Older age and a higher CSF/blood viral load ratio were associated with compartmentalization, consistent with independent replication in the CNS. Cell tropism was assessed using pseudotyped reporter viruses to enter a cell line on which CD4 and CCR5 receptor expression can be differentially induced. In a subset of compartmentalized cases , the CNS virus was able to infect cells with low CD4 surface expression, a hallmark of macrophage-tropic viruses, and intermediate compartmentalization early was associated with an intermediate CD4 entry phenotype. Transmission of multiple variants was observed for 5 children; in several cases, one variant was sequestered within the CNS, consistent with early stochastic colonization of the CNS by virus. Thus we hypothesize two pathways to compartmentalization: early stochastic sequestration in the CNS of one of multiple variants transmitted from mother to child, and emergence of compartmentalized variants later in infection, on average at age 13.5 months, and becoming fully apparent in the CSF by age 18 months. Overall, compartmentalized viral replication in the CNS occurred in half of children by year three.HIV-1 subtype B replication in the CNS can occur in CD4+ T cells or macrophages/microglia in adults. However, little is known about CNS infection in children or the ability of subtype C HIV-1 to evolve macrophage-tropic variants. In this study, we examined HIV-1 variants in ART-naïve children aged three years or younger to determine viral genotypes and phenotypes associated with HIV-1 subtype C pediatric CNS infection. We examined HIV-1 subtype C populations in blood and CSF of 43 Malawian children with neurodevelopmental delay or acute neurological symptoms. Using single genome amplification (SGA) and phylogenetic analysis of the full-length  Genetically compartmentalized human immunodeficiency virus type 1 (HIV-1) subtype B populations can be variably detected in the cerebrospinal fluid (CSF) of adults. Compartmentalization is indicative of local CNS replication, and late in disease is linked to HIV-associated dementia (HAD). Compartmentalized viral populations can comprise either CCR5-using T cell-tropic or macrophage-tropic virus. Little is known about CNS infection in children or the ability of subtype C HIV-1 to evolve macrophage-tropic variants. We examined viral populations in the blood and CSF of HIV-1 subtype C-infected children. We found an intermediate level of compartmentalization in about half of the children under 18 months of age. About 50% of children older than 18 months had clearly compartmentalized virus in the CSF/CNS, and in some cases CSF virus evolved a low CD4 entry phenotype. In some of the children two variants were transmitted from the mother. In several of these cases one of the transmitted viruses was replicating in the CNS while the other was found predominantly in the blood/periphery. Our results suggest that compartmentalized CSF/CNS populations can be detected in up to 50% of children by year three, either established early in the infection or through sequestration of a transmitted variant within the CNS. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can occur shortly after transmission, and compartmentalized HIV-1 variants, genetically distinct from the virus in the blood, can be detected in the cerebrospinal fluid (CSF) in some individuals throughout the course of infection. Detection of compartmentalized CSF viral populations during primary infection suggests that compartmentalization can occur early in the absence of overt neurological symptoms Compartmentalized HIV-1 subtype B CNS populations can be either CCR5 (R5)-using T cell-tropic or macrophage-tropic Information on the genetic and phenotypic characteristics of HIV-1 within the CNS of infants and young children is scarce. HIV-1 CNS disease is often an AIDS-defining illness in children env gene env gene was determined for each amplicon and phylogenetic analysis was completed.We examined viral populations in paired peripheral blood and CSF samples collected from 43 HIV-infected Malawian children presenting with either neurodevelopmental delay or acute neurologic symptoms . Subjects were infected with HIV-1 subtype C, were ART naïve, and ranged in age from 3 to 35 months. Blood viral loads ranged from 4,344 to >80,000,000 copies/mL and CSF viral loads ranged from 56 to 4,745,000 copies/mL. The virological characteristics for each subject are summarized in P value<0.05 and a genetically distinct CSF population with a bootstrap value ≥40; we used a relatively low bootstrap value in this exploratory analysis because of the overall low diversity of the viral population in these children. Intermediate populations were defined by a Slatkin-Maddison P value>0.05 but with visual evidence of a minor CSF subpopulation of ≥4 CSF amplicons and a bootstrap value ≥40. Equilibrated populations were defined by a Slatkin-Maddison P value>0.05 and no evidence of a minor or major CSF population  had equilibrated viral populations in their blood and CSF . For theIn 11 out of 43 subjects (26%), an intermediate condition existed where the peripheral blood and CSF HIV-1 populations were not uniformly equilibrated and contained a minor CSF subpopulation . Six of Significant genetic compartmentalization was detected between the blood and CSF populations in 12 out of 43 subjects (28%)  indicatiP = 0.05) and intermediate subjects (P = 0.005)  or intermediate subjects (P = 0.001)  . As the = 0.001) , consistBayesian Evolutionary Analysis by Sampling Trees (BEAST) As the majority of subjects were probably infected at or around the time of birth, we were able to depict the occurrence of compartmentalization as a function of time . Before For 5 subjects for whom the predicted TMRCA was substantially greater than the age of the subject , furtherMacrophage tropism of HIV-1 is associated with the ability to infect cells expressing low levels of CD4 Our study design involves cross-sectional sampling and thus has several limitations, especially with regard to inferring temporal relationship. However, there is a wide distribution of ages of the subjects within the enrollment criteria allowing us to compare between different age groups and to draw correlations based on age. Also, the virus carries the history of longitudinal evolution in its sequence, thus allowing us to infer dates of bottlenecks in the history of viral replication. While cross-sectional analyses are inherently limited, we have some basis for suggesting temporal relationships in the observed phenomenon. Based on our results we hypothesize four distinct states to describe the relationship between virus in the CSF/CNS and virus in the blood/periphery. The first state has no genetic evidence for HIV-1 replication in the CNS, wherein the only virus detected in the CSF is genetically similar to that in the blood and is typically present at 1% or lower of the level in the blood, possibly due to some import or spill pathway from the blood into the CSF. The second state occurs prior to 18 months of age and involves minor compartmentalization of the CSF viral population, suggesting some local replication in the CSF/CNS but not to a level where the viral load increases in the CSF. In 10–20% of these children there is evidence for the initial evolution of virus that can use lower levels of CD4, potentially on a path to becoming macrophage-tropic. The third state occurs in about half of the children older than 18 months of age, and in this state the viral population in the CSF shows strong evidence of genetic compartmentalization, indicative of local replication and evolution within the CNS. This is also accompanied by a higher relative viral load in the CSF due to the local production of virus well above the low level that is imported from the periphery. In about 10–20% of the children with compartmentalized virus in the CSF, we identified variants that had evolved to use low levels of CD4, which we presume indicates that the virus was now growing in macrophages and/or microglia within the CNS. The fourth state involves multiple variant transmissions from mother to infant of which one variant preferentially replicates in the CNS and another replicates in the periphery. As HIV-1 replication in the CNS can contribute to neurological disease, further research should determine whether the ability to detect different states of CSF viral populations within the CNS of young children could guide strategies to monitor and prevent neurodevelopmental disorders in HIV-infected children.env genes tested from three of these subjects . In these cases it appears that compartmentalization is established after the initial stages of infection, with the compartmentalized virus emerging on average at 13.5 months but with outgrowth in the CNS only becoming apparent in the CSF approximately 18 months after birth. Thus we can identify two distinct pathways to compartmentalization: early sequestration of a transmitted virus in the CNS, or the later establishment of independently replicating virus that originates in the periphery. The compartmentalized lineage in the intermediate group appeared earlier after birth (mean 5.1 months) compared to the compartmentalized lineage in the compartmentalized group (mean 13.5 months) . This may be an indication that the intermediate state represents susceptibility to viral replication in the CNS but that there is a subsequent bottleneck that defines the CNS population.We can infer several other features of compartmentalization by comparing the age of the infant to the inferred age of the viral population using BEAST for those infants infected with a single variant. In the remaining 10 infants with compartmentalization who were infected with a single variant, the age of the CSF/CNS compartmentalized viral population was significantly less than the age of the entire viral population when compared to the age of the infant , neopterin, IP-10, and neurofilament light subunit (NFL). Production of these inflammatory markers has been observed in animal models env protein phenotypic analyses. Blood plasma and CSF HIV-1 viral loads (copies/mL) were determined by the UNC Chapel Hill Center for AIDS Research Virology Core.The study was approved by the Institutional Review Boards of the University of North Carolina at Chapel Hill and the University of Malawi College of Medicine in Blantyre. Permission to participate in the research study was obtained for all children through written informed consent by the caregiver. All subjects included in this study were HIV-1 subtype C-infected children between 3 and 35 months of age. HIV-1 infection was verified at time of enrollment by a positive PCR for HIV DNA/RNA if <18 months of age or two positive rapid HIV antibody tests after age 18 months. Samples were collected at a one pre-HAART baseline visit for all subjects. CSF and blood plasma samples were used for viral genetic compartmentalization and env gene through the 3′ LTR U3 end was conducted as previously described 5′-TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′; nt 9607 to 9636), and EnvA 5′-TGAGGCTTAAGCAGTGGGTTCC-3′; nt 9591 to 9612), were used for the first and second rounds of PCR, respectively. PCR amplicons were sequenced from the start of env through env gp41, gp160 end (HXB2 numbering of positions 6110–8833). Chromatograms with double peaks, indicating amplification from more than one cDNA template, as well as sequences with frameshift mutations resulting in premature stop codons, were excluded from analysis.Subtype C HIV-1 RNA was isolated from blood plasma and CSF samples as previously described env genes were performed using ClustalW DNA sequences alignments of −5 substitutions/site/generation was fixed under a strict clock model, as determined by calculation of inter-patient percent difference in the plasma nucleotide sequence. The HKY nucleotide substitution model had estimated base frequencies and a gamma-distributed rate heterogeneity (4 gamma categories). A coalescent Bayesian Skyline tree prior with a Piecewise-constant skyline model was used (4 groups). The MCMC algorithm was run for 30 million generations, logging every 1000 and with a 10% burn-in. The results from at least two independent runs were combined, and the effective sample size for all estimates was >200. A generation time of 1.5 days was used for estimation of time to the MRCA A Bayesian Markov Chain Monte Carlo (MCMC) approach, as implemented in BEAST v.1.6.1 5′ CACCGGCTTAGGCATCTCCTGTGGCAGGAAGAA-3′; nt 5950–5982) and EnvN env amplicons were then gel purified using the Qiagen gel extraction kit (QIagen). Purified HIV-1 env genes (50 ng) were cloned into the pcDNA3.1D/V5-His-TOPO expression vector (invitrogen) using the pcDNA 3.1 directional TOPO expression kit (Invitrogen) and the entire cloning reaction (6 µl) was transformed into MAX Efficiency Stbl2 competent cells (50 µl) as per the manufacturer's instructions. Bacterial colonies were screened for correct insertion of the HIV-1 env gene using colony PCR, and DNA was extracted from 3–6 positive colonies using the Qiaprep spin miniprep kit (Qiagen).SGA amplicons, selected based on the subject's phylogenetic tree structure, were re-amplified from the first-round nested PCR product using the Phusion hot start high-fidelity DNA polymerase (Finnzymes) and the primers EnvAClon (293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/ml of penicillin and streptomycin. 293-Affinofile cells env expression vector and the pNL4-3.LucR-E- HIV-1 backbone . Prior to transfection, 293T cells were seeded at a density of 4.8×105 cells/well in 6-well tissue culture plates coated with 10% poly-L-lysine. Transfection medium was replaced five hours post-transfection with fresh culture medium and the cells were incubated at 37°C for 48 hours, after which viral supernatants were filtered with 0.45 µM filters (Millipore) and stored at −80°C.Each Env-pseudotyped luciferase reporter virus was generated using the Fugene 6 transfection reagent and protocol (Roche) to co-transfect 293T cells with an QuantiBRITE beads (BD Biosciences).293-Affinofile cell 4 cells/well. 18–24 hours later, expression of CD4 and CCR5 was induced at two conditions in triplicate: CD4high/CCR5high  and CD4low/CCR5high (0 ng/ml doxy and 5 µM ponA). 18 to 24 hours later, the induction medium was removed and gently replaced with 100 µl of fresh, warmed culture medium containing env-pseudotyped virus. The infection plates were spinoculated Env-pseudotyped luciferase reporter viruses were first titered in triplicate in a 96-well plate format on 293-Affinofile cells env nucleotide sequences determined in this study have been deposited in GenBank under accession numbers KC186127-KC187733.The HIV-1 Figure S1No relationship between viral load and subject classification. Relationships between blood or CSF viral loads and virological classifications were assessed using the Mann-Whitney test in GraphPad Prism 4. Horizontal bars represent median values. (a) Comparisons between blood viral load and CSF compartment classification  (b) Comparisons between blood viral load and TMRCA classification. (c) Comparisons between CSF viral load and TMRCA classification.(TIF)Click here for additional data file.Figure S2Compartmentalized subject 4004 exhibiting three or more transmitted viruses. Phylogenetic and sequence analysis of plasma and CSF HIV-1 populations for subject 4004. (a) Neighbor-joining tree. CSF sequences are labeled with solid blue circles, and plasma sequences (PL) are labeled with solid red triangles. Bootstrap values ≥40 are indicated (*) at the appropriate nodes. Genetic distance is scaled at the bottom of the figure (0.001) and indicates the number of nucleotide substitutions per site between env sequences. The subject's age is noted, as well as the TMRCA for the transmitted and compartmentalized populations. The CNS sequestered population is represented by an open black circle. (b) Highlighter plot of aligned env plasma and CSF sequences, generated at www.hiv.lanl.gov. The HXB2 base number is indicated on the x axis, and the sequence identifier is indicated on the y axis. Base changes are indicated by the following ticks on the highlighter plot: A, green; T, red; G, orange; and C, blue. Sequestered populations resulting from the transmitted viruses are separated by heavy black lines. One variant was sequestered within the CSF (top) and additional variants were established within the blood (middle). Another variant was also isolated from the CSF (bottom), but the population was not maintained.(TIF)Click here for additional data file.Figure S3Intermediate subject 3017 exhibiting two transmitted viruses. Phylogenetic and sequence analysis of plasma and CSF HIV-1 populations for subject 3017. (a) Neighbor-joining tree. Sequences from the CSF are labeled with solid blue circles, and plasma sequences (PL) are labeled with solid red triangles. Bootstrap values ≥40 are indicated (*) at the appropriate nodes. Genetic distance is scaled at the bottom of the figure (0.001) and indicates the number of nucleotide substitutions per site between env sequences. The subject's age is noted, as well as the overall TMRCA. BEAST was unable to assign a TMRCA to the internal nodes due to recombination in the population. The CNS sequestered population is represented by an open black circle. (b) Highlighter plot of aligned env plasma and CSF sequences, generated at www.hiv.lanl.gov. The highlighter plot characteristics are the same as those stated in env genetic diversity detected in both the plasma and CSF populations. The transmitted CSF variant was maintained within the CNS, generating a minor CSF population with small local replication, accounting for the intermediate state observed for this subject.(TIF)Click here for additional data file.Figure S4Intermediate subject 4048 exhibiting greater than two transmitted viruses. Phylogenetic and sequence analysis of plasma and CSF HIV-1 populations for subject 4048. (a) Neighbor-joining tree. Sequences from the CSF are labeled with solid blue circles, and plasma sequences (PL) are labeled with solid red triangles. Bootstrap values ≥40 are indicated (*) at the appropriate nodes. The phylogenetic tree characteristics are the same as those stated in env plasma and CSF sequences, generated at www.hiv.lanl.gov. The highlighter plot characteristics are the same as those stated in env genetic diversity detected in both the plasma and CSF populations. The transmitted CSF variant was maintained within the CNS, generating a minor CSF population with small local replication, accounting for the intermediate state observed for this subject.(TIF)Click here for additional data file.Figure S5Equilibrated subject 4055 exhibiting two transmitted viruses. Phylogenetic and sequence analysis of plasma and CSF HIV-1 populations for subject 4055. (a) Neighbor-joining tree. Sequences from the CSF are labeled with solid blue circles, and plasma sequences (PL) are labeled with solid red triangles. Bootstrap values ≥40 are indicated (*) at the appropriate nodes. Genetic distance is scaled at the bottom of the figure (0.001) and indicates the number of nucleotide substitutions per site between env sequences. The subject's age is noted, as well as the overall TMRCA. (b) Highlighter plot of aligned env plasma and CSF sequences, generated at www.hiv.lanl.gov. The highlighter plot characteristics are the same as those stated in env genetic diversity detected in both the plasma and CSF populations. Both variants were maintained within the blood and CSF, accounting for the equilibrated state observed for this patient.(TIF)Click here for additional data file.Figure S6No contamination was observed between subjects. Neighbor-joining phylogenetic tree . env sequences from the CSF are labeled with solid blue circles and env sequences from the blood plasma are labeled with solid red triangles. Genetic distance is indicated at the bottom of the figure and indicates the number of nucleotide substitutions per site between env sequences. Each subject ID is indicated.(TIF)Click here for additional data file.


The appearance of Aβ42 peptide deposits is admitted to be a key event in the pathogenesis of Alzheimer's disease, although amyloid deposits also occur in aged non-demented subjects. Aβ42 is a degradation product of the amyloid protein precursor (APP). It can be catabolized by several enzymes, reabsorbed by capillaries or cleared into cerebrospinal fluid (CSF). The possible involvement of a decrease in CSF turnover in A4β2 deposit formation is up to now poorly known. We therefore investigated a possible relationship between a reduced CSF turnover and the CSF levels of the A4β2 peptide.To this aim, CSF of 31 patients with decreased CSF turnover were studied. These patients presented chronic hydrocephalus communicating or obstructive, which required surgery . Nine subjects had idiopathic normal pressure hydrocephalus (iNPH), and the other 22 chronic hydrocephalus from other origins (oCH).The Aβ42 peptide concentration was measured by an ELISA test in 31 ventricular CSF samples and in 5 lumbar CSF samples from patients with communicating hydrocephalus.The 5 patients with lumbar CSF analysis had similar levels of lumbar and ventricular Aβ42. A significant reduction in Aβ42 ventricular levels was observed in 24 / 31 patients with hydrocephalus. The values were lower than 300 pg/ml in 5 out of 9 subjects with iNPH, and in 15 out of 22 subjects with oCH.The decrease of CSF Aβ42 seems to occur independently of the surgical hydrocephalus aetiology. This suggests that a CSF reduced turnover may play an important role in the decrease of CSF Aβ42 concentration. Alzheimer's disease (AD) is characterized by the presence of diffuse and senile dense-core plaques. Senile plaques may also be seen in non-demented elderly patients. According to the « amyloid theory », the appearance of Aβ42 deposits is considered as the crucial event in the evolution of AD .Aβ42 can be reabsorbed by capillaries via specific receptors, the most charaterized being LRP-1 . In AD pIn vitro, Aβ42 monomers, incubated into CSF, build spontaneously oligomers and then fibrils; these phenomena are time-dependant ,7. ExperSeveral human types of chronic hydrocephalus with altered CSF turnover are known, such as idiopathic Normal Pressure Hydrocephalus (iNPH), and those following meningeal haemorraghe or related to surgery for Sylvius acqueduct stenosis, or brain tumors. iNPH is a rare disease (about 1 case for 200000 people), described in 1965, and characterized by a clinical triad of mental alteration, gait disturbance and sphincter dysfunction .In order to investigate a possible involvement of a decrease in CSF turn-over on Aβ42 clearance, we studied the ventricular levels of this peptide in patients suffering from chronic hydrocephalus, which required surgery . We compared lumbar and ventricular CSF levels in some patients with communicating hydrocephalus, we found ventricular levels are decreased in iNPH and similar to those measured in patients suffering from chronic hydrocephalus, communicating or obstructive.The prospective study was performed on a panel of 31 consecutive patients , aged from 28 to 86 years . All of them were affected with chronic hydrocephalus from various aetiologies, which required either a ventriculostomy or a ventriculo-peritoneal shunt. In Phase Contrast MRI, all the patients had ventricular dilatation and periventricular edema due to transependymal resorption of CSF; the patients with communicating hydrocephalus were also characterized by hyperdynamic aqueductal CSF flow .3 of CSF were aspired, as usual, in order to check the position or the functionality of the shunt. After sampling for cyto-bacteriological analysis, residual CSF was immediately aliquoted in polypropylene tubes placed on ice, and transported to the Biochemistry Laboratory, where it was centrifuged at 4000 × g for 10 min., then stored at - 80°C. This study was achieved according to the French bioethic rules for clinical studies on tailing samples. All the patients gave first their agreement for this protocol.The experimental protocol was the following: during the surgery, 3-4 cmThe respective diagnoses for these patients were: 9 iNPH, and 22 chronic hydrocephalus from other origins (oCH): 3 related to a meningitis or a meningeal haemorrhage, 4 due to stenosis of the Sylvius aqueduct, 10 from benign or malignant tumor origin, 2 following a meningioma surgery, 2 due to a Arnold-Chiari syndrome or a neonatal hydrocephaly and one secondary to the rupture of a dermoid cyst. Five out of these patients  also benefited of a prior subtractive lumbar tap.The concentrations of Aβ42 were measured using a commercial ELISA test . With this method, the intra-assay variability coefficient is less than 6%. The range for the titration zone is 125-2000 pg/ml. CSF lumbar Aβ42 levels higher than 500 pg/ml are considered as normal [t test corrected for unequal variance. Correlations between values of Aβ42 measured in lumbar and ventricular CSF, were analyzed using the Pearson's test. P < 0.05 were considered as significant.Ventricular Aβ42 values in the two groups  were compared using the Student' There was no correlation between Aβ42 levels and age. In the 5 patients with lumbar CSF analysis, lumbar and ventricular Aβ42 levels  were found similar and highly correlated . The ventricular and lumbar protein concentrations are often different, depending upon their origin. Indeed, brain proteins are considered to generally exhibit a higher level in intraventricular CSF, whereas blood proteins are more concentrated in lumbar CSF ,14. HoweThere are no available reference values well established for Aβ42 ventricular levels in healthy individuals. Therefore, we measured Aβ42 levels in lumbar and ventricular CSF of 5 patients with chronic communicating hydrocephalus and we found that ventricular and lumbar Aβ42 values are not significantly different. Despite the small sample size, this fact suggests the ventricular CSF Aβ42 concentrations measured in this study seem to be relevant for a comparison with those existing in lumbar CSF of control subjects.In iNPH, several studies -19 demonSeveral hypothesis might be put forward to explain the decrease in Aβ42 levels. First, oligomer formation, favoured by the altered CSF turnover in NPH, could partially mask the antigenic sites of the peptide, especially the C-terminal part, which is hidden inside the hydrophobic core of the aggregate. This therefore could lead to an underestimation of the Aβ42 levels when using an ELISA technique . Second,et al, investigating the relationship between amyloid neuropathology and post-mortem CSF Aβ42 levels in an autopsy sample of 155 patients with dementia found that a high number of plaques is associated with reduced levels of ventricular Aβ42 [However, an intra-cerebral sequestration of amyloid protein most likely occurs. Strozyk lar Aβ42 .in vivo using PET scanning with PIB [et al [et al [via LRP-I [The « B compound of Pittsburgh » (PIB), a thioflavine derivative, which is a ligand of Aβ42, is able, after i.v. injection, to cross the blood brain barrier and bind to Aβ peptides. Molecular neuroimaging allows to detect amyloid pathology with PIB . Severalwith PIB ,28 usingB [et al  demonstrl [et al  also shol [et al , which cia LRP-I  and loweia LRP-I ; this suIt is interesting to note, based on data derived from the ADNI database, an inverse correlation between ventricular size and CSF Aβ42 levels is observed in controls, MCI and AD, which also supports the hypothesis that the CSF turnover could play an important role in the regulation of the CSF Aβ42 peptide concentration .Our study shows that ventricular and lumbar levels of Aβ42 are not significantly different in surgical communicating hydrocephalus. Patients with iNPH exhibit a frequent decrease in ventricular CSF Aβ42 peptide levels, which is in good agreement with decreased lumbar levels previously described. This is consistent with an intra-cerebral sequestration of the peptide resulting from the reduced CSF turnover. Similarly, patients with surgical chronic hydrocephalus of other origins show an impairment of CSF turnover and also exhibit a decrease in Aβ42 ventricular levels. Thus, the decline in CSF Aβ42 concentration is not specific of iNPH and seems independent of the surgical hydrocephalus aetiology. This study suggests that the reduced CSF turnover is a key event in the decrease of Aβ42.CSF: cerebrospinal fluid; iNPH: idiopathic normal pressure hydrocephalus; oCH: chronic hydrocephalus of other origins; PIB: B compound of PittsburghThe authors declare that they have no competing interests.JMS conceived the study, led this project and drafted the manuscript with PJ and JCM. JP, AF, PT, DL, OG, and JMS selected patients. JP, AF, PT and DL removed CSF during surgery. NL, AMB, JCM performed assays. OG performed the statistical analysis. All authors read and approved the final manuscript. All the authors contributed equally to this work.


Allograft/xenograft rejection is associated with “passenger leukocyte” migration from the organ into recipient lymph nodes. In Study 1, we attempted to deplete leukocytes from potential kidney “donor” pigs, using two regimens of total body irradiation. A dose of 700 cGy was administered, followed by either 800 cGy (“low-dose”) or 1,300 cGy (“high dose”) with the kidneys shielded. Neither regimen was entirely successful in depleting all leukocytes, although remaining T and 8 cell numbers were negligible. Study 2 was aimed at providing an indication of whether near-complete depletion of leukocytes had any major impact on kidney allograft survival. In non-immunosuppressed recipient pigs, survival of a kidney from a donor that received high-dose irradiation was compared with that of a kidney taken from a non-irradiated donor. Kidney graft survival was 9 and 7 days, respectively, suggesting that depletion had little impact on graft survival. The lack of effect may have been related to (i) inadequate depletion of passenger leukocytes, thus not preventing a direct T cell response, (ii) the presence of dead or dying leukocytes (antigens), thus not preventing an indirect T cell response, or (iii) constitutive expression of MHC class II and B7 molecules on the porcine vascular endothelium, activating recipient T cells. Donor-derived passenger leukocytes can initiate graft rejection in some settings, whereas in others they contribute to graft acceptance –3. In a survival . In animlografts . In contlografts . + dendritic cells present in a donor organ is a poor predictor of graft outcome, and survival of solid organ allografts is more closely related to the density of the donor dendritic cell network within the graft [The absolute number of MHC class IIhe graft . Prior iThe primary aim of the present study, therefore, was to determine whether a sufficient dose of irradiation could be administered to the “donor” pig to kill the leukocytes without seriously damaging the kidney (to be transplanted). The secondary aim was to determine whether complete or near-complete depletion of passenger leukocytes delayed rejection after kidney Tx.Total body irradiation of the donor of 700 cGy with a further 800 cGy or 1,300 cGy with the kidneys shielded proved insufficient to deplete all leukocytes. However, the function of the irradiated kidney and any possible protection from rejection was tested by Tx into a recipient pig without immunosuppressive therapy.n = 3 in each group) and as sources of kidneys (n = 3) for Tx into recipients (n = 3). All animal care and procedures were in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health .Outbred large white landrace pigs , aged 4–10 months, weighing 20–50 kg, were used to assess two irradiation regimens  and received TBI of 700 cGy (350 cGy to each side) and then, on the same occasion, received a further 800 or 1300 cGy (400 or 650 cGy to each side) with both kidneys shielded. The dosages were determined from a review of the literature . The pigin vitro assays throughout the course of the experiment.In all pigs, the right internal and external jugular veins were cannulated with silastic catheters, and the cannulae tunneled under the skin to the back, where they were brought out through the skin. These allowed access for drug administration and blood withdrawal for monitoring of renal function, tacrolimus levels (where indicated), and μmol/L (>10 mg/dL) indicated failure of the kidney.Life-supporting kidney Tx was carried out to leave no doubt that the survival of the pig depended on the function of the transplanted kidney. The right kidney of the donor pig was perfused with 2L cold (4°C) University of Wisconsin solution before nephrectomy. The recipient pig underwent right nephrectomy and kidney Tx. End-to-side anastomoses were carried out between the donor renal artery and recipient aorta and the donor renal vein and recipient inferior vena cava. The donor ureter was implanted into the recipient bladder. A double-J stent was placed from the pelvis of the kidney graft to the recipient bladder to maintain patency of the ureteric anastomosis. When the kidney graft had shown good urine output for >30 min, the native contralateral ureter was doubly ligated. Graft function was monitored daily by serum creatinine. A serum creatinine of >884 In one recipient pig, during the operative procedure (Day 0) immunosuppression was initiated with tacrolimus  at 0.1 mg/kg/day by i.v. infusion. Thereafter, tacrolimus was given i.m. at a dose of approximately 0.05 mg/kg ×2 daily, the dose being adjusted to maintain a 12 h blood trough level of 10–15 ng/mL. Blood levels of tacrolimus were monitored daily to ensure the dosage was optimum. This therapy was continued for 42 days. No other immunosuppressive therapy was administered.Complete WBC counts on pig blood were carried out using automated methodology at the University of Pittsburgh Medical Center Central Laboratory, Presbyterian Hospital, Pittsburgh, PA.After euthanasia of the pigs, necropsies were performed through a midline abdominal incision. Kidney specimens were taken at necropsy, placed in formalin, stained with hematoxylin and eosin, and examined microscopically. Kidney allograft rejection (acute cellular rejection) was scored by standard pathologic criteria according to the cooperative clinical trials in transplantation (CCTT) classification system and incorporated into the revised Banff criteria , 12.3) for flow cytometry or in mixed lymphocyte reaction (MLR) medium  for MLR assay.PBMC from donor, recipient, and third-party pigs were isolated from heparinized blood by centrifugation over Ficoll-Paque PLUS , as described previously . The totDonor and third-party pig PBMCs were stored in 10% DMSO RPMI with 20% FBS and kept at −80°C for 24 h before being stored in liquid nitrogen. For MLR, the cryopreserved donor and third-party pig PBMCs were thawed by a rapid rewarming procedure in a water bath at 37°C and washed with 10% FBS RPMI medium.μL) was lysed ×2 using ACK lysing solution to obtain pig WBCs, which were washed with PBS, resuspended with FACS buffer, and aliquoted into FACS tubes (80 μL/tube). Detection of T, B, CD4+, and CD8+ cells was determined by incubating the pig WBC with, respectively, 10 μL mouse anti-pig CD3ε-FITC mAb at a dilution of 1 : 64, mouse anti-human CD21-PE mAb at 1 : 16, mouse anti-pig CD4a-PE mAb at 1 : 16, or mouse anti-pig CD8a-PE mAb at 1 : 64. . The samples were washed twice, and the cells resuspended with 200 μL of FACS buffer. Data acquisition was performed with FACScan  using Cell Quest software. Dead cells were excluded from the analysis by side-scatter and propidium iodide staining . Data analysis was carried out using WinMDI software.Fresh heparinized pig blood (400 6 cells/mL. Pig PBMCs (0.4 × 106 cells/well) were incubated with an equal number of irradiated stimulator cells (0.4 × 106 cells/well) in 10% CPSR-3 RPMI medium at 37°C and 5% CO2 using 96-well round-bottom plates  [137 irradiator. Each assay was performed in triplicate. After 4 days, 10 μL of 3H-thymidine-labeling medium  was added to each well. Cells were harvested after 18 h on glass-fiber filter mats with a cell harvester and were analyzed by beta counting on a liquid scintillation counter. Responder pig PBMCs were stimulated with autologous, donor, and third-party pig PBMC. The results were recorded as stimulation index (ratio of the response compared to that of autologous cells).PBMC were isolated from fresh heparinized pig blood, as described above, to a concentration of 4 × 10ork, NY) . Stimulan = 3) or high-dose  irradiation. All pigs were euthanized 60 or 72 hours after irradiation for examination of the kidneys.An attempt was made to determine (i) the optimum dose of irradiation that would deplete leukocytes without damaging the kidneys, and (ii) the optimum period that should elapse before the kidneys should be excised for Tx. Pigs received either low-dose  into a nonimmunosuppressed recipient.A kidney from an not to be siblings.In Experiments n = 3) or high-dose (n = 3) irradiation indicated that the number of WBC, T cells, and B cells decreased significantly within 24 h of irradiation  for almost 5 months after discontinuation of tacrolimus immunosuppression  within 7 days  within 9 days  were significantly depleted by both dosages of irradiation tested and particularly by the higher dose (700 + 1300 cGy). By 72 h after TBI, T and B lymphocyte depletion from the blood was total except for an occasional polymorphonuclear leukocyte. The dose of irradiation selected may not have depleted all T and B cells in the thymus and/or spleen and/or bone marrow; one of the limitations of our study is that we did not monitor for leukocytes in these organs. At the higher dose of irradiation, minor injury to the kidneys was seen on histological examination, possibly related to inadequate protection of the kidneys. The pathophysiology of radiation injury of the kidney is poorly understood, and its presentation can be acute and irreversible or subtle, with a gradual progressive dysfunction over years [The irradiated pigs in the present study were euthanized 60–72 h after irradiation to prevent irradiation-related complications. All cell types measured in the blood  the donor and recipient pigs were closely related, for example, siblings, or (ii) the herd was sufficiently inbred to allow kidney Tx in the absence of graft rejection, particularly in the presence of a 42-day course of tacrolimus.kidney allograft if a short course of immunosuppressive therapy is administered. Kidney allografts between the Massachusetts General Hospital (NIH) [class I mismatched miniature swine that receive a 12-day course of cyclosporine are uniformly accepted [full-mismatch barrier is rejected, but tolerance can be achieved if high-dose tacrolimus replaces cyclosporine [The pig is known to be a relatively easy Tx model in which to develop tolerance to a al (NIH)  MHC two-accepted . Using tosporine . In the not siblings . This indicated that irradiation of the donor (in the dosage administered) did not prevent rejection (possibly because depletion of donor passenger leukocytes had been incomplete or because antigens from dead leukocytes were presented by recipient antigen-presenting cells). Furthermore, in the absence of a course of tacrolimus, operational tolerance was not achieved after kidney Tx between members of this herd.When no immunosuppressive therapy was administered to pigs that received a kidney from either a nonirradiated donor or irradiated donor, acute cellular rejection developed within 7–9 days. In both cases, the donor and recipient pigs were The present study also suggests that MLR data in pigs may not be indicative of graft outcome, as illustrated by In view of the difficulty in completely depleting passenger leukocytes in the donor and the lack of any obvious difference in survival between the two kidney allografts, we did not feel that there was any purpose in pursuing the project further.ex vivo. However, there might be several problems with this approach. Lymphocyte numbers significantly decrease after irradiation of pigs in vivo or of pig blood in vitro in a dose-dependent manner, but reduction of lymphocyte numbers after irradiation in vivo proceeds much faster than after irradiation in vitro [in vivo than in vitro or ex vivo. To have an optimum effect, irradiation depends on high oxygen content in the tissues [ex vivo kidney, and this would reduce the effect of irradiation on the passenger leukocytes within the kidney. In view of a lack of background and experience in this approach, it would probably require several experiments to elucidate the optimum dosage if the organ was to be subjected to irradiation ex vivo.It would be possible to excise the kidney from the donor pig and irradiate it in vitro . Further tissues , 23. TheCombining irradiation of the donor with the administration of a cytotoxic agent, for example, cyclophosphamide  might rIn summary, TBI at the doses administered was well tolerated by the pigs, but was not completely successful in depleting all passenger leukocytes, and this may not have completely prevented a direct T-cell response. Furthermore, we could not exclude presentation of donor antigens from dead or dying passenger leukocytes transplanted with the graft by recipient antigen-presenting cells, and therefore the indirect response may have been active.detrimental to kidney graft survival, though the results do not support this conclusion.Although kidney Tx was carried out in only one pig that had received a kidney from an irradiated donor, graft survival was only minimally prolonged compared with that of a nonirradiated kidney. We conclude that the irradiation regimens used in the present study were insufficient to deplete all leukocytes and did not prolong kidney graft survival after Tx. Further investigation of means of effectively depleting passenger leukocytes in large animals is required. The study did not rule out that TBI of the donor may be constitutively, at least in some vascular beds [+ T cells. They also constitutively express porcine B7 molecules [+ cells just as efficiently as hematopoietic cells. It may be, therefore, that, even in the complete absence of passenger leukocytes in a transplanted pig organ allograft, acute rejection may still develop unless immunosuppressive therapy is administered for a period of time.However, one other factor has to be considered in assessing the results. Porcine endothelial cells express MHC class II molecules lar beds , 26, indolecules , indicatIf pigs are to be used for these studies, it would be essential to have donors and recipients known to be of different MHC, and some immunosuppressive therapy would appear to be required, though the intensity and duration of the therapy would require careful investigation, making the model a difficult one. As it is so relatively easy to induce tolerance in a pig  it may be that the pig is not the ideal animal model in which to explore this phenomenon.


Normocellular bacterial meningitis is rarely observed in adult patients. We here report two cases of adult patients with pneumococcal meningitis with a normal cerebrospinal fluid leukocyte count and review eight other cases in the literature.Streptococcus pneumoniae, which was detectable with Gram staining. She survived after prompt treatment, but her hearing loss remained. Case 2 was a 62-year-old Japanese man with a history of laryngeal cancer who was transferred to our emergency department after an acute onset of delirium and rapid progression to septic shock. As in Case 1, cerebrospinal fluid examination showed a normal leukocyte count despite the presence of S. pneumoniae, which was detectable with Gram staining. Within 1 hour of arrival, he developed hypotension and subsequent cardiopulmonary arrest, and resuscitation was unsuccessful.Case 1 was a 34-year-old Japanese woman with a history of splenectomy who presented with pyrexia, nausea, headache, and loss of hearing in her right ear. She was in a hypotensive state with no neck stiffness and had a normal mental status at the initial presentation. She became progressively disoriented during out-patient management. A cerebrospinal fluid examination showed a normal leukocyte count despite the presence of These cases imply that a normal leukocyte count in the cerebrospinal fluid does not exclude the possibility of bacterial meningitis. Gram staining of cerebrospinal fluid and immediate administration of antibiotics should be performed in all patients with suspected bacterial meningitis. Lumbar puncture and examination of the cerebrospinal fluid (CSF) are essential steps in the diagnosis of meningitis. Typically, the CSF shows significant neutrophilic pleocytosis , a decreA 34-year-old Japanese woman presented to our hospital one morning because of nausea, headache, and loss of hearing in her right ear, all of which had developed a few hours before visiting our hospital. She also stated that she had a fever of 39.7°C, which had suddenly developed after suffering chills two nights previously. She had a history of splenectomy as a result of a traffic accident, but had not received a pneumococcal vaccine. She had received oral cephalosporin (cefditoren pivoxil) and an antipyretic analgesic (loxoprofen sodium hydrate), which were prescribed at a nearby clinic, for the fever on the day before her hospital visit.Streptococcus pneumoniae. Penicillin G was initially administered in the emergency department, and combination therapy with cefotaxime and vancomycin was started after admission. CSF examination on day 2 of hospitalization showed marked pleocytosis , an increased total protein level (270mg/dL), and a decreased glucose level (13mg/dL). Two blood samples for culture were obtained simultaneously with the start of intravenous antimicrobial therapy in the emergency department, but no bacteria were cultivated. The drug susceptibilities of S. pneumoniae grown in the CSF cultures were determined by the disk dilution method and showed susceptibility to penicillin (≥20mm), erythromycin (≥21mm), and levofloxacin (≥17mm). She recovered after a 3-week treatment with these antimicrobial agents and was discharged on day 23. Her hearing loss remained.On initial physical examination, she was afebrile (36.6°C), but in a hypotensive state (88/55mmHg) with tachycardia (120 beats/minute). There was no obvious deficiency in her mental status nor was neck stiffness detected at initial presentation, but she became progressively disoriented  during out-patient management. She was admitted to the intensive care unit and treated with fluid replacement and a vasopressor. Blood tests showed an elevated leukocyte count  with a left shift, elevated C-reactive protein level (27.3mg/dL), and low platelet count . Examination of a CSF sample obtained 90 minutes after presenting to the hospital demonstrated a normal cell count (2/μL) without red blood cells. The CSF was clear, and the concentrations of glucose and total protein in the CSF were 56mg/dL (blood glucose level: 92mg/dL) and 40mg/dL, respectively  and showed susceptibility to ampicillin (≤0.5μg/mL), cefotaxime (≤0.5μg/mL), and meropenem (≤1μg/mL), but resistance to penicillin (0.12μg/mL) and erythromycin (≥2μg/mL).A 62-year-old Japanese man with a history of treatment for laryngeal cancer 7 years previously was transferred to the emergency department after an acute onset of delirium. He had developed a fever the previous day. He did not receive antimicrobial therapy before being transferred. Neither neck stiffness nor paralysis was apparent on physical examination. Blood tests revealed a low leukocyte count (2480/μL), elevated C-reactive protein level (33.0mg/dL), and low platelet count . CSF examination demonstrated a normal cell count (1/μL) without red blood cells. A low CSF glucose level of 8mg/dL (blood glucose level: 69mg/dL) and a high total protein level of 125mg/dL were present. Gram staining of the CSF showed numerous Gram-positive cocci that were proven to be S. pneumoniae, which was detectable with Gram staining. Both patients had an acute onset of fever accompanied by neuropsychological signs and symptoms and rapid progression to septic shock. Clinical evaluations were performed immediately after hospital presentation in both cases, and lumbar puncture was performed less than half a day after neurological symptoms appeared in Case 1. The findings in these cases indicate that the CSF can appear normal, especially when a lumbar puncture is performed soon after the start of symptoms. The first patient survived with hearing loss after prompt treatment, but the second patient died prior to administration of antibiotics.In both cases in this report, CSF examinations showed normal leukocyte counts despite the presence of To the best of our knowledge, only eight cases of adult patients with pneumococcal meningitis with a normal CSF cell count have been previously reported -9. The cet al. found that a CSF leukocyte count of <1000/μL was correlated with morbidity and mortality in patients with pneumococcal meningitis [In adult patients with bacterial meningitis, a low white blood cell count in the CSF is an important risk factor for a poor prognosis . Weisfelningitis  and suggS. pneumoniae microorganisms, is also a poor prognostic factor in patients with bacterial meningitis [In addition to a low CSF cell count, a positive CSF Gram stain result, indicating a high number of ningitis . Of the ningitis ,6, and tThe present and previous cases suggest that bacterial meningitis should be considered in adult patients suspected to have bacterial meningitis, even if the initial CSF cell count is normal, and that Gram staining of the CSF and prompt administration of an adequate dose of antibiotics should be performed with a second CSF examination the following day. This approach is important because the CSF cell count may not be elevated in the first CSF examination, and the presence of Gram-positive cocci in the CSF is associated with high mortality and morbidity in patients with normocellular pneumococcal meningitis.Written informed consent was obtained from the patient described in Case 1 for publication of this case report, and a copy of the written consent is available for review by the Editor-in-Chief of this journal. Although the patient described in Case 2 died of pneumococcal meningitis, the hospital ethics committee confirmed that written informed consent could be waived because the family agreed to the anonymous use of the patient’s data for clinical research purposes. A copy of the certification of approval is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.YK and MS were involved in the clinical diagnostic evaluation and management. HS, HN, and YT analyzed and interpreted the patient data and were major contributors in writing the first draft. All authors reviewed and approved the final manuscript.


A variety of carbohydrate diol derivatives were prepared from their benzylidene derivatives in excellent yield.Clean deprotection of carbohydrate derivatives containing benzylidene acetals and benzyl ethers was achieved under catalytic transfer hydrogenation conditions by using a combination of triethylsilane and 10% Pd/C in CH In our synthetic endeavor we sought to explore the catalytic potential of a combination of Et3SiH and 10% Pd/C in the deprotection of a benzylidene group in the carbohydrate derivatives, without the formation of unwanted byproducts. We report herein our findings on the removal of benzylidene acetal using a combination of triethylsilane and 10% Pd/C  was treated with a combination of a varied quantity of triethylsilane (1.0–5.0 equiv of the substrate) and 10% Pd/C (10–20 mg per 100 mg substrate) in CH3OH at room temperature. It was observed that the use of a total 3.0 equiv of triethylsilane and 10% Pd/C (10 mg/100 mg of substrate) in CH3OH at room temperature furnished the clean removal of the benzylidene group in a 87% yield in 30 min. Generalizing the reaction conditions, a series of benzylidene acetal containing monosaccharide and disaccharide derivatives, with different functional groups present in them, were treated with the optimized reagent system, and clean removal of benzylidene acetal was observed in all cases. The effect of triethylsilane and 10% Pd/C on the removal of benzylidene acetal is presented in In a set of initial experiments, methyl 2,3-di-O-benzyl groups under catalytic transfer hydrogenation conditions has been developed by using a combination of triethylsilane and 10% Pd/C. The efficacy of this methodology is comparable to the conventional hydrogenation involving hydrogen gas and Pd/C, whereas it does not require handling of a flammable gas. Being operationally simple and high yielding, these reaction conditions will certainly be accepted as a useful alternative to those currently existing in this area.In summary, the efficient deprotection of carbohydrate derivatives containing benzylidene acetal and 1  and 10% Pd(OH)2/C (50 mg) in CH3OH (5 mL) was added Et3SiH  portionwise, and the reaction mixture was stirred at room temperature for an appropriate time, given in 3OH. The combined filtrate was concentrated under reduced pressure to give the crude product, which was passed through a short pad of SiO2 with EtOAc as eluant to give pure compound 2 .To a solution of compound File 11H NMR and 13C NMR spectra.Analytical data of new compounds,


Background. The Cryptococcus neoformans polysaccharide capsule is a well-characterized virulence factor with immunomodulatory properties. The organism and/or shed capsule is postulated to raise intracranial pressure (ICP) in cryptococcal meningitis (CM) by mechanical obstruction of cerebrospinal fluid (CSF) outflow. Little is known regarding capsule phenotype in human cryptococcosis. We investigated the relationship of ex vivo CSF capsular phenotype with ICP and CSF immune response, as well as in vitro phenotype.Methods. In total, 134 human immunodeficiency virus (HIV)-infected Ugandan adults with CM had serial lumbar punctures with measurement of CSF opening pressures, quantitative cultures, ex vivo capsule size and shedding, viscosity, and CSF cytokines; 108 had complete data. Induced capsular size and shedding were measured in vitro for 48 C. neoformans isolates.Results. Cryptococcal strains producing larger ex vivo capsules in the baseline (pretreatment) CSF correlated with higher ICP (P = .02), slower rate of fungal clearance (P = .02), and paucity of CSF inflammation, including decreased CSF white blood cell (WBC) count (P < .001), interleukin (IL)-4 (P = .02), IL-6 (P = .01), IL-7 (P = .04), IL-8 (P = .03), and interferon γ (P = .03). CSF capsule shedding did not correlate with ICP. On multivariable analysis, capsule size remained independently associated with ICP. Ex vivo capsular size and shedding did not correlate with that of the same isolates grown in vitro.Conclusions. Cryptococcal capsule size ex vivo is an important contributor to virulence in human cryptococcal meningitis. Cryptococcus neoformans, an opportunistic fungus causing infection in immunocompromised hosts, is the most common cause of adult meningitis in Sub-Saharan Africa [C. neoformans in cerebrospinal fluid (CSF), as well as its most important virulence factor [n Africa , 2 and an Africa . A distie factor , is its e factor , whose he factor .2 [During mammalian infection, the capsule undergoes dramatic changes in size, structure, and proportion relative to total cell volume, stimulated by factors such as iron limitation and physiologic CO2 , 8. Shed2 , which m2 , by mech2 .C. neoformans capsule in virulence have used laboratory strains in animal models. In an immunocompetent murine model, acapsular mutants were avirulent [Many studies demonstrating the importance of the virulent , 13. In virulent . The capvirulent . A mucoivirulent , and shovirulent .C. neoformans capsular phenotype in human cryptococcal meningitis (CM) is associated with the propensity to develop raised ICP and capacity to elicit a CSF inflammatory response. Given in vitro capsular phenotype is often assumed to represent in vivo phenotype, our second objective was to correlate ex vivo phenotype in human CSF with in vitro phenotype of corresponding clinical isolates grown in culture.The cryptococcal capsule has not been well characterized in vivo in human infection. Our study's objective was to test the hypothesis that diversity in the C neoformans capsular phenotype and clinical and immune parameters in the human host.Using serial CSF samples and clinical isolates collected and examined prospectively in an African clinical trial cohort, we present the first study to describe the relationship between ex vivo 2O. The trial had approval from the Research Ethics Committees of Makerere University, University of Minnesota, Mulago Hospital, and Uganda National Council for Science and Technology.Specimens were collected from a prospective cohort at Mulago Hospital, Kampala, Uganda, as a nested sub-study of the Cryptococcal Optimal ART Timing (COAT) trial . Inclusion criteria were human immunodeficiency virus (HIV)-infected, ART-naive persons ≥18 years presenting with a first CM episode confirmed by CSF culture and/or CrAg testing. Written informed consent was provided. Participants had serial lumbar punctures (LPs) on days 1, 7, and 14 of treatment with amphotericin B deoxycholate (0.7–1.0 mg/kg/day) and fluconazole (800 mg/day). CSF opening pressure (OP) was measured at each LP, up to 55 cm HC. neoformans were routinely grown at 37°C on either Sabouraud dextrose agar (SDA), or in Sabouraud dextrose broth (SDB) with shaking at 150 rpm, unless otherwise specified.CSF supernatants and cryptococcal isolates (taken as a sweep across multiple colonies) were cryopreserved at −80°C prior to shipment to St. George's University, London (SGUL). 5), and 100 μL of each dilution inoculated onto SDA. Plates were incubated at 30°C for ≤14 days, and colonies counted at the lowest dilution showing discernible colonies, multiplying by the dilution to give colony-forming units (CFU) per mL CSF. Rate of clearance of infection, or early fungicidal activity (EFA), was determined by subject-specific linear regression of log10-transformed serial cultures by day of cryptococcal therapy, as described elsewhere [Quantitative cultures were performed by serial 10-fold dilution  semi-quantitatively, by performance of serial 2-fold dilutions of CSF, starting at 1:250. CSF cytokines were measured via Luminex  for interleukin 1β, interleukin 2, interleukin 4 (IL-4), interleukin 5, interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10, interleukin 12, interleukin 13, interleukin 17, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interferon γ (IFN-γ), macrophage chemoattractant protein 1, macrophage inflammatory protein 1β , tumor necrosis factor α (TNF-α), and vascular endothelial growth factor (VEGF).For ex vivo analysis fresh CSF was counter-stained with India ink, and capsule thickness , hereafter referred to as capsule size, measured within 1 hour of LP, using a 40× objective and an eyepiece grid micrometer . Digital photography via microscope C-mount documented views for external quality assurance.C. neoformans isolates were grown overnight in SDB. The following day 20 μL of culture was inoculated into 5 mL capsule-inducing media  and incubated at 37°C with 10% CO2 for 48 hours. Cells were harvested by centrifugation at 950 × g for 5 minutes, counter-stained with India ink, imaged using 40× objective, and measured using ImageJ software (v1.44o). Mean capsular parameters were measured for 30–50 cells ex vivo and 50 cells in vitro.For in vitro analysis, Cryptococcus neoformans isolates were inoculated into 10 mL SDB, grown at 37°C until optical density (OD)600 was 0.7–1.1 and centrifuged twice at 950 g for 5 minutes. Supernatants were maintained at 4°C short-term or −80°C long-term. GXM concentration was determined on in vitro culture supernatants and corresponding patient CSF supernatants using the CrAg EIA . Briefly, 50 μL of specimen was added to 96-well plates coated with anti-GXM monoclonal antibody. Using specimen diluent, whole CSF was diluted 1:10, and culture supernatants diluted 1:100 and titrated using 1:5 dilutions. A positive control of 10 ng/mL cryptococcal serotype A GXM was included . EIA titers were calculated using the highest 450 nm absorbance value falling within the acceptable range (0.265–2.55). Final GXM concentration was determined by comparison of OD450 against the positive control. For comparison of in vitro-in vivo shedding, in vitro titers were normalized to OD600 of 1, and CSF titers normalized to a CSF fungal burden of 1 log10CFU/mL.C. neoformans isolates were inoculated into 10 mL SDB and incubated overnight at 30°C. Cultures were added to 500 mL minimal media , and incubated for 11 days at 30°C with shaking. Cultures were centrifuged at 4000 g for 15 minutes (at 4°C), supernatant removed, and centrifuged again at 15 000 g for 15 minutes (4°C). Supernatant was passed through a 0.22 μM Millipore vacuum filter and concentrated using an Amicon filter (100 kDa cut-off). The GXM “jelly” was removed from the membrane and lyophilized in preparation for viscosity measurements.GXM purification was performed as described elsewhere . Brieflyr(t), using the formula:BTk is thermal energy, τ is lag-time , a is particle radius, and η fluid viscosity.Viscoelastic properties of CSF supernatants and GXM solutions were measured using 2 microrheology techniques: passive video particle tracking and optical tweezers. The first was performed using an upright BH2 microscope  equipped with a Prosilica GV640M complementary metal-oxide semiconductor camera and 100× objective. Carboxylic-acid-modified polystyrene spheres (1 μm) were used as probes. Images were stored as AVI files (25 frames/sec). An average 30 particles per frame were tracked simultaneously in 10 minute movies, which were processed using a particle-tracking MATLAB code (University of Glasgow). CSF supernatant viscosity was measured by mean square displacement 2O (concentration range 10−3 mg/mL–10 mg/mL). Viscoelastic properties of GXM solutions were measured using both the analytical method and the optical tweezers setup described in Tassieri et al [Microrheology measurements with optical tweezers were performed on purified GXM dissolved in ultrapure Hri et al . The linunction  :where  2 test, and continuous used the Kruskal-Wallis test. Linear regression was used to estimate the relationship of capsule size with EFA and CSF opening pressure. All CSF parameters significantly associated with CSF OP on univariate linear regression analysis (P ≤ .05), were combined into a multivariable model to investigate factors independently associated with ICP. Despite lack of significant univariate association in this study, baseline CSF fungal burden was also included in the multivariable model due to its previously described association with ICP [Data were analyzed using SAS version 9.3  and Stata v11 . Correlations were assessed with Spearman rank correlation (ρ), categorical comparisons used the χwith ICP . Changes10 CFU/mL), and minimal CSF inflammation (median 10 lymphocytes/μL CSF).Among 134 HIV-infected persons with CM, 362 serial LPs were performed. At the baseline diagnostic LP, prior to antifungal therapy, 124 persons had CSF OP recorded and 122 persons had capsular parameters measured in real-time on fresh CSF. Complete data were available for 108 persons for capsule size, LP OP, and CSF cytokines. Table C. neoformans isolate in vitro was performed on the first 38 participants. In CSF, median (IQR) GXM concentration was 4.8 (1.28–16.27) μg/mL. Using CrAg lateral flow assay (LFA) titers (n = 119) as a semi-quantitative measure of capsular shedding into CSF, mean capsule size correlated with CrAg shedding , including after adjustment for fungal burden .Using intra-subject mean capsule size, the median (IQR) ex vivo capsular size for the cohort was 5.3 (4.6–6.3) μm, with capsule comprising almost two-thirds (65%) of total cell diameter. Quantification of baseline CSF GXM and GXM shedding of its corresponding r = 0.52, P < .001) and ex vivo GXM shedding  but not with CSF protein .CSF viscosity was measured in a total of 38 samples, of which 15 were at baseline. Median viscosity relative to water was 1.1. CSF viscosity in baseline and serial CSF samples correlated with fungal burden . By stratifying the cohort into OP categories, it became apparent that this effect was particularly driven by the patients with extremely high OP >40 cm H2O . Although only 3.6% (441/12 197) of all the cryptococcal cells measured in human CSF in all subjects at all time points were large cells , 15 persons accounted for 80% (352/441) of all the large cells observed. In these subjects, large cells accounted for up to 62% of all cells measured. The cell enlargement was due to both capsular  and cell body enlargement  and thus not consistent with cryptococcal titan cells observed in mice . 13 of 14 with OP recorded had OP>30 cm H2O, and 9 of 14 (64%) suffered extremely high OP >40 cm H2O, compared to only 29% in those without large cells . In the much smaller subset of patients with CSF GXM and CSF viscosity measurements, there were no significant correlations between ICP and GXM shedding  nor CSF viscosity . There were also no associations between ICP and in vitro capsule size (P = .59) or in vitro shedding (P = .31).Using CrAg LFA titers, there was no correlation between ICP and CrAg shedding in CSF  and 3 with normal ICP (open symbols). At physiologic concentrations equivalent to those found in CSF (10−3 mg/mL), high and normal ICP strains showed no differences in GXM viscosity.Figure r = − 0.33, P < .001) and the cytokines IFN-γ , IL-4 , IL-6 , IL-7 , and IL-8 . Figure P < .001). This was also true for total diameter , largely driven by the increased capsule size.Average ex vivo capsule size inversely correlated with CSF WBC count . In prior studies, higher levels of IFN-γ were associated with more rapid clearance of infection [P = .04).An increase in capsule size was associated with a slower rate of clearance of infection, adjusted for baseline fungal burden. The EFA for the cohort was −0.34 lognfection , 25. ThiP = .59), altered mental status (P = .37) or 2-week mortality (P = .88).There was no significant association of capsule size with CD4 count (P≤.05) with opening pressure were mean capsule size, CSF protein, TNF-α, IFN-γ, IL-6, and IL-8. A multivariable regression model predicting OP was run using data from 97 patients with complete data for all of these parameters; CSF fungal burden was also included. Mean capsule size remained independently associated with CSF OP  , cell body diameter , or proportion of large cells  over time.Mixed effects models of change in parameters over time were analyzed among 74 persons with >2 observations over 2 weeks of antifungal therapy. There were no significant changes in capsular size , were CSF OP , CSF WBC count  and CSF protein .Variables showing significant decrease over time, in addition to fungal burden . Mean capsular diameters were larger and more heterogeneous ex vivo than following in vitro culture in capsule-inducing media, median (range) 4.7 (0.9–9.5) vs 2.0 (1.1–4.3) μm, respectively. In addition, no correlation was observed between ex vivo and in vitro GXM shedding , even after adjustment for fungal burden in CSF .Induced capsule size in vitro was measured for 58 serial isolates from 41 patients and compared to ex vivo measurements from corresponding CSF samples. There was no correlation between ex vivo and in vitro capsular size . Based on these findings, the relationship between capsule size, paucity of CSF inflammation, fungal burden, and raised ICP seems to be one of inter-related variables with multidirectional potential interactions, resulting in a particular clinical presentation , techniques that have been successfully applied in exploring other etiologies of communicating hydrocephalus , 39. BeyGiven the lack of in vitro-ex vivo correlations, our study illustrates the importance, as well as the challenges, of studying the relative contribution of virulence factors such as the cryptococcal capsule to clinical presentation in vivo, specifically in humans where, unlike in animal models, one is unable to standardize for host genetic and immune background, fungal inoculum, or timing of infection. Our finding of a significant independent association between capsular size and raised ICP, after adjustment for potential confounders, is thus likely to have pathophysiologic implications. To show causation, one would need to replicate these findings using a selection of highly encapsulated clinical strains, together with appropriate controls, in the rat ICP model.C. neoformans strains have significant and important clinical associations. Strains producing larger capsules were associated with raised ICP as well as poorer CSF inflammatory response and slower rate of fungal clearance.In conclusion, we have demonstrated that phenotypic variation occurs in clinical cryptococcal strains, and that differences in the in vivo capsule phenotype of infecting


We have examined the alleles of eleven minor histocompatibility antigens (MiHAs) and investigated the occurrence of immunogenic MiHA disparities in 62 recipients of allogeneic hematopoietic cell transplantation  with myeloablative conditioning performed between 2000 and 2008 and in their HLA-matched sibling donors. Immunogenic MiHA mismatches were detected in 42 donor-recipient pairs: in 29% MiHA was mismatched in HVG direction, in another 29% in GVH direction; bidirectional MiHA disparity was detected in 10% and no MiHA mismatches in 32%. Patients with GVH-directed HY mismatches had lower both overall survival and disease-free survival at 3 years than patients with compatible HY; also higher incidence of both severe acute GvHD and extensive chronic GVHD was observed in patients with GVH-directed HY mismatch. On contrary, GVH-directed mismatches of autosomally encoded MiHAs had no negative effect on overall survival. Results of our study help to understand why posttransplant courses of allo-HCT from siblings may vary despite the complete high-resolution HLA matching of a donor and a recipient.  Despite the improved matching of donor-recipient pairs that was possible after the implementation of high-resolution methods of molecular HLA typing, the better outcomes of transplantations are still limited by high number of complications: graft versus host disease (GVHD), engraftment problems (lack or loss of engraftment), and relapse [The allogeneic hematopoietic cell transplantation  still remains a curative treatment of many severe diseases, especially hematooncological malignancies. The successful donor search is one of the most important factors deciding about the feasibility of transplantation. It starts with search among the patient's siblings as the HLA-matched sibling donor is regarded as the optimal one. The odds ratio for HLA compatibility in siblings is 1 : 4. The probability of having a matched sibling donor by a particular patient is determined by the formula 1 − (0.75) relapse .HLA matching remains the most important factor influencing both donor selection and transplantation outcomes. However, research of the human genome revealed that polymorphism of nucleotides in genes that are non-HLA related  may also determine the individual immunological phenotype of donor-recipient pairs, thus influencing GVHD, infections, and overall survival . Minor hThe impact of antigens independent from Major Histocompatibility complex on transplantation results was first observed by Counce et al. in 1950s . They exMinor histocompatibility antigens are polymorphic peptides consisting of 9–12 amino acids. After binding to the antigen recognition site of either class I or class II HLA molecules present on a cell surface MiHAs can be recognized by T-lymphocytes. Thus the occurrence of MiHA depends on the presence of specific HLA antigens, which is called the MHC restriction. MiHAs are encoded by either autosomal chromosomes or by Y-chromosome –9. DispaMost MiHA possess only one immunogenic allele, which is sufficient to induce MiHA immunogenicity . Up to dThere are two patterns of MiHAs' tissue distribution: restricted and broad. Autosomal HA-3, HA-8, and most of MiHAs encoded by Y-chromosome are present in most tissues, including those crucial for GVHD: skin, intestines, and liver , 12. MosDetection of MiHA bases most often on genomic typing with PCR-SSP method. The assessment of detected immunogenic disparities is simplified by the online availability of Leiden University Medical Center's dbMinor database . DispariThe aim of this study was to determine MiHA alleles and genotypes enabling to detect their immunogenic disparities in sibling donor-recipient pairs and to explore their influence on the results of allo-HCT.62 patients: 34 women and 28 men of median age 38 (range 14–59) years, who received allo-HCT from siblings in the Department of Hematology and Bone Marrow Transplantation, Medical University of Silesia, Katowice, Poland, in years 2000–2008, entered the study. The indication for transplantation was acute myeloid leukemia (45 pts), acute lymphoblastic leukemia (14 pts), chronic myeloid leukemia in chronic phase, myelodysplastic syndrome, and resistant non-Hodgkin's lymphoma (1 pt each). Donors were 30 women and 32 men of median age 35 (range 14–60) years. Median followup was 3 (0.04–10) years.2 i.v., fludarabine 150 mg/m2 i.v. TBI dose was 12 Gy. Bone marrow was the source of hematopoietic cells in 40 patients, G-CSF-stimulated peripheral blood in 10 and both  in 12 patients. Details of transplanted cells are presented in 2 i.v. on day +1 and 10 mg/m2 i.v. on days +3 and +6. Methylprednisolone at dose 2 mg/kg i.v. was the first line therapy of aGVHD symptoms. The criteria defined by Glucksberg were used for the grading of aGVHD; the diagnosis and severity of cGVHD were determined according to NIH  criteria established in 2005 [Conditioning treatment was myeloablative , reduced intensity , or nonmyeloablative (BuFlu: busulfan + fludarabine in 2 pts). Cumulative doses of drugs used in conditioning were busulfan 16 or 8 mg/kg p.o., cyclophosphamide 120 mg/kg i.v., treosulfan 42 g/m in 2005 .DNA of patients and siblings was isolated from peripheral blood in the Biomolecular Laboratory of the Department of Hematology and BMT, Medical University of Silesia. Alleles of 11 autosomal and Y-chromosome encoded MiHAs were analyzed with PCR-SSP method for each donor-recipient pair in the Immunogenetics and HLA Laboratory of the Regional Blood Center in Katowice with the use of Dynal AllSet+ Minor Histocompatibility Antigen Typing Kit, according to a methodology recommended by Leiden University Medical Center. Products obtained in PCR-SSP reaction were analyzed on agarose gel and each detected allele encoding MiHA was translated into a specific letter code. dbMinor database of LUMC was used to determine the number, direction, and tissue distribution of MiHA mismatches on the base of MiHA alleles and HLA antigens of respective donor-recipient pairs. The study has been approved by the responsible Ethical Committee of Medical University of Silesia.P < 0.05 were considered statistically significant.Median, minimal, and maximum values were used to show numeric parameters of donor-recipient groups. Statistical analysis of MiHA mismatches' impact on transplantation outcomes was conducted in accordance to recommendation of EBMT . MiHA miImmunogenic MiHA mismatches were detected in 42 68%) donor-recipient pairs; 20 (32%) pairs had no mismatched MiHAs. Unidirectional HVG-directed disparities were observed in 18 (29%) pairs  and GVH-directed MiHA disparities were observed in another 18 (29%) pairs . In 6 (10%) pairs bi-directional (both HVG and GVH in the same donor-recipient pairs) MiHA mismatches were observed. The direction of MiHA mismatches is presented in 8% donor-P = 0.011), as presented in P = 0.045), as presented in Analysis of overall survival showed unfavorable impact of GVH-directed Y-chromosome encoded MiHA mismatches (P = 0.05), as shown in GVH-directed mismatches of Y-chromosome encoded MiHA influenced unfavorable the disease free-survival (P = 0.037), which is presented in Serious (grade III or IV) acute GVHD was observed in 24 patients and it was influenced by Y-chromosome encoded GVH-directed MiHA mismatches  and with “restricted” pattern of tissue distribution (0.29(0.18–0.45) versus 0(0-0), P = 0.028). These data are presented in The relapse following allo-HCT was observed in 15(24.2%) patients. Lower risk of relapse was observed in patients with HVG-directed MiHA mismatches: both autosomal (0.28(0.18–0.44) versus 0(0-0), Minor histocompatibility antigens belong to genetic factors which may vary between the donor and the recipient despite identical HLA and thus they may influence allo-HCT results. Knowledge of MiHA alleles and genotypes enables to detect their disparities, which could be helpful not only in optimal matching of a donor/recipient pair and in understanding transplant results, but also it may create a chance to the use of MiHA in immunotherapy aiming to improve patients' survival –54. The P = 0.011) and DFS (P = 0.05). Y-chromosome encoded MiHA represents MiHA with “broad” tissue distribution. Attack of donor's T-lymphocytes on recipients' tissues precipitated by HY mismatch could explain the increased occurrence of severe forms of acute and chronic GVHD, leading to earlier deaths of recipients. In our study recipients of allo-HCT from siblings did not receive anti-thymocyte globulin, what probably influenced the worse course, including the fatal course of their GVHD. We have shown that GVH-directed mismatches of HY correlate significantly with serious (III or IV) aGVHD and extensive cGVHD. These results correspond to the reported influence of sex difference on transplant outcomes, especially in the case of female donor to male recipient (FDMR) transplants [P = 0.05) [We have observed the unfavorable impact of GVH-directed mismatches of Y-chromosome encoded MiHAs on OS  , 63. TheThe analysis of GVH-directed mismatches of autosomal MiHAs, oppositely to HY, showed favorable trend to increase the OS, which was 76% in a mismatched versus 53% in a compatible groups at a 4-year posttransplant. Unlike GVH-directed HY disparities, those of autosomal MiHAs did not increase the occurrence of serious GVHD in our study, which contributed to the better survival. There are reports describing the role of autosomal MiHAs in GVHD: for example, higher risk of aGVHD in the case of autosomal HA-1 incompatibility was reported in Tunisian group of 60 sibling donor-recipient pairs . Others One could expect that disparities of MiHAs with broad tissue distribution present in the host should precipitate the posttransplant reaction of donor's lymphocytes and induce the GVHD. Unexpectedly, the tissue distribution of neither GVH- nor HVG-directed MiHA mismatches did not influence the incidence of GVHD. Much lower probability of relapse following allo-HCT was observed by us in patients with HVG-, but not with GVH-directed MiHA mismatches. This finding, although intriguing, needs further confirmation as we do not find a reasonable explanation for this result. Japanese group found that GVH-directed HA-1 mismatches were associated with lower risk of relapse . SimilarResults of our study help to explain why posttransplant courses of allo-HCT from siblings may vary despite complete high-resolution HLA-match and why cells interactions between the donor and the recipient may lead to serious complications.GVH-directed HY mismatch significantly increased the occurrence of serious acute GVHD and extensive chronic GVHD and finally caused decreased overall survival. GVH-directed mismatches of autosomally encoded MiHAs had no negative effect on overall survival, which in fact was even longer. Findings of our study help to explain why the occurrence of immunological complications and in consequence final results of allo-HCT from high-resolution HLA-matched sibling donors are variable.


Gas chromatography/negative-ion chemical-ionization mass spectrometry (GC/NICI-MS) is the most sensitive and robust method for quantifying these compounds, which is essential for CSF samples because abundance of these compounds in CSF is very low. The present study revealed potential interferences on the analysis of F2-IsoPs and F4-NPs in CSF by GC/NICI-MS due to the use of improper analytical methods that have been employed in the literature. First, simultaneous quantification of F2-IsoPs and F4-NPs in CSF samples processed for F4-NPs analysis could cause poor chromatographic separation and falsely higher F2-IsoPs values for CSF samples with high levels of F2-IsoPs and F4-NPs. Second, retention of unknown substances in GC columns from CSF samples during F4-NPs analysis and from plasma samples during F2-IsoPs analysis might interfere with F4-NPs analysis of subsequent runs, which could be solved by holding columns at a high temperature for a period of time after data acquisition. Therefore, these special issues should be taken into consideration when performing analysis of F2-IsoPs and F4-NPs in CSF to avoid misleading results.F We also evaluated whether the peaks from the retained substances could overlap with the peaks for quantification of F2-IsoPs, F4-NPs, or [2H4]-15-Ft2-IsoP  at their corresponding masses. Finally, the beneficial effect of additional holding of the column at a high temperature after regular data acquisition in the method setting on removing retained peaks was investigated. In this report, we first investigated whether simultaneous analysis of F2-IsoPs and F4-NPs, which were designated as L-CSF, M-CSF, and H-CSF for low levels, medium levels, and high levels of these compounds, respectively. L-CSF sample was from patients with normal pressure hydrocephalus, whereas M-CSF and H-CSF samples were from patients with aneurysmal subarachnoid hemorrhage (aSAH). These CSF samples were pooled from specimen collected during our previous study on aSAH that have been published -15-Ft2-IsoP [2H4]-8-Ft2-IsoP to avoid interferences peaks from endogenous substances in urine samples [2H4]-15-Ft2-IsoP and [2H4]-8-Ft2-IsoP were purchased from Cayman Chemical, which were designated as 8-iso prostaglandin Fα2-d4  and iPFα2-IV-d4 , respectively, in the catalog of Cayman Chemical. These organic solvents in vials containing the two internal standards purchased from Cayman Chemical were evaporated and resuspended in a fixed amount of ethanol followed by the calibration of true concentration of the internal standard in the ethanol solution by the method of Milne et al. -1e et al. . In this18 columns was dried very briefly with 2 g anhydrous sodium sulfate in glass vials before being processed by silica columns. The analyte in final eluate from silica columns was dried, converted to PFB esters by using PFBB and DIPE, purified by thin-layer chromatography (TLC), and extracted by ethyl acetate according to the methods of Milne et al. -15-Ft2-IsoP or [2H4]-8-Ft2-IsoP added into the samples with the ratio of the peak height of F2-IsoPs to that of the corresponding internal standard. Settings and instrumentation of GC/NICI-MS used was the same as what we have previously described -15-Ft2-IsoP was used with 1.5 mL of CSF. For M-CSF and H-CSF, 1 mL and 0.6 mL of samples, respectively, were used and diluted to 1.5 mL with water before adding into 3 mL of water. The whole process and principle are similar to those for analysis of free F2-IsoPs in body fluids. One difference was that the washing solvent for silica SPE columns in the second SPE was ethyl acetate -15-Ft2-IsoP added into samples with the ratio of the peak area of F4-NPs at m/z 593.5, which was defined by the range of peaks of oxidized DHA, to that of [2H4]-15-Ft2-IsoP.  The procedures for analysis of free Flication , which wlication , except  acetate  and ethystandard , 13 instf F4-NFs . In thist-test with SPSS software (SPSS Inc.). The Levene test was also conducted to determine whether p values should be obtained under the assumption of equal variance or unequal variance. Statistical significance was considered when p values were smaller than 0.05.  The significance of the difference for the data between the 2 groups was evaluated using a two-tailed independent 2-IsoPs and F4-NPs for the same CSF sample processed for F4-NPs analysis was appropriate, we first analyzed F2-IsoPs and F4-NPs simultaneously for L-CSF, M-CSF, and H-CSF samples processed for F4-NPs analysis from three independent replicates of each sample. The results of F2-IsoPs quantification were then compared with those for the corresponding three CSF samples processed for F2-IsoPs analysis in a separate experiment. As shown by 2-IsoPs from samples processed in two different ways relative to the values from samples processed for F2-IsoPs were small for L-CSF (5.2%) and M-CSF (1.7%) but were very high for H-CSF (66%). Despite of having the small difference for values of F2-IsoPs quantified from two different methods (5.2%), there was statistical significance between values of F2-IsoPs for L-CSF processed for F2-IsoPs analysis versus for F4-NPs analysis. It could be a systematic difference due to amount of CSF samples or internal standards used in two different operations. On the other hand, due to wider TLC scraping ranges for F4-NPs analysis, the abundance of the peaks adjacent to the peak of F2-IsoPs became much higher for all samples tested. The chromatograms at m/z 569.4 of H-CSF processed in two different ways are shown to illustrate such alteration  at the end of chromatograms of Sample-3 to Sample-5 and Wash-1 to Wash-2  in the range of retention time for F4-NPs quantification or the large peak (peak e) at the end of chromatograms rises markedly for Sample-3 to Sample-5 and remained in high abundance for Wash-1 and Wash-2 , while only some peaks with low abundance showed up in Wash-1 and Wash-2 after injections of plasma samples at m/z 573.4 . Because the amount of internal standard monitored at m/z 573.4 was very large, such low-abundant residual peaks should not affect the quantification of the internal standard. The same pattern of examination described in analysis . The comanalysis . Interes4-NPs, there was no peak with appreciable amount of abundance at m/z 569.4 during this second holding time ). The abundance of the retained peaks in the chromatograms at m/z 573.4 was also very low ).  The chromatograms during the second holding were also monitored for the same processed CSF, plasma, and urine samples used in 4-NPs in CSF at m/z 593.5, we previously needed to conduct multiple injections of undecane to monitor the conditions of GC columns, which was a troublesome process. To eliminate this problem during sample analysis in a more efficient way, we have incorporated additional holding of the column at 280°C, the highest temperature tolerated by the column recommended by the manufacturer, into the method for each injection without the detector on, which could reduce contamination of the ion source. The holding time was determined by the degree of improvement on removing retained peaks, which was confirmed by the absence of obvious peaks in the subsequent washes and disappearance of retained peaks during the second holding as what monitored in  To avoid potential interferences of the retention effect on the subsequent analysis of F4-NPs quantification of the 5 consecutive CSF samples without and with the additional 30-min holding were compared  in high abundance at m/z 593.5 from the retention of the previous plasma sample with the retention time in the range of that for F4-NPs peaks , which employed the same samples described in Figure 3, along with chromatograms at 593.5 having broader range of retention time than what shown in Figure 6.Click here for additional data file.


GDAP1), which is involved in the Charcot-Marie-Tooth disease (CMT), the most commonly inherited peripheral neuropathy, encodes a protein anchored to the mitochondrial outer membrane. The phenotypic presentations of patients carrying GDAP1 mutations are heterogeneous, making it difficult to determine genotype-phenotype correlations, since the majority of the mutations have been found in only a few unrelated patients. Locus-specific databases (LSDB) established in the framework of the Human Variome Project provide powerful tools for the investigation of such rare diseases.The ganglioside-induced differentiation-associated protein 1 gene (GDAP1 gene. The GDAP1 LSDB has adopted the Leiden Open-source Variation Database (LOVD) software platform. This database, which now contains 57 unique variants reported in 179 cases of CMT, offers a detailed description of the molecular, clinical and electrophysiological data of the patients. The usefulness of the GDAP1 database is illustrated by the finding that GDAP1 mutations lead to primary axonal damage in CMT, with secondary demyelination in the more severe cases of the disease.We report the development of a publicly accessible LSDB for the GDAP1 LSDB, which is part of the mitodyn.org portal of databases of genes incriminated in disorders involving mitochondrial dynamics and bioenergetics, should yield new insights into mitochondrial diseases.Findings of this nature should lead to a better understanding of the pathophysiology of CMT. Finally, the  MFN2) [MIM:608507] and the ganglioside-induced differentiation-associated-protein 1 (GDAP1) [MIM:606598]. GDAP1 mutations are responsible: for CMT4A [MIM:214400]  [DNM1L associated with encephalopathy with lactic acidosis [MIM:603850]  , and DNM:603850] . Since tThe authors declare that they have no competing interests.JC and MF initiated the project, created the database, monitored data collection and drafted the manuscript. AC participated in writing the article and the analysis of results. DB, CV, VP, PR and MF supervised the project and revised the manuscript. All authors have given final approval of the version to be published.


We describe here a two-year-old male who required urgent laparotomy to relieve a strangulated small bowel caused by internal herniation around an intra-abdominal testis. This clinical presentation has not been reported previously. A 2-year-old boy presented with a 3-day history of diffuse abdominal cramping, nausea, vomiting, constipation, and progressive abdominal distension. There was no perinatal history of exposure to toxins, and prenatal ultrasound revealed no abnormalities.Physical examination showed that the child was dehydrated, with a diffusely distended and tender abdomen, but no erythema or crepitus. His right scrotum was empty, with no testis present in his groin, while the contralateral testis was normal. Plain abdominal radiology revealed typical features of low-mechanical small bowel obstruction with numerous gas-fluid levels . AbdominWe performed urgent laparotomy through a right transverse supraumbilical incision that revealed an ischemic loop of terminal small bowel herniating below an intra-abdominal testis residing freely in the peritoneal cavity, with its gubernaculum adherent to the terminal ileum . We perfPostoperatively, the patient recovered from surgery without incident and was discharged from hospital after 3 days. Followup in our outpatient clinic 3 months later revealed a normal-sized right testis properly residing in the scrotum.Failure of a testis to reside properly within the scrotum after birth usually leads to increased rates of infertility and malignancy. Other dangers include increased risks of torsion and trauma, in addition to the psychological impact of an absent testis , 2. Intestinal obstruction due to internal herniation is rare, reported in only 1% of patients with these obstructions . IntestiWe report what is, to our knowledge, the first case of a young child with an acutely strangulated small bowel that internally herniated around an intra-abdominal testis and was obstructed by it. It remains unclear whether the testis is an intra- or extraperitoneal organ. Although adult anatomy texts favor an extraperitoneal position, intraperitoneal testes have been observed in patients with gastroschisis, spigelian hernia, and testicular torsion . Moreover, it has been suggested that, instead of following the processus vaginalis, the testis actually pulls out the peritoneal pouch into the scrotum , 8. In c