COVID-19 is a clinical syndrome ranging from mild symptoms to severe pneumonia that often leads to respiratory failure, need for mechanical ventilation, and death. Most of the lung damage is driven by a surge in inflammatory cytokines . Blunting this hyperinflammation with immunomodulation may lead to clinical improvement. GM-CSF is produced by many cells, including macrophages and T-cells. GM-CSF-derived signals are involved in differentiation of macrophages, including alveolar macrophages (AMs). In animal models of respiratory infections, the intranasal administration of GM-CSF increased the proliferation of AMs and improved outcomes. Increased levels of GM-CSF have been recently described in patients with COVID-19 compared to healthy controls. While GM-CSF might be beneficial in some circumstances as an appropriate response, in this case the inflammatory response is maladaptive by virtue of being later and disproportionate. The inhibition of GM-CSF signaling may be beneficial in improving the hyperinflammation-related lung damage in the most severe cases of COVID-19. This blockade can be achieved through antagonism of the GM-CSF receptor or the direct binding of circulating GM-CSF. Initial findings from patients with COVID-19 treated with a single intravenous dose of mavrilimumab, a monoclonal antibody binding GM-CSF receptor α, showed oxygenation improvement and shorter hospitalization. Prospective, randomized, placebo-controlled trials are ongoing. Anti-GM-CSF monoclonal antibodies, TJ003234 and gimsilumab, will be tested in clinical trials in patients with COVID-19, while lenzilumab received FDA approval for compassionate use. These trials will help inform whether blunting the inflammatory signaling provided by the GM-CSF axis in COVID-19 is beneficial. Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a clinical spectrum ranging from asymptomatic/pauci-symptomatic forms to severe pneumonia leading to respiratory failure, need for mechanical ventilation, and death . TherefoIn a recent report, patients with COVID-19 needing intensive care unit (ICU) admission showed a cytokine profile similar to that of secondary hemophagocytic lymphohistiocytosis with increased levels of several inflammatory cytokines  . AdditioData supporting the role of hyperinflammation in sepsis-related acute respiratory distress syndrome (ARDS) are derived from a sub-group analysis of a phase 3 randomized controlled trial of IL-1 receptor antagonist (anakinra), which showed significant survival benefit in patients treated with anakinra compared to placebo . IL-1β iin vivo activation of CAR T-cells  and an IL-1 receptor blocker  improve COVID-19 pneumonia outcomes. The identification and treatment of hyperinflammation using existing therapies with understood safety profiles that are either in clinical development or approved for other indications represent a valid option to cope with the immediate need to reduce the rising mortality of COVID-19.in vitro  are essential to clear respiratory microbes , 53, andCSF2RA or CSF2RB, the genes encoding the GM-CSF-Rα and GM-CSF-Rβ chains  because AMs require GM-CSF to differentiate . The pooβ chains .+ T lymphocytes were rapidly activated in the lung to pathogenic T helper (Th) 1 cells and generated GM-CSF and IL-6. This potent pro-inflammatory environment strongly induced CD14+CD16+ monocytes, which also released GM-CSF and IL-6, further worsening the cytokine storm. These aberrant and numerous GM-CSF+-IL-6+ cells may enter the lungs and explain the detrimental actions provided by hyperinflammation in the most severe and even fatal cases , sargramostim and molgramostim, were investigated in humans –67. Sarge events . The stue events . In anote events .In the early phases of viral infections, GM-CSF's role may be protective as it helps limit virus-related injury. For this reason, an inhaled formulation of sargramostim is being tested in patients with COVID-19-related acute hypoxic respiratory failure (NCT04326920).In later stages of COVID-19, the severity of the illness appears to be driven by the inappropriate release of several cytokines, such as IL-6 and GM-CSF. These mediators are involved in the inflammatory lung injury, predisposing patients to respiratory failure and eventually ARDS. Therefore, inhibition of GM-CSF signaling may be a reasonable treatment in this stage of disease. This is supported by pre-clinical data in CRS showing that GM-CSF blockade reduced CAR T-cell therapy-related toxicity by preventing CRS development without affecting its therapeutic activity .post-hoc analysis of these studies has shown that the administration of mavrilimumab was associated with a significant downregulation of the macrophage-derived chemokine C-C motif chemokine ligand 22 (CCL22) and IL-6  of mavrilimumab-treated patients by day 28 compared to 27% (n = 7/26) of control-group patients  are in development and are currently under investigation mainly for the treatment of RA. The principal clinical trials both completed and ongoing are described in Because GM-CSF is a key mediator in pulmonary homeostasis, there is the theoretical concern that inhibition of GM-CSF signaling by either binding to GM-CSF or blocking the receptor may result in dysfunctional AMs, leading to PAP and development of new infections. Fortunately, there has yet to be a case of PAP reported with the use of anti-GM-CSF monoclonal antibodies. This may be due to the fact that patients with autoimmune PAP have to reach a “critical threshold” of neutralizing antibodies to develop the disease, and the doses currently being utilized in clinical trials may not reach this threshold . This maAs COVID-19 pneumonia is likely to be aggravated by a cytokine storm, immunomodulation gained importance as a possible therapeutic strategy to this disease. A wealth of IL-6 and IL-1 blockade trials are ongoing and results are awaited. However, an approach targeting hyperinflammation upstream of IL-1 and IL-6 as well as neutrophils and macrophages may be envisioned through GM-CSF signaling. GM-CSF is an immunomodulatory cytokine that may help to clear respiratory microbes by stimulating AMs. A clinical trial with a hrGM-CSF, sargramostim, will be conducted in COVID-19 patients with the rationale that it may help clear the SARS-CoV-2 earlier in the disease course. However, in the later phase of COVID-19 lung injury, the marked elevation in GM-CSF levels as part of the cytokine storm during the onset of COVID-19 pneumonia suggests that GM-CSF may actually be deleterious at this stage of the disease. Blocking GM-CSF signaling could therefore be an effective therapeutic strategy by reducing the cytokine storm, which leads to the progression of acute respiratory failure in patients with hyperinflammation. Multiple clinical trials with inhibition of the GM-CSF pathway are either ongoing or under development.AB, AV, and AA conceived the manuscript. AB and AV drafted manuscript, figure, and tables. TW, EL, PC, BC, PR, KH, LK, BV, LD, and AA critically revised the manuscript. All authors approved the final version.AB and AV received a travel grant from Kiniksa Pharmaceuticals Ltd. to attend the 2019 AHA Scientific Sessions . TW was on a research advisory board for GSK. PC was on a scientific advisory board for Kiniksa Pharmaceuticals. AA has served as a consultant to Kiniksa Pharmaceuticals Ltd. The remaining 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.


GDAP1. In our approach, the wild-type and pathogenic variants of human GDAP1 gene were expressed in yeast. Then, a growth rate and mitochondrial morphology and function of GDAP1-expressing strains were studied. Also, the mutant GDAP1 proteins localization and functionality were assessed in yeast. We have shown, that GDAP1 was not only stably expressed but also functional in yeast cell, as it influenced morphology and function of mitochondria and altered the growth of a mutant yeast strain. What is more, the various GDAP1 pathogenic sequence variants caused the specific for them effect in the tests we performed. Thus, the proposed model is suitable for validating the pathogenic effect of known GDAP1 mutations and may be used for testing of unknown sequence variants found in CMT patients.The question of whether a newly identified sequence variant is truly a causative mutation is a central problem of modern clinical genetics. In the current era of massive sequencing, there is an urgent need to develop new tools for assessing the pathogenic effect of new sequence variants. In Charcot-Marie-Tooth disorders (CMT) with their extreme genetic heterogeneity and relatively homogenous clinical presentation, addressing the pathogenic effect of rare sequence variants within 80 CMT genes is extremely challenging. The presence of multiple rare sequence variants within a single CMT-affected patient makes selection for the strongest one, the truly causative mutation, a challenging issue. In the present study we propose a new yeast-based model to evaluate the pathogenic effect of rare sequence variants found within the one of the CMT-associated genes,  In the past, molecular genetic testing was limited to single genes. Nowadays, whole exome sequencing (WES) is applied, the result of which is a series of DNA sequence variants even for the Mendelian diseases . For exaGDAP1 (ganglioside induced differentiation associated protein 1)   .600) was determined and cultures were diluted with water to OD600 ∼1. Subsequently, aliquots of 10-fold serial dilutions of cells were spotted on solid media plates (as indicated) supplemented as indicated. Plates were incubated at 28 °C for the indicated number of days.Yeast were grown at 28 °C or 30 °C in YPD medium , YPG medium  or YPG + 0.2% glucose, in minimal synthetic medium with glycerol ) or in complete synthetic medium (SC) ) either solid or liquid. For growth tests, mitochondrial observations and mitochondrial DNA escape assays, a mixture of several yeast colonies, obtained after transformation, were grown overnight in liquid media. For growth tests the optical cell density  as a template and primers providing restriction sites  and additional codon stop for GDAP1 m1 and ligation obtained alleles into p425-PTDH3 vector. The m2 , m3 , m4 , m5 , m6 , m7 , and m8  mutations were introduced by site directed mutagenesis into cDNA GDAP1 gene.The p425-PGDAP1 (NM_018972) Human Untagged Clone, using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme) in accordance with the manufacturer’s instructions.Site directed mutagenesis was performed on the OriGene plasmid: pCMV6-XL5 with Primers for mutagenesis were designed using Quick Change Primer Design (Agilent) on-line software  fluorescence microscope equipped with a DS-5Mc camera (Nikon). Images were collected using Lucia General 5.1 software . The same fields were viewed by differential interference contrast (DIC) optics.To observe the localization of GDAP1, yeast cells were grown overnight in SC-leu-his media at 28 °C and shifted into SC gly-leu-his for 4 h. Cells were fixed by incubation for 25 min in 3.7% formaldehyde. They were collected by centrifugation and washed four times in buffer B . Then they were resuspended in buffer B supplemented with 0.2 mg/mL Zymolyase 20T  and 1% 2-mercaptoethanol and incubated for 1 h at 37 °C. The resulting spheroplasts were washed twice in buffer B and were spotted onto polylysine-coated slides. They were permeabilized by incubation in buffer F  supplemented with 0.1% Triton-X-100 for 15 min. The slides were rinse with buffer F and saturated in buffer F for 1 h. Then they were incubated with rabbit polyclonal anti-GDAP1 antibody  diluted 1:200 in buffer F for 2 h. They were washed with buffer F and incubated with TRITC-conjugated porc anti-rabbit IgG antibody  diluted 1:60 for 2 h. Then slides were rinsed with buffer F and nucleus was stained by DAPI  for 2 min. After being washed with water, slides were mounted in mounting medium (DAKO). Samples were viewed using LSM 780 Axio Observer Z.1 confocal microscope . Images were collected using Zen 2012 black edition software (Zeiss). The confocal microscopy observations were performed in Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Centre, PAS.Yeast cells were grown overnight at 28 °C in SC -leu medium. Protein extracts were prepared after disrupting cells with acid-washed glass beads in 2 × urea electrophoresis sample buffer  supplemented with protease inhibitor cocktail . Samples were analyzed by standard Western blotting using rabbit polyclonal anti-GDAP1 (Abcam) or rabbit polyclonal anti-Histone H3 (Abcam) antibodies and secondary anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibodies (Sigma-Aldrich), followed by enhanced chemiluminescence .GDAP1 variants. The transformants were grown in minimal synthetic medium with glycerol-leu overnight and aliquots were spread on SC-trp or diluted and spread on YPD to count colony forming units. The percentage of cells able to grow on SC–trp was calculated for each type of transformants.DNA escape from mitochondria to the nucleus was assayed as in . BrieflyCells were grown overnight in YPG medium, diluted, and spread on a medium containing glycerol (non-fermentable) as the main source of carbon and with a limited amount of fermentable glucose (0.2%). We counted the fraction of small colonies (the respiratory deficient) whose growth was limited due to reduced levels of available fermentable glucose in relation to the total number of colonies.GDAP1 gene sequence variants, we have selected the eight most representative for CMT4A disease in the Polish families diagnosed and observed for at least 5 years in the Warsaw Neuromuscular Unit . The main selection criterion was our detailed knowledge of the phenotypes associated with each mutation that have been extensively characterized in our laboratory [GDAP1 m1 to m8 (GDAP1 gene mutations. It was shown that the GDAP1 m4 mutation (Glu222Lys) is inherited in both dominant and recessive ways. The last mutation, that we called GDAP1 m1, results in a C-terminal truncation of the GDAP1 protein (to 287 amino acid residues). This mutation is representative for the two recurrent nonsense GDAP1 gene mutations i.e. Glu163X and Ser194X  affected the growth of yeast cultures on rich YPD  were also stably expressed in wild-type yeast cells , resulted in GDAP1 proteins that did not localize to mitochondria  genome. Such yeast is unable to grow without tryptophan supplementation. During the propagation of such yeast, mtDNA escapes from the mitochondria to the nucleus where it integrates and complements the defective nuclear TRP1 allele allowing cells to grow on the media without tryptophan [In the previous experiment we have shown that expression of the cDNA from the human reported ,14. Takireported ,28, we dreported . Thus, wl genome ,30 in a yptophan ,31.GDAP1 gene mutation. The greatest mitochondrial DNA escape rate was observed when the cDNA of GDAP1 m5 (resulting in the Leu239Phe substitution) was expressed (GDAP1 m2 (Gly327Asp substitution within the TMD). The effect of expressing other GDAP1 variants was not strong. There were no statistically relevant differences in mtDNA escape rates between cells bearing empty plasmid or cDNA of the GDAP1 m1, m3, m4, m6, m7, and m8 variants. Altogether, these results show that although the expression of GDAP1 does not affect the growth of yeast cells, it does affect their physiology, in this instance the mitochondria. The changes are so extensive that they can be observed. This supports the idea of using a yeast model to study pathologies caused by mutations in the GDAP1 gene.The idea of this experiment may be summarized by the following simple association: more extensive abnormal mitochondria leads to a greater rate of DNA escape and reflects the severity of the xpressed B. A modeGDAP1 caused a significant increase in the number of cells which are unable to grow on non-fermentable carbon source containing media (p < 0.05) and thus are respiratory deficient  and was not altered by GDAP1 m3, m4, and m5 variants  and loss of mitochondrial DNA suppress this accumulation [GDAP1 or its variants which results accumulation of Rho- cells could be helpful for growth of csg2Δ at stress conditions. We found that the tunicamycin and calcium ion (Ca2+) sensitivity of the csg2Δ strain is suppressed by expression of wild-type the GDAP1 allele  mutation reported in numerous patients. However, several mutations are poorly documented. For example, the GDAP1 m6 (His123Arg) mutation was found only in two families. Thus, testing of pathogenicity is even more necessary in instances such as the GDAP1 m6 variant [GDAP1 m4 (Glu222Lys) mutation. The GDAP1 m4 mutation has been shown by us to segregate with an extremely mild phenotype in the case of autosomal dominant CMT but in combination with the GDAP1 m5 (Leu239Phe) mutation it causes severe CMT with autosomal recessive traits [GDAP1 m5 mutation that resulted in the highest observed level of mtDNA escape. This may indicate a separate and specific mechanism of action for the GDAP1 m5 mutation. The GDAP1 m8 (Ala156Gly) mutation, which segregates with a CMT with dominant inheritance traits and a moderate clinical pathology, also results in a moderate pathogenic effect in our yeast growth assay. In our study, the GDAP1 m4 (Glu222Lys) mutation has the lowest pathogenic effect  may generate an average of 2 or 3 variants within CMT genes with ambiguous pathogenic effect [The model established here is shown to be suitable for validating the pathogenic effect of known


To study intrathecal B-cell activity in leucine-rich, glioma-inactivated 1 (LGI1) antibody encephalitis. In patients with LGI1 antibodies, the lack of CSF lymphocytosis or oligoclonal bands and serum-predominant LGI1 antibodies suggests a peripherally initiated immune response. However, it is unknown whether B cells within the CNS contribute to the ongoing pathogenesis of LGI1 antibody encephalitis.Paired CSF and peripheral blood (PB) mononuclear cells were collected from 6 patients with LGI1 antibody encephalitis and 2 patients with other neurologic diseases. Deep B-cell immune repertoire sequencing was performed on immunoglobulin heavy chain transcripts from CSF B cells and sorted PB B-cell subsets. In addition, LGI1 antibody levels were determined in CSF and PB.Serum LGI1 antibody titers were on average 127-fold higher than CSF LGI1 antibody titers. Yet, deep B-cell repertoire analysis demonstrated a restricted CSF repertoire with frequent extensive clusters of clonally related B cells connected to mature PB B cells. These clusters showed intensive mutational activity of CSF B cells, providing strong evidence for an independent CNS-based antigen-driven response in patients with LGI1 antibody encephalitis but not in controls.Our results demonstrate that intrathecal immunoglobulin repertoire expansion is a feature of LGI1 antibody encephalitis and suggests a need for CNS-penetrant therapies. Indeed, very little is known about B cells that participate in the autoimmune response against LGI1, either in the periphery or CSF. Here, we applied deep B-cell immune repertoire sequencing (DIRS) on sorted peripheral blood (PB) B-cell subsets and CSF and found strong evidence for intrathecal antigen-driven immune responses in patients with LGI1 antibody encephalitis. These observations inform disease biology and suggest CNS B cells as a candidate therapeutic target in these patients.Although CSF LGI1 antibodies are detected in around 90% of cases, this condition is infrequently associated with CSF lymphocytosis or oligoclonal bands.8 As controls, 2 patients with other noninflammatory neurologic diseases from the same center were included in the study and their PB and CSF samples collected accordingly.Six patients with LGI1 antibody encephalitis from the University of California, San Francisco (UCSF) Autoimmune Encephalopathy Clinic underwent collection of paired PB (40 mL) and 10–25 mL of CSF. B-cell subsets were isolated as described previously.The study was approved by the Institutional Review Board of the UCSF. Written informed consent was obtained from all participants in the study.8 B-cell subsets were sorted using a FACS Aria III  into naive (CD19+IgD+CD27−), unswitched memory (CD19+IgD+CD27+), switched memory (CD19+IgD−CD27+CD38−), double negative (CD19+IgD−CD27−), and plasmablasts/plasma cells (CD19+IgD−CD27hiCD38hi). Sorted cells were immediately lyzed in RLT buffer  and stored at −80°C. To preserve the far lower CSF lymphocyte frequencies, unfractionated pelleted CSF cells were studied.Ficoll-density separated peripheral blood mononuclear cells were stained with the following antibodies: CD19 (APC Cy7), immunoglobulin D (IgD) (PE Cy7), CD27 (Qdot605), CD38 (PerCP Cy5.5), and CD3 (Pacific blue) as previously described.9 with modifications to sequence human samples. In brief, total RNA was isolated from CSF cells and PB B-cell subsets, followed by reverse transcription into complementary DNA (cDNA). Next, immunoglobulin G (IgG) heavy chain variable region (VH) and immunoglobulin M (IgM) VH were amplified by PCR using the following primers: IgG 3′ primer: 5′-GGGAAGACSGATGGGCCCTTGGTGG-3′; IgM 3′ primer: 5′-GCTCGTATCCGACGGG-3′; an equimolar mix of 7 VH family 5′ primers: VH1: 5′-GAARRTYTCCTGCAAGGYWTC-3′; VH2: 5′-CACRCTGACCTGCACCKTCTC-3′; VH3: 5′-KARACTCTCCTGTRCAGCCTB-3′; VH4: 5′-GTCCCTCACCTGCRCTGTCTM-3′; VH5: 5′-GARGATCTCCTGTAAGGGTTC-3′; VH6: 5′-CTCACTCACCTGTGCCATCTC-3′; VH7: 5′-GAAGGTTTCCTGCAAGGCTTC-3′. PCR conditions were (1) 95°C, 60 seconds; (2) 95°C, 30 seconds; 66.6°C, 30 seconds; 72°C, 60 seconds (33 or 45 cycles); and (3) 72°C, 7 minutes. Specific PCR products were gel purified and mixed to create 15 pM cDNA libraries, which were analyzed by Ion Torrent semiconductor sequencing.Sequencing work flow was performed as previously described,9 Briefly, CDR3 amino acid sequences were determined using a custom-made pipeline adapted from the AbMining tool,10 and identified CDR3 regions were related to IGHV and IGHJ germline genes using IgBlast. To calculate SHM profiles, sequencing reads with identical CDR1 to CDR3 nucleotide sequences were grouped as nonredundant (unique) reads, and SHMs were quantified for this entire region based on the alignment of reads with germline gene segments.IGHV and IGHJ gene segment usage, complementarity determining region (CDR)1-3 amino acid sequence, and number of somatic hypermutation (SHM) events were determined as previously described.8 Briefly, clonally related Ig-VH sequences were identified based on H-CDR3 similarity (hamming distance of H-CDR3 amino acid sequence less than 2) and usage of identical Ig germline segments. For lineage analysis, only Ig-VH sequences with in-frame H-CDR3 and which spanned at least from the 5′ end of H-CDR1 to the 3′ end of H-CDR3 with a contiguous reading frame were used; IgTree11  was used to map the lineage.8 Putative germline nodes are inferred, and lineage intermediates not found by DIRS were calculated by IgTree.Compartmental connectivity via bicompartmental clustering of IgM-VH or IgG-VH from CSF and PB B-cell subsets was performed as previously described.All next-generation sequencing data and computer code other than software packages are available from the corresponding author on reasonable request.12 None of the 6 patients showed a CSF lymphocytosis or oligoclonal bands on routine CSF analysis. There was no abnormal enhancement on brain MRI in any of the patients following the administration of gadolinium, consistent with no substantial blood-brain barrier (BBB) opening. DIRS of IgG-VH and IgM-VH was performed from paired CSF and B-cell subsets sorted from peripheral blood mononuclear cell samples of the 6 LGI1 antibody encephalitis patients, and a median of 600,707 sequences per sample  were generated. As expected, circulating naive B cells had limited somatic mutations and as cells acquired the postgerminal center marker CD27, and class-switched to IgG, mutations accumulated  unique sequences. In this analysis, each unique sequence was represented by a dot and a line joining neighboring sequences represented a mutational (Hamming) distance of 1 in their CDR3 region . By defiIn contrast to all LGI1 antibody encephalitis cases, 2 patients ultimately diagnosed with noninflammatory neurologic diseases (1 with headache and 1 with chronic migraine), which were analyzed following the same protocol, did not show comparable intrathecal B-cell activity. In 1 of the 2 controls, we were unable to amplify enough CSF IgG RNA for sequencing, indicating very few CSF IgG transcripts; in the other, we were able to detect 43 unique sequences in the CSF, which were parts of shared CSF/PB clusters compared with 990.3  in LGI1 antibody encephalitis. Shared clusters in the control were overwhelmingly PB dominated and had on average 1.2 CSF IgG sequences (range 1–3), whereas shared clusters in patients with LGI1 antibody encephalitis had larger CSF fractions with a mean of 8.1 CSF IgG sequences .Taken together, DIRS demonstrated high sensitivity in detecting an intrathecal B-cell response and marked interconnectivity between the PB and CSF compartments in LGI1 antibody encephalitis, particularly of the more mature B-cell subsets. However, the antigen specificity of the clonally expanded intrathecal B cells remains unclear. Peripheral B-cell expansions infrequently reached the CSF, but those which did commonly lead to intrathecal B-cell expansions, consistent with secondary intrathecal B-cell receptor (BCR) diversification in LGI1 antibody encephalitis but not in noninflammatory controls.Here, we examined the potential paradox between the limited detectable CSF activity in routine clinical assessments , alongside the presence of pathogenic CNS-active autoantibodies in patients with LGI1 antibody encephalitis. For the first time, using DIRS, we demonstrate highly active intrathecal B-cell activity in LGI1 antibody patients and show that B-cell repertoires, particularly from postgerminal center B cells on both sides of the BBB, may actively mutate and mature in patients with LGI1 antibody encephalitis.Because, for methodological reasons, our study could not address the antigen specificity of the intrathecal B-cell response, it remains to be determined whether these expanded clones recognize LGI1-specific epitopes, and it is likely that some of these represent non–LGI1-specific B cells. LGI1-specific CSF B cells are more likely to exist in the patients in which intrathecal LGI1 antibodies were detectable. However, it cannot be excluded that LGI1 antibodies have passively diffused to the CSF.13 and accordingly, we did not observe this phenomenon in noninflammatory controls. Yet, these observations appear different to the more limited intrathecal expansions observed in neuromyelitis optica spectrum disorder and the very low mutational load in CSF B cells from patients with NMDA receptor (NMDAR) antibody encephalitis.15 Hence, it may be that several different immunologic mechanisms operate across these autoantibody-mediated conditions. Our study did not aim to examine the antigen-specific population or correlate intrathecal B-cell responses with clinical outcome. Of interest, it has recently been observed that intrathecal LGI1 antibody synthesis correlates with a poorer prognosis.16 However, the lack of CSF LGI1 antibodies in some of our patients with striking intrathecal SHM may suggest that DIRS provides a more sensitive measure of B-cell activity.In our studies, we found PB plasmablast/plasma cell IgG-VHs, which underwent SHM and were related to expanded clusters of CSF B cells. This pattern closely resembles findings in MS, suggesting that it may be a generic mechanism across CNS autoimmune conditions,17 However, we also found evidence for extensively activated IgM-expressing peripheral B cells, which share similar or identical BCR heavy chains to intrathecal IgG-expressing B cells. The immunopathologic relevance of this IgM response is unknown, but may indicate an ongoing germinal center reaction-mediated stimulation of B cells as has been proposed in patients with NMDAR antibody encephalitis, where IgM NMDAR-reactive autoantibodies can be detected.19In general, the majority of autoimmune encephalitis syndromes are considered to be IgG mediated.20 Our findings suggest that plasmablasts and plasma cells (which do not express CD20) are active in the CSF, and therefore, novel drugs that actively target these prolific protein synthesizers in the CNS compartment might be more effective than selective anti-CD20 treatments. Conversely, given that affinity maturation is an ongoing process in LGI1 antibody encephalitis and could result in increasingly efficient pathogenic antibodies, therapeutic depletion of CD20-expressing B-cell populations early in this disease might prevent development of chronic or progressive clinical phenotypes. Accordingly, early diagnosis of intrathecal immune activation in patients with mild clinical signs would be highly desirable.A major question is whether interrupting these responses could potentially mitigate disease activity, prevent relapses, and improve long-term cognitive outcomes. Yet, only a subset of patients with LGI1 antibody encephalitis have been reported to respond favorably to rituximab, which targets—mainly peripheral—CD20-expressing B cells and has little effect on LGI1 antibody production.


Borrelia spirochaete via phagocytosis, oxidative bursts, and hydrolytic enzymes. However, their importance in Lyme neuroborreliosis (LNB) is unclear. Neutrophil extracellular trap (NET) formation, which is associated with the production of reactive oxygen species, involves the extrusion of the neutrophil DNA to form traps that incapacitate bacteria and immobilise viruses. Meanwhile, NET formation has recently been studied in pneumococcal meningitis, the role of NETs in other central nervous system (CNS) infections has previously not been studied. Here, cerebrospinal fluid (CSF) samples from clinically well-characterised children (N = 111) and adults (N = 64) with LNB and other CNS infections were analysed for NETs (DNA/myeloperoxidase complexes) and elastase activity. NETs were detected more frequently in the children than the adults (p = 0.01). NET presence was associated with higher CSF levels of CXCL1 (p < 0.001), CXCL6 (p = 0.007), CXCL8 (p = 0.003), CXCL10 (p < 0.001), MMP-9 (p = 0.002), TNF (p = 0.02), IL-6 (p < 0.001), and IL-17A (p = 0.03). NETs were associated with fever (p = 0.002) and correlated with polynuclear pleocytosis . We show that neutrophil activation and active NET formation occur in the CSF samples of children and adults with CNS infections, mainly caused by Borrelia and neurotropic viruses. The role of NETs in the early phase of viral/bacterial CNS infections warrants further investigation.Neutrophils operate as part of the innate defence in the skin and may eliminate the  Borrelia burgdorferi sensu lato and transmitted to humans by the Ixodes ricinus tick, is the most serious manifestation of Lyme disease (LD) [Borrelia constitutes a common cause of bacterial infection of the central nervous system (CNS) among children and adults in Sweden [erythema migrans [Lyme neuroborreliosis (LNB), which is caused by a complex of Gram-negative spirochaetes called ase (LD) . Borrelin Sweden ,3,4,5 ann Sweden . Accordin Sweden , mononucn Sweden  but also migrans —the earlNeutrophils are an essential component of the innate immune system, in that they limit locally the pathogen load through phagocytosis and the production of hydrolytic enzymes and chemokines, thereby orchestrating the subsequent adaptive immune response against the bacteria . Beyond Streptococcus suis meningitis [N = 3) and acute viral meningitis (N = 4) [The activities and roles of NETs are mostly unknown in the context of LD and human CNS infections overall. Nevertheless, NETs have been detected in the skin samples of Borrelia-infected, tick-bitten mice and have been shown to entrap and kill the spirochaetes despite a tick saliva-mediated decrease in the production of reactive oxygen species by the neutrophils . In addiningitis  and in a (N = 4) .Neutrophils and their activation products are important early during infection, and they most likely affect the course and outcome of many diseases. With LNB, children tend to seek medical care earlier than adults owing to their having higher frequencies of meningeal symptoms and facial nerve palsy ,4,21. ThN = 28), tick-borne encephalitis , enteroviral meningitis , other viral infection , and other non-infectious disorders with neurological or CNS symptoms without pleocytosis  was defined as the presence of: (i) symptoms attributable to LNB, (ii) mononuclear pleocytosis in the CSF, and (iii) intrathecally produced anti-Borrelia antibodies. All the children in the ‘possible LNB’ group (N = 8) had symptoms that were attributable to LNB and pleocytosis in the CSF but had neither detectable intrathecally produced anti-Borrelia antibodies nor clinical signs or laboratory evidence of other infections. These children all responded well to antibiotic treatment. Definite and possible LNB (N = 28) were referred to collectively as ‘LNB patients’. All samples were drawn before the initiation of antibiotic treatment, and the CSF samples for evaluation of NETs were frozen at −70 °C until analysis. All the children were evaluated for intrathecal anti-Borrelia antibody production (Borrelia burgdorferi-specific IgG and/or IgM), using the flagella antigen-based, enzyme-linked immunosorbent assay  in the IDEIA Lyme Neuroborreliosis Kit  [6 cells/L. The children were followed for assessment of recovery 2 months after admittance to hospital.The clinical characteristics of the children are shown in EVM; N = , other vire, UK) . An indeN = 32), unspecified LD (N = 6), TBE (N = 3), other viral meningitides , and other non-infectious disorders with neurological or CNS symptoms  was defined as the presence of: (i) symptoms attributable to LNB; (ii) mononuclear pleocytosis in the CSF; and (iii) intrathecally produced anti-Borrelia antibodies. In addition, the level of CSF-CXCL13 was high (>100 pg/mL) in all the patients, supporting the diagnosis [N = 3) had symptoms attributable to LNB and mononuclear pleocytosis in the CSF, and they showed neither clinical signs nor laboratory evidence of other infections. Increased levels of CSF-CXCL13 supported the diagnosis. The patients with definite and possible LNB ( N = 31) were collectively referred to as the ‘LNB patients’. Unspecified LD is a common manifestation in the highly Borrelia-endemic Åland Islands , where a large proportion of the population has anti-Borrelia antibodies in the serum and in the CSF due to previous exposure. Unspecified LD is defined as the presence of: (i) new symptoms suggestive of LD ; (ii) significantly increased anti-Borrelia antibody levels in the serum; (iii) a clear effect of antibiotic treatment for B. burgdorferi on current symptoms; (iv) a lack of pleocytosis and no increase in CSF-CXCL13; and (v) neither clinical signs nor laboratory evidence of other infections. Pleocytosis in the CSF was defined as ≥5 × 106 cells/L. The CSF samples from adults were handled in the same manner as those from the children, i.e., frozen at −70 °C until analysis. CSF samples were analysed for anti-Borrelia IgG antibodies using the recomBead Luminex-based assay . An index >0.3 was considered as positive, indicating intrathecal production of anti-Borrelia antibodies, in line with the manufacturer’s instructions. The adult patients were followed for the assessment of recovery 3 weeks and 6 months after admittance to hospital. The clinical characteristics of the adult patients are shown in iagnosis . Patient6 cells per well, and stimulated with 20 nM PMA for 3 h. The wells were then washed twice with culture medium before incubation with 20 U/mL of the restriction enzyme AluI  at 37 °C for 20 min to cleave the NETs. The samples were centrifuged at 300× g for 5 min and NET-rich supernatants were pooled, aliquoted, and stored at −80 °C. Phorbol-12-myristate-13-acetate -induced NETs were used to create the standard curve for the ELISA. To induce NETs, neutrophils were isolated through Percoll density centrifugation, as described previously . The neuPLUS; Roche Diagnostics GmbH, Mannheim, Germany) for 2 h. Standards and samples were run in duplicate. For the standard curve, an 8-point dilution series with 2-fold dilutions of the PMA-induced NETs was used, and two plasma samples  served as positive controls. After incubation, a substrate for peroxidase  was added for 40 min before the plate was read at 405 nm in a VersaMax ELISA microplate reader . All the samples were interpolated from the standard curve using the sigmoidal 4-parameter logistic regression equation and reported as arbitrary units (a.u.). Non-detectable samples were given the value of 0 a.u. Analysis of NETs in the 175 CSF samples, which originated from the 111 children and 64 adults, was performed at the Division of Drug Research, Linköping University  [The NET remnant ELISA was performed as previously described , althoug Sweden) .To confirm NET formation, 28 representative CSF samples (from 26 children and 2 adults) were also analysed for NE activity at the Friedrich-Alexander-Universität Erlangen-Nürnberg . For measurement of NE activity in the CSF, 100 µL of CSF were added to 100 µL PBS plus 25 µL of 1 M fluorogenic substrate MeOSuc-AAPV-AMC  plus 25 µL of 3.3 mM sivelestat  or PBS in black 96-well plates , as previously described [® assays . The Human Magnetic Luminex® assay was used to measure the levels of CXCL1, CXCL6, CXCL8 (IL-8), CXCL10, and MMP-9, whereas a high-sensitivity assay (Luminex® Performance Assay) was used for the quantification of TNF, IL-6, and IL-17A. The m.f.i. value of the respective analyte was obtained using FLEXMAP 3D , and the Bio-Plex Manager ver. 6.2 software  was used for data analysis. One of the 11 NET-positive samples was not available for the high-sensitivity assay. The samples were run in two different dilutions at a minimum, and the concordance between dilutions was checked carefully when concentrations had to be determined from different dilutions. Concentrations above the range of quantification were assigned the value of the expected concentration of the highest standard. An m.f.i. value below the lowest standard concentration was assigned a value of 0. The cytokines and chemokines were selected based on their associations with neutrophils and/or NETosis [CSF samples from 13 NET remnant-positive children  and 25 age- and sex-matched NET remnant-negative children  were analysed for chemokines and cytokines using two different magnetic Luminex NETosis ,30,31,32U-test was applied in comparisons between two groups with independent observations. The Kruskal–Wallis test was used for comparisons of more than two groups and if the Kruskal–Wallis test result was significant, Dunn’s post hoc test was performed. For correlation analyses, Spearman’s rank correlation coefficient (rs) was employed. In all the analyses, a p-value < 0.05 was considered statistically significant.Data were analysed using the GraphPad Prism ver. 8.01 software . Chi-square or Fisher’s exact test was used to compare discrete variables in 2 × 2 contingency tables. D’Agostino and Pearson omnibus normality tests were used to determine whether or not the data for continuous variables had a Gaussian distribution. For a non-Gaussian distribution of the data, the Mann–Whitney The study protocol was approved by the Regional Ethics Review Board in Uppsala  and the local Ethics Committee of the Åland Health Care, Finland .p = 0.01); in 14 (13%) children, whereof 7 (6%) with LNB, 4 (4%) with EVM, 2 (2%) with TBE, and 1 (1%) with idiopathic peripheral facial nerve palsy (IPFP) without pleocytosis (belonged to the OVI group). Only one adult patient, with meningitis and mononuclear pleocytosis caused by Varicella zoster virus (VZV), had detectable NETs (5 a.u.). The range of NETs levels were as follows: NET-positive patients with LNB, 2–18 a.u. ; NET-positive patients with EVM, 38–2643 a.u. ; in the two NET-positive patients with TBE, 39–74 a.u.; and in the single NET-positive patient with IPFP, the level was 17 a.u. The proportions of NET-positive patients in the LNB (25%), TBE (67%), EVM (57%), and OVI (25%) groups did not differ significantly. Higher levels of NETs were observed among patients with fever (p = 0.002). The NETs levels correlated significantly with the total white blood cell counts in the CSF samples     (p = 0.03); and the number of polynuclear cells in CSF among paediatric patients showed an inverse significant correlation with the duration of neurological symptoms .NETs were detected in the CSF samples of significantly more children than adults (< 0.001) A, and wi 0.0001) B as well 0.0001) C, althouctively) D,E. Chils = 0.81, p < 0.0001) . The levels of NETs and NE activity correlated significantly (r 0.0001) F. p = 0.72). All seven children with EVM were fully recovered at the two-month follow-up, four of whom were NET-positive. Among the three patients with TBE, one of the two patients who recovered was NET-positive, as was the patient who did not recover. Two of the four OVI cases had recovered at follow-up, whereas none had detectable NETs in the CSF. Only two patients in the group with other neurological and non-neurological disorders with CNS symptoms were NET-positive, and they had not recovered at follow-up.As we only found one NET-positive adult patient, we focused on evaluating NETs in relation to the disease prognosis for children . The vasN = 13) and NET-negative (N = 24 or 25) CSF samples, respectively. The total number of samples within the range of quantification is shown in p < 0.001), CXCL8 (p = 0.003), IL-17A (p = 0.03), TNF (p = 0.02), MMP-9 (p = 0.002), CXCL1 (p < 0.001), CXCL6 (p = 0.007) and CXCL10 (p < 0.001) (N = 16); IL-6 (p < 0.001), CXCL8 (p = 0.001), IL-17A (p = 0.028), TNF (p < 0.001), MMP-9 (p = 0.009), CXCL1 (p = 0.002), CXCL6 (p = 0.002), and CXCL10 (p = 0.001). Furthermore, all the cytokines/chemokines, with the exception of IL-17A, were detected at significantly higher levels in the CSF samples from children with pleocytosis (N = 24) (p < 0.001). A strong and significant correlation was noted between the CSF levels of IL-6, CXCL8, TNF, MMP-9, CXCL1, CXCL6, CXCL10 and NETs, NE, and total CSF cell count, as well as mononuclear and polynuclear pleocytosis, respectively (All eight cytokines/chemokines were detected in at least one sample among the NET-positive (< 0.001) A–H and fectively . The levIn recent years, interest in NETs has shifted from their roles in innate immune defence to their pathogenic importance in several clinical conditions, such as autoimmune diseases, cancer, cardio-vascular morbidity, and most recently, in infections ,33. In tThe levels of NETs showed strong variability between the different patient groups, ranging from 2 to 2000 a.u. Interestingly, the highest levels of NETs were detected in children with EVM and TBE, who also had the highest median levels of polynuclear cells. Children with EVM also had the highest median levels of NETs/neutrophil-associated cytokines and chemokines in their CSF samples . In addition, the levels of NETs correlated significantly with the numbers of polynuclear cells, i.e., including neutrophils, which were absent in the paediatric controls (Other disorders) and were overall present at lower levels in the adult patients with longer disease duration, which might explain the absence of NETs in these samples. The only adult who possessed detectable NETs had VZV meningitis with a symptom duration of several weeks and few polynuclear cells in the CSF.The median pleocytosis values were comparable among children with LNB and EVM, although the proportion of polynuclear cells was higher in the latter group. This may be due to the fact that all the patients with EVM had meningeal signs and, consequently, a significantly shorter disease duration before admittance to hospital, as compared to the patients with LNB. In line with this, the numbers of polynuclear cells in the CSF were found to be inversely correlated with the duration of symptoms.in vitro stimulation of neuronal cells in response to B. burgdorferi spirochaetes [B. burgdorferi and enterovirus. To clarify further the presence of neutrophils in these CNS infections, we analysed several potent neutrophil-activating cytokines and chemokines in the NET-positive samples. The results showed increased levels of almost all the measured cytokines/chemokines, and these levels strongly correlated with both poly- and mononuclear pleocytosis.The finding of neutrophil activation in both LNB and EVM is intriguing, since these infections have similarities with respect to both clinical presentation and CSF cellular profile, which may include neutrophil predominance in the early phase . Severalochaetes . Thus, tB. burgdorferi [The chemokine CXCL10, also called interferon (IFN) γ-inducible protein 10 (IP-10), is produced by innate immune cells, such as neutrophils, and it stimulates chemotaxis of mononuclear cells into the CNS in response to meningitis/encephalitis caused by, for instance, enterovirus , B. burggdorferi , and TBEgdorferi . Furthergdorferi . Interesgdorferi  in the Egdorferi , activatgdorferi . The higgdorferi  and in ogdorferi . B. burgdorferi in the early phase of LD, are required for the spirochaetes to establish infection. OspB have been shown to inhibit the phagocytosis and oxidative burst of human neutrophils and may interfere with complement activation [Borrelia sp. from phagocytosis by mononuclear phagocytes, and also to some extent by neutrophils [The paucity of NETs in the CSF of the adult patients in general, and in the adult LNB patients in particular, might be attributable to a delay between innate immune activation and CSF analysis through lumbar puncture, since the adult patients had longer duration of symptoms at admittance. While the reason for the modest neutrophil activation in LNB is unknown, it might involve an inhibitory effect of the spirochaetes on early pathogen recognition and elimination. Indeed, outer surface proteins (Osp), which are major virulence factors expressed on the surface of tivation . OspC istrophils .Since extracellular traps are not produced exclusively by neutrophils, being also produced by mononuclear cells, termed monocyte or macrophage extracellular traps (METs) ,53, we cWe are unable to draw firm conclusions regarding the levels of NETs in relation to disease recovery in any of the patient groups, except in the paediatric LNB group, in which the occurrence of NETs at admission was shown not to be associated with poor clinical outcome at the two-month follow-up. The strengths of the present study are that it is hypothesis driven and has a systematic design, in addition to having well-characterised patients and concordant results. However, the uneven distribution of patients in the different groups and the heterogeneity within the group of other disorders are limitations that made comparisons of the results between patients more difficult.We demonstrate the presence of NETs in the CSF samples collected from patients with LNB and viral CNS infections and show that NETs strongly correlate with the presence of pleocytosis and neutrophil-stimulating cytokines/chemokines in the CSF. Further studies of human neutrophils and their activation products are warranted, so as to elucidate their pathogenic and prognostic roles in acute bacterial and viral infections of the CNS.


Rationale: The high expression of Galectin-3  in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging.Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab')2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab')2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3  gene expression in each macrophage of several subtypes present in murine or human plaques.Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab')2 and 89Zr-DFO-Gal3-F(ab')2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab')2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts.Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab')2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease. Atherosclerosis is a multifactorial chronic inflammatory disorder characterized by a disturbed equilibrium of immune responses and lipid accumulation, leading to plaque development in vivo is a challenging objective with the ultimate goal to develop pharmacological and/or therapeutic strategies to prevent lethality in vivo, imaging approaches are highly relevant to facilitate decisions by the medical community when and how to apply invasive therapies to prevent clinical sequelae of the disease. Molecular imaging offers potential opportunities to develop diagnostic approaches to assess the pathobiology of atherosclerotic plaques. Improved understanding of the molecular and biological processes has stimulated the development of imaging probes, which may aid to identify high-risk atherosclerotic lesions and to apply individually tailored interventions Visualization of atherosclerotic plaques and in particular identification of the high-risk lesions prone to rupture Monocyte-derived macrophages are major inflammatory cells associated with atherosclerotic lesions and are recognized as key pathophysiologically important immune cells. Therefore, macrophages are gaining attention as imaging targets in atherosclerosis 2 fragment derivative have been developed and used for non-invasive imaging of Gal3-expressing tumors i.e. subset of macrophages and to a lesser extent other Gal3 positive cell types, using a Gal3-specific probe in apolipoprotein E-knockout (ApoE-KO) mice.Galectin-3  is a member of the lectin family, which is known to be involved in multiple aspects of inflammatory cell pathology 2 mAb, we show that nuclear imaging has the potential to visualize specific biological activities associated with plaque progression and/or instability, and that Gal3 expression in atherosclerotic plaques may be used as a novel biomarker to identify patients at high risk of cardiovascular events or to monitor the impact of treatment aimed at plaque stabilization. Using fluorophore coupled Gal3-F(ab')2 mAb, we delineated the distribution of the probe within the plaques with high resolution, providing initial evidence of the potential sensitivity of the proposed imaging approach.Using zirconium-89 labeled Gal3-F(ab')in vivo and ex vivo imaging studies. Mice were on high fat Western diet  for approximately 22 weeks. Age-matched C57BL/6 mice  were used as controls. Control mice were fed a normal chow diet. Experiments were approved by the local animal care committee  and were in accordance with the German Animal Welfare Act.Adult ApoE-KO mice  were used for 2 mAb was produced by pepsin digestion of a well-characterized rat anti-Gal3-mAb  binding to an amino-terminal common epitope of human and mouse Gal3 Gal3-F(ab')89Z was carried out as previously reported  and radiolabeling with reported  20.via incubation in specific media and in presence of activating substances and cytokines  using a Plan Fluor (Kodak) 20× lens. Another set of cells was incubated with 10 nM of 89Zr-DFO-Gal3-F(ab')2 mAb for 1 h at RT, or co-incubated with 100-fold excess of unlabelled full-length rat anti Gal3 mAb , referred as blocker (2). Data are shown as percentages of cell-associated radioactivity over total radioactivity added. The experiments were performed in triplicate.CD14-positive monocytes were isolated from peripheral blood mononuclear cells (PBMCs) obtained from healthy donors using positive selection technology . Successive differentiation in M1 and M2 macrophages was achieved ytokines  23. Diff blocker . After in = 12) and control (n = 6), were used for in vivo and ex vivo imaging. Mice were kept sedated with 1.5-2% isoflurane during injections and PET/CT scans. Static images were acquired 48 h after i.v. injections of 89Zr-DFO-Gal3-F(ab')2 mAb  using the Inveon small animal PET/CT scanner  with an acquisition time of 20 min. Images were reconstructed using Siemens Inveon software, which employs a 3-dimensional ordered subsets expectation maximum (OSEM3D) algorithm without attenuation and scatter correction. To confirm tracer uptake specificity, a group of ApoE-KO mice (n = 3) was co-injected with a 100-fold excess of unlabeled full-length rat anti Gal3 mAb, and referred as ApoE-KO blocked.Two groups of mice, ApoE-KO  were dissected longitudinally, stained with Sudan-IV for neutral lipids using a previously published method 2) in whole aorta.Tracer biodistribution and organ accumulation analyses were evaluated as previously described 2 mAb diluted in 300 µl saline solution, embedded in a mixture of Tissue-Tek and black ink (3.2% v/v) inside a cylinder of 4.5 cm diameter, and placed into the -80 °C freezer overnight. Cryosections were obtained using a Leica CM 3500 cryostat  at distances of 20 μm at -17 °C. A highly sensitive fluorescence imaging system was mounted onto the cryostat and macro tissue sections imaged every 5 slices (i.e. 100 μm), as reported previously Four mice  were sacrificed 48 h after i.v. injection of 100 µg of Cy5.5-Gal3-F(ab')Whole body imaging was performed on 2 ApoE-KO mice and one healthy mouse, while from the remaining ApoE-KO mouse slices of interest at 10 μm thickness were obtained with a LEICA CM 1950 cryostat. These slices were processed for H&E staining and morphological analyzed with a Zeiss M2 Axio Imager microscope , while from neighboring unstained slices fluorescent  images were captured.n = 2) were stained for CD68 and Gal3 as described previously and in To visualize macrophages, atherosclerotic lesions of the aorta from ApoE-KO mice  used for the production of the F(ab')For CD68 and α-SMA staining, mouse anti CD68  and mouse anti-α-SMA  mAbs were used according to the manufactory instructions at concentrations ranging ~10-30 µg/mL. 4-µm-thick longitudinal cross-sections along the axis of the aorta were prepared from formalin-fixed and paraffin-embedded tissues. For Gal3 immunostaining on human and mouse tissue sections, an antigen retrieval microwave treatment in 0.01 M citrate buffer pH 6.0, at 750 Watt for three cycles (3 minutes each) was applied as previously described For indirect immunoperoxidase, mAbs were incubated at 4°C for 30-45 min in a moist chamber. After washing in PBS the sections were incubated with a secondary goat anti-mouse IgG antiserum-HRP-conjugated (Dako) at dilution 1:200 for 30 min. The enzymatic activity was visualized using 3,3'-diamino-benzidine  as the chromogen.In order to compare expression of Gal3 in the ApoE-KO model with that in human plaques, 42 surgical samples of human plaques obtained from arteries during surgery (19 women and 23 men) or post-mortem tissues (5 cases), Gal3, CD68 or α-SMA staining were used as described for murine aortas. Images were recorded using an Aperio CS2 ScanScope image capture device  and analyzed using Aperio Slide Viewing software . Morphological analyses and immunohistochemistry were performed in blind and independently by two experienced pathologists. The study on surgical human samples was carried out according to the ethical guidelines of the Declaration of Helsinki. Specific approval was also obtained from the institutional scientific board and ethical committee of Sant'Andrea Hospital (Prot. CE nr. 8391/2013).n = 3). Plaques were removed using a dissection microscope and cut into small pieces, then digested with enzyme cocktail  for 40 min at 37°C with slow shaking. Single cell suspensions were stained with fixable viability dye and the panleukocyte marker antibody CD45 (Ebioscience). Total single live CD45+ cells were sorted by FACSAriaTM II. The RNA library of total CD45-positive (CD45+) live cells was constructed using the Chromium Single Cell 5' Library & Gel Bead Kit (10X Genomics) and sequencing was performed by 26+ 91 bp paired-end next generation sequencing (Illumina). The sequencing raw data was demultiplexed with the cellranger mkfastq pipeline, reads were aligned to mouse mm10 reference transcriptome using STAR alignment built in Cell Ranger (version 3.1.0). All 578 macrophages from total plaque CD45+ cells were sub-clustered for 2000 highly expressed genes of each cell using non-linear dimensional reduction (tSNE) algorithm. Three macrophage subtypes were designated following a previous report using marker gene profiles Plaques were dissected from aortas of aged (> 78 weeks) ApoE-KO mice  Human plaque single cell sequencing raw data was downloaded from a publicly available databank .Data are expressed as means ± standard deviation (SD). The Mann-Whitney U test was used to compare two variables. A 2 mAb and Cy5.5-Gal3-F(ab')2 mAb conjugates showed a fluorochrome/protein ratio of 5.6 ± 0.1 and 3.6 ± 0.1, respectively. Labelling of DFO-Gal3-F(ab')2 mAb with 89Zr resulted in a 74 ± 4% radiochemical yield and a radiochemical purity of 98.5 ± 0.5% after purification. The specific activity of the probe was 26.0 ± 2.0 MBq/nmol.The AlexaFluor488-Gal3-F(ab')2 mAb 2 mAb radioactivity was observed for M2 (M2a/M2c) macrophages compared to the M1 and M0 subsets, respectively 2 mAb to the M2 (M2a/M2c) macrophages was target-specific, as it could be decreased ~8-fold by co-incubation with a 100-fold excess of non-labelled rat anti-Gal3 mAb  displayed significantly increased uptake of AlexaFluor488-Gal3-F(ab')89Zr-DFO-Gal3-F(ab')2 mAb for ApoE-KO and control mice are shown in Figure in vivo. In contrast, no signals were seen in the aortas of control mice. These findings were confirmed by ex vivo PET/CT images that showed 89Zr-DFO-Gal3-F(ab')2 mAb binding in the same area 2 mAb in ApoE-KO mice are presented in Online in vivo due to physiologic uptake of the Gal3 antibody by the liver, but visualized when analyzed ex vivo by PET imaging  compared to aortic tissue from control mice ((6.8 ± 0.8) × 105, QL/mm2)  Figure B. Applic2 mAb confirmed accumulation of the Gal3 antibody fragment in atherosclerotic lesions s Figure A. This ws Figure . Despites Figure B insert.ex vivo PET imaging of excised aortas (+) macrophages and other myeloid cells located in the fibrous cap layer and shoulder region of plaques , indicating ongoing involvement of Gal3 in tissue remodeling  cells including smooth muscle cells, myofibroblasts , scattered foamy macrophages, and some endothelial cells (which are involved in re-endothelization of the plaque) also showed positive signals for Gal3  macrophages. To discriminate TREM2high from resident-like (Res-like) macrophages, the MRC1 (CD11b) gene was utilized as an index marker for M2 macrophages while inflammatory macrophages (M1) were defined by CCR2 gene expression high M2 macrophages express higher levels of Gal3 transcripts when compared to M1 macrophages both in murine and human plaques in vivo  and alternatively activated macrophages, and found that Gal3 expression was significantly higher in M2 macrophages with respect to the classically activated cells in vitroin vitro binding results on macrophage subpopulations are consistent with these observations, and show approximately 3-fold higher uptake of 89Zr-DFO-Gal3-F(ab')2 mAb on M2 (M2a/M2c) macrophages as compared to non-activated and classically activated cells. Moreover, scRNA-seq analyses of plaque-resident macrophages indicate that a portion of TREM2high M2 macrophages express higher level of lgals3/Gal3 transcripts when compared to M1 macrophages both in murine and human plaques. These data indicate that Gal3-F(ab')2 mAb-based tracers preferentially bind to M2 macrophages. However, the pathological role of alternative macrophages in atherosclerosis progression and their contribution to plaque (in)stability remains to be fully understood et al., deletion of Gal3 in ApoE-KO mice was associated with smaller plaques with reduced necrotic cores and collagen content Our observations support the hypothesis that plaques harbor several Gal3Different experimental imaging approaches, like ultrasound molecular imaging (USI), fluorescent imaging, intravascular photoacoustic imaging, magnetic resonance imaging (MRI) associated to experimental probes have been studied for this purpose 18F-FDG) has become a mainstay of metabolic imaging in atherosclerosis 18F-FDG by macrophages is unspecific and it is taken up by the same membrane transporters that take up glucose in all metabolically active cells. In addition, this approach has limitations in the specific setting of vascular imaging, including physiological uptake in the left ventricular myocardium that impairs imaging of coronary arteries, interaction with blood glucose metabolism, and the need for an extended fasting period before imaging. Due to the high affinity of 18F-fluoride to inorganic hydroxylapatite and its ability to localize noninvasively in bone metastasis or soft tissue dystrophic calcification, 18F-sodium fluoride (18F-NaF)-PET has also been investigated as a potential noninvasive imaging methodology to identify calcified tissue deposits in plaques with high-risk characteristics 18F-NaF versus 18F-FDG in cancer patients, Derlin and co-workers reported contrasting results about accumulation of both tracers in calcified lesions, with colocalization of 18F-NaF and 18F-FDG occurring in only 6.5% of the arterial lesions studied 18F-NaF uptake, it has been suggested that fluoride uptake probably occurs in dying foam cells, or in the senescent, dying, foam cells when cell metabolism is reduced or absent Different PET and single photon emission computed tomography (SPECT) radiotracers have been investigated for imaging of plaques 89Zr-DFO-Gal3-F(ab')2 mAb over above mentioned tracers is that our probe binds specifically and with high sensitivity to major actors of plaque evolution and vascular repair. This methodology may therefore allow real time imaging of different phases of plaque maturation.The main advantage of using 89Zr-labeled F(ab')2 fragment for studying plaques could be challenging. To overcome this problem, production of a chimeric/human F(ab') anti-Gal3 mAb with optimized plasma half-life for clinical usage is in progress in our laboratory. Second, the fluorescent dye which was used to label Gal3-F(ab')2 mAb was primarily used to study the intra-plaque distribution of the antibody fragment with higher resolution, which is not achievable by autoradiography. Intravascular fluorescence molecular imaging (FMI) is an emerging technology that enables imaging of specific molecular events within rupture-prone plaques Some limitations associated with the current study require further considerations. First, due to typical late imaging time points and extended patient radiation exposure, the clinical use of an The role of Gal3 in physiological and pathophysiological processes has led to several distinct galectin inhibitors, both for experimental purposes and potential clinical use as novel therapeutics In conclusions, our findings indicate that PET imaging of plaques by targeting Gal3 appears to be feasible. Clinical translation of these imaging approaches holds promise to improve the management of patients affected by plaques at high risk of lethal clinical outcomes of the disease.Supplementary figures and tables.Click here for additional data file.


Aldh1a2, which is responsible for testicular RA synthesis, while Cyp26b1, which is responsible for testicular RA metabolism, was still expressed even after germ cell depletion, suggesting an alteration of the RA synthesis/metabolism ratio. These observations suggested that RA insufficiency was one of the causes of the defective donor spermatogenesis. Indeed, repetitive RA administrations significantly improved donor spermatogenesis to produce fertile offspring without any side effects. These findings may contribute to improving fertility preservation techniques for males, especially to prevent iatrogenic infertility induced by chemotherapy in prepubertal cancer patients.Germ cell depletion in recipient testes is indispensable for successful transplantation of spermatogonial stem cells. However, we found that such treatment had an adverse effect on spermatogenesis of orthotopically transplanted donor testis tissues. In the donor tissue, the frequency of stimulated by retinoic acid (RA) 8 (STRA8) expression was reduced in germ cells, suggesting that RA signalling indispensable for spermatogenesis was attenuated in germ cell-depleted recipient testes. In this context, germ cell depletion diminished expression of testicular  Undifferentiated spermatogonia are induced to differentiate into differentiating spermatogonia by retinoic acid (RA). This molecule also mediates the next differentiation step, namely differentiation of differentiating spermatogonia into spermatocytes3. These processes require support from the germline niche consisting of Sertoli and Leydig cells in addition to the basement membrane5. The former cell type was found by Enrico Sertoli as ‘nurse cells’ for germ cells, which produces GDNF and form a blood-testis barrier, both of which are indispensable for proper spermatogenesis6. The latter cell type found by Franz Leydig synthesises androgens depending on luteinising hormone to regulate Sertoli cell functions7. Recent reports have demonstrated that residential macrophages, peritubular myoid cells, and neighbouring lymphatic epithelial cells also have some effects on spermatogenic processes11. Moreover, the hypothalamic-pituitary axis further controls Leydig cell functions via luteinising hormones to produce testosterone12. Oestradiol synthesised from testosterone by aromatase in Leydig cells is also dispensable for proper spermatogenesis14.Spermatogonial stem cells (SSCs), a subset of undifferentiated spermatogonia, maintain their population by glial cell line-derived neurotrophic factor (GDNF)-mediated self-renewal15. In particular, although adult patients can avoid complete loss of fertility by freeze stocking of their own sperm before cancer therapy, this strategy is not applicable to prepubertal patients whose testes are too immature to produce haploid germ cells applicable to microinsemination17. Considering the recent strategic improvements in cancer therapy, cancer survivors suffering from male infertility will continue to increase.Although spermatogenesis can be impaired because of several reasons, cancer treatments such as anti-cancer drugs, irradiation, and orchiectomy have a significant effect on the fertility prognosis18. This technique is applicable to transplantation of frozen and allogenic SSCs20. Although successful xenotransplantation of rat and hamster spermatogonia into infertile nude mice has been achieved for offspring production, offspring has not been obtained from dogs, rabbits, cattle, pigs, baboons, and humans even in allogenic transplantation21. In vitro spermatogenesis is a recently established technique to restore spermatogenesis. Sato and colleagues developed a testis culture system in which testis pieces from pup/adult mice are cultured on an agarose gel block for more efficient oxygen uptake by the tissues at the liquid–air interface23. However, this technique is not applicable to mammals other than mice24.Thus far, there are three techniques to restore spermatogenesis: spermatogonial transplantation, in vitro spermatogenesis, and testis piece transplantation. Spermatogonial transplantation is able to restore the fertility of infertile male mice26. Moreover, successful offspring production has already been achieved in mice, rabbits, pigs, and primates by this technique29.Based on these contexts, it seems that testis piece transplantation is the most practical technique to restore male fertility in mammalian species at present. Indeed, this technique has already been applied to not only functional analysis of mutant mouse testes, but also restoration of spermatogenesis in other mammalian species31. Therefore, in the present study, we examined why depletion of recipient germ cells affects the spermatogenesis of orthotopically transplanted testis pieces, and found that recipient germ cell depletion caused RA insufficiency that compromised the spermatogenesis of orthotopically transplanted donor testis pieces.However, we found that germ cell depletion by the anti-cancer drug busulfan compromises the spermatogenesis of orthotopically transplanted donor testes, whereas testis pieces transplanted into healthy testes result in proper spermatogenesis. This observation is inconsistent with previous observations that germ cell depletion dramatically enhances colonisation, SSC-originating spermatogenesis, and offspring production28, we found that the number of GFRA1+ undifferentiated spermatogonia and the frequency of seminiferous tubules carrying MCAM+ spermatogonia were comparable between testis pieces transplanted into busulfan-treated testes and those in control recipients  constitutively (green mice) were transplanted into the testicular interstitium of recipient testes with or without germ cell depletion induced by busulfan Fig. . Althougnts Fig. a–d. More3. Testosterone produced by Leydig cells is also indispensable for the progression of meiosis33. It has been demonstrated that busulfan-treated testis produce testosterone35, whereas RA is synthesised by the combination of testicular cells including germ cells36. Based on this context, we next focused on RA signalling in transplanted testis tissues and found that the frequency of tubules containing STRA8+ differentiating spermatogonia/pre-leptotene spermatocytes was reduced significantly  . In addition, the population of MCAM+ cells was not affected. In the busulfan group (n = 7), 97.1 ± 1.4% of tubules contained MCAM+ haploid germ cells, whereas these cells were found in 99.6 ± 0.2% of tubules in the busulfan + RA group (n = 4) . Concomitantly, Johnsen’s score count was also improved by this treatment .As expected, RA administration improved the production of haploid germ cells Fig. a. In the+ undifferentiated spermatogonia in donor tubules , whereas the busulfan + RA group carried 0.17 ± 0.04 cells/tube (n = 4) . In addition, tubules with an unusual germ cell arrangement were occasionally seen in the busulfan + RA group  developed to the two-cell stage within 24 h of culture. After transfer into the oviducts of four pseudo-pregnant females, 27 live pups were born  of busulfan at which germ cell-mediated RA synthesis is considered to be diminished completely. Such treatment might diminish the production of testosterone. Moreover, oestradiol, another sex steroid synthesized from testosterone by CYP19A/aromatase in Leydig cells and indispensable for proper spermatogenesis, might be affected by busulfan14. Considering these observations, RA insufficiency might also indirectly compromise spermatogenesis via modulated production of sex steroids in Leydig cells.The present study did not investigate testosterone. Previous studies have demonstrated that busulfan treatment does not alter testicular testosterone49. In addition, repetitive administrations of the GnRH analog support recovery from busulfan damage for spermatogenesis50. These findings suggest that gonadotropin exerts adverse effects on the recovery process of spermatogenesis. In this context, busulfan treatment distinctly elevates LH and FSH levels in the pituitary and serum of rats51. Therefore, gonadotropin suppression in recipient might improve spermatogenesis in orthotopically transplanted donor testis pieces.A GnRH analog supports colonisation of SSCs transplanted into infertile rodent testes by suppressing gonadotropin release from the pituitary grandRarg in undifferentiated spermatogonia both in vitro and in vivo, and germ cell depletion upregulates testicular Gdnf38. Although the number of GFRA1+ spermatogonia was not changed in our present study, Gdnf upregulated in recipient testes might suppress Rarg expression in GFRA1+ spermatogonia of donor testes to suppress production of differentiating spermatogonia. Germ cell-derived PDGFA is also important for Leydig cell functions and proper spermatogenesis52. Restructuring of the molecular network by controlling these factors is beneficial for proper spermatogenesis in transplanted testis pieces.Altered expression of several factors might have also contributed to the reduced spermatogenesis in transplanted testis pieces. Our previous study demonstrated that GDNF suppresses 53. In addition, RA alters the epigenome status of several cell types54. Moreover, alteration of the epigenome status in sperm results in developmental abnormalities of offspring55. However, the present study demonstrated that ectopic RA treatment did not cause any developmental defects in offspring.RA causes various developmental defects in vertebrates because it plays fundamental roles in gastrulation, axis formation, and organ formation during development because of cell fate disruption39. This technique has already been employed to assist human reproduction, and no overt abnormalities have been reported to date17. A combined technique including testis piece transplantation, RA treatment, and microinsemination might be practical to restore spermatogenesis, especially in prepubertal cancer patients.The microinsemination technique was primarily developed by Yanagimachi and colleagues in 1995 using mice56, most studies have employed subcutaneous transplantation, and other transplantation sites remain to be elucidated. The present study employed intratesticular transplantation and found that depletion of recipient germ cells compromised the RA production/metabolism ratio and reduced spermatogenesis of donor testis pieces transplanted into recipient testis. RA treatment supported the spermatogenesis of donor testis pieces when the size of the donor testis piece was insufficient for the size of the recipient testis after treatment with the anti-cancer drug. These findings may contribute to the treatment of prepubertal cancer patients to recover fertility after anti-cancer therapy. However, there are still many parameters  to be considered when applying testis piece transplantation to assisted reproductive technology.Although it has been reported that recipient conditions affect the spermatogenesis of transplanted testis piecesThe institutional animal care and use committee of Shinshu University approved all animal experimentation protocols . All experiments were performed in precise accordance with the manual provided by the institutional animal care and use committee of Shinshu University.57. Testis pieces collected from newborn mice (1.5–4.5 days postpartum) were transplanted into recipient testes. Briefly, recipient mice were anesthetised with isoflurane  and a small cut was made in the tunica albuginea of each animal using fine forceps. A single testis piece was inserted into the testicular parenchyma25. In some cases, recipient testes were treated five times with all-trans retinoic acid  dissolved in a 10% ethanol-sesame oil  with 8.5-day intervals just prior to sacrifice . Seven-week-old mice with/without treatment with 44 mg/kg body weight busulfan  at 3 weeks of age, which depletes germ cell in testes, were used as recipients. Male C57BL/6 Tg14(act-EGFP)OsbY01 ‘green’ mice were used as testis donors2 fragment ; rhodamine Red-X-conjugated anti-rat IgG .Recipient mice were sacrificed at around 146 days after transplantation, and donor recipient testes carrying donor testis pieces were collected. After overnight fixation in 4% paraformaldehyde/PBS (−) at 4 °C followed by overnight immersion in 30% sucrose/PBS (−), testes were embedded in Tissue-Tek OCT compound  to prepare 8 µm-thick cryosections. After 10 min of treatment with 0.1% Triton-X 100 (Sigma-Aldrich) in PBS (−) at 4 °C followed by blocking with 10% donkey serum , the sections were incubated with primary antibodies overnight at 4 °C. Sections were further incubation with secondary antibodies at room temperature for 1 h. Nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich). Images were captured by confocal laser scanning microscopy  or All-in-one fluorescence microscopy . Collected images were joined to reconstruct an entire image of testicular cross-sections. Antibodies used in this study were: a rat anti-mouse CD146 monoclonal antibody ; rhodamine-conjugated peanut agglutinin ; goat anti-rat GFRA1 antibody ; rat anti-mouse GATA1 monoclonal antibody ; Alexa Fluor 647-conjugated donkey anti-rabbit IgG ; Alexa Fluor 647-conjugated donkey anti-rat IgG F(ab′)®-RNA I Super G . First-strand cDNA was synthesised using ReverTra Ace® qPCR RT Master Mix with gDNA Remover . TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus)  in combination with a Thermal Cycler Dice® Real Time System TP800 was used for quantitative PCR. Reaction conditions were as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s, followed by a melting curve program. Each qRT-PCR was run in duplicate. Cycle threshold values were calculated by a second derivative maximum method using Thermal Cycler Dice® Real Time System Software Version 5.10 (Takara Bio Inc.). The standard curve method was applied to determine the absolute quantity. A standard cDNA stock for absolute quantitation was prepared from the PCR products using a FastGene™ Gel/PCR Extraction Kit . For data evaluation, values of the control group were set to 1.0 after normalisation to Hprt expression levels. Primer sequences are listed in Table Total RNA was prepared using Sepasol39. In brief, donor testes were recovered and transported overnight at 4 °C before ICSI using a previously published method for transportation of epididymides58.To assess the integrity of haploid germ cells in donor testes treated five times with retinoic acid injection, round and elongated spermatids were collected and subjected to intracellular sperm injection (ICSI)59. Resultant zygotes were cultured for 24 h and then transferred into the oviducts of pseudopregnant female mice . Offspring were delivered by caesarean section.Round and elongated spermatids were collected from donor testis pieces by simple repeated pipetting of seminiferous tubules and injected into oocytes of B6D2F1 mice (Japan SLC) using a Piezo-driven micropipetter  as described previouslyP < 0.05 was considered statistically significant.Results are presented as means ± SEM. Significance of differences between means for single comparisons was determined by the Student’s t test. Supplementary information


Toilet training (TT) is an important marker in a child's physical and psychosocial development. The present study aimed to evaluate aspects associated to delayed TT.We interviewed 372 parents of children who had completed TT up to 48 months before the interview. The questionnaires were applied at school exits when parents went to pick their children up and at public parks. Questions included demographics, aspects related to TT, dysfunction voiding symptom score and evaluation of constipation.The interviews were performed at a mean of 15.3±10.4 (0 to 47) months after the end of TT. Girls accounted for 53% of the sample. The mean age at finishing TT was 31.6±9.3 months and similar in both genders (p=0.77). TT occurred before school entry in 45.7% of the children and medical advice for TT was sought only by 4.8% of the parents. No association was observed of age at completing TT and presence of lower urinary tract symptoms (LUTS) (p=0.57) and/or constipation (p=0.98). In the univariate analysis, prematurity  and mothers who work outside their household  were associated to delayed TT.Children completed TT at a mean of 2 years and 7 months of age. The age of completing TT was not related to LUTS and/or constipation. Premature children and those whose mothers work outside the home finish TT later. Toilet training (TT) is defined as the ability to independently start and complete micturition and defecation. It is an important marker in a child's normal development, being a challenging process not only for the children but also for their parents. In the past decades an increase in the age of toilet training has been observed –3. The AData on acquisition of bladder and bowel control is conflicting with studies showing no differences between genders, and others demonstrating an earlier control in girls –6. AcquiMany different factors have been associated with the age of TT, such as age of the mother, parental education level , workingThe literature on TT is scarce and shows many controversies. We hypothesized that child development and familial environment may influence in the age a child completes TT. Therefore, the aim of the present study is to evaluate personal and household factors associated with toilet training as well as if there is any correlation between bladder and bowel dysfunction and the age TT is completed.A questionnaire was applied to parents of children who had completed TT no more than 48 months before the interview. Parents were interviewed in public areas, such as parks or in front of schools and the interview lasted about 10 minutes. Children whose parents refused to participate, presenting any urogenital disorder, who were using any medications or presented with any disease known to interfere on bladder or bowel function, severe intellectual or motor disability, and those who had not finished TT were not included in the study. Age at which toilet training was completed was defined as the age that the child had full bladder and bowel control, without any failure in holding urine or stool during day and nighttime.The institutional review board on ethics approved the study (protocol number 1.757.676) and all parents signed an informed consent.The questionnaires included: 1) demographic questions on gender, socioeconomic status, neonatal history, marital status of the parents, if both parents worked full time or not; 2) aspects related to TT ; 3) currData were expressed as means±SD, medians and interquartile ranges, or absolute values and fractions. The Student t or Mann-Whitney U test was used to compare continuous variables while categorical variables were compared using the chi-square or Fisher's exact test.Analysis of risk factors for delayed toilet training was performed by multivariable binary logistic regression with stepwise forward likelihood ratio method. Odds ratios (OR) were reported with a 95% confidence interval. Tests of significance were two-tailed, and significance was defined by p <0.05.All tests were 2-sided with p <0.05 considered statistically significant and were performed using MedCalc Statistical Software version 18.11.6.Of the 400 questionnaires applied, 372 were included in the study. Of the 28 not included, 15 were due to refusal of parents or caregivers to participate in the study, and the remaining did not fulfill inclusion criteria. The mean time between TT and the interview was 15.3±10.4 (0 a 47) months. Girls represented 53% of the sample. All other demographic data are described on The mean age at the moment of the interview was 46.5 months  and the mean age for TT was 31.6±9.3 months (median: 30.0 months), being 31.1±9.4 for girls and 32.2±9.2 for boys (p=0.20). No differences were found between TT for bowel (day and night) and bladder (day and night) according to gender or type of TT .We used the 75-percentile value that was 36 months to divide the sample for binary further analysis.Evaluating the family, age of the mother (p=0.10) and father (p=0.66), schooling of the householder (p=0.27), socioeconomic class (p=0.47) had no impact on the age of completing TT. The only familial factor associated with a delayed TT was mothers that work outside of their household (p <0.001) .Analyzing the association between age of TT and bladder and bowel dysfunction (BBD) it was observed that age at the end of TT had no impact on the development of LUTS (p=0.56) nor functional constipation  .In a univariate analysis, only prematurity  and mothers who work outside household  were associated with a delayed TT. The same factors remained as independent predictors in the multivariate analysis .Toilet Training is an important step in a child's development, and it depends on child's growth and maturation. As in many other developmental delays seen on a preterm born child , we haveVoluntary control of micturition is mediated by the cerebral cortex that is responsible for facilitating and inhibiting the micturition reflex. Therefore, the ability to gain bladder control is a complex developmental process that can be influenced by anatomical, physiological, cultural, and behavioral conditions , 17. ConSince our data were collected interviewing parents up to 48 months after their children completed TT, we did not inquire about the age of starting training due to a considerable memory bias.Despite the fact that most studies show no association between these two variables, working status of the parents, especially of the mother, is currently identified as the major cause of a delayed TT , 11. In Besides the working status of the mother, the use of disposable diapers and the availability of more information on the Child-Oriented Approach for TT have also been reported as related to this increasing age for completing TT , 19. TheThere is a widespread believe that girls end TT at an earlier age. Shum et al. studying the sequential of acquisition of TT skills described that normal developed girls achieve nearly all skills prior to boys  and few One of the greatest debates related to TT is whether an earlier or later TT is associated to BBD, although few studies have investigated these associations. As data present herein, other studies found no relationship between age or method use for TT , 2, 23 wEvaluating the presence of functional constipation, we also found that age of acquiring bladder control is not related to constipation, which is in agreement with the findings of Wald et al. . On the There are some limitations to this study. Although we tried to evaluate children who had finished their TT no later than 48 months, since a memory bias could have influenced the results, for this matter we may have missed long-term problems, such as BBD, related to TT. We also failed to evaluate age at the beginning and time taken to complete TT.Children completed TT at two and a half years of age. Both girls and boys completed TT at a similar age. Premature children and those whose mothers work outside their household are at increased risk of completing TT at a later age. We could not demonstrate association between age at completing TT to LUTS or functional constipation..


Meningoencephalocele is a rare and potentially lethal disease, requiring early diagnosis and treatment. A 30-year-old male patient was diagnosed with a massive meningoencephalocele. His medical history included cerebrospinal fluid (CSF) rhinorrhea since the age of 7 years, which was attributed to right eye mining during infancy due to Coats disease. Following failed attempts of open surgical repair and CSF diversion during childhood, no further attempts of surgical management were made. He presented a long history of recurrent episodes of meningitis, resulting in long-lasting hospitalization in the intensive care unit. Eventually, he underwent surgical repair through an endoscopic multilayer approach. Subsequent endoscopic and radiological assessment demonstrated no recurrence during a follow-up period of one year. Endoscopic treatment is highly successful. An effective and definite surgical repair is of paramount importance in order to avoid life-threatening complications, improve patient’s and caregiver’s quality of life, and avoid unnecessary health-system costs. Encephalocele or cranium bifidum is a neural tube defect characterized by the protrusion of brain tissue and overlying meninges through a bony deficit either along the midline of the cranial vault or at the base of the skull . In the Our aim is to underline the high efficiency of the endoscopic surgical treatment and the significance of early diagnosis and effective, definite surgical treatment by presenting an interesting case of a massive, neglected, postoperative meningoencephalocele.A 30-year-old man was transferred to the Department of Otolaryngology - Head and Neck Surgery of Evangelismos General Hospital, Athens, Greece, due to a massive meningoencephalocele, resulting in meningitis and coma. His surgical history included right eye mining during infancy due to Coats disease. At the age of 7 years, he developed CSF rhinorrhea, which was attributed to his previous operation. Five years later, he underwent open surgical repair in order to close the deficit and control the CSF leak. Four days postoperatively, the CSF rhinorrhea reappeared. He underwent lumbar diversion twice, one and nine years later respectively, due to persistence of the CSF leak, which resulted in meningitis. Despite efforts, CSF rhinorrhea never ceased, and the patient suffered recurrent episodes of meningitis, with the last one leading to coma and long-lasting admission in the intensive care unit.Nasal endoscopy revealed a cystic/polypoid lesion adherent to the middle meatus of the right nasal cavity, causing complete obstruction Figure . Clear fThe patient underwent extended endoscopic endonasal surgery under general anesthesia in order to resect the right anterior skull-base meningoencephalocele and repair the CSF leak. Intraoperatively, the root of meningocele and the bony defect were recognized using a navigator with real-time MRI fusion. The meningoencephalocele was gradually dissected and completely removed using bipolar diathermy Figure . The skuThe patient had an uneventful postoperative course in the regular ward and was discharged 48 hours postoperatively. During a follow-up period of 12 months, the patient presented with no CSF rhinorrhea, and subsequent endoscopic and imaging assessment demonstrated no recurrence Figures .Meningoencephalocele and CSF rhinorrhea can be iatrogenic, congenital, acquired (due to trauma or erosion), or idiopathic in origin . The posThe main symptom of meningoencephalocele is CSF fluid leak -2. CSF lImaging  and nasal endoscopy confirm the diagnosis, evaluate the underlying cause, and localize and characterize the defect site prior to surgical repair .The main targets of meningoencephalocele treatment are to avoid relapse and cease the CSF leak . ConservEndoscopic technique offers reduced postoperative morbidity and reduced length of hospitalization usually 48 hours), which were confirmed in our case, and avoids the complications of craniotomy, such as anosmia and intense headaches  hours, w. The aveEndoscopic treatment of meningoencephalocele is a highly successful, minimally invasive treatment option with low morbidity. Preoperative imaging and planning by experienced head and neck surgeons are of paramount importance in order to select the appropriate reconstructive method and to achieve the optimal outcome for the patient. Effective, definite and timely surgical repair is crucial in order to avoid life-threatening complications, improve patient’s and caregiver’s quality of life, and avoid unnecessary health-system costs.