Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability.Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification.The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in The online version of this article (10.1186/s13007-018-0363-y) contains supplementary material, which is available to authorized users. Plants have emerged as alternative expression systems for the production of pharmaceutical proteins –4. WhileN-glycosylation differs mainly in the attachment of α-fucose and β-xylose to the core N-glycan, while N-glycans in human and mammalian cells present α-fucose and no xylose is attached. Further maturation of the plant core N-glycan typically results in a biantennary structure, occasionally with terminal Lewis A epitopes. Terminal Lewis A epitopes are rarely observed in human proteins, but are widely distributed in plant glycoproteins. Maturation of human N-glycans will present a multi-antennary structure with two or more terminal branches, further extended with galactose and sialic (neuraminic) acid [N-glycosylation patterns in plants have focused on retaining the recombinant protein in the ER to avoid further modification of the core N-glycan with α-fucose, β-xylose and Lewis A, abolishing expression of glycosyltransferases responsible for the attachment of α-fucose and β-xylose, and further humanization has included the expression of human β-galactosyltransferase and heterologous enzymes required to generate sialylation, which are present in human cells but absent in plants [With regard to the differences in glycosylation patterns between humans and plants, plant ic) acid , 11. Majhuman β1,-galactosn plants .O-glycosylation, patterns differ significantly between plants and human cells, and specifically from the human mucin-type O-glycosylation. In plants, protein O-glycosylation is characterized by the presence of O-glycans attached to hydroxyprolines (Hyp) and Ser residues. Typically, Hyp residues in plants are decorated with arabinogalactan polysaccharides or arabinose oligosaccharides, and Ser residues with galactose [O-glycosylation is characterized by the attachment of N-acetylgalactosamine  to Ser or Thr residues, which can be further elongated with other sugars to form specific and highly complex glycans. Although mucin-type O-glycosylation is widely present in human proteins, plants lack the enzymatic machinery to perform this specific glycosylation [In the case of alactose , 15, 16.sylation , 15, 16.O-glycosylation in plants to produce mucin-type glycans were reported. Among the recent achievements, Daskalova et al. [O-glycosylated human Mucin 1 peptide derivative, which was detected exclusively as a glycoform in Nicotiana benthamiana. This was achieved by co-expressing the Yersinia enterocolitica UDP-GlcNAc 4-epimerase, in charge of converting UDP-GlcNAc to UDP-GalNAc in the cytoplasm, and a Caenorhabditis elegans UDP-GlcNAc/UDP-GalNAc transporter, responsible for the transport of the sugar donor to the Golgi lumen, together with the human GalNAc transferase 2.Recently, a few, but significant, advances in engineering a et al.  successfO-glycosylation in N. benthamiana, using a similar approach. They transiently expressed a Pseudomonas aeruginosa UDP-GlcNAc 4-epimerase, together with the human GalNAc-T 2 and 4, and a human 3.5 tandem repeat of Mucin1. The derivative mucin peptide was GalNAc-O-glycosylated with up to three and five GalNAc residues, when expressed with GalNAc-T2 and GalNAc-T 2 together with GalNAc-T 4, respectively. The mucin-type O-glycosylation was also demonstrated by mass spectrometry on a tandem repeat of MUC16 and on interferon α2b. These authors [O-glycosylation in Arabidopsis thaliana and Nicotiana tabacum BY-2 cells, and encountered a high degree of proline hydroxylation and Hydroxyproline linked arabinosides, plant-specific O-glycosylation, on the substrate model protein.In 2012, Yang et al.  were als authors  were alsO-glycosylated at a single Thr residue. It has been successfully expressed in CHO cells, Escherichia coli, yeast and plants [O-glycosylated, while the yeast-derived variant is decorated with mannose residues. Several studies have shown the impact of glycosylation on protein stability [The human Granulocyte-Colony Stimulating Factor (G-CSF) is a cytokine that stimulates the production, proliferation, differentiation and activation of neutrophil stem cells, raising the levels of neutrophils in the blood stream, and thus protecting the organism against bacterial, fungal and viral infections –22. Thisd plants –32. Nevetability –37, poinN. benthamiana plants, via Agrobacterium infiltration, together with the genes required for the synthesis of N-acetylgalactosamine -O-glycosylation, allowing the production of a plant-derived human-specific O-glycosylated G-CSF.In this project, our objective is to transiently express the human Granulocyte-Colony Stimulating Factor as a model pharmaceutical protein in Agrobacterium tumefaciens strain AGL-1.The coding sequence corresponding to the human G-CSF variant 2 (NCBI: NM_172219) was chemically synthesized  in three different fragments to build the variants used in this study. The three fragments consisted of the nucleotide sequences corresponding to the secretory signal peptide (pUC57 Nat-SP), the mature G-CSF fragment with a mutated glycosylation site that encodes an Ala, instead of the native Thr, and an extra 5′-end Met (pUC57 G-CSF A), and a 5′-end enterokinase coding sequence fused to the mature G-CSF fragment with the native glycosylation site (pUC57 EkG-CSF). The Sec-G-CSF coding sequence was built by digesting the pUC57 Nat-SP with NcoI and Bsu361 and using the digested fragment to replace the 5′-end EkG-CSF fragment of the pUC57 EkG-CSF, which was released with the same restriction enzymes, producing the pUC57 Sec-G-CSF. The Cyt-G-CSF coding sequence was built by releasing the 3′-end of the pUC57 G-CSF A with SapI cutting upstream the non-glycosylation site and downstream the stop codon. The same restriction enzyme was used to digest the pUC57 EkG-CSF, releasing the same fragment but containing the native glycosylation site (Thr), and the released fragment was cloned into the SapI opened pUC57 G-CSF A, generating the pUC57 Cyt-G-CSF with the glycosylation site. The Zera-G-CSF coding sequence was built by digesting the pUC57 EkG-CSF with NcoI and SacI, releasing the whole fragment, and ligating it into a pUC18 ZeraF1V vector available in the lab , by releCymbidium ringspot tombusvirus [Agrobacterium strain AGL-1 was used for pGWB 642, and Agrobacterium strain EHA105 was used for p19, GFP, and STtmd:mRFP constructs. The binary vectors used to generate O-glycosylation were as follows: pH7WG2 GNE, containing the Y. enterocolitica gne gene coding for UDPGlcNAc-4 epimerase; pH7WG2 GT, containing the C. elegans UDPGlcNAc/UDPGalNAc Transporter gene; and pH7WG2 GNT2, containing the human GalNAc Transferase 2 coding sequence, previously published [Agrobacterium strain AGL-1.Other constructs used in this work were the suppressor of post-transcriptional gene silencing p19 from busvirus  to increbusvirus , and STtbusvirus . AgrobacAgrobacterium tumefaciens strains were cultured to an optical density at 600 nm (OD600) of 0.5–0.8. The cells were then collected by centrifugation at 1000 g for 30 min. The pellets were resuspended in Agro-infiltration solution  to a final OD600 of 1.0, followed by incubation at room temperature for 1 h, with gentle agitation. The suspension was then used for needle-less infiltration of the abaxial leaf epidermis through the stomata of N. benthamiana plants [a plants .Protein subcellular visualization was determined by imaging the abaxial epidermal cells of leaf samples, with an Olympus LSM FV1200. Different lasers allowed for imaging of the different fluorescent tag fusion proteins. For GFP imaging, the tag was excited with a 488 Argon laser and detected at 500–545 nm. For eYFP imaging, the tag was excited at 515 and detected at 530–545 nm. For mRFP imaging, the tag was excited at 559 nm with a He/Ne laser and detected at 570–545 nm. The Imaris software  was used to generate 3D images from z-stack confocal images. Line-sequential scanning mode was used for co-localization imaging.2. Collected leaf discs were pulverized in a Mixer Mill  for 1 min at 30 Hz, in previously frozen homogenizer blocks. Pulverized tissue was spun down for 1 min at 1000 g and 300 μL Protein Extraction Buffer  or Reducing Extraction Buffer  were added to the sample. Samples were vigorously vortexed 3 times for 5 s and centrifuged at 20,000g for 15 min at 4 °C. Cleared supernatant was transferred to a new tube and TSP  was quantified using the Bio-Rad Bradford [Four leaf discs (approximate fresh weight of 30 mg) from at least three biological replicates per sample were collected with a 7 mm diameter cork borer, put into a 2 ml tube with three 2.3 mm zirconia/silica beads  and frozen in liquid NBradford  protein Serological assays were performed by resolving the protein sample in a sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF or Nitrocellulose membrane. Recombinant proteins were detected with a 1:5000 dilution of the primary mouse anti-c-Myc monoclonal antibody  or 1:5000 dilution of the primary rabbit anti-GCSF polyclonal antibody , and 1:3000 dilution of the goat anti-mouse IgG HRP-conjugated secondary antibody  or 1:3000 dilution of the goat anti-rabbit IgG-conjugated secondary antibody . In the case of lectin blots, a 1:1000 VVA-HRP conjugate  was used. Blotted membranes were visualized with the enhanced chemiluminescence (ECL) detection kit , following the manufacturer recommendations, and imaged with the DNR Bio-Imaging System MicroChemi . Band analysis was performed using the TotalLab TL 100 software .Protein extract was transferred to a spin column, and anti-c-Myc beads were added to the column, following the manufacturer instructions . The sample was incubated at 4 °C for 1 h, with gentle agitation end-over-end. The column was briefly centrifuged for 10 s, and the flow through was recovered. Three washes were performed by adding 200 μL of the washing buffer provided with the kit, and briefly centrifuging. Finally, 20 μL of elution peptide was added to the column, the sample was incubated for 5 min at 4 °C, and the purified protein was recovered by centrifuging the column for 10 s. Elution was repeated twice.−1 and 10 µL of the protein digest was injected. 97% mobile phase A  was decreased to 90% over 3 min. Peptides were eluted with a linear gradient from 10 to 35% mobile phase B (LC/MS Optima acetonitrile 0.1% formic acid) over 21 min followed by 35–90% over 3 min and maintained for 8 min. The nanospray voltage was set at 2.1 kV, capillary temperature 275 °C, and S-lens RF level 55. The Q-Exactive was operated in top 10 data-dependent acquisition mode with a full scan mass range of 400–2000 m/z at 70,000 resolution, automatic gain control (AGC) of 1e6 and maximum injection time (IT) of 250 ms. The MS/MS scans were acquired at 17,500 resolution, AGC of 2e5, maximum IT of 50 ms, intensity threshold of 8e4, normalized collision energy of 27 and isolation window of 1.2 m/z. Unassigned, singly and > 4 charged peptides were not selected for MS/MS and a 20 s dynamic exclusion was used. The Thermo .raw files were converted to mascot generic format using Proteowizard v2 [N. benthamiana proteome  using X! tandem [SDS-PAGE excised bands were Trypsin/Chymotrypsin (6.25 mg/L each) digested  at 30 °Cizard v2  and the ! tandem  search a! tandem  interfac! tandem . A 3 ppmp ≤ 0.05).The statistical analyses were performed with the Minitab 18 software . A one-way analysis of variance (one-way ANOVA) was performed followed by Tukey test to find significance differences between means  coding sequence, including its native secretory signal sequence, was used to build the corresponding expression cassettes, in order to target the model protein to the secretory pathway  [To explore the feasibility of using way Fig. . Two oth peptide . The dif(CymRSV) .Fig. 1ScN. benthamiana leaves were monitored from 2 to 8 dpi (days post-infiltration). Sec-G-CSF:eYFP was detected in the ER (endoplasmic reticulum), as a multiple dot-like pattern indicating a potential localization in the Golgi, as well as in the apoplast  as a Golgi marker, which would suggest trafficking of secretory G-CSF along the secretory network  comprised by the Y. enterocolitica UDP-GlcNAc/GalNAc 4-epimerase, the C. elegans UDP-GlcNAc/GalNAc transporter, and the human GalNAc-Transferase 2, to allow the mammalian-specific GalNAc-O-glycosylation of G-CSF. In this case, the eYFP tag was replaced by a C-terminal c-Myc4x tag (Sec-G-CSF:cMyc) for detection and purification purposes. Expression of Sec-G-CSF:cMyc was determined by western blot with a molecular weight of 28 kDa , which is specific for GalNAc-O-T/S moieties, was used as probe  derived from the peptide where the Thr-163 is localized, as indicated by the characteristic HexNAc product ion at m/z 204  Figs. : Fig. S3A. niger , 66, 67,A. niger , 49–52, A. niger , 66–68. O-glycosylated G-CSF in plants [It must also be noticed that the accumulation levels obtained with the secretory G-CSF were the lowest in comparison to the other two variants (Cyt-G-CSF and Zera-G-CSF). Co-expression with the glycosylation machinery decreased the accumulation levels of Sec-G-CSF:cMyc even more  Fig. . Such den plants .O-glycosylated G-CSF in N. benthamiana plants via agroinfiltration, offering an alternative system for the production of this pharmaceutical protein with its native mammalian-specific post-translational modification. Further studies would be required to explore elongation of the glycosylation moiety and approaches to increasing accumulation of this recombinant glycoprotein.In conclusion, we have successfully expressed GalNAc-Additional file 1: Figure S3. Impact of extraction buffer on G-CSF. A Western Blot detection of Sec-G-CSF:eYFP and Cyt-G-CSF:eYFP extracted with PBST0.1 % (gels on the left) or under reducing and denaturing extraction conditions (SDS-DTT) (gels on the right). B Band quantification of Western blot detected proteins. Samples collected at 4, 6 and 8 dpi. Four leaf discs from different leaves were collected from each biological sample. 20 μL TSP of PBST0.1 % treated sample or equivalent volume of SDS-DTT treated sample were loaded on the gel. Black arrows denote monomeric G-CSF:eYFP variant. p19: negative control. eGEHK: protein standard. Proteins were detected with GFP antibody. Band quantification of Western blot detected proteins (Graph). Columns denoted with a different letter are significantly different (p ≤ 0.05) using one-way ANOVA and followed by Tukey test. Error bars are standard deviation of the meansAdditional file 2: Figure S2. Secretory and Cytoplasmic G-CSF can be extracted with a PBS-based extraction buffer, but Zera-G-CSF can only be extracted under denaturing and reducing conditions. Western Blot detection of G-CSF:eYFP variants transiently expressed in N. benthamiana plants. Samples collected at 4 dpi. Upper panel: Plant 1; Lower panel: Plant 2. Sec-G-CSF:eYFP , Cyt-G-CSF:eYFP , Zera-G-CSF:eYFP , eYFP , p19 negative control. Four leaf discs from two different plants were collected for each sample. 50 µg TSP of PBST samples or equivalent volume of SDS-DTT samples were loaded on the gel. Black arrows denote Zera-G-CSF:eYFP. Gray arrows denote Sec-GCSF-:eYFP and Cyt-G-CSF:eYFP. Red asterisks denotes a faint band corresponding to Zera-G-CSF:eYFP extracted with PBST extraction buffer and its corresponding proper extraction with SDS-DTT extraction buffer. Proteins were detected with anti GFPAdditional file 3: Figure S1. Compartmentalization of GalNAc attachment to target proteins in the secretory pathway. GalNAc-Transferases are traditionally referred to be localized in the Golgi Apparatus (Cis-Golgi), but recent studies suggest also ER localization, thus proposing attachment of GalNAc in subregions of ER and proximal Golgi compartment. GalNAc-T, GalNAc transferase. ER, Endoplasmic Reticulum. ERGIC, intermediate ER-Golgi Compartment. Grey box, GalNAcAdditional file 4: Figure S4. MS/MS identification of glycosylated Sec-G-CSF:cMyc-derived peptide. A Schematic illustration of released peptide after Trypsin/Chymotrypsin in gel digestion of c-Myc purified Sec-G-CSF:cMyc expressed alone or co-expressed with the O-glycosylation machinery. Native glycosylation site (Thr-163) is denoted by underlining. B Extracted ion chromatograms of predicted QQMEELGMAPALQPTQGAMPAFASAF peptide derived from Sec-G-CSF:cMyc expressed alone (upper panel), not being detected; or co-expressed with the O-glycosylation machinery (lower panel), where it was detected. C MS/MS of c-Myc purified and Trypsin/Chymotrypsin in gel digested Sec-G-CSF:cMyc-derived peptide (QQMEELGMAPALQPTQGAMPAFASAF) co-expressed with the O-glycosylation machinery, showing identified b- and y-ions. Modified Thr-163 is marked in red. Complete list of detected product ions is shown in Supplementary Table S1Additional file 5: Table S1.y- and b- product ions detected in GalNAc-O-glycosylated QQMEELGMAPALQPTQGAMPAFASAF derived peptide and associated mass errors


Chlorocebus aethiops sabaeus). Consistent with the human disease, T2D monkeys have increased plasma and CSF glucose levels as they transition from normoglycemia to PreD and diabetic states. Although plasma levels of acylcarnitines and amino acids remained largely unchanged, peripheral hyperglycemia correlated with decreased CSF acylcarnitines and CSF amino acids, including branched chain amino acid (BCAA) concentrations, suggesting profound changes in cerebral metabolism coincident with systemic glucose dysregulation. Moreover, CSF Aβ40 and CSF Aβ42 levels decreased in T2D monkeys, a phenomenon observed in the human course of AD which coincides with increased amyloid deposition within the brain. In agreement with previous studies in mice, CSF Aβ40 and CSF Aβ42 were highly correlated with CSF glucose levels, suggesting that glucose levels in the brain are associated with changes in Aβ metabolism. Interestingly, CSF Aβ40 and CSF Aβ42 levels were also highly correlated with plasma but not CSF lactate levels, suggesting that plasma lactate might serve as a potential biomarker of disease progression in AD. Moreover, CSF glucose and plasma lactate levels were correlated with CSF amino acid and acylcarnitine levels, demonstrating alterations in cerebral metabolism occurring with the onset of T2D. Together, these data suggest that peripheral metabolic changes associated with the development of T2D produce alterations in brain metabolism that lead to early changes in the amyloid cascade, similar to those observed in pre-symptomatic AD.Epidemiological studies suggest that individuals with type 2 diabetes (T2D) have a twofold to fourfold increased risk for developing Alzheimer’s disease (AD), however, the exact mechanisms linking the two diseases are unknown. In both conditions, the majority of pathophysiological changes, including glucose and insulin dysregulation, insulin resistance, and AD-related changes in Aβ and tau, occur decades before the onset of clinical symptoms and diagnosis. In this study, we investigated the relationship between metabolic biomarkers associated with T2D and amyloid pathology including Aβ levels, from cerebrospinal fluid (CSF) and fasting plasma of healthy, pre-diabetic (PreD), and T2D vervet monkeys ( Rates of type 2 diabetes (T2D) and Alzheimer’s disease (AD) are reaching epidemic proportions and are expected to continue to rise over the next several decades . T2D is de novo lipogenesis, making BCAAs a potential biomarker of metabolic disease (Branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential amino acids necessary for protein synthesis, but when found in excess, impact energy homeostasis . Recent  disease . BCAAs a disease . NeverthAcylcarnitines are byproducts of mitochondrial fatty acid, amino acid and glucose catabolism that serve as useful biomarkers of metabolic changes . ChangesChlorocebus aethiops sabaeus), which develop neuropathological changes consistent with human AD pathology including increased amyloid plaque burden, elevated cortical tau levels and paired helical filament tau immunoreactivity, Aβ-related vascular impairment, reduced cerebral metabolism, regional atrophy, decreased CSF Aβ42 and increased CSF tau levels , pre-diabetic (PreD), and diabetic (T2D) monkeys to explore the relationship between T2D and amyloid pathology. We utilized a cohort of aging vervet monkeys . Veterinary and research staff categorized the monkeys as either healthy , pre-diabetic , or type-2 diabetic  according to repeated fasting glucose measurements and American Diabetes Association criteria  and in compliance with the USDA animal Welfare Act and Animal Welfare Regulations .40 and Aβ42 levels from CSF samples were assayed using sandwich ELISAs as previously described  targeted against amino acids 45–50 (HJ2) or 37–42 (HJ7.4), respectively. For detection, both Aβ40 and Aβ42 used a biotinylated monoclonal antibody against the central domain (HJ5.1B), followed by incubation with streptavidin-poly-HRP-40. Assays were developed using Super Slow TMB (Sigma) and the plates read on a Bio-Tek Synergy 2 plate reader at 650 nm.Aβescribed . BrieflyGlucose and lactate measurements within the plasma and CSF were quantified using a YSI 2900 analyzer as previously described . A detair. To determine the relative relationship between CSF Glucose, CSF Aβ42, and plasma lactate and CSF analytes, we transformed each data point to represent its value relative to the control group mean . Data are represented as means ± SEM. Tukey’s post hoc tests were used when appropriate.Data were analyzed using one-way ANOVA and correlations were determined by Pearson’s correlation coefficient, p < 0.0001, F = 39.17], but there were no differences in fasting insulin levels  = 10.39] and CSF glucose concentrations  compared to normoglycemic controls, while PreD had intermediate values. Data analysis revealed that plasma and CSF glucose levels had a strong positive correlation , which is consistent with observations from preclinical rodent models  = 4.544], however, no differences in CSF lactate concentrations were observed  = 5.030], and CSF Aβ42 concentrations . Interestingly, both CSF Aβ40 and CSF Aβ42 were highly correlated with CSF glucose  and plasma lactate  but not with plasma insulin  = 11.04], total AA , essential AA , aromatic AA , and basic AA  , suggesting peripheral metabolic perturbations were present, although the differences in CSF amino acids (p = 0.0219 and p = 0.0524).Because plasma amino acid and acylcarnitine levels are linked to metabolic dysfunction in T2D , the lev= 8.421] . In the = 8.421] . No diff= 8.421] . Interes= 8.421] , there wno acids , valine p = 0.0208, F = 6.139], but no differences in plasma total acylcarnitine levels  = 5.764]. However, due to variability in the control monkeys, medium- and long-chain acylcarnitine concentrations were consistently lower in T2D monkeys, but the difference did not reach significance  to symptomatic AD, and increases the rate of amyloid accumulation in the brain . MoreoveIn the current study, we demonstrated that T2D monkeys have lower CSF acylcarnitine and amino acid concentrations , 3. SeveAnother explanation for the difference in CSF AA levels could be that the T2D brain is overconsuming amino acids as fuel or rapidly increasing protein synthesis. While glucose is the primary source of energy for the brain, the brain can readily use fatty-acids as energy substrates; however, this typically occurs with decreased glucose availability, such as fasting or starvation . This coThe data presented here show that in the progression from healthy to PreD to T2D, the brain moves into a state of altered metabolism that results in an increase in glucose and lowering of amino acids and acylcarnitines in the CNS. Increased cerebral metabolism drives Aβ production and accelerates Aβ aggregation, which reciprocally escalates the disease cascades in T2D and AD. These findings shed further light on the metabolic link between T2D and amyloid pathology and how T2D progression could lead to AD-related pathology and cognitive decline.All datasets generated for this study are included in the manuscript and/or Animal subjects: The animal study was reviewed and approved by Wake Forest University Institutional Animal Care and Use Committee.KK, DM, SM, CN, and OI conceived on the experimental design. KK, MP, WM, CN, OI, and SM performed the experiments. SD, KK, CN, OI, DM, and SM analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lumbar puncture (LP) is a common way of collecting cerebrospinal fluid (CSF) both in the clinic and in research. In this extension of a study on the relationship between sleep deprivation and CSF biomarkers for Alzheimer’s disease, we investigated CSF biomarker dynamics in relation to rebound sleep after sleep deprivation. Two LPs were performed within 3 days in 13 healthy volunteers. We noticed an unexpected sharp rise in biomarker concentrations in the second sample and therefore repeated the experiment, but without sleep intervention, in four additional individuals. The findings were similar in these subjects, suggesting an inherent methodological problem with repeated LPs. The result corroborates findings in studies with repeated CSF collection via indwelling lumbar catheters, and needs to be addressed in, for instance, pharmacodynamic studies employing these techniques. Lumbar puncture (LP) is a routine procedure used in the clinical setting, as well as in research, to collect cerebrospinal fluid (CSF). A wide range of biomarkers can be measured in CSF, making LP a useful procedure in diagnostics of and research on traumatic injuries, infections, autoimmune conditions, bleeding and other CNS pathologies. More specifically, LP has emerged as a key tool in the diagnosis of neurodegenerative disease .We have previously examined the link between partial sleep deprivation and biomarkers for Alzheimer’s disease (AD) in both CSF and plasma , 3. In tIn a previously published study , 13 healIn the follow-up experiment, four healthy adults Table  with theg for 10 min, aliquoted and stored in 0.5 mL aliquots at − 80 °C pending analysis within 1 h after sampling.CSF samples were collected by LP at the L3/L4 or L4/L5 interspace with a 22gx90mm Sprotte™ needle, by an experienced neurologist. The needle type was chosen to minimize the risk of post-LP headache [CSF Aβ38, Aβ40 and Aβ42 concentrations were measured using MSD Abeta Triplex . CSF total tau (T-tau) and phosphorylated tau (P-tau) concentrations were measured using INNOTEST sandwich enzyme-linked immunosorbent assays . CSF neurofilament light (NF-L) concentration was measured using the NF-Light ELISA . CSF YKL-40  concentration was measured using the Human Chitinase 3-like 1 Quantikine ELISA Kit . CSF glial fibrillary acidic protein (GFAP) concentration was measured using an in-house ELISA . All meaActiGraph GT3X+ devices were worn on the nondominant wrist, throughout the experiment. Data from the devices was used to validate a normal sleep pattern with at least 8 h of bedtime. Actigraphy data was reviewed with the ActiLife software and analyzed with the Sadeh algorithm , 8.Statistical analysis was done using IBM SPSS version 25.0. Significance was calculated by two-tailed paired sample t-test and alpha was set to 0.05.In conjunction with our earlier experiment, we investigated the effect of recovery sleep on a set of AD biomarkers Fig. . An unexThe rise in the concentration of several AD biomarkers suggests an inherent methodological problem with repeated LPs within a few days. Studies analyzing repeat CSF samples taken through an indwelling lumbar catheter also report marked increases in Aβ levels, up to > 100% of baseline levels during the first 12 h, and then fluctuating around double baseline levels for 36 h . A plausIn the present study, we found similar, but less pronounced (only 12 mL of CSF was tapped at the first LP) increases in CSF levels of brain biomarkers, which may have similar explanations. Contributing explanations may be CSF leakage after the LP into the under-pressured epidural space; an explanation supported by the finding that subjects with post-procedure headache (that is known to be caused by CSF leakage) had a more marked increase in CSF concentrations of Aβ than those without this complication . A limitWe would now like to alert the research community of an apparent effect of repeated LPs on CSF biomarker concentrations. It is also worth noting that we used a “non-traumatic” needle type. Using standard needles might have aggravated the biomarker changes even further. These results are important to consider when designing studies with repeated LPs. They may also be of relevance in studies where spinal catheters are placed to examine dynamic changes in CSF composition.An LP in itself may have profound effects on CSF protein concentrations if samples are collected by repeated LPs 3 days apart. This confounding influence needs to be taken into account in CSF biomarkers studies with repeat LPs, such as pharmacodynamic Phase I trials.


Unlike pediatric kidney donors, there is no literature regarding the growth of pediatric donor pancreatic transplant grafts. Our center prospectively followed three pediatric donor grafts after transplant by measuring two dimensions of the graft at postoperative day one and then at one, two, and three months post-transplant surgery with the hypothesis that the grafted pancreas would not grow like pediatric kidney donors given the fundamental physiologic differences between these two organs. Two grafts were stable to minimally larger in size, the third case decreased in size. Interestingly, all patients had an excellent clinical response with normalization of HbA1c. Further study will be required to understand the natural history of pancreatic transplants from a pediatric donor. Volumetric assessment with magnetic resonance imaging (MRI) is proposed as the next step for better evaluation of graft size. Approximately 1.25 million Americans have type I diabetes (T1D) with over 40,000 new diagnoses each year [Donor pediatric kidneys grow following transplantation . The aimThree adult recipients received pediatric donor pancreatic grafts as part of a multi-organ SKP with standard vascular anastomoses to the right common iliac artery and distal vena cava. Enteric drainage was used with the pancreas head–up and side-to-side ileal-duodenal anastomosis. The standard protocol was to analyze serial sonographic measurements of the transplants for three months to determine if pancreatic organ growth occurred. Measurements were obtained by three independent radiologists and averaged for assessment.Case 1The first case was a 24-year-old woman with a 13-year history of T1D complicated by end-stage renal disease (ESRD) requiring hemodialysis. At the time of surgery, she weighed 61.9 kg and received a pancreas from a seven-year-old, 25 kg donor as part of her SKP. The patient had normal endogenous pancreatic function within 24 hours, independent of exogenous insulin with normal amylase and lipase function. At six months post-op, she had a normalized HgbA1c of 5.1% and C-peptide increased from <0.1 to 4.2 ng/mL.A small peripancreatic fluid collection seen in the one and two-month follow-up scans resolved by three months; presumptive diagnosis was hematoma. Sonographic measurements of the transplanted pancreas were taken postoperatively, showing a size of 10.4 cm x 2.9 cm (CC x AP). Three-month follow-up average measurements showed minimal enlargement with measurements of 9.9 cm x 3.4 cm (CC x AP).Case 2The second case was a 55-year-old woman with T1D complicated by gastroparesis and ESRD requiring peritoneal dialysis. At the time of surgery, she weighed 49 kg and received a pancreas from a seven-year-old, 35 kg donor as part of her SKP. This patient also had near immediate normal endocrine and exocrine pancreatic function. By six months after transplant, pre-transplant HgbA1c of 7.2% dropped to a post-transplant Hgb A1c of 5.4% and C-peptide increased from <0.1 to 1.3 ng/mL.Immediate postoperative sonographic size of the transplant pancreas was 9.5 cm x 3.6 cm (CC x AP). Three-month follow-up showed a decrease in the size of the transplant to 6.1 cm x 1.8 cm (CC x AP). The sonographic appearance of the pancreatic allograft remained normal throughout all studies, with no complicating events.Case 3A 47-year-old male with T1D complicated by ESRD and diabetic retinopathy received a SKP. At the time of surgery, he weighed 56 kg and the weight of the six-year-old pediatric pancreatic donor was 25 kg. His postoperative course was complicated by a small bowel obstruction requiring lysis of adhesions and an internal hernia requiring reduction, within one month after his initial transplant surgery. His endocrine and exocrine function were normal after surgery. At six-months follow-up, his preoperative HgbA1c of 8.4% had dropped to 4.6% and C-peptide increased from <0.1 to 2.5 ng/mL.Immediate postoperative sonographic size of the transplant pancreas was 7.8 cm x 1.6 cm (CC x AP). Three months postoperatively, his transplanted pancreas was 8.0 cm x 1.9 cm (CC x AP).All three patients had normal endocrine and exocrine function postoperatively, and have maintained normal function to date (more than one-year follow-up) despite the mismatched donor–recipient sizes. Recent studies have shown superior metabolic results as well as similar or decreased complication rates compared to adult donors with transplantation of a pediatric donor pancreas into an adult recipient -6. A stuAs early as 1974, infant rat kidneys were found to grow at the same rate and to the same ultimate size, whether grafted in infants or adults . These rThere is no published information on pancreatic graft size following transplantation. Our case series showed no appreciable growth up to three months post op with measurements within a few millimeters difference. In fact, the gland decreased in size in case #2 Table . The resThe largest limitation of this study is the fact that a precise sonographic measurement of a grafted pancreas is very difficult. The organ is curved and three dimensional while our study attempts to use a two-dimensional measurement Figure . We attePancreas organ underutilization from organ donors has been a historical barrier. This has led several institutions, including ours, to begin grafting non-traditional pediatric donors with adult recipients. Functional results in our patients have been a success despite the mismatch in donor-recipient size. Because pediatric donor kidneys grow rapidly after transplant, we were curious to study how pediatric donor pancreases would respond after transplant. As more transplant cases like these take place, further research is required to better understand the conundrum of how the pediatric organ functions sufficiently in adult patients despite the lack of growth typically seen in other transplanted pediatric organs.


Parents can influence offspring dispersal through breeding site selection, competition, or by directly moving their offspring during parental care. Many animals move their young, but the potential role of this behavior in dispersal has rarely been investigated. Neotropical poison frogs (Dendrobatidae) are well known for shuttling their tadpoles from land to water, but the associated movements have rarely been quantified and the potential function of tadpole transport in dispersal has not been addressed. We used miniature radio-transmitters to track the movements of two poison frog species during tadpole transport, and surveyed pool availability in the study area. We found that parental males move farther than expected by the distance to the nearest pool and spread their offspring across multiple pools. We argue that these movement patterns cannot be fully explained by pool quality and availability, and suggest that adaptive benefits related to offspring dispersal also shape the spatial behavior of parental frogs.The online version of this article (10.1007/s10682-019-09994-z) contains supplementary material, which is available to authorized users. The local physical and social environment can have a strong influence on animal dispersal  Wells . Poison We used miniature radio-transmitters to track parental frog movements during tadpole transport in two poison frog species with male parental care, but otherwise contrasting reproductive strategies. We were particularly interested in establishing whether the frogs use the nearest pool available. Tadpole transport requires energy and time, and may increase exposure to predators and reduce mating opportunities  and Dendrobates tinctorius . Both species are diurnal, breed throughout the rainy season, and are locally common but allopatric throughout most of their range . We monitored an area of approximately 30 ha of rainforest bordering a pastureland on one side. We mapped all terrestrial water bodies found during our study with a GPS/GIS device  and ArcPAD 10 (ESRI) software. Some stream sections were not accessible and were mapped by extrapolating mapped parts of the stream. Water bodies were found opportunistically, mostly when tracking tadpole-transporting frogs. Most water bodies consisted of series of partially separated pools in the stream bed that were intermittently connected by flowing water after heavy rainfalls . We monitored an area of approximately 4 ha of terra-firme rainforest. We mapped all visible water bodies and trees that were climbed by tadpole-transporting frogs. All pools visited by frogs that were accessible for inspection contained tadpoles of Dt and were considered as suitable tadpole deposition sites. In addition, we considered all trees that were climbed by tadpole-transporting frogs as potential deposition sites. Ameerega trivittata males (n = 9) were all captured during tadpole transport. One frog was recaptured on two consecutive tadpole transport events. Dendrobates tinctorius males were captured either during tadpole transport (n = 5) or were already tagged before tadpole transport (n = 6) as part of another study. From these 11 Dt males, two frogs were observed on two and three consecutive transport events. One individual was tracked on two different years. Frogs were kept inside a plastic bag or a net-cage for 15 to 105 min for the preparation and fitting of the transmitter, but handled only for a few minutes at a time. Frogs were equipped with miniature radio transmitters  attached externally using a waistband or a harness made of silicone materials . In two cases tracking was terminated due to skin injuries from tag attachment. The mapping was done using a combination of GPS and local references established with traditional survey methods  and R statistical software (http://www.R-project.org/). For each tadpole transport event, we calculated (1) total duration, (2) cumulative path length, (3) straight-line distance from home to the farthest tadpole deposition site used , and (4) straight-line distance to the nearest identified pool site . We did not quantify the home ranges or territories in this study, but approximated the origin of the tadpole transport by a single location termed “home reference” within the presumed home territory. Because most frogs were first located on the way to or at the pools, we used the last point of their homing trajectory as their home reference . When the exact starting location of the tadpole transport was known or when homing tracking was terminated prematurely due to predation or injury, we used the first point of the trajectory as the home reference. The distances to pools for each frog were calculated from their respective home references. To estimate the cumulative path travelled for incomplete trajectories, we added the straight-line distance from the last point of the trajectory  to the location of the first observation. Because of the incomplete tadpole transport trajectories, the total duration and the distances of the transport are very conservative and likely underestimated. We performed a Mann–Whitney–Wilcoxon Test to compare the total durations, observed pool distance, and cumulative distances between the two species, and a paired Wilcoxon Signed-Rank Test to compare the observed and nearest pool distance for each species.Data analysis and visualization were done in QGIS v2.14  and deposited them in standing water pools in a partially dried-up stream bed (n = 6) or flooded meadow on the edge of the forest (n = 2). Frogs visited and deposited tadpoles in one or two separate water bodies within a single transport event. Dendrobates tinctorius transported 1 to 2 tadpoles and deposited them in water-filled tree holes at 1–10 m above ground (n = 5), presumed tree holes above 10 m and thus out of sight (n = 6), standing water inside fallen trunks (n = 3), and palm bracts (n = 1). Dendrobates tinctorius visited up to three deposition sites within one transport event. When transporting two tadpoles (n = 4) frogs deposited both tadpoles in the same pool. When the same Dt male was observed during more than one transport event (n = 3) frogs deposited tadpoles in different sites during each event.We successfully tracked eight At and 15 Dt tadpole transport events  Fig. . AmeeregAmeerega trivittata used tadpole deposition sites farther from home areas than Dt ; traveled longer cumulative paths ; and spent longer time away from the home area .AT ± SD = 52 ± 41 m, range 3–126 m; Wilcoxon paired test: VAT = 28, p = 0.02; mean pool distanceDT ± SD = 19 ± 14 m, range 5–64 m; Wilcoxon paired test: VDT = 55, p = 0.006; Fig. Males of both species traveled significantly longer distances than the distance to the nearest available pool Supplementary material 2 (DOCX 2140 kb)Supplementary material 3 (CSV 3 kb)Supplementary material 4 (XLSX 24 kb)Supplementary material 5 (XLSX 20 kb)Below is the link to the electronic supplementary material.


Candida parapsilosis (C. parapsilosis) is a known pathogen in device-associated CNS infections and has been reported in both, infants and adults.Meningitis is a common and life-threatening infection of the central nervous system (CNS) in infants with long-term and disabling sequelae like hydrocephalus. Hydrocephalus is treated by diverting cerebrospinal fluid (CSF) either to another body cavity (via CSF shunt) or externally (via CSF drain) which are prone to infection. Though rare, C. parapsilosis and fluconazole was started. Despite treatment, CSF continued to grow C. parapsilosis through day 10. The EVD was removed, and an Ommaya reservoir along with the ventricular catheter was placed for better interventricular antibiotic administration. After day 13 CSF became sterile. Ommaya reservoir was removed, fluconazole was continued for three weeks, and a ventriculoperitoneal shunt was placed five weeks later.A six-month-old male infant was brought to the hospital with disproportionate head enlargement of three months duration. Magnetic resonance imaging (MRI) was suggestive of gross asymmetrical hydrocephalus. An external ventricular drain (EVD) was placed, and vancomycin and meropenem were started. Four weeks later, he developed a fever with a blocked EVD. Repeat MRI revealed gross asymmetric dilatation of left lateral ventricle along with pneumocephalus in the right periventricular region. A right temporoparietal craniotomy with drainage of a multiloculated abscess was done along with the removal of right EVD and placement of left EVD. CSF showed pan-susceptible C. parapsilosis has been increasing in prevalence, especially in immunocompromised hosts, infants, and in patients with indwelling catheters. Amphotericin B or fluconazole is the usual treatment with excellent outcomes and no mortality. This case underscores the need for suspicion of C. parapsilosis as a cause of device-associated CNS infections.The device-associated CNS infections are insidious with nonspecific manifestations making diagnosis difficult.  Meningitis of bacterial or nonbacterial origin is a common and life-threatening infection of the central nervous system (CNS) in infants. With the advent of modern medicine, the mortality rate has decreased with a proportionate increase in the risk for long-term and disabling sequelae. Meningitis in infants can lead to various postinfectious sequelae among which hydrocephalus is common. It can result from blockage of the cerebrospinal fluid (CSF) flow at the aqueduct of Sylvius or the outlets of the fourth ventricle, obstruction of flow within the subarachnoid spaces, or an impediment to CSF absorption.Candida parapsilosis (C. parapsilosis) is a known pathogen in device-associated CNS infections and has been reported not only in infants but also in adults [Hydrocephalus requires hospital admission and is treated by diverting CSF either to another body cavity (via CSF shunt) or externally (via CSF drain). Both CSF shunts and CSF drains are prone to infection with higher infection rates noted in those undergoing successive shunt revisions. Gram-positive cocci account for a majority of these cases, but Gram-negative and positive bacilli, fungi, and antimicrobial resistant bacteria have also been reported -2. Fungin adults .C. parapsilosis in an infant with hydrocephalus is being reported. A case of device-associated CSF infection by A six-month-old male infant, with a significant past medical history of neonatal meningitis on the second day of life, was brought with complaints of disproportionate head enlargement for three months duration. Initial magnetic resonance imaging (MRI) was suggestive of gross asymmetrical hydrocephalus with obstruction at the level of the aqueduct, and no signs of ependymal thickening Figure .Ventricular tap was done, and CSF was received for cytology, biochemical analysis, and culture. Cultures were sterile, and there were no features of infection.For intra-cranial pressure reduction, an external ventricular drain (EVD) was placed, and intraventricular vancomycin (10 mg 12 hourly) was started along with parenteral vancomycin (120 mg 8 hourly) and meropenem (240 mg 8 hourly). Serial CSF monitoring was continued.Four weeks later, the child developed a fever. It was noted that the EVD had blocked and a repeat MRI scan revealed gross asymmetric dilatation of left lateral ventricle along with air-fluid level in right periventricular region suggestive of pneumocephalus. The child was managed by right temporoparietal craniotomy and excision of multiloculated abscess done along with the removal of right EVD and placement of left EVD.The CSF samples received showed features of infection and Gram-positive budding yeast was seen on a direct stain Figure .Candida parapsilosis was isolated from culture and was susceptible to all antifungals. Fluconazole (50 mg 24 hourly) was started, and serial monitoring of CSF continued. Despite treatment, daily CSF samples continued to grow C. parapsilosis through day 10. The EVD was removed, and an Ommaya reservoir along with a ventricular catheter was placed for better intraventricular antibiotic administration (vancomycin 10 mg 12 hourly). CSF samples taken 13 days and onwards were sterile. Clinically, the infant became afebrile and stable.Antifungal therapy was continued for three weeks. The Ommaya reservoir was removed, and a ventriculoperitoneal shunt was placed five weeks later. Three months later, the infant remains asymptomatic.The device-associated CNS infections have nonspecific manifestations and have an insidious nature, which makes diagnosis difficult. Retrograde infection is the most likely mechanism of infection of CSF drains. Microorganisms may enter the device by tracking from the exit site alongside the device, gaining access to the fluid column that drains CSF. CSF shunts and CSF drains are prone to infection with a reported incidence rate varying from 4% to 17% [Bacteria remain the most prevalent cause of device-associated CNS infections. Though fungi are rare causes, the growing evidence suggests that fungal infections should be a differential in device-associated infections. Past studies have shown a varying incidence of shunt infections caused by fungi. Chiou et al.  in a retAs in the present case, literature -9 mentioC. parapsilosis has been increasing in prevalence, especially in immunocompromised hosts, neonates, and in patients with indwelling catheters [As fungal infections are rare causes of device-associated CNS infections, fungi are initially not considered as the implicating pathogen. The only definitive diagnostic test is the direct observation and culture of the CSF. atheters .C. albicans remains the most important pathogen, followed by C. parapsilosis and C. glabrata with symptoms appearing as early as one week to as delayed as one year [As reported by earlier studies, one year -6. Amphoone year , 5-7, 9.Candida parapsilosis is the most common fungus isolated from human hands, and given its ability to transfer horizontally, it can contaminate medical devices with ease. A study reported hand colonization of more than 25% of healthcare workers in a community hospital with C. parapsilosis [psilosis . Thus, iC. parapsilosis as a cause of device-associated CNS infections.This case underscores the need for suspicion of C. parapsilosis has been increasing in prevalence, especially in immunocompromised hosts, neonates, and in patients with indwelling catheters. It has a high affinity for parenteral nutrition, frequently colonizes the hands of healthcare workers, and forms a biofilm on prosthetic surfaces and central venous catheters. Extraventricular drainage, therapy with amphotericin B or fluconazole (intravenous or intraventricular), and insertion of a new shunt remain the principal components of the treatment regimen for pediatric fungal shunt infections. There is no established recommendation for the duration of treatment of pediatric fungal shunt infection or the role of other newer antifungal drugs. This case underscores the need for suspicion of C. parapsilosis as a cause of device-associated CNS infection.Device-associated CNS infections are insidious with nonspecific manifestations making diagnosis difficult.


Tenderness of masseters and temporalis can be considered a relevant tool for diagnosis of myo-type craniofacial pain disorders, but a limit of pain score systems is that they are based on subjective pain perception. Surface electromyography (sEMG) is a noninvasive and reliable tool for recording muscle activity. Therefore, we investigated whether a correlation exists between tenderness on masseters and temporalis, assessed by subjective pain scale, and muscles activity, evaluated by sEMG, in patients with painful temporomandibular disorder (TMD) and concurrent tension-type headache (TTH).  A cross-sectional study on fifty adult volunteer patients with TMD and TTH, who underwent tenderness protocol according to Diagnostic Criteria for TMD (DC/TMD) guidelines, was conducted followed by sEMG recording of temporalis and masseters. Pearson's correlation was performed to investigate the correlation between muscular activity and subjective pain scores. y = 1 + 1.2 · x; r2 = 0.62; p < 0.0001), particularly in the temporalis, was observed. Segregation of data occurred according to tenderness and sEMG values. At the highest pain score, the mean sEMG absolute value was higher at the temporalis than the masseters.  An overall moderate correlation between muscle tenderness and sEMG values (NCT02789085. Our study provides evidence that subjective pain perception can be objectively quantified at a magnitude proportional to pain severity. At greater tenderness scores, higher sEMG activity at the level of temporalis could help discriminate clinically prevalent TTH versus prevalent TMD. sEMG confirms to be an accurate tool to reliably objectify the subjective perception of pain. When combined with clinical evaluation and patients' symptoms, sEMG increases diagnostic sensitivity in the field of myo-type craniofacial pain disorders. This trial is registered with  Pain and muscle dysfunctions are considered keystone symptoms in a temporomandibular joint disorder (TMD)  and are Surface electromyography (sEMG) is a noninvasive technique for recording muscle activity. It is considered a reliable and complementary tool for clinical diagnosis of myogenous TMD and for the study of muscle function –15. In aTo our knowledge, only a few studies  report oTo this aim, we investigated the correlation between tenderness on masseters and temporalis, assessed by subjective pain scale (NS) using validated criteria for TMD  and objectified muscle activity, evaluated by sEMG, in patients with diagnosis of TMD and concurrent TTH.This cross-sectional study was conducted in accordance with the Declaration of Helsinki. The Committee on Ethics in Science of the University of L'Aquila  approved the study, and the written informed consent was obtained from each subject and electronically stored as suggested by our institutional guidelines. All procedures were completed between February and November 2016 at the Division of TMD and Orofacial Pain at University of L'Aquila .Fifty volunteer patients  evaluated with RDC/TMD  who fulfIn order to confirm TTH, each subject completed a diagnostic headache diary for 4 weeks .Each enrolled subject underwent tenderness protocol followed by sEMG recording, as further specified in the next paragraphs.According to the newly evidence-based Diagnostic Criteria for TMD (DC/TMD) , pain waAccording to the abovementioned criteria, one point for each palpation area of temporalis  and masseters  was tested, for a total of 12 points (six for each side). The palpation was carried out simultaneously at the left and right sides using both hands. For each muscle, only the highest pain score out of the three measurements was recorded for the analysis.Finger pressure (1.0 kg) was calibrated twice using a single-hand mechanical algometer  prior to palpation –24. EachPalpation was performed with the subjects in a horizontal supine position on a bed with their eyes closed, after 10 minutes of acclimation. Room temperature (21°C) and relative humidity (50%) remained constant. Any external or internal noise sources were controlled. Enrolled patients self-reported the most frequent headache site as occipital, temporal, mixed , or spread.The same operator (DP) performed the tenderness protocol and registered the induced-pain values in an electronic spreadsheet, reporting the highest pain score for each muscle .μV) were used as indices of the signal amplitude [According to the literature , 25–27, mplitude .Three consecutive sEMG rest position tracks, with a duration of 15 seconds each, were acquired, and any trace with interposing phasic event, such as swallowing, voluntary movement, or clenching, was discarded. The sEMG recording procedure was subsequent to the palpation, 5 minutes after placing the electrodes. The recording procedure was carried out in the same room where the tenderness protocol was performed.Procedure for sEMG registration was performed by one examiner (AM) in a blinded fashion. For each studied muscle, the average between the three recordings, which was generated by the software and expressed as microvolts (mV), was reported in a spreadsheet for differential statistics.Parametric approach was used for a differential statistic since collected data revealed normal distribution with the Shapiro–Wilk test. Relation between sEMG and palpation-induced pain values was evaluated by Pearson's correlation.p < 0.05. The R software was used for statistical analysis [Level of significance was set at analysis . Moreovey = 1 + 1.2 · x; r2 = 0.62; p < 0.0001)  .y = 0.93 + 1.3x; r2 = 0.695; p < 0.0001—masseters: y = 1.1 + 1.1x; r2 = 0.514; p=0.0062)  .y = 0.54 + 1.4x; r2 = 0.776; p < 0.0001), a moderate correlation for LAT , a moderate-low correlation for RMM , and a moderate correlation for LMM   .The hierarchical cluster analysis paired muscles and relative pain perception using an euclidean correlation (columns) When considering the sEMG findings as a whole , segregap < 0.001)  at pain scores between 0 and 2 did not significantly differ between the ATs and MMs groups. Indeed, at the highest pain score (=3), the mean sEMG absolute value was 5.16 ± 0.47 mV and 4.73 ± 0.30 mV at the ATs and the MMs, respectively (< 0.001) .Our study demonstrates three important findings. First, sEMG findings in individuals with TMD and concurrent TTH correlate with temporal and masseter tenderness, and this correlation is particularly strong at the level of ATs. Second, sEMG confirms to be an accurate tool to reliably objectify the subjective perception of pain. Third, at greater tenderness scores, higher sEMG activity at the level of ATs compared to MMs could help discriminate clinically prevalent TTH vs prevalent myo-type TMD .With reference to the first point, tenderness at the ATs and MMs has been documented in individuals suffering from TMD and TTH . InteresAccording to this, segregation of data occurred based on tenderness and sEMG values, with a direct relationship between the two parameters . The preComorbidity of TMD and TTH is well documented in the literature , 38 and Our results also demonstrated significantly higher absolute values of sEMG findings at greater tenderness scores at the level of ATs compared to MMs. Although little differences in mV amplitude between ATs and MMs may occur, as expression of different skin impedance at the two sites , and altThis study has some limitations that deserve to be mentioned. First, the lack of control group of healthy subjects since it is a cross-sectional study; data were extrapolated by using software as RMS and finally the sEMG measurement was made only after muscle palpation, and these data indicate only a correlation between subjective pain and sEMG values, thus the generalizability deserves caution.In conclusion, pure myo-facial TMD without TTH may be characterized by the lack of such a manifest activation of ATs. The clinical meaningfulness of these observations needs to be evaluated in the light of further knowledge, particularly studies looking at the therapeutic response to different treatment approaches.


Spermatogonial stem cell transplantation (SSCT) is a promising therapy in restoring the fertility of childhood cancer survivors. However, the low efficiency of SSCT is a significant concern. SSCT could be improved by co-transplanting transforming growth factor beta 1 (TGFβ1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGFβ1-induced MSCs.n = 10) and a combination of SSCs and TGFβ1-treated MSCs . Both transplanted groups and a fertile control group (n = 7) were allowed to mate naturally to check the reproductive efficiency after transplantation. Furthermore, the testes from transplanted males and donor-derived male offspring were analyzed for the epigenetic markers DNA methyltransferase 3A (DNMT3A) and histone 4 lysine 5 acetylation (H4K5ac).A mouse model for long-term infertility was used to transplant SSCs , even after injecting half of the number of SSCs in MSi-SSCT. The litter sizes after SSCT (3.7 ± 3.7) and MSi-SSCT (3.7 ± 3.6) were similar but differed significantly with the control group . The number of GFP+ offspring per litter obtained after SSCT (1.6 ± 0.5) and MSi-SSCT (2.0 ± 1.0) was also similar. The expression of DNMT3A and H4K5ac in germ cells of transplanted males was found to be significantly reduced compared to the control group. However, in donor-derived offspring, DNMT3A and H4K5ac followed the normal pattern.The overall tubular fertility index (TFI) after SSCT (76 ± 12) was similar to that after MSi-SSCT (73 ± 14). However, the donor-derived TFI after MSi-SSCT (26 ± 14) was higher compared to the one after SSCT (9 ± 5; Co-transplanting SSCs and TGFβ1-treated MSCs results in reproductive efficiency as good as SSCT, even after transplanting half the number of SSCs. Although transplanted males showed lower expression of DNMT3A and H4K5ac in donor-derived germ cells, the expression was restored to normal levels in germ cells of donor-derived offspring. This procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure safety in the long term. Over the last decades, the survival rate after childhood cancer has increased tremendously , 2. HoweAs mesenchymal stem cells (MSCs) secrete paracrine factors which have anti-apoptotic, anti-inflammatory, and anti-oxidative properties –19, theyThe highly complex and dynamic process of epigenetic reprogramming is a crucial step during spermatogenesis. Any abnormality is likely to cause infertility, and the risk of inheriting an altered epigenome resulting in phenotypic defects in offspring cannot be neglected , 23.After SSCT in mice, the expression levels of DNA methyltransferase (DNMT) 1 and 3A and the DNA methylation patterns were not different between controls and first- and second-generation offspring . MoreoveTo investigate the reproductive efficiency and safety of MSi-SSCT, we performed mating experiments and assessed litter sizes and number of (donor-derived) pups. Furthermore, we evaluated the reproductive safety by detecting the expression pattern of DNMT3A, which is involved in paternal imprinting , and his6 cells/flask with 10 ng/ml recombinant mouse TGFβ1  in OriCell™ mouse MSC basal medium  in a humidified incubator with 5% CO2 at 37 °C for 15–21 days. The medium was changed every third day, and cells were passaged after reaching 80% confluency  to obtain a concentration of 10–20 × 106 cells/ml. For MSi-SSCT (n = 10), TGFβ1-treated MSCs at passage 5 or 6 were resuspended in the injection medium to obtain a concentration of 10–20 × 106 cells/ml. GFP+ testicular cells (including SSCs) and MSCs were mixed in 1:1 volume. The target was to inject 2 × 105 cells (10 μl) per testis. Non-treated and non-transplanted mice (n = 7) were used as fertile controls  and control (n = 7) mice was housed with two female mice for a period of 3 months. Litter sizes and numbers of GFP+ and RFP+ pups were recorded. To confirm the origin of the progeny , the pups were analyzed with a royal blue (excitation 440–460 nm for GFP) and green (excitation 510–540 nm for RFP) led flashlight . All pups were checked for anatomical features, response to external stimuli, and reflexes  at 10 and 15 days after birth [Two months after transplantation, each of the transplanted group  were blindly analyzed to assess the overall TFI (percent of tubules containing spermatogenesis) and the donor-derived TFI (percent of tubules containing donor-derived spermatogenesis) [At the end of the mating experiments (5 months after transplantation), recipient males were sacrificed by cervical dislocation. Both transplanted mice and donor-derived offspring were examined thoroughly by a standardized postmortem protocol at anatomical and histological levels . All majgenesis) .+ tubules per spermatogenic stage were analyzed  followed by a 5-min wash in PBS. Endogenous peroxidases were blocked in 3% hydrogen peroxide for 30 min. For DNMT3A staining, the antigen retrieval step was carried out in a microwave (350 W) with citrate buffer for 10 min, whereas H4K5ac did not require antigen retrieval. For both stainings, non-specific antibody binding was blocked by 5% normal goat serum for 1 h. After each step, the sections were washed in PBS for 5 min. The primary antibodies for DNMT3A  and H4K5ac  were added at a dilution of 1:250 and 1:1500, respectively, and incubated overnight at 4 °C. For the negative controls, PBS was added instead of the primary antibody. The next day, the sections were washed three times with PBS after which the secondary antibody was added for 1 h at room temperature. The sections were again washed three times with PBS. The visualization of the staining was done with 3,3′-diaminobenzidine (30 s). The sections were immersed in PBS and counterstained with hematoxylin for 1 min. The sections were dehydrated in a mounting series of alcohol  and in xylene. Acrytol mounting medium was added before placing the coverslip. For transplanted males, at least ten GFP+ pups were analyzed by the Mann-Whitney test. TFI in offspring, litter sizes, and the percentage of tubules expressing DNMT3A or H4K5ac were analyzed by one-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons . P values < 0.05 are considered statistically significant.(Donor-derived) TFI (mean ± SD) and data of GFPP = 0.002). Moreover, TFI in offspring after SSCT (89 ± 3%) and MSi-SSCT (87 ± 4%) was similar to that of control  and MSi-SSCT (2.0 ± 1.0)  and reproductive organs. Their location and number were found to be normal as well as the color, size, shape, consistency, and texture. No anomalies were detected.SSCT was successful in 85% 17/20) of the testes, while MSi-SSCT was successful in 75% (15/20) of the testes. However, in both transplantation groups, one mouse died after transplantation. These mice were excluded from the analysis. RFP-derived spermatogenesis was never observed. GFP-derived spermatogenesis was found in 47% (7/15) of the successfully injected testes after SSCT and in 62% (8/13) after MSi-SSCT. The overall TFI after SSCT (76 ± 12%) did not differ from the one after MSi-SSCT (73 ± 14%), but the donor-derived TFI after MSi-SSCT (26 ± 14%) was higher compared to the one after SSCT . H4K5ac was detected in spermatocytes and round spermatids but not in elongated spermatids. Interestingly, pre-leptotene spermatocytes also showed H4K5ac in the control group but not in any of the transplanted groups. Overall, H4K5ac showed a similar pattern in transplanted males and control mice, but in transplanted males, the expression was lower from stage VII onwards (P < 0.001) leptotene spermatocytes. The expression pattern was similar between control and donor-derived offspring Fig. . OverallPreviously, we showed that the co-transplantation of MSCs improved the efficiency of SSCT. However, before MSi-SSCT can become a clinical application, the efficiency and safety need to be confirmed as well. Although MSCs did not trans-differentiate towards germ cells, their paracrine factors might have played a role in restoring the damaged SSC niche and thus improving the efficiency of transplantation . Whether+ MSC-derived pups were not observed. Indeed, also in our previous study, RFP+ MSC-derived spermatogenesis could not be observed, although few MSCs co-expressed the germ cell marker MVH [RFPrker MVH .P = 0.002). This difference was not seen in the proportions of GFP+ pups. Thirty percent (2.0/6.7) of the pups were GFP+ after MSi-SSCT, and 22% (1.6/7.2) were GFP+ after SSCT (P > 0.05). However, it has to be mentioned that only two SSCT and one MSi-SSCT transplanted male produced GFP+ pups. This could be due to the fact that donor-derived spermatogenesis was in competition with spontaneously recovered or MSC-induced endogenous spermatogenesis. If an SSC niche is occupied with an endogenous SSC, it cannot harbor a transplanted SSC, as the niche can only contain one SSC [Although TFI was similar for SSCT and MSi-SSCT, the donor-derived TFI after MSi-SSCT was higher than that after SSCT. Moreover, the donor-derived TFI after MSi-SSCT accounted for 36% of the total TFI whereas it only accounted for 12% after SSCT genetic inheritance to the next generations , 23. The+ pups.Aberrations in sperm DNA methylation are correlated with poor sperm quality and impaired fertility . Moreovev mice). In contrast to the present model, somatic cells from W/Wv mice have never supported spermatogenesis, which may have interfered with epigenetic modification. SSCT using fresh SSCs resulted in significantly higher donor-derived TFI (35%), compared to using cryopreserved SSCs, which resulted only in a donor-derived TFI of only 9% (P < 0.001) [Although we reported H4K5ac up to leptotene spermatocytes before , we only< 0.001) . This co< 0.001) . Harsh c< 0.001) , which m< 0.001) , it woulAlthough our data look promising for a future clinical application, we have to be aware that much more epigenetic marks are being reprogrammed during gametogenesis and that the method used in this study is semi-quantitative. For the exact quantification of epigenetic modifications, recent improvements in single-cell analytics are promising. Epigenetic mechanisms can now be explored in-depth using advanced techniques , even on single-cell level (for review ). MoreovAlthough MSCs offer an enormous regenerative potential, they also pose some difficulties for clinical implementation such as their diverse sources, individual variability, and lack of standardized protocols for in vitro maintenance and differentiation . These dIt would have been interesting to evaluate the transplantation of TGFβ-treated MSCs alone. In our previous study , we inclAnother limitation of the study is that testicular cell suspensions  were transplanted instead of isolated SSCs. Although we acknowledge that SSC sorting could have improved SSCT efficiency . ParreirMoreover, in a future clinical application, xeno-derived products, like fetal calf serum, need to be replaced by human-derived alternatives .Co-transplanting SSCs and TGFβ1-treated MSCs reach the reproductive potential of SSCT alone even after transplanting half the number of SSCs. Although low expression of DNMT3A and H4K5ac were observed in donor-derived gem cells, normal levels were restored in offspring. However, extensive epigenetic analyses are needed in order to ensure reproductive safety.


Definitive diagnosis of meningitis is made by analysis of cerebrospinal fluid (CSF) culture or polymerase chain reaction (PCR) obtained from a lumbar puncture (LP), which may take days. A timelier diagnostic clue of meningitis is pleocytosis on CSF analysis. However, meningitis may occur in the absence of pleocytosis on CSF.Areas of Uncertainty: A diagnosis of meningitis seems less likely without pleocytosis on CSF, leading clinicians to prematurely exclude this. Further, there is little available literature on the subject.Ovid/Medline and Google Scholar search was conducted for cases of CSF culture-confirmed meningitis with lack of pleocytosis. Inclusion criterion was reported cases of CSF culture-positive or PCR positive meningitis in the absence of pleocytosis on LP. Exclusion criteria were pleocytosis on CSF, cases in which CSF cultures/PCR were not performed, and articles that did not include CSF laboratory values.A total of 124 cases from 51 articles were included. Causative organisms were primarily bacterial (99 cases). Outcome was reported in 86 cases, 27 of which died and 59 survived. Mortality in viral, fungal and bacterial organisms was 0, 56 and 31%, respectively. The overall percentage of positive initial CSF PCR/culture for viral, fungal and bacterial organisms was 100, 89 and 82%, respectively. Blood cultures were performed in 79 of the 124 cases, 56 (71%) of which ultimately cultured the causative organism. In addition to bacteremia, concomitant sources of infection occurred in 17 cases.Meningitis in the absence of pleocytosis on CSF is rare. If this occurs, causative organism is likely bacterial. We recommend ordering blood cultures as an adjunct, and, if clinically relevant, concomitant sources of infection should be sought. If meningitis is suspected, empiric antibiotics/antifungals should be administered regardless of initial WBC count on lumbar puncture.The online version of this article (10.1186/s12879-019-4204-z) contains supplementary material, which is available to authorized users. Meningitis is a serious acute infection of the meninges that can be caused by bacteria, viruses, parasites, or fungi . DefinitThe purpose of this paper is to review the published literature to describe themes associated with CSF culture-proven meningitis or CSF PCR positive in the absence of pleocytosis on initial LP. There are no conflicts of interest in this report.2- chi-square.Throughout this paper, several abbreviations are used, which include: AIDS - Acquired Immune Deficiency Syndrome; CSF - cerebrospinal fluid; LP - lumbar puncture; PCR - polymerase chain reaction; WBC - white blood cell; ΧWe conducted a search of Ovid/Medline and Google Scholar using a combination of keywords and controlled vocabulary representing the concepts “meningitis,” “cerebrospinal fluid,” and “absence of pleocytosis.” The reference lists of each included article were also searched for relevant citations not identified in the search.3 in cases 28 days of age or younger, CSF WBC ≤ 9 cells/mm3 for cases 29–56 days of age, and CSF WBC ≤ 5 cells/mm3 in cases over 57 days of age [3 in cases 28 days of age or younger, > 9 cells/mm3 for cases 29–56 days of age and CSF WBC > 5 cells/mm3 in cases over 57 days of age; other exclusion criteria were cases where CSF cultures were not performed, and articles that did not include CSF laboratory values.All studies were reviewed by title and abstract and then by full text. Studies were included in this paper if they reported cases of CSF culture/PCR positive meningitis in the absence of pleocytosis on LP. Absence of pleocytosis was defined as CSF WBC ≤ 19 cells/mms of age . ExclusiWe did not exclude based on age. Thus, this literature review encompasses patients of all ages, including neonates. The review includes both immunocompetent and immunosuppressed patient populations. We did not place restrictions on the years that articles were published. Articles in all languages were reviewed with the assistance of translators when indicated.The Ovid/Medline search yielded a total of 682 articles, 23 of which were duplicates. Of these articles, 535 were excluded as they were not found to be relevant by title or abstract. The remaining articles were assessed full-text for possible inclusion. Of these 124 remaining articles assessed by full text, 77 were excluded. Titles from references of the 124 articles screened by full text were also assessed for possible inclusion. We reviewed 36 articles from references, 4 of which met inclusion criteria. A total of 4 articles from references of full-text screened articles and 47 articles from Ovid/Medline search were included for a total of 51 articles  was performed to compare categorical data. Age ranged from the first day of life to 86 years. We defined neonate from age 1–28 days, pediatric 29 days – 17 years, adult 18–64 years, and senior as 65 years and over. Forty-one cases were female, 43 cases were male, and 40 cases did not have gender reported. Across all age groups, most cases were bacterial (99 of 124). However, viral and fungal infections also caused 16 and 9 cases, respectively. The most common bacterial organisms reported were N. meningitides (25 cases), S. pneumoniae (22 cases), and H. influenzae (17 cases). The most commonly reported viral organism was Enterovirus (12 cases), and the most commonly reported fungal organism was Cryptococcus sp (7 cases). Males were more likely to present with bacterial and fungal meningitis, while females were more likely to present with viral meningitisTables N. meningitides, E. coli, Cryptococcus, and S. Aureus.All cases had no pleocytosis on the initial LP. A repeat LP was performed in 37 of the 124 cases, and WBC on LP was reported in 33. Four of these 33 cases (12%) still did not yield CSF pleocytosis. The organisms involved in this phenomenon were Outcome in terms of death, survival, or unknown is summarized in Table Cryptococcus sp was the most commonly reported organism , with mortality in 57% of these cases. Further, these patients most likely had co-morbidities. The overall reported mortality of bacterial infections was 31%. The four most commonly reported bacterial organisms were N. meningitides, S. pneumoniae, H. influenzae, and E. coli, with mortality at 33, 33, and 21%, and 33%, respectively. There was one reported case dual infection by Clostridium & Bacteroides sp., which resulted in death.Mortality varied between types organisms. Across the age groups, adults were most likely to die. No patient that was afflicted with viral meningitis died. Fungal organisms yielded the highest mortality at 56%. Of the fungal infections, Table H. influenzae.There were 19 cases in which the initial CSF culture was negative. In 13 of these cases, blood cultures were taken at the time of the initial LP, and 11 were positive on initial blood draw. The 2 with initially negative blood cultures returned positive on repeat blood culture, and both cultured H. influenzae.Blood cultures were performed in 79 of the 124 cases. Of these 79 cases, 56 (71%) ultimately cultured the causative organism. Fifty-five were bacterial, and one was fungal. Fifty-three cultured the causative organisms at the time of the initial blood culture. The 3 blood cultures that did not initially return positive were all the bacterium S. pneumoniae. There were 3 cases reported in which patients had arthrocentesis which cultured the same causative organism on CSF. One patient had a positive urine culture, which grew P. mirabilis. This patient had a co-morbidity of a chronic indwelling foley catheter which was clogged on initial presentation. Lastly, there were two cases of peritoneal fluid which cultured E. coli. Both had a history of alcohol abuse. In these 17 cases, if blood cultures were performed, they were positive except for the case of C. albicans.In addition to blood cultures, concomitant sources of infection for causative organism were reported in 17 cases Table . There wAs above, the WBC on LP was within normal limits at the time of the first lumbar puncture. Other CSF markers that could indicate meningitis is elevated protein and depressed CSF glucose/serum glucose. In adults, the generally accepted normal range for protein in CSF is 15–45 mg/dL . UnfortuWe analyzed the serum glucose and protein level in all patients to assess if there were instances if there was normal CSF markers for WBC, CSF glucose, and CSF protein. In our study, of the cases in which all markers were recorded, all CSF markers were within normal limits in 3 cases aged 1–56 days, 24 cases aged 56 days – 18 years, 12 adult patients, and 1 senior patient.Meningitis in the absence of pleocytosis on CSF is rare. However, it has been reported in both adult and pediatric patients.N. meningitides, the patient had a co-morbidity of diabetes mellitus and positive blood culture and joint fluid culture. The patient with E. coli suffered from chronic ethanol abuse and had a positive blood and peritoneal fluid culture. We suspect that in these 2 cases meningitis developed secondary to seeding of the bacterium from another source, and this delay in time in addition to a chronic immune compromised state may have resulted in the patient being unable to mount an immune response at the time of repeat LP. This is further supported by both patients having a negative initial CSF culture, indicating that the organisms may have not truly inoculated the CSF at the time of initial LP. The patient with Cryptococcus sp suffered from acquired immune deficiency syndrome (AIDS), and likely the patient’s immunocompromised state prevented proper leukocytosis. Lastly, the patient with S. aureus had a history of congenital hydrocephalus with VP shunt with revision. It is unclear why this patient did not mount a leukocytosis on repeat LP.While most cases had CSF pleocytosis on repeat LP, 4 cases still had lack of pleocytosis on repeat lumbar puncture. In the case of Cryptococcal meningitis were immunocompromised (5 with Acquired Immune Deficiency Syndrome (AIDS)), 1 with lupus with CD4 count of 75 yet but negative for Human Immunodeficiency Virus, and 1 with stage IV Hodgkin’s Lymphoma with pancytopenia), which likely increased mortality. Further, empiric coverage for meningitis may have targeted bacterial infections and not fungal infections, further increasing mortality in this subset. Viral infection portended the best prognosis, which is likely reflective of viral meningitis tending to be self-limited with good outcome [Herpes simplex virus is a notable exception, the one reported case survived [Mortality varied between organisms, which may be due to several reasons. Fungal infection portended the poorest prognosis with an overall mortality in 56% of cases. All 7 cases with  outcome . While Hsurvived , 59. HowUltimately, 100% of CSF cultures returned as positive at the time of second lumbar puncture. As above, the initial CSF cultured positive for causative organism in 105 of the 124 cases (85%). There are several possibilities why the initial CSF culture would have been negative. First, patients may have received IV antibiotics prior to LP, temporarily sterilizing the CSF and resulting in a negative culture. Second, the CSF draw might not have been sufficient to culture. Third, patients may have developed meningitis secondary to another source such as bacteremia, which had not seeded into the CSF prior to the time of the initial LP . Fourth,H. influenzae, had no growth on the initial blood culture. We hypothesize that the bacterium had not yet seeded into the bloodstream from the original source of infection at the time of initial blood culture. If clinically relevant, other sources of infection, such as joint fluid, peritoneal fluid, pleural fluid and urine should be cultured for possible causative organism.Blood cultures had greatest clinical utility with bacterial infection. Only 1 bacterium, We recognize several limitations in this literature review. While to our knowledge this is the largest review of available literature of meningitis in the absence of pleocytosis, is likely that this phenomenon is underreported. One possible cause of underreporting is that these cases meningitis may be overlooked due to seemingly normal appearing CSF biochemistry. Thus, our sample size is still relatively small, and results may not be generalizable. Further, some articles did not present all data we sought to analyze. We also recognize that viral meningitis without pleocytosis may be classified as headache and no further CSF analysis performed. If confirmatory CSF PCR is not performed, underreporting will occur. As this was a literature review derived from pooled data, articles were subject to varying reported clinical information, methods, results, and timelines.N. meningitides, another Enterovirus, and another Cryptococcus sp in patients with AIDS, thus increasing the number of these organisms reported [H. influenzae B in childhood has caused a decline in this organism. Many of the articles that reported this were written before vaccination, and, therefore, results may not reflect what is seen in current practice.Another limitation of the study is that articles were subject to selection bias. One prospective study targeted reported , 33, 44.reported , 30. Onereported , 30. ThuThere were also a variety in the immunocompetent state of the patient. This study includes oncology patients who may have been neutropenic secondary to chemotherapy, AIDS patients who were immunocompromised secondary to CD4 deficiency, and patients who were immunocompromised due to extremes of age. Thus, laboratory findings, clinical course, morbidity and mortality in these populations may not be generalizable in an immunocompetent population.Mortality and co-morbidities that may have affected it were also underreported. Lastly, the administration of antibiotics before LP, which could have caused negative cultures, could have occurred but was not reported.Although rarely reported, meningitis in the absence of pleocytosis may occur. While bacterial organisms are most commonly implicated, it may also occur in viral or fungal infections. It occurs in either gender nearly equally and may occur at any age range. Viral infection portends a favorable prognosis. Mortality may be increased with dual infection, fungal infection and immunocompromised state. Given the high mortality associated with bacterial and fungal meningitis in the absence of pleocytosis, clinicians should maintain a suspicion of meningitis in the absence of initial CSF pleocytosis when the clinical picture is highly suggestive and consider appropriate antibiotic/antifungal therapy while awaiting culture results and/or a repeat LP. It has been hypothesized that lack of pleocytosis may occur due to increased severity of disease with rapid progression, leaving insufficient time for an adequate inflammatory response to be mounted prior to obtaining CSF , 19. FurH. influenzae. CSF from initial LP may not culture the organism, and a repeat LP may be indicated to determine the causative organism. If meningitis is present, pleocytosis is likely to be present on repeat LP. However, if patients are severely immunocompromised, have significant co-morbidities, or have meningitis secondary to inoculation of organism from another primary source, a pleocytosis still may not occur. If clinical suspicion suggests meningitis in a patient who is exhibiting a rapid, fulminant course, we recommend empiric treatment for meningitis prior to performing the LP, as this course of action may be life-saving.Since bacterial meningitis can occur secondary to underlying bacteremia from other sources, we suggest ordering blood cultures routinely in suspected cases. Blood cultures may need to be repeated, especially in cases of Additional file 1:Summary table of patient age, gender, CSF results, blood cultures and outcome . (XLSX 3