Our results also suggest that drugs that inhibit or decrease tau phosphorylation may slow cognitive decline in individuals with very mild dementia or delay the appearance of memory problems in elderly individuals with low CSF Aβ42 levels. Finally, we believe genome-wide association studies of CSF tau/ptau181 levels should identify novel genetic variants which will likely influence rate of progression of AD.Alzheimer's Disease (AD) is a complex and multifactorial disease. While large genome-wide association studies have had some success in identifying novel genetic risk factors for AD, case-control studies are less likely to uncover genetic factors that influence progression of disease. An alternative approach to identifying genetic risk for AD is the use of quantitative traits or endophenotypes. The use of endophenotypes has proven to be an effective strategy, implicating genetic risk factors in several diseases, including anemia, osteoporosis and heart disease. In this study we identify a genetic factor associated with the rate of decline in AD patients and present a methodology for identification of other such factors. We have used an established biomarker for AD, cerebrospinal fluid (CSF) tau phosphorylated at threonine 181  and polymorphisms in APOE as risk factors. These findings have led to a better understanding of the underlying disease mechanisms. However, half of all AD cases have no known genetic risk factors for disease. Most studies are designed to identify variants associated with risk or age at onset, but rarely cover other important facets of AD, such as disease progression or duration. In this study we have used an established AD biomarker  to find genetic variants that influence levels of ptau181 in the cerebrospinal fluid. This novel and powerful approach has allowed us to identify a genetic factor located in the regulatory subunit of the calcineurin that is also strongly associated with rate of progression of AD. This study is important because it defines a strategy to find novel genetic factors influencing different facets of AD pathobiology including risk, onset and progression.Alzheimer's disease (AD) is the most common neurodegenerative disease affecting more than 4.5 million people in the US. Genetic studies of AD have previously identified pathogenic mutations in three genes ( APOE4), present in 50% of late onset AD (LOAD) cases, has been convincingly demonstrated to influence risk for LOAD. The traditional method for searching for genetic risk factors involves the comparison of genes in AD cases and non-demented elderly controls. AD is a complex and multifactorial disease, and as a result very large datasets have been necessary to identify these genetic risk factors Genetic studies have helped to further our understanding of the pathogenic mechanism of several diseases, including AD. To date only the ε4 allele of apolipoprotein E  181) are increased in AD 181 levels appear to be specific to AD Both Aβ and tau protein play an important role in AD, are detectable in cerebrospinal fluid (CSF) in all individuals, and have been used as biomarkers for diagnosis 181, then determined the effects of those variants on AD risk, onset and rate of progression.Tau activity depends on its state of phosphorylation 181 levels was evaluated by ANCOVA in 353 CSF samples from the Washington University Alzheimer Disease Research Center (WU-ADRC-CSF) . The association of rs1868402 with CSF ptau181 levels showed the best fit in the dominant model, with minor alleles carriers showing significantly higher CSF ptau181 levels  with rs1868402 (181. Based on the linkage disequilibrium (LD) in PPP3R1, we selected rs1868402 and rs6546366 for replication. The remaining eleven SNPs that were significant after multiple test correction were also selected for replication   and 2. Crrection . The SNPs1868402 , also shlication .P = 0.005 ptau181, 181 levels. In the replication CSF series, rs6546366 (PPP3R1), showed no association with CSF ptau181 levels (181 levels is probably due to a lower level of LD with rs1868402 in the replication series (r2 = 0.65) compared with the LD between these two SNPs in the WU-ADRC-CSF series (r2 = 0.80). This result indicates that rs1868402, or another unknown variant in LD with rs1868402, is the variant that drives the association with CSF ptau181 levels.In a replication series of 493 independent CSF samples from ADNI (ADNI-CSF) and University of Washington (UW)  and 2 on1 levels . The lac181 after correcting for the covariates . None of the other SNPs were significant after Bonferroni correction . The difference in CSF Aβ42 threshold levels between the WU-ADRC-CSF and ADNI-CSF series is due to different antibodies and procedures used to measure the CSF levels . The nominally significant p-value in the high Aβ stratum may indicate a moderate effect on CSF ptau181 levels in healthy individuals, but it is clear that when AD pathology is present the effect of this SNP is more marked.It has been demonstrated in our longitudinal data and that of others that the increase in CSF tau and ptauvels see . The dat181 levels in individuals with low CSF Aβ42 levels in the WU-ADRC-CSF+ADNI-CSF samples, which is similar to the variability explained by other SNPs and endophenotypes 42 group there are individuals diagnosed with DAT  and non-demented individuals  with possible Aβ deposition in the brain and brain atrophy (presymptomatic AD) 42 stratum 80% of the samples (n = 242) had a CDR = 0.Rs1868402 explains 4.62% of the variability in CSF ptauP = 0.10, P = 0.19 The premise of this endophenotype-based approach is that a SNP, such as rs1868402 that shows strong, replicable association with an important AD biomarker should also modulate risk, onset and/or progression of AD. We tested whether rs1868402 influences risk for AD, age at onset and disease progression. We found no association between rs1868402 and risk for AD , and an initial CDR of 0 or 0.5. Individuals with CDR 0 and 0.5 were selected to maximize the amount of information on progression per individual and to avoid possible ceiling effects from individuals who began the study with advanced levels of dementia.To examine disease progression we used two longitudinal datasets: 109 subjects from WU-ADRC-CSF (399 observations) and 150 subjects from ADNI-CSF (620 observations). Association with rate of progression was evaluated by comparing the change in sum of boxes of the CDR (SB-CDR) per year (slope) by genotype including age at the first visit, gender, 181 levels showed an increase of 0.58 SB-CDR per year, which is six-fold faster than the rate seen in individuals homozygous for the allele associated with low CSF ptau181 levels  with a Pcombined = 1.96×10−05. In addition, we also used the ADNI samples with no CSF data  also shows association with PPP3R1 mRNA levels and in the same direction in the publicly available GEO GSE8919 P = 0.015).Since rs1868402 is located in a region that encodes a tau phosphatase, we tested next whether the SNP is associated with 181 levels and faster rate of progression of AD. We failed to detect evidence for association with risk for disease or age at onset. This is consistent with the known pathobiology of AD, in which Aβ aggregation and deposition is an early preclinical event, followed by increased CSF tau and ptau181 levels and tau pathology during the clinical phase of the disease. Under this model, factors that affect tau phosphorylation and aggregation may be expected to modify disease progression but not risk for disease, while genes that influence Aβ aggregation, such as APOE442 levels and risk/age at onset of disease but not CSF tau/ptau181 levels or rate of progression of disease. 42 and tau variation therefore allow identification of genetic factors that influence different components of the disease process.In the present study we have used a novel and powerful endophenotype-based approach to identify a novel genetic factor implicated in AD. Most genetic studies in AD have focused on identifying genetic factors that modulate risk or age at onset of disease Our results suggest that rs1868402, or another variant in LD with it, may reduce calcineurin expression/activity leading to an increase in tau phosphorylation increasing tau pathology and neurodegeneration in individuals with Aβ deposition. The resulting increase in tau-related pathology would then increase the rate of progression of AD. Several studies provide support for a role of calcineurin in AD pathogenesis. Inhibition of calcineurin in mouse brains by cyclosporin A or FK506 or rat brain by antisense oligonucleotides led to enhanced tau phosphorylation 42 levels also influence risk and age at onset (e.g. APOE) but variants associated with CSF ptau181 levels have a greater impact on rate of progression .The cerebrospinal fluid discovery series includes 353 individuals enrolled in longitudinal studies at the WU-ADRC. CSF collection and AβRisk for disease and age at onset analyses were analyzed in a total of 1106 late-onset AD (LOAD) cases and 1216 age-gender-ethnicity matched non-demented controls . These s42, as explained in the Statistical Analyses section.Association of rate of progression of dementia with genetic variants was tested in two longitudinal series from the WU-ADRC and ADNI. The WU-ADRC-CSF series includes 109 individuals with clinical data from at least two time points starting with a CDR of 0 or 0.5 in the first interview, and a diagnosis of DAT (dementia Alzheimer Type) at the last visit. There are an average of 3.8 observations per individual, which varies from 2 to 14 with an average follow up time of 3.2 years. The second series with longitudinal data is the ADNI series: 459 individuals (236 with CSF data and another 223 with no CSF data) have had a clinical examination at least two time points with an average of 4.1 observations per individual, which varies between two and six observations, however the average follow up time is only 1.9 years. To study the association with progression rate in the CSF samples we analyze only the 150 samples with low (<192pg/ml) CSF Aβ2>0.8), based on CEU-HapMap data, were selected for each of these genes. We used Pupasuite software Based on bibliographic data, we selected 384 SNPs in the most relevant tau kinases, phosphatases, and in other genes implicated in other posttranslational modifications of tau, or tau degradation Expression studies were carried out using cDNA obtained from the parietal lobe of 82 AD cases and 39 non-demented individuals (CDR = 0) obtained through the WU-ADRC Neuropathology Core  PPP3R1 (ABI: C_12044272_10) and cyclophilin A (ABI: 4326316E) were used to quantify the gene expression levels. Each real-time PCR run included within-plate duplicates and each experiment was performed, at least twice for each sample. Real-time data were analyzed using the comparative Ct method. The Ct values of each sample were normalized with the Ct value for the housekeeping genes, GADPH and cyclophilin, and were corrected for the PCR efficiency of each assay Total RNA was extracted from the parietal lobe of 82 AD cases and 39 non-demented individuals, using the RNeasy mini kit (Qiagen) following the manufacturer's protocol. cDNAs were prepared from the total RNA, using the High-Capacity cDNA Archive kit (ABI). Gene expression was analyzed by real-time PCR, using an ABI-7500 real-time PCR system. Real-time PCR assays were used to quantify 181 values were log-log transformed to approximate a normal distribution. Analysis of the covariance (ANCOVA) was used to test for association between genotypes and CSF ptau181 levels. In order to identify the covariates that affect CSF ptau181 levels, we performed a stepwise discriminant analysis including CDR, age, gender and APOE genotype. CDR, age, and APOE genotype were identified as significant covariates in the WU-ADRC-CSF series and, CDR and APOE genotype in the replication series (this series has a narrower age range than the WU-ADRC-CSF series). These covariates were included in the respective ANCOVA. Each SNP was tested using an additive model with minor allele homozygotes coded as 0, heterozygotes coded as 1, and major allele homozygotes coded as 2. When the additive model was significant after multiple test correction, dominant and recessive models were tested to determine whether the y were a better fit. Because the CSF ptau181 levels in the WU-ADRC-CSF, ADNI-CSF and UW samples were measured using different platforms  we were not able to combine the raw data, rather we combined the residual values of the CSF ptau181 obtained after correcting for the covariates. No significant differences in the residuals from the different series were found, indicating that the differences in the CSF levels due to the different platforms were corrected by using the residuals.CSF ptau181 levels were evaluated using a False Discovery Rate (FDR) filter of 0.1 to correct for multiple testing 181 levels in the combined samples. In this case, the threshold for Bonferroni correction for the combined sample is 181 levels, we calculated the Odds Ratio (OR) of this SNP by comparing its frequency in the highest vs lowest quartile of the residuals for CSF ptau181 levels.Multiple test correction: Initial tests for association of SNPs with CSF ptauAPOE, gender, age and series as covariates. Association with AAO was carried out using the Kaplan-Meier method and tested for significant differences, using a log-rank test. Association with rate of disease progression was evaluated as described previously APOE, initial CDR, CSF ptau181 and Aβ42 levels. Because the association between rs1868402 and CSF ptau181 was driven by individuals with low CSF Aβ42 levels, association with progression in the CSF datasets was analyzed only in individuals with low CSF Aβ42 levels . One-tailed P-values were calculated, because a priori predictions were made based on the associations with CSF ptau181 levels. We also used the GEO dataset GSE8919 2 = 0.75) and PPP3R1 gene expression. In this dataset there are genotype and expression data from 486 Late onset Alzheimer Diseases cases and 279 neuropathologically confirmed controls. We only extracted the normalized PPP3R1 mRNA levels and the genotype data for rs12713636. Genotypes for rs12713636 were used because rs1868402 was not included in this dataset.Association between cDNA levels, tau pathology (Braak tangle stage) and genotypes were carried out using ANCOVA. Stepwise discriminant analysis was used to determine the significant covariates . The ADNI was launched in 2003 by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, the Food and Drug Administration, private pharmaceutical companies and non-profit organizations, as a $60 million, 5-year public-private partnership. The Principal Investigator of this initiative is Michael W. Weiner, M.D. ADNI is the result of efforts of many co-investigators from a broad range of academic institutions and private corporations, and subjects have been recruited from over 50 sites across the U.S. and Canada. The initial goal of ADNI was to recruit 800 adults, ages 55 to 90, to participate in the research -approximately 200 cognitively normal older individuals to be followed for 3 years, 400 people with MCI to be followed for 3 years, and 200 people with early AD to be followed for 2 years.” For up-to-date information see www.adni-info.org.Data used in the preparation of this article were obtained from the ADNI database  for the raw and residuals CSF ptau181 levels for the WU-ADRC-CSF, ADNI-CSF and UW series is shown. A. Raw CSF ptau181 levels for the WU-ADRC-CSF series by rs1868402 genotype. CC+CT: 64.45±2.52. TT: 57.20±2.49 pg/ml. B. Raw CSF ptau181 levels for the ADNI-CSF series by rs1868402 genotype. CC+CT: 35.95±1.85. TT: 30.22±1.44 pg/ml. C. Raw CSF ptau181 levels for the UW series by rs1868402 genotype. CC+CT: 67.46±3.53. TT: 61.42±2.71 pg/ml. D. Residuals CSF ptau181 levels for the WU-ADRC-CSF series by rs1868402 genotype. CC+CT: 0.17±0.07. TT: −0.22±0.08. E. Residuals CSF ptau181 levels for the ADNI-CSF series by rs1868402 genotype. CC+CT: 0.13±0.09. TT: −0.16±0.08. F. Residuals CSF ptau181 levels for the UW series by rs1868402 genotype. CC+CT: 0.10±0.08. TT: −0.12±0.09.Minor allele carriers of rs1868402 present significantly higher CSF ptau(0.06 MB DOC)Click here for additional data file.Figure S2PPP3R1 SNPs significantly associated with CSF tau levels in the ADRC series. Color represents D′ = 1 and numbers correspond to r2.Linkage disequilibrium among (0.16 MB DOC)Click here for additional data file.Figure S3181 levels are also associated with rate of progression. Average progression rate by genotype with the 95% confidence interval. Solid lines represent the average progression rate. Dotted lines represent the 95% confidence interval. The lines for the different genotypes are color code. A. Minor allele carriers of rs1868402, are associated with higher CSF ptau181 levels, and show a 6-fold faster progression than homozygotes for the major allele  in individuals from the WU-ADRC-CSF with low CSF Aβ42 levels (<500pg/ml). B. rs3785883 genotypes do not have significantly different progression rates P = 0.057. The genotype frequency distribution for rs3785883 with disease progression is most likely not significant due to the low statistical power. AA carriers show a CDR-SB of 1.01, AG of 0.47 and GG 0.26 (p = 0.057).Genetic variants associated with CSF ptau(0.23 MB DOC)Click here for additional data file.Table S1Genes and SNPs/gene genotyped in this study. The official and the most common alias of the gene, the activity related to tau, chromosomal position, and gene size in Kb are shown. A. Tag SNP. SNPs that capture 80% of the common sequence diversity within the gene. B. Only validated SNP with a minor allele frequency >0.1. C. Number of SNPs that passed quality controls.(0.09 MB DOC)Click here for additional data file.Table S2PPP3R1 associated with CSF ptau181 levels in the discovery series (WU-ADRC-CSF). SNPs associated with CSF ptau181 levels after FDR correction are shown. MAF = Minor Allele Frequency. A: Dominant model. B: Recessive model.SNPs in (0.03 MB DOC)Click here for additional data file.


Granulocyte-colony stimulating factor (G-CSF)-producing cancer has been reported to occur in various organs, especially the lung. However, G-CSF-producing colorectal cancer (CRC) has never been reported in the English literature.A 57-year-old man was admitted for the surgical removal of a rectal cancer. Some hepatic tumors in the liver were revealed concurrently, and their appearance suggested multiple liver metastases. Low anterior resection was performed. with the help of histopathological examination and immunohistochemical studies, we diagnosed this case to be an undifferentiated carcinoma of the rectum. After the operation, the white blood cell (WBC) count increased gradually to 81,000 cells/μL. Modified-FOLFOX6 therapy was initiated to treat the liver metastases, but there was no effect, and peritoneal dissemination had also occurred. The serum level of G-CSF was elevated to 840 pg/mL . Furthermore, immunohistochemistry with a specific monoclonal antibody against G-CSF was positive; therefore, we diagnosed this tumor as a G-CSF-producing cancer. The patient died from rapid growth of the liver metastases and peritoneal dissemination 2 months after surgery.This is the first case of G-CSF-producing rectal cancer, and its prognosis was very poor. Granulocyte-colony stimulating factor (G-CSF)-producing cancer has been reported to occur in the lung , stomachA 57-year-old man was admitted to our hospital with lower abdominal pain in June 2007. Barium enema and colonoscopy revealed an ulcerative tumor in the rectum Figure , which, An ulcerated hard tumor was present in the rectum Figure . Histopaet al., reported a case of early-stage gastric cancer that presented as a well-differentiated adenocarcinoma that changed to a poorly differentiated adenocarcinoma at the advanced stage, and acquired the ability to produce G-CSF [G-CSF-producing cancer has been reported to occur in various organs, especially in the lung. Histologically, more than half of the reported cases of G-CSF-producing lung cancer have been large cell carcinoma . Howeverce G-CSF . In our ce G-CSF , and ouret al., reported that median values of G-CSF in CRC patients were significantly higher than those in healthy subjects [The mechanism by which certain CRCs produce G-CSF has not been clarified. Mroczko subjects . Furthersubjects ,13.et al., showed that G-CSF production by transitional cell carcinoma of the bladder augments autocrine growth, which may in part explain the poor prognoses [et al., reported that 56.5% of primary ovarian carcinomas co-expressed G-CSF and the G-CSF receptor (G-CSFR); potential autocrine and/or paracrine loops involving G-CSF and its receptor occur in over 90% of primary ovarian carcinomas [et al., reported that the G-CSFR was expressed in 59% of CRCs [et al., reported that G-CSF stimulates angiogenesis and promotes tumor growth [et al., showed that the production of GM-CSF by squamous cell carcinoma cell lines was closely related to their in vitro invasiveness and MMP activity [Tachibana rognoses . Savaresrcinomas . As ment of CRCs . Therefor growth . In addiactivity . For theIn summary, we present the first case of G-CSF-producing rectal cancer. Its prognosis was very poor, and mFOLFOX6 therapy had no effect. But, we believe that further investigation may make it clear that molecular targeted therapies for G-CSF may become part of the treatment paradigm.CRC: colorectal cancer; G-CSF: granulocyte-colony-stimulating factor; GM-CSF: granulocyte-macrophage colony stimulating factor; ELISA: enzyme-linked immunosorbent assay; G-CSFR: granulocyte-colony-stimulating factor receptor; CT: Computed tomography; MRI: Magnetic resonance imaging; mFOLFOX: Modified-FOLFOX6.The authors declare that they have no competing interests.HT, SM, NO, ST and HT carried out the clinical examination and operation, HT, AY, HS, and YA performed the pathological analysis, HT, HF and TW participated in the design of the study, HT and MS conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.


The levels of retinol-binding protein 4 (RBP4) – the carrier protein for Vitamin A in plasma – are tightly regulated under healthy circumstances. The kidney, the main site of RBP4 catabolism, contributes to an elevation of RBP4 levels during chronic kidney disease (CKD) whereas during chronic liver disease (CLD) RBP4 levels decrease. Little is known about RBP4 isoforms including apo-RBP4, holo-RBP4 as well as RBP4 truncated at the C-terminus (RBP4-L and RBP4-LL) except that RBP4 isoforms have been reported to be increased in hemodialysis patients. Since it is not known whether CLD influence RBP4 isoforms, we investigated RBP4 levels, apo- and holo-RBP4 as well as RBP4-L and RBP4-LL in plasma of 36 patients suffering from CKD, in 55 CLD patients and in 50 control subjects. RBP4 was determined by ELISA and apo- and holo-RBP4 by native polyacrylamide gel electrophoresis (PAGE). RBP4-L and RBP4-LL were analyzed after immunoprecipitation by mass spectrometry (MALDI-TOF-MS).RBP4 isoforms and levels were highly increased in CKD patients compared to controls (P < 0.05) whereas in CLD patients RBP4 isoforms were not different from controls. In addition, in hepatic dysfunction RBP4 levels were decreased whereas the amount of isoforms was not affected.The occurrence of RBP4 isoforms is not influenced by liver function but seems to be strongly related to kidney function and may therefore be important in investigating kidney function and related disorders. Retinol–binding protein 4 (RBP4) is a 21 kDa plasma protein, which is mainly secreted from the liver and adipose tissue and is known to transport retinol (ROH) in the blood. The binding of ROH to RBP4 guarantees the homeostatic regulation of plasma ROH levels, which are an essential aspect for a variety of physiological processes . RecentlIn healthy individuals RBP4 is mainly synthesized in the liver and secreted into the circulation in a 1:1:1 complex with ROH (holo-RBP4) and transthyretin (TTR) ,8. The bRBP4 has been reported to occur in different isoforms in serum, namely holo-RBP4 (RBP4 bound to ROH) and apo-RBP4, which remains after the release of ROH into the target cell. In addition, little is known about RBP4 isoforms resulting from the truncation of RBP4: RBP4-L, which is truncated at one C-terminal leucine molecule (Leu-183), and RBP4-LL, which is truncated at a second leucine molecule (Leu-182 and Leu-183). The relative amounts of apo-RBP4 are increased in rats during acute renal failure and RBP4-L and RBP4-LL have been shown to be increased in hemo-dialysis patients ,20,21. IAnthropometric and clinical characteristics of controls, CLD patients and CKD patients are shown in Table Standard tests of liver function such as alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) concentrations showed increased levels in the CLD group compared to the values in the controls (P < 0.001). The levels of ALT, GGT and ALP were also increased in the CKD group compared to controls (P < 0.01). However, AST and ALT were markedly lower in the CKD compared to the CLD group (P < 0.001).Compared to controls, RBP4 levels were lower in CLD (P < 0.001), but highly elevated in patients with CKD  as well as to CKD (P < 0.001). The CKD group showed the highest value of the RBP4 to ROH ratio compared to controls as well as to the CLD group (P < 0.001). An excess of RBP4 over ROH indicates an elevation in free RBP4 and thus apo-RBP4 (unbound ROH). This is supported by the significant correlation of apo-RBP4 and the RBP4-ROH ratio . The molar ratio of RBP4 to TTR was increased in CLD and CKD compared to controls . Conversely, apo-RBP4 was detected in higher quantities in CKD compared to controls and CLD patients , ALT , ALP  and GGT . AST was correlated with holo-RBP4 (r = 0.330) and inversely with apo-RBP4 . In addition, AST levels were inversely correlated with RBP4-L (r = -0.421) and RBP4-LL . ALP was inversely correlated with RBP4-L .With regards to kidney function, there was a correlation between serum creatinine and RBP4 levels (r = 0.633), apo-RBP4 (r = 0.674), RBP4-L (r = 0.494) and RBP4-LL (r = 0.438) as well as ROH . Holo-RBP4, in contrast, was inversely correlated with serum creatinine . In CLD, serum creatinine was correlated with RBP4 (r = 0.535), RBP4-L  and ROH levels .This study was designed to investigate the effect of CLD and CKD on RBP4 isoforms and to identify factors influencing and/or generating RBP4 isoforms. We were able to show that the relative amount of RBP4 isoforms  was increased in CKD patients, but not in CLD patients, in comparison to controls. Our results also show that RBP4 levels were significantly elevated in serum of CKD patients compared to both, CLD patients and controls. In contrast, RBP4, TTR and ROH levels were significantly decreased in CLD patients, as compared to CKD patients and controls.Jaconi et al. [i et al.  investigi et al. ,17. To di et al. , respecti et al. ,17 and ni et al. , the phyi et al. . In addii et al. .Additionally, we confirmed that RBP4, TTR and ROH levels in various liver diseases were markedly depressed, particularly in patients with c2-cirrhosis or hepato-cellular carcinoma, which is in accordance with results of previously published studies ,26-28. TIn patients with CKD, the levels of RBP4 were markedly elevated and therefore the molar ratio of RBP4 to TTR was increased. In healthy states, TTR is present in a 3–5 fold molar excess in plasma and the serum RBP4/TTR ratio is approximately 0.4 whereas in CKD patients an increase in the RBP4/TTR molar ratio up to 1.06 has been reported ,18,29,30The kidneys play an important role in the recycling of RBP4 since RBP4 catabolism is disturbed in CKD patients ,31. AccoThe alterations in RBP4 metabolism during CKD are of interest in relation to T2DM since T2DM patients are exposed to increased oxidative stress which has been reported to be linked to endothelial dysfunction . It is kThe results of the study show that the disturbed catabolism of RBP4 in CKD results in an increase in RBP4 isoforms including apo-RBP4, RBP4-L and RBP4-LL – whereas the generation of RBP4 isoforms is not influenced by liver function. However, both CKD and CLD do influence serum RBP4 levels. Since the increase in RBP4 isoforms was not observed in patients suffering from various CLD, the important physiological function of the kidneys in that context is emphasized and it may be suggested that impaired catabolism of RBP4 in the kidneys leads to an accumulation of RBP4 isoforms in serum. These results support the hypothesis that the C-terminal truncation of RBP4 may be specific during CKD.Sera of 50 healthy subjects were obtained from the Department of Clinical Nutrition, German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany. The inclusion criteria for healthy subjects were no known diagnosis of any kidney, liver or metabolic disease, such as obesity/adiposity, diabetes or hypertension, and no drug intake.2) and severely decreased (< 30 ml/min/1.73 m2) eGFR were included [Sera of 45 patients with CKD were obtained from the Department of Medicine IV, Charité Campus Benjamin Franklin, Berlin, Germany. Subjects were characterized according to their estimated glomerular filtration rate (eGFR) which was calculated according to the MDRD formula . In the included .2).Sera of 63 patients with CLD were obtained from the Department of Medicine IV, Charité Campus Virchow, Berlin, Germany. Of these patients, 10 were diagnosed with fibrosis METAVIR stage 0 – 1.5, 12 with fibrosis METAVIR stage 2 – 2.5, 9 with METAVIR stage 3, 7 with fibrosis METAVIR stage 4, 10 with hepato-cellular cancer and 7 with c2 cirrhosis. Cirrhosis diagnosis was made depending on histopathologic, clinical and laboratory findings. Staging was differentiated according to fibrosis: Stage 1 = zone 3 perisinusoidal/pericellular fibrosis, focal or diffuse; stage 2 = focal of diffuse periportal fibrosis together with zone 3 perisinusoidal/pericellular fibrosis; stage 3 = focal and diffuse bridging necrosis together with perisinusoidal/pericellular fibrosis and portal fibrosis; stage 4 = Cirrhosis. Body mass index (BMI) was calculated by the formula: weight (kg)/height, solvent B methanol:methyl-tert-butyl-ether:water .For separation and quantification of ROH a gradient reversed-phase HPLC system was used as previously described . BrieflySerum levels of RBP4 and TTR were measured by ELISA using polyclonal rabbit anti-human antibodies for RBP4 and Prealbumin  as previously described ,41. DeteFor immunoprecipitation 10 μl of serum sample was incubated with an equal amount of Sephadex G 15 and 5 μl polyclonal rabbit anti-human RBP4  at room temperature for 18 hours, centrifuged at 13,000 rpm for 20 min at room temperature. After removing the supernatant, the protein-antibody complex was washed twice with PBS and once with HEPES. The samples were then applied on the MALDI target using 2 μl of sample. Afterwards, 2 μl saturated sinapinic acid solution was placed on a serum drop and dried. The matrix solution contained 1 mg sinapinic acid and an equal amount of 1% trifluoroacetic acid and acetonitrile. MALDI mass spectra were obtained using a Reflex II MALDI-TOF mass spectrometer  which was performed in a linear mode at 20 k acceleration voltage. For ionisation, a nitrogen laser  was used. For optimisation of the mass spectra, the laser was aimed either at the central area of the sample or at the outmost edge of the crystal rim. All spectra were measured using external calibration. As the ionization efficiencies of non-truncated RBP4, RBP4-L and RBP4-LL are similar, the peaks in the mass spectra reflect the relative amounts of RBP4-L and RBP4-LL . Therefo® software (Bio-Rad). The relative amounts of apo- and holo-RBP4 per lane are expressed as per cent of total intensity of each lane. However, since apo- and holo-RBP4 are the only visible bands, the sum of the relative quantities of both isoforms equals 100% per lane.Relative amounts of holo-RBP4 and apo-RBP4 in serum were assessed by using nondenaturating polyacrylamide gel electrophoresis (PAGE) with subsequent immunoblotting analysis. Under these conditions retinol remains bound to RBP4 and due to the higher molecular weight of holo-RBP4 (+ 286 Da), two bands may be detected. The PAGE was performed according to Siegenthaler and Saurat with slight modifications . BrieflyResults are shown as medians and interquartile ranges. Statistic calculations were performed using SPSS 14.0 . The Kruskal-Wallis test was used to test for significant differences in continuous variables between the groups. If there was a significant effect, Mann-Whitney U-rank test was performed to describe differences in proportions between cases and controls. Spearman rank correlation coefficients were used to test the association between laboratory parameters and variables of ROH-RBP4 transport complex. Values of P < 0.05 were regarded as significant.CKD: Chronic kidney disease; CLD: Chronic liver disease; MALDI-TOF-MS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry; RBP4: Retinol-binding protein 4; T2DM: type 2 diabetes.The authors declare that they have no competing interests.SKF and BN carried out sample analysis and prepared the manuscript. MOW and AHFP recruited blood samples of the control group. TB, BS, MT and WZ recruited blood samples of patients. AH, JR reviewed the manuscript and FJS created the study design. All authors have read and approved the final version of the manuscript.


Xenopus laevis, that had been enucleated (eye removed) during early larvallife. This rejection did not occur in intact frogs or in those enucleated in later larval or adultlife. Whereas the splenocytes from intact frogs did not proliferate in response to a co-culturedsyngeneic lens, those from frogs that had been enucleated at any of the larval stages, or evenafter metamorphosis, proliferated intensely. Both of these responses were shown to be thymus-dependent. In conclusion, it was demonstrated that the frog immune system rejectedeven syngeheic lenses by enucleation in early larval life and that it began to recognize thesyngeneic lenses by lymphoid proliferation after enucleation, even in later life.We have reinvestigated an important issue in the amphibian immunology that has not beensettled for years since the pioneer work of Triplett, concerning the necessity of being exposedto organ-specific antigens early in development. It was found that syngeneic lenses wererejected by frogs,


Torsion of an undescended testis is uncommon. Torsion of a cryptorchid testicle presents a nonspecific symptomatology. Clinical suspicion indicates emergent surgical exploration, irrespective of Doppler ultrasound with its inherent false negative results. Management of the contralateral testis is controversial. We emphasize the need of a complete physical examination of the child who goes to the emergency room with nonspecific symptoms of abdominal pain and ipsilateral empty hemiscrotum to rule out torsion of a cryptorchid testicle. Herein, we report a one-year-old infant with missed torsion of undescended left testis. The patient was a one-year-old infant that presented with 3-day history of poor feeding, restlessness, progressive left inguinal swelling, and tenderness. In physical examination, a swollen left inguinal area with redness, point tenderness, and ipsilateral empty hemiscrotum was noted. The parents noted that they had been informed about the presence of a left UDT in their infant 3 months before. In the sonographic evaluation of testes, the right testis was normal, but the inguinal left testis was edematous, and no testicular circulation was found in Doppler evaluation of it. The patient underwent surgical exploration with left inguinal incision under the diagnosis of missed testicular torsion. After opening of layers, necrotic deep extravaginal testicular torsion that was located at the deep inguinal area was noted . Due to Cryptorchidism constitutes a common congenital surgical problem encountered in males in pediatric urology. The incidence of cryptorchidism or maldescended testis in full-term neonates is estimated to be from 2.7% to 5.9% at birth, but decreases to 1.2% or 1.8% by age 1 year . WilliamTorsion of an undescended testis is a relatively rare phenomenon that should be suspected, diagnosed, and treated without delay. With improved recognition of this entity and earlier referrals of patients with undescended testes by primary care physicians, this entity might eventually be prevented. As for the diagnosis of torsion in normal descended testis, there is no one history, physical, laboratory, or radiological finding that might predict testicular salvageability. This can only be determined at surgical exploration and is independent of the degree of torsion. In addition, when it is remembered that close to 2% of all males have an undescended testis, the risk of torsion in an undescended testis clearly needs to be made more evident to pediatric urologists and pediatricians.


Sir,Midazolam belongs to a class of medications called benzodiazepines and it is used before medical procedures or before anesthesia for surgery to cause drowsiness and sleep, relieve anxiety, and prevent any memory of event. Midazolam may cause side-effects, some of which may be serious, such as agitation, restlessness, uncontrollable shaking of a part of the body, stiffening and jerking of the arms and legs, and aggression. This paradoxical excitement occurs in <1% of all patients receiving midazolam, which may be reversed with flumazenil, avoiding increasing the level of sedation.1 In this A 38-year-old man who had no previous history of psychotic or neurotic disorders occasionally used cocaine and did not use narcotics. He was previously intervened with colonic diverticulitis using the technique of Hartman, and was now admitted to carrying out reconstruction of the intestinal transit. After surgery, he was consistently awake, aware, calm and without focal neurologic changes until he suffered from respiratory depression by opiates, used as analgesic medication, and, therefore, was reintubated with 15 mg midazolam after the procedure. After 2 h, the patient did not respond to requests to open his eyes. The Glasgow Coma Score was 8–10 points and he had significant psychomotor agitation, without neurological focus, which did not lessen even after the administration of haloperidol and dipotassium clorazepate. During that time, we excluded other causes of agitation because the vital signs remained stable and hypoxia, hypercapnia, hypertension, and increased intracranial pressure could be excluded. He did not complain of any pain or discomfort, hypoglycemia was ruled out, and the electrolyte imbalance did not exist to justify the patient’s symptoms. It is decided to administer flumazenil 0.5 mg intravenously, immediately after which the patient became conscious, oriented and calm, the paradoxical reaction was terminated, and he had no recollection of the events. Computed tomography of the brain was not performed because the patient was fully awake after the administration of flumazenil.Benzodiazepines stimulate the effect of gamma-aminobutyric acid (GABA) in the ascending reticular activating system. Paradoxical reactions have been described for many drugs that interact with the GABA receptor, includin4Flumazenil is a benzodiazepine antagonist that cannot just reverse sedation, hypnosis, and respiratory depression induced by the benzodiazepine, but several reports have also demonstrated its role in the successful treatment of benzodiazepine-induced paradoxical reactions. The onseAlthough delirium caused by midazolam or propofol in different patients has been reported, there are cases of delirium caused by midazolam alone and by propofol in the same patient.In conclusion, this case report demonstrates that paradoxical reactions to midazolam can be treated with small doses of flumazenil. Moreover, saying that since the sedoanalgesia is an integral part of the management of critical patients in intensive care units and emergency services, is essential to know the clinical guidelines for evidence-based management of these drugs.


Mycobacterium liflandii causes a fatal frog disease in captive anurans. Here we report, to our knowledge, the first epizootic of mycobacteriosis in a European colony of clawed frogs , previously imported from a United States biologic supply company. Our findings suggest the emerging potential of this infection through international trade. Mycobacterium fortuitum, M. chelonae, M. marinum, and M. xenopi are some of the mycobacteria that infect amphibians, causing subcutaneous nodules, edema, and chronic wasting  medium  in a European research laboratory. With the rising popularity of this vertebrate laboratory model and the foreseen establishment of stock centers for mutant or transgenic animals, the epizootiology of this emerging disease must be defined so that preventive measures may be instituted.We investigated an epizootic of S. tropicalis, the African tropical clawed frog. The Department of Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology of Ghent University, Belgium, had imported S. tropicalis frogs from a supplier in the United States in September 2004. Within 5 weeks, some animals became lethargic with signs similar to those described by Trott et al.: loss of diving reflex, bloating, and ulcerative skin lesions  was used to type M. liflandii . Each of the specimens was divided into 2 equal parts, half for histopathologic analysis, and half for preparation of decontaminated suspensions for culture and microscopic examination  and for 1 test. All tadpoles and water samples showed negative results for all tests.All visually affected specimens showed positive results for at least 1 organ and for at least 2 of the following tests: microscopy (Ziehl-Neelsen staining), in vitro cultivation , ISHistopathologic evaluation showed many acid-fast bacilli (AFB) in the oviduct lumen . NumerouM. liflandii in all frogs: by growth on charcoal medium, by restriction fragment length polymorphism, and by sequence analysis. Isolate M05–0456 had a similarity value of 100% with M. liflandii (GenBank accession no. AY845224.1). Growth on charcoal medium can be considered as an additional identification criterion for M. liflandii because growth on charcoal is better than on LJ medium . None of the laboratory staff who handled the anurans exhibited any signs of a mycobacterial disease.We identified the causative pathogen as M. liflandii infection was reported by Trott et al. in 2004 in pipid frog colonies in the United States .The genetic and phenotypic identification of M. liflandii infection in different S. tropicalis breeding companies in the United States  in a research colony at North Carolina State University . We do not know at this stage whether the importation of frogs contaminated by M. liflandii represents a danger for wild or autochthonous frogs. Further investigation is required to establish this.We propose examining the oocytes of newly imported frogs as an intervening noninvasive screening method on a regular basis, noting that all affected frogs reported in both intercontinental epizootics were females (


Complement activation is increased in Alzheimer's disease (AD), but its significance is unclear. The objective of this study was to determine the relationship between complement activation and cognition during the development of AD.iC3b, C9, Bielschowsky, and Gallyas staining was performed on aged normal (n = 17), mild cognitively impaired (n = 12), and AD (n = 17–18) inferior temporal gyrus specimens. Plaques were counted in 10× fields with high numbers of Bielschowsky-stained plaques. One-way ANOVA was used to determine between-group differences for plaque counts and measures of cognitive function, and linear regression was used to evaluate global cognition as a function of Bielschowsky-stained plaques. Terms for iC3b- and C9-stained plaques were then added sequentially as additional predictors in a "mediation analysis" model.p ≤ 0.01). C9 staining was present on some diffuse plaques, as well as on neuritic plaques. Bielschowsky-stained and complement-stained plaque counts were highly correlated, and were negatively correlated with cognitive measures. When the Bielschowsky plaque count was used as a predictor, its correlations with cognitive measures were statistically significant, but when iC3b and C9 plaque counts were added as additional predictors, these correlations were no longer significant. This loss of significance was attributed to multicollinearity, i.e., high correlations between Bielschowsky-stained and complement-stained plaque counts.Complement was detected on plaques in all groups, and on neurofibrillary tangles only in AD specimens. iC3b, C9, and Bielschowsky-stained plaque counts increased 2.5- to 3-fold in AD vs. other groups  and late-stage (C9) complement activation occurs on neocortical plaques in subjects across the cognitive spectrum; contrary to previous reports, C9 is present on some diffuse plaques. Because of high correlations between complement-stained and Bielschowsky-stained plaque counts, quantitative assessment of the extent to which complement activation may mediate the relationship between plaques and cognitive function could not be performed. Additional studies with animal models of AD (if late-stage complement activation can be demonstrated), or possibly a trial in AD patients with an inhibitor of late-stage complement activation, may be necessary to determine the significance of this process in AD. Complement activation is a major inflammatory process which assists in the removal of microorganisms and cellular debris and the processing of immune complexes. Complement immunoreactivity is present in the Alzheimer's disease (AD) brain on amyloid-beta (Aβ) – containing plaques, neurofibrillary tangles (NFTs), neuropil threads, and dystrophic neurites -4. ActivIn contrast to late-stage AD, few studies have examined complement activation during the development of the disease. Lue et al.  reportedHistological sections  of 4% paraformaldehyde-fixed, paraffin-embedded inferior temporal gyrus (Brodman area 20) were obtained from post-mortem brain specimens from non-cognitively impaired, aged normal (AN) subjects n = 17), individuals with mild cognitive impairment , and subjects with AD (n = 17–18). These groups were based upon clinical diagnosis. Summary statistics , and global neuropathology score) and measures of cognitive functioning for these subjects are shown in Tables 7, indiviModified Bielschowsky staining , Gallyas2O2/10% methanol in TBS for 30 min to eliminate endogenous peroxidase activity, rinsed in TBS with 0.1% Triton X-100 , then treated with TBS with 0.4% Triton, 1% bovine serum albumin (BSA), and 20% normal horse serum  for 30 min. The specimens were then incubated overnight at 4ºC with mouse monoclonal anti-human iC3b  or goat anti-human C9 . Negative controls, performed for each specimen, consisted of substituting mouse myeloma MOPC-21  and normal goat serum  for anti-iC3b and anti-C9 antisera, respectively. After rinsing the sections in TBS-T, biotinylated horse anti-mouse IgG (for iC3b staining) or biotinylated horse anti-goat IgG (for C9 staining)  was applied at room temperature for 90 min, followed by rinsing in TBS and then incubation in avidin-biotin-horseradish peroxidase conjugate  for 1 hr. After washing in TBS, the sections were developed with 3,3'-diaminobenzidine (DAB)/H2O2 with nickel enhancement . iC3b- and MOPC-21-stained sections were developed for 10 min, and C9- and NGS-stained sections were developed for 20 min. Sections were then dehydrated in ethanol baths to xylene and coverslipped with Cytoseal-60 Mounting Medium .Procedures were similar to those reported previously , with slIn Bielschowsky-stained sections, images from eight 10×-objective fields from areas of high plaque densities were captured with a Nikon Eclipse 80i microscope equipped with a Nikon Digital DXM1200 camera. Nikon ACT-1 (version 2) image analysis software  was used to find the same fields in iC3b- and C9-stained serial sections, and additional images were then captured. The numbers of plaques in each of these images  were subsequently counted by one investigator (DAL) in a blinded fashion.Neuronal immunoreactivity in iC3b- and C9-stained sections was scored, in a blinded fashion, on a scale from 0 to 3, with 0 = no complement immunoreactivity, 1 = slight complement immunoreactivity, 2 = moderate complement immunoreactivity, and 3 = extensive complement immunoreactivity. Two observers (DAL and DMC) independently scored the specimens, and then the results were compared. When specimen scores differed (less than 10% of the specimens) the specimens were re-evaluated and a consensus was reached as to the appropriate score.2: AN, 1.35 ± 0.89; MCI, 1.00 ± 0.46; AD, 9.39 ± 2.93). Evaluation of NFT complement staining was therefore limited to specimens with NFT counts ≥ 3/mm2 . These sections were scanned with the 40× objective by an observer blinded to the diagnosis for each specimen, and a description of NFT complement staining was recorded for each.Blinded observation of the Bielschowsky- and Gallyas-stained sections suggested that few or no NFTs were present in many of the specimens. This was confirmed by NFT counts that were subsequently obtained from the Rush ADC  was employed, controlling for subject age, gender, and education, to determine statistical significance between groups for Bielschowsky, iC3b, and C9 plaque counts, global neuropathology, global cognition, and the summary measures for each of the five cognitive areas referred to earlier. Post-hoc testing was performed, when necessary, via Tukey's HSD. Kruskall-Wallace (non-parametric) ANOVA was used to evaluate between-group differences for numbers of iC3b- and C9-stained neurons. Spearman rank correlations were performed to determine the strength of association between each of these variables. For plaque analyses, mean plaque counts from eight 10× fields per specimen were used; a preliminary analysis confirmed the legitimacy of including mean plaque counts, rather than counts from individual fields, in the analysis. Linear regression was employed to model global cognition as a function of global pathology and of Bielschowsky-, iC3b-, and C9-stained plaques, using these terms as individual predictors and global cognition as the outcome. Terms for iC3b-stained plaques, C9-stained plaques, and the combination of iC3b- and C9-stained plaques were then added sequentially as additional predictors in a "mediation analysis" model to determine if they would influence the strength of association between Bielschowsky plaque counts and global cognition. The analysis was subsequently repeated using each of the measures of cognitive function  as the outcome. These regression analyses were also adjusted for subject age, gender, and educational level. The significance level for all analyses was set at AD and MCI subjects were older than AN subjects, but no significant differences were present between groups for educational level or PMI. The global neuropathology scores of the AD group differed from AN and MCI subjects, but were similar between the latter two groups. AD subjects differed from the other groups with regard to last MMSE score, global cognition, episodic memory, semantic memory, working memory, and perceptual speed. Visuospatial ability was different only between AD and AN subjects.p = 0.0002, 0.0116, and 0.0130 for Bielschowsky, iC3b, and C9 staining, respectively) . Assessment of plaque morphology (diffuse vs. neuritic) in Bielschowsky-stained plaques which precisely aligned with complement-stained plaques in serial sections indicated that C9 immunoreactivity was occasionally present on diffuse plaques, as well as on neuritic plaques, in all three groups of specimens . All three types of plaque counts were highly correlated with global neuropathology .Bielschowsky, iC3b, and C9 plaque counts were moderately inversely correlated with global cognition and with the five measures of cognitive ability , were also significant vs. perceptual speed.When Bielschowsky plaques, iC3b plaques, C9 plaques, and global neuropathology were used as individual predictors, statistical significance was present vs. global cognition, episodic memory, semantic memory, working memory, and visuospatial ability as outcomes. These predictors, with the exception of global pathology , though far less than when Bielschowsky-stained plaques were used as the only predictor (p = 0.0004). The loss of statistical significance for the association of Bielschowsky-stained plaques with cognition, when iC3b- and C9-stained plaque counts were added as additional predictors, was attributed to multicollinearity. Multicollinearity exists, and is irrespective of sample size, when predictor variables are highly correlated with each other [When terms for iC3b-stained plaques, C9-stained plaques, and the combination of iC3b- and C9-stained plaques were sequentially added as additional predictors to the regression analysis model examining the association of Bielschowsky-stained plaques with cognition , the ch other ; in this2. Complement immunoreactivity on NFTs was observed only in AD specimens, within apparently intact neurons , then it is unclear why the MAC should be present on normal-appearing neurons. This staining could represent sublytic concentrations of C5b-9, which have been suggested to be neuroprotective . Our finOur results demonstrate the limitations of studies with human brain specimens for determining the role of complement activation in AD. Although the extent of this process in the brain correlates strongly with cognitive loss, differentiating its effects from the effects of AD neuropathology on cognition is difficult. Studies in animal models of AD may eventually be of value in resolving this issue if MAC deposition can be demonstrated to the same extent in these models as in AD. A trial in AD patients with an inhibitor of late-stage complement activation such as Eculizumab  , a humanEarly-stage (iC3b) and late-stage (C9) complement activation occurs on low numbers of plaques in the inferior temporal gyrus in both aged normal individuals and subjects with mild cognitive impairment. Complement staining on plaques is highly correlated with total plaque counts, and both of these parameters are inversely associated with measures of cognitive function. But the strong association between the numbers of complement-stained plaques and total plaque counts results in multicollinearity, which prevents a quantitative assessment of the extent to which complement activation may mediate the association of plaques with cognition during the development of AD.The author(s) declare that they have no competing interests.DAL performed the immunocytochemical staining, plaque counts, assessment of neuronal complement immunoreactivity, and wrote the manuscript. DMC assisted in data collection  and assessment of neuronal complement staining, generated the figures, performed some of the statistical analyses, and assisted with the writing of the manuscript. DAB assisted in experimental design, obtaining of brain specimens and clinical and post-mortem data, statistical analyses, and provided input during the drafting of the manuscript. All authors read and approved the final manuscript.


Patients suspected of having swallowing disorders, could highly benefit from simple diagnostic screening before being referred to specialist evaluations. The article analyzes various instrumental methods of dysphagia assessment, introduces surface electromyography (sEMG) to carry out rapid assessment of such patients, and debates proposed suggestions for sEMG screening protocol in order to identify abnormal deglutition.Subject related books and articles from 1813 to 2007 were obtained through library search, MEDLINE (1949–2007) and EMBASE (1975–2007).Specifics steps for establishing the protocol for applying the technique for screening purposes , the requirements for diagnostic sEMG equipment, the sEMG technique itself, and defining the tests suitable for assessing deglutition  are presented in detail. SEMG is compared with other techniques in terms of cost, timing, involvement of radiation, etc.According to the published data, SEMG of swallowing is a simple and reliable method for screening and preliminary differentiation among dysphagia and odynophagia of various origins. This noninvasive radiation-free examination has a low level of discomfort, and is simple, time-saving and inexpensive to perform. The major weakness of the method seems to be inability for precise diagnostic of neurologically induced dysphagia.With standardization of the technique and an established normative database, sEMG might serve as a reliable screening method for optimal patient management but cannot serve for proper investigation of neurogenic dysphagia. Swallowing disorders comprise an interdisciplinary phenomenon. Practitioners in various fields of medicine, such as otorhinolaryngology, neurology, general medicine, gastroenterology, head and neck surgery, dentistry and facial surgery, pediatrics and psychiatry deal with these disorders regularly, but family doctors and emergency department personnel might well be the first physicians to evaluate these patients.Dysphagia is a difficult or abnormal swallowing, which can include nasopharyngeal regurgitation and aspiration . It is aThe above described combination of voluntary and involuntary stages in deglutition makes evaluation of swallowing pathophysiology a difficult and sometimes timely and expensive process. Different diagnostic techniques were proposed for it. 20 years ago, in 1988, in a general description of evaluation of swallowing pathophysiology, the list of these techniques was as follows: still X-ray, computerized axial tomography (CAT), magnetic resonance imaging (MRI), indirect laryngoscopy, pharyngeal manometry, scintigraphy, ultrasound, videofluoroscopy . ElectroAt that moment, disadvantages for each of the above mentioned methods were well known. In brief, they are:barium esophagram – uses x-irradiation, needs a facility and personnel, no dynamic futures;air contrast esophagram – the same as above ,9;manometry – cannot diagnose visible lesions, unpleasant, can be unvalid because of movement of larynx ,11;manofluorography – not widely available, costly ,13;FEESST – requires complicated equipment, not widespread, expensive ,15;bolus scintigraphy – cannot see patient anatomy, uses isotopes ;ultrasonography – segmental, cannot present panoramic anatomic detail, expensive ;VESS – the phases of swallowing cannot be seen directly, and also aspiration that occurs during the swallow, the function of the cricopharyngeus muscle cannot be directly assessed .VFSS – relies on x-irradiation, needs radiology equipment and personnel, expensive ;Currently VFSS is the most commonly used tool in the assessment of oropharyngeal dysphagia, and it is considered the gold standard in the dysphagia workup. But in addition to above mentioned drawbacks, VFSS does not always identify neuromuscular abnormalities in pharyngeal or laryngeal physiology.In addition to the above, one can see that all these methods hardly can be used for rapid screening purposes. Comparison between swallowing studies options in respect to radiation, timing and cost is shown in the Table Dysphagia occurs in approximately 14% of patients in acute care setting an up to 50% of patients in nursing homes . Its pre• reliable• preferably noninvasive• preferably radiation-free• inexpensive• time saving• providing both qualitative and quantitative data• simple and easy to operateIn general, aim of a screening procedure is to separate patients into a "passed"- vs. "failed" group, specifically into groups with normal and abnormal deglutition. Screening so far is a filter to decide whether further clinical diagnostics or treatment is necessary. In case of dysphagia, however, the second step after normal-abnormal screening is needed. This step might answer the question to what specialist a dysphagic patient should be referred to: neurologist? ENT-physician? dentist? gastroenterologist? psychiatrist? This second screening stage is actually more practicable comparing with the first passed-failed one because initial dysphagia detection starts with a history taking.Swallowing is a muscular process. Its mechanism, by which food is transmitted to the stomach, is a complex action involving 26 muscles and five cranial nerves . This faDespite numerous studies of SEMG activity of face and neck muscles during swallowing made in the 1990s -30, lackUp to date, however, we face various technical approaches to SEMG usage in evaluation of swallowing process. While the normative database for SEMG assessment of deglutition has been established, the next step might be establishment of standards for this diagnostic procedure, i.e. the protocol. Large variation in examination techniques, strategies, interpretations and diagnostic criteria have been found among electromyographers and it is suggested that the value of SEMG studies of deglutition may be further improved by international standardization.SEMG is to be clearly separated from needle electrode examination EMG . Needle Reliability of the method was proved in a series of research using SEMG for diagnostic purposes in oral ,39, pharIf a diagnostic method is offered to be used in different areas of medicine but for one purpose, the standard protocol is a must. The need for established protocols in SEMG investigations is well understood -48, and • protocol application• protocol requirements for diagnostic equipment• protocol technique• protocol tests• normative database and standard analysisAs it was mentioned above, the act of swallowing is described as consisting of four stages, as the oral stage was divided into oral initial  and oral final stages . This stSurface SEMG devices are non-invasive, radiation-free, generally inexpensive and as simple as standard EKG equipment . Four-chAny other SEMG device with similar characteristics can be used but one quality has to be insured: the SEMG record has to be full-wave rectified and low-passed filtered in a way to look like a single EKG-looking line. SEMG records with numerous closely packed spikes are almost impossible to interpret rapidly. If the four-channel device is used, the investigation can be performed in 3–5 minutes in cases when we have full cooperation of a patient.Four muscle groups are to be examined during the evaluation -36:(1) for oral phase: the orbicularis oris superior and inferior (OO),(2) for oral phase: the masseter (MS),(3) for pharyngeal phase: the submental muscle group (SUB) which includes the anterior belly of the digastric, mylohyoid, and geniohyoid, all covered by the platysma,(4) for pharyngeal and initial oesophageal phases: (INF), the infrahyoid group, thyrohyoid, and the laryngeal strap muscles also covered by the platysma.Protocol electrode positions might be as follows -36:(1) Two bipolar stick-on surface electrodes are to be applied at the right or left angle of mouth, one electrode above the upper lip, and another electrode below the lower lip (OO-location);(2) Two electrodes are to be placed parallel to the masseter muscle fibers on the left or right side of the face, preferably on the opposite side from the OO-location (MS-location);(3) Two surface electrodes are to be attached to the skin beneath the chin on the right or left side of midline to record SUB myoelectrical activity over the platysma (SUB-location);(4) Two electrodes are to be placed on the left or right side of the thyroid cartilage to record from the laryngeal strap and infrahyoid muscles (INF-location).when OO-location is uninformative, but some general muscular-neurologic disease is suspected, the fourth electrode can be attached above a muscle not involved in deglutition, for example, above m. trapezius Fig. . The samth century .,42.39,42It is estimated that 15 million patients suffered from a swallowing disorder during each year in the United States alone ,82. ThesSurface SEMG of swallowing is a simple and reliable method for screening and initial evaluation of dysphagia and odynophagia complaints of various origins. This noninvasive radiation-free examination has low level of discomfort, simple, time-saving and inexpensive. With proper standard technique and established normative database surface SEMG can serve as a reliable screening method for assessment of dysphasia of unknown origin in order to refer a patient to a neurologist or some other proper specialist and proper further investigation. The findings are the impetus for further study regarding the mechanisms of muscle activity changes in disorders affecting swallowing.Written informed consent was obtained from the patient for publication of accompanying image. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.General concept, data collection, assessment, writing.


A over B, B over C, C over D and D over E, via reinforcement feedback. Transitive responding based on the hierarchical structure A > B > C > D > E is then tested with the novel BD pair. We and others have argued that successful BD performance by animals – and even humans in some implicit studies – can be explained by simple reinforcement learning processes which do not depend critically on the hippocampus, but rather on the striatal dopamine system. We recently showed that the benzodiazepene midazolam, which is thought to disrupt hippocampal function, profoundly impaired human memory recall performance but actually enhanced implicit TI performance . We posited that midazolam biased participants to recruit striatum during learning due to dysfunctional hippocampal processing, and that this change actually supported generalization of reinforcement values. Greene (2007) questions the validity of our pharmacological assumptions and argues that our conclusions are unfounded. Here we stand by our original hypothesis, which remains the most parsimonious account of the data, and is grounded by multiple lines of evidence.The transitive inference (TI) task assesses the ability to generalize learned knowledge to new contexts, and is thought to depend on the hippocampus . Animals or humans learn in separate trials to choose stimulus  B develops a higher associative strength then stimulus D, and that transitive responding can be achieved simply by comparing these implicit values [Our interpretation of our findings was based on the following premises. When humans are prevented from becoming explicitly aware of the hierarchy they can still perform better than chance at the novel "inference" test using an implicit reinforcement learning system . Severalt values -5. Thus t values  .AB and DE, which can be solved purely on the basis of reinforcement values, and worse performance on the conjunctive inner pairs BC and CD; (iii) substantially enhanced generalization of reinforcement values in the transitive test [We therefore posited that midazolam would disrupt the hippocampus -18, therive test . These rive test . Finallyive test , midazolive test , when thive test , consistive test .In his critique, Greene argued that our theory entails three critical assumptions that are "required but not met"   and the associated improvement in TI, are related specifically to hippocampal deactivation. However, our central hypothesis is that the critical brain system supporting reinforcement-based learning in the TI and other feedback learning tasks is the striatal dopamine system, and to the best of our knowledge there is no evidence to suggest this system is inhibited by midazolam, either at the neural level or in learning tasks that depend on this system. Moreover, there is some evidence in rats that the two main peptide markers of activity in the direct and indirect pathways of the basal ganglia  are actstration ,27, as astration . These estration ,9. As onstration .Greene also questions whether midazolam disrupts hippocampal function at all. That is, despite the dense expression of benzodiazepene receptors in the hippocampus , some huFurthermore, Greene also asserts that our theory requires midazolam to "deactivate the hippocampus to the point that it no longer functions in a mnemonic capacity". This is clearly not the case – while the low dose used in our study robustly impaired memory encoding/recall, this memory was far from obliterated altogether. These findings are similar to other studies purporting that midazolam impairs hippocampal function and substantially degrades, but does not eliminate, episodic memory ,17,31. IFinally, Greene claims that our argument requires midazolam to affect explicit but not implicit memory, but that there are a few studies showing some impairments in implicit memory. We note that first, there is substantial evidence to suggest that midazolam has a far greater proportional impairing effect on explicit than implicit memory -38. OtheTo help further resolve this controversy, midazolam effects on hippocampal activity ought to be examined in situations for which the hippocampus is thought to be critical according to the same computational principles used to motivate our original hypothesis. It would be particularly informative to explore the effects of midazolam on a contrast between conjunctive and elemental memory encoding in the TI or other task showing hippocampal sensitivity to conjunctions . Indeed,a priori theoretical predictions. Greene's commentary does reaffirm that alternative opinions are valid and that as is always the case, further research is necessary.Our original study was motivated by an explicit theoretical and neurocomputational framework for why this pattern should be observed, and is consistent with other psychological and mathematical analysis. In contrast, while legitimately questioning some of our assumptions, Greene provides no alternative explanation for the finding that midazolam substantially improved TI performance in our study. Until such an interpretation arises, with supporting data, the most parsimonious explanation of our data is that motivated by TI: transitive inferenceThe author(s) declare that they have no competing interests.M.J.F., R.C.O. and T.C wrote the paper.


These data together with UK G-CSF drug costs were combined to develop a simple cost-effectiveness model, based on direct costs. Given the current cost of G-CSF, it would only be cost-effective among patients in which high rates of hospital stay due to neutropenia or infection are expected. Alternatively, if the cost could be reduced then all patients may be able to obtain the benefits. However, the evidence that prophylactic HGFs are clinically worthwhile is clear.Combination chemotherapy used to treat patients with aggressive non-Hodgkin's lymphoma is associated with neutropenia and subsequent infection, hospital admission and treatment delays. Haematopoietic growth factors (HGF) can prevent neutropenia and improve quality of life. We undertook a meta-analysis of six randomised and one nonrandomised trials to quantify the effect in previously untreated patients, and a simple cost-effectiveness analysis. The trials compared HGF plus chemotherapy with chemotherapy alone. In total, there were 779 patients aged between 15 and 82 years. Haematopoietic growth factors was associated with a statistically significant 44% reduction in the incidence of severe neutropenia (neutrophil count <0.5 × 10 Intensive chemotherapy can be highly effective in the treatment of aggressive non-Hodgkin's lymphoma (NHL). However, this form of treatment is associated with neutropenia that can result in infection and subsequent hospital admission, treatment delays and chemotherapy dose reduction. Haematopoietic growth factors (HGF) can be used to prevent neutropenia and its consequences in untreated patients with advanced NHL. We here present a meta-analysis based on the controlled trials of the clinical effectiveness of such growth factors, when used as a primary prophylaxis. We also present a simple cost-effectiveness analysis.A systematic review of the literature was performed to identify clinical trials that compared HGF plus chemotherapy with chemotherapy alone. The literature search covered several medical databases; Medline, Embase, Cancerlit, Cochrane library, the UICCR Trials Register and the publication databases of the European Haematology Association and the American Society of Hematology. Keywords used were ‘lymphoma’, ‘growth factors’, ‘G-CSF or GM-CSF’ and ‘trial’.μg kg day−1 in five trials the incidence of severe neutropenia  tumour remissionthe proportion of patients surviving to 2, 2.5 or 5 yearsInformation on the following outcomes were obtained for each treatment group from each published report, where available:For each outcome and trial, the relative risk and 95% confidence interval (CI) was calculated by comparing the proportion (or incidence) in the G-CSF group with the proportion (or incidence) in the control (no G-CSF) group. A statistical test for heterogeneity was performed for each outcome to assess whether the relative risks were significantly different between the trials . In the μg kg day−1  the percentage of patients who, if not given HGF, would be hospitalised  and (ii) the number of times each patient could be hospitalised during five treatment cycles . We estimated the percentage reduction in the published list price of G-CSF that would be needed in order for the health service cost to be cheaper than if it were not used. The analysis was also performed assuming that the dose of G-CSF could be reduced from the standard dose of 5 to 2 The reduction in hospital admission due to infectionThe reduction in length of stayTwo results from the meta-analysis of the clinical outcomes were used:The cost of G-CSF per patient was taken as £4406  and the cost of G-CSF is added.P>0.16).P<0.001) and a 43% reduction in the number of patients with a clinically relevant infection . As a consequence, there was a 60% reduction in the number of hospital admissions due to infection  and if a patient on G-CSF were admitted they spent about half the time in hospital . Administering G-CSF also had an effect on patients experiencing a treatment delay; there was a 60% reduction in the number of patients experiencing a treatment delay for any reason  and an 80% reduction in the number of patients whose delay was due to neutropenia .G-CSF was associated with a statistically significant 44% reduction in the incidence of severe neutropenia , those reporting on complete or partial remission combined (P=0.99) or those reporting on survival (P>0.90). There was no evidence that the use of G-CSF influenced tumour remission or survival; the pooled relative risks were close to unity and none were statistically significant.From the section above, in patients given G-CSF compared to those who were not, the relative risk for hospital admission due to infection was 0.4 and the ratio for the length of stay was 0.53. These estimates are used in the following cost-effectiveness analysis.μg kg day−1 (63%). Similarly, the higher the chance of an individual patient being hospitalised the less the reduction in the list price of G-CSF.The results of the meta-analyses show that the use of HGFs such as G-CSF has a significant effect on several important clinical outcomes associated with the management of patients with aggressive NHL. They result in far fewer patients with neutropenia and as a consequence fewer patients with infection, fewer who are hospitalised due to infection and fewer whose chemotherapy treatment has to be delayed. There was however no evidence of an improvement on tumour remission or survival.Our simple cost-effectiveness analysis, based on direct costs alone, suggests that using G-CSF as a primary prophylaxis for chemotherapy-induced neutropenia would be more expensive to the service provider than not using it, a similar conclusion found by others in Canada and Italy (The evidence shows that the routine use of HGFs is worthwhile. Although they may seem to be expensive to the health service, it would be unsatisfactory to choose financial saving over patient health.In 1999, there were 9014 new cases of NHL in the UK  and it i


Studies on the zebrafish model have contributed to our understanding of severalimportant developmental processes, especially those that can be easily studied in theembryo. However, our knowledge on late events such as gonad differentiation in thezebrafish is still limited. Here we provide an analysis on the gene sets expressed inthe adult zebrafish testis and ovary in an attempt to identify genes with potentialrole in (zebra)fish gonad development and function. We produced 10 533 expressedsequence tags (ESTs) from zebrafish testis or ovary and downloaded an additional23 642 gonad-derived sequences from the zebrafish EST database. We clustered thesesequences together with over 13 000 kidney-derived zebrafish ESTs to study partialtranscriptomes for these three organs. We searched for genes with gonad-specificexpression by screening macroarrays containing at least 2600 unique cDNA insertswith testis-, ovary- and kidney-derived cDNA probes. Clones hybridizing to only oneof the two gonad probes were selected, and subsequently screened with computationaltools to identify 72 genes with potentially testis-specific and 97 genes with potentiallyovary-specific expression, respectively. PCR-amplification confirmed gonad-specificityfor 21 of the 45 clones tested . Our study, which involvesover 47 000 EST sequences and specialized cDNA arrays, is the first analysis of adultorgan transcriptomes of zebrafish at such a scale. The study of genes expressed inadult zebrafish testis and ovary will provide useful information on regulation of geneexpression in teleost gonads and might also contribute to our understanding of thedevelopment and differentiation of reproductive organs in vertebrates.


The incidence of undescended testis (UDT) along with hypospadias varies from 6 to 31%. The simultaneous repair of UDT and hypospadias is rarely done. Herein, we present a novel technique to use processes vaginalis as a vascular cover for neourethra in a hypospadias patient with UDT. We have done urethroplasty and orchiopexy simultaneously. This is the first report concerning the use of processes vaginalis to reinforce the urethra.Simultaneous repair of hypospadias and undescended testis.Both the patients withstood the procedure well. Postoperative period was uneventful. Patients passed urine in single stream without any fistula.In patients of undescended testis with hypospadias, simultaneous repair with the processes vaginalis flap is an ideal technique with good results. Processes vaginalis is good vascular cover for neourethra. Repair of undescended testis with hypospadias is rarely done simultaneously and there are more chances of fistula formation as there is no adequate vascular cover for the neourethra. The objective of this technique was to find new surgical technique to achieve this aim.Patient with undescended testis with hypospadias.A 12-year-old boy presented with proximal penile hypospadias and bilateral palpable undescended testis. Patient had mild chordee and length of penis was adequate. Patient was evaluated for intersex and karyotype was 46 XY. Genitoscopy revealed evidence of large prostatic utricle. After release of chordee, tube pedicle urethroplasty (Duckett's) was done. The required 5-cm long tube was constructed on number 10 feeding tube. We routinely do proximal anastomosis first before making the tube.To prevent complications such as fistula, the urethral repair needs vascular cover. At this juncture, we planned to do orchidopexy along with the repair of hypospadias. Inguinal crease incision was taken on right side and herniotomy done close to the internal ring. After mobilizing the cord structure proximally, testis was brought down into scrotum along with its processes vaginalis . Testis A 3-year-old child of partial androgen insensitivity syndrome was operated by masculizing genitoplasty. Hypospadias repair was done with tube pedicle (ducketts).The repair was covered with processes vaginalis which was brought down along with left orchidopexy. Post operative period was uneventful and patient passed single stream urine on day 10 of operation. Patient is on regular follow-up without any complication.Both the patients stood the procedure well and after removal of catheter passed urine in single stream without any fistula. Patients are on follow-up for about six months with no complications.The incidence of the undescended testis along with hypospadias varies from 6% to 31%.[There are various methods for protective intermediate vascular cover to the neourethra to prevent fistula formation, Snow described the use of Tunica vaginalis wrap. Retik waIn patients of hypospadias associated with the undescended testis, tunica vaginalis is not available to use as a protective layer for hypospadias repair. As scrotum is not well developed because of undescended testis, one cannot get easily the dartos fascia for cover of urethral repair.et al, had described method of using gubernaculum as vascular cover after dissecting it from the testis and leaving its anomalous distal attachment to the pubic tubercle intact, but the limitation of gubernaculum is that it is not long enough to cover whole urethra.[Yamataka  urethra.We are presenting two cases where we could develop processes vaginalis flap and we could do orchidopexy and reinforce neourethra with processes vaginalis with good results. Our technique is useful to cover the anastomosis and suture line as well as allow for the simultaneous repair of UDT. Processes vaginalis is long and can be easily brought down with testis with good vascular supply. To achieve adequate mobilization of processes vaginalis, herniotomy should be done at internal ring level.There are no reports in literature regarding the use of processes vaginalis as vascular cover and this simple technique will reduce the complication rate of hypospadias repair with UDT.


Granulocyte-colony-stimulating factor (G-CSF) functions as a hematopoietic growth factor and it is responsible for leukocytosis. G-CSF-producing tumors associated with leukocytosis include various types of malignancies.We report the case of a 72-year-old man with dedifferentiated liposarcoma characterized by dedifferentiated components of malignant fibrous histiocytoma (MFH)-like features in addition to well-differentiated lipoma-like liposarcoma, arising from his upper arm. Preoperative laboratory data showed leukocytosis . The serum level of G-CSF was also elevated . Nine days after the surgery, the leukocytosis was relieved  and the elevated serum G-CSF level was significantly decreased . One month after the surgery, leukocytosis gradually began to appear again. Three months after the surgery metastatic lung lesions were confirmed, and the patient subsequently died of respiratory problems. In the English literature regarding soft-tissue tumors with leukocytosis, including the current case, we could review a total of 6 cases of liposarcoma with leukocytosis. The subtype of these 6 liposarcoma cases was undifferentiated liposarcoma, comprising dedifferentiated liposarcoma in 4 cases and pleomorphic liposarcoma in 2 cases.Since the only other soft-tissue tumor that was associated with leukocytosis was MFH, and since MFH is characterized by the absence of any specific differentiation, we would like to propose a possible association between G-CSF-producing soft-tissue tumors and an undifferentiated liposarcoma lineage, such as dedifferentiated liposarcoma or pleomorphic liposarcoma. Granulocyte-colony-stimulating factor (G-CSF) enhances differentiation along the neutrophil lineage, and accelerates maturation of metamyelocytes into mature neutrophils. Consequently, G-CSF is known to function as a hematopoietic growth factor and it is known to be responsible for leukocytosis. G-CSF-producing tumors associated with leukocytosis include various types of malignancies. In epithelial cancers, the expression of G-CSF has been associated with poor differentiation and invasiveness -3. HowevA 72-year-old man who suffered from a tumor in his upper arm presented to us Figure . Body teThe blood data of white blood cells and serum level of G-CSF are summarized in Figure Expression of G-CSF  failed to be detected not only in the well-differentiated lipoma-like liposarcoma components, but also in the dedifferentiated components, immunohistochemically (data not shown).The leukocytosis seen in the current case with dedifferentiated liposarcoma seemed to be due to stimulation by the tumor-produced G-CSF, on the basis that both the leukocytosis and the elevated level of G-CSF disappeared after the tumor was resected. In a previous report, leukocytosis appeared at an advanced stage, as characterized by poorly differentiated adenocarcinoma, not in the early stage, as characterized by well-differentiated adenocaricnoma, in the same lesion of gastric cancer. In that gastric cancer case, immunopositivity of G-CSF was only seen in the poorly differentiated adenocarcinoma, not in the well-differentiated adenocarcinoma. It is thus possible that histological changes may influence the acquisition of G-CSF-producing ability . In the In the English literature, including the current case, there have been 6 cases of liposarcoma accompanied by leukocytosis and associated with G-CSF expression -12. GendMFH has been reported to be accompanied by leukocytosis, previously -6. The dIn epithelial cancers, G-CSF expression has been associated with poor differentiation and invasiveness -3. MoreoIn this report, we present a case of dedifferentiated liposarcoma with leukocytosis. The leukocytosis seemed to be associated with an elevated level of serum G-CSF. We would like to propose a possible association between G-CSF-producing soft-tissue tumors and an undifferentiated liposarcoma lineage, such as dedifferentiated liposarcoma or pleomorphic liposarcoma.The author(s) declare that they have no competing interests.AS drafted the manuscript. HM performed the immunohistochemical study. TY, HM, KT, SM and YO participated in the design of the study. YI conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The central nervous system is considered a sanctuary site for HIV-1 replication. Variables associated with HIV cerebrospinal fluid (CSF) viral load in the context of opportunistic CNS infections are poorly understood. Our objective was to evaluate the relation between: (1) CSF HIV-1 viral load and CSF cytological and biochemical characteristics ; (2) CSF HIV-1 viral load and HIV-1 plasma viral load; and (3) CSF leukocyte count and the peripheral blood CD4+ T lymphocyte count.Statistix 7.0 software .Our approach was to use a prospective collection and analysis of pre-treatment, paired CSF and plasma samples from antiretroviral-naive HIV-positive patients with cryptococcal meningitis and assisted at the Francisco J Muñiz Hospital, Buenos Aires, Argentina (period: 2004 to 2006). We measured HIV CSF and plasma levels by polymerase chain reaction using the Cobas Amplicor HIV-1 Monitor Test version 1.5 (Roche). Data were processed with r = 0.4, 95% CI = 0.13-0.63, p = 0.01). No correlation was found with the plasma viral load, CSF protein concentration and cryptococcal antigen titer. A positive correlation was found between peripheral blood CD4+ T lymphocyte count and the CSF leukocyte count .Samples from 34 patients were analyzed. CSF leukocyte count showed statistically significant correlation with CSF HIV-1 viral load (Cryptococcus neoformans.Our study suggests that CSF leukocyte count influences CSF HIV-1 viral load in patients with meningitis caused by  Invasion of the central nervous system (CNS) occurs early in the course of HIV-1 infection, but the exact mechanisms of HIV-1 entry to the brain are still under debate . AlthougHowever, the blood-brain barrier is permeable to immune cells, which has led to the proposal that HIV-1 might be transported to the CNS by infected immune cells (Trojan horse hypothesis) . The bioHIV-1 is found in the CSF of most infected individuals at all stages of the disease, including primary infection and the asymptomatic and symptomatic  phases ,7,8. It HIV-1 infection also induces a humoral immune response in the CNS, as measured by an increased immunoglobulin G index. The highest levels of CSF neopterin are found in infected patients with CNS opportunistic infections or AIDS dementia complex, although asymptomatic carriers may also show moderately increased levels ,9-11. ThIn patients without opportunistic infections, CSF HIV-1 viral load depends mainly on the plasma viral load and the CSF leukocyte count ,12. HoweCytomegalovirus encephalitis, progressive multifocal leukoencephalopathy, and tuberculous meningitis) that were analyzed together [However, these studies included a low number of patients with different CNS opportunistic infections , but this appears not to be the case in symptomatic patients, possibly due to the low CD4+ T cell counts found in this latter population . HoweverIn vitro studies have demonstrated that a clinically relevant concentration of CCPA enhances HIV-1 production in H9 cells and peripheral mononuclear cells [in vitro interaction, it is necessary to investigate the potential correlation between this microorganism burden (CCPA titers) and CSF HIV-1 viral load in a clinical setting.ar cells . ConsideCryptococcus neoformans. The objectives were to evaluate the relationships between: CSF HIV-1 viral load and CSF cytological and biochemical characteristics ; CSF HIV-1 viral load and HIV-1 plasma viral load; and CSF leukocyte count and peripheral blood CD4+ T lymphocyte count.In this context, we designed an observational prospective investigation to describe the factors that may influence CSF HIV-1 viral load in patients with meningitis caused by We conducted a prospective single-centre observational non-comparative study. We prospectively collected and analyzed pre-treatment, paired CSF and blood samples from 34 antiretroviral-naive HIV-1 positive patients with culture-confirmed cryptococcal meningitis at the Francisco J Muñiz Infectious Diseases Hospital in Buenos Aires, Argentina (period: 2004 to 2006). All procedures were in accordance with this institution's ethical standards and with the Helsinki Declaration of 1975, as revised in 1983.Cryptococcus neoformans or evidence of another simultaneous CNS infection were excluded.CSF cytological and biochemical characteristics, including CCPA titer, were analyzed. All samples were routinely cultured on Saboureaud agar plates to detect fungal pathogens, and in other media to detect mycobacteria (Lowenstein-Jensen) and common aerobic bacteria (blood agar). Patients with positive cultures for pathogens other than Cryptococcus Antigen Detection System, Immuno-Mycologics, Norman, OK) following the manufacturer's instructions. Peripheral blood CD4+ T lymphocyte count was determined by flow cytometry .For each patient, we measured CSF HIV-1 and plasma viral loads in the same assay by polymerase chain reaction using the Cobas Amplicor Monitor Test version 1.5  following the manufacturer's instructions. CCPA titer was measured by latex agglutination . Scatter plots were made using SPSS 15.0 software .Linear regression analysis was performed to evaluate the relationships between: CSF HIV-1 RNA viral load and CSF leukocyte count, protein concentration, CCPA titer, and HIV-1 plasma viral load; and between CSF leukocyte count and peripheral blood CD4+ T lymphocyte count. Data were processed with Cytomegalovirus encephalitis, cerebral toxoplasmosis, or Chagas' encephalitis). Therefore, the samples from 34 patients were used for the final analysis. The median (interquartile range) age was 35 years (32-42), and 74% of patients were male.Paired, pre-treatment CSF and blood samples from 37 HIV-1 infected patients with culture-confirmed cryptococcal meningitis were collected. CSF samples from three patients were excluded due to the diagnosis of a simultaneous opportunistic CNS disease (either 3 (11-43). The CSF cytological and biochemical characteristics were as follows: leukocyte count, 10 cells/mm3 (4-23); glucose, 39 mg/dL (32.7-50); protein concentration 0.75 g/L (0.48-1.06); and CCPA titer, 1/100 dilutions (1/10-1/1000). Eighty-five percent of patients had a positive CSF India ink examination.All data are presented as the median (interquartile range), unless otherwise specified. CD4+ T lymphocyte count was 24 cells/mm10 copies/mL (4.96-5.87) and 4.83 log10 copies/mL (3.77-5.47), respectively . There was no evidence of a statistical correlation between plasma and CSF viral load.HIV-1 plasma viral load was higher than CSF viral load, with values of 5.43 logr = 0.4, 95% CI = 0.13-0.63, p = 0.01), as shown in Figure p > 0.05) between CSF HIV-1 viral load and protein concentration, or CCPA titer , as shown in Figure Finally, a positive correlation was found between peripheral blood CD4+ T lymphocyte count and the absolute CSF leukocyte count  may harbour HIV-1 and thus constitute an exogenous source of the virus, contributing to the viral load at this site .3 and the median CD4 T cell count of our population was 24 cells/mm3[Our study also shows that viral load in cryptococcal meningitis is higher in plasma than in CSF, although no correlation was found between the plasma and CSF HIV-1 RNA viral loads. This is not unexpected, considering that such a correlation was described in patients with CD4 counts of >200 cells/mmcells/mm3,12,21.In vitro studies have shown that antigens from certain opportunistic organisms, such as CCPA from Cryptococcus neoformans, may promote viral replication [lication . This suin vivo. To the best of our knowledge, this is the first study to assess the relationship between the levels of an opportunistic pathogen in CSF and HIV-1 viral load in a clinical setting.Therefore, our study tentatively suggests that the microorganism burden associated with a given CCPA titer may not be a determining factor of CSF HIV-1 viral load Cryptococcus neoformans, peripheral blood CD4+ T lymphocyte counts influence the cellular response in the CSF.A positive correlation was found between peripheral CD4+ T lymphocyte count and CSF leukocyte count. This finding is unexpected, considering the suppressed immune systems and modest CSF pleocytosis of our patient population. These results suggest that, despite the advanced level of immunodeficiency observed in patients with meningitis by Our study has several limitations. First, we used CCPA titer in the linear regression analysis to evaluate the correlation between the disease burden of this pathogen and CSF HIV-1 RNA viral load. Although previous reports demonstrated a strong correlation between cryptococcal colony-forming units in quantitative cultures and this antigen titer as measures of microorganism load in CSF , the firCryptococcus neoformans [Second, we did not measure cytokines in the CSF. Some molecules, such as TNF-α, IFN-γ, IL-6, and IL-8, are negatively correlated with baseline colony-forming units of oformans , but no Cryptococcus neoformans, and cannot be extrapolated to other CNS infections, such as Cytomegalovirus encephalitis, tuberculous meningitis, progressive multifocal leukoencephalopathy, or cerebral toxoplasmosis.Third, our results are representative only of patients with meningitis caused by Finally, the observational design of this study  precluded the inclusion of a control group of asymptomatic subjects  with which to compare CSF viral loads.The present study shows that CSF HIV-1 viral load in patients with cryptococcal meningitis is positively correlated with CSF leukocyte count, and not with plasma viral load. The CSF cellular response may depend in part on the peripheral blood CD4+ T lymphocyte count, despite the advanced level of immunodeficiency observed in these patients, as a positive correlation was found between both variables. Further investigations are needed to elucidate the relationship between other CNS opportunistic infections and CSF HIV-1 viral load.The authors declare that they have no competing interests.DMC was responsible for the design of the study, patient enrolment, data analysis, and writing of the manuscript. AMC was responsible for the proceedings performed in the Virology Unit, and was co-writer of the manuscript. HR was responsible for patient enrolment. AA and RN were responsible for proceedings performed in the Mycology Unit. MBB undertook design of the study, and was supervisor of the proceedings performed in the Virology Unit, and co-writer and final supervisor of the manuscript. JAB undertook design and general supervision of the study, and was co-writer and final supervisor of the manuscript.