in hippocampal function. Age-related reduction in CSF BDNF was independent of inheritance of


Epitheloid hemangioendothelioma (EHE) is a vascular tumor of intermediate malignancy that arises in soft tissues, liver, lung, bone, and spleen. The hepaA 39-year-old woman presented with right upper quadrant abdominal pain of six months duration. She denied any fever, chills, or malaise. On physical examination, there was painless hepatomegaly. Laboratory values, including serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), bilirubin, and alpha-fetoprotein (AFP) levels, were all normal; there was increased alkaline phosphatase (620 U/l) and gamma-glutamyl-transpeptidase (300 U/l) and mild hypoalbuminemia (3 g/dl). She underwent abdominal CT, which revealed conglomerate mass lesions suggestive of metastases in the liver. MRI of the abdomen was then performed on a 1.5-T unit  using T1W, T2W, and dynamic gadolinium-enhanced sequences. Innumerable small, rounded, predominantly peripheral hepatic lesions, measuring between 1 and 3 cm in size, were identified throughout segments 2 and 3 as well as in the subcapsular right lobe of the liver. These were centrally isointense on T2W and hypointense on T1W images, with an unusual peripheral rim of T2 hyperintensity . There wHepatic EHE is a rare clinical entity that was first described by Weiss and Enzinger in 1982. Its etioAmong all radiological modalities, MRI provides the best tissue contrast and helps in the diagnosis of EHE. It depicts the internal architecture of hepatic EHE better than CT scan. The MRI 7The differential diagnosis includes cholangiocarcinoma, fibrolamellar hepatocellular carcinoma, and metastatic carcinoma.7 These c6Two histological types, nodular and diffuse, have been described. Infiltration into sinusoids and into intrahepatic veins, resulting in luminal narrowing and obliteration with polypoidal projections, are characteristic features of hepatic EHE.36 Demons36Multifocal hepatic EHE has a better prognosis and better long-term survival than any other hepatic malignancy. The typical MRI features should raise a suspicion in the correct clinical setting and an appropriately guided biopsy will usually give the answer, thus avoiding more complicated diagnostic or therapeutic interventions.


Post-traumatic rhinnorhea due to large frontobasal fractures remains a difficult entity to treat. The authors report the case of a 9-year-old boy who had persistent CSF rhinnorhea due to extensive frontobasal fractures and who was managed with transcranial extradural surgery with titanium mesh placement and only pedicled pericranial flap. The appropriate timing as well as type of surgical intervention in patients with persistent post-traumatic CSF leaks remains unclear. Many surgical approaches – both intracranial and extracranial – may be successful, depending on patient factors and the anatomy of the fistula. The goal of neurosurgical management of CSF rhinorrhea is to prevent external deformity of the skull, seal the CSF leak, and to avoid chronic sinusitis. The best method of closing frontobasal defects is still a matter of debate, especially if there is a need for reconstructive surgery.–5 We repA 9-year-old boy presented to us with complaints of watery nasal discharge from right nostril after seven months following fall from 20 feet. He had suffered four episodes of high grade fever in the intervening period. On presentation, he was fully conscious without any neurological deficits. An MRI cisternography was done which showed extensive bilateral cribriform plate defects with a doubtful defect from the sellar floor into the sphenoid sinus . The patA lumbar drain was placed prophylactically and CSF drainage was continued till third post operative day.The remainder of the hospital course was uneventful and the patient could be discharged on the sixth post operative day and remains asymptomatic at last follow up.The use of titanium mesh in skull base surgery has previously been reported in craniofacial and cranial vault procedures.–4 Its usBadie evaluateOur case shows that the use of titanium mesh is a safe and feasible method for reconstructing large anterior skull base.


Adhesive dressing application to the phlebotomy site is a norm that is usually followed by all blood banks as postdonation care. The purpose is to provide an environment conducive for healing i.e. prevent infection. However whether we need a medicated antiseptic dressing is of doubtful value. The risk of infection at phlebotomy site is estimated to be 1 in 200,000,[An erythematous rash developed in one of our first time blood donor, exactly over the area covered under the region of “medicated area” of the adhesive dressing applied post phlebotomy. The donor observed this rash and itching 24 hours after removing the dressing. There was no history of any drug allergy during donor screening .The adhesive dressing was medicated with nitrofurazone (0.2%w/w), which is reported to be a known contact medicament-allergen.45 The do4Non-medicated dressings can be considered to avoid such allergies because phlebotomy site is always prepared with an antiseptic and sterile needles are used. This case highlights the need of active donor hemovigilance program so that delayed donor adverse events can be managed appropriately to the satisfaction of the donor.


Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients.In sex-mismatched transfers, functionality of female donor leukocytes was not affected in male recipients. In contrast, male donor leukocytes were greatly affected in the female recipients. In MHC-mismatches, recipients of different MHC backgrounds, or mice of different strains, showed a greater negative impact on donor leukocytes than sex-mismatches. The negative effects of sex-mismatch and MHC-mismatch on donor leukocytes were additive. Old donor leukocytes performed worse than young donor leukocytes in all settings including in young recipients. Young recipients were not able to revive the declining function of old donor leukocytes. However, the function of young donor leukocytes declined gradually in old recipients, suggesting that an aged environment may contain factors that are deleterious to cellular functions. The irradiation of donor leukocytes prior to transfers had a profound suppressive effect on donor leukocyte functions, possibly as a result of impaired transcription. The cryopreserving of donor leukocytes in liquid nitrogen had no apparent effect on donor leukocyte functions, except for a small loss of cell number after revival from freezing.Despite the functional suppression of donor leukocytes in sex- and MHC-mismatched recipients, as well as old recipients, there was a therapeutic time period during the initial few weeks during which donor leukocytes were functional before their eventual rejection or functional decline. The eventual rejection of donor leukocytes will likely prevent donor leukocyte engraftment which would help minimize the risk of transfusion-associated graft-versus-host disease. Therefore, using leukocytes from healthy donors with high anti-cancer activity may be a feasible therapeutic concept for treating malignant diseases. Spontaneous regression/complete resistant (SR/CR) mice are a line of cancer-resistant mice that are capable of resisting large doses of transplanted lethal cancer cells -3. The bThese prior studies suggest that it may be possible to develop a similar leukocyte transfer platform for humans if we can identify humans with anti-cancer activity similar to the SR/CR mice to serve as leukocyte donors. However, the prior adoptive transfers of donor leukocytes in these mice were considerably different from what would be necessary in human cancer patients. The leukocyte transfers in mice were done in MHC-matched and relatively young recipients . In the First, it is not known how much of an effect a sex mismatch would have on the donor leukocytes. It is possible for a donor leukocyte treatment to involve donors that are a different sex than the recipient. While female donor leukocytes in a male recipient are not expected to cause substantial problems, it is reasonable to expect some issues with male donor leukocytes in female recipients because of the existence of unique proteins associated with y-chromosome gene expression . HoweverSecond, in the human setting, a long-term engraftment of donor leukocytes, especially donor T lymphocytes, should be avoided in order to minimize the possibility of transfusion-associated graft-versus-host diseases (TA-GVHD) . The lonThird, the transfers in the prior studies were done primarily with young mice as the recipients and donors . The sceIn this paper, we describe the results of studies that determine the impact of sex, MHC, and age on the therapeutic effects of SR/CR donor leukocytes in a lethal cancer model.6 S180 cells, 24 hours after the adoptive transfer, to evaluate the anticancer activity of the transferred leukocytes. The surviving mice were then challenged with 1 × 106 S180 two more times with 5-6 week intervals between the injections. As expected, the sex- and MHC-matched controls showed 100% overall survival after all three S180 challenges . Both groups of WT mice were challenged with 1 × 106 S180 cells 24 hours after the designated adoptive transfers. Even with the slightly reduced number of leukocytes delivered after cryopreservation, both groups showed identical survival (100%) after a challenge with S180  . S180 cells were propagated in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in 5% carbon dioxide, or maintained by serial passage in wild-type (WT) C57BL/6 mice as ascites tumors. C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from The Jackson Laboratory and Charles River, respectively. SR/CR mice in C57BL/6 (H-2b) and BALB/c (H-2d) backgrounds were bred at the animal resource program (ARP) facility of Wake Forest University School of Medicine. All mice were housed in plastic cages covered with individual air filter tops, containing corncob bedding, allowed free access to water and chow diet, and exposed to a 12-h fluorescent light/dark cycle. All procedures performed on the mice were in compliance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Wake Forest University and the National Institutes of Health Guide for the Care and Use of Animals.6) that are necessary to transfer the SR/CR phenotype (unpublished data). The number of transferred cells was matched within each experimental group based on a cell count or approximate spleen size and ranged from 51 × 106 to 102 × 106 or 1 to 1.5 spleens per recipient. The exception to this range and matching criteria were the cryopreservation adoptive transfer experimental group which had an approximately 2 × 106 difference between the control and treated group (7-9 × 106 for each recipient). The pooled donor leukocytes were then treated, if applicable, and injected intraperitoneally (IP) into recipient mice. One day after the leukocyte transfers, recipient mice were challenged with 1 × 106 S180 to verify resistance. As previously shown [For adoptive transfers, donor leukocytes were harvested from the spleens and bone marrow of sacrificed SR/CR mice, pooled in phosphate buffered saline and counted. The various experimental setups used one donor mouse per recipient with the exception of the mismatch experimental setup, which used one donor per recipient for the male donors and approximately 1.5 donors per recipient for the female donors due to differences in the spleen sizes. The number of leukocytes transferred to each donor was well above the minimum number of transferred leukocytes  with a cesium irradiator.For cryopreserving, donor leukocytes were resuspended in a cryopreserving media consisting of 90% FBS +10% Dimethyl Sulfoxide (DMSO) and stored first in a -80°C freezer for approximately one day before being transferred to liquid nitrogen for approximately one month. Before transfers, the stored leukocytes were thawed in a 37°C water bath, washed two times with phosphate buffered saline to remove any remaining fetal bovine serum, and counted via trypan blue exclusion.At desired time points of the studies, mice were sacrificed. The lungs, livers, intestines, kidneys, spleens and other tissues of potential interest were dissected and stored in 10% neutral buffered formalin. The collected tissues were embedded in paraffin. Sections of the tissues were stained with hematoxylin and eosin and examined for histological abnormalities.The authors declare that they have no competing interests.JS helped conceive of the study, participated in its design and coordination, helped perform the adoptive transfers, irradiation, and histological analysis and drafted the manuscript. MB helped design the studies, perform the adoptive transfers, and with manuscript and figure preparation. GR helped design the studies, perform the adoptive transfers, and helped with manuscript preparation. JK performed the histological evaluation of the stored tissues and helped with manuscript preparation. AS helped design the studies, perform the adoptive transfers, and with manuscript preparation. JA helped perform the adoptive transfers and animal care and helped with manuscript preparation. MW helped design the studies, helped with the histological evaluation, and with manuscript preparation. ZC helped conceive and design the studies and with drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/328/prepub


We present an interesting intracardiac electrogram of a dissimilar atrial rhythm in a patient of bi-atrial orthotopic cardiac transplant. Seventy eight year old man with prior history of bi-atrial orthotopic cardiac transplant for end stage ischemic cardiomyopathy presented with symptomatic typical counter-clockwise atrial flutter. He underwent successful ablation of cavo-tricuspid isthmus in the donor atria followed by dual chamber pacemaker implantation for a prolonged HV interval > 100 msecs. While attempting to implant right atrial pacing lead a dissimilar rhythm was recorded from the donor and the recipient atria. While the donor right atrium was in the sinus rhythm at the rate of 64 beats per minute (940 milliseconds), the recipient atrium was in a regular tachycardia at a rate of 300 beats (200 milliseconds) per minute suggestive of atrial flutter . There wThe donor atrium in patients with bi-atrial cardiac transplant tends to lie in a more septal and posterior position requiring steerable catheters to assure proper lead positioning. While the two atria tend to be electrically isolated, breakthrough conduction of atrial arrythmias of recipient atria across the suture line into the donor atrium has been reported . BidirecIt has been noted that in many bi-atrial transplanted hearts the recipient atrium is often in some form of supraventricular tachycardia. Sinus rhythm in the recipient atrium and atrial flutter in the donor atrium has also been reported ,4.The case highlights the importance of knowing the type of cardiac transplant procedure the patient has undergone to facilitate the proper catheter and pacemaker lead placements in these patients.


Hepatologists assay liver enzymes and cardiologists structural heart proteins in serum to diagnose and monitor their patients. This way of thinking has not quite made it into the memory clinics yet, in spite of the availability of validated cerebrospinal fluid biomarkers for key pathological events in the brain in neurodegeneration. Here, we argue that a spinal tap should be considered in all patients who seek medical advice for memory problems and list the highly relevant clinical questions CSF analyses can address. Memory problems may be caused by a wide range of neuropsychiatric diseases, including Alzheimer's disease (AD), vascular dementia (VaD), dementia with Lewy bodies, frontotemporal dementia (FTD), to mention a few . CognitiPatients with memory complaints most often undergo extensive clinical and neuropsychological assessments, and often also one or more brain imaging investigations. We argue that CSF analysis should be considered in the diagnostic work-up of all patients with memory problems to answer a number of highly relevant questions discussed below. Fear of spinal tap-related side-effects should not preclude CSF analyses, since complications are very rare in the elderly, provided that regular precautions well known to any trained physician are taken –4.β (Aβ1-42). Low CSF levels indicate retention of Aβ1-42 in the brain parenchyma [β1-42 may be seen Creutzfeldt-Jakob disease (CJD), also in the absence of significant amounts of brain amyloid pathology [The robust association of brain amyloid pathology with AD makes this question highly relevant. The easiest and most cost-effective way of giving it a reliable answer is to analyse CSF for the 42 amino acid form of amyloid renchyma –8. This renchyma –11. Low athology .Tau expression is high in nonmyelinated cortical axons where it serves as a microtubule-stabilizing protein . HyperphElevated CSF levels of hyperphosphorylated tau (P-tau) protein are the most specific finding suggesting an ongoing AD process in the brain . P-tau lPathogenic processes that damage axons in the cortex result in increased CSF levels of total tau . T-tau is a dynamic marker of the intensity of the axonal degeneration/damage: the more pronounced increase, the more intense degenerative process, the faster disease progression . Very hiβ1-42), tangle pathology (P-tau) and cortical axonal degeneration (T-tau) identify AD with dementia and prodromal AD in patients with MCI with 75–95% sensitivity and specificity [Together, CSF biomarkers of amyloid pathology . This type of axonal degeneration is frequently seen in VaD –39, FTD The best-established biomarker for the integrity of the blood-brain barrier is the ratio of the albumin concentration in CSF to serum . Strictly speaking, the CSF/serum albumin ratio is a direct measure of the blood-CSF barrier . HoweverBasic CSF examinations of inflammatory activity, including white blood cell count and general signs of IgG or IgM production within the CNS are generally negative in AD and other primary neurodegenerative diseases . Distincβ1-42), neurofibrillary pathology (P-tau), cortical axonal degeneration/damage (T-tau), sub-cortical axonal degeneration/damage (NFL), blood-brain barrier function  and neuroinflammation . The biochemical data should be interpreted with great care together with the whole clinical picture, as well as findings in neuropsychological testing and neuroimaging investigations. Typical changes in relation to different diagnostic entities are summarised in Robust answers to the clinically relevant questions listed above are warranted in the professional evaluation of any patient with memory complaints. Valid CSF biomarkers are available for amyloid pathology (A


Twenty patients with progressive metastatic solid tumours were entered into a study to evaluate the biological effects and toxicity of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF was given as half-hour intravenous infusions during two 10-day phases of daily treatments (separated by 10 days without GM-CSF) and over a final phase of 20 days of alternate day infusions. Doses were escalated in steps from 0.3 to 60 micrograms kg-1 day-1 between successive patient groups. Significant increases (P less than 0.005) of total leucocyte, neutrophil and eosinophil polymorph counts were seen over the periods of daily infusions  at dose levels of 10 micrograms kg-1 and above. Counts produced at 30 micrograms kg-1 were significantly higher than at 10 micrograms kg-1 (P less than 0.025). Toxic side effects of GM-CSF included mild transient pyrexias, bone pain and pruritus. The maximum tolerated dose was 60 micrograms kg-1, which produced severe toxicity in 80% of patients. The toxicity at this dose included pericarditis and dyspnoea ascribed to a 'capillary-leak' syndrome. One patient receiving 60 micrograms kg-1 died as a result of a pulmonary embolus. Seven patients with previously rapidly progressive metastatic tumours experienced stabilisation of disease while receiving GM-CSF and one patient with a previously heavily pretreated metastatic soft tissue sarcoma underwent a greater than 50% reduction of tumour volume. Patients undergoing chemotherapy may benefit both from a reduction of the myelosuppressive effects of cytotoxic agents and from an antitumour effect if GM-CSF is incorporated into future regimens.


Modulation of the immune response against tumour cells is emerging as a valuable approach for cancer treatment. Some experimental studies have shown that secretion of colony stimulating factors by cancer cells reduces their tumorigenicity and increases their immunogenicity probably by promoting the cytolitic and antigen presenting activities of leukocytes. We have observed that human colon cancer cells (HT-29) are able to secrete granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor when stimulated with cytokines (IL-1β and TNF-α). In this study we assessed, for the first time, the effects of several anticancer drugs on colony stimulating factor release or apoptosis in HT-29 cells. Cytokine-induced release of granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor was significantly increased by cisplatin and 6-mercaptopurine. Taxol only increased macrophage-colony stimulating factor release while reduced that of granulocyte-colony stimulating factor. No changes in colony stimulating factor secretion were observed after treatment with methotrexate. Only cisplatin and taxol induced apoptosis in these cells. Secretion of colony stimulating factors by colon cancer cells may contribute to the immune host response against them. Anticancer drugs such as cisplatin and 6-mercaptopurine increase colony stimulating factor secretion by cytokine stimulated cancer cells probably through mechanisms different to those leading to cell apoptosis, an effect that may contribute to their anti-neoplasic action.British Journal of Cancer (2002) 86, 1316–1321. DOI: 10.1038/sj/bjc/6600240www.bjcancer.comCancer Research UK© 2002  Treatment of cancer widely relies on the use of cytotoxic drugs. Anticancer drugs interfere with different steps involved in the processes of cell division and so damage cancerous cells. In many instances these drug effects are now known to be via influences on the apoptotic pathway. In addition, there is now an increasing interest in manipulating the body's immune response against cancer cells. Indeed, genetic therapies aimed at increasing the secretion of certain cytokines by cancer cells seem to have potential as a mode of increasing the host response against tumour progression . In partIn addition to their recently noted effects on cancer cells, CSFs such as G-CSF, GM-CSF or macrophage-CSF (M-CSF) have multiple biological activities. These biological activities include the ability to stimulate the proliferation and differentiation of multipotential bone marrow progenitor cells into mature granulocytes and macrophages  and grown in monolayers to confluency. Cells were serum starved for 24 h prior to use. In preliminary studies, cells were stimulated with different combinations of cytokines  plus tumour necrosis factor α  was selected as the optimal treatment for a consistent release of CSFs. The effects of these cytokines on CSF secretion were evaluated in the presence or absence of different concentrations (10−11–10−4 M) of 6-mercaptopurine (6-MP), cisplatin, taxol, colchicine or methotrexate . In some experiments, cells were treated with cycloheximide (10−6 M) at the time of, or 6 h before, cytokine addition. Medium was removed 24 h after cytokine treatment for the measurement of CSFs. Each treatment was tested in triplicate, on a minimum of three separate occasions.HT-29 cells (Human Caucasian Colon adenocarcinoma Grade II) were obtained from the European Collection of Animal Cell Culture. Cells were seeded at a density of 2×103 in PBS, 100 μl well−1), 100 μl of standard or undiluted cell culture supernatant was added and incubation continued for a further 2 h. The wells were subsequently incubated with the respective detection antibodies  and streptavidin peroxidase . Finally, 100 μl of substrate solution (1:1 mixture of tetramethylbenzidine and hydrogen peroxide) was added and the reaction stopped 30 min later by addition of 50 μl of 1 N H2SO4. Absorbance was then read at 450 nm (λ correction 550 nm) and the concentrations of CSFs calculated from the standard curves.The concentrations of GM-CSF, G-CSF and M-CSF were measured in cell culture supernatant using specific ELISAs constructed from commercially available components. In brief, 96-well plates were coated with the correspondent capture antibodies . Following 1 h blocking  by ELISA  according to the manufacturer's recommendations. Briefly, the medium was removed and the cells lysed in 200 μl of the lysis buffer provided. The lysate was centrifuged and 20 μl of supernatant added to a streptavidin-coated microtiter plate with a mixture of mouse biotin-labelled anti-histone and mouse peroxidase-conjugated anti-DNA. After 2 h-incubation period, the plate was washed and the retained peroxidase determined photometrically with ABTS  as a substrate. Optical density was measured at 405 nm (λ correction 492 nm).−1 of MTT. After incubation for a further 30 min at 37°C the medium containing the MTT was removed and the cells dissolved in 100 μl dimethyl sulphoxide (DMSO). The extent of MTT conversion to formazan was quantified by measurement of optical density at 550 nm with a wavelength correction of 650 nm.Cellular respiration was assessed by mitochondrial-dependent reduction of 3--2,5-diphenyltetrazolium bromide  to formazan. At the end of the experiment, medium was removed from the cells and replaced with 100 μl of warm (37°C) medium containing 0.2 mg ml−1). This response was significantly enhanced by co-treatment with TNF-α at a concentration devoid of any secretory effect per se (10 ng ml−1). The combined action of IL-1β plus TNF-α was further enhanced by interferon-γ (1000 U ml−1) or interleukin-6 (2 μg ml−1)    although−6–10−4 M both) induced significant increases in GM-CSF, G-CSF and M-CSF secretion by cytokine treated HT-29 cells  or colchicine (10−11–10−4 M), which induces microtubule depolymerisation. Pre-treatment or co-treatment with cycloheximide inhibited cytokine-induced release of GM-CSF and M-CSF but not that of G-CSF  increased GM-CSF and M-CSF secretion whilst it decreased that of G-CSF .The functional effects of granulocytes, lymphocytes, and macrophages are important in patients with malignancies because of the abilities of these cells to exhibit antitumour activities. Because of these abilities, different immunotherapy strategies have been assayed for cancer treatment. In experimental studies tumour regression follows increases in the local release of different cytokines that would induce the recruitment and activation of immune cells . Indeed,In the present study we confirm our previous observations that human colon cancer epithelial cells have the ability to produce GM-CSF, G-CSF and M-CSF in response to cytokines commonly found in the inflammatory responses (IL-1β and TNF-α), including those that accompany tumour formation. Moreover, we have shown, for the first time, that the secretion of CSFs by colon cancer cells stimulated with these cytokines is modified by different anticancer drugs.In our study the purine analogue 6-MP and the DNA-damaging agent cisplatin acted similarly. They both increased the secretion of all three CSF by cytokine-stimulated HT-29 cells. Colchicine, which binds to a site on ß-tubulin and causes microtubule depolymerisation, also increased the release of GM-CSF and M-CSF, but decreased the release of G-CSF. Taxol, which inhibits microtubule depolymerisation, similarly increased M-CSF but decreased G-CSF production and did not modify GM-CSF secretion. By contrast methotrexate, an inhibitor of dihydrofolate reductase, failed to affect the release of any CSF.The aim of antineoplasic treatment with these drugs is to impair mitosis in cancer cells, block progression through the cell cycle, and so promote apoptosis. However, cancer cells often develop resistance to chemotherapy, which may be drug-specific or mediated by more general mechanisms such as the mutation of p53 suppressor oncogene observed in HT-29 cells (de novo protein synthesis, while G-CSF secretion does not. Taken together these findings suggest that G-CSF secretion in response to cytokines occurs via a tubulin driven release of pre-formed G-CSF.Taxol and colchicine both modify microtubule formation and both drugs reduced G-CSF secretion. However, the effects of taxol and colchicine on GM-CSF and M-CSF release were different. Colchicine induced a significant increase in the release of both cytokines. By contrast, taxol only increased M-CSF secretion and only at concentrations that were much higher than those necessary to modify microtubule function. Experiments performed in the presence of cycloheximide suggested that the cytokine-induced release of GM-CSF and M-CSF by HT-29 cells depends on Previous studies have shown that cisplatin and taxol modify the function of immune cells. For example, treatment with these drugs induces an increase in the secretion of pro-inflammatory cytokines by leukocytes, especially monocytes and macrophages, which is usually accompanied by an enhanced activity and cytotoxicity against cancer cells . The prein vivo and whether anticancer drugs act preferentially in cancer cells.Secretion of CSFs by cancer cells has important consequences for tumour progression. For instance, in murine models of colon adenocarcinoma, melanoma or acute myeloid leukaemia transfection of cancer cells with genes to overexpress G-CSF or GM-CSF results in reduced tumorigenicity . Rejecti


In previous experiments, we have demonstrated that limb buds engrafted duringembryonic life at E4, between MHC-mismatched chick embryos, are not only toleratedafter birth, but induce in the recipient a state of split tolerance toward cells expressingthe donor MHC haplotype: donor's skin grafts are permanently tolerated while aproliferative response of host's T cells is generated in MLR by donor–type blood cells. Ifthe same experiment is performed, using quail embryo as a donor and chick as arecipient, acute rejection of the quail limb starts during the first two weeks after birth,thus suggesting that the peripheral type of tolerance induced in these experiments canbe obtained only in allogeneic but not in xenogeneic combinations.Phasianidaefamily. In contrast, xenogeneic combinations involving more distant species  resulted in strong and early rejectionfrom both recipients. As a whole, quails exhibit a greater ability than the chick tobecome tolerant to antigens presented peripherally from early developmental stages. Inadult quails, however, skin grafts performed in either direction  are rejected according to a similar temporal pattern. Moreover, lymphocytes ofboth species are able to respond equally well to quail or chick IL-2. Several hypothesesare envisaged to account for these observations. It seems likely that this type oftolerance is directly related to antigenic load because the load in chick to quail wingchimeras is larger than that in quail to chick chimeras. This view is supported by theprotracted delay in graft rejection observed when two quail wing buds instead of oneare grafted into chickens.We report here the unexpected result that when a chick limb bud is grafted into aquail at E4, it is tolerated and, like allogeneic grafts in chickens, induces adult skin-grafttolerance without modifying the MLR response. Similar results were obtained withgrafts from another closely related species of bird, the guinea fowl from the


Antiphospholipid antibodies (aPL) have been found in the blood of patients with systemic andneurological disease. The rare reports of aPL in cerebral spinal fluid (CSF) have been limited mostly toIgG and IgM anticardiolipin (aCL). Our published finding of IgA aPE in the CSF of a young strokevictim prompted us to establish “normal” CSF aPL values for a panel of aPL, which included aCL,antiphosphatidylserine (aPS), antiphosphatidylethanolamine (aPE) and antiphosphatidylcholine (aPC).CSF samples were tested by ELISA for IgG, IgM and IgA aPL. In addition, the CSF samples weretested for activity in the presence and absence of phospholipid (PL) binding plasma-proteins. A total of24 data points were obtained for each CSF sample.We tested 59 CSF samples obtained from 59 patientswho were undergoing evaluation for systemic or neurologic diseases. All CSF samples had normalprotein, glucose and cell counts. Ten of the 59 CSF samples (17%) had elevated aPL optical density(OD) values an order of magnitude higher than the other 49 CSF samples for one or more aPLspecificity and/or isotype. One CSF sample had both PL-binding protein dependent and independentIgG aPE activity. Another CSF sample showed both IgG aPE and aPC reactivity. The remaining eightCSF samples showed single aPL findings; IgG aPE (5), IgG aPC (1), IgG aCL (1) and IgM aPC (1).Seven of 10 patients with elevated CSF values were females. As expected, most “normal” aPL ODvalues were substantially lower in CSF than those we have reported in blood samples from volunteerblood donors.


Sir,We would like to highlight the importance of checking the portable ventilator before usage for each patient. The Oxylog 2000 ventilator  is being


Arachnoid cyst (AC) fluid has not previously been compared with cerebrospinal fluid (CSF) from the same patient. ACs are commonly referred to as containing "CSF-like fluid". The objective of this study was to characterize AC fluid by clinical chemistry and to compare AC fluid to CSF drawn from the same patient. Such comparative analysis can shed further light on the mechanisms for filling and sustaining of ACs.Cyst fluid from 15 adult patients with unilateral temporal AC  was compared with CSF from the same patients by clinical chemical analysis.p = 0.02), and significantly reduced concentrations of total protein , of ferritin  and of lactate dehydrogenase  in AC fluid relative to CSF.AC fluid and CSF had the same osmolarity. There were no significant differences in the concentrations of sodium, potassium, chloride, calcium, magnesium or glucose. We found significant elevated concentration of phosphate in AC fluid (0.39 versus 0.35 mmol/L in CSF; AC fluid is not identical to CSF. The differential composition of AC fluid relative to CSF supports secretion or active transport as the mechanism underlying cyst filling. Oncotic pressure gradients or slit-valves as mechanisms for generating fluid in temporal ACs are not supported by these results. Threeyst wall ,8, 2) flyst wall , and 3) yst wall . It is cet al. [In their study of clinical chemical analysis of cyst fluid in pediatric patients, Sandberg et al.  describeet al. . This fiA total of 15 patients  with unilateral, temporal AC were included. Cyst type and sidedness are summarized in table ®) was used as the neuromuscular blocking agent. A burr hole was made with a high-speed drill immediately posterior to the sphenoid wing in order to gain access to the anterior and most basal aspects of the middle cranial fossa. The dura and the underlying cyst membrane were punctured through the burr-hole with a 23 G, 25 mm long syringe connected to an Optidynamic® spinal fluid manometer . After pressure equilibration and registration, a cyst fluid sample (3 - 5 ml) was collected using the manometer tube as a siphon. The sample was immediately transferred to a sterile centrifuge tube for centrifuging and further analytic processing as described below (sample handling). After this procedure, a standard craniotomy with a microsurgical resection and fenestration of the cyst membranes was performed. Before opening the medial cyst wall and thus communicating the cyst interior to the basal arachnoid space/CSF, all cyst fluid was aspirated to avoid cyst fluid contamination of the CSF.Details for the surgical procedure have previously been given elsewhere -14 but aAfter opening the medial cyst membrane that covered the basal structures , thus creating communication to the basal cisterns and the posterior fossa, a CSF-sample was collected with a pre-cut baby-feeding catheter #6, connected to a 10 ml syringe. The catheter was placed below the tentorium via the tentorial slit and fluid was aspirated gently from the posterior fossa. The collected CSF was transferred to centrifuge tubes and processed in an identical manner to the cyst fluid.x g to remove cells and cell debris. The supernatant was transferred to new polypropylene tubes and immediately stored on dry ice prior to long term storage at -80°C. Such sample handling has previously been demonstrated to reduce degradation of components and cell lysis, which may change the composition of the sample [The samples were transferred to polypropylene tubes  and centrifuged for five min at 450 e sample .Samples were thawed for analysis at room temperature and transferred to pre-marked analysis tubes for laboratory analysis.The samples were analyzed at Laboratory for Clinical Biochemistry, Haukeland University Hospital, 5021, Bergen, Norway. The laboratory is accredited by Norwegian Accreditation (accreditation number "TEST 231") as a testing laboratory and complies with the requirements of NS-EN ISO 15189. Clinical chemistry analysis was performed on a Modular Analytics System by Roche Diagnostics . The analytical coefficient of variation (CV) is noted in parenthesis for each analyte; Sodium (CV 1%), potassium (CV 2%) and chloride (CV 2%), were measured by ion-selective electrode on an ISE 1800 module. Magnesium (CV 2.5%), phosphate (CV 3%), calcium (CV 2%), bilirubin (CV 6%) lactate dehydrogenase (CV 2.5%), protein (CV 1.8%) glucose (CV 2.5%), triglycerides (CV 3%) and iron (CV 2%) were measured by photometic assays on a P 800-module. Ferritin (CV 5%) was measured by an ECLIA (electrochemiluminescence) immunoassay on an E 170-module. Osmolarity (CV 1.5%) was measured by freeze point depression on a Fiske Micro-Osmometer . Immunoglobulins (CV 2-5%) were measured by nephelometry with system specific N antisera to Human Immunoglobulins  on a BN Pro Spec System P-values were calculated utilizing Pearson's product moment correlation test. Statistical analysis was performed using the statistics software package R version 2.10.1 .For each of the thirteen patients without previous operations or known injuries to the AC, the differences in concentration between cyst fluid and corresponding CSF were calculated. A paired T-test was then utilized to check if the mean ratio was equal to one. Due to slight hemolysis of two CSF samples (patients 6 and 10), data from these patients were omitted from the statistical analyses for lactate dehydrogenase, ferritin and protein. Two patients (patient 3 and 4) were described clinically as different from the others, and were compared separately with the thirteen native patients using a two-sample t-test assuming equal variances. Correlation between lactate dehydrogenase, ferritin and protein was determined by correlation analysis as a control against a possible contamination of the samples with blood. p = 0.02), while the concentration of total protein , lactate dehydrogenase  and ferritin  was significantly lower in AC relative to CSF. Bilirubin, iron, triglycerides and immunoglobulins were below quantification limit for analysis setup. Patient 12 was identified as an extreme outlier for protein measurement and was excluded from this analysis.Chemical analysis of AC fluid and CSF, obtained during elective surgery for arachnoid cysts from 15 patients, was performed in a routine hospital laboratory. The results from the measurements are presented in table p = 0.006), ferritin and protein was 0.36 (p = 0.312) and lactate dehydrogenase and protein was 0.34 (p = 0.34). The protein concentrations in the cyst fluid from the two clinically different patients  and 3.54 g/L (previous operation)) were significantly elevated relative to the others (p < 10-16).Correlations were calculated between possible blood contamination parameters ferritin, lactate dehydrogenase and protein on the CSF samples without observed blood contamination (n = 11). The correlation between ferritin and LD was 0.77 , ferritin  and lactate dehydrogenase  would be expected to pass freely through a slit in the cyst membrane. However, the ratio between cyst fluid and CSF for these three protein complexes was reduced (dependent on size), from 0.73 for protein  to 0.31 for ferritin. Our findings are therefore contradictive of a slit valve mechanism underlying the filling of temporal AC.et al [The skewed distribution of phosphate could imply a selective or active transport of fluid and solutes over the AC membranes. This is consistent with previous findings of morphological and enzyme ultracytochemical structures in the wall of arachnoid cysts assumed to be capable of fluid secretion, as reported by Go et al . Furtheret al , and a set al . The phoet al , due to et al .et al [As we have only studied temporal cysts, we cannot generalize the assumption of an active transport mechanism to all ACs. For other locations, other fillings mechanisms may well exist, such as a slit-valve in supracellar cysts ,19. In oet al  found thA limitation for this study is the small number of samples. Applied on such a dataset, the T-test is vulnerable to outliers such as the excluded patient 12 in protein concentration (outlier plots not shown). Nonetheless, our results can support reflections around the filling mechanisms for arachnoid cysts. As far as the authors know, this is the first publication of AC fluid analysis matched with CSF from the same patient.The chemical composition of AC fluid found in this study does not support an oncotic pressure or valve mechanism as responsible for filling an AC. Due to the pattern of differences, we postulate that the filling mechanism for temporal AC is by either a selective or active transport mechanism or a secretion from the cyst-lining cells.The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.MB applied for ethical approval, collected the samples, processed the samples for analysis, processed the data and drafted and edited the manuscript. KGW is the senior neurosurgeon and operated on the patients, devised sampling technique and drafted and edited parts of the manuscript. RJU applied for ethical approval, organized clinical chemical analysis and participated in editing of the manuscript. ACK worked on the biobanking design of the study, discussion around and editing of the manuscript. OAH did the statistical analysis and statistics interpretation. MAM contributed on writing the discussion part and the intellectual interpretation of the data. FSB worked on biobanking design and the editing of the manuscript. CAH conceived of this study, operated on the patients and helped draft and edit the manuscript. All authors read and approved the final manuscript.


Hmga1-knockout mice) developed a type 2-like diabetic phenotype, in which cell-surface insulin receptors were dramatically reduced (below 10% of those in the controls) in the major targets of insulin action, and glucose intolerance was associated with increased peripheral insulin sensitivity. This particular phenotype supports the existence of compensatory mechanisms of insulin resistance that promote glucose uptake and disposal in peripheral tissues by either insulin-dependent or insulin-independent mechanisms. We explored the role of these mechanisms in the regulation of glucose homeostasis by studying the Hmga1-knockout mouse model. Also, the hypothesis that increased insulin sensitivity in Hmga1-deficient mice could be related to the deficit of an insulin resistance factor is discussed.We previously showed that mice lacking the high mobility group A1 gene (RBP4) gene and protein expression in living cells of both human and mouse origin. Then, by employing the Hmga1-knockout mouse model, we provide evidence for the identification of a novel biochemical pathway involving HMGA1 and the RBP4, whose activation by the cAMP-signaling pathway may play an essential role for maintaining glucose metabolism homeostasis in vivo, in certain adverse metabolic conditions in which insulin action is precluded. In comparative studies of normal and mutant mice, glucagon administration caused a considerable upregulation of HMGA1 and RBP4 expression both at the mRNA and protein level in wild-type animals. Conversely, in Hmga1-knockout mice, basal and glucagon-mediated expression of RBP4 was severely attenuated and correlated inversely with increased Glut4 mRNA and protein abundance in skeletal muscle and fat, in which the activation state of the protein kinase Akt, an important downstream mediator of the metabolic effects of insulin on Glut4 translocation and carbohydrate metabolism, was simultaneously increased.We first show that HMGA1 is needed for basal and cAMP-induced retinol-binding protein 4 (RBP4 gene expression in vivo. Further, they provide evidence for the identification of a novel biochemical pathway involving the cAMP-HMGA1-RBP4 system, whose activation may play a role in glucose homeostasis in both rodents and humans. Elucidating these mechanisms has importance for both fundamental biology and therapeutic implications.These results indicate that HMGA1 is an important modulator of  RBP4 gene show increased insulin sensitivity, and normalizing increased RBP4 serum levels improves insulin resistance and glucose intolerance [Insulin resistance is a metabolic condition found relatively frequently among humans with chronic hyperinsulinemia and in experimental animal models with defective insulin signaling -3. RecenHMGA1 is a small basic protein that binds to adenine-thymine (A-T) rich regions of DNA and functions mainly as a specific cofactor for gene activation . HMGA1 bHMGA1 gene, caused a type 2-like diabetic phenotype, in which, however, impaired glucose tolerance and overt diabetes coexisted with a condition of peripheral insulin hypersensitivity [ob/ob mice [Cdk4 knockout mice with defective pancreatic beta cell development and blunted insulin secretion [Hmga1-deficient mice could be due to a deficit, in these animals, of RBP4 is supported by our data. Herein, by employing the Hmga1-knockout mouse model, we provide compelling evidence for the identification of a novel biochemical pathway involving HMGA1 and RBP4, whose activation by the cAMP pathway may play an important role in maintaining glucose metabolism homeostasis in vivo, in both rodents and humans. The importance of HMGA1 in RBP4 gene transcription was substantiated in Hmga1-deficient mice, in which loss of HMGA1 expression considerably decreased RBP4 mRNA abundance and RBP4 protein production.As part of an investigation into the molecular basis regulating the human insulin receptor gene, we previously showed that HMGA1 is required for proper insulin receptor gene transcription ,12. Moresitivity . Concomi/ob mice , as wellecretion . The hypRBP4 gene promoter at the transcriptional level. To test this possibility, HepG2 human hepatoma cells and mouse Hepa1 hepatoma cells were cotransfected transiently with mouse RBP4-Luc reporter plasmid plus increasing amounts of the HMGA1 expression vector. As shown in Figure RBP4-Luc activity in both cell types and this effect occurred in a dose-dependent manner. Consistent with these results, RBP4 mRNA abundance was increased in cells overexpressing HMGA1 and was reduced in cells pretreated with siRNA targeting HMGA1  assay, showing that binding of HMGA1 to the endogenous RBP4 locus was increased in whole, intact HepG2 and Hepa1 cells naturally expressing HMGA1, and was decreased in cells exposed to siRNA against HMGA1 , a standard concentration for c-AMP induction experiments [RBP4 mRNA levels increased starting at 3 h, peaking at 24 h and then declining, suggesting a transient transcriptional stimulation. To establish whether HMGA1 was required for basal and cAMP-dependent RBP4 transcription, we transfected the HMGA1 expression vector in Hepa1 cells treated or not with Br-cAMP and RBP4 protein levels were analyzed 48 h later by Western blot. As shown in Figure RBP4 gene expression was confirmed in transient transcription assays in Hepa1  cells, in which overexpression of HMGA1 caused an increase in both basal and cAMP-induced Luc activity from the mouse RBP-Luc reporter plasmid . Reduced RBP4 mRNA levels in liver and adipose tissue paralleled the decrease in RBP4 serum levels as detected by Western blot analysis of serum samples from age- and body weight-matched mice with diverse genotypes  and Hmga1-null (Hmga1-/-) animals . After glucagon injection, hepatic levels of cAMP increased to 1.50 μmol/g tissue in control mice, compared with 1.48 and 1.52 in Hmga1-/- and Hmga1+/- mice, respectively. Results similar to those shown in the inset of Figure As a measure of the glucagon efficacy in glucagon-injected mice, a liver biopsy was taken before and after glucagon injection, and cAMP levels in liver were determined for both control and e Figure , inset. Hmga1-knockout mice [Hmga1-deficient mice could be mediated by the HMGA1-RBP4 system, we carried out quantitative measurements of Glut4 mRNA transcript abundance. Examination by qRT-PCR showed a significant increase of Glut4 transcripts in both skeletal muscle and adipose tissues from Hmga1-deficient mice compared with controls , indicating that in isolated adipocytes treated with Br-cAMP, Glut4 mRNA was decreased in all three genotypes. A positive correlation of RBP4 levels with markers of lipid metabolism adversely affecting insulin sensitivity has been reported recently in both clinical and experimental studies [Hmga1-knockout mice had lower levels of serum free fatty acids , which might contribute to their improved insulin sensitivity.Systemic insulin resistance has been associated with elevation of serum RBP4, whereas genetic and pharmacological interventions aimed at decreasing serum RBP4 levels enhance insulin action and improve insulin sensitivity [out mice . To veris Figure . Accordis Figure , clearlytabolism , was inc by cAMP , endocri studies ,20. HmgaHmga1-deficient mice. As shown in Figure Hmga1 mutants. The reduction in Akt phosphorylation in these genotypes correlated inversely with RBP4 serum levels in the same animals .To demonstrate that increased insulin sensitivity in mutant mice was directly due to HMGA1 regulation of RBP4, we determined the effect of recombinant RBP4 administration on Akt phosphorylation and Glut4 protein expression in skeletal muscle from s Figure  and paras Figure , indicatHmga1-deficient mice during insulin-tolerance test (ITT) [Hmga1 mutants, we have determined the effect of RBP4 administration on the glucose fall induced by insulin in these genotypes during ITT. As shown in Figure Hmga1 mutants caused a less dramatic fall in blood glucose levels, lessening the hypoglycemic response to intraperitoneal insulin observed in the saline-injected animals. Thus, taken together, our findings consistently support the role of HMGA1 as a key element in the transcriptional regulation of genes involved in glucose metabolism and add new insights into the compensatory mechanisms that may contribute to counteract insulin resistance in vivo. By directly regulating RBP4 gene transcription, HMGA1 enhances peripheral insulin sensitivity, ensuring glucose uptake in skeletal muscle. This, if on one hand might represent an adaptive mechanism to ameliorate insulin resistance in animals with a disadvantageous metabolic risk profile, on the other might indicate that the cAMP/HMGA1-mediated RBP4 expression during fasting (when glucagon peaks) may act physiologically to reduce insulin sensitivity in peripheral tissues, thereby contributing to the maintenance of euglycemia under this condition. This was supported by the observation that after an overnight fasting period (12–16 h) plasma glucose concentration in wild-type mice was higher than that of Hmga1-deficient mice .Consistent with the condition of insulin hypersensitivity, we previously reported that the glucose-lowering effect of exogenous insulin was enhanced in st (ITT) . To suppHMGA1 gene, severely decreased insulin receptor expression  and phosphorylation in the major targets of insulin action, largely impaired insulin signaling, and reduced insulin secretion, producing a type 2-like diabetic phenotype in which defects in both peripheral insulin sensitivity and pancreatic beta-cell insulin secretion were coexpressed simultaneously [Hmga1-deficient mice during ITT, and the glucose infusion rate necessary to maintain euglycemia was higher in mutant mice during hyperinsulinemic-euglycemic clamp [Hmga1-knockout mouse. The existence of signaling pathways promoting glucose uptake and utilization in peripheral tissues through mechanisms that are independent of insulin has been postulated before, on the basis of experimental observations supporting the existence of molecular circuits/pathways that can compensate for the decrease in insulin-stimulated glucose uptake in vivo, in both animal models and human patients with type 2 diabetes [Hmga1-deficient mice [Hmga1-mutant animals, in which the acute first-phase insulin secretory response was severely blunted [Hmga1-knockout mice [We have previously shown that loss of HMGA1 protein expression, induced in mice by disrupting the aneously . Howeveric clamp , supportdiabetes -23. Howeent mice . Loss ofent mice . As in t blunted , indicatout mice , indicatHmga1-knockout mice RBP4 levels are considerably decreased in serum and in whole liver and adipose tissue extracts, strictly linking HMGA1 and RBP4 expression. We propose that HMGA1 deficiency adversely affects RBP4 expression and this, in animals with a disadvantageous metabolic risk profile like that observed in the Hmga1-knockout mouse model, might reflect an adaptive mechanism to increase glucose uptake and glucose disposal. Consistent with the results obtained in Hmga1-deficient mice, RBP4 was considerably reduced in cells of both human (HepG2) and mouse (Hepa1) origin readily expressing RBP4, following perturbation of endogenous HMGA1 protein expression in cells treated with siRNA against HMGA1. Conversely, an increase in RBP4 mRNA abundance was observed in both cell lines following forced expression of HMGA1, consistently supporting a role for HMGA1 in the transcriptional activation of the RBP4 gene. These findings were substantiated further by ChIP analysis, showing that HMGA1 indeed binds to the RBP4 locus in intact living cells.Downregulation of Glut4 in adipose tissue is a typical feature of insulin-resistant states, such as obesity and type 2 diabetes . It has RBP4 gene expression [RBP4 gene transcription via the cAMP signaling pathway may be physiologically relevant. One important physiological condition in which intracellular cAMP increases is in response to low glucose availability. In this metabolic setting, a concomitant predominance of circulating counter-regulatory hormones, in particular pancreatic glucagon acting via the cAMP pathway, induces glycogenolysis and gluconeogenesis in the liver, which produce and release hepatic glucose in the blood. In this regard, the cAMP-element-binding protein (CREB) has been identified as a critical transcriptional checkpoint which, in response to cAMP, promotes hepatic glucose output through the synergistic activation of distinct transcriptional effector pathways, which include the PPAR gamma coactivator 1 (PGC1) and the NR4A orphan nuclear receptors [Signal transduction pathways which raise intracellular cAMP have been reported to have a potential role in the regulation of pression . Althougeceptors .RBP4 mRNA and protein expression, along with an increase of both intracellular cAMP and HMGA1 levels. Glucagon effects were attenuated in Hmga1-deficient mice, supporting a distinct role for HMGA1 in the regulation of RBP4 gene expression and functionally linking this two genes. As a consequence of the functional link between HMGA1 and RBP4, a significant increase in Glut4 mRNA and protein was observed in both skeletal muscle and adipose tissues from Hmga1-deficient mice compared with controls. An inverse relationship between RBP4 and Glut4 has been described previously, in the adipose-Glut4-/- mouse, in which the decrease in Glut4 expression that occurs in the fatty tissue of this mutant genotype is accompanied by increased expression and secretion of the fat-derived RBP4 [RBP4 expression is genetically impaired due to the lack of HMGA1 and Glut4 is increased in both muscle and fat, suggesting that abnormalities in RBP4 and/or metabolites of the vitamin A metabolism may directly affect whole-body insulin action and peripheral insulin sensitivity. In support of this possibility, identification of regulatory single nucleotide polymorphisms in the RBP4 gene associated with type 2 diabetes has been recently reported [Hmga1-deficient mice confirm that an inverse correlation indeed exists between RBP4 and insulin sensitivity in vivo, in this animal model of diabetes, lending support to previous hypotheses that lowering RBP4 levels would be helpful in ameliorating insulin resistance, at least in mice.In this paper, we report that systemic injection of glucagon to wild-type control mice caused an increase in reported ,28, whilreported . AlthougRBP4 gene. Apart from the intrinsic biological interest in elucidating the mechanisms leading to improvement in insulin sensitivity, a clear understanding of the molecular process involved is of potential importance in the development of new therapeutic strategies for patients with metabolic disorders such as obesity, diabetes, and other insulin resistant states.Overall, our findings provide mechanistic insight into the regulation of glucose uptake and disposal in peripheral tissues, and support further the role of HMGA1 as a molecule that is likely to be an important emerging factor in the transcriptional activity of genes implicated in the maintenance of glucose homeostasis and metabolic control, such as the insulin receptor gene -13, the RBP4 gene expression and protein function and represents an important novel mediator of glucose homeostasis in vivo.We propose that HMGA1 can serve as a modulator of both RBP4-Luc reporter plasmid was obtained by cloning the NheI/XhoI 1427-bp sequence of the mouse RBP4 promoter (-1417 to +10) into pGL3 (Promega). This fragment was amplified from genomic DNA using the following modified primers: 5'-TTGCTAGCATGGCTAAGGTGCTTGTTGAAA-3', 5'-TTCTCGAGCACACCCACTCCATCTCACC-3' and the integrity of this construct was checked by DNA sequencing. RBP4-Luc reporter plasmid, together with either the control vector plasmid or expression plasmid encoding HMGA1 [RBP4-containing vector was cotransfected into Hepa1 cells with the expression plasmid pcDNA1 containing the HMGA1 cDNA in the antisense orientation [HMGA1 [HMGA1 siRNA as described previously [RBP4 sequence were used for PCR amplification of immunoprecipitated DNA (30 cycles), using PCR ready-to-go beads (Amersham Pharmacia Biotech). PCR products were electrophoretically resolved on 1.5% agarose gel and visualized by ethidium bromide staining.The ng HMGA1 , was trang HMGA1 . Renillaentation . Small in [HMGA1  was traneviously . FormaldHmga1-deficient and wild-type mice aged 6–9 months were studied. The generation of these animals and many of the physiological characteristics of the mice have been described in detail [Hmga1-knockout mice (n = 12–16 per genotype) using the NEFA C kit (Wako).Male n detail . All aniHMGA1 (NM_016660.2) , RBP4 (NM_011255.2) , Glut4 (NM_009204) , and RPS9 (NM_029767.2)  were designed according to sequences from the GenBank database. A real-time thermocycler  was used to perform quantitative PCR. In a 20-μl final volume, 0.5 μl of the cDNA solution was mixed with SYBR Green RealMasterMix (Eppendorf), and 0.3 μM each of sense and antisense primers. The mixture was used as a template for the amplification by the following protocol: a denaturing step at 95°C for 2 min, then an amplification and quantification program repeated for 45 cycles of 95°C for 15 s, 55°C for 25 s, and 68°C for 25 s, followed by the melting curve step. SYBR Green fluorescence was measured, and relative quantification was made against the RPS9 cDNA used as an internal standard. All PCR reactions were done in triplicate.For qRT-PCR, total cellular RNA was extracted from tissues using the RNAqueous-4PCR kit and subjected to DNase treatment (Ambion). RNA levels were normalized against 18S ribosomal RNA in each sample, and cDNAs were synthesized from 2 μg of total RNA using the RETROscript first strand synthesis kit (Ambion). Primers for mouse Western blot analysis was performed to analyze HMGA1 and RBP4 protein expression in whole-cell liver and fat extracts from normal and mutant mice, using polyclonal specific antibodies raised against HMGA1  and RBP4For systemic administration of exogenous glucagon, mice were injected in the peritoneal cavity with human glucagon (1 mg/kg body weight) or saline after 3 h of fasting. At this dose, the peak increase of plasma glucagon in all genotypes was ~96% ± 10% above pre-injection levels, reflecting similar previous observations in rodents . At diffRBP4 cDNA cloned into a pET3a expression vector was a kind gift from JW Kelly (The Scripps Research Institute). Based on previously published methodology [RBP4 protein expression vector was transformed into the BL21 strain of Escherichia coli (Stratagene), expanded in suspension culture and induced for 6 h with 1 mM isopropyl-D-thiogalactopyranoside to stimulate protein expression. Bacteria were pelleted and lysed by osmotic shock [Human hodology , RBP4 pric shock . From thic shock . Proteinin vivo, in Hmga1-deficient mice, heterozygous and homozygous Hmga1 mutants, were intraperitoneally injected twice daily  with 200 μg of purified human RBP4 (13 μg/g body weight per mouse) for 7 days. This resulted in a daily average serum level of human RBP4 similar to that of control mice (see Figure To determine whether elevation of RBP4 affected insulin hypersensitivity P < 0.05 was considered significant.The ANOVA test was used to evaluate the differences between the groups of mice. For all analyses, Akt: protein kinase B; Br-cAMP: 8-bromo cAMP; cAMP: cyclic adenosine monophosphate; CREB: cAMP-element-binding protein; Glut4: glucose transporter-4; HEK-293: human embryonic kidney-293; Hepa1: mouse hepatoma; HMGA1: high mobility group A1; ITT: insulin-tolerance test; PGC1: PPAR gamma coactivator 1; qRT-PCR: quantitative Real-Time PCR; RBP4: retinol-binding protein 4; siRNA: small interfering RNA; TCA: trichloroacetic acid; TTR: transthyretin.in vivo data from normal and mutant mice. GB, AL, and VC performed certain aspects of the assays detailed in Figures EC and FP performed qRT-PCR studies as well as transient transfections with reporter and expression vectors, and participated in the design of the study. SI was involved in Western blotting studies and assisted FP in performing transfections with siRNA. ILP participated in Western blotting and performed cloning studies. DP, EG, GDS and AF participated in the analysis and discussion of the


We analyzed data from the baseline assessment of a large intervention project to describe typical handwashing practices in rural Bangladesh, and compare measures of hand cleanliness with household characteristics.We randomly selected 100 villages from 36 districts in rural Bangladesh. Field workers identified 17 eligible households per village using systematic sampling. Field workers conducted 5-hour structured observations in 1000 households, and a cross-sectional assessment in 1692 households that included spot checks, an evaluation of hand cleanliness and a request that residents demonstrate their usual handwashing practices after defecation.Although 47% of caregivers reported and 51% demonstrated washing both hands with soap after defecation, in structured observation, only 33% of caregivers and 14% of all persons observed washed both hands with soap after defecation. Less than 1% used soap and water for handwashing before eating and/or feeding a child. More commonly people washed their hands only with water, 23% after defecation and 5% before eating. Spot checks during the cross sectional survey classified 930 caregivers (55%) and 453 children (28%) as having clean appearing hands. In multivariate analysis economic status and water available at handwashing locations were significantly associated with hand cleanliness among both caregivers and children.A minority of rural Bangladeshi residents washed both hands with soap at key handwashing times, though rinsing hands with only water was more common. To realize the health benefits of handwashing, efforts to improve handwashing in these communities should target adding soap to current hand rinsing practices. The WHO estimates that 3.8 million children aged under five die each year from diarrhoea and acute respiratory tract infections . InterveThe Government of Bangladesh (GoB) and UNICEF with the support of the Department for International Development (DFID) of the British Government instituted the Sanitation, Hygiene Education and Water supply-Bangladesh project (SHEWA-B) in 2007. This project, which targets some 30 million underserved people, is among the largest intensive handwashing, hygiene/sanitation and water quality improvement programs ever attempted in a developing country. It aims to contribute towards achieving the Millennium Development Goals (MDGs) relating to water and sanitation.Understanding usual handwashing practices in rural Bangladesh is an important baseline assessment for the program. One of the major challenges with assessing handwashing behavior is that no measure has proven to be both practical and valid ,8. AlthoThis evaluation assessed the pre-intervention baseline of handwashing practices across rural Bangladesh. The objective of this analysis was to describe typical handwashing practices in rural Bangladesh and assess the association of measures of hand cleanliness with hygiene associated household characteristics.upazilas) in 19 districts. The method for selecting the evaluation population has been described previously [In the initial two years from 2007, the SHEWA-B project targeted interventions for 19.6 million underserved populations from 68 sub-districts , availability of separate soap for handwashing, availability of spare soap at household, use of improved latrines, availability of handwashing locations, availability of water and soap at handwashing locations, and respondents' economic status ,11,14,15To construct multivariate models we included as covariates those variables significantly associated (p < 0.05) with the outcome. We used an exchangeable correlation structure for all generalized estimated equations analyses to account for the clustering measures within different villages. We used STATA for Windows, Version SE/10  for the generalized estimated equations modeling.The data collectors sought informed consent before enrolling a household in the evaluation. In seeking consent, the data collectors described the objective of the evaluation, and noted the benefit of the intervention program and its evaluation for the rural Bangladeshi communities. Data collectors clarified that there was no special benefit to participating but also no health risks. They noted that confidentiality of data would be maintained, that participation for the study was voluntary, and refusing to participate would not affect the family getting any intervention services. Because this was an evaluation of a public health program rather than a primary research project, the program evaluation was not reviewed by a human subjects research panel i.e. we did not seek a waiver from a human subjects research panel.The field team completed structured observations in 1000 households (100%) and cross sectional surveys in 1692 (99%) of the 1700 targeted households. Among the households in the cross sectional survey, 671 (40%) had a child aged 3 to 5 years old. The field team assessed a handwashing demonstration for 946 (93%) of 1021 targeted mothers/caregivers and 203 (30%) of 671 targeted 3-5 year old children. The other children were unwilling or unable to demonstrate handwashing. The field staff evaluated hand cleanliness of mothers/caregivers for 100% of evaluated households (1692) and for 96% of targeted children (1628).The majority of households (94%) were headed by a male. Approximately one third of mothers 31%) and fathers (37%) lacked formal education. Almost half (45%) of the households had an electricity connection and 29% owned a mobile phone  or washed one hand only (46%). Among those who did wash both hands, most washed with only water before eating, before feeding a child, after defecation and after cleaning a child who defecated  and 453 children (28%) as having clean appearing hands. In bivariate analysis, 5 of 8 household characteristics--spare soap available at household, use of an improved latrine, water available at handwashing locations, soap available at handwashing location and economic status, were associated with mothers' hand cleanliness. These same five household characteristics plus having separate soap available at the household for handwashing was also associated with child hand cleanliness in bivariate analysis (Table In multivariate analysis, two of these variables -- household wealth as assessed by principal component analysis and water availability at handwashing locations - remained significantly associated with the hand cleanliness for both mother and child table . The oddResidents of these rural Bangladeshi communities commonly washed their hands with only water, but they infrequently washed both hands with soap. Similar findings have been noted in other settings ,19. SincAlthough handwashing with soap after fecal contacted events is dangerously low, it is much higher than handwashing before feeding a child or eating. Bangladeshi residents conceptualize the need for handwashing quite differently after fecal contact than before feeding a child or eating . BehavioAlthough handwashing with soap after fecal contact was more common among mothers/caregivers than other categories of household members; but similar to findings in other studies ,22, thisHand cleanliness as assessed by field workers is a potential low cost method to evaluate hand hygiene practices. In this evaluation observed hand cleanliness of the palms and finger pads was independently associated with wealth and the presence of water at the most convenient place to wash hands in household. Indicators of handwashing are commonly strongly associated with measures of socioeconomic status ,26,27. IThe observed behavior of child mothers/caregivers within this study may not represent their actual behavior because the presence of an observer can change the handwashing behavior of subjects ,28. We aThe proportion of Bangladeshi residents in these rural communities who washed their hands with soap at key times was consistently low. There is a deeply-rooted belief among Bangladeshi people that water is a potent purifying agent. Accordingly, hands which have been rinsed with water alone and which show no visible contamination are considered to be clean . MoreoveThe authors declare that they have no competing interests.Conception and design of the study: AKH, CT, AB, RJ, SA, SPL; Analysis and interpretation of data: AKH, SPL; Writing paper: AKH, SPL; Contribution to reagents/materials: AB, CT, RJ, SA. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/545/prepub


The acceptor photobleaching fluorescence resonance energy transfer (FRET) method is widely used for monitoring molecular interactions in cells. This method of FRET, while among those with the simplest mathematics, is robust, self-controlled and independent of fluorophore amounts and ratios.AccPbFRET is a user-friendly, efficient ImageJ plugin which allows fully corrected, pixel-wise calculation and detailed, ROI (region of interest)-based analysis of FRET efficiencies in microscopic images. Furthermore, automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing FRET evaluation software.Despite of the widespread applicability of the acceptor photobleaching FRET technique, this is the first paper where all possible sources of major errors of the measurement and analysis are considered, and AccPbFRET is the only program which provides the complete suite of corrections – for registering image pairs, for unwanted photobleaching of the donor, for cross-talk of the acceptor and/or its photoproduct to the donor channel and for partial photobleaching of the acceptor. The program efficiently speeds up the analysis of large image sets even for novice users and is freely available. Fluorescence resonance energy transfer (FRET) is a powerful technique that can be applied to study nanoscale intra- and intermolecular events and interactions of molecules in situ in biological systems . In asseE:Donor photobleaching FRET, exploiting the decrease of excited state lifetime and consequential protection from photodestruction in the presence of FRET was the first quantitative approach applied to microscopy ,15,20 anA measurement that exploits this proportionality is facilely implemented in confocal microscopy, thus providing the option of distinguishing various molecular association states even at the subcellular level. The method is also applicable to the ever-spreading family of green fluorescent protein (GFP) derivatives .Image manipulation and analysis in biological research are often performed with the free ImageJ package . In spitThe plugin was written in Java v1.6, and tested with ImageJ version 1.38×.Ei, j)  and FD2 are the donor fluorescence values of the pixel  before (1) and after (2) photobleaching the acceptor, and FA1 the acceptor fluorescence for the same pixel before photobleaching. All F values are background corrected throughout. α, γ, δ and ε are correction factors that are described below.where α is calculated asIn some cases, photobleaching of the acceptor is not complete. As shown by van Munster et al. , the aveFA2 and FA1 are intensities in the acceptor channel in pixels above threshold of the donor and acceptor labeled sample, before (1) and after (2) photobleaching.where γ for unwanted photobleaching of the donor during the image acquisition procedure [The correction factor rocedure ,26 can borFDd1 and FDd2 are donor fluorescence intensities of donor only (Dd) samples in pixels above threshold before (1) and after (2) photobleaching the acceptor, and the ⟨⟩ signs denote mean value. Since FRET protects the donor from photobleaching [where leaching , this faδ and ε), the possibility to calculate the factor on a pixel-by-pixel basis and then average pixels above threshold for raw data, or, alternatively, to average the raw data for these pixels and then calculate an average correction factor.The program offers  and FAa1 are signals in the donor and acceptor channels in pixels above threshold of an acceptor only labeled sample, before (1) photobleaching the acceptor.where ε for such acceptor-photoproduct is calculated asIn some cases, photobleaching the acceptor can yield a photoproduct with distinct absorption and emission properties, which can contribute to the post-bleach donor signal, resulting in the overestimation of FRET efficiency . The cororFDa2 and FAa1 are intensities in the donor and acceptor channels in pixels above threshold of an acceptor only labeled sample, before (1) and after (2) photobleaching.where γ, δ and ε requires taking images with the same photobleaching protocol on samples labeled with donor only and acceptor only, which usually need to be taken anyway. To correct for shifts in the x-y plane, the images are registered using the Fast Hartley Transform algorithm [The calculation of the constants lgorithm  implemenWith our program AccPbFRET, which can be found in the additional file [see Additional file Other important issues with acceptor photobleaching FRET is correcting for bleaching of the donor, for the cross-talk of the acceptor and/or its provisional photoproduct to the donor channel, and partial photobleaching of the acceptor, which are also solved by AccPbFRET.The steps of creating the FRET image are enumerated in the main program window, so the user only needs to follow the instructions , the program opens images sequentially in the chosen directory, and only threshold setting(s) and creation of the FRET image need to be done manually. The upper left corner (1/6 × 1/6 of the image) is considered automatically as background.The AccPbFRET plugin provides an easy to use graphical interface, which leads the user through the evaluation process, and does not require cumbersome pre-setting of various parameters. It allows correcting for bleaching of the donor, for the cross-talk of the acceptor and its photoproduct to the donor channel and for partial acceptor photobleaching. Furthermore, automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing evaluation software.• Project name: AccPbFRET• Project home page: • Operating system(s): platform independent• Programming language: Java• Other requirements: ImageJ 1.38× (bundled with Java 1.6.0_02) or higher, screen resolution 1280 × 900 or higher• License: free software• Any restrictions to use by non-academics: noneGV conceived and coordinated the project. JR, JS and GV participated in software design and testing. JR implemented, and JR and GV tested the software. JR drafted the manuscript, JS and GV helped to create the final version. All authors read and approved the final manuscript.Source code and example images. This file contains the AccPbFRET.java source code and some example LSM and TIFF image files together with explanations.Click here for file


The lengthof time the recipient remained euglycemic was used to measurethe ability of the transgenes to protect the graft fromautoimmune destruction. Although the results of these cotransfectionsgave little evidence of a synergistic relationship,they were useful to show that gene combinations can be used to more efficiently protect islet grafts from diabetogenicT cells.Islet transplantation therapy would be applicable to awider range of diabetic patients if donor islet acceptanceand protection were possible without systemic immunosuppressionof the recipient. To this aim, gene transferto isolated donor islets ex vivo is one method that hasshown promise. This study examines the combined effect ofselected immunomodulatory and anti-inflammatory genesknown to extend the functional viability of pancreatic isletgrafts in an autoimmune system. These genes, indoleamine2,3-dioxygenase (IDO), manganese superoxide dismutase(MnSOD), and interleukin (IL)-1 receptor antagonist protein(IRAP), were transferred to isolated NOD donor isletsex vivo then transplanted to NOD


Psychological Science, 17, 700–707). The authors demonstrated that intravenous midazolam, which is thought to inactivate the hippocampus, may enhance TI performance. Three critical assumptions are required but not met: 1) that deactivations of other regions could not account for the effect 2) that intravenous midazolam does indeed deactivate the hippocampus and 3) that midazolam influences explicit but not implicit memory. Each of these assumptions is seriously flawed. Consequently, the suggestion that implicit TI does not depend upon the hippocampus is unfounded.Recent advances have led to an understanding that the hippocampus is involved more broadly than explicit or declarative memory alone. Tasks which involve the acquisition of complex associations involve the hippocampus whether the learning is explicit or implicit. One hippocampal-dependent implicit task is transitive inference (TI). Recently it was suggested that implicit transitive inference does not depend upon the hippocampus (Frank, M. J., O'Reilly, R. C., & Curran, T. 2006. When memory fails, intuition reigns: midazolam enhances implicit inference in humans.  The capacity to learn complex stimulus relations and express them flexibly is thought to require the hippocampus . While tA recent study demonstrated that the administration of intravenous midazolam improved TI performance in human participants  which waIn studies where midazolam has been shown to have selective effects on the hippocampus, non-human animals receive midazolam as a direct injection into the hippocampal tissue . The priOf the regions shown in human imaging studies to be deactivated by normal concentrations of intravenous midazolam, the hippocampus is not among them. Two studies attempting to discover an effect of midazolam on rCBF in the hippocampus found no such effects ,24. CleaFurthermore, were it the case that midazolam selectively deactivates the hippocampus, one would expect the effects of midazolam on TI  to yieldGiven the widespread effects of intravenous midazolam in regions of cortex other than the hippocampus, it is natural to question whether the effects of midazolam are limited to explicit memory. Several studies show that midazolam influences not just explicit, but also implicit memory performance ,28. WhilThe existing data do not permit claims about selective effects if intravenous midazolam on the human hippocampus. Rather, the effects of intravenous midazolam involve numerous regions of cortex, including frontal and parietal regions. The assertion that intravenous midazolam selectively deactivates the hippocampus is at best premature and is more likely to be incorrect. Therefore, there is no adequate substantiation for the claim that TI is improved by hippocampal deactivation .The author(s) declare that they have no competing interests.