Importantly, cerebral expression area of P2RY12+and TMEM119+ homeostatic microglia and the branches of P2RY12+ homeostatic microglia were increased in the SCF+G-CSF-treated aged APP/PS1 mice. SCF+G-CSF treatment also decreased NOS-2 and increased IL-4 in the brains of aged APP/PS1 mice. Moreover, the loss of MAP2+dendrites and PSD-95+post-synapses and the accumulation of aggregated tau in the brains of aged APP/PS1 mice were ameliorated by SCF+G-CSF treatment. Furthermore, the density of P2RY12+ microglia was negatively correlated with Aβ deposits, but positively correlated with the densities of MAP2+ dendrites and PSD-95+ puncta in the brains of aged APP/PS1 mice. These findings reveal the therapeutic potential of SCF+G-CSF treatment in ameliorating AD pathology at the late stage.Alzheimer’s disease (AD), characterized by the accumulation of β-amyloid (Aβ) plaques and tau neurofibrillary tangles in the brain, neuroinflammation and neurodegeneration, is the most common form of neurodegenerative disease among the elderly. No effective treatment is available now in restricting the pathological progression of AD. The aim of this study is to determine the therapeutic efficacy of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in aged APPswe/PS1dE9 (APP/PS1) mice. SCF+G-CSF was subcutaneously injected for 12 days to 25-month-old male APP/PS1 mice. We observed that SCF+G-CSF treatment reduced the Aβ plaques in both the cortex and hippocampus. SCF+G-CSF treatment increased the association of TREM2 Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly, which leads to huge economic costs and social burden worldwide . In the (25-35) chronically activates microglia, resulting in persistent neuroinflammation [The pathological hallmarks of AD are characterized by the presence of amyloid beta (Aβ) plaques, intracellular neurofibrillary tangles, and neuro-inflammation , 4. The ammation . It has ammation . Therefoammation -11. Overammation -16. A loammation , leadingammation , 16-19. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally demonstrated as essential hematopoietic growth factors regulating the proliferation, differentiation and mobilization of hematopoietic stem cells (HSCs) , 21. SCFIn clinical studies, decreased levels of SCF and G-CSF in the plasma have been observed in AD patients , 31. It In the present study, 25-month-old APP/PS1 mice received 12-days of SCF+G-CSF injections. As an initial study exploring pharmaceutical strategy for AD at such an old age, we have demonstrated that SCF+G-CSF treatment reduces Aβ deposits, ameliorates neuroinflammation, and increases the densities of dendrites and synapses in the brains of the very old APP/PS1 mice.The experiments have been carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals in the United States. All experimental procedures have been approved by the Animal Care and Use Committees of State University of New York Upstate Medical University (IACUC#369).APPswe/PS1dE9 (APP/PS1) transgenic mice co-expressing Swedish double mutations (K595N/M596L) of amyloid precursor protein (APP) together with mutant human presenilin protein 1 (PS1) carrying the exon-9-deleted variant (PS1-dE9) were useA total of 20 male APP/PS1 mice were prepared for this study (n=10/group). At the age of ~25 months, nine of them remained. These aged APP/PS1 mice and five age-matched C57BL/6J wild type (WT) mice  were used in this study. The APP/PS1 mice were randomly divided into two groups and subcutaneously injected with recombinant mouse SCF  and recombinant human G-CSF  (SCF+G-CSF) (n =4) or vehicle solution  (n =5) once a day for 12 days. Recombinant SCF and G-CSF have been shown to pass through the blood-brain barrier in intact animals . The dosMice were transcardially perfused with phosphate buffered saline (PBS) followed by 4% buffered formaldehyde . The brains were removed, placed in 4% buffered formal-dehyde overnight at 4°C, and then cryoprotected by immersion in 30% sucrose. Coronal brain sections (30μm thick) were cut with a microtome . Two or three brain sections from each brain were processed for immunohistochemistry or histochemistry. The free-floating method was used for immunohistochemistry. Briefly, to minimize endogenous mouse IgG background staining when using mouse primary antibodies, sections were incubated with mouse on mouse (MOM) solution  for 1 h at room temperature. After being washed with PBS (3 × 5 min), nonspecific binding was blocked by 10% normal donkey serum in 1% bovine serum albumin  and 0.25% Triton X-100  for 1 h at room temperature. Brain sections were then incubated with primary antibodies overnight at 4°C. The primary antibodies include mouse anti-β-amyloid (1:1000) , mouse anti-β-amyloid (1:1000) , rabbit anti-Iba1  , goat anti-Iba1 , rabbit anti-P2RY12  , rabbit anti-MAP2 (microtubule associated protein 2) , mouse anti-PSD-95 (postsynaptic density protein 95) , sheep anti-TREM2 (triggering receptor expressed on myeloid cells-2) , NOS2 , IL-4 , NeuN , GFAP , CD68 , TMEM119 , and AT8 . After being washed with PBS (3 × 10 min), the sections were incubated with Alexa Fluor 488-, 594- or 647-conjugated donkey anti-mouse, rabbit, goat or sheep secondary antibodies  for 2 h at room temperature in the dark. After being washed with PBS (3 × 10 min), sections were mounted with Vectashield antifade mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) .2O for 2 min. The sections were then put into 100μM X-34 working solution (pH=10) for 10 min at room temperature in the dark. After being washed with ddH2O (5 × 2 min), the sections were differentiated for 2 min with 0.2% NaOH. After PBS washing (2 × 5 min), the sections were then incubated with Col-IV antibody  overnight at 4°C and incubated with Alexa Fluor 594-conjugated donkey anti-rabbit for 2h at room temperature following the standard immunohistochemistry protocol described above. Finally, the sections were mounted with Vectashield antifade mounting medium without DAPI . Brain sections with the omission of primary antibodies served as negative controls  and Collagen-IV staining, Tris-EDTA Buffer  was used for antigen retrieval before staining. Briefly, after being washed with PBS for 5 min, the sections were put into pre-heated antigen retrieval buffer (500 ml) at 90°C for 10 min and then cooled to room temperature. After PBS washing (3 × 5min), the sections were rinsed with ddHcontrols .To determine the Aβ deposition in the brain, images were obtained using a Zeiss-inverted microscope  and quantified using ImageJ software. Images were taken from eight selected areas in the cortex  and 6 selected areas in the hippocampus  of each brain section using a 20× or 40× objective lens . Plaque +senile plaques within Iba1+ cells surrounding the senile plaques in each image field was calculated. The Aβ uptake volume was determined by dividing the Iba1+/4G8+colocalization volume by the total Iba1+ cell volume. For quantification of Iba1, TREM2, CD68, P2RY12, TMEM119, AT8, NOS-2 and IL-4 immunopositive staining, images  were taken with the Zeiss LSM780 confocal microscope using the 40× lens. After the images were stacked with the maximum approach, the immunopositive staining of Iba1, TREM2, CD68, P2RY12, TMEM119, AT8, NOS-2 and IL-4 was quantified by dividing the total immunopositive staining area by the area of the image field using ImageJ.For the rest of immunohistochemistry quantification, four random areas in the cortex and 1 × 3 tile scan areas in the CA1 region  of each hemisphere were selected to capture images from each brain section using a confocal microscope (Zeiss LSM780) with a 20× or 40× objective lens . In the + immunostaining in the cortex and CA1 was determined by dividing the MAP2 positive area by the area of the image field of the entire cortex or selected CA1 region using Image J. For quantification of PSD-95 staining, images (1024 × 1024 pixels) were taken by using the Zeiss LSM780 confocal microscope with the 40× lens. The number of PSD-95-labeled puncta in the cortex and CA1 was counted automatically with a threshold of 20 pixels in ImageJ.For quantification of MAP2 staining, images were captured with the Zeiss LSM780 confocal microscope using the tile scan feature with an average grid size of 3 × 5 taken with the 40× objective at a resolution of 1024 × 1024 pixels. The MAP2+ Aβ deposition and Col-IV+ blood vessels, images were taken with the Zeiss LSM780 confocal microscope using the 20× lens. The X-34 and Col-IV positive staining were quantified by dividing X-34 and Col-IV positive area by the area of the image fields in the cortex or CA1 using ImageJ.For quantification of X-34For all image analysis, the background fluorescence of secondary antibodies was established as a threshold to be subtracted from the positive staining during data analysis using ImageJ.+ Aβ deposition and P2RY12+ cells, between P2RY12+ cells and MAP2+ dendrites, and between P2RY12+ cells and PSD-95+ synapses. All data are expressed as mean ± S.E.M. Statistical significance was considered p<0.05.Using Prism 6.0 software, comparisons among three groups were analyzed by one-way ANOVA followed by Fisher's LSD test, and the comparisons between two groups were analyzed by unpaired student’s t-test. Pearson correlation analysis was used to evaluate the correlative association between X-34+Aβ deposits in the cortex and hippocampus of APP/PS1 mice, while SCF+G-CSF treatment significantly reduced the percentage area of 4G8+Aβ deposits in both the cortex (p<0.01) and hippocampus (p<0.05) (+Aβ plaques in the cortex was significantly decreased in the SCF+G-CSF-treated APP/PS1 mice as compared to the vehicle controls (p<0.001) (+Aβ plaques in the cortex and hippocampus (p<0.05)  . In addip<0.001) . A similp<0.001) . Moreove(p<0.05) . These fFibrillar Aβ is the primary constituent of amyloid plaques in the brain of AD , 40. X-3Since vascular impairment has been shown to be involved in Aβ accumulation and AD progression , we thenAltogether, our findings suggest that SCF+G-CSF treatment in aged male APP/PS1 mice leads to remarkable reductions in cerebral Aβ load, which may be the result of increased removal of Aβ deposits in the brain by SCF+G-CSF intervention. The SCF+G-CSF-reduced Aβ load may be associated with, but not dependent on, its effects on maintaining vasculature in the brains of aged APP/PS1 mice.+cells, triple immunofluorescence staining of Iba1, TREM2 and 6E10 was performed in brain sections of APP/PS1 mice. We observed that SCF+G-CSF treatment significantly increased the TREM2 expression around the 6E10+Aβ plaques in the brains of APP/PS1 mice as compared to the vehicle controls (p<0.05)    (Supplem(p<0.05) . Further(p<0.05)  (Supplem(p<0.05) . These d+ cells and Aβ plaques and the volume of engulfed Aβ in Iba1+ cells in the brains of APP/PS1 mice using immunofluorescence double staining and confocal imaging. As shown in + cells with 4G8+ plaques (p<0.05). Moreover, 3-dimensional (3D) analysis revealed that the volume of 4G8+ Aβ within the Iba1+ cells was significantly increased by SCF+G-CSF treatment (p<0.05)  .+ Aβ with CD68+ compartment. We found that CD68+ cells around the 4G8+ plaques were significantly increased in the cortex of SCF+G-CSF-treated APP/PS1 mice (p<0.05)  (CD68 is a transmembrane glycoprotein and belongs to the family of lysosome-associated membrane proteins and the family of scavenger receptors . CD68 is(p<0.05) . In addi(p<0.05) .+ and CD68+ microglia/macrophages surrounding the senile plaques and promotes phagocytic clearance of Aβ by the Iba1+ and CD68+ microglia/macrophages in the brains of aged APP/PS1 mice.These findings demonstrate that SCF+G-CSF treatment enhances the accumulation of Iba1+/Iba1+ resting microglial cells immediately next to the Aβ plaques were significantly decreased in the brains of SCF+G-CSF-treated APP/PS1 mice as compared to the vehicle controls (p<0.01)  . SCF+G-CSF treatment significantly increased the branches of the P2RY12+ microglia in the brains of APP/PS1 mice as compared to the vehicle control APP/PS1 mice (p<0.05)  . By cont(p<0.05) . Since t(p<0.05) .To further verify the contribution of SCF+G-CSF treatment in maintaining/restoring the homeostatic state of microglia, we used another homeostatic microglial marker, TMEM119, to detect the homeostatic microglia. We observed that the expression of TMEM119 in the cortex of APP/PS1 mice was significantly decreased as compared to the WT mice (p<0.001) . SCF+G-CThese findings demonstrate that SCF+G-CSF treatment leads to increases in microglial homeostasis in the brains of aged APP/PS1 mice.To further evaluate the effects of SCF+G-CSF intervention on the inflammatory reactivity in the brains of aged APP/PS1 mice, we quantified the expression of pro-inflammation factor, NOS-2, and anti-inflammation factor, IL-4, in the brains of aged APP/PS1 mice through immunohistochemistry. We observed that the expression of NOS-2 was significantly increased in both the cortex (p<0.001) and hippocampal CA1 region (p<0.001) of aged APP/PS1 mice as compare to the WT controls . SCF+G-C+) (+) (+) (+) (+) (+) , Iba1  and GFAP (astrocyte marker). We observed that NOS-2 was expressed in the neurons (NeuN+)  and micr+) (+)  in both  (+) (+) . IL-4 wa (+) (+) , microgl (+) (+)  and astr (+) (+)  in both + dendrites were significantly decreased in both the entire cortex (p<0.01) (+ dendrites in the brains of aged APP/PS1 mice were significantly restored by SCF+G-CSF intervention in both the entire cortex (p<0.05) (+ puncta in both the cortex (p<0.001) (Dendritic degeneration and synaptic loss induced by Aβ neurotoxicity and chronic inflammation are the major causes for the cognitive impairments in AD -58. Buil(p<0.01)  and hipp(p<0.01)  of aged (p<0.05)  and CA1 (p<0.05) . Further(p<0.05)  and CA1 (p<0.05)  of the ap<0.001)  and CA1 p<0.001)  of aged + tau pathological structures in the cortex of APP/PS1 mice (+ puncta) was not significantly different between SCF+G-CSF-treated and vehicle control APP/PS1 mice (+ puncta in the cortex as compared to the vehicle control APP/PS1 mice (p<0.001)  mice . To detePS1 mice . The numPS1 mice . Howeverp<0.001) . This ob+ resting microglia and X-34+ fibrillar Aβ plaques in both the cortex    and hipp p<0.05)  of the A+microglia and MAP2+ dendrites in the cortex     and hipp p<0.01) . Further p<0.01)  and hipp p<0.01)  of the AUsing a widely-used mouse model of cerebral amyloidosis in AD research, the present study has demonstrated the therapeutic efficacy of combined SCF and G-CSF treatment in aged male APP/PS1 mice. Our data have revealed that SCF+G-CSF treatment reduces diffuse and fibrillar Aβ deposits, increases the association of microglia/macrophages with senile plaques, and enhances Aβ uptake by microglia/macrophages in the brains of aged APP/PS1 mice. Furthermore, SCF+G-CSF treatment increases homeostatic microglia and ameliorates inflammatory status in the brains of aged APP/PS1 mice. Systemic administration of SCF+G-CSF also reverses the loss of dendrites and synapses and reduces pathological tau in the brains of aged APP/PS1 mice. These data suggest that SCF+G-CSF treatment ameliorates the pathological severity in the brain of aged APP/PS1 mice.Microglia are the resident macrophages in the central nervous system. Microglial cells constantly survey their local microenvironment, control neurogenesis and synaptic generation, and contribute to the maintenance of brain homeostasis. Microglia also detect the first signs of pathogenic invasion and tissue damage and support tissue repair . Iba1 isin vitro study reveals that M-CSF treatment increases microglial proliferation and phagocytosis of Aβ in primary cultured adult human microglia [Hematopoietic growth factors have been demonstrated to effectively reduce Aβ load in the brains of APP/PS1 mice. In addition to SCF and G-CSF, macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are also the family members of hematopoietic growth factors. Systemic injection of M-CSF  or GM-CSicroglia . In a pricroglia . It has icroglia , while cicroglia . In a lo+ microglia and bone marrow-derived Iba1+ macrophages have been proposed to be involved in the clearance of Aβ [+ Aβ in the Iba1+ microglia/macrophages is increased by SCF+G-CSF treatment. CD68 is the marker for detecting lysosomes of microglia and macrophages [+ Aβ in CD68+lysosomes of microglia/macrophages is increased by SCF+G-CSF treatment in aged APP/PS1 mice. These findings suggest that SCF+G-CSF treatment-reduced Aβ load in the brains of aged APP/PS1 mice is modulated by enhanced Aβ uptake and degradation (Aβ clearance) through phagocytosis by microglia/macrophages. Similar findings have been reported in the M-CSF study showing that Iba1+ microglia-internalized Aβ in the brain of APP/PS1 mice is increased by systemic injection of M-CSF [in vitro study also demonstrates the efficacy of M-CSF in enhancing the ability of microglia to phagocytize and degrade fibrillar Aβ [Although the precise mechanism underlying the SCF+G-CSF-reduced Aβ load remains unclear, our data suggest that increasing phagocytic removal of Aβ by microglia/macrophages is involved in the SCF+G-CSF-reduced Aβ load in the brain of aged APP/PS1 mice. Both resident Iba1ce of Aβ , 75, 76.rophages -48. Our of M-CSF . An in v+ microglia/macrophages in transgenic mouse models of AD [+ microglia/macrophages surrounding the Aβ plaques, while TREM2 could not be detected in the Iba1+ cells distant from the plaques in the brains of APP/PS1 mice. This observation is in line with the findings reported by other investigators [+ microglia/macrophages surrounding the senile plaques, suggesting that TREM2 upregulation is involved in SCF+G-CSF-enhanced Aβ clearance by the Iba1+ microglia/macrophages in the senile plaques. In addition to SCF+G-CSF-increased TREM2 expression in the Iba1+ microglia/macrophages surrounding the Aβ plaques and SCF+G-CSF-increased CD68-expressing microglia/macrophages around the Aβ plaques, it is worth noting that SCF+G-CSF treatment also leads to reduced P2RY12+/Iba1+ homeostatic microglia in the vicinity of the Aβ plaques. Our observations suggest that SCF+G-CSF treatment in aged APP/PS1 mice enhances the transformation of homeostatic microglia into phagocytic DAM phenotype in the area next to the Aβ plaques. This SCF+G-CSF-enhanced microglial phenotype transformation occurring particularly at the location surrounding the Aβ plaques may be necessary to reinforce Aβ removal and restrict Aβ-induced pathology. A recent transcriptomic study has shown that the population of DAM contains both pro-inflammatory DAM and anti-inflammatory DAM. The anti-inflammatory DAM express phagocytic genes. Using a pharmacological approach to promote anti-inflammatory DAM or inhibit pro-inflammatory DAM leads to enhanced Aβ clearance in 5xFAD mice [Recent studies have demonstrated that TREM2 plays a vital role in mediating phagocytosis and degradation of Aβ by Iba1ls of AD , 44. Losls of AD , 78. It ls of AD , 79. Ourtigators . The finFAD mice . It woul+ microglia have been found in the cerebral parenchyma in mouse models and AD patients [+ microglia in both the cortex and hippocampus of aged APP/PS1 mice. While the consequences of P2RY12 downregulation in AD are not fully understood, P2RY12 expression in microglia has been proposed to have protective effects in the context of AD [+ dendritic density and PSD-95+post-synaptic puncta in the brains of aged APP/PS1 mice. Notably, SCF+G-CSF treatment increases the P2RY12+microglia in the cortex and hippocampus, and augments P2RY12+/Iba1+ cells distant from the Aβ plaque regions in aged APP/PS1 mice. Our findings indicate that SCF+G-CSF treatment improves the recovery of homeostatic phenotype of microglia and ameliorates pathological severity in the brains of aged APP/PS1 mice. The SCF+G-CSF treatment-enhanced recovery of homeostatic microglia in the brains of aged APP/PS1 mice is further supported by our findings in analyses of TMEM119 immunoreactivity and the branches in P2RY12+microglia. The reduced TMEM119+homeostatic microglia and decreased branches of the P2RY12+ homeostatic microglia in the cortex of aged APP/PS1 mice are reversed by SCF+G-CSF treatment. It requires further examinations to elucidate how SCF+G-CSF treatment enhances the recovery of homeostatic microglia in the brain of aged APP/PS1 mice. We propose that interventional molecules/chemicals targeting Aβ clearance and modulating microglial transition into homeostatic phenotype would be helpful therapeutic approaches to restrict pathological progression in AD.As stated earlier, resident microglia play a vital role in the maintenance of brain homeostasis. Losing the homeostatic phenotype in microglia is thought to be linked to pathological severity in the context of AD , 67. P2Rpatients , 67. In xt of AD . In mousxt of AD . These fMicroglial activation has been thought to be essential for the clearance of accumulated Aβ in the brains of transgenic mouse models of AD , 85. How+ dendrites and PSD-95+ post-synapses are decreased in 26-27-month-old APP/PS1 mice as compared to age-matched WT controls. SCF+G-CSF treatment ameliorates the loss of MAP2+ dendrites and PSD-95+ synapses in the cortex and hippocampal CA1 of aged APP/PS1 mice. The precise mechanism underlying the SCF+G-CSF-increased dendrites and synapses in aged APP/PS1 mice remains unknown. SCF+G-CSF treatment in aged APP/PS1 mice enhances Aβ removal and reduces neuroinflammation, suggesting that the SCF+G-CSF-ameliorated pathological severity in the brain of aged APP/PS1 mice may be beneficial to the maintenance of dendrites and synapses. In addition, our previous study has demonstrated a direct effect of SCF and G-CSF in supporting neurite extension. SCF+G-CSF synergistically enhances neurite extension and neural network structure formation in cultured primary cortical neurons [+ dendrites and PSD-95+ synapses in the ipsilesional cortex [+ homeostatic microglial area is positively correlated with the MAP2+ dendritic area and PSD-95+ synaptic puncta, indicating that the SCF+G-CSF-increased resting microglia and SCF+G-CSF-improved homeostasis of brain microenvironment may play supportive and reparative roles in maintaining dendrites and synapses in the brains of aged APP/PS1 mice. Finally, intracellular pathological tau aggregation in neurons has been shown to accelerate post-synapse loss in the dendrites [+ puncta) in the cortex of aged APP/PS1 mice is reduced by SCF+G-CSF treatment. Further studies are needed, however, to explore how SCF+G-CSF treatment in aged APP/PS1 mice restricts pathological tau aggregation.It has been documented that Aβ accumulation and chronic neuroinflammation-induced dendritic loss play a vital role in cognition impairments in APP/PS1 mice , 92-94. l cortex . Thus, Sl cortex . On the l cortex -104. In endrites . Aβ inteendrites , 107. Spendrites . Our finThe limitations of the present study are the lack of a biochemistry approach to validate our observations in immunohistochemistry and the lack of a control group with SCF+G-CSF treatment in age-matched WT mice to determine whether the SCF+G-CSF-ameliorated neuroinflammation and synaptic loss are the results of decreasing Aβ load.In total, the present study provides the evidence that SCF+G-CSF treatment increases Aβ clearance, promotes recovery of homeostatic microglia, decreases inflammation, reduces aggregated tau, and restricts the loss of dendrites and synapses in the brains of aged male APP/PS1 transgenic mice. SCF+G-CSF-enhanced DAM accumulation surrounding the Aβ plaques for removing pathological Aβ, together with the SCF+G-CSF-enhanced recovery of homeostatic microglia distant from the Aβ plaques for maintaining dendrites and synapses are the unique modulation processes to restrict AD pathology and promote brain repair in the context of AD. Our results indicate that the combination of two hematopoietic growth factors, SCF and G-CSF, may have therapeutic potential for AD, even at the late-stage.www.aginganddisease.org/EN/10.14336/AD.2020.0201.The Supplemenantry data can be found online at:


Otitis media with effusion (OME) is a common inflammatory disease that primarily affects children. OME is defined as a chronic low-grade inflammation of the middle ear (ME), without any signs of infection and with effusion persisting in the ME for more than 3 months. The precise pathogenesis is, however, not fully understood. Here, we comprehensively characterized and compared the host immune responses (inflammatory cells and mediators) and the overall microbial community composition (microbiota) present in matched middle ear effusion (MEE) samples, external ear canal (EEC) lavages, and nasopharynx (NPH) samples from children with OME. Haemophilus spp. and Corynebacterium propinquum in MEE was accompanied by proinflammatory mediators, whereas their presence in NPH was accompanied by anti-inflammatory mediators. For Turicella and Alloiococcus, we found exactly the opposite results, i.e., an anti-inflammatory profile when present in MEE, whereas their presence in the the NPH was accompanied by a proinflammatory profile. Together, our results indicate that immune responses in children with OME are highly niche- and microbiota-driven, but gender-based differences were also observed, providing novel insight into potential pathogenic mechanisms behind OME.Otitis media with effusion (OME) is a common inflammatory disease that primarily affects children. OME is defined as a chronic low-grade inflammation of the middle ear (ME), without any signs of infection and with effusion persisting in the ME for more than 3 months. The precise pathogenesis is, however, not fully understood. Here, we comprehensively characterized and compared the host immune responses (inflammatory cells and mediators) and the overall microbial community composition (microbiota) present in matched middle ear effusion (MEE) samples, external ear canal (EEC) lavages, and nasopharynx (NPH) samples from children with OME. Female patients had significantly increased percentages of T lymphocytes and higher levels of a wide array of inflammatory mediators in their MEE compared to that of male patients, which were unrelated to microbiota composition. The relative abundances of identified microorganisms were strongly associated with their niche of origin. Furthermore, specific inflammatory mediators were highly correlated with certain bacterial species. Interestingly, some organisms displayed a niche-driven inflammation pattern in which presence of  Otitis media (OM) is a common inflammatory condition of the middle ear that predominantly affects children. With more than 700 million cases annually, OM accounts for a majority of pediatric emergency department visits and antibiotic prescriptions worldwide . OM presPseudomonas aeruginosa, Staphylococcus aureus, Turicella otitidis, Klebsiella pneumoniae, and Haemophilus spp. in MEE, as well as in the NPH, in children with AOM , a patient group highly prone to develop chronic purulent OM  through the Eustachian tube to the ME. This route is supported by the observation that colonization of specific bacteria in the NPH is associated with increased risk of OM, and is possibly even a prerequisite for particular infections . A stronwith AOM . Remarkawith AOM . A microulent OM , 11, butulent OM , 13. Howulent OM , 15.Inflammation is a hallmark of OM and may be initiated when bacteria are recognized by the host. Thus, initiation of inflammation may be caused by infection, yet the role of microbes in perpetuating the inflammation in OME is debated . The spe21–The current literature is confounded by unclear definitions of OM/OME and is most often based on pooled samples from both ears, although the two ears possibly could differ in microbiota composition and active immunological processes at play . MoreoveHaemophilus influenzae as the most commonly identified species , followed by Staphylococcus aureus , coagulase-negative staphylococci , Alloiococcus otitidis , Moraxella catarrhalis , Streptococcus pyogenes , and Staphylococcus spp.  (Table S1). In NPH samples, M. catarrhalis  and H. influenzae  predominated, followed by Streptococcus pneumoniae , Streptococcus pyogenes , S. aureus , and Neisseria spp.  comprising in total 88.9% of all NPH samples analyzed (Table S1).Out of 53 patients eligible for the study, 36 patients were enrolled, and middle ear effusions (MEEs), external ear canal (EEC) lavage and nasopharynx (NPH) samples were collected when possible. Out of the 36 patients, nine had bilateral dry ears at the time of surgery and were hence excluded from all analyses . The median age of the remaining 27 patients was 58 months . In total, 14 patients (51.9%) were male and 13 (48.1%) were female . There was no significant difference between the age at myringotomy for boys and girls. Clinical microbiological analyses identified microorganisms in 31.5% of the MEE samples analyzed and revealed decontam R package, a total of 5,938,330 reads remained for analysis . These reads were binned into 140 OTUs, representing 73 taxonomic genera from 8 phyla. The observed number of species and the Shannon diversity index were significantly higher within NPH samples compared to those in MEE or EEC lavage samples  , as identified by the frequency method from the  samples . Furthert genera  and D. Iacterium  and D.Moraxella (5), Corynebacterium propinquum (6), Streptococcus (10), Haemophilus (4), and Dolosigranulum (9) . MEE and EEC lavage samples had overall similar microbiota profiles that were dominated by one or two of the following taxa: Alloiococcus (1), Staphylococcus epidermidis (2), Turicella (3) and Haemophilus (4) .Hierarchical clustering at the OTU level showed that NPH samples had a distinct microbiota profile, largely separated from those of MEE and EEC lavage samples, and that was characterized by high microbial diversity dominated by Staphylococcus epidermidis (2), Haemophilus (4), and Moraxella (5), which were consistently present between niches (Haemophilus (4) was present in both niches, although with a higher, albeit nonsignificant, abundance in MEE samples. In contrast, Corynebacterium propinquum (6) and Dolosigranulum (9) were significantly more abundant in NPH samples, whereas Alloiococcus (1), Turicella (3), and Turicella (60) abundances were associated with MEE samples (Staphylococcus epidermidis (2), cluster 2 with Alloiococcus (1), cluster 3 with Corynebacterium propinquum (6) and Dolosigranulum (9), cluster 4 with Haemophilus (4), cluster 5 with Moraxella (5), and cluster 7 with Turicella (3). In contrast, clusters 6 and 8 were not strongly associated with any biomarker species and are referred to here as Mixed_cluster_A and Mixed_cluster_B, respectively .Hierarchical clustering showed the presence of eight distinct microbiota profiles, which were mainly driven by the abundance of eight biomarker species  and B. Mn niches . Haemoph samples . To asse+] cells) and IL-6 concentrations than those in NPH samples . Conversely, NPH samples displayed significantly higher percentages of cytotoxic T lymphocytes (CD8+ cells) and B lymphocytes (CD19+ cells), as well as higher concentrations of the proinflammatory mediators TNF-α and IL-1β than those in MEE samples (Table S3). Memory T lymphocytes predominated over naive lymphocytes in all samples, although the percentages of naive CD8+ cytotoxic T lymphocytes were higher in MEE samples than in NPH samples (Table S3).Next, we assessed the overall inflammatory profiles (inflammatory cells and mediators) in MEE and NPH samples. MEE samples had nonsignificantly higher percentages of neutrophils (CD66b-positive  and 18 girls [50%]) with a median age of 61 months  were enrolled. Among these, nine patients (three boys [33%] and six girls [67%]) were excluded due to bilateral dry ears. The median age of the excluded patients was 70 months. See Fig. S1 for overview of the inclusion scheme and Table S1 for characteristics of the included patients. Study permission was obtained from the Research Ethics Committee in Lund (Dnr 2012/11), and all work was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each child’s legal guardians.Prior to myringotomy, the ears were rinsed from cerumen and flushed with 1 ml sterile saline. This external ear canal (EEC) lavage fluid was collected for microbiome analyses. The ear canals were subsequently washed once with 70% denatured alcohol. After incision of the tympanic membrane, the middle ear effusion (MEE) was aspirated into a sterile trap, and 1 ml sterile saline was used to clear the suction catheter. Nasopharyngeal (NPH) swabs were collected and swirled in 1 ml sterile saline. See Fig. S1B for a summary of sample collection procedure. Besides use for the analyses in this study, all MEE and NPH samples were also routinely sent to the Department of Clinical Microbiology, Skåne University Hospital, for identification of bacteria.g for 10 min. The supernatants were collected and stored at −80°C until used for analyses of inflammatory mediators. Red blood cells in the cell pellets were lysed using ammonium-chloride-potassium (ACK) lysing buffer (Thermo Fisher Scientific). The cells were washed, and the total number of cells in the samples, as well as the percentage of viable cells, was calculated by the trypan blue exclusion assay.Aliquots of all samples were immediately frozen at −80°C for subsequent microbiome analyses. Cells were collected by centrifugation at 350 × Freshly isolated cells were stained with relevant antibodies for a total of 20 min at room temperature and analyzed by flow cytometry. Due to various sample amounts (number of cells in each sample), we were not able to perform all analyses on all samples and patients. The antibodies (with dilutions in parenthesis) used were as follows: CD14-fluorescein isothiocyanate (FITC) clone M5E2 (1:10), HLA-DR-phycoerythrin (PE) clone G46-6 (1:20), HLA-DR-allophycocyanin (APC) clone L243 (1:50), CD86-PE clone IT2.2 (1:20), CD33-APC clone WM53 (1:10), CD3-FITC clone HIT3a (1:25), CD4-APC clone RPA-T4 (1:25), CD8-PE HIT8a (1:25), CD8-APC clone RPA-T8 (1:25), CD25-FITC clone 2A3 (1:10), CD127-PE clone HIL-7R-M21 (1:20), CD45RA-FITC clone HI100 (1:10), CD45RO-PE clone UCHL1 (1:10), CD19-PE clone HIB19 (1:20), CD138-APC clone MI15 (1:15), CD183-PE clone 1C6/CXCR3 (1:25), EpCAM-FITC clone EBA-1 (1:25), CD66b-PE clone G10F5 (1:20), CD64-APC clone 10.1 (1:20), CD294-FITC clone BM16 (1:25), CD209-APC clone DCN46 (1:15), CD203c-PE clone NP4D6 (1:25), CD196-PE clone 11A9 (1:20), CD161-FITC clone DX12 (1:10), and CD56-APC clone B159 (1:10), all from BD Biosciences. Cells were analyzed using an FACSCalibur flow cytometer . All analyses were performed using 7-AAD dead exclusion stain (BD Biosciences).After thawing on ice, MEE and NPH cell-free samples were analyzed using Bio-Plex and a cytometric cytokine bead array (CBA). (i) For Bio-Plex analysis, all samples were supplemented with 0.5% bovine serum albumin (BSA) (from a 10% stock) before analysis. The Bio-Plex Pro Human Cytokine 27-plex assay (Bio-Rad Laboratories) with two additional analytes was used according to the manufacturer’s instructions. The entire panel includes FGF-basic, eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF, with M-CSF and IL-18 added separately to the panel. (ii) The levels of IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70 were also assessed using a human inflammatory cytokine cytometric bead array . IL-12p70 was largely undetectable by this method and hence was excluded from further analyses.decontam R package . Data from two groups were compared by the Mann-Whitney U test, whereas data comparing more than two groups were analyzed by Kruskal-Wallis with Dunn’s multiple-comparison test. The nonparametric Spearman’s test was used for rank correlation analysis. A vegan  were generated where appropriate. A P value and a q value of 0.05 was considered significant, unless otherwise stated.For the analysis of the microbiome and its correlation with the immune response, we used R version 3.5 (R Core Team) within RStudio v1.2, primarily with the packages vegan , phylosephyloseq , metagenenomeSeq , microbicrobiome , and ggp, to test for the significance of niche and other environmental factors  on the overall microbiota composition. The optimal number of clusters was estimated using the average silhouette width and the Calinski-Harabasz index (fpc R package). To confirm with an unsupervised quantitative method which biomarker OTUs were associated with the clusters, we used a random forest algorithm (VSURF R package). Associations between the biomarker species abundance and either the NPH niche or the ME niche, as well as associations between biomarkers and anti- to proinflammatory ratio, were assessed using the metagenomeSeq package. Associations between the biomarkers and clusters were assessed on the log-transformed relative abundance of the biomarkers by linear models, corrected by niche, and followed by Tukey’s post hoc test. The “envfit” function from the vegan package was used to fit the immune response through the expression of inflammatory mediators onto the microbiota nMDS ordination. This function performs multivariate analysis of variance (MANOVA) and linear correlations for categorical and continuous variables, respectively. The significance of fitted vectors was assessed using permutation (999) of environmental variables and corrected by niche. We used a Spearman’s rank correlation to determine any correlations between cytokines and cluster biomarkers and the “correlation.heatmap” function from the microbiome R package for visualization. For Spearman’s rank correlations, a q value of ≤0.2 was considered significant  plot and hierarchical clustering dendrogram based on the Bray-Curtis dissimilarity matrix were used to visualize differences in the overall microbial community composition between niches. We performed PERMANOVA tests using the “adonis” function (nificant .


Exploring the neurochemical continuum between frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) with respect to monoamines and kynurenines in cerebrospinal fluid (CSF) and serum, may be useful to identify possible new research/therapeutic targets. Hence, we analysed monoamines and kynurenines in CSF and serum derived from patients with FTD (n = 39), ALS (n = 23), FTD-ALS (n = 4) and age-matched control subjects (n = 26), using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) with electrochemical detection (ECD) and liquid chromatography tandem mass spectrometry, respectively. We noted a shared dopaminergic disturbance in FTD and ALS when compared to CONTR, with significantly increased serum DA levels and decreased DOPAC concentrations, as well as decreased DOPAC/DA ratios in both disease groups. In CSF, significantly reduced DOPAC concentrations in FTD and ALS were observed as well. Here, a significant increase in DA levels and decrease in DOPAC/DA ratios was only found in FTD relative to CONTR. With respect to the kynurenine pathway (KP), we only found decreased HK/XA ratios, indicative for vitamin B6 status, in serum of ALS subjects compared to FTD. The dopaminergic commonalities observed in FTD and ALS might relate to a disturbance of dopaminergic nerve terminals in projection areas of the substantia nigra and/or ventral tegmental area, although these findings should first be confirmed in brain tissue. Lastly, based on the results of this work, the KP does not hold promise as a research/therapeutic target in FTD and ALS.The online version of this article (10.1007/s11064-020-03002-5) contains supplementary material, which is available to authorized users. C9ORF72, present in approximately 30–47% [Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders demonstrating genetic and neuropathologic overlap. The most common shared genetic mutation is a GGGGCC expansion in y 30–47% , 2 and 2y 30–47% , 3 of fay 30–47% . Consequy 30–47% . Next toy 30–47% . Likewisy 30–47% , 12.Although this clinicopathologic overlap has been well covered in the scientific literature, less is known about (monoaminergic) neurotransmitter disturbances in ALS. As this condition is currently treated with strategies primarily targeting glutamatergic neurotransmission and oxidative stress using riluzole and edaravone, respectively –15, offel-tryptophan (TRP), not only 5-HT and other related monoamines, but also compounds of the KP could possibly represent an interesting means to investigate the neurochemical continuum of FTD-ALS.Due to its implication in (neuro-)inflammation, the kynurenine pathway (KP) has received increasing attention in immune-related diseases such as cancer , 21, HIVC9ORF72, three patients had a GRN mutation, two subjects had a MAPT mutation and two other patients had a mutation in either TBK1 or VCP. A summary of neuropathologically confirmed cases and related genetics in the FTD group can be found in Fig. This retrospective study used CSF and serum samples derived from the NeuroBioBank of the Institute Born-Bunge (NBB-IBB no. BB190113), Wilrijk). Monoamines and metabolites were analysed in CSF samples of patients suffering from probable FTD (n = 39) , FTD-ALS90113, WiLumbar puncture was performed at the L3/L4 or L4/L5 intervertebral space to collect a total volume of 16.5 mL, which was divided across five fractions of 4.5 mL, 1.5 mL, 1.5 mL, 4.5 mL and 4.5 mL, respectively, in polypropylene vials  . Serum wg, 25 min, 4 °C). Serum and CSF were brought onto these columns, followed by centrifugation at 4 °C for 40 min at 14,000×g. The resulting CSF filtrate was diluted 1:2, and either 1:7 or 1:10, while the filtered serum was diluted 1:5 and 1:25 or 1:30. This RP-UHPLC system operated at an isocratic flow rate of 75 μL/min. The Decade II electrochemical detector was equipped with a thin layered electrochemical VT03 flow cell fitted with a glassy carbon 0.7 mm working electrode and an in situ Ag/AgCl (ISAAC) reference electrode. Samples of 5 μL were loaded with an Alexys AS 110 Autosampler. Separation was achieved using a short 15 cm Acquity Column  [An optimized and validated reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) system with electrochemical detection (ECD) was used to determine (nor)adrenaline and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). The sample preparation consisted of a purification on Amicon® Ultra 0.5 Centrifugal Filters , which were washed twice beforehand with 450 µL buffer while centrifuging  .l-kynurenine (KYN), 3-hydroxykynurenine (HK), anthranilic acid (AA), kynurenic acid, xanthurenic acid (XA), quinolinic acid, picolinic acid, and nicotinic acid, were determined at the department of Laboratory Medicine of the University Medical Center Groningen by liquid chromatography in combination with isotope dilution tandem mass spectrometry (LC–MS/MS) essentially as described by [13C-labeled internal standards of the KP compounds under investigation. These mixtures were extracted with Strata X-A 96-well plates with a pore size of 33 µm and sorbent mass of 30 mg/well  and eluted with 3 M HCl in 1-butanol. Finally, 1 µL was injected into an Acquity UHPLC  equipped with a Phenomenex Luna column , and coupled to a XEVO TQ-S MS/MS system . KYN/TRP ratios were calculated as a measure for TDO and IDO activity [Concentrations of TRP, ribed by . In shoractivity  and HK/Xactivity .One-way ANOVA was applied for continuous demographic parameters such as age and storage time. In case the assumption of homogeneity of variances was violated, Welch’s ANOVA was applied. Fisher’s Exact tests were applied to investigate the association between categorical variables such as disease category, gender and medication use.U statistics. Correction for multiple testing was applied using the Benjamini–Hochberg procedure. The results for the FTD-ALS group are presented solely as median concentrations, as the size of this group was very small and often even reduced to n = 2 or n = 3. Therefore, we opted not to include monoaminergic and kynurenergic concentrations of this group in statistical tests for group comparisons. Lastly, Spearman’s rank correlation analysis was used to assess the relationship between storage time and concentrations of monoamines and kynurenines. Again, Benjamini–Hochberg corrections were applied to account for multiple testing. All statistical analyses were performed using SPSS version 25.0 for Windows. Figures were created with GraphPad Prism version 6 for Windows .Nonparametric statistics were applied to test inter-group differences of monoamines and kynurenines due to the non-normal distribution of these parameters in CSF and serum. Kruskal–Wallis analyses were applied as omnibus tests, while post-hoc analyses were performed with Mann–Whitney Table P < 0.05 for DOPAC; H(2) = 17.963, P < 0.001 for DA; H(2) = 12.392, P < 0.05 for DOPAC/DA and H(2) = 7.252, P < 0.05 for HVA/DA) and serum (H(2) = 12.116, P < 0.05 for DOPAC, H(2) = 16.850, P < 0.001 for DA; and H(2) = 19.438, P < 0.0001 for DOPAC/DA). A general decrease in DOPAC values was observed in FTD and ALS subjects compared to the CONTR group. Increased DA concentrations in CSF were found in the FTD versus the CONTR and ALS groups. In serum, DA levels were also highly significantly increased in the FTD subjects compared to CONTR individuals, while a similar result was observed between the CONTR and ALS group. Analysis of the DOPAC/DA ratio revealed a disturbed dopaminergic catabolism in FTD versus the CONTR group, evidenced by significantly lower CSF and serum DOPAC/DA ratios in the former category. Furthermore, serum DOPAC/DA ratios were severely decreased in ALS subjects compared to healthy CONTR subjects  = 9.017,  = 9.017,P < 0.05), with significant differences after post-hoc correction indicating higher MHPG levels in FTD and ALS compared to the CONTR group  = 8.833, oup Fig. . All resP < 0.05), however, intergroup comparisons for CSF TRP with post-hoc Mann–Whitney U tests did not remain significant following Benjamini–Hochberg corrections for multiple comparisons , with lower serum HK/XA ratios in the ALS versus the FTD group  = 6.088, P = 0.056), but none of the monoaminergic differences remained statistically significant. In contrast, a significant difference was found for CSF TRP across groups (H(2) = 6.548, P < 0.05).When equivalent analyses were performed to detect differences in monoamine/kynurenine content only in subjects who were free of psychotropic medication and who did not take riluzole nor supplements containing vitamin B, a trend for increased CSF DA levels in FTD and ALS compared to CONTR could be observed (H(2) = 5.751, P < 0.0001 and rs(73) =  − 0.330, P < 0.05), whereas a positive association between storage time and 5-HIAA (rs(89) = 0.286, P < 0.05) as well as 5-HIAA/5-HT ratios (rs(87) = 0.346, P ≤ 0.001) was noted. In serum, negative correlations were noted between DA levels and storage time (rs(82) =  − 0.242, P < 0.05)). As for kynurenines, CSF TRP and serum HK/XA ratios decreased with increasing storage time (rs(81) =  − 0.232, P < 0.05 and rs(81) =  − 0.241, P < 0.05, respectively).We found that CSF MHPG and DA were negatively correlated with storage time (rs(89) =  − 0.409, 11C-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (11C-CFT), a cocaine analogue targeting DA transporters (DAT), in the putamen of FTD subjects was also observed in another study [123I-N-(3-iodopropen-2-yl)-2β-carbomethoxy-3β-(4-chlorophenyl)-tropane (123IPT), a cocaine analogue binding specifically to DATs [123IPT and decreased binding of 11C-CFT in ALS and FTD, might reflect a dysfunction of dopaminergic nerve terminals, possibly related to presynaptic reuptake mechanisms mediated by DAT. This might explain our observation of increased CSF DA levels in these disease groups. The decreased DOPAC concentrations and DOPAC/DA ratios may then be a result of impaired DOPAC synthesis due to dopaminergic neuron dysfunction. Exploring the observed dopaminergic alterations in brain tissue of FTD and ALS subjects is mandatory to assess the validity of this hypothesis.A general dopaminergic disturbance was noted in FTD and ALS compared to CONTR subjects, largely corresponding to previous reports. Single photon emission computed tomography has shown decreased frontal uptake of a dopamine 2 receptor (D2) postsynaptic ligand, as well as a relative frontal hypoperfusion in FTD versus CONTR and subjects with Alzheimer’s disease . Reduceder study . In addi to DATs . Further to DATs . Taken tl-DOPA values ≤ 5 nmol/L and 10 nmol/L, were included. We calculated DA ratios and DOPAC ratios in CSF for the disease groups included in our study versus the control group (CONTR), as well as identical ratios for the PD group versus CONTR in the previous report [It would also be interesting to compare the dopaminergic disturbance in the FTD-ALS continuum with that observed in Parkinson’s disease (PD). In a previous study on dopaminergic alterations in CSF of synucleinopathy patients , among ws report . We obses report , the PD/In this respect, it is clear that the extent of the dopaminergic disturbance is most evident in PD, with severely decreased CSF DOPAC and DA concentrations compared to CONTR. This finding of lowered CSF DOPAC and DA concentrations in PD are supported by several other studies , 41, eveThe dopaminergic alterations in serum of FTD and ALS versus CONTR subjects resembled those in CSF, which is surprising since DA nor DOPAC readily cross the blood–brain barrier (BBB) , 43. HowThe use of psychotropic medication may have influenced our findings since the dopaminergic alterations could not be detected between FTD, ALS or CONTR in a medication-free subgroup. However, given the low number of observations in this analysis (n = 3–9 per group), type II errors cannot be ruled out.The longer storage time in the CONTR group might have led to a false positive result regarding the increased MHPG and DA levels in FTD compared to the CONTR group. Thus, the absolute DA levels in CSF should be considered carefully, while the DOPAC concentrations and the dopaminergic turnover in CSF remain unaffected by storage time.Based on the therapeutic strategies applied in FTD, we expected to find serotonergic (dis)similarities, in the ALS-FTD continuum. However, our results suggest a shared dopaminergic disturbance in CSF and serum, except for CSF DA levels. This finding may shift the research in these neurodegenerative conditions to DA-based approaches.In previous studies investigating the KP in ALS, KYN levels, neuroprotective and neurotoxic KP compounds appeared to be altered in this condition , 26. It The results of subjects who did not take psychotropic medication, riluzole or vitamin B supplements did indicate altered CSF TRP levels between FTD, ALS and CONTR. It is difficult to speculate about the (patho)physiological meaning of this result, as lowered KYN/TRP ratios would indicate a downregulation of IDO/TDO activity, but no such results could be observed. In addition, no serotonergic differences were observed in FTD and ALS versus the CONTR group, indicating that TRP metabolism was not shifted towards the synthesis of serotonergic compounds in these groups. Lastly, our observation of decreased serum HK/XA ratios in ALS compared to FTD could have been influenced by storage time. Another compound of the KP for which we found significant differences, CSF TRP, was also influenced by storage time. Based on the results of this study, we suggest that the KP offers limited therapeutic value/research potential for the FTD-ALS continuum.Although the SN was shown to be affected in FTD and ALS , 49, theIn conclusion, we found that ALS and FTD share a dopaminergic disturbance in serum, while these changes are also partly present in CSF. This could possibly be caused by dopaminergic terminal dysfunction in  projection areas of the SN and VTA. The compounds of the KP did not appear to be altered in FTD or ALS.Supplementary file1 (DOCX 15 kb)Supplementary file2 (DOCX 18 kb)Below is the link to the electronic supplementary material.


Concordantly, CSF levels of SOD increased from day 1 to 7 after SAH  without becoming significantly different compared to controls . Mean plasma TAC at day 1 (d1: 77.87 ± 49.72 µmol/L) was not statistically different compared to controls . TAC remained unchanged from day 1 to 7  in plasma. TAC in CSF steeply declined from day 1 to 7 in patients with SAH becoming significantly different from controls at days 3 and 7 . Decreased SOD levels in plasma and CSF are associated with a worse neurological outcome 6 weeks  and 6 months  after SAH. Increased plasma TAC correlated with a worse neurological outcome 6 weeks (mRS: p = 0.001/GOSE p = 0.001) and 6 months (mRS p = 0.001/GOSE p = 0.001) after SAH. Conclusion: In our study, a reduction in the antioxidative enzyme SOD and elevated TAC were associated with a poorer neurological outcome reflected by mRS and GOSE at 6 weeks and 6 months after SAH. A lower initial SOD CSF concentration was associated with the late deterioration of cognitive ability. These findings support the mounting evidence of the role of oxidative stress in early brain injury formation and unfavorable outcome after SAH.Background: Subarachnoid hemorrhage (SAH) is a devastating disease with high morbidity and mortality. Hypoxia-induced changes and hemoglobin accumulation within the subarachnoid space are thought to lead to oxidative stress, early brain injury, and delayed vasospasm. This study aimed to evaluate the antioxidant status and its impact on neurological outcome in patients with aneurysmal SAH. Methods: In this prospective observational study, 29 patients with aneurysmal SAH were included (mean age 54.7 ± 12.4). Blood and cerebrospinal fluid (CSF) samples were collected on days (d) 1, 3, and 7. In addition, 29 patients without intracranial hemorrhage served as controls. The antioxidant system was analyzed by glutathione peroxidase  and total and free glutathione-sulfhydryl  in the plasma. Superoxide dismutase  and total antioxidant capacity  were measured in the serum and CSF. Clinical data were compiled on admission . Neurological and cognitive outcome , Glasgow Outcome Scale Extended (GOSE) and Montreal Cognitive Assessment (MoCA)) was assessed after 6 weeks (6 w) and 6 months (6 m). Results: Plasma levels of SOD increased from day 1 to 7 after SAH (d1: 1.22 ± 0.36 U/L; d3: 1.25 ± 0.33 U/L,  Spontaneous subarachnoid hemorrhage (SAH), a devastating form of hemorrhagic stroke, accounts for 5–7% of all strokes and is associated with an exceptionally high disease-specific burden ,2. In toRecently, early brain injury (EBI) mechanisms have been strongly linked to morbidity and mortality after aSAH . EBI comThis study aims to elucidate the interrelation of TAC, SOD, GSH, and GSH-Px measured in plasma and cerebrospinal fluid (CSF) with the neurological and cognitive outcome in patients with SAH.Patient population: From February 2017 to February 2019, 29 consecutive patients  who required an extra-ventricular drainage after aSAH were included in our study. The patients’ age ranged from 35 to 89 years (55 ± 12.4 years). An extra ventricular drain was placed on admission to our neurosurgical intensive care unit. Patients were followed-up until six months after discharge or until death occurred. CSF and clinical data were collected and analyzed prospectively. All patients received a thorough clinical examination on admission and before discharge from our hospital. Computed tomography (CT) was used to analyze hemorrhage characteristics and calculate the Fisher score. Neuromonitoring included continuous ICP measurement and transcranial doppler ultrasound. The clinical data and baseline characteristics are summarized in g at 4 °C for 5 min. The supernatant was frozen, and aliquots were stored at −80 °C until analysis.Sampling procedure: CSF was obtained through external ventricular drainage on days 1, 3, and 7 after SAH. Concordant blood samples were obtained via an arterial cannula. The CSF of controls was obtained from either lumbar drainage placed for normal pressure hydrocephalus evaluation or the intraoperative opening of CSF spaces in meningioma and schwannoma patients. Samples were collected in a sterile plastic tube. The tube was centrifuged at 1500× ® (TAS/TAC) Kit, KC5200, Immundiagnostik AG, Germany). SOD (Superoxide Dismutase Assay Kit (No. 706002) Cayman Chemical, Ann Arbor, MI, USA) and GSH-Px (Glutathione Peroxidase Kit (No. RS 504), Randox Laboratories Ltd., Crumlin, UK) were measured via photometry. GSH was measured using high-performance liquid chromatography (HPLC).Measurement of analytes in the blood and CSF: TAC was measured in the plasma and CSF using a decolorization technique, where the radical cation 2,2′–azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) was generated by reaction with potassium persulfate just before reaction with the plasma sample  with Bonferroni‘s multiple comparison post hoc test using GraphPad Prism version 8.4.2 for macOS, GraphPad Software, La Jolla, CA, USA. A value of Clinical presentation: The median GCS on admission was 14, the median Hunt and Hess Classification (H&H) was 3, and the median Fisher score was 4. Of 29 patients, 13 developed symptomatic vasospasms. In six of these patients, DCI was confirmed. Six weeks after SAH, median mRS was 3 and median GOSE was 5. After 6 months, the median mRS was 1 and GOSE was 6. The median MOCA score 6 months after SAH was 28.p = 0.95; free GSH: 67.13 ± 28.47 mg/L, p = 0.79) and controls . After SAH, no differences in the plasma levels of total GSH  and free GSH  were detectable between day 1 and days 3 or 7, respectively. Likewise, no statistically significant differences were found between GSH-Px levels measured at day 1, 3, and 7 in patients with SAH  and controls .Plasma glutathione (GSH) and GSH-Px in patients with SAH and controls: No statistically significant differences were found in total and free GSH between patients with SAH  and were significantly higher compared to controls (1.11 ± 0.27) at day 7 (p < 0.001). Concordantly, CSF levels of SOD increased from day 1 to 7 after SAH  without becoming significantly different compared to controls   .p = 0.25). TAC plasma levels remained unchanged from day 1 to 7 . In similar fashion, CSF TAC levels were not different in patients with SAH (d1: 261.7 ± 29.15 µmol/L) compared to controls  at day 1. Nevertheless, TAC levels in CSF steeply declined from day 1 to 7 in patients with SAH becoming significantly different from controls at days 3 and 7  (TAC in plasma and CSF from patients with SAH and controls: Mean plasma levels of TAC at day 1 (d1: 77.87 ± 49.72 µmol/L) were not statistically different compared to controls  .p = 0.0001; plasma p = 0.027/GOSE: CSF p = 0.001; plasma p = 0.001) and 6 months  after SAH. As the only analyte, lower SOD levels in CSF were also related to cognitive decline 6 months after SAH (MoCA p = 0.001). The antioxidative capacity in CSF after SAH showed no significant correlation with the neurological (mRS and GOSE) or cognitive outcome (MoCA) at 6 weeks and 6 months after hemorrhage. In contrast, increased plasma TAC correlated with a worse neurological outcome 6 weeks (mRS: p = 0.001/GOSE p = 0.001) and 6 months (mRS p = 0.001/GOSE p = 0.001) after hemorrhage (p < 0.0001) and 0.9615  respectively.Correlation of TAC, SOD, and GSH/GSH-Px with clinical outcome: Decreased SOD levels in plasma and CSF are associated with a worse neurological outcome 6 weeks   was statistically significantly associated with increased plasma TAC levels (mRS: = 0.001) .p = 0.045, GOSE p = 0.035/Fisher scale: mRS p = 0.065, GOSE p = 0.12). In contrast, poor initial H&H and GCS correlated with higher SOD plasma levels . CSF and plasma TAC levels on day 1 did not correlate with symptomatic vasospasms, while their occurrence was associated with decreased CSF levels of TAC at day 7 (p = 0.049).Antioxidant capacity in CSF and plasma did not correlate with impaired cognitive outcome 6 weeks and 6 months after SAH. Poor clinical condition at the time of admission  and amount of subarachnoidal blood  were not predictors of impaired TAC, but were individually associated with poor neurological outcome after 6 months  is a free radical scavenger and is supposed to contribute to elevated serum TAC levels. UA has been independently correlated with mortality in septic patients. . High UA2O2 from O2−, which is scavenged by GSH-Px to prevent the accumulation of the more dangerous OH− [−, subsequent oxidative stress, and consecutive EBI [− accumulation. In our study, the resulting increase in the SOD/GSH-Px ratio is not correlated with a detrimental neurological outcome. Still, it might contribute to impaired TAC and thus could add to neurological deficits after SAH.SOD and GSH-Px are among the most essential enzymatic scavengers during physiological cellular respiration . SOD prorous OH− . SAH learous OH− . This chtive EBI . In our tive EBI . Lower ctive EBI . A higheGiven the delayed nature of the changes leading to oxidative stress after SAH, it should come as no surprise that the initial GCS or Hunt and Hess Classification showed no correlation with decreased antioxidative capacity, but rather represent more independent predictors of an unfavorable outcome. However, higher Hunt and Hess grades correlated with increased SOD plasma levels. This increase might indicate SAH-related oxidative stress and the consequent increase in SOD activity and the SOD/GSH-Px ratio. During the time after SAH, this increase in oxidative stress might lead to an overpowering of the antioxidant defense mechanisms, which becomes evident in a declining TAC. This circumstance is highlighted by the fact that TAC concentrations within plasma and CSF at day one after hemorrhage were not associated with the occurrence of vasospasms during the curse of the disease. However, in patients with symptomatic vasospasm a significantly lower TAC was detected within the CSF at day 7. Although the occurrence of oxyhemoglobin within the subarachnoid space apparently contributes to oxidative stress, this is not reflected by the Fisher grade in our study. A possible explanation for this might be found in the lack of a more refined quantification of subarachnoid blood, rather than the small effects reflected within the antioxidant systems.However, as the outcome after SAH is heterogenous and is influenced by a plethora of different factors, the rather small number of patients is a clear limitation of our study. The presented patient collective represents rather mild types of SAH with a median GCS of 14 and a more favorable outcome. It is speculated that more severe forms of SAH might lead to more differentiated results. Nevertheless, in the light of mounting evidence implicating oxidative stress in the pathophysiology of SAH induced brain injury, targeted therapies have become the focus of continued research efforts. So far, there are promising clinical and pre-clinical results of free radical scavengers such as docosahexaenoic acid, resveratrol, apigenin, paeoniflorin, and ebselen ,39,40,41In our study, increased circulating TAC and decreased SOD plasma and CSF levels were associated with an unfavorable neurological outcome at both 6 weeks and 6 months after SAH. The initial H&H and GCS scores were correlated with higher levels of SOD in the plasma and CSF, indicating higher oxidative stress in patients with more severe bleeds. Furthermore, symptomatic vasospasm was associated with lower concentrations of TAC within the CSF 7 days after hemorrhage.


Chlamydia spp. have been detected in the class Amphibia with C. pneumoniae being the predominant species involved. Chlamydiae have been linked to mass mortality events, thereby representing significant pathogens that deserve attention with respect to worldwide amphibian decline. We here present six cases of chlamydiosis and asymptomatic chlamydial infections in different frog species from three ex situ amphibian conservation facilities. Clinical signs predominantly characterised by regurgitation, chronic wasting, lethargy and suspended breeding were associated with C. pneumoniae infection. Despite various treatment regimens, it was not possible to clear infections. However, intra vitam diagnostics succeeded from skin, faeces and urine for the first time.A number of different  Chlamydia is the sole genus in the family Chlamydiaceae and currently contains thirteen valid taxa  suffering from a severe, chronic, mononuclear pneumonia, non-regenerative anaemia and pancytopenia represents the first chlamydiosis case from the wild. This frog was found to be infected with a ‘homeothermic‘C. pneumoniae strain giving rise to the question whether amphibians may play a role for zoonotic infections  in a zoological collection , densely planted with tropical vegetation. Maintenance conditions included a twelve-hour night/day interval with temperatures between 20 and 27 °C during the day unless otherwise stated. An automatic sprinkling system was used to achieve a relative humidity above 70%.Triprion (Anotheca) spinosus, originally imported from Costa Rica housed together with eight lemur leaf frogs . The Triprion group was later transferred to the quarantine unit of zoo B. Additionally, there was a second terrarium with a group of eight captive-bred, 3-year old fringed leaf frogs (Cruziohyla craspedopus), originally imported from Peru. A third terrarium contained a group of 17 captive-bred 1–3-year old casque-headed treefrogs (Triprion petasatus). All three terraria were standing directly next to each other, but drains were closed off each by a turncock to prevent wastewater bypass. The Triprion petasatus terrarium was further equipped with an additional ultraviolet and heating device  to achieve daytime maximum temperatures of up to 35 °C. In zoos A and B, each a group of White’s tree frogs, Ranoidea (Litoria) caerulea, and long-nosed horned frogs (Megophrys nasuta), 2 females and one male, were kept without any clinical abnormalities under the same conditions like in the private collection. Zoo B also took over the group of Triprion spinosus from the private collection.The private collection comprised five captive-bred approx. 4-year old coronated tree frogs, Batrachochytrium dendrobatidis, B. salamandrivorans, ranavirus, Brucella spp. and by faecal examination for parasites and yielded—except for the Strongyloides sp.—only negative results (data not shown).All the mentioned frog species of the three collections were initially checked during quarantine and when abnormalities were observed by specific qPCRs for the presence of Chlamydia-specific immuno-histochemistry (IHC). Briefly, chlamydiae were detected by the indirect immunoperoxidase method. Tissues examined included skin, muscle, bone, eye, brain, heart, lung, liver, spleen, kidney, intestine, mesothelium, testis and ovary. For antigen retrieval, slides were incubated in 0.025% proteinase K  in phosphate buffered saline (pH 7.4) for 5 min at 37 °C. A monoclonal antibody against Chlamydia-LPS  was used as primary antibody, HRP-conjugated goat anti-mouse immunoglobulins  as secondary antibody and 3-amino-9-ethlycarbazole as chromogen. Sections were counterstained with hemalaun.A full necropsy was performed on deceased frogs from this study. For histopathological examination, small slices of multiple organs were fixed in buffered 4% formalin, processed by standard methods and embedded in paraffin. Sections were stained with hematoxylin–eosin (HE). Additional sections were further processed for 2 for 3–4 days. Tissue samples from liver, spleen and kidney were homogenised and ultrasonicated in 2–5 mL SPGA  stabilising buffer. After a 15 min centrifugation at 500 × g, 30–300 µL of the supernatants were inoculated into 4–6 Track bottles and centrifuged at 3400 × g at 37 °C for 60 min. The bottles were incubated for 2 h and EMEM was replaced by serum-free UMDCK medium (Lonza) with an antibiotic cocktail containing nystatin, gentamicin and vancomycin. Medium was renewed after 18 h. Two to 3 days after inoculation, the coverslips were fixed with methanol and cells were stained with fluorescence-labeled monoclonal antibodies from the IMAGEN Chlamydia kit (Oxoid).For chlamydia isolation, buffalo green monkey (BGM) cells in Eagle’s Minimal Essential Medium  with 2 mmol glutamine and 5% fetal calf serum were seeded into Trac bottles with coverslips  and incubated at 37 °C with 5% COBrucella selective media  as well as in enrichment broth for the isolation of Salmonella.Samples from living frogs  or various tissue samples  obtained during necropsies were directly cultured on 5% Columbia sheep blood (SBA) and water-blue metachrome-yellow lactose agar according to Gassner  at 20 °C aerobically for up to 48 h. Concomitant bacterial isolates were identified by MALDI-TOF mass spectrometry . Tissue samples were also inoculated onto For the molecular detection of chlamydiae, 250 mg tissue samples as mentioned above for bacterial cultivation were homogenised in 1 mL of phosphate buffered saline  using a FastPrep-24 Classic bead beating grinder and Lysing Matrix tubes with ceramic beads . Homogenates were centrifuged briefly to sediment tissue fragments and DNA was extracted from 200 µL supernatant via IndiMag Pathogen Kit on the IndiMag 48 Magnetic bead based nucleic acid extractor  according to the manufacturer’s instructions.Chlamydia typing was performed with a commercial qPCR assay  based on the murB gene (species level C. pneumoniae). An almost full-length 16S rRNA gene (> 1500 bp) was amplified using the primers 16S F (5′GCGTGGATGAGGCATGCAA′3) and 16S R (5′GGAGGTGATCCAGCCCCA′3) described by Everett  under default conditions  and ranavirus by PCR, respectively, employing previously published protocols  spinosus and Agalychnis lemur: The group of coronated treefrogs regularly laid fertilised clutches until summer 2018. Whereas the lemur leaf frogs did not show any clinical signs or abnormalities, the Triprion group stopped calling and breeding, lost their bright skin colouration and developed a faded and dry skin with regular ecdysis and consistently regurgitated insect prey items within 48 h of feeding. At this stage, the group was transferred to the quarantine unit of zoo B in February 2019 in order to improve breeding success.Cruziohyla craspedopus: The group regularly laid fertilised clutches until summer 2018. Similarly, frogs stopped calling and breeding with no externally visible signs like weight loss and skin abnormalities, except sporadically regurgitating insect prey items. All animals survived but without any breeding success.Triprion petasatus: As these frogs are very seasonal, calling cannot be observed throughout the year and cannot be exploited for assessment of clinical disease status. Breeding could be induced every year. The group was active, but often regurgitated insect prey items since summer 2018 and emaciated significantly. However, the group also contained subjects with an adipose nutritional status. All these frogs were still active at night. Ten frogs died, but only two were available for post mortem examinations due to high-grade decomposition; the rest of the animals survived. A low-grade nematode infestation with Strongyloides sp. was noted which turned out to be refractory to oral levamisole and ivermectin treatment regimens (data not shown).2.Zoo A:Ranoidea (Litoria) caerulea: During 2016 and 2017, 13 White’s tree frogs died. Prior to death, three animals displayed clinical signs including emaciation, oedema on legs and abdomen, neurological signs, and bloating  into the dorsal lymph sac at 5 mg/kg SC q 24 h were conducted. In the private collection, treatment baths were initially (in March 2019) carried out to treat Agalychnis lemur, Cruziohyla craspedopus and Triprion petasatus with tetracycline  and subsequently—due to repeated pathogen detection in April 2019—with enrofloxacin .In zoo B, parenteral treatments were initiated in the coronated tree frogs during first Triprion spinosus from zoo B, but not in tissues of Litorea caerulea from zoo A. One of the four Triprion spinosus examined had chlamydial inclusions in the skin, the kidney and the intestine, and one each in the kidney and the lung only. In all of these tissues, few chlamydial inclusions were present in single cells, which were most likely macrophages. Positive cells were located in the superficial dermis of skin, in the interstitium of kidney and lung, and in the lamina propria and the submucosa of the intestine  values above 30, only few samples were processed for chlamydial culture. All cultures gave negative results .Frogs from this study  in most cases. An overview of Chlamydia PCR results and sample data is presented in Table Frogs from all three collections were found to be infected with o low Ct 0–41 in mTriprion petasatus  clustered together in another clade  and ranavirus by PCR (data not shown).All samples were tested negative for the presence of chytrid fungus  it was concluded that they belong to five genotypes (A–E), from which genotypes A, B, and C are among others associated with frogs (Mitchell et al. Interestingly, further analysis particularly of the Chlamydia spp. especially C. pneumoniae, but also so far unclassified species must be considered in the differential diagnosis of infectious amphibian diseases. Hence, we observed in our study an association between clinical signs of infertility and chronic wasting and pathological findings with infections caused by chlamydial organisms in frogs. Treatment should be only initiated, if chlamydia are confirmed as causative pathogens, since current treatment options have a poor prognosis due to possible persistence of this pathogen. Although C. pneumoniae is considered a ‘monomorphic pathogen’ with high intraspecific genetic similarity and human-to-human infections are frequently observed, C. pneumoniae isolates from amphibians do not seem to have a pronounced zoonotic potential.The aim of the present study is to raise awareness about chlamydial infections in amphibians, especially frogs, and to suggest a targeted analysis in routine diagnostics. Among the


Little is known about the histology of contralateral descended testes in boys with unilaterally absent testis. We investigated whether absence of one testis is associated with abnormal tissue architecture of the solitary contralaterally descended testis.For this retrospective study, we examined the results of biopsies of the contralateral descended testis in 43 boys with monorchidism. Data from 26 control testes from boys of matching ages were selected from results published in 1977 and 2009. During surgery, any nubbins were removed. In each case, the scrotal testis was biopsied, and the testis fixed by subdartos pouch or suture.Of the 43 affected boys, 23 had normal testicular histology in the contralateral descended testis, whereas 20 (46%) had abnormal histology. Eight of the abnormal biopsies matched the criteria for high infertility risk. Samples from three boys in this latter group revealed a Sertoli-cell-only phenotype. Immunohistochemical assays were positive for steroidogenic acute regulatory (STAR) protein in Leydig cells and spermatogonia. STAR expression was stronger in the monorchid group with normal testicular histology.Almost half of the patients with unilateral absent testis were at risk for subfertility or infertility. Our results emphasize the need for testicular biopsy of the solitary testis in boys with monorchidism to appropriately assess infertility risk. A unilaterally absent testis (monorchidism) is thought to be caused by prenatal or antenatal testicular torsion. The absent testis may be explained either by a twisted undescended gonad or twisted normal gonad that vanished because of events following complete descent. Therefore, vanishing testes and their descended counterparts may not be subject to the endocrinopathy that affects cryptorchid testes because torsion, the probable cause of monorchidism, is thought to occur along the path into the scrotum . Of noteFollowing approval by our institutional ethical committee, we conducted a retrospective database review that identified 43 boys who had undergone surgery for non-palpable testis. Inclusion criteria consisted of a preoperative diagnosis of a nonpalpable undescended testis and either the presence of a testicular nubbin or absent testis with blind-ending gonadal vessels and vas deferens at surgical exploration. During surgery, all testicular remnants (nubbins) detected were removed. Vanishing testes were confirmed during inguinal surgeries. In addition, the scrotal descended testis was biopsied in each case and fixed by subdartos pouch or suture.Boys with monorchidism with or without a nubbin that completely lacked testicular tissue were included in the study. Investigated patients neither had chromosomal anomalies nor “re-do” surgeries performed earlier in life. Four boys with normal and 3 boys with abnormal testicular histology were hormonally treated prior to the surgery. All tissue samples were immediately fixed in 2% glutaraldehyde in phosphate buffer, embedded in epoxy resin, sectioned at a thickness of 1 μm, and stained with toluidine blue. Histological analysis was performed using light microscopy at 60× total magnification. For each biopsy, at least 100 seminiferous tubules were examined. Histological data included the total number of germ cells, the number of A dark (Ad) spermatogonia, and the presence of primary spermatocytes. Exclusion criteria were age > 18 years and missing data for any of the histological variables. Germ cell counts from the contralateral descended testis group and from a historical control group were compared. For the latter, data from 26 control testes, most of them obtained post-mortem, were selected from data published and age-matched .16 testicular biopsies were analyzed (8 in each group). Fresh performed slides were blindly evaluated by two examiners experienced in immunohistology.Immunohistochemistry was performed for steroidogenic acute regulatory (STAR) protein which is important in steroid hormone synthesis. It enhances the metabolism of cholesterol into pregnenolone by mediating the transfer of cholesterol to the inner mitochondrial membrane . RNA-binFor immunohistochemical analysis, epon was removed from the tissue sections. The sections were treated with 2% bovine serum albumin to reduce non-specific binding and then incubated with the primary antibody overnight at 4 °C. All samples were washed with phosphate-buffered saline between incubations. We used an antibody targeting the steroidogenic acute regulatory (STAR) protein , labeled with horseradish peroxidase-polymer  to detect the binding of the primary antibody. The chromogenic reaction was developed by adding a freshly prepared solution of 3,3-diaminobenzidine solution . The DAB reaction was terminated by washing in Tris-buffered saline . To allow visualization of testicular cells, the samples were counterstained with toluidine blue. Antibody binding was indicated by a brown precipitate. Different cell types were identified based on their nuclear morphology and position within the gonad. Immunohistochemistry experiments were performed at least three times on samples from at least eight patients from each group, and only those with identical results between experiments for each sample were included in the study. Controls for non-specific binding of the secondary antibody were performed in all experiments by omitting the primary antibody; these experiments consistently yielded no signal within the seminiferous epithelium or the interstitial space. Experimental design, biomaterials and treatments, reporters, staining, imaging data, and image characterization were performed in compliance with the minimum information specification for immunohistochemistry experiments .p < 0.01 was considered to indicate significance.The software package StatXact 6.30  was used to apply the Mann–Whitney U test of unpaired data. In addition, Fisher’s exact test was applied, and Biopsy data were available for 43 prepubertal patients with absent unilateral testes. The mean total spermatogonia count was 2.3 ± 1.5 , and the mean Ad spermatogonia count for the whole group was 0.127 ± 0.129 (95% CI 0.008–0.16) per tubular cross section. Boys with monorchidism whose testis showed normal histology had a mean age of 2.79 ± 3.8 (95% CI 0.96–4.62) years at the time of surgery, which did not differ significantly from that of boys with monorchidism and abnormal testicular pathology (2.8 ± 1.85 [95% CI 1.58–4.08] years).n = 23) of the patients in our cohort showed normal testicular histology. The total mean count for germ cells was 2.9 ± 1.41 (95% CI 2.3–3.5) per tubule, whereas for Ad spermatogonia it was 0.21 ± 0.12 (95% CI 0.15–0.26) per tubule  of the affected boys. Mean values for both total germ cell count (1.18 ± 1.04 [95% CI 0.6–1.6] germ cells/tubule) and Ad spermatogonia (0.003 ± 0.002 [95% CI 0.0018–0.0035] per tubular cross-section) were reduced (Fig. We did identify different degrees of testicular pathology in 46% (STAR immunoreactivity was observed exclusively in the cytoplasm in a granular pattern regardless of the tissue type studied. STAR was found in germ cells and Leydig cells. It was strongly expressed in testes with normal histology [4+] but only weakly so [1+] in testes with abnormal histology Figs.  and 3.FIn one study covering cases from 1985 to 1991, absent testes were diagnosed in 10% of 1225 patients examined for cryptorchidism . PatholoThe monorchid testis in boys with a unilateral testicular nubbin is generally believed to be normal and leave prospective fertility intact , 5. Of n\Few reports have described the quality of the testicular tissue of the contralateral descended testis from boys with a unilateral testicular nubbin , 4. KrafIn this study, we found that 46% of the patients with a congenitally absent testis on one side have an elevated risk of infertility. Therefore, boys with an absent testis should be examined by a contralateral testicular biopsy at the time of elective fixation of the solitary testis. The result will justify the application of a therapeutic regime to reduce the risk for impaired fertility in these boys. In cases in which a biopsy is not performed, the family should be advised that a sperm quality assessment should be sought for the patient in adulthood.