The primary aim of this study was to determine age-stratified rates of co-existing bacterial meningitis in children with urinary tract infection (UTI). The secondary aims of this study were to determine the causative pathogens of UTI, and the clinical features and outcome of children with co-existing meningitis.Analysis of data collected over a nine-year period at a tertiary pediatric hospital in Australia. Study population: children below 16 years of age with culture-confirmed UTI and a paired CSF sample.Escherichia coli (67.4%), Enterococcus faecalis (8.4%), Klebsiella oxytoca (3.5%) and Klebsiella pneumoniae (3.5%). Only two  of 163 neonates (between 0 and 28 days of age) with UTI had co-existing meningitis. Both presented with pyrexia, irritability and lethargy, and recovered uneventfully with antibiotic treatment. There were no cases of co-existing meningitis among 499 infants (between 29 days and 12 months of age) with UTI (95% CI: 0.00–0.74%), or any of the 86 children aged 12 months or over (95% CI: 0.00–4.20%).A total of 748 episodes in 735 cases were included in the final analysis. The commonest pathogens causing UTI were These findings indicate that clinicians should have a low threshold to perform a lumbar puncture in neonates with UTI, as the risk of co-existing meningitis is not insignificant in this age group. In contrast, beyond the neonatal period, the risk is small and a more selective approach is warranted. Urinary tract infection (UTI) is common in infants and young children. Between 1% and 15% of young children presenting to hospital with pyrexia have an underlying UTI et al. published in 1972, which is frequently referred to in this context, suggests that co-existing meningitis is very common in infants with UTI A study by Bergstrom Accurately determining the rate of co-existing meningitis in children with UTI is clinically highly relevant. In the absence of reliable data, it remains uncertain whether young children with UTI should always have a lumbar puncture to rule out co-existing meningitis The primary aim of our study was to determine accurate, age-stratified rates of co-existing meningitis in children with culture-proven UTI. Secondarily the study aimed to determine the spectrum of bacteria causing UTI in children, and to describe the clinical features and outcome of children with co-existing meningitis.st Jan 2001 to 1st Jan 2010). The following data were extracted from the hospital microbiology department database: patient age, date and type of sample, and bacterial culture results. In patients who potentially fulfilled the case definition of UTI and co-existing meningitis, additional clinical and laboratory data were extracted from the patient's record by one of the investigators.The study was conducted at the Royal Children's Hospital Melbourne, the largest pediatric referral center in the state of Victoria, Australia (estimated population 1.19 million children between 0 and 18 years of age). We analyzed data collected over a nine-year period  sample obtained within 48 hours of the urine sample being collected. Cases were identified by matching all children with a positive urine culture result during the study period with CSF culture results.2 colony forming units (CFU)/ml from a urine sample obtained by suprapubic aspirate or catheterization, ii) ≥103 CFU/ml from a clean catch urine sample or, iii) ≥105 CFU/ml from a bag urine sample. In cases with more than one UTI, these were included in the analysis as separate episodes if they occurred more than 14 days apart. For each episode, only the result of the first positive urine culture was included in the data analysis. In the event that two different bacterial pathogens were isolated from two separate urine samples during one episode, the culture result of the more reliable sample was included in the final analysis (suprapubic aspirate>catheter urine>clean catch urine>bag urine).In this study, ‘neonate’ was defined as a patient between 0 and 28 days of age; ‘infant’ was defined as a patient between 29 days and 12 months of age. UTI was defined as: i) a pure growth of a single bacterial pathogen with ≥10Micrococcus species, Bacillus species, coagulase-negative staphylococci and viridans streptococci isolated from CSF were considered to be contaminants CSF pleocytosis was defined as a CSF white blood cell (WBC) count on microscopy of >19/µl during the first month of life, and >9/µl thereafter, based on recently published data Episodes with urine cultures that showed mixed growth (ie two or more organisms in one sample) were excluded, as these are likely to represent contaminated samples. Episodes with urine samples growing fungal organisms  were also excluded.Bacillus subtilis . Presence of antibiotics leads to inhibition of bacterial growth resulting in a zone of clearing around the inoculated CSF, which is assessed after overnight incubation at 35°C.CSF samples were routinely tested for antimicrobial activity to identify samples which may yield false-negative culture results due to the presence of antibiotics administered prior to the CSF being obtained, as previously described with minor modifications Data analysis and the construction of the figures were done using Prism  and Excel . Nonparametric statistics were used for age-related data, as these data were not normally distributed. The ‘exact’ method was used to calculate 95% confidence intervals (CI). A two-sided chi-square test was used to compare the rate of co-existing meningitis between different age groups.The study was approved by the Human Research Ethics Committee of the Royal Children's Hospital Melbourne (HREC 30062A).A total of 735 cases who fulfilled the inclusion criteria were identified. Thirteen of these cases had two episodes of UTI during which CSF had been obtained. None of the cases had three or more episodes. Therefore, a total of 748 episodes were included in the final analysis. In 607 (81.1%) episodes, urine and CSF samples had been obtained on the same day, in 114 (15.2%) episodes within one day, and in 27 (3.6%) episodes within two days. The most common type of urine sample was catheter urine (31.1%), followed by clean catch urine (28.2%), and suprapubic aspirates (20.9%) .Escherichia coli was by far the commonest isolate, accounting for 67.4% of positive urine cultures, followed by Enterococcus faecalis (8.4%), Klebsiella oxytoca (3.5%) and Klebsiella pneumoniae (3.5%).The spectrum and frequency of bacterial pathogens isolated from urine cultures are summarized in Staphylococcus aureus was detected in both the urine and the CSF sample. The patient received two weeks of intravenous flucloxacillin and gentamicin. Case 2 was a 19-day-old male neonate who presented with pyrexia, poor feeding, irritability and lethargy. A catheter urine sample was obtained and a lumbar puncture done 12 hours later. The CRP was within normal range (<8 mg/L), but the full blood count revealed neutrophilia . A pure growth of E. coli was detected in both the urine and the CSF sample; an assay for E. coli K1 antigen in CSF was also positive. The patient received two weeks of ceftriaxone and gentamicin, followed by a further two weeks of ceftriaxone monotherapy. Both patients had an uncomplicated clinical course, and were discharged without evidence of neurological sequelae.There were two cases of UTI with definite co-existing bacterial meningitis. Case 1 was a male neonate who had been diagnosed with bilateral renal dysplasia antenatally. On day 15 of life he presented with pyrexia, poor feeding, irritability and lethargy. The serum C-reactive protein (CRP) concentration was elevated (36 mg/L); the peripheral blood WBC was within normal limits. A catheter urine sample was obtained and a lumbar puncture was done at the same time. A pure growth of In an additional four patients, pathogenic bacteria were isolated from the CSF sample, but these did not fulfill the case definition for definite co-existing meningitis.Enterococcus faecalis was isolated from urine obtained by suprapubic aspirate. The CSF sample did not show pleocytosis , RBC 18,750/µl, protein 1.18 g/L, glucose 2.6 mmol/L) and no antimicrobial activity was detected. Micrococcus species were grown from CSF within 48 hours. The following day, a scanty growth of E. faecalis was detected in CSF enrichment cultures. In a 40-day-old infant with congenital posterior urethral valves, a urine sample obtained by suprapubic aspirate grew E. coli; the CSF grew scanty mixed E. coli and E. faecalis , RBC 6,410/µl, protein 0.42 g/L, glucose 2.5 mmol/L). In both cases CSF findings were not suggestive of meningitis making contamination of the CSF sample likely.In two cases, it is likely that the children did not have meningitis. In an 8-day-old, previously healthy term neonate, E. coli, while group B streptococci were isolated from blood cultures and the CSF. In a 16-month-old child with a ventriculo-peritoneal shunt, E. coli was isolated from a urine sample (sampling method not stated on the request form), while CSF cultures grew S. aureus.In the other two cases the children had bacterial meningitis, but their urine sample was likely contaminated. In a two-month-old infant with Down's syndrome and complex cardiac malformation, a catheter urine sample grew Antimicrobial activity assays were done on the CSF samples from 730 (97.6%) episodes . In 658 et al. that exclusively investigated patients between 29 and 60 days of age To our knowledge this study is the first to provide age-stratified data on co-existing meningitis in children of all ages with UTI. Our study is considerably larger than any previous study in this area with the exception of the recent study by Schnadover et al. is frequently quoted as evidence that young children with UTI are at significant risk of co-existing meningitis The aforementioned study by Bergstrom et al., several other studies have investigated co-existing meningitis in children with UTI Since the publication by Bergstrom The majority of previous studies have exclusively focused on infants under three months of age Ginsburg and McCracken, who reported a study in 100 infants with UTI, found that the proportion of positive blood cultures correlated inversely with age While it could be argued that universal lumbar puncture would be the safest strategy in infants with UTI, this procedure is uncomfortable and can be associated with rare, but potentially serious complications, such as brainstem herniation, introduction of infectious organisms into the CSF and epidermoid tumors In our study, both patients with co-existing meningitis were neonates who presented with features suggestive of meningitis, including pyrexia, poor feeding, irritability and lethargy. However, clinical features are insufficiently sensitive to reliably rule out meningitis in neonates and typical meningeal signs are often absent in this age group It is important to highlight that our study only includes data from patients who had paired urine and CSF results. Patients with UTI who did not have a lumbar puncture were not included, as the presence or absence of meningitis cannot be established. Consequently, our data are more likely to overestimate rather than underestimate the risk of co-existing meningitis, as clinicians are likely to have done a lumbar puncture preferentially on ‘sicker’-appearing children.E. coli accounting for more than two thirds of infections Enterococcus, Klebsiella and Enterobacter species were also commonly implicated The spectrum and frequency of bacterial pathogens causing UTI in our study is consistent with previous pediatric studies, with The strengths of our study include the large sample size, the age-stratification of data and the quality of the urine samples included. More than half of the urine samples were obtained by suprapubic aspirate or catheterization, which are less prone to contamination than other sampling methods Limitations of our study include its retrospective nature. We cannot exclude with absolute certainty the possibility that some patients had received antibiotics prior to the CSF sample being obtained, thereby potentially rendering their CSF culture false-negative, as we did not have data regarding the timing of the lumbar puncture and the initiation of antibiotic treatment. However, in the large majority (81.1%) of episodes CSF cultures were obtained on the same day as the urine sample. Also, antimicrobial activity was detected in the CSF in only 9.9% of the episodes. The vast majority of these patients had a normal CSF WBC count, while only ten had CSF pleocytosis. In only four of these could no alternative diagnosis be established; in none of these patients were microscopy and biochemical findings characteristic of bacterial meningitis. By assessing these aspects in detail we have limited the number of potentially false-negative CSF culture results, which adds to the robustness of our data. Notably, only one previous study, which included only 106 patients, has attempted to identify patients who had received antibiotics prior to lumbar puncture Identification of co-existing meningitis in children presenting with UTI is critically important, as failure to detect CNS infection can result in partial treatment of meningitis with potentially severe long-term consequences


Idiopathic normal pressure hydrocephalus (iNPH) is a treatable cause of dementia, gait disturbance, and urinary incontinence in elderly patients with ventriculomegaly. Its unique morphological feature, called disproportionately enlarged subarachnoid-space hydrocephalus (DESH), may also be a diagnostic feature. Lipocalin-type prostaglandin D synthase (L-PGDS) is a major cerebrospinal fluid (CSF) protein produced by arachnoid cells, and its concentration in the CSF is reportedly decreased in iNPH. L-PGDS acts as a prostaglandin D2-producing enzyme and behaves as a chaperone to prevent the neurotoxic aggregation of amyloid beta (Aβ) implicated in Alzheimer’s disease, a major comorbidity of iNPH. The aim of this study was to confirm the L-PGDS decrease in DESH-type iNPH and to clarify its relationship with clinico-radiological features or other CSF biomarkers.We evaluated 22 patients  referred for ventriculomegaly without CSF pathway obstruction, and conducted a CSF tap test to determine the surgical indication. CSF concentrations of L-PGDS, Aβ42, Aβ40, and total tau (t-tau) protein were determined using enzyme-linked immunosorbent assays. Clinical symptoms were evaluated by the iNPH grading scale, mini-mental state examination, frontal assessment battery (FAB), and timed up and go test. The extent of DESH was approximated by the callosal angle, and the severity of parenchymal damage was evaluated by the age-related white matter change (ARWMC) score.p = 0.013 and p = 0.003, respectively). L-PGDS and t-tau showed a significant positive correlation . Among the clinico-radiological profiles, L-PGDS levels correlated positively with age , callosal angle , and ARWMC scores , but were negatively correlated with FAB scores .L-PGDS and t-tau levels in CSF were significantly decreased in DESH patients compared to non-DESH patients (Our data support the diagnostic value of L-PGDS as a CSF biomarker for iNPH and suggest a possible interaction between L-PGDS and tau protein. In addition, L-PGDS might work as a surrogate marker for DESH features, white matter damage, and frontal lobe dysfunction. Normal pressure hydrocephalus (NPH) is a cause of treatable dementia, gait disturbance, and urinary incontinence in elderly patients with ventriculomegaly. However, the diagnostic strategy is fairly anecdotal, and ultimately dependent on successful cerebrospinal fluid (CSF) shunt surgery, particularly for idiopathic NPH (iNPH) ,2. PatieAlthough the clinical features may be fairly nonspecific, we must consider a CSF tap test when encountering elderly patients with dilated cerebral ventricles . AnalysiIn this study, we analyzed the clinical, radiological, and CSF profiles of 22 consecutive patients who were referred to our institute for possible iNPH. We found significant differences in the arachnoidopathic marker lipocalin-type prostaglandin D synthase (L-PGDS) between DESH and non-DESH patients. Moreover, we found that this marker was correlated with the cognitive profiles, neurodegenerative CSF markers, white matter damage scores, and tight high convexity.Twenty-two patients  diagnosed with possible iNPH according to Japanese guidelines [Clinical evaluations of gait, cognition, and incontinence were performed before and 24 h after the CSF tap test, using the timed up and go test (TUG) , iNPH gr3 were excluded. All CSF samples were aliquoted and stored in polypropylene tubes at -80°C until biochemical analysis. For the CSF biomarkers, concentrations of L-PGDS, total tau (t-tau), amyloid beta (Aβ)1–42 (Aβ42), and Aβ1–40 (Aβ40) were estimated. L-PGDS levels were measured with a standardized in-house enzyme-linked immunosorbent assay (ELISA) method, as previously reported [Lumbar puncture was performed in the L3–L4 or L4–L5 interspace. A 10–30-mL CSF sample was collected and gently mixed to avoid gradient effects. CSF samples with cell counts > 5/mmreported . As a coreported . The CSFA 3.0-Tesla magnetic resonance imaging (MRI) system  was used. Three-dimensional T1-weighted fast-field echo images  and T2-weighted turbo spin echo images  were obtained in sections parallel to the anteroposterior commissure plane, covering brain regions from the base of the cerebellum to the vertex. All MRI evaluations were done by the first author. Evans index was calculated as the maximal width of the frontal horns/maximal width of the inner skull . For appp < 0.05 was considered statistically significant.Values are given as means and standard deviations. We compared DESH and non-DESH group parameters using the Wilcoxon signed-rank test. Comparisons among the two groups with ventriculomegaly  and the control group were done by one-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls multiple comparison test. The relationships among demographical, radiological, and laboratory data were evaluated by Spearman correlation tests. All statistical analyses were performed using GraphPad Prism 5.01 , and p = 0.01). Eight out of 10 DESH patients showed a positive tap test response. Of these patients, 7 underwent shunt operation, and 6 responded positively to the shunt. On the contrary, only 5 out of the 12 non-DESH patients were tap test positive, with 3 undergoing surgery, and 2 being shunt responders (Table p = 0.04/0.01). Among the CSF biomarkers, t-tau and L-PGDS were significantly lower in the DESH group . The 8 patients with AChE inhibitor prescriptions were slightly older  and had higher CSF tau levels  compared to patients without AChE inhibitor prescriptions. However, their tau levels were still low compared to the institutional values for AD patients .According to the radiological criteria, 10 of the 22 patients showed typical DESH patterns  and non-ventriculomegalic controls, we recruited two control groups for L-PGDS and neurodegenerative markers. As shown in Figure p < 0.001). Age and callosal angle correlated positively with both t-tau  and L-PGDS . L-PGDS also correlated positively with ARWMC scores  and negatively with FAB scores . ARWMC scores were negatively correlated with MMSE  and FAB  scores. Compared to other CSF biomarkers, Aβs were not correlated with clinico-radiological features at the p < 0.005 level.To elucidate the relationship between clinico-radiological features and CSF biomarkers further, a correlation analysis was performed. As shown in Table In this study, we confirmed the usefulness of MRI-based (DESH-based) diagnostic schemes and recognized the lower success rate of tap tests in non-DESH ventriculomegaly. As for the CSF biomarkers, we confirmed that patients with DESH-type iNPH had significantly lower L-PGDS and t-tau levels compared to non-DESH. Moreover, we recognized the positive correlation between L-PGDS and t-tau, both of which correlated positively with the callosal angle and age of ventriculomegalic patients.In addition to our small sample size, this study, similar to previous clinical studies on iNPH, has several further limitations that were related to the patients’ comorbidities . We inclSeveral reports have suggested that the CSF biomarker t-tau plays a role in the neurodegenerative mechanisms underlying iNPH development. Tau is a microtubule-associated protein that promotes and stabilizes microtubule assembly, and is primarily located in the axons of neuronal cells ,19. IncrIn regards to L-PGDS, its decrease has been attributed to arachnoidopathy  particularly in secondary NPH after subarachnoid hemorrhage ,26. In oL-PGDS is considered a dual functional protein, i.e., it acts as a prostaglandin D2-producing enzyme and as a lipophilic ligand-binding protein . Its lipIn summary, we found two potential CSF biomarkers, t-tau and L-PGDS, for distinguishing DESH-type iNPH from non-DESH type. Moreover, their concomitant decrease and positive relationship could indicate an unknown mechanism underlying iNPH development. In addition, we propose L-PGDS as a surrogate marker of DESH features, white matter damage, and frontal lobe dysfunction. The same two markers did not predict tap test results.Aβ: Amyloid beta; AChE: Acetylcholinesterase; AD: Alzheimer’s disease; ARWMC: Age related white matter change; CSF: Cerebrospinal fluid; DESH: Disproportionately enlarged subarachnoid-space hydrocephalus; ELISA: Enzyme-linked immunosorbent assay; FAB: Frontal assessment battery; iNPH: Idiopathic normal pressure hydrocephalus; iNPHGS: iNPH grading scale; L-PGDS: Lipocalin-type prostaglandin D synthase; MMSE: Mini-mental state examination; MRI: Magnetic resonance imaging; TE: Echo time; TR: Repetition time; t-tau: Total tau; TUG: Timed up and go test; VD: Vascular dementia.None of the authors have financial disclosures to declare.NNi: designed the study, carried out the clinical and MRI evaluations, performed the statistical analysis, and drafted the manuscript. NNa, MM, NJ, and KU carried out the immunoassays. HT, AO, SM, and KO helped coordinate the study. MM, YU, and MI performed critical revisions of the manuscript for important intellectual content. All authors read and approved the final manuscript.


Vanishing testes syndrome is often referred to as testicular regression syndrome (TRS) in the recent medical literature. The most characteristic histological findings are presence of a fibrovascular nodule with associated hemosiderin-laden macrophages and dystrophic calcification. Residual testicular tubules are found in less than 10% of cases, with prevalence being unrelated to age at surgery. Presence of seminiferous tubules and viable germ cells in testicular remnant tissue has been reported in some series. TRS theoretically carries a potential for malignant degeneration in the long term and therefore removal of any remnant is a common practice to eliminate this risk. However, no case series has reported germinal dysplasia or intratubular germ cell neoplasia in any of the specimens taken from these patients.Conflict of interest:None declared. Testicular regression syndrome (TRS) or ‘vanishing testis’ is acondition which is considered to be due to the subsequentatrophy and disappearance in fetal life of an initially normal testis. The preTRS or vanishing testis syndrome may be seen in less than 5% of cryptorchidism cases . CryptorAbsence of testis, intra-abdominal testis, inguinal or intra-abdominal vanishing testis are reasons for nonpalpable testis ,12. VaniLou and co-workers reported a series of patients with nonpalpable testes, of which 64% had cryptorchidism. Of these, 22.5% had complete absence of testis, vas deferens and epididymis, 15% had only a blindly-ending vas deferens, and 67.5% had a blindly-ending vas deferens with blood vessels in the inguinal canal .Hormonal and mechanical factors have been implicated in the descent of the testes, but a specific factor has not yet been identified for the pathogenesis of undescended or nonpalpable testis. Although the etiology, pathophysiology, and prognosis of TRS and that of the viable undescended testis might be different, TRS is thought to be the result of late antenatal or perinatal vascular thrombosis, torsion, or endocrinopathy ,17,18,19Histopathological comparison of the contralateral testis from patients with unilateral vanishing testis and undescended testis was performed by Huff et al  and theyTesticular regression has been observed in association with genetic abnormality such as microdeletion of the Y chromosome and boys with persistence of Mullerian duct structures have been found to undergo TRS after birth . AdditioIn the vast majority of cases, TRS appears to be a sporadic occurrence, and the patients are otherwise normal with no significant family history. However, there are now several reports of TRS either in association with other defects, including severe mental retardation in chromosomally normal siblings, or occurring in several members of the same family, suggesting a possible genetic basis for the condition in some subjects ,26. The clinical manifestations of testicular regression can be associated with the embryological sequence and can be classified into early and late embryonic, and early, mid and late fetal or early neonatal events . This diTRS patients usually exhibit a male phenotype. However, although uncommon, the phenotypic spectrum associated with TRS may vary from normal male with unilateral nonpalpable testis through phenotypic male with micropenis, to phenotypic female . The pheEmbryonic TRS has been considered to be part of the clinical spectrum of partial 46, XY gonadal dysgenesis . Most ofClinically, a unilateral nonpalpable testis may be associated with TRS, cryptorchidism, retractile testis, or testicular agenesis. Laparoscopy is widely used to distinguish among these conditions while avoiding open abdominal surgery. When, in the setting of a nonpalpable testicle, vas deferens and spermatic vessels are visualized exiting the internal inguinal ring on laparoscopy, a groin exploration may be carried out to identify and remove the testicular tissue remnant associated with a vanished testis .Diagnosis of TRS is based on a clinically nonpalpable testis with a blind-ending spermatic cord located within the retroperitoneum or exiting a closed internal inguinal ring. Usually, a small fibrotic nodule is found at the end of this spermatic cord. Fibrosis, dystrophic calcification, and hemosiderin deposition in association with identifiable testicular/paratesticular structures are also features of TRS . A number of criteria have been proposed to ascertain the diagnosis of the vanishing testis syndrome. These criteria are: 1) no testis palpated during an examination under anesthesia, 2) visualizing blind-ending spermatic vessels within the retroperitoneum or visualizing spermatic vessels and vas deferens exiting a closed internal inguinal ring. In gross descriptions of the specimens, a spermatic cord with a small mass of firm, fibrotic tissue at one end, as well as elements of the vas deferens, spermatic artery and venous plexuses have been reported. Smith et al  in theirHistopathologic examination has confirmed the presence of germ cells in 11% of testicular remnants. Viable germ cells or seminiferous tubules have been reported in 0 to 16% of excised testicular remnants ,13,33. ALe Conte first recognized the tumor potential of the undescended testis in 1851 . Since tThere is a high risk of malignant transformation in intraabdominal testes especially when associated with external genitalia anomaly and chromosomal anomaly. A testis tumor may develop even after a successful orchiopexy for undescended testis. However, to date, it is not clarified whether vanishing testis and true undescended testis have same etiopathogenesis and risks. The concern in this situation raises the question about the malignant degeneration of residual germinal tissue in the testicular remnants.Theoretically, presence of testicular tissue in testicular remnants indicates a potential for malignancy in the long term. Histopathological examination of testicular remnants in nonpalpable testes demonstrated presence of seminiferous tubules and viable germ cells in 0 to 16% of the reported series ,6,10,36.Nonpalpable testes are a heterogeneous group; their assessment for differential diagnosis needs to include subsets of absent testes, intra-abdominal testes, and testes or testicular remnants located in the inguinal canal that were missed on physical examination. Because of this heterogeneity, laparoscopy has emerged as a useful tool in the localization and assessment of the nonpalpable testis. Various series report success rates of 90% to 95% in the localization of nonpalpable testes with laparoscopy ,38,39.While diagnostic laparoscopy is a safe and effective method of investigating patients with a nonpalpable testis, a simpler, less invasive and less costly initial step is to first explore the scrotum. Failure to detect the gonad in the scrotum in a patient with spermatic cord duct structures may occur in two situations: the testis has regressed or the surgeon has not yet localized it. The pathologist may play a critical role in the management of these patients. 95% of the testes are localized at or below the internal inguinal ring, therefore, pathologic evaluation of the spermatic cord and confirmation of the testis as regressed in tissues removed at primary inguinal exploration can reassure the surgeon of the correct diagnosis. In the majority of the cases, this pathologic evaluation can also eliminate the necessity for further surgical intervention or for radiologic evaluation. Some authors argue that routine exploration of the groin is required only if laparoscopy reveals an open internal ring with normal vessels, while this exploration is unnecessary if the findings show hypoplastic vessels entering a closed ring . Others There is controversy regarding the optimal management of the testicular remnant in cases of TRS. Some urologists recommend surgical exploration, either laparoscopically or via inguinal/scrotal approach, whereas others believe that these procedures are unnecessary ,33,39. TThe histological evaluation of inguinal testicular remnants suggests that these testes became ischemic and atrophied during descent in utero or shortly after birth. The optimal management of the testicular remnants is controversial. Some authors recommend routine excision of remnants, whereas others believe that this is superfluous. A review of the current literature has not identified any report of testicular germ cell tumors which appear to arise from TRS.


In hemodialysis patients, elevated plasma troponin concentrations are a common finding that has even increased with the advent of newly developed sensitive assays. However, the interpretation and relevance of this is still under debate.In this cross-sectional study, we analyzed plasma concentrations of sensitive troponin I (TnI) and troponin T (TnT) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality.In all of the enrolled patients, plasma TnI or TnT was detectable at a median concentration of 14 pg/ml (interquartile range: 7–29) using the Siemens TnI ultra assay and 49 pg/ml (31–74) using the Roche Elecsys high sensitive TnT assay. Markedly more patients exceeded the 99th percentile for TnT than for TnI . In a multivariate linear regression model, TnT was independently associated with age, gender, systolic dysfunction, time on dialysis, residual diuresis and systolic blood pressure, whereas TnI was independently associated with age, systolic dysfunction, pulse pressure, time on dialysis and duration of a HD session. During a follow-up period of nearly two years, TnT concentration above 38 pg/mL was associated with a 5-fold risk of death, whereas elevation of TnI had a gradual association to mortality.In hemodialysis patients, elevations of plasma troponin concentrations are explained by cardiac function and dialysis-related parameters, which contribute to cardiac strain. Both are highly predictive of increased risk of death. At this point, the analytical precision of conventional troponin measurements is reduced, and the coefficient of variation (CV) can reach 10–20% th percentile have been developed and have shown improved accuracy in the diagnosis of MI th percentile Typically, plasma troponin concentrations in ESRD patients are slightly elevated above the 99With the advent of sensitive assays, cardiac troponins are now detected to a larger extent in hemodialysis patients raising questions about interpretation and relevance to nephrologists and cardiologists alike. The increased sensitivity and resolution of the assays, however, also improve the chance to identify factors that are associated with elevated plasma troponin concentrations, such as loss of residual renal function This cross-sectional, prospective multicenter study was conducted in stable, ambulatory hemodialysis patients from four dialysis centers in southwest Germany between September 2009 and April 2010. Patients without evidence of an acute illness and cardiac event or procedure within two months were included in the study after providing written informed consent. Patients with cardiac diseases leading to increased plasma troponin concentrations independent of ESRD, such as amyloidosis, were excluded. The study was approved by the local ethics committee of the University hospital Tuebingen.th percentile at 40 pg/ml, and a CV of less than 10% at 30 pg/ml, as specified by the manufacturer. Plasma concentrations of troponin T were measured using an automated Roche assay on an Elecsys 2010 system with a detection limit of 2 pg/ml, a 99th percentile at 14 pg/ml, and a coefficient of variation of less than 10% at 13 pg/ml Plasma concentrations of troponin I and troponin T were measured in three independent samples collected within two weeks. Each sample was collected prior to the start of a dialysis session. Blood was collected in lithium-heparinized tubes , cooled at 4°C, and centrifuged within 4 hours. The sera were stored at −80°C until further analysis. Plasma concentrations of troponin I were measured using the Troponin I Ultra assay on a Siemens ADVIA Centaur system  with a detection limit of 6 pg/ml, a 99The following data were extracted from each patient: residual diuresis, as measured by 24 h urine collection: single pool Kt/V , interdialytic weight gain, predialytic systolic and diastolic blood pressure , dialysis access and membrane, length of time on dialysis, blood pump flow and shunt flow measured using a Transonic system . The left ventricular (LV) systolic function was classified from available echocardiography examinations in which class 1 indicated normal function, 2 was mildly reduced, and 3 was moderately or severely reduced. Determination of the LV systolic function was done at the discretion of the cardiologist and not standardized. Echocardiography was available in 84% of all patients within one year (plus-minus) relative to study enrollment.http://www.wessa.net/. Univariate and multivariate proportional hazards were calculated to analyze the risk ratios of each predictor and the independence of the predictors. Data analysis was performed using the statistical software package JMP 10.0.1 .Three samples were available in 87% of the patients and were averaged to calculate the arithmetic mean without excluding possible outliers. For further analysis, plasma troponin concentrations and continuous clinical data were log transformed to approximate normal distribution. The association of the plasma troponin concentrations with clinical or dialysis-related factors was analyzed by univariate parametric correlation. Multivariate linear regression analyses were performed to identify independent determinants of the plasma troponin concentrations. Selection of the variables entering the model were derived from forward, stepwise multiple linear regression, and all variables with a p-value <0.05 were included in the multivariate linear regression models. The residuals of each model were tested for normality. Averaged values of the deceased patients were compared to those from the surviving patients using t-Test or Wilcoxon's test. Kaplan-Meier curves were generated after stratification into tertiles of the variable according to its distribution. The follow-up period started on the first day of blood draw and was censored as of 31 December 2011. The diagnostic performance was analyzed using receiver-operator curves (ROC or c-index) and the best cut-off value was considered as the maximal difference of sensitivity and 1-specificity (Youden index). C-Index was calculated using Wessa, P. (2012), Free Statistics Software, Office for Research Development and Education, version 1.1.23-r7, Of the n = 250 available patients that were treated in the participating centers, n = 239 were included in the study. The reasons for exclusion of the n = 11 patients were the following: patients declined to participate (n = 6), deaths occurred following the initial plasma collection (n = 2), cardiac amyloidosis (n = 2) and a recent enrollment in dialysis (n = 1). The characteristics of the study cohort are provided in Cardiac comorbidities were present in a large proportion of the patients. Ischemic heart disease was the most frequent condition (31%). Systolic LV dysfunction was found in 26% of all of the patients .th percentile at 14 pg/ml, while only 14% of the patients had a plasma TnI concentration above the 99th percentile at 40 pg/ml. Expressed as multiples of the 99th percentile, the TnT concentration was elevated 3.5 fold (interquartile range: 2.2 to 5.2) compared to 0.4 fold for TnI , 44 patients died corresponding to an annual mortality rate of 9.2%. Compared to surviving patients, deceased patients were significantly older , were more likely to have LV dysfunction  and had lower diastolic blood pressure . They had higher plasma CRP and parathormone concentrations . Plasma troponin T and I concentrations were also significantly (p<0.0001) higher in deceased patients  compared to surviving patients  was above the 99th percentile.On average, patients in this cohort had a TnT value that was 3.5-fold greater than the 99The study is limited by the paucity of cardiac parameters that were collected, which might have contributed to an unexplained variability of roughly 70% and 50% in plasma troponin I and troponin T concentrations during multivariate modeling. Survival analyses might be limited by the low mortality during the follow-up period. This study focused on nephrological parameters that are commonly available and accessible during hemodialysis treatment, such residual diuresis or shunt flow. Systolic left ventricular function served as a surrogate for cardiac status and showed a high correlation to the plasma troponin concentration. Other cardiac morbidities, such as valvular disease or atrial fibrillation were also contributors of the plasma troponin concentration. Again it should be emphasized that association studies cannot prove causality between the studied parameters and elevated troponin concentrations and cannot distinguish between cause and consequence.In summary, elevations of plasma troponin concentrations in hemodialysis patients measured with sensitive assays are influenced by cardiac function and dialysis-related parameters. Both are strong predictors of mortality in the short-term and are useful markers to identify patients at risk, however, troponin T is more retained during ESRD compared to troponin I.


Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT) is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM):blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg) and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan). We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.  Haematopoietic stem cell transplantation (HSCT) is used to treat several genetic disorders, where a diffusible factor delivered by donor cells can complement the disease. Both the dose of protein or enzyme delivered by donor cells and the level of donor chimerism achieved are important to achieve maximal correction, as illustrated in the lysosomal disease Mucopolysaccharidosis I (MPS I) Hurler . HSCT is6), but only in permissive strain combinations, whilst C57BL/6 recipients are resistant to this method of transplant tolerance generation [6) and 7Gy thymic irradiation (TI) can achieve 20-35% donor chimerism, but only 10-15% if 3.5Gy is used [6) and sirolimus can generate 60-80% donor chimerism, but only 10-30% with lower non-myeloablative busulfan doses [6) [6), and resulting donor chimerism (1-40%) is lower than regimens involving myeloablation [Graft rejection involves multiple mechanisms , but theneration . In more is used ,13, and  is used . Combinian doses . Costimuoses [6) -20. Furtoses [6) , whilst oses [6) ,23. Regimerism 1-0% is lowablation ,24. Thesablation , therefoIn summary, few RIC protocols for murine allogeneic HSCT achieve >80% donor chimerism, and those that do include moderate myeloablation, multiple immune suppressing agents and high donor cell doses ,16,18,26In order to improve the stringency of existing allo-transplant regimens we therefore developed a fully MHC mismatched mouse model of HSCT using low CBA donor cell doses into C57BL/6 recipients. This model requires fully myeloablative busulfan conditioning combined with T cell co-receptor blockade of signal 1 for long-term graft acceptance. Having identified that higher syngeneic chimerism in C57BL/6 recipients was associated with an increased ratio of donor to recipient haematopoietic cells in BM initially after transplant, we then compared published methods for improving donor to recipient cell number in the BM niche including ACK2 , G-CSF  or high We chose to develop RIC regimens using busulfan, because it is considered less toxic than irradiation; it is myeloablative but not immune suppressive and has specific action against primitive HSCs ,13. It iTo determine the effect of these altered SDF-1α levels on donor cell migration to the BM, we performed syngeneic HSCT and determined the number and percentage of donor cells that homed to BM . Signifi6 syngeneic BM cells [6 cells have average donor chimerism of 4% after 2 weeks, which increases to 52% by 12 weeks post-transplant, whilst recipients of 10x106 cells have 12% after 2 weeks, and 76% by 12 weeks post-transplant  or Balb/c (H2Dd) donors to C57BL/6 recipients (H2Db) would be appropriate as C57BL/6 recipients have increased stem cell numbers [6 cells from either CBA or Balb/c donors led to rejection in all C57BL/6 recipients, and at 2 weeks post-transplant 70-80% of the peripheral blood was recipient T cells, demonstrating a large T cell response to the allogeneic cells , increasing the potential number of donor cells reaching the niche, allowed early engraftment in 4/5 recipients, but only 1 retained long-term donor chimerism and gained tolerance to donor cells , allogeneic HSCT (rejection), or allogeneic HSCT with anti-CD4/CD8/CD40L mAb treatment (successful engraftment) . The num6 cells. This gradual increase has been observed by others; in syngeneic HSCT with 20mg/kg busulfan and 20-28x106 cells, low initial engraftment increased to 70-90% by 12-17 weeks post-transplant [Many murine RIC HSCT regimens use high cell doses, but in the clinic patients often receive a dose only just sufficient to reconstitute the haematopoietic system . Using cansplant ,36. Thisansplant ,36. Unliansplant ,38. This6 cells, costimulation blockade generates mixed chimerism [Studies of RIC HSCT have shown that Tregs are important in generation, but not maintenance, of tolerance ,39. Therhimerism , but T chimerism . It has himerism , and thihimerism , which ihimerism ,17,18,21Myeloablation with irradiation has been reported to increase SDF-1 production in BM ; howeverAddition of anti-CD40L costimulatory blockade to anti-CD4/CD8 mAb treatment allows reduction of busulfan to 25mg/kg. Anti-CD40L mAb is widely used in mouse models of RIC allogeneic HSCT, but it causes perturbation of blood coagulation and thromboembolic effects in non-human primates and humans . However6 cells [The RIC regimen developed by Graca et al also includes pre-treatment of recipients with anti-CD4/CD8 mAb 4 weeks before transplant, which increases memory and regulatory cells . Recipie6 cells ,16. Howe6 cells . In conclusion we have used a stringent mouse model of allogeneic HSCT that requires full myeloablation and immune suppression for engraftment, to compare RIC regimens. We have determined that although in syngeneic transplant increasing the ratio of donor: recipient cells in the BM niche positively influences engraftment, improvements in initial engraftment, either by creating more space in the BM niche, or by delivering more donor cells, are ineffective in RIC for allo-transplant. To achieve long-term high donor chimerism in allogeneic transplant using clinically relevant cell doses and a non-myeloablative busulfan dose requires immune blockade, of T cell activation via both signal 1 and 2. Neither signal 1 or 2 blockade alone was sufficient to achieve allotransplant acceptance thus highlighting the importance of T cell signalling in the transplant setting. Addition of costimulatory blockade was critical for reduction of busulfan doses, which is informative for the clinic, where CTLA4Ig (Abatacept/Belatacept) is already approved for costimulatory blockade  but is nk) and Balb/c (H2Dd) , C57BL/6  and B6.SJL-PtprcaPepcb/BoyJ  mice were maintained at the University of Manchester Biological Services Unit, in a 12/12h light/dark cycle and constant temperature with access to food and water ad libitum. All animal procedures were approved by the University of Manchester Ethical Review Process Committee, and carried out in accordance with the Animals (Scientific Procedures) Act, 1986 (UK) and Home Office licence PPL40/3117.CBA (H2DC57BL/6 recipients (6-12 weeks old) (n=3-7) received conditioning regimens. MAC used 10Gy TBI (2 x 5Gy separated by 3-4 hours) on the day of transplant, or 125mg/kg busulfan , (5 x 25mg/kg/day over 5 days) via intraperitoneal (IP) injection finishing on day -1 to transplant -51. RIC Some groups received non-depleting mAbs that block CD4 (YTS177), CD8 (YTS105) and CD40L (MR1), produced from hybridoma cell lines kindly provided by Prof. H. Waldmann ,40. The Standard mAb treatment was 1mg each of anti-CD4 and anti-CD8 via IP injection on days 0, 2 and 4 relative to transplant, with additional use as stated. Baytril oral solution, (0.25mg Enrofloxacin/ml) was given for the duration of mAb treatment and 1 week afterwards to avoid infections.Additional factors were tested in combination with 25 or 75mg/kg busulfan and anti-CD4/CD8 mAb treatment; 500μg ACK2 (eBioscience) on day -7 via intravenous (IV) injection , 4μg G-CDonor BM was isolated as previously described . Donor ck or H2Db as relevant, and lineage staining against CD19 (B cells), CD11b (monocytes) and CD3 (T cells) in 5% solutions of ToPro3 iodide (Invitrogen). Haematopoietic chimerism was quantified by measuring the percentage of donor: recipient cells in peripheral blood (PB), BM or spleen using flow cytometry . Donor and recipient cells were distinguished using antibodies (BD Biosciences) against CD45.1, CD45.2, H2Din vivo cytotoxicity assay was modified from Yamazaki et al [7) were stained with 10µM Celltracker Orange CMTMR or 5µM Celltrace CFSE (Invitrogen), according to the manufacturer’s instructions, washed, mixed in a 1:1 ratio and delivered via IV injection to transplant recipients at >20 weeks post-transplant. After 20 hours, PB and splenocytes were collected and the donor (CFSE) and recipient (Celltracker Orange) ratio determined using flow cytometry. Tolerised recipients retain close to a 1:1 mix of donor and recipient stained splenocytes, whilst donor rejection is indicated by the reduction or absence of stained donor splenocytes. Results are presented as percentage of donor cells/percentage of recipient cells; >0.6 indicates tolerance, <0.6 indicates non-tolerance.The ki et al . SplenocPB collected with sodium citrate anti-coagulant was centrifuged at 1000g to isolate plasma. BM was prepared , then ceStatistical significance was determined using one-way ANOVA, applying Tukey’s multiple comparisons test and assuming significance where p<0.05 of a studentised range of Q. Figure S1In syngeneic transplant, conditioning with 125mg/kg busulfan or 10Gy irradiation leads to equivalent long-term chimerism. (A) HSCT recipients (CD45.1) were conditioned with 125mg/kg busulfan (BU) or 10Gy total body irradiation (TBI) before receiving 2x106 syngeneic (CD45.2) donor bone marrow cells (n=5). (B) Mean percentage donor chimerism in peripheral blood over time is shown; error bars represent standard deviation.(TIF)Click here for additional data file.Figure S2In allogeneic transplant, 125mg/kg busulfan is insufficient for engraftment. (A) HSCT recipients (C57BL/6) were treated with 125mg/kg busulfan before allogeneic HSCT with 10x106 CBA (n=3) or Balb/c (n=6) donor bone marrow cells. (B) The mean percentage contribution of donor and recipient T cells, monocytes and B cells to peripheral blood at 2 weeks post-transplant is displayed.(TIF)Click here for additional data file.Figure S3Combined signal 1 and 2 T cell blockade allows long-term allogeneic engraftment with reduced intensity conditioning. (A) HSCT recipients (C57BL/6) were treated with 75mg/kg busulfan before transplant with 10x106 CBA donor cells, along with along with 1mg anti-CD4, anti-CD8 and anti-CD40L mAb on days 0, 2 and 4 (n=5). (B) Donor chimerism in peripheral blood and the mean percentage contribution of donor and recipient T cells, monocytes and B cells to peripheral blood at 2-20 weeks post-transplant, and the invivo cytotoxicity assay results from >20 weeks post-transplant are displayed. The contribution of different lineages to peripheral blood and the invivo cytotoxicity results are separated for the mice with and without donor chimerism. (TIF)Click here for additional data file.


Oligoclonal bands (OCB) are the most widely used CSF test to support the diagnosis of MS and to predict conversion of clinically isolated syndrome (CIS) to multiple sclerosis (MS). Since OCB tests are based on non-quantitative and difficult to standardise techniques, measurement of immunoglobulin kappa free light chains (KFLC) may represent an easier to use quantitative test.KFLC were measured in CSF and serum of 211 patients using ELISA. These include patients without any inflammatory central nervous system reaction , MS (n = 20), viral CNS infections , neuroborreliosis  and other bacterial CNS infections . Furthermore a cohort of 77 patients with CIS, including 39 patients that remained CIS over follow-up of two years (CIS-CIS) and 38 patients that developed MS over the same follow-up time (CIS-MS).CSF-serum ratio of KFLC (Q KFLC) was elevated in all patients with MS, 86.8% of patients with CIS-MS and 61.5% of patients with CIS-CIS. It was significantly elevated in CIS with presence of OCB (p<0.001). Q KFLC significantly correlated with other CSF variables such as CSF leukocyte count , CSF CXCL13 levels  and also intrathecal IgG synthesis  as determined by nephelometry and quotient diagram. OCB were detected in 66.7% of CIS-CIS and in 92.1% of CIS-MS.Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS. In most patients who develop multiple sclerosis (MS), the disease initially presents with a first relapse-like episode known as clinically isolated syndrome (CIS) Several studies indicated that elevated immunoglobulin kappa free light chains (KFLC) and lambda free light chains (LFLC) in the CSF may offer a quantitative tool to support the diagnosis of MS Here, we provide 1) CSF reference values for KFLC based on a highly sensitive ELISA to evaluate the relevance in MS, CIS and pathogen-related CNS diseases and 2) a systematical analysis of the prognostic relevance of KFLC regarding the occurrence of further relapses in a large and clinically well-defined cohort of patients with CIS. We compare the prognostic relevance of KFLC in CIS to MRI Barkhof criteria 211-paired CSF and serum samples from the Department of Neurology, University of Ulm (Germany) were analysed. These included 77 patients with CIS collected in a prospective study with a follow-up time of two years, as previously described The inflammatory control groups consisted of 17 patients with neuroborreliosis (NB) .MRZR  was determined as previously described (23) using an enzyme-linked immunosorbent assay (ELISA) according to the instructions as supplied by the manufacturer . Quantitative expression of the intrathecal immune response was based on calculation of the CSF/serum quotients (Q) of specific antiviral IgG antibodies, and the intrathecal synthesis of antibodies was detected by calculation of the corresponding antibody indices (AI). AI values ≥1.5 were considered to be indicative of intrathecal IgG synthesis against the respective antigen, and MRZR was considered positive if two or more AI values were ≥1.5.CXCL13 was measured using ELISA  according to the instructions as supplied by the manufacturer. Samples of 50 ml CSF and 50 ml serum were used for the ELISA. Details on the application of the assay for CSF have been published previously by our group CSF leukocyte count (cells/cu.mm), total protein (g/L), lactate (mmol/L), the albumin CSF/serum concentration ratio , immunoglobulins G, A and M were obtained as previously described OCB were detected by isoelectric focusing (IEF) on agarose gels followed by immunoblotting using an IgG-specific antibody staining. Paired CSF and serum adjusted for protein concentrations were applied in the same assay as described earlier Immunoglobulin Kappa and Lambda Free Light Chains (KFLC and LFLC) were measured by quantitative enzyme-linked immunosorbent assay (ELISA) (BioVendor) according to the instructions supplied by the manufacturer. All samples were stored at −80°C until analysis according to the sampling and storage protocol of BioMS MRI scans of the brain and spinal cord were performed on a 1.5 tesla whole-body MRI  according to a previously fixed protocol including T1-weighted spin-echo (SE) axial slices with and without application of gadolinium-DTPA as well as T2-weighted SE axial slices. Barkhof criteria Statistical analysis was performed using SPSS . Differences of FLC between groups were analyzed using Kruskal-Wallis test followed by single pairwise comparisons (Mann-Whitney U-test). The Correlation was analysed using Spearman rank order correlation. To test for normality, the Kolmogorov-Smirnov-test was applied. Sensitivity was calculated as , specificity was calculated as . The positive predictive value (PPV) was calculated as , and the negative predictive value (NPV) as . For all diagnostic values the exact 95% confidence intervals were given Patients with MS and CIS showed significantly elevated CSF KFLC levels as compared with NIND (p<0.001) . CSF KFLIn NIND, CSF KFLC correlated significantly with serum KFLC  and also significantly with CSF-serum ratio of albumin   . TherefoQ KFLC was significantly elevated in cases with presence of CSF oligoclonal IgG bands (OCB)  . Q KFLC In cases of CIS, Q KFLC was positive , CSF CXCL13 levels  and also intrathecal IgG synthesis . Q KFLC was significantly higher in CIS with positive OCB as compared to CIS without OCB p<0.001, . SimilarAll patients with MS showed elevated Q KFLC, 90% of them showed positive OCBs.Of all markers investigated, OCB showed the highest sensitivity for conversion of CIS to MS (92.1%), which could not be improved by adding any of the other parameters . KFLC weIncreasing recognition of the pathogenetic relevance of B lineage cells in MS encouraged the evaluation of B cell-associated biomarker candidates Ig free light chains (FLCs) are “sideline products” of the Ig synthesis by B lymphocytes Our observation of elevated KFLC in the CSF of patients with MS and pathogen related CNS diseases support previous studies alb, which is a widely accepted quantitative measure of blood-CSF barrier function Due to a significant correlation of CSF KFLC with serum KFLC and CSF-serum ratio of albumin  we introduce reference values of Q KFLC in relation to QAs a new aspect, we analyse the prognostic relevance of KFLC and other parameters regarding the occurrence of further relapses in a large and clinically well-defined cohort of patients with CIS. Here, we showed OCB to have the highest sensitivity (92.1%) and highest negative predictive value (81.3%) for conversion of CIS to MS as compared to Barkhof criteria (highest specificity (88.2%)). We observed Q KFLC to be nearly as sensitive as CSF OCB in predicting conversion of CIS to MS .The correlation of Q KFLC with CSF leukocyte count, intrathecal IgG synthesis and the B cell attracting chemokine CXCL13 possibly is in line with the polyspecific intrathecal B cell response in MS So far, OCB are the most widely used CSF test to support the diagnosis of MS and to predict conversion of CIS to MS. OCB s in CSF that are absent in serum indicate intrathecal IgG synthesis. In the classification by Andersson et al 1994 The significant correlation of Q KFLC with positive OCB  supportsIn conclusion, our data indicates the relevance of KFLC as a marker of polyspecific IgG response in the CSF of MS and CIS patients. Similar to oligoclonal bands KFLC add information in predicting conversion of patients with CIS to MS. Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS. Additionally, these findings underline the relevance of CSF parameters in MS and CIS. Although CSF analysis is no longer a mandatory part of diagnostic criteria in MS


Spontaneous CSF leakage is now recognized as a secondary chronic daily headache (CDH) and its characteristic feature is a postural (orthostatic) headache. Despite occipital headaches being common, only one published case report exists of occipital neuralgia associated with significant cerebral descent from CSF leakage. Our case is the first one, occurring without prominent cerebral descent. Potential pathogenic mechanisms are discussed.To report a spontaneous CSF leak as an unusual cause for occipital neuralgia.Case report.A 51-year-old woman with CDH for 3 years presented with bilateral, pressure-like headache aggravated by valsalva and alleviated by lying down. Neurologic exam, brain MRI/MRV/MRA and cervical MRI were all unremarkable. Considering a New Daily Persistent Headache (NDPH), amitriptyline was started but failed. Her headache changed to bilateral occipital, shock-like pains, lasting seconds to minutes superimposed on a “spongy, numb” occipital sensation but without postural component. Occipital nerve block resulted in a dramatic response. Repeat brain MRI showed dural enhancement with subtle “brain sagging”. An empiric lumbar epidural blood patch failed. Spinal MRI showed extradural fluid collections in the lower thoracic region. Conventional computed Tomography (CT) myelogram confirmed a mid-thoracic CSF leak. Subsequent dynamic CT myelography confirmed the location of fast CSF leakage at T4-T5 secondary to a disc extrusion. A decompressive laminectomy with repair of the CSF leak completely aborted her headaches.Occipital neuralgia secondary to CSF leakage may be an under-diagnosed phenomenon. Pressure to the C2-C3 nerve roots during prominent cerebral descent may be the underlying headache cause. In cases without significant cerebral descent such as ours, traction of the C2-C3 nerve roots or the occipital nerve may be a better explanation. Other mechanisms, however, are likely role players as occipital neuralgia is a rare happening in the setting of a spontaneous CSF leak.


Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker.RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants.The linear range of the assay was 7.81–500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1% and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 – 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA  = 1.05× ELISA – 3.09, while the Cusum linearity p-value was >0.1 and the mean bias determined by the Altman Bland test was 1.2%.The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker. The evolution of proteomics over the past decade has seen it mature from its nebulous and esoteric origins into a facet of science with a tangible objective; to develop and apply targeted mass spectrometric assays for the analysis of human proteins The majority of these nouveau technologies that are competing for clinical acceptance are based on the fundamental principle of the ELISA; immuno-affinity recognition and capture of a target for subsequent quantitative detection. However, the adaptation of these approaches to incorporate mass spectrometry (MS) for detection has been at the very least challenging, largely due to the polarized philosophies regarding the application of MS. The majority of these technologies subscribe to a bottom-up approach; in which the proteins are proteolytically digested,  for subsequent surrogate peptide detection Other assay features that need to be considered are the general performance characteristics of the ELISA that have been repeatedly demonstrated over the course of its existence. Only very recently have potential clinical proteomics approaches begun to address this issue, by benchmarking the characteristics of their MS based assay against available ELISA for the same target The simplest remedy to this issue is to adopt a top-down approach, which differs from bottom-up in that the intact protein target is detected instead of a surrogate peptide. Since this alternative proteomic philosophy omits proteolytic digestion, top-down assays are comparatively simpler, much faster and less expensive. Moreover, this approach has the unique ability to both qualitatively and quantitatively evaluate mass shifted variants in a single analysis. Neither bottom-up proteomic approaches nor classical ELISA possess this capability. This ability to detect protein micro-heterogeneity has only very recently become a topic of discussion within proteomics Presented is a unique top-down proteomic method for the analysis of Retinol Binding Protein 4 (RBP4), a clinically significant biomarker for the detection of insulin resistance and type 2 diabetes Not only does this novel assay provide Total RBP4 concentration measurements, but individual values of protein variants that are found within human plasma samples. This unique approach to MS quantification forgoes the need to generate costly isotopically labeled peptides, and its generic nature can serve as a template for the quantitation of other protein targets. Moreover, the utility of this quantitative approach allows for the expedited development of cost effective MS assays. This RBP4 assay described was fully characterized and its quantitative performance was benchmarked against a commercially available ELISA.The novel proteomic method described for the targeted analysis of RBP4 utilized the MSIA approach; immuno-affinity protein enrichment and capture followed by mass spectrometric (MS) detection of the eluted protein target. This approach has been previously utilized in the analysis of numerous proteins from a variety of different biological matrices. As described here, the front end sample processing is configured as an affinity pipette tip (MSIA-Tip) ®, Inc. The polypropylene 96-micro titer plates (deep well and standard) used were from Greiner Bio-One. Premade 10 mM HEPES-buffered saline with 3 mM EDTA and 0.005% (vol/vol) polysorbate 20 (HBS-EP) used was provided by Biacore while purified human urinary RBP4 standard (huRBP4), bovine beta-lactoglobulin standard (b-Lac), tween 20, sinapic acid and all other chemical reagents were obtained from Sigma-Aldrich. The reference RBP4 ELISA was purchased was the Immun Diagnostik RBP4 ELISA kit from ALPCO.The MSIA-Tips were provided by Intrinsic Bioprobes Inc. The antibodies used in the MSIA technology were polyclonal anti-RBP4 from Dako and polyclonal anti-beta-lactoglobulin purchased from GeneTexAll samples used in this study were purchased from PromedDx . Heparinized plasmas from a total of 45 individuals were obtained and arrived de-identified as to ensure the privacy and protection of the individuals who provided samples. Five samples were used for the method characterization studies, while the remaining 40 were used in the method comparison. The samples consisted of a mixture of controls  and individuals diagnosed with acute myocardial infarction . Ethnicity information regarding the samples was unavailable. Samples were stored frozen at −80°C until ready for use.The RBP4-MSIA method utilized a seven point calibration curve and a single control point. Calibrants were prepared by diluting the purified huRBP4 in HBS-EP buffer so that the adjusted concentrations used (accounting for the sample dilution factor) ranged from 7.81 to 500 ug/mL. The control sample was prepared in the same fashion as calibrants but at a concentration of 100 ug/mL. In the standard runs, plasma samples were serially diluted 1∶100 in HBS-EP buffer and a 50 uL aliquot was then used in each analytical sample, however, this sample volume was varied during the linearity determination (described accordingly below). The bovine b-Lac, which is utilized as the internal reference standard (IRS) for assay quantitation, was prepared by serially diluting a 1 mg/mL stock solution in HBS-EP to a final concentration of 25 ug/mL. The selection of the IRS as bovine b-Lac was based on several criteria that included reagent (antibody and antigen) cost and availability, mass spectrometric utility , as well as the fact it is exogenous to human plasma. These factors made bovine b-Lac as an ideal IRS for this quantitative RBP4 assay. The analytical samples were then prepared by aliquoting sample (diluted plasma or standard) into individual wells of a 96-deep well micro titer plate already containing a 50 uL aliquot of the IRS . All samples were then diluted to 1 mL with sample diluent (HBS-EP that contains 0.1% tween 20).+1  = 18,278.2). Calibrated spectra were then processed and normalized to the integral of the IRS signal in each spectrum. Quantitative aspect of the RBP4 MSIA is shown in The MSIA-Tips utilized here were prepared using standard protocols as previously described The quantitation of endogenous retinol binding protein 4 was achieved by summing the integrals of all RBP4 forms observed within each sample. This summed or total integral is then applied to the established line equation generated by the calibrants within each run. The resulting concentration is for the total amount of RBP4 present in each sample. The total RBP4 concentration was used in the studies to determine the performance characteristics of the assay.We performed a series of studies to characterize the performance of the RBP4 MSIA and determine the standard specifications for clinically applied immunoassays. The reproducibility of the assay was determined by comparing MSIA results of standard curves over an eleven day period . The intPrior to these studies, assay development included the establishment of the limit of detection (LOD) and the limit of quantification (LOQ). The LOD was defined as 3 SD of the lowest normalized back ground signal obtained from blank samples, while the LOQ was delineated as the lowest concentration of analyte that could produce a quantitative MSIA signal with a CV<10%. The LOD and LOQ were calculated by repeatedly analyzing standard replicates over a period of 3 months.Precision of the assay was also evaluated by the linearity of dilution. This was assessed by running MSIA on serially diluted plasma samples. The dilution consisted of taking a high RBP4 concentration sample and diluting with increasing proportions of a plasma sample with a low RBP4 concentration . Preliminary RBP4 concentrations were determined by ELISA. Each dilution was prepared in quadruplicate and the RBP4 concentration in each was determined by MSIA. The assay linearity was evaluated by performing a polynomial regression  to determine the degree of non-linearity of the set. The acceptable level of non-linearity was selected to be <10% .Percent recovery experiments were also performed to assess the accuracy of quantification. This was run on three different plasma samples that were established, by ELISA, as having low endogenous RBP4 concentrations. Sample aliquots were either prepared neat or underwent standard addition with three increasing concentration levels of RBP4 standard . RBP4 measurements were taken by MSIA and the percent recovery between the expected and the measured was calculated and averaged for each concentration level. These results are presented in The RBP4 MSIA was referenced to the Immun Diagnostik RBP4 ELISA. A total of 40 plasma samples were utilized in this study with endogenous RBP4 concentrations between 6.7 ug/mL and 182.9 ug/mL as determined by the Immun Diagnostik RBP4 ELISA in a preliminary screening. The RBP4 MSIA method and the ELISA reference studies  were performed on the same day. The ELISA analyses were performed according to the instructions provided by the manufacturer  and the absorbance readings were taken on Cary 50 UV/Vis spectrophotometer (Varian).Data were analyzed using Microsoft Excel 2003 and Analyse-it® .The application of MSIA for RBP4 analysis was consistently able to detect the target as well as produce spectra that distinguished between samples containing different concentrations of RBP4. The internal calibration of each mass spectrum by the IRS produced intact RBP4 m/z values that had a mass accuracy within 0.014% (<150 ppm). The average resolution observed during these studies was ∼900 (FWHM), which is more than adequate to resolve the signals produced by the known variants of RBP4. Combined with the immuno-affinity capture, this was sufficient for confirmation of the identity of the detected analyte.2 = 0.996, intercept  = 0.523 and a slope  = 0.996 . The developed calibration curves were linear throughout the established range, demonstrating a R = 0.996 . Analyse2 = 0.990. Since the non-linearity tolerance is satisfied, the assay exhibits linear characteristics confirming that the sample matrix does not influence the analysis at lower analyte concentrations. The percent recovery studies demonstrated that there was no observable sample matrix interference at the three spiked RBP4 concentration levels  in the test plasma samples. As shown in The RBP4 MSIA quantitative values were referenced to the Immun Diagnostik immunoassay results for the 40 plasma samples (run in duplicate), which ranged from 6.7 to 182.9 ug/mL (0.318 umol/L to 8.68 umol/L). In the ELISA measurements, two samples had results that were above the range of the assay, so they were repeated. The repeat analyses were performed using a 2-fold dilution of the sample and the measured concentration was doubled to account for the dilution. The repeats confirmed that these samples had the two highest RBP4 concentrations and the repeat values were integrated into the data set. All other measurements were performed in a single determination.The same samples were then analyzed for RBP4 by MSIA . The MSIA quantitation showed two samples as having RBP4 values that were below the establish LOQ, therefore were repeated but with a 2-fold increase in the amount of plasma sample used. The measured RBP4 concentrations were then mathematically corrected by dividing by 2. The MSIA repeats confirmed the low concentration of RBP4 in these two samples, therefore the repeat RBP4 measurements were integrated into the data set. All other MSIA measurements were acquired in a single run.The method comparison was performed by referencing the MSIA RBP4 concentrations to the ELISA measurements. These values were plotted and a Passing & Bablok regression was applied +1 , des-L RBP4+1  and des-LL RBP4+1  being routinely reported. Some samples include minor forms of RBP4 that have been less notably observed in human plasma +1 . The data generated from this sample population used in the method comparison study again confirmed the presence of these same species in varying degrees of abundance in some samples.The MSIA approach to protein analytics also allows for the detection and identification of protein heterogeneity (variants) based on changes in their molecular weights. The intact protein MS results demonstrated that gross heterogeneity in endogenous RBP4 was present in the samples analyzed. This is illustrated in The ability of this approach to detect RBP4 variants is not solely qualitative. Since the determination of the total RBP4 concentration included the contribution of all RBP4 species detected in each sample, each variant can also be quantitatively segregated. This was achieved by determining the percent contribution of each variant to the total integral in each sample. This fraction was then applied to the sample total RBP4 concentration to calculate the concentration of the each specific variant within each. A list of the RBP4 variant concentrations are presented in This top-down approach to immuno-affinity mass spectrometry clearly demonstrated that it has the ability to rapidly quantify RBP4 from human plasma over a wide range of concentrations. The described approach to protein quantitation, based on the use of an exogenous IRS that is co-extracted by a multiplexed antibody system, has been previously discussed The RBP4 MSIA demonstrated in this study is both high throughput and automated , and since the MS analyses are performed on the intact immuno-affinity captured protein there is no need for time consuming and costly proteolytic digestion. Not only does the omission of this step reduce cost, decrease sample complexity and provide a large time savings over other quantitative proteomic approaches that are competing for clinical acceptance As a clinical biomarker of insulin resistance and type 2 diabetes


Liver transplantation is the only life-saving therapeutic option for end-stage liver disease. Progressive donor organ shortage and declining donor organ quality justify the evaluation of the leverage of the Donor-Risk-Index, which was recently adjusted to the Eurotransplant community’s requirements (ET-DRI). We analysed the prognostic value of the ET-DRI for the prediction of outcome after liver transplantation in our center within the Eurotransplant community.291 consecutive adult liver transplants were analysed in a single centre study with ongoing data collection. Determination of the area under the receiver operating characteristic curve (AUROC) was performed to calculate the sensitivity, specificity, and overall correctness of the Eurotransplant-Donor-Risk-Index (ET-DRI) for the prediction of 3-month and 1-year mortality, as well as 3-month and 1-year graft survival. Cut-off values were determined with the best Youden-index. The ET-DRI is unable to predict 3-month mortality (AUROC: 0.477) and 3-month graft survival (AUROC: 0.524) with acceptable sensitivity, specificity and overall correctness . Logistic regression confirmed this finding . Determined cut-off values of the ET-DRI for these predictions had no significant influence on long-term patient and graft survival .The ET-DRI should not be used for donor organ allocation policies without further evaluation, e.g. in combination with relevant recipient variables. Robust and objective prognostic scores for donor organ allocation purposes are desperately needed to balance equity and utility in donor organ allocation. Expansion of the organ donor pool by using grafts with reduced quality has become reality in transplant centers around the world due to increasing donor organ shortage and raising numbers of patients on the waiting lists. This has led to an increased awareness of the associated risks for patient and graft survival -16. ThusA relevant body of scientific literature in the bio-statistical world agrees on the outstanding value of the receiver operating curve analysis for the determination of the value of clinical prognostic scores -22. TherThis study is a timely contribution to the urgently needed debate on prognostic models in liver transplantation with potential impact on donor organ allocation policies. The recent liver transplant scandals in Germany revolve around waiting list manipulations that affected donor organ allocation directly and undermined public trust in current donor organ policies .The variables’ frequencies, range and percentages used in the ET-DRI of our study population are summarized in Table Major results of the analyses of the ET-DRI’s predictive capabilities for the study endpoints are summarized in Table With an AUROC of 0.477 (95%-CI: 0.390-0.564) the ET-DRI failed to predict 3-month mortality after liver transplantation in our study population  (see Table With an AUROC of 0.524 (95%CI: 0.447-0.601) the ET-DRI failed to predict 3-month graft survival after liver transplantation in our study population . In binary logistic regression, which was performed with adequate model goodness of fit , the ET-DRI was no statistically significant risk factor for graft loss within one year after transplantation (p = 0.163). Kaplan-Meier analysis of graft survival above versus below the determined cut-off level (1.84) did not reach statistical significance .Since the focus of this paper is prognostic estimation rather than hypothesis testing, strictly speaking power calculations are not directly relevant. The rationale for a sufficient sample size in the presented study is the fact that all 95%-CIs of the AUROCs are sufficiently narrow to exclude 0.700, the nominal criterion value for utility .c-index above 0.700 for the prediction of graft survival in thousands of liver transplant recipients .Regional share was defined by Braat et al. as allocation within Germany and national share as allocation within Eurotransplant land .This is a single centre analysis complemented by ongoing data collection.The setting of the study is a university hospital in Germany within the Eurotransplant community.Included were all consecutive liver transplants performed in adult recipients (minimum age 18 years). Excluded were all combined transplants (e.g. combined liver and kidney transplantation) and living-related organ donor transplants.DRI: Donor risk index; ET-DRI: Eurotransplant-donor risk index; AUROC: Area under the receiver operating characteristic; MELD: Model of end-stage liver disease; ECD: Extended donor criteria.The authors have no competing interests to declare. They did not receive any funding for this work.BR, AK and HS designed the study and accumulated the data. BR, AK, AG and HS analyzed and interpreted the data and drafted the manuscript. All authors read and approved the final manuscript.