Alzheimer’s disease (AD) is characterized by the pathological deposition of amyloid-β (Aβ) protein-containing plaques. Microglia and astrocytes are commonly attracted to the plaques by an unknown mechanism that may involve cell adhesion. One cell adhesion family of proteins, the cadherins, are widely expressed in the central nervous system. Therefore, our study was designed to map the expression of cadherins in AD mouse brains. A particular focus was on plaques because diverse mRNA-species were found in plaques and their surrounding area in brains of AD patients.In this study, we used in situ hybridization to visualize cadherin expression in brains of two mouse models for AD (APP/PS1 and APP23).sense probes. Further experiments with variations in probe length, probe sequence, molecular tag and experimental procedure lead us to conclude that cRNA probes bind generally and in an unspecific manner to plaques.A variable number of plaques was detected in transgenic brain sections, depending on the probe used. Our first impression was that the cadherin probes visualized specific mRNA expression in plaques and that endogenous staining was unaffected. However, control experiments revealed unspecific binding with We demonstrate unspecific binding of cRNA probes to plaques in two mouse models for AD. The widespread and general staining of the plaques prevented us from studying endogenous expression of cadherins in transgenic brain by in situ hybridization.The online version of this article (doi:10.1186/s12952-016-0065-9) contains supplementary material, which is available to authorized users. With more than 25 million people affected worldwide, AD is one of the most common age-dependent neurodegenerative disorders . Its patsense probes, demonstrated that the plaque staining was unspecific. Additional controls with probes that varied in length, sequence and the molecular tag, confirmed the unspecific character of the RNA probe binding to the plaques.The aim of the present study was to investigate the expression of multiple types of cadherins and to detect possible alterations in brains from mouse models of AD. We used the in situ hybridization technique to map mRNA expression in the brain and other organs. With this very popular and highly useful method, we obtained plaque staining of different intensities, depending on the individual probe used. The initial conclusion drawn from this differential staining pattern was that the cadherin probes bind to plaques in a specific manner. However, careful control experiments, including the use of All procedures were performed in accordance with institutional guidelines and national laws on the treatment of animals in research. We used the minimum number of animals necessary to produce reliable scientific data.For the present study, 6 hemizygous APP/PS1 mice  and 4 wild-type littermates  were used. Differences in the expression patterns between the genders were not detected. In the present study, we show results for advanced amyloidosis at 6 months of age and for a severe state at 12 months of age only. Data for 3-months and 9-months old mice are not provided in the present work (unpublished data) in order to avoid repetitions of similar results. In addition, brains from 18.5-months old APP23 mutant mice  were used. Both mouse lines  have a C57BL/6 background and were originally generated by the groups of Dr. Mathias Jucker and Dr. Matthias Staufenbiel 12;13]. In the APP/PS1 line, human KM670/671NL-mutated APP (= Swedish double mutation) and L166P-mutated PS1 are coexpressed under the murine Thy-1 promotor. In this mutation, amyloid deposits can be detected from 2–4 months of age onward [. In the Template RNA was isolated from the brain of a male C57/BL6J adult mouse by using TRIzol reagent according to the manufacturer’s instructions . Obtained RNA concentration was measured by a spectrophotometer.®II-TOPO vector by using the TOPO TA Cloning Kit (Invitrogen), following the manufacturer’s instructions. Successfully integrated fragments were sequenced by a commercial company  by using specific internal primers. Sequences were verified by using the NCBI-BLAST program [Primer pairs for shortened Pcdh8 probes were designed by using the Lasergene® Genomics Suite Software . In order to obtain fragment 1 (F1), which contains the first 812 bp of the open reading frame, the following primers were used: Forward: ATT TAG TCT CTG CTG GGT GCT CTC and reverse: GGG CGC CGA AGG TGA AC. Another primer pair  was used to obtain a second fragment (F2), which represents the last 848 bp of the open reading frame. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed with the ONE Step RT-PCR Kit  with the following parameters: Reverse transcription at 50 °C for 30 minutes, inactivation of the transcriptase at 95 °C for 15 minutes, followed by 30 cycles of amplification . The correct size of the generated fragments was verified by agarose gel electrophoresis. Fragments were eluted by MinElute Extraction Kit (Qiagen) and cloned into a customized pCR program .sense and antisense probes were produced with the DIG RNA Labeling Kit or Fluo RNA Labeling Kit , respectively, according to the manufacturer’s instructions. Quick Spin columns (Roche Diagnostics) were used for purification of the probes. Their correct size was verified by agarose gel electrophoresis.The plasmids listed in Table APP/PS1 mice were deeply anesthetized with chloroform and decapitated for brain dissection. APP23 mice were anesthetized with an intraperitoneal overdose injection of pentobarbital (300 mg/kg body weight) and the brains were dissected. Collected brains were fresh frozen in 2-methyl butane chilled to about −40 °C on dry ice and stored at −80 °C. For cryosectioning, frozen brains of APP/PS1 and APP23 were embedded in Tissue-Tek® OCT™ compound , cut at 20 μm thickness on a refrigerated microtome and collected on SuperFrost/Plus slide glasses . To obtain a neuroanatomical overview, thionin staining was carried out on adjacent sections within each brain series, as described previously .Brain sections were fixed in 4% formaldehyde (FA) diluted in phosphate-buffered saline (PBS) at 4 °C for 30 minutes and subsequently washed with PBS. For blocking of unspecific antibody binding, sections were incubated for 1 hour at room temperature with 2% sheep serum and 1% Triton-X diluted in PBS. Primary antibody α-3552 against Aβ  was diluted 1:1000 in blocking solution and applied overnight at 4 °C. Sections were again washed with PBS and incubated with secondary antibody Alexa Fluor® 488 α-rabbit IgG (Invitrogen) diluted 1:1000 in blocking solution, for three hours at room temperature. Final differentiation was performed by PBS washes. Brain sections were mounted in Mowiol solution .All of the mentioned solutions were supplemented with 1 mM CaCl2 and 1 mM MgCl2. Frozen brain sections were thawed for 30 minutes at 37 °C, followed by retrieval in HEPES-buffered salt solution  and immediately put in ice-cold HBSS-buffer. Sections were fixed in 4% PFA/HBSS for 30 minutes at 4 °C and washed twice with Tris-buffered saline (TBS), followed by TBS supplemented with 0.1% Triton X (TBT). Thereafter, slides were incubated in blocking solution  for 1 hour at room temperature. Primary antibodies α-Pcdh10 , RIKEN, Kobe, Japan.), diluted 1:300, and α-3552, diluted 1:1000, were applied in blocking solution overnight at 4 °C, followed by washing steps of TBS and TBT. As secondary antibodies, Biotin-SP-conjugated AffiniPure α-rat  and Goat anti-Rabbit IgG (H + L) Cy5  were used in the same blocking solution and applied for 2 hours at room temperature. Brain slices were then washed again with TBS and TBT and incubated with Alexa Fluor® 488 conjugate-streptavidin  in blocking solution for 2 hours at room temperature. After several washing steps with TBS, sections were counterstained with Hoechst 33342 (Thermo Fisher Scientific) to visualize nuclei and mounted in Mowiol solution.2) for ten minutes. Labeled mRNA was visualized by incubating the sections with the substrates 0.03% nitroblue tetrazolium salt  and 0.02% 5-bromo-4-chloro-3-indolyl-phosphate, ρ-toluidine salt (Fermentas) in NTM solution for one to three days at room temperature. The reaction was stopped by washing in H2O, followed by a rinse with TE buffer. Staining was differentiated by using ethanol and xylenes. Finally, slides were mounted in Entellan .The in situ hybridization procedure followed established protocols , 16. BraThe procedure was the same as the one described above, but we excluded formamide from all washing steps, i.e. sections were treated in the 2× SSC solution only. However, formamide was kept in the hybridization solution.Brain sections were pretreated with 10 μg/ml RNase A in PBS for 30 minutes at 37 °C, followed by three washes with PBS at 37 °C. RNase A was inactivated by incubation with 0.3 U/μl RiboLock (Fermentas) in PBS for 10 minutes at RT, followed by PBS washes. Subsequently, the in situ hybridization was performed exactly as described above.Digital photomicrographs of the brain sections were taken with a light transmission and fluorescence microscope  and a digital camera . Digitized fluorescence signal was converted to grayscale pixel values by a computer. Contrast and brightness of the images were adjusted for optimal display of the staining patterns by using the Photoshop software .For identification of different brain areas, neuroanatomical nomenclature and abbreviations, an adult mouse brain atlas  was consantisense cRNA probes. In the present work, we show exemplary staining patterns only for cadherin-2 (Cdh2), Cdh11, protocadherin-8 (Pcdh8) and Pcdh10 in transgenic 12-months-old APP/PS1 mice and wild-type littermates. In Fig. We analyzed the expression patterns of several cadherins with sense cRNA probes of the chosen cadherins revealed a high rate of plaque detection in transgenic APP/PS1 mice. This result suggested unspecific plaque staining. To confirm this possibility, adjacent frontal hippocampal sections of transgenic and wild-type APP/PS1 mice were stained with sense and antisense probes . To study whether the unspecific plaque binding of the probes depended on probe length or on specific probe regions, we asked whether shorter  cRNA probes for the cadherin sequences resulted in less or even absent unspecific plaque staining. For this purpose, we generated two partial probes from the open reading frame of Pcdh8 and hybridized them with sections from APP/PS1 mice  stage of plaque maturation . The lowsense probes tend to detect the plaques: Due to the lack of specific binding, the substrate reaction is stopped only after plaque staining has developed. Alternatively or in addition, the reduced endogenous staining might also originate from the high number of plaques, which may capture probes and therefore reduce the amount of probe available for detection of endogenous mRNA. A third possibility for the variation in plaque staining may be a variation in hybridization efficiency of the different probes. While some probes may tend to have a higher binding affinity to their specific sequences, other probes may show less binding specificity and therefore attach more prominently to the plaques.Whereas some probes stained nearly all plaques, other probes tended to detect only a few plaques. This differential staining pattern was similar in both transgenic mouse lines and contributed to our initial impression that the staining patterns were specific for individual cadherins. We offer the following possible explanations for this finding: The reduced endogenous staining might originate from differences in the time-course of the substrate reaction between brain sections treated with different probes. On the one hand, for cadherins that are expressed at high levels in the brain, the specific staining will appear fast during the substrate reaction, while the staining of plaques will emerge more slowly. Thus, substrate reaction will be stopped early when endogenous staining has appeared; plaque staining will be less intense. On the other hand, if endogenous expression is weak, the specific staining will appear more slowly, while the staining of plaques emerge faster, resulting in apparently more intense plaque staining when the substrate reaction is stopped. This possibility could also explain why all sense and antisense oligonucleotides against Aβ [In general, it is believed that shorter fragments of RNA and DNA increase unspecific binding which may explain why oligonucleotides with a length of ~30 bp adhere strongly to plaques, as shown in a study of brain sections of AD patients . Similarainst Aβ . In our Another possible cause for the binding of cRNA probe to the plaques may be the molecular tag of the probe, which might be prone for binding to plaques. To detect the probe, we used digoxigenin, a steroid with a large three-dimensional structure. Digoxigenin is commonly used as a tag for in situ hybridization and immunohistochemistry. To exclude the possibility that digoxigenin caused the unspecific binding, we generated a Pcdh10 probe with the same sequence but with fluorescein, another commonly used molecular tag. This probe turned out to have the same affinity to plaques as the digoxigenin-labelled probe, although fluorescein has a different three-dimensional structure. An investigation with radiolabeled oligonucleotide probes showed similarly unspecific binding to plaques on post-mortem tissue of AD patients . TherefoIn the study of Pardue and colleagues , severalHere we described the phenomenon of unspecific binding of cRNA probes to plaques in transgenic brains of two mouse models for AD. Due to the interference by unspecific probe adhesion, it was not possible to investigate alterations in cadherin expression in the transgenic mouse models for AD. One way to approach the investigation of cadherin expression in transgenic AD-mouse lines is the use of protein detection by immunohistochemistry (Additional file


Bilateral paramedian thalamic infarctions (BPTIs) due to artery of Percheron occlusion are known to cause hypersomnia. However, the role of hypocretin-1, a wake-promoting peptide that is located at the lateral hypothalamus, in hypersomnia in these patients remains unclear.To clarify the role of hypocretin-1 in hypersomnia in patients with BPTIs, hypocretin-1 levels in the cerebrospinal fluid (CSF) were measured in 6 patients with BPTIs: 2 with rostral midbrain involvement (BPT+RMI) and 4 without midbrain involvement (BPT-MI).CSF hypocretin-1 levels were decreased in 2 patients with BPT+RMI and were within normal ranges in 4 patients with BPT-MI. Hypersomnia was noted in all the patients. In one BPT+RMI patient, hypersomnia was improved within 2 weeks and decreased CSF hypocretin-1 levels were reversed (acute phase (on day 9), 109.2 pg/mL; chronic phase (at 3 months), 323 pg/mL), whereas another BPT+RMI patient who displayed coma in the acute phase had decreased CSF orexin levels (107 pg/mL) at day 49 and exhibited severe disability.Hypocretin deficiency was not involved in hypersomnia observed in BPT-MI patients; however, CSF hypocretin-1 levels were reduced in BPT+RMI patients. Reduced CSF hypocretin-1 levels in the chronic phase may possibly predict a poor clinical outcome in patients with Percheron artery infarction. Decreased levels of CSF hypocretin-1 have been described in cases of symptomatic narcolepsy such as hypothalamic lesions due to neuromyelitis optica or multiple sclerosis and tumors.,4Hypocretin-1 (orexin-A) is a wake-promoting peptide that is located at the lateral hypothalamus and that plays pivotal roles in maintaining not only wakefulness but also feeding behavior, reward processes, and motivated behaviors.,6 Lazzaro et al studied 37 patients with AOP infarction and identified 4 different patterns of AOP infarction: bilateral paramedian thalamic with rostral midbrain (43%), bilateral paramedian thalamic without midbrain (38%), bilateral paramedian and anterior thalamic with midbrain (14%), and bilateral paramedian and anterior thalamic without midbrain (5%). AOP occlusion results in bilateral paramedian thalamic infarction with or without the involvement of the rostral midbrain and/or anterior thalamus likely due to great individual variability in the paramedian arteries. AOP infarction shows a clinical triad including altered mental status, such as coma, drowsiness and hypersomnia; vertical gaze palsy; and memory impairment.–8 Sleep/wake disturbances are inevitably observed in patients with AOP infarction from stroke onset or become evident following recovery from coma. The long-term follow-up outcome is generally good, except in cases with midbrain involvement.The thalamic vascular supply is classically categorized into 4 territories: anterior, paramedian, inferolateral, and posterior territories with variation and overlap. Artery of Percheron (AOP) is an uncommon variant of the paramedian arteries characterized by a single dominant perforating artery supplying the bilateral paramedian thalami. However, whether hypocretin is involved in hypersomnia in AOP infarction has not been well studied. To clarify the role of hypocretin in hypersomnia in patients with AOP infarction, we assessed CSF hypocretin-1 levels in 6 patients with AOP infarction.Narcolepsy–cataplexy associated with decreased CSF hypocretin-1 (167 pg/mL) was reported previously in a 23-year-old man who presented a large hypothalamic stroke following the removal of a craniopharyngioma at the posterior hypothalamus.2,12 The clinical and radiological characteristics of the patients and their relation to the measured CSF hypocretin-1 levels were reviewed retrospectively. The CSF samples were stored at −80°C until analysis. The measurement of hypocretin-1 in the CSF was performed using radioimmunoassay kits  as described previously. The intraassay variability was 4.3%, and the detection limit was 40 pg/mL. CSF hypocretin-1 levels of ≤110 pg/mL, 110 to 200 pg/mL, and ≥200 pg/mL were defined as low, intermediate, and normal, respectively. Either the patients or family members provided written informed consent for lumbar puncture and CSF hypocretin-1 measurements. The ethics committee of Akita University School of Medicine permitted our study. We classified the patients with AOP infarction into 2 groups based on the following magnetic resonance imaging (MRI) findings: bilateral paramedian thalamic infarctions (BPTIs) with rostral midbrain involvement (BPT+RMI) and BPTI without midbrain involvement (BPT-MI). Additionally, the involvement of the anterior thalamic nucleus was assessed.Between 2004 and 2015, we identified 6 patients with AOP infarction in whom the CSF hypocretin-1 levels were measured at Akita University School of Medicine.3Table Decreased CSF hypocretin-1 levels were observed in 2 of 2 (100%) patients with BPT+RMI, while CSF hypocretin-1 levels were within normal ranges in 4 patients with BPT-MI. All patients with AOP infarction exhibited hypersomnia: patients 2 to 6 showed hypersomnia from stroke onset, whereas patient 1 showed hypersomnia following coma recovery. Vertical gaze palsy was observed in 2 patients (100%) in the BPT+RMI group and in 2 patients (50%) in the BPT-MI group. In 1 BPT+RMI patient (patient 2), hypersomnia was improved within 2 weeks and decreased CSF hypocretin-1 levels were reversed , whereas another BPT+RMI patient (patient 1) who displayed coma in the acute phase had decreased CSF orexin levels (107 pg/mL) at day 49 and exhibited severe functional disability.4,6 Our primary findings are that the CSF hypocretin-1 levels were not reduced in the patients with BPT-MI but were reduced in the patients with BPT+RMI, who likely had ischemic lesions extending to the lateral hypothalamus. These findings indicate that hypocretin-1 deficiency is not generally involved in increases in sleep needs and hypersomnia observed in patients with AOP infarction when the lesions are restricted to the paramedian thalami. Compared with patients with narcolepsy, who often show undetectable levels of CSF hypocretin-1, decreases in the levels of this neuropeptide were relatively mild in the 2 patients with BPT+RMI.We performed a retrospective analysis of the CSF hypocretin-1 levels in 6 patients with AOP infarctions over a 10-year period. Although likely underestimated, the prevalence of AOP is only in 0.1% to 2% of all strokes and 4% to 18% of thalamic strokes.,8 In the study by Hermann et al, despite improvement in hypersomnia during a follow-up, repeated polysomnography showed no change in the sleep structure except for an increase in the sleep spindle, suggesting that different mechanisms are involved in maintaining wakefulness and sleep structure and that an impaired wakefulness maintenance system is responsible for subjective sleepiness rather than sleep structure changes. In this regard, from our intriguing findings that CSF hypocretin-1 deficiency was not observed in patients with AOP infarction without midbrain involvement, impairment in the wakefulness regulatory systems other than hypocretin-1 could contribute to “hypersomnia” in the acute phase of AOP infarction. Bassetti et al reported normal CSF hypocretin-1 levels (265 pg/mL) in thalamic stroke with subjective sleepiness .Hypersomnia in paramedian thalamic stroke is called “pseudo-hypersomnia” and is marked by increased stage N1 and decreased stage N2 and spindles during sleep; however, REM sleep is not affected.,15 CSF hypocretin-1 levels appear normal in patients with lesions restricted to paramedian thalami due to AOP infarctions because the lesions are not involved in the hypocretin cell bodies. In our study, patient 2 (BPT+RMI) showed reversible subjective sleepiness in association with a reversal of decreased CSF hypocretin-1 levels , indicating impairment of hypocretin projections, but not of hypocretin cell bodies, during the acute phase. In contrast, patient 1 showed decreased CSF hypocretin-1 levels during the chronic phase (107 pg/mL), displaying residual sleepiness and memory disturbance with a resultant poor functional outcome. Among 12 patients with PTIs , clinical outcomes were favorable in the right-sided lesions but were poor in the bilateral and left-sided lesions, which were associated with residual cognitive deficits found in the bilateral and left-sided lesions. Consistent with those study results, patient 1 with anterior thalamus lesions had residual cognitive impairment and poor functional recovery in our study. Additionally, because hypocretin has a central role in maintaining not only the sleep/wake cycle but also reward processes and motivated behaviors, the decreased CSF hypocretin-1 levels in the chronic phase, which resulted from impaired hypocretin cell bodies, could possibly predict a poor clinical outcome in patients with BPT+RMI.Hypocretin-1-producing neurons are located in the lateral hypothalamus and adjacent perifornical regions, and these neurons heavily project to the rostral brainstem.The study limitations include a retrospective setting and a lack of statistical comparison due to the small number of patients examined. We could not correlate the sleep study results with the CSF hypocretin-1 levels in this study. Further prospective studies with larger number of patients will be necessary to investigate the relationship between subjective sleepiness and CSF hypocretin-1 levels, and these should involve sleep studies and hypocretin-1 measurements at multiple points to determine how baseline hypocretin-1 levels predict functional prognosis.In conclusion, our pilot study showed that hypocretin deficiency was not involved in hypersomnia observed in patients with BPT-MI; however, the CSF hypocretin-1 levels were reduced in patients with BPT+RMI.


Pogostemon cablin (Blanco) Benth. The present study aims to investigate the potential benefit of PO against IBD in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis model. PO treatment by enema significantly brought down the disease activity index (DAI) of the TNBS-challenged rats, which was manifested by the ameliorated inflammatory features including ulceration, adhesion, and edema. Hematoxylin-eosin (HE) staining and immunohistochemistry analysis showed that PO effectively relived colon damage by restoring epithelium, and more importantly, by inhibiting the infiltration of pro-inflammatory Th1 and Th17 cells in the colon. Additionally, PO inhibited the activity of myeloperoxidase and secretion of inflammatory cytokines including IFN-γ, IL-12p70, IL-17A, and IL-10. Together with our previous findings, the present data indicated that the anti-IBD effect of PO probably related to its direct inhibition on Th cell proliferation and suppression of the cytokines secretion. These results highlighted the potential of PO as a promising candidate to relieve IBD.Inflammatory bowel disease (IBD) is a chronic immune-related disease mainly caused by the disequilibrium of T helper (Th) cell paradigm? Pogostone (PO) is one of the major chemical constituents of  Most of the current remedies have been designed to directly control or remiss the inflammatory response of IBD, which is mainly a combination of anti-inflammatory agents , glucocorticosteroids, immunomodulating drugs , and more recently the biological neutralizer . Nevertheless, physicians and researchers keep seeking resolutions for IBD due to the unsatisfied efficacy and adverse drug reactions, such as parkinsonism, gastrointestinal, and liver adverse effects (Inflammatory bowel disease (IBD) is a chronic immune-related disease that mainly involves the colon. There are two main entities of IBD, the ulcerative colitis (UC) and the Crohn’s disease (CD), depending on their specific pathological characteristics in terms of intestinal distribution, histological appearance, and some clinical complications . Inciden effects .de novo differentiation of naïve T cells  Benth. is a valuable medicinal and aromatic plant with thousands-of-years history for the treatment of sunstroke-related headache, fever, emesis, and even coma. Numerous experimental studies has proved that the efficacies of P. cablin include anti-inflammation and analgesia , is one of the major chemical constituents of P. cablin. It has been revealed that PO is responsible for most activities of P. cablin, including those of antibacterial  induction is a widely used chemical approach for its reproducibility and ease of development. Thus, in the present study, a TNBS-induced experimental colitis model was employed to investigate the potential benefit of PO against IBD.Nowadays, more and more endeavor has been focused on the effective natural product from the wisdoms of traditional medicines in IBD studies. In traditional Chinese medicine, nalgesia , immunornalgesia , anti-emnalgesia , and so acterial  and antiacterial . Given tacterial , it is rad libitum. The animals were acclimatized for 7 d prior the experiment. The handling of animals and all experimental procedures performed were approved by the Committee for Animal Care and Use at GZUCM and in accordance with the Guide for the Care and Use of Laboratory Animals.Thirty-six Sprague-Dawley rats  were provided by Laboratory Animal Center of Guangzhou University of Chinese Medicine  All animals were housed at 20 ± 2°C with a humidity of 50% ± 5% in a 12 h light/dark cycle with food and water P. cablin as described previously (P. cablin was authenticated by one of the authors (Pro. Xiaoping Lai) at the School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, where a voucher specimen (No. 101009) was deposited. Chemical structure and purity of the obtained PO were identified and determined as reported previously  was isolated from the aerial parts of eviously . P. cableviously .TM Simplex Bead Set Reagent Kits was provided by eBioscience .2,4,6-Trinitrobenzenesulfonic acid, cetyltriethyl ammonium bromide, 3,3′-dianisidine, were purchased from Sigma (United States). Chloral hydrate was purchased from Daomao Chemical reagent factory . Salicylazosulfapyridine (SASP) was purchased from Shanghai pharmaceutical Co. Ltd. . Rat ProcartaPlexColitis was induced in accordance with the method as described previously . Brieflyn = 6), SASP group  and three PO groups (n = 6 in each group). From the day of colitis induction, rats of PO groups were administered with different doses of PO by enema, once a day  for 7 days. PO was suspended in 5% sodium carboxymethylcellulose (CMC-Na). SASP was used as a positive control at a dose of 200 mg/kg daily by enema as well. Equal volume of solvent was given to both the Normal and Model groups. Throughout the experiments, rats were monitored for body weight loss. Twenty-four hours after the last PO administration, animals were sacrificed, and the colons were removed, dissected, and opened lengthwise for further analysis.The TNBS-challenged rats were divided in to Model group (Table 1), the DAI was evaluated as sum of the grade scores of weight loss, stool consistency, and occult/gross bleeding  to positive area (IOD/pixel) were used for analyzing the results of immunohistochemistry by Image Pro Plus 6.0 software .Tissue sections for immunohistochemistry were prepared from formalin-fixed, paraffin-embedded colon tissue. Stains against CD4 , IFN-γ , IL-17A  and Foxp3  were performed according to the kit protocol . Briefly, the slides were deparaffinized. Antigen retrieval for IFN-γ and IL-17A were carried out by incubation in 10 mM sodium citrate buffer with 0.1% Tween 20 in 100°C water bath for 20 min, while those for CD4 and Foxp3 were performed by incubation in EDTA buffer (pH 8.0) via microwave heating. Endogenous peroxidase in the tissue was blocked by incubation with 3% Hg for 20 min at 4°C. The MPO enzymatic reaction was assessed by mixing 20 μL supernatant with 180 μL reaction buffer , and immediately scanned at 460 nm for 2 min at time point of 0 s, 40 s, 80 s, 120 s. The MPO enzyme activity (U/g) = (ΔA460/11.3 × (g/0.2), in which ΔA460 = [(OD120s - OD40s) - (OD80s - OD0s), g means tissue weight.Neutrophil infiltration into the colon was assessed indirectly by measuring MPO activity. The MPO activity assay was performed as described previously . The cleg for 20 min at 4°C. Protein content in the supernatant was quantified with BCA protein kit . TGF-β was measured with TGF-β ELISA kit , and IL-17A, IL-10, IL-4, IL-12p70, IFN-γ were determined by ProcartaPlexTM Simplex Bead Set Reagent Kits  according to the manufacturer’s instruction.About 30 mg colon tissue was minced, homogenized in chilled PBS with proteinase inhibitor cocktail  for 30 s, and centrifuged at 18,000 × post hoc LSD test was applied for difference analysis under homogeneity of variance, if not, a Dunnett’s test would be applied. #p < 0.05 and ##p < 0.01 as compared to Normal group; ∗p < 0.05 and ∗∗p < 0.01 as compared to Model group.Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS 18.0. A Figure 1A, all the TNBS-challenged rats did not exhibited evident body weight loss compared with their normal counterparts. However, after the TNBS challenge, model group developed significant colitis signs , which were characterized by lack of vitality, shaggy hair, loose and bloody stools, and a highest DAI scoring at 3.2 ± 1.1 (p < 0.01). By contrast, PO treatment reanimated the suffering rats, and turned their stools to be mild soft but less bloody. DAI were also brought down by PO treatment, scoring at 2.2 ± 1.2 (20 mg/kg), 1.3 ± 0.62 , 2.3 ± 0.4 (80 mg/kg), respectively, suggesting that PO, especially the 40 mg/kg dosage, relieved TNBS-induced colitis.As showed in Figures 2A,C), TNBS challenge did not affect the colon length of the suffering rats. However, colons from the Model groups exhibited apparent inflammatory features, such as ulceration, severe adhesion, and thickening, which scored up to 9.2 ± 0.86 . As a clinical immunosuppressant, SASP (200 mg/kg) significantly relieved ulceration and colon wall edema, but did not eliminate adhesion, resulting in a score of 8.2 ± 1.6 . PO (40 mg/kg), corresponding to the results of DAI evaluation, remarkably ameliorated the aforementioned inflammation characteristics, including adhesion, ulceration, and edema, which scored at 5.9 ± 1.8 . Since both results from DAI assessment and macroscopical assessment indicated that 40 mg/kg PO exhibited the biggest benefit against TNBS-induced colitis, samples from the 40 mg/kg PO group were used for the further studies.Macroscopical assessment including colon length (from anus to cecum) and injuries scoring was employed to assess the protection of PO against colonic damage. As indicated by the results . By contrast, the score of SASP group was 8.8 ± 1.9, of which the colon exhibited less epithelium shedding, ulceration and leukocyte infiltration. Regarding to PO treatment (40 mg/kg), it was found that, in addition to the remission in epithelium, colons from the PO group showed much better crypt form and fewer vessel proliferations , suggesting that the colitis-induced colon lesion was significantly ameliorated by PO.Hematoxylin-eosin staining was applied to provide more details about the effect of PO. As revealed in +) existed in the TNBS-challenged rats , as indicated by the significant higher ratio of IOD to positive area . Both SASP and PO inhibited Th cell infiltration, and PO especially made a remarkable reduction (p < 0.01).Given that Th cell plays a cardinal but dual role in the IBD development, we employed immunohistochemistry to investigate the Th cell infiltration in the inflammatory colon tissue. It was found that large number of Th cell (CD4+) and Th17 (IL-17A+) are the pro-inflammatory ones that drive the development of IBD, while regulatory T cell  is the one that suppresses the inflammation, thus it is necessary to make a more detailed observation for their infiltration in the colon. In accordance with the increase of total Th cell, Th1, Th17, and Treg cells obviously increased in the colon tissue of Model group with higher ratios of IOD to positive area , and those of Th1 (IFN-γ+) and Th17 (IL-17A+) increased much more than that of Treg . Interestingly, both SASP and PO evidently suppressed the excess infiltrations of pro-inflammatory Th1 and Th17 (p < 0.01), but did not apparently affected the distribution of Treg cell.Among the Th cell subsets, Th1  were extremely higher than that of normal group . By contrast, those of SASP and PO (40 mg/kg) groups were remarkably decreased to 0.15 ± 0.077 U/mg tissue (p < 0.05) and 0.12 ± 0.040 U/mg tissue (p < 0.01).Myeloperoxidase (MPO) is a heme protease that is secreted by neutrophile granulocyte, monocyte and macrophage, and serves as a marker of neutrophile granulocyte activity. As showed in Figures G, TNBS challenge strikingly elevated the levels of IL-4, IL-12p70, IL-10, IL-17A, and TGF-β (p < 0.01). In comparison, both SASP and PO remarkably suppressed most of them. In brief, SASP made significant suppressions on IFN-γ, IL-12p70, and IL-17A, while PO decreased the pro-inflammatory cytokines including IFN-γ, IL-12p70, IL-17A, as well as the anti-inflammatory IL-10, which was interestingly quite accordance with the depression on Th cell infiltration. Nevertheless, TGF-β was not affected by both reagents. Together with the results from the macroscopical and microscopical assessments, it can be speculated that the anti-inflammatory effect of PO on IBD possibly attributes to suppression on the Th-cell immune response.Changes of the inflammatory cytokines profile in the colon revealed more details about the activity of PO. As showed in Inflammatory bowel disease is characterized by an increased infiltration of leukocytes into the inflamed intestinal mucosa, thus resulting in a persistent inflammation. Pathologically, the mucosa injuries caused by leukocytes infiltration include ulceration, necrosis, edema, and even blooding, which not only impair the nutrients intake and metabolism in the guts, but also induce abnormalities in peristalsis and submucosal enteric nervous system. Therefore, typical signs of IBD always consist of weight loss, abdomen pain with diarrhea, fecal occult blood, and bloody stools, which have been employed as criteria for assessment in clinical and in animal researches . TNBS isAs afore-mentioned, leukocyte trafficking plays a central role in the immunopathogenesis of IBD, and inhibition of leukocyte trafficking to the gut mucosa has been recognized as an important target for the development of IBD drugs . MPO is + as symbol) is an effector Th cell that is characterized by secretion of IFN-γ, IL-2 and IL-12 to mediate cytotoxic immune response involved in cellular immunity and hypersensitivity; while Th2 (IL-4+ as symbol) features in secreting IL-4, IL-5, and IL-6 to promote B cell and antibody production for humoral immunity. Accumulating clinical studies has pointed that the imbalance of Th1/Th2 paradigm are important in chronic inflammatory process of IBD (+ as symbol) has been heavily implicated in tissue-specific immune pathology not only in the murine models of IBD, but also in human CD and UC (+) and Th17 (IL-17A+) but not that of Treg. These results indicate that inhibition on Th cell contributes to the protective effect of PO against TNBS-induced colon injury.Among the offensive leukocyte giving rise to IBD, Th cells are the most concerned factors that contribute to its complexity . Accordis of IBD , and it s of IBD . Th17 , Th2 (Gata3 and Stat6), Th17 (Ror-gama t and Stat3), and Treg (Foxp3) , suggesting that the restrains of PO on Th1 or Th17 cell infiltration had no concern with the regulation on Th cell differentiation into specific functional subset. In our previous study, PO was demonstrated to be able to directly block T cell proliferation via down-regulation of cyclin E, cyclin B, and CDK1 and the subsequent S-phase arrest without toxicity on the cell vitality, exhibiting a blunt on the T cell-associated immune response (in vitro and in vivo (Collectively, our data revealed that PO as an protect animal against experimental IBD via inhibiting the infiltration of pro-inflammatory Th1 and Th17 cells, suppressing MPO activity and decreasing inflammatory cytokines, i.e., IFN-γ, IL-12p70, IL-17A, and IL-10. Nevertheless, PO did not affected mRNA expression of the specific transcript factor for Th1 (response . Given tresponse , our fin in vivo , highligConceived and designed the experiments: XZ and XL. Performed the experiments: JS, CL, XY, GY, and JD. Analyzed the data: JS, CL, ZS, HZ, and JC. Drafted and revised the manuscript: JS, CL, XZ, and XL.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.


Many studies have focused on the challenges of small molecule uptake across the blood–brain barrier, whereas few in-depth studies have assessed the challenges with the uptake of antibodies into the central nervous system (CNS). In drug development, cerebrospinal fluid (CSF) sampling is routinely used as a surrogate for assessing CNS drug exposure and biomarker levels. In this report, we have studied the kinetic correlation between CSF and serum drug concentration–time profiles for five humanized monoclonal antibodies in rats and cynomolgus monkeys and analyzed factors that affect their CSF exposure.CSFTmax) in one to several days in both rats and monkeys. Antibody serum and CSF concentration–time curves converged until they became parallel after CSFTmax was reached. Antibody half-lives in CSF (CSFt½) approximated their serum half-lives (serumt½). Although the intended targets of these antibodies were different, the steady-state CSF to serum concentration ratios were similar at 0.1–0.2% in both species. Independent of antibody target and serum concentration, CSF-to-serum concentration ratios for individual monkeys ranged by up to tenfold from 0.03 to 0.3%.Upon intravenous (IV) bolus injection, antibodies entered the CNS slowly and reached maximum CSF concentration (CSFt½ of the antibodies was largely determined by their long systemic t½ (systemict½).Upon systemic administration, average antibodies CSF-to-serum concentration ratios in rats and monkeys were 0.1–0.2%. The  CSFTmax), antibody CSF half-life (CSFt½) and the length of time required for achieving steady-state antibody levels between CSF and systemic circulation. In this study, we report the kinetic relationships of CSF versus serum concentration–time profiles of five humanized monoclonal antibodies in rats and cynomolgus monkeys following systemic administration and factors that affect their CSF antibody exposures. These findings have important implications for assessing CNS antibody drug kinetics in humans.The blood brain barrier (BBB) is the single most significant obstacle that impedes the delivery of therapeutics to targets in the central nervous system (CNS). For medications that interact with targets in the CNS, it is essential for therapeutic and/or safety reasons to obtain accurate kinetic relationships between CNS and systemic circulation levels. It is also important to understand the factors that affect this relationship in order to project CNS drug levels based on their systemic concentration–time profiles. In practice, cerebrospinal fluid (CSF) is the most accessible and widely used sampling matrix for measuring drug and biomarker levels in the CNS. CSF sampling is inconvenient, and therefore requires rigorous study design to minimize the number of time points that need to be collected. Several excellent reviews highlight the scientific rationale and utility of using CSF as a surrogate for assessing CNS drug exposure levels –5. AlthoIn this study, we tested five human monoclonal antibodies (mAb). All five antibodies are highly selective and have subnanomolar affinities for their respective targets. Antibody A (BIIB054) preferentially binds to aggregated forms of α-synuclein and binds human, non-human primate (NHP), and rat α-synuclein with similar affinity. Antibody B (399 H0/L0) recognizes JC virus, which is found in humans , but is 2 asphyxiation and immediately followed by CSF collection via cisterna magna puncture and blood collection via cardiac puncture. The rats used to evaluate antibody E were adult male Brown Norway rats (average weight 150–200 g). The procedures were essentially the same. For brain and spinal cord collection, perfusion was performed promptly following CSF/blood collection with 15 mL of saline via a peristaltic pump inserted into the left ventricle. Whole blood samples were left on the bench for about 15–30 min and spun at 3000 rpm (approximately 1000×g) for 10 min to isolate serum. All samples were stored at − 80 °C until analysis. All experimental procedures were approved and monitored by Biogen’s Institutional Animal Care and Use Committee (IACUC).Rat PK studies. Adult male Sprague–Dawley rats (250–300 g)  were housed at constant temperature (22 ± 2 °C) and relative humidity (50–70%) under a regular light/dark schedule . Food and water were freely available. The dose administration route was either by tail vein  or intraperitoneal injection (mAb E). At various time points after dose administration, the animals were euthanized by COStudies in NHP were carried out at SNBL  with approved IACUC accreditation. Briefly, young adult cynomolgus monkeys (2.5–4 kg), were individually housed in cages that complied with the Animal Welfare Act, corresponding guidelines and standard operating procedures (SOPs) of SNBL. Approximately 1 week prior to dose administration, a single lumbar intrathecal catheter  was surgically implanted to allow repeated CSF sampling. Prior to dose administration, CSF was collected from the catheter for baseline measurements. The intravenous (IV) dose was administered via primed butterfly infusion lines over 1–2 min. At pre-selected sampling time points, blood (1 mL) and CSF (0.5 mL) were collected from restrained, conscious animals. Serum and CSF samples were prepared as described above for rat studies. CSF samples were visually examined for blood contamination and contaminated samples were excluded.http://www.southernbiotech.com). Briefly, for the analysis of antibody A–D samples, 96-well ELISA plates were coated with the capture antibody, monkey-adsorbed goat anti-human IgG, at 1 µg/mL. The plates were blocked and washed, and standards (STDs), quality controls (QCs), or study samples containing test articles were added to the plate. The plates were again washed, and a 1:7500 dilution (50 ng/mL) of enzyme conjugated detection antibody (goat anti-human IgG-HRP), was added to the wells. After washing, the HRP substrate tetramethylbenzidine (TMB) was added. The HRP reaction was stopped following the addition of 1 N H2SO4 and the absorbance at 450 nm was measured on a microplate reader. Standard calibration curves were generated and the concentrations of the controls and samples were interpolated from their respective standard curves. On average, the quantitation ranges of the ELISAs were between 16 and 4400 ng/mL. Lower limits of quantitation were generally 16 ng/mL for CSF and 50 ng/mL for serum samples. Antibody E samples were quantified as previously described [http://www.certara.com).The concentrations of antibodies A–E in serum and CNS specimens were measured using enzyme-linked immunosorbent assays (ELISAs). A generic sandwich ELISA format similar to the one described  was optiescribed . PharmacCSFTmax ranging between 6 and 48 h. Serum and CSF concentration–time curves became parallel after CSFTmax. CSF antibody concentrations were approximately 1000-fold lower than serum concentrations. Since antibody E was dosed by intraperitoneal (IP) injection in Brown-Norway rats, the serum and CNS tissue concentration–time profiles for CSF, brain, and spinal cord are plotted separately in Fig. serumTmax was observed 6–24 h after dosing. The CSFTmax, brainTmax and Spinal_ cordTmax were achieved 1–2 days following serumTmax. Like the data shown in Fig. CSFTmax. Concentration–time curves in brain (for antibodies A and E) and spinal cord (for antibody E) paralleled those in CSF, although the antibody concentrations in these CNS tissues were slightly higher than in CSF , B (10 mg/kg), C 20 mg/kg) and D (20 mg/kg) in rats following IV administration are shown in Fig. 0 mg/kg, 0 mg/kg aCSFTmax, and CSF antibody concentrations on average were approximately 1000-fold lower than serum concentrations. Figure CSFt½ and serumt½ were similar, but the CSF-to-serum concentration ratios varied from 0.03 to 0.32%. The variation in CSF-to-serum ratios was random and occurred in all studies irrespective of intended target and dose strength.Antibodies A, C and D were also evaluated in cynomolgus monkeys. The serum and CSF concentration–time profiles are plotted in Figs. erumsAUC), and long half-lives (t½) in both serum and CNS tissues. Following IV administration, antibodies appeared in the CSF gradually, reaching maximum CSF concentration (CSFCmax) between 16 and 48 h in rats and 2–3 days in cynomolgus monkeys (CSFTmax). For all five antibodies, serum and CSF concentration–time curves became parallel 2–3 days after dose administration, with CSF concentrations roughly one thousand times lower than the corresponding serum concentrations and independent of dose. For example, antibody A had a CSFAUC-to-serumAUC ratio of 0.15 ± 0.07% at 20 mg/kg (N = 4) and 0.16 ± 0.08% at 100 mg/kg (N = 11). Antibody C had an average CSFAUC-to-serumAUC ratio of 0.09 ± 0.06% at 30 mg/kg (N = 12) and 0.10 ± 0.05% at 100 mg/kg (N = 6). Taking into consideration low and high dose groups, antibody A had relatively long serumt½ of 18 ± 3 days and CSFt½ of 21 ± 6 days in monkeys (N = 15). On the other hand, antibody C had a relatively short serumt½ of 6 ± 2 days and CSFt½ of 8 ± 3 days in monkeys (N = 18).Results of non-compartmental analysis (NCA) of the pharmacokinetic parameters for the five antibodies characterized in Figs. Rin is the rate of passage into the CNS in amount per time; CLout is the total clearance out of the CNS in volume per time; CSFC and serumC denote the steady-state drug concentrations in CSF and systemic circulation, respectively; PSA to B and PSB to A represent directional first order kinetics across BBB from apical to basolateral and from basolateral to aoical, respectively; and Clefflux, Clmetabolism and Qconvection represent the individual clearance mechanisms indicated.Upon entering the CNS, there are four possible clearance mechanisms: 1) movement back across the BBB and/or BCSFB via a first order non-specific process; (2) active/facilitated transport out of the CNS by efflux transporters located at the membrane sites of the BBB and/or BCSFB, an energy-requiring process against the antibody concentration gradient; (3) biotransformation/metabolism, and (4) convection carried by brain ISF/CSF flow and drainage. For mass balance at steady-state, solute concentration in the CNS is determined by the rate entering the CNS divided by the sum of the four possible clearance mechanisms out of the same compartment. To simplify the analysis, we treated the brain tissue and CSF as one fast equilibrium homogeneous compartment as shown in Eq.  movementWithout consumption of energy, passive diffusion or facilitated transcytosis requires a favorable concentration gradient and occurs only from high to low concentrations. Therefore, passive diffusion out of the CNS is not a feasible clearance mechanism for antibodies. Thus far, there has been no reported evidence for a high efficiency efflux pump or massive brain specific catabolism for antibodies or human IgGs. Unlike the other clearance mechanisms described above, convection by ISF/CSF flow does not require a favorable concentration gradient, specific transporter, or energy and is not structurally restricted by stoichiometry. If one assumes that convection is the most plausible and efficient mechanism for CNS removal of antibodies, Eq. CNSV is the apparent volume of distribution for CNS, CSFC and serumC denote the antibody concentrations in CSF and serum, respectively, and PSproduct and Qconvection are independent parameters that govern entering and exiting the CNS. Entering CNS (PSproduct) requires overcoming the BBB/BCSFB, whereas exiting CNS (Qconvection) to a large extent does not. Both processes are independent of systemic clearance mechanisms.In this model, the major factors that determine CNS exposures of therapeutic antibodies are two independent processes: an antibody’s ability to cross the BBB, and clearance dominated by the mechanism of convection, which is closely associated with the brain’s ISF/CSF production rate. Assuming rapid equilibrium across the various CNS compartments and CSF antibody concentration as a surrogate for CNS drug levels, the pharmacokinetic modeling of CNS antibody concentration can be simplified to Eq. CSFAUC-to-serumAUC ratios ranged from 0.03 to 0.23%.The apparent linearity of the predicted vs. experimentally measured serum and CSF data for antibody A in Fig. The CSF-to-serum ratio of 0.1–0.2% that we measured for five independent antibodies in rats and NHPs agrees well with published results for other antibodies. CSF-to-total serum IgG ratios of 0.1–0.2% in healthy humans have been reported by several groups –13. Simi125I-labeled antibody was significantly reduced in FcRn−/− mice, the brain-to-plasma exposures remained unchanged at 0.22% in wildtype and FcRn−/− mice. Similar to what we observed in rats and NHP, the brain concentration–time profiles paralleled the plasma profiles in wildtype and FcRn−/− mice  [There are many studies exploring the mechanisms by which antibodies cross the BBB . Garg anlasmat½) . Other plasmat½) –23. Unlilasmat½) . In inveAlthough CSF is largely produced at the choroid plexus (CP) and secreted into the four ventricles of the brain, recent studies indicate there are other sources of CSF –27. As iPSproduct and Qconvection are two independent processes that cannot be simplified by inter-compartmental clearance and are independent of systemic clearance mechanisms. Large inter-subject variations in CNS antibody exposures need to be taken into consideration even if there is little variation in a drug’s serum concentration–time profile. Variation can be introduced at the CNS level by an individual’s PSproduct/Qconvection ratio. From a pharmacokinetic modeling point of view, these two parameters are highly correlated and difficult to differentiate other than through a change in their ratio. That said, the PSproduct value of an antibody can be assessed using in vitro methods [CSFTmax for BAN2401 is > 24 h and that CSFC levels have not yet peaked 24 h after dose administration. CSF-to-serum ratios of greater than 0.1–0.2% have also been reported for other antibody therapies in individuals with AD [For the factors that determine the CNS exposure of an antibody,  methods  and an i methods . On aver methods . The CSF methods –36, norm methods , concurr methods , 41 and  methods , 43, and methods , 44 and  methods , 46. Alt methods , 46. BAN methods . These d with AD , 48, 49. with AD , 13, con with AD , 46.CSFTmax and steady-state CSF-to-serum concentration ratios are observed. These should be taken into consideration when designing CSF sampling schemes. In patients with reduced CSF production, either due to disease or concurrent medications, higher CSF drug exposures should be expected. Unfortunately, the factors that affect CSF production rates and, more importantly, the subsequent impact on their ability to clear CNS waste have not yet been fully investigated and/or appreciated.Linking CSF production rates with CNS clearance of antibodies potentially has broad applications, since it enables the estimation of antibody kinetics between the CSF and systemic circulation and would allow for rational design of CSF sampling schedules and projection of CNS drug exposures in selected patient groups. For example, for antibodies, delayed CSFTmax around 24–72 h after IV administration, and that CSF and serum concentration–time curves become parallel after CSFTmax, with average CSF-to-serum ratios of 0.1–0.2%. Prior to achieving CSFTmax, CSF-to-serum ratios are not accurate indicators of CNS uptake or steady-state CSF-to-systemic concentration ratios. For antibodies with long systemic t½s, serumt½ determines CSFt½. These studies should aid in the design of future studies to better understand factors that contribute to the elimination of antibodies from the CNS.In summary, from an extensive analysis of the pharmacokinetics of five human antibodies in rats and monkeys, we show that antibodies enter the CNS slowly, with


Encephalitozoon cuniculi was transmitted from an infected donor to 3 solid organ recipients, 1 of whom died. Encephalitozoon cuniculi was detected by tissue PCR in the central nervous system of the deceased kidney recipient and in renal allograft tissue from both kidney recipients. Urine PCR was positive for E. cuniculi in the 2 surviving recipients. Donor serum was positive for E. cuniculi antibodies. E. cuniculi was transmitted to 3 recipients from 1 donor. This rare presentation of disseminated disease resulted in diagnostic delays. Clinicians should consider donor-derived microsporidial infection in organ recipients with unexplained encephalitis, even when gastrointestinal manifestations are absent.In April 2014, a kidney transplant recipient in the United States experienced headache, diplopia, and confusion, followed by neurologic decline and death. An investigation to evaluate the possibility of donor-derived infection determined that 3 patients had received 4 organs  from the same donor. The liver recipient experienced tremor and gait instability; the heart/kidney and contralateral kidney recipients were hospitalized with encephalitis. None experienced gastrointestinal symptoms.  Each year in the United States, ≈30,000 solid organ transplants are performed (https://optn.transplant.hrsa.gov/) per policy and reviewed by the Network’s ad hoc Disease Transmission Advisory Committee, which categorizes each by the likelihood of disease transmission. Through representation on this advisory committee, the Centers for Disease Control and Prevention (CDC), with support from state and local health departments, leads investigations of select cases of public health importance. In April 2014, CDC was notified of a renal transplant recipient hospitalized with signs and symptoms of encephalitis , PCR, and transmission electron microscopy. Serum immunofluorescence antibody testing (IFA) was performed on available serum samples.Encephalitozoon cuniculi reference strain cultures were suspended in phosphate-buffered saline (PBS) at a concentration of 108 spores/mL. We added 10 μL of this suspension to each well (106 spores/well), allowed the slides to air dry, and stored them at −80°C before use.We inoculated recipient urine specimens onto monkey kidney cells (Vero E6) and human embryonic lung fibroblasts by using established methods  in PBS with 3 μL/mL Evans Blue  counterstain. After another 30-min incubation at 37°C, the slides were again washed 3 times in PBS, dried, and mounted with glycerol mounting media. Serum titers were determined by observing the slides under a fluorescence microscope. Antibody titers were considered positive at cutoffs of >1:16 for immunocompromised and >1:64 for immunocompetent persons  and negative (titer 1:32) control serum obtained from the CDC free-living amebic infections laboratory. The serum samples were then diluted to 1:4,096 by 2-fold dilution. We added 10 μL from each dilution to the wells of the previously made E. cuniculi serum at a 1:1,000 dilution as previously described . The microsporidia IHC assay used rabbit anti–For transmission electron microscopic examination, we evaluated a paraffin-embedded section by on-slide embedding as previously described . DNA was extracted from the formalin-fixed paraffin-embedded tissues by using a QIAamp DNA Mini Kit (QIAGEN) as previously described , the patient remained well without symptoms of microsporidial infection. The liver recipient was a man with hepatitis C–associated cirrhosis and hepatocellular carcinoma. At the time of transplantation, he received intravenous methylprednisolone to induce immunosuppression; at the time of discharge, he received tacrolimus, prednisone, and mycophenolate mofetil to maintain immunosuppression. Thirteen weeks after transplant, the patient visited an outpatient clinic and reported bilateral upper extremity tremor, which was thought to result from elevated tacrolimus levels. A week later, the patient was readmitted to the hospital after an outpatient visit for right lower extremity swelling and shortness of breath; the ultimate diagnosis was deep vein thrombosis and pulmonary embolism. During this hospitalization, the recipient reported lightheadedness, headache, blurry vision, and continued tremor. Because of his neurologic symptoms and identification of microsporidiosis in the deceased right kidney recipient, this patient was also empirically given albendazole. The recipient declined lumbar puncture. Subsequently, microsporidia were identified in a urine specimen by trichrome staining performed at the patient’s local hospital , panel A culture , panel B culture , panel CThe donor was a middle-aged woman, originally from Mexico but a resident of the United States for several decades. In December 2013, she experienced recurrent and persistent headache, which she attributed to a known history of migraine. The headaches persisted and progressed over several weeks to include nausea, pulsatile tinnitus, and diplopia. Approximately 4 weeks later, she sought care at an emergency department, where brain computed tomography and magnetic resonance imaging revealed several arteriovenous malformations and a right internal carotid saccular aneurysm. She underwent endovascular repair with stenting and coil embolization. This repair was complicated by several episodes of intracranial hemorrhage requiring craniotomy. Despite surgical intervention, her neurologic function continued to decline, and she was eventually declared brain dead. No other symptoms were reported before organ donation. Review of her medical record revealed no reports of gastrointestinal illness during her hospitalization. Interviews with next of kin revealed no clear risk factors for microsporidiosis , no gastrointestinal illness before her death, and no travel outside the United States within the previous 12 months.Because no autopsy was performed on the donor, the only donor specimen available for retrospective testing was archived serum. This sample was tested by microsporidia IFA; a positive titer of 1:2,048 suggested active infection  and use of a modified trichrome stain , panel AE. cuniculi might be the cause of isolated severe neurologic microsporidiosis, as occurred in these 3 patients. Furthermore, given that animal studies have shown that encephalitozoonosis can cause vasculitic manifestations, including aortitis  and the likelihood of underlying donor infection on the basis of clinical course or characteristics alone. However, when a similar infectious syndrome develops in Enterocytozoon bieneusi, another common species of microsporidia affecting humans, is not. Although immunocompetent persons may require no treatment or short-course therapy  was not performed as part of the public health investigation, and we are thus unable to further investigate strain relatedness. However, we were able to confirm infection with the same species of microsporidia in all 3 recipients and the donor, which is highly suggestive of donor-derived transmission.The findings of this investigation are subject to limitations. Because no donor tissue was available for testing, we were unable to differentiate whether the donor’s neurologic complaints and radiographic findings were caused by microsporidial disease or whether she had subclinical disseminated infection. Additionally, the IFA result interpretation has not been standardized for the serologic diagnosis of microsporidiosis. Last, sequencing of Identifying donor-derived disease transmission events and ensuring appropriate treatment and management of recipients requires extensive collaboration. This investigation, as did other donor-derived disease transmissions described by CDC, required close cooperation among clinicians, laboratory scientists, the organ procurement organization, and public health agencies. Clinicians should maintain a high index of suspicion for donor-derived infections and should report any suspected or potential events to the Organ Procurement and Transplantation Network.


GDAP1 impede the protein’s role in mitochondrial dynamics. In silico analysis reveals sequence similarities of GDAP1 to glutathione S-transferases (GSTs). However, a proof of GST activity and its possible impact on membrane dynamics are lacking to date. Using recombinant protein, we demonstrate for the first time theta-class-like GST activity for GDAP1, and it’s activity being regulated by the C-terminal hydrophobic domain 1 (HD1) of GDAP1 in an autoinhibitory manner. Moreover, we show that the HD1 amphipathic pattern is required to induce membrane dynamics by GDAP1. As both, fission and GST activities of GDAP1, are critically dependent on HD1, we propose that GDAP1 undergoes a molecular switch, turning from a pro-fission active to an auto-inhibited inactive conformation.Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) cause severe peripheral motor and sensory neuropathies called Charcot-Marie-Tooth disease. GDAP1 expression induces fission of mitochondria and peroxisomes by a currently elusive mechanism, while disease causing mutations in  GDAP1 are associated with the hereditary motor and sensory neuropathy Charcot-Marie-Tooth (CMT) disease67In vitro studies revealed that recessively inherited mutant forms of GDAP1 (rmGDAP1) exhibit reduced fission-promoting activity, whereas dominantly inherited mutant forms (dmGDAP1) interfere with mitochondrial fusion4Ganglioside-induced differentiation associated protein 1 (GDAP1) is an integral, tail-anchored protein of the mitochondrial outer membrane (MOM) and the peroxisomal membrane121234In silico analyses predict that GDAP1 is a glutathione S-transferase (GST)111214141511in vitro and in vivo where it is capable of substituting for the loss of GDAP1 in the central nervous system of GDAP1-deficient mice18192051112in vitro. We discovered that GST activity is regulated by the hydrophobic domain 1 (HD1), which exerts an autoinhibitory function. HD1 could adopt an amphipathic pattern and this amphipathic pattern is necessary to induce remodelling of organelles-mimicking liposomes by Gdap1. We propose a model of action with two different, interconvertible conformations of GDAP1: a GST active state with an exposed HD1 mediating mitochondrial and peroxisomal fission, and a GST-inactive state caused by an autoinhibitory binding mode of HD1.We show here that highly purified recombinant human GDAP1 is indeed a GST enzyme, and we demonstrate specific GSH-conjugating activity GST enzymes critically depend on dimerization in order to be catalytically activeWe next tested whether the C-terminal hydrophobic domains of GDAP1 are dispensable for dimerization. GDAP1 lacking the TMD (GDAP1 T318X) or lacking the TMD and HD1 (GDAP1 T288X) were co-expressed as human and murine truncated proteins in HEK-293T cells . Co-immu11Recombinant, full-length or truncated GDAP1 expressed in bacteria is not soluble (data not shown), consistent with previous reportsp-Nitrobenzylchloride, and 1,2-Epoxy-3-(4-nitrophenoxy)propane, but not to the classic GST-substrate 1-Chloro-2,4-dinitrobenzene  to break the amphipathic structure , C-terminal truncated recombinant GDAP1 proteins without the C-terminal transmembrane domain (TMD) are readily expressed and soluble. More interestingly, we found that only GDAP1 lacking both its C-terminal TMD and HD1 has GSH-conjugating activity. By size-exclusion chromatography (SEC) and immunoprecipitation experiments we confirm that GDAP1 with HD1 but without the TMD also forms dimers. We conclude that loss of GST-activity in the presence of HD1 is not caused by a protein dimerization deficit and hypothesize that HD1 might have an auto-inhibitory function in regulating the enzyme activity of GDAP1.We provide here first experimental evidence that GDAP1 has a GSH-conjugation activity typical for theta-class GSTs. While full-length GDAP1 cannot be purified in soluble form from in silico analysis, which predicted an amphipathic pattern for HD1, a structure known for its membrane-remodeling activities. Indeed, the addition of in vitro translated full-length GDAP1 to liposomes mimicking the peroxisomal or outer mitochondrial membrane composition lead to a deformation of these liposomes. Incubation oft the same liposomes with GDAP1 with a scrambled HD1 sequence or GDAP1 lacking the TMD does not alter the liposomal shape. This corroborates our previous findings showing that the HD1 is essential to mediate GDAP1-induced mitochondrial and peroxisomal fission3in vitro translated full-length GDAP1 did not prove to have GST activity (not shown) and the GST-active recombinant GDAP1 is not targeted to membranes as it lacks the tail-anchor domainTo understand the potential role of HD1 in GDAP1 we performed Implementing our new findings together with results from previous reports23410GDAP11819In the absence of high-resolution atomic structures of GDAP1, experimental prove for our proposed model is currently lacking. Notwithstanding, evidence exists that is consistent with our model, including the observed properties of point mutated disease-associated forms of GDAP1. Recessively inherited mutations cluster within the coding region of the N-terminal, cytosolic GST-N, GST-C, and interdomain of bona fide GST enzyme, changing its conformational state upon activating stimuli, which regulates the release of the autoinhibitory HD1 domain to execute fission by interaction with the organelle’s outer leaflet.Expressing rmGDAP1 is associated with a loss or markedly reduced fission capacity, while dmGDAP1 still induce fission but block or delay mitochondrial fusion14GDAP1 or MFN2, which converge on sensing of redox stress conditions and result in the regulation of mitochondrial morphology and intracellular Ca(2+) levels. However the precise conditions stimulating these proteins in vivo and the resulting molecular and cellular reactions remain to be elucidated.Besides the induction of mitochondrial and peroxisomal fission, GDAP1 has been linked to the regulation of intracellular Ca(2+) homeostasis27HEK-293T cells were cultured and transfected with Lipofectamine 2000 (Invitrogen) as described previously1Human GDAP1 (hGDAP1) missense mutations and truncations were cloned into the pcDNA3.1 expression vector as described previously11in vitro translation of GDAP1 wildtype and mutant forms was performed using the TNT T7 Quick Coupled reticulocyte system  as described previouslyProteins were expressed as described in ref. 30 μl wet Protein A-Sepharose beads  were incubated overnight with 5 μl GDAP1 antibody serum. Transfected HEK-293T were harvested in lysis buffer ), centrifuged for 5 min at 800 g and the cleared supernatant was than incubated with the antibody coupled beads for 2 h. Beads were washed four times with lysis buffer, and subsequently the samples were boiled at 95 °C in SDS-loading buffer and subjected to immunoblotting analysis.For all GST activity assays 50 μg of recombinant protein  in a total reaction volume of 200 μl using the conditions listed in 33in vitro translate or were left untreated for 4 hours on ice, mixed 1:1 with 2% phosphotungstic acid (pH 7.2) and immediately placed on Formvar-coated nickel grids  for 4 minutes. The liquid was removed and the grids were dried for 2 hours under vacuum and examined in blinded fashion with a Zeiss EM 109S electron microscope . On the electronic pictures all separate liposomes were selected with the magic wand tool in Adobe Photoshop and the circularity of the objects was determined.Liposomes were prepared using 5 mM stock solutions in methanol:chloroform (1:4) of phosphtidycholine , L-α-phosphatidylethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine , L-α-phosphatidylinositol (PI), cardiolipin (CA). Lipids were purchased from Avanti Polar Lipids Inc., if not indicated differently. To prepare liposomes in the desired composition, 20 μl lipid-mixture  and the amphipathic pattern was determined by the modeling software How to cite this article: Huber, N. et al. Glutathione-conjugating and membrane-remodeling activity of GDAP1 relies on amphipathic C-terminal domain. Sci. Rep. 6, 36930; doi: 10.1038/srep36930 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting. 20 of 21 recipients of B10.D2 BMC but only 13 of 18 of Balb/c BMC and 13 of 20 of B10.A BMC developed stable long-term multilineage chimerism (p < 0.05 for each donor strain versus B10.D2). Significantly superior donor skin graft survival was observed in successfully established long-term chimeras after mHAg matched BMT compared to mHAg mismatched BMT (p < 0.05). Both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative BMT and costimulation blockade is negatively influenced by minor antigen disparities.        Mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation (BMT) with costimulation blockade (CB), but varying success rates have been reported with distinct models and protocols. We therefore investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance. C57BL/6 (H2 Donor specific tolerance through mixed chimerism can be achieved in various animal models by nonmyeloablative BMT and CB . SuccessmHAg are polymorphic non-MHC proteins that are able to induce a T cell response due to allelic variations between donor and recipient . mHAg pl1R) and endothelin type A receptor (ETAR), have been shown to correlate with an increased incidence of acute rejection and inferior long-term graft survival in kidney and heart allografts  versus 5.20% ± 0.17 in naïve BL6 mice [n = 2]). Compared to Balb/c chimeras, a similar degree of deletion was observed in B10.D2 chimeras . Interestingly, transplantation of B10.A  bone marrow led to a significantly more pronounced early deletion compared to Balb/c and B10.D2 . In contrast irrelevant Vβ8.1/2+ CD4+ T cells were not deleted in either group after BMT, indicating the specificity of the deletion for superantigens presented by the donor  but still was significantly less pronounced than in recipients of B10.A BMC  may influence the extent and kinetic of deletion of donor-reactive T cells.Peripheral and central clonal deletion are important mechanisms for the induction and maintenance of tolerance in models of mixed chimerism induced by BMT plus CB . Notablyhe donor . Ten weeIn this study we provide evidence that mHAg disparities play a decisive role in the induction and maintenance of tolerance in recipients conditioned with nonmyeloablative BMT and CB, in which mHAg both impede the engraftment of BM and promote the rejection of donor skin in successfully established mixed chimeras.Tolerance induction through donor BMT depends on two, mechanistically separate, events: successful engraftment of donor BM and durable tolerization of all relevant donor antigens in the context of donor chimerism. The findings presented herein indicate that mHAg influence both of these events. The frequency of successful long-term chimeras was higher in the absence of mHAg disparities when the MHC haplotype was the same . These results are in line with findings that MHC-matched BM is rejected in sublethally irradiated mice [Only some of the minor antigens that drive alloreactivity in this strain combination  have been identified, with H60 provoking a particularly strong reaction . H60 is Unexpectedly, significantly fewer mice developed long lasting multilineage chimerism after receiving BM from B10.A compared to B10.D2 donors (both mHAg matched to recipient). Since the only difference between B10.A and B10.D2 is the H2 haplotype these results suggest that the MHC haplotype per se influences chimerism induction. Distinct H2 haplotypes are known to stimulate varying numbers of alloreactive T cells and thus exhibit a varying degree of immunogenicity , which aWith regard to the second event, mHAg disparities increased the rate of skin graft rejection in successfully established chimeras . In addition, the surviving skin grafts of Balb/c but not B10.D2 donors exhibited histological signs of chronic inflammation. Thus, mHAg disparities can drive chronic rejection in the presence of stable mixed chimerism. Several groups attributed tissue specific antigens, which are not present in the BM but the skin, for this state of so-called “split tolerance” . CriticaThis study reveals that mHAg disparities have a negative impact on BM engraftment and tolerance maintenance in a nonmyeloablative, CB-based chimerism model. Preclinical tolerance protocols should encompass mHAg disparities to reflect the clinical setting and need to induce mechanisms capable of durable tolerization of donor mHAg.


Severe hypoxia develops close to the necrotic core of advanced human atherosclerotic plaques, but the energy metabolic consequences of this hypoxia are not known. In animal models, plaque hypoxia is also associated with depletion of glucose and ATP. ATP depletion may impair healing of plaques and promote necrotic core expansion. To investigate if ATP depletion is present in human plaques, we analyzed the distribution of energy metabolites  in intermediate and advanced human plaques.Snap frozen carotid endarterectomies from 6 symptomatic patients were analyzed. Each endarterectomy included a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). ATP, glucose, and glycogen concentrations were lower in advanced (ICA) compared to intermediate plaques (CCA), whereas lactate concentrations were higher. The lowest concentrations of ATP, glucose and glycogen were detected in the perinecrotic zone of advanced plaques.Our study demonstrates severe ATP depletion and glucose deficiency in the perinecrotic zone of human advanced atherosclerotic plaques. ATP depletion may impair healing of plaques and promote disease progression. Cells need oxygen and nutrients to produce ATP required for ion pumps, migration and intracellular metabolism. In the artery wall, oxygen and nutrients are supplied via diffusion from luminal blood and vasa vasorum , 2. In aATP depletion within atherosclerotic plaques may promote progression of atherosclerosis . In suppIn the current study, we mapped concentrations of ATP, glucose, glycogen and lactate within human atherosclerotic plaques. Our results demonstrate severe cellular ATP depletion and lack of glucose in advanced human plaques, in particular close to the necrotic core. These results support the ATP depletion hypothesis.Energy metabolites were measured in human plaques from carotid endarterectomies. The endarterectomies were obtained from the Gothenburg Atheroma Study Group biobank. The patients had a high grade symptomatic stenosis (≥70% stenosis of the carotid artery) and underwent carotid endarterectomy at the Sahlgrenska University Hospital  during 2005 or 2006. Patient characteristics and time from last symptom until surgery are shown in It is known from previous studies that plaque severity (AHA type) varies within carotid plaques. Typically, plaque histology is more advanced upstream of the carotid bifurcation, in the internal carotid artery (ICA), than in downstream portions of the same plaque, in the common carotid artery (CCA) . To obtaPlaque histology was analyzed by Mayer’s HTX and eosin. Frozen 8 μm sections were incubated in Mayer’s HTX (Histolab Products AB) for 90 seconds and in eosin for 60 seconds, after which they were dehydrated in ethanol and mounted with Pertex (Histolab Products AB). Each H&E-stained section was classified by a pathologist (LMA) using AHA classification .Macrophage and smooth muscle cell content was analyzed by immunofluorescence staining of acetone fixed frozen 8 μm sections. Macrophages were labeled with an anti-CD68 antibody . Secondary detection was made using donkey Fab fragment anti mouse 488  after which a directly conjugated anti-α-actin antibody  was added. Isotype controls were made using mouse IgG1 . Cell nuclei were stained using DAPI. Slides were scanned using a Metafer Slide Scanning Platform (MetaSystems GmbH) with a 10x objective. Quantification of CD68, α-actin, necrotic core area, cellularity, and intimal thickness was performed in ImageJ .Neutral lipid accumulation was assessed by staining one cryosection (8 μm) of each sample with Oil red O. Sections were fixed in 2% paraformaldehyde in PBS during 4 minutes, rinsed in tap water, treated with 20% isopropanol in water for 30 seconds, after which oil red O working solution was added during 7 minutes. The sections were treated with 20% isopropanol in water during 30 seconds, and rinsed in tap water for two minutes. The slides were treated with Mayers HTX (Histolab Products AB) during 1 minute, rinsed in tap water, distilled water, and finally mounted with Mowiol.3 to 106 times higher than the extracellular concentration. Therefore, ATP assessment almost exclusively reflects intracellular ATP.ATP, glucose, glycogen and lactate was measured by bioluminescence imaging according to previously published protocols . BiolumiData are expressed with median values in all graphs. p values were calculated using unpaired t-test , WilcoxoTo investigate energy metabolism in human atherosclerotic plaques, we analyzed snap frozen human carotid endarterectomies. Each endarterectomy contained a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). Plaque severity varied within each plaque and plaques in the common carotid artery were intermediate  whereas plaques in the internal carotid artery were advanced  , in agreFirst, we investigated if there was a difference in intimal concentrations of energy metabolites between intermediate and advanced segments of carotid plaques . The meaWe then investigated how energy metabolites are distributed within advanced plaques. Hypoxia in advanced plaques was mainly present in the perinecrotic zone, as indicated by high expression of hexokinase II; a glycolytic enzyme upregulated by hypoxia  13]. Th. Th13]. In this study, we demonstrate depletion of energy metabolites in advanced human atherosclerotic plaques. The most pronounced energy depletion was detected adjacent to the necrotic core.ATP was severely depleted in advanced plaques, in particular in the perinecrotic zone adjacent to the necrotic core. Accumulation of dead macrophages in the necrotic core promotes lesion instability and is a key event in atherosclerosis progression. Macrophages in plaques die either via apoptosis or necrosis . ApoptosATP depletion may also impair lipid removal from plaques. Macrophage foam cells in plaques need ATP to transfer intracellular lipids to HDL particles and subsequent clearance via lymph vessels . Macrophex vivo [In addition to ATP depletion, advanced plaques are also depleted in glucose, especially in the perinecrotic zone. Glucose depletion is most likely due to excessive glycolysis in hypoxic macrophage foam cells in combination with insufficient glucose supply from luminal blood and vasa vasorum , 17. ATPex vivo .Another consequence of excessive glycolysis is accumulation of lactate. Accumulation of lactate decrease tissue pH and low pH increases intimal lipid retention by increasing affinity of atherogenic lipoproteins for proteoglycans . In addiOne limitation of our study is that bioluminescence imaging is an in vitro method and analysis of snap frozen tissue is required to reflect the in vivo situation . Human eIn conclusion, the advanced human plaque is ATP-depleted, deprived of oxygen and glucose, and accumulates lactate. In this respect, the advanced plaque has features of a chronic, non-healing wound , 22. Res


Surface electromyography (sEMG) is a popular research tool in sport and rehabilitation sciences. Common study designs include the comparison of sEMG amplitudes collected from different muscles as participants perform various exercises and techniques under different loads. Based on such comparisons, researchers attempt to draw conclusions concerning the neuro- and electrophysiological underpinning of force production and hypothesize about possible longitudinal adaptations, such as strength and hypertrophy. However, such conclusions are frequently unsubstantiated and unwarranted. Hence, the goal of this review is to discuss what can and cannot be inferred from comparative research designs as it pertains to both the acute and longitudinal outcomes. General methodological recommendations are made, gaps in the literature are identified, and lines for future research to help improve the applicability of sEMG are suggested. R2 = 0.98)  is a common research tool used to investigate a wide range of research questions across various disciplines. sEMG is perhaps most useful for providing insight into how the neuromuscular system behaves. In its simplest sense, sEMG is a highly-sensitive voltmeter that detects depolarizations and hyperpolarizations  that occur on the sarcolemma (muscle fiber membrane). These depolarizations are necessary for, and precede, the contraction of a muscle. Among other disciplines, the utilization of sEMG is popular in sport and rehabilitation sciences, with the number of studies including the terms “EMG” and “exercise” increasing exponentially since 1950  to 100%  . With appropriate processing and based on sufficiently reasonable approximations, sEMG may provide information on the timing and degree of muscles' excitation  or average rectified value (ARV)] under non-fatiguing conditions  relative to its maximum ability to produce force actively Figure . Althougneural excitation; in other words, sEMG is a measure of muscle excitation. Activation and excitation differ in that excitation is a precursor to activation . To estimate force sharing, one must utilize musculoskeletal modeling rather than Collectively, sEMG studies that aim to draw conclusions about muscle force production, muscle activation, or mechanisms of force production are problematic when based solely on sEMG amplitude. Oftentimes, in order to draw mechanistic conclusions, more advanced sEMG processing and modeling techniques are needed. At face value, at best, sEMG amplitude is strictly indicative of muscle excitation.Longitudinal outcomes are undoubtedly the most applicable, clinically relevant measures that a sport or rehabilitation scientist can provide to a practitioner. In this section, we discuss two commonly-measured and sought after outcomes: muscular strength and hypertrophy. Measuring these outcomes, however, can be quite difficult and time-consuming  and hypertrophy, the presence of hypertrophy without muscle excitation, and differential hypertrophy with similar excitation, which are explained herein.Acutely, it has been postulated that maximum motor unit recruitment, a primary constituent of the sEMG signal, is important for stimulating hypertrophy , with loads corresponding to 80% of each participant's MVIC strength, measured at each participant's optimal joint angle, over a period of 6 weeks. Only participants who trained at long muscle lengths experienced increases in quadriceps muscle volume  and 90% (concentric) of maximum. A related study could explore the influence of sEMG amplitude on hypertrophy while keeping constant muscle length and contraction velocity. Moreover, it is important to note that absence of evidence is not evidence of absence; although sEMG has not been established to be predictive of hypertrophy, it does not mean that no relationship exists. Some indirect evidence loosely suggests that sEMG amplitude may have a potential role in predicting hypertrophy: When comparing rectus femoris sEMG amplitude in single-joint exercises (knee extension) to that elicited by multi-joint exercises (simultaneous hip and knee extension), it is clear that the former elicits much greater sEMG amplitudes  have also been shown to have the ability to increase strength , it will provide the state of the muscle over longer periods of time. When dealing with short bursts of excitation, the implications are less clear and more caution should be used when interpreting results , tissue conductivity, and signal nonstationarity  are taken into account Farina, . LongituPerhaps one of the most common types of comparisons made in sport and rehabilitation sciences are within-subject, within-muscle comparisons. For example, researchers will compare sEMG amplitudes elicited during different exercises  must be taken into account; tissue conductivity will necessarily be different between muscles, due to differences in geometry and dynamics Farina, . It shouIf signals are properly normalized and appropriate considerations are made Table , one mayMuch like comparing between-muscles, there are a number of concerns pertaining to whether or not individuals in a group, especially symptomatic ones, have the ability to maximally excite a muscle during a normalization trialIn more homogeneous populations, one can use sEMG to understand mechanisms for differences in function. For example, authors have used maximum M-wave normalized sEMG amplitudes to help us understand differences in strength between individuals (Trezise et al., The considerations for between-subject and between-muscle comparisons above both apply to this category, but are amplified due to the larger potential for differences to exist.sEMG can be useful for comparing co-contraction between populations—such young and old individuals—to understand how differences in function—such as strength—may arise (Macaluso et al., Challenging the applicability of sEMG can be viewed as bittersweet. While it is quite humbling that we do not truly understand what we are measuring or its implications, this also opens the door for high quality, impactful research. Ultimately, longitudinal outcomes are likely of greatest interest to practitioners (Halperin et al., Factors other than muscular effort influence the myoelectric signal, including muscle length, contraction mode, contraction speed, etc. Comparing sEMG signals between different exercises that do not control for these variables should be avoided.Even when the sEMG signal adequately represents the force of the muscle, caution should be exercised when concluding that a certain exercise will be better for increasing strength or hypertrophy due to other factors that influence these adaptations.Comparing normalized sEMG values between individuals with and without pain should be viewed cautiously. Changes in the normalized sEMG value over time cannot indicate changes in excitation because the normalized value can be influenced by excitation during the normalization contraction or during the measured exercise. Normalizing to maximum M-wave amplitude can, to an extent, help diminish such effects.Within-subject, within-muscle comparisons of the sEMG signal across different exercises may be able to provide insight into muscular force production, provided the previously mentioned controls are made.sEMG is a useful tool for gaining insight into the neuromuscular system, musculoskeletal modeling, and basic science work, but its practical applicability is limited at present. Researchers wishing to produce applicable research pertaining to longitudinal adaptation should prioritize longitudinal studies rather than acute, cross-sectional sEMG work (Halperin et al., AV, IH, GT, and TV conceived the manuscript. AV, IH, GL, GT, and TV wrote, reviewed, and approved the final version of the manuscript. AV and TV created the figures. AV and IH constructed the tables.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.