Many clinicians have concerns about the safety of atopic dermatitis (AD) treatments, particularly in children requiring long-term daily maintenance therapy. Topical corticosteroids (TCS) have been widely used for >5 decades. Long-term TCS monotherapy has been associated with adverse cutaneous effects including atrophy, rebound flares, and increased percutaneous absorption with potential for adverse systemic effects. Topical calcineurin inhibitors (TCIs), tacrolimus and pimecrolimus, available for 1–2 decades, are not associated with atrophy or increased percutaneous absorption after prolonged use and have much lower potential for systemic effects. However, since 2006 TCIs have carried a controversial Boxed Warning based on a theoretical risk of malignancy  that has limited TCI use for standard-of-care maintenance therapy.A comparative systematic search of PubMed was done for long-term (≥12 week) clinical trials of TCS or TCI treatment in patients <12 years with AD. Citations were reviewed for inclusion based on MeSH terms, abstracts, and relevant article text. Studies were excluded if they did not encompass subjects <12 years, or were <12 weeks’ duration, retrospective, meta-analyses, or limited to anecdotal case reports.Of 27 trials meeting criteria, 21 included 5825 pediatric patients treated with TCIs, and 6 included 1999 patients treated with TCS. TCS studies were limited to low- to mid-potency products, and all but one study lacked a vehicle control. Eight TCI studies were vehicle-controlled, and safety data were well reported, with ≤5 % of patients reporting discontinuation due to adverse effects (DAEs). Cutaneous and systemic adverse events (AEs) were similar in TCI and vehicle groups, with no reports of lymphoma. Safety data in TCS trials were less well reported. DAE incidence was addressed in just 2 trials, and systemic and cutaneous AEs were mostly unreported.Data supporting long-term use of TCIs are robust, documenting safety and efficacy, while data supporting long-term TCS use are limited to low- to mid-potency products. Our review identifies a lack of information on the safety of commonly prescribed, long-term monotherapy with mid- to high-potency TCS in pediatric AD, and supports standard-of-care maintenance therapy with TCIs and intermittent use of low- to mid-potency TCS for flares. Atopic Dermatitis (AD) is a chronic, pruritic inflammatory skin disease that occurs most frequently in children. It is the most common chronic pediatric inflammatory skin disease, affecting 12.5 % of US children (aged 0–17 years) from 2009 to 2011, an increase of 5.1 % from 1997 to 1999 , 4. ThisTCS, which are considered to be first-line treatment for AD flares, have been FDA-approved for a variety of grandfathered indications since 1955. Their mechanism of action, though not well understood, is multifaceted and includes broad-spectrum impact on immune and skin barrier function. Despite their demonstrated efficacy in AD, TCS are associated with a known potential for cutaneous atrophy-related adverse effects such as telangiectasia, striae and purpura, as well as focal hypertrichosis, hypopigmentation and perioral dermatitis –14. ThesIn 2000–2001, TCIs were approved in the US for “short-term and noncontinuous chronic treatment of AD in nonimmunocompromised individuals who have failed to respond adequately to other topical prescription AD treatments” , 16. TacSince then, no clear link has been demonstrated between TCI use and lymphoma risk, despite almost a decade of clinical and epidemiological studies, post-marketing surveillance, and monitoring of reports to the FDA Adverse Event Reporting System (AERS). Recent published reviews and/or meta-analyses that assess lymphoma risk of TCIs based on the last decade of clinical experience conclude that there is no evidence that TCI use is associated with increased risk of lymphoma Table  18–39].–39.18–39To assess the safety of TCS and TCI use in children, we performed a comparative systematic literature search for published, long-term clinical trials of TCS or TCI treatment in pediatric patients with AD.The search strategies we used to identify published, long-term ≥ 12 week) clinical trials of TCI and TCS in pediatric patients (<12 yrs of age) with AD are shown in Figs. 2 week clFinally, we excluded studies that did not include safety data, were less than 12 weeks’ duration, or did not include pediatric patients (<12 yrs of age).Long-term safety data from the TCI and TCS trials that met inclusion criteria were summarized descriptively; statistical analyses were not performed due to the inconsistency in reporting of these data. We limited the focus to safety data that we judged to be most relevant to long-term AD treatment: discontinuations due to AEs, cutaneous AEs , and systemic AEs . We did not present the incidence of minor application site reactions  that typically occur early in treatment and resolve, potentially atopic or allergic events  that are common in patients with AD and are generally not related to treatment, or events with unclear origin that are unlikely to be treatment-related .n = 756) [The PubMed queries resulted in 221 TCI citations and 487 TCS citations  . In thisTreatment varied according to individual study designs, but typically a brief twice-daily regimen to control the initial flare was followed by a maintenance period of intermittent, as-needed treatment for flares  vs mid-potency TCS  , and 1 lTwo additional studies that met inclusion criteria for TCS safety summary were actually TCI studies in which TCS treatment was an active comparator: one was a double-blind trial of tacrolimus vs low-potency TCS (hydrocortisone) vs no treatment , and theWe did not identify any published long-term pediatric trials of high-potency TCS.All except 3 of the tacrolimus studies prohibited TCS use; 1 study permitted TCS use per protocol during the first 4 weeks , 1 permin = 91), 4-year study (n = 391), and the 5-year Petite study (n = 1205).The TCI studies ranged in duration from 12 weeks to 5 years (260 weeks) and included 5825 pediatric patients with mild to severe AD  . In the studies that reported the incidence of specific AEs, the terminology used for the AEs was not consistent between studies.N = 1370; 0.03 %, n = 524; 0.1 %, n = 846), pimecrolimus (N = 4455), low-potency TCS (N = 400), mid-potency TCS (N = 257), low- or mid-potency TCS (N = 1342), vehicle + TCS rescue , vehicle (N = 355), and no treatment .Safety data are summarized in Table To increase clarity and interpretability, AEs were categorized as either cutaneous events  or systemic events . A list of the AE terms that were included in each category is provided in the footer of Table Fourteen of 21 TCI studies reported the incidence of discontinuation due to AEs  event . Almost In the 5-year Petite study, up to 17 % of pimecrolimus and TCS patients reported some type of systemic bacterial infection, and 17 % reported influenza-like illness . Up to 3The Petite study investigated the effects of up to 5 years of AD treatment on growth rate and immune system function in a large population of infants, and found no difference in growth rate between the pimecrolimus and TCS treatment groups . SimilarPrior to the Petite study, there were few published studies that evaluated the effects of long-term TCS treatment on immune system or HPA-axis function in pediatric patients. In a 44-week study of mid-potency TCS, 2 patients from a small cohort that were administered a cosyntropin stimulation test (CST) had reduced serum cortisol responses . In a 28None of the TCI and TCS trials we identified in our systematic literature search assessed glaucoma or cataracts.There were no reports of lymphoma in any of the TCI and TCS trials, including the 5-year Petite Study  were published in or after 2006. More recent trials tend to have improved protocols with standardized and validated methods for assessing safety data. This is especially true following the establishment by the NIH in 2000 of a clinical trial registry , and FDA-mandated expansions in 2007 of registry requirements, including disclosure of study design. Further, journal criteria for publication continue to become more rigorous, requiring more clear and complete reporting of data as well as clinical trial registration prior to publication.According to current treatment guidelines, low-to-mid potency TCS should be used as first-line, short-term treatment of flares , 8, 78. Our review supports the long-term safety of TCIs and low-to-mid potency TCS in pediatric patients with AD. Long-term treatment with TCIs and intermittent use of low-to-mid potency TCS was generally well tolerated in 27 trials of >5800 and >1900 pediatric patients, respectively, with no evidence of cutaneous atrophy or cumulative systemic exposure and no reports of lymphoma. This reflects preclinical animal studies that detected malignancy signals only after systemic TCI exposure was high enough to cause immune suppression. This exposure was much higher than the negligible systemic exposure detected after twice daily topical administration  , 79–84.Many recent meta-analyses and reviews have also contributed to the body of evidence that has filed to detect increased lymphoma risk with TCIs –39. One A decade’s worth of clinical experience, epidemiological data, postmarketing surveillance, and adverse event database monitoring have failed to demonstrate a causal relationship between TCI use and malignancy, yet TCI labelling continues to include a Boxed Warning. The biggest impact of the warning is to limit patient access to the most well-studied medications for long-term maintenance AD treatment, especially in children.This comprehensive literature review supports the long-term safety of TCI and low- to mid-potency TCS therapy in children with AD, with no evidence of cutaneous atrophy or cumulative systemic exposure and no reports of lymphoma. We found comparatively limited data on the long-term safety of mid- to high-potency TCS. Our findings are not reflected by the current TCI labelling and Boxed Warning; therefore we hope our review facilitates the rescindment of the Boxed Warning.


This has been implicated as a pathogenic mechanism of mitochondrial dysfunction but has yet to be validated in patients. 12S m62A rRNA methylation, by the mitochondrial transcription factor 1 (TFB1M) enzyme, occurs at two successive nucleotides (m.1584A and m.1583A) in close proximity to m.1555A>G. We examined m62A methylation in 14 patients with m.1555A>G, and controls, and found all detectable 12S rRNA transcripts to be methylated in both groups. Moreover, different RNA samples derived from the same patient  revealed that only transformed cells contained some unmethylated 12S rRNA transcripts, with all detectable 12S rRNA transcripts derived from primary samples m62A-methylated. Our data indicate that TFB1M 12S m62A rRNA hypermethylation is unlikely to be a pathogenic mechanism and may be an artefact of previous experimental models studied. We propose that RNA methylation studies in experimental models should be validated in primary clinical samples to ensure that they are applicable to the human situation.The mitochondrial DNA mutation m.1555A>G predisposes to hearing loss following aminoglycoside antibiotic exposure in an idiosyncratic dose-independent manner. However, it may also cause maternally inherited hearing loss in the absence of aminoglycoside exposure or any other clinical features (non-syndromic hearing loss). Although m.1555A>G was identified as a cause of deafness more than twenty years ago, the pathogenic mechanism of this mutation of ribosomal RNA remains controversial. Different mechanistic concepts have been proposed. Most recently, evidence from cell lines and animal models suggested that patients with m.1555A>G may have more 12S rRNA N6, N6–dimethyladenosine (m One in 520 people carries a maternally inherited mitochondrial DNA (mtDNA) mutation, m.1555A>G, which predisposes to irreversible hearing loss following aminoglycoside antibiotic exposure . HoweverMT-RNR1 gene, which encodes the mitochondrial ribosomal 12S RNA (12S rRNA). The m.1555A>G 12S rRNA mutation is located in the ribosomal aminoacyl-tRNA-acceptor site (A-site), where the correct tRNA is matched to the correct mRNA codon during protein synthesis .,10.9,10)et al. hypothesized that the nuclear-encoded mitochondrial transcription factor 1 (TFB1M) enzyme may play a role in m.1555A>G hearing loss  methylation of two successive adenosine nucleotides near the 3′ end of small rRNA subunits in almost all species .Fi.Fi62A r62A-methylated at the equivalent of m.1584A terminates at 42 bp, i.e. the presence of m62A methylation does not allow further extension  primer extension on a template m62A-methylated at the equivalent of m.1584A and m.1583A showed that it is not possible to detect m.1583A m62A methylation in this situation  m62Supplementary Material, Fig. S2A) (n = 3), calculated using area under the curve. Analysis of a control ρ0 206 cell line, lacking mtDNA, showed no primer extension, confirming primer specificity to the mitochondrial transcriptome .Consistent with earlier findings by others, we found HeLa 12S rRNA and a 143B.TK− cell line to be partially methylated  . Mean pe62A-methylated at m.1584A in both groups . We found that the absolute quantity of 12S rRNA transcript in 10 µg of RNA varied between individuals, but in all cases, no unmethylated transcript could be detected using our fluorescent assay . Individuals with mild hearing loss and severe hearing loss all showed full 12S m62A rRNA methylation at position m.1584A with no detectable unmethylated transcripts.Primer extension analysis of primary lymphocyte RNA from 14 patients with m.1555A>G  but not the m.1555A>G genotype  compared with a control cell line (a 1 : 2 ratio of methylated : unmethylated transcripts)  . Mouse mscripts) ,24.TFB1M gene on chromosome 6 ; GTP binding protein 3 (GTPBP3); and 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU). MTO1, GTPBP3 and TRMU are tRNA modification enzymes, involved in the metabolism of mitochondrial tRNAs (mt-tRNAs), particularly the post-transcriptional modification at position 34 on five specific mt-tRNAs  and otoacoustic emissions (OAE) at 6 weeks: ABR showed normal click-evoked responses, and OAE were clearly demonstrable on the left, less so on the right. At age 2 years, she was re-tested and found to have a moderate-to-severe hearing loss. Hearing loss was profound in the left ear by 3.5 years and in the right ear by 5.5 years. She received a unilateral cochlear implant aged 10 years with a second implant at 15 years. Clinical examination of both siblings was normal. She was diagnosed with m.1555A>G aged 4 years, at the same time as her brother, who was then aged 7 years.In Family 2, case II-1 is the first child of unrelated Sudanese parents and was born in the UK at 42 weeks of gestation following a normal pregnancy and uneventful vaginal delivery. She failed newborn hearing screening and was diagnosed with bilateral moderate to severe SNHL by ABR aged 2 months. At 10 months, she had a pyrexial illness with lymphadenopathy and was subsequently diagnosed with autoimmune neutropaenia. She received six doses of co-amoxiclav intravenously, but no aminoglycosides. She was diagnosed with m.1555A>G aged 3 years 8 months and underwent bilateral cochlear implantation at this age because her hearing loss was profound. She has never received aminoglycosides.Case II-2 is her younger brother. Delivery was normal but, in view of prolonged rupture of membranes (PROM), he was given five doses of benzyl penicillin (147 mg) and two doses of gentamicin (14.7 mg), the first at birth and the second 24 h later according to standard protocols for PROM. He failed newborn hearing screening aged 3 weeks, and hearing loss was confirmed at 4 weeks. ABR testing at 3 months showed bilateral profound hearing loss. He was tested and found to have m.1555A>G at 13 months and received bilateral cochlear implants aged 2 years but has had no other hospital admissions. Clinical examination was normal in both siblings. Both parents have normal hearing up to and including 8 kHz, but mother has a maternal history of hearing loss starting in the fourth decade .Family 3 consists of twins, their older brother and their mother who are Ghanaian in origin. The first child was born following a normal pregnancy and delivery. In the twin pregnancy, there were no complications before 36 weeks when there was PROM. Both twins were delivered by Caesarian section because of a maternal history of uterine fibroids requiring myomectomy. Twin 1 was a female weighing 3.01 kg, and Twin 2 (the proband), a male weighing 2.89 kg. In view of PROM, both twins were given Benzyl Penicillin and Gentamicin according to protocol for PROM. They received two doses, levels were normal and medication was stopped on Day 3. Both twins passed the newborn hearing screen.Twin 2 had normal speech development, but he was found to have a hearing loss on school entry at 4 years. Current testing at age 8 years shows him to have a moderate asymmetrical hearing loss, worse in the high frequencies [maximum 60 decibels hearing level (dBHL) at 2 kHz in the right ear and 50 dbHL at 4 kHz in the left ear]. Following discovery of hearing loss in Twin 2, Twin 1 who had expressive language delay at age 7 years was tested; she has a mild–moderate high-frequency hearing loss, milder than her brother. The 20-year-old brother of the proband has normal hearing. The mother has a mild bilateral hearing loss, which reaches moderate in the high frequencies. In the family history, there is also reported childhood-onset hearing loss in the maternal uncle and aunt , and adult-onset hearing loss in the maternal grandmother and maternal great uncle.m RNA oligonucleotide was incubated with 1 µl of 50–100 nm 5′-Ned-labelled primer and 20 units of RNAse Inhibitor (NEB) at 55°C for 20 min. Twenty units of Avian Myeloblastosis Virus (AMV) Reverse Transcriptase (NEB), ×10 reaction buffer, 20 units of RNase Inhibitor (NEB) and 1 µl 500 nm dNTP stock (containing ddGTP in place of dGTP) were added, and the reaction mixture was incubated overnight at 42°C in a final volume of 10 µl. One microliter was mixed with 8.95 µl Hi-Di Formamide (Applied Biosystems) and 0.05 µl GeneScan Rox size standard (Applied Biosystems) and run on an ABI3130 Genetic Analyser (Applied Biosystems). GeneMarker software (SoftGenetics) was used for fragment analysis. Reactions were performed in triplicate.Methylation was detected using a primer extension protocol Fig. . Unmethy62A .The 38-bp, HPLC-purified, 5′ Ned-labelled DNA primer used was 5′ ACATCATCATCATCATCATCATTCGGTTCGTCCAAGTG 3′. A 25-bp tail sequence was added to make the primer 38 bp in length, the minimum reliable length that is compatible with fragment separation on the ABI3130 sequencer. The synthetic, HPLC-purified, model RNA oligonucleotides used (Dharmacon) were as follows: 5′ AAGUGUACUGGAAAGUGCACUUGGACGAAC 3′ for the unmethylated RNA template, 5′ CUGGAdmAAGUGCACUUGGACGAACC 3′ for the template methylated at the equivalent of m.1584A, 5′ CUGGdmAAAGUGCACUUGGACGAACC 3′ for the template methylated at the equivalent of m.1583A and 5′ CUGGdmAdmAAGUGCACUUGGACGAACC 3′ for the template methylated at the equivalent of m.1584A and m.1583A. dm in the oligonucleotide sequence represents mGG 3′)  and concentrated using 3 K centrifugal units (Millipore). m.1555A>G was detected using restriction digestion and Sanger sequencing. First, a 339-bp fragment was amplified by PCR (forward primer 5′ GCTCAGCCTATATACCGCCATCTTCAGCAA 3′ and reverse primer 5′ TTTCCAGTACACTTACCATGTTACGACT0 206 cell lines were a kind gift from Dr J.-W. Taanman. The HeLa and 143B.TK− cell lines were cultured in high-glucose DMEM supplemented with 10% FBS. The 143B.TK− ρ0 cell line was supplemented with 50 μg/ml uridine. Cells were free of mycoplasma infection (Promocell).Control and m.1555A>G primary fibroblast cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (FBS) (Sigma) and 50 µg/ml uridine (Sigma). Fibroblasts were isolated using an established isolation protocol in the absence of aminoglycoside antibiotics. Control and m.1555A>G lymphoblastoid cells were cultured in high-glucose RPMI (Life Technologies) supplemented with 10% FBS (Sigma). The cancerous bone 143B.TK- and 143B.TK-ρm 5′-Ned-labelled primer at 55°C for 20 min. Twenty units of AMV Reverse Transcriptase (NEB), ×10 reaction buffer, 20 units of RNase Inhibitor (NEB) and 1 µl of 500 nm dNTP stock (containing ddGTP in place of dGTP) were added. The reaction was incubated overnight at 42°C, desalted using 3K-centrifugal filters (Millipore), vacuum desiccated and re-suspended in 8.95 µl HiDi Formamide (Applied Biosystems), 1 µl dH2O and 0.05 µl GeneScan Rox size standard (Applied Biosystems). Reactions were run and analysed as described previously. Samples were analysed in triplicate.Total RNA (10 µg) was incubated with 4 nHMG online.Supplementary Material is available at This work was supported by UCL Grand Challenges, Sparks Children's Charity, Great Ormond Street Hospital Children's Charity and the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Trust. Funding to pay the Open Access publication charges for this article was provided by University College London.


Few things are more important to an older adult than safety and high health-related quality of life. Equally relevant is the ability of those surrounding such an adult to understand that individual’s functionality. However, achieving this reality is not simple because most people do not have experience living with older adults who have multiple, complex or even distinct health problems, including hearing loss.Although a large proportion of adults retain good hearing throughout life, age-related hearing loss (A-RHL) is quite common among elderly individuals While no treatment is currently capable of restoring lost hearing, sophisticated experimental and human studies into hearing restoration have been conducted over the last decade rd carbon from the end of the carbon chain 71013162225Omega-3 FAs, primarily eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are considered essential fatty acids 7With regard to hearing loss, the importance of omega-3 FA and fish consumption on the risk of A-RHL was elegantly demonstrated by Gopinath et al. Hearing loss in the elderly significantly impacts everyday life. Due to the aging of populations in developed and developing countries


TCs were found in perivascular connective tissue and smooth muscle bundles in sham oviduct, with typical ultrastructural features , and specific immunophenotype of CD34-positive/vimentin-positive/c-kit-negative. However, in EMs-affected oviduct tissue (grade III), extensive ultrastructural damage , significant decrease or loss of TCs and interstitial fibrosis were observed, together with elevated level of iNOS, COX-2, LPO and estradiol, thus suggestive of inflammation and ischaemia-induced TCs damage. Based on TCs distribution and intercellular connections, we proposed that such damage might be involved in structural and functional abnormalities of oviduct, such as attenuated intercellular signalling and oviduct contractility, impaired immunoregulation and stem cell-mediated tissue repair, 3-D interstitial architectural derangement and tissue fibrosis. Therefore, TCs damage might provide a new explanation and potential target for EMs-induced tubal damage and fertility disorders.Women with endometriosis (EMs) have unexplained infertility. The recently identified telocytes (TCs) might participate in the maintenance of structural and functional integrity of oviduct tissue, but so far the involvement of TCs in EMs-affected oviduct tissue and potential impact on fertility capacity remain unknown. By an integrated technique of haematoxylin and eosin staining,  Endometriosis (EMs) is an inflammatory, ischaemic, hyperestrogenic condition associated with many clinical manifestations, particularly in women of childbearing age Telocytes  exist in interstitial space of many normal tissues of mammalian and human organs We suggested that, chronic exposure to pelvic micro-environments, which is characterized by overproduced inflammatory factors ad libitum before experiments. Rats were obtained from the Medical Experimental Animal Administrative Committee of Soochow university , and handling procedures meet the guidelines of the Institutional Ethics Review Board of Soochow university.Three-month-old virgin female Sprague–Dawley rats (200–250 g) were used, all with regular 4- to 5-day estrous cycle before and after surgery, as determined by vaginal cytology The autotransplantation rat model of EMs-affected oviduct tissue was surgically constructed in mature female rats in oestrus 3) were fixed in 4% formalin and embedded in paraffin. Transverse serial sections (5 μm) were processed for haematoxylin and eosin, in situ immunohistochemistry (IHC) and immuofluorescent staining. The rest of fresh tissue (1 mm3) was processed for transmission electron microscopy (TEM). Healthy oviduct taken from the sham group served as control.At 2 months, all rats were killed and oviduct segment with grade III ectopic endometriotic vesicles was harvested in situ IHC staining. The primary antibodies were rabbit anti-rat polyclonal CD34 , mouse anti-rat monoclonal vimentin , rabbit anti-rat polyclonal c-kit . Images of the same areas of interest in consecutive sections were observed by light microscope. Omission of the primary antibodies served as the negative controls.Sections from sham group (5 μm) were subjected to procedures for Firstly, for quantitative determination of cytotoxic substance, single-labelled fluorescent IHC staining was used. Briefly, sequential sections (5 μm) from both groups were exposed to primary antibodies: rabbit anti-rat polyclonal iNOS , mouse anti-rat polyclonal COX-2 , rabbit anti-rat polyclonal LPO , and rabbit anti-rat polyclonal estradiol . Then FITC-goat anti-rabbit/mouse IgG  was added. Finally, sections were coverslipped with antifade mounting medium . Immunofluorescence intensity was quantitatively analysed by laser confocal scanning microscopy . Total fluorescent intensity per selected area was calculated by multiplying the number of pixels/area with the area mean intensity. More than 500 cells obtained from three separate microscopic fields were analysed for each marker in situ double-labelled immuofluorescent staining was applied. Briefly, sequential 5-μm sections from both groups were exposed to the primary antibodies in pairs : CD34 (1:100), vimentin (1:100), c-kit (1:100). Then CY3-goat anti-rabbit IgG for CD34 , FITC-goat antimouse IgG for vimentin , TRITC-goat anti-rabbit IgG for c-kit  were added . Finally, counterstained with DAPI  and mounted with antifade medium . Sections were then observed with fluorescence microscope . The number of TCs was identified in ten EMs-affected and ten sham samples, respectively, in a double-blinded manner (one section per sample), by correlating unique morphology (piriform/spindle/triangular-shaped cell body and extremely long and thin prolongations) with immunofluorescence, together with well-defined nuclei under 10 randomly selected microscopic high-power fields in the merged images per section , and statistically analysed Secondly, for further distinguishing, localization and precise counting of TCs, 3) from both groups were subjected to routine epon-embedding procedure Fresh oviduct fragments . P < 0.05 was considered statistically significant.All values were expressed as mean ± SD. i) hyperplasia and disturbance of capillaries within the wall  increased lymphocyte infiltration and increased contents of fibre, suggestive for chronic inflammation and interstitial fibrosis  inosis Fig.. Howeverosis Fig..In particular areas on consecutive sections from sham oviduct, these cells with similar TCs morphology: stellate-shaped cells with prolonged cell body located around the vessels, demonstrated positive expression for CD34 Fig. and vimeP = 0.000; Fig.P = 0.000; Fig.P = 0.000; Fig.P = 0.002; Fig.Firstly, in EMs-affected oviduct tissue, significant up-regulation of all the markers was found as compared with the sham group, respectively: iNOS , emerged from cell body and extend to various directions. More detailedly, Tps are made by an alteration of thin segments (podomers) and thick segments (podoms). In addition, Tps accommodate the organelles, such as mitochondria, rough endoplasmic reticulum, cytoskeletal elements, caveolae and microvesicles Fig. and B.Telocytes frequently established homocellular contacts with their Tps, or make close contact with various types of vicinity cells through heterocellular junctions. TCs were resident dominantly around capillaries; two or three layers of TCs run parallel to each other and/or formed a sheath with their long prolongations enfolding the vascular endothelial cells, together with homocellular junctions through their Tps, or heterocellular junctions with fibrocyte and pericytes Fig.. MoreoveConversely, in EMs-affected oviduct tissue, which contained typical ectopic endometriotic glands with abundant secretory granules Fig., TCs disEndometriosis is a hyperestrogenic gynaecological disease characterized by the presence of endometrium outside the uterine cavity, affects an estimated 8–10% of women in their reproductive age and causes fertility problems in industrialized countries Conventionally, stromal cells in oviduct tissue mainly include myocyte, dendritic cells, macrophages, MCs, plasma cells, eosinophils, lymphocytes, plus interstitial cells of Cajal (ICC), which were the pacemaker cells regulating oviduct rhythmic peristalsis Nevertheless, most of current studies were focused on TCs in normal tissues, except a very few reports in cardiac, skin and Crohn's disease via the bio-functions that have been proposed based on their distribution and ultrastructure in different organs, as follows, although none of them has been certainly proved yet.Recent genetic and proteomic analysis showed that lung TCs were completely different from fibroblasts Firstly, homocellular contacts between TCs themselves Fig. and heteThen, ultrastructure damage, loss of TCs Fig.–E and elSecondly, the existence of SC niches, which were supported or guarded by TCs and their Tps, with intercellular connections between them Fig., indicatetc.), with intercellular different signalling mechanisms Thirdly, TCs usually embedded into bundles of collagen and elastic fibres, connected or combined different structural components of interstitium into an integrate system with their extremely long and thin Tps in present study and previous literatures i) dysregulation of intercellular signalling, including immunoregulation/immunosurveillance, pacemaker activity, pelvic pain of EMs, (ii) impaired SC-mediated oviduct repair and/or regeneration capacity, and formation of interstitial fibrosis, (iii) impaired 3-D extracellular architecture reorganization, which were structural basis for intercellular signalling, tissue repair/remodelling and homeostasis.Taken together, although the exact function(s) of TCs is (are) still not well defined, our results demonstrated a broad involvement of TCs, accompanied by the fibrotic remodelling in EMs-affected oviduct wall and might contribute to derangement of tissue architecture: (Nevertheless, in EMs, what exactly happened on the pathway, by which TCs mediate cell interactions with other structural components of oviduct, remains undefined. And in future steps, animal models with defects in oviduct TCs networks still need to be developed to clarify the real functional consequences of TCs damage on reproductive activities. Also, it would be more valuable to perform high-throughput technologies such as comparative proteomics and bioinformatics analysis between disease-affected and -unaffected tissue, to provide new insights into potential roles of TCs in oviduct pathophysiology. And, additionally, whether TCs damage simultaneously occurred at uterine level and affects fertility capacity in EMs may be another critical issue. Finally, like emerging treatments aimed at promoting regeneration and reparation of injury-induced acute myocardial infarction by using TCs transportation


Callithrix jacchus). We examined 20 genes whose expression in the cochlea has already been reported. The deafness genes GJB3, CRYM, GRHL2, DFNA5, and ATP6B1 were expressed in marmoset cochleae in patterns different from those in mouse cochleae. Of note, all those genes are causative for progressive hearing loss in humans, but not in mice. The other tested genes, including the deafness gene COCH, in which mutation recapitulates deafness in mice, were expressed in a similar manner in both species. The result suggests that the discrepancy in the expression between rodents and primates may account for the phenotypic difference. This limitation of the rodent models can be bypassed by using non-human primate models such as the marmoset.Hearing impairment is the most frequent sensory deficit in humans. Deafness genes, which harbor pathogenic mutations that have been identified in families with hereditary hearing loss, are commonly expressed in the auditory end organ or the cochlea and may contribute to normal hearing function, yet some of the mouse models carrying these mutations fail to recapitulate the hearing loss phenotype. In this study, we find that distinct expression patterns of those deafness genes in the cochlea of a non-human primate, the common marmoset ( Deafness is one of the most common types of congenital impairment, and at least half of which are caused by hereditary mutations. In some patients, hearing loss is progressive, with hearing loss developing gradually during childhood or youth after the acquisition of speech abilities. In these post-lingual hearing loss cases, a hearing aid or cochlea implant is effective because the auditory cortex has already developedAs of 2015, the genes responsible for hearing loss have been identified in 99 autosomal recessive565Callithrix jacchus), which exhibits more human-associated traits than Old World monkeys, including a richer variety of vocalizations and verbal communicationTherefore, we wanted to use non-human primates to investigate the anatomy of hearing organs and the pathophysiology of hearing loss. In particular, we were interested in a small New World monkey species, the common marmoset , CRYM, GRHL2 (DFNA28), DFNA5, and ATP6B1  with hearing loss), all of whose pathophysiology remains unknown because their rodent models did not recapitulate the deafness phenotype. We found that these genes had distinct expression patterns, in which the proteins were distributed in the cochlea differently in primates and rodents. We also examined the expression profiles of COCH (DFNA9) and CONNEXIN26 , which have mouse models that recapitulate the deafness phenotype. We found that those proteins were expressed in a pattern similar to that reported for rodents.In this study, we established a successful immunohistochemical approach for the common marmoset and examined the expression patterns of 20 genes in the inner ear, genes whose expression has already been investigated in rodent or human cochleae . The genTaken together, our results suggest that the discrepancy of the expression between rodents and primates accounts for the phenotypic difference between them, and that the limitations of rodent models in researching human hereditary hearing loss could be bypassed by using non-human primate models such as the common marmoset.Few studies1214+/K+ ATPase α1, and CTGF. The lateral wall of the cochlea is composed of type I–V lateral wall fibrocytes and three layers of cells in the stria vascularis . The cell types of the lateral wall fibrocytes are defined by their spatial distribution and structure16+/K+ ATPase α1 immunoreactivity was observed in type II, IV, and V fibrocytes and marginal cells. CTGF immunoreactivity was observed in type I, II, III, IV, and V fibrocytes, the basilar membrane, and the spiral limbus , CX30, CTGF, AQUAPORIN 1 (AQP1), CALDESMON, CARBONIC ANHYDRASE II (CAII), Na-K-Cl COTRANSPORTER 1 (NKCC1), Nal limbus .In the organ of Corti, immunoreactivity for MYOSIN 7a (MYO7a) and CALBINDIN was observed in three rows of outer hair cells and one row of inner hair cells. SOX2, a well-established supporting cell marker, was observed in some supporting cells: Hensen’s cells, Deiters’ cells, inner pillar cells, outer pillar cells, inner phalangeal cells, and border cells . In spir+/K+ ATPase α1, MYO7a, SOX2, and β-III TUBULIN in the cochlea of common marmosets was similar to that previously reported in mouse and other rodents. On the other hand, two proteins, CTGF and PERIPHERIN, were expressed differently between marmosets and rodents: CTGF expression was observed in all types of fibrocytes in the common marmoset, whereas its expression in mice is limited to type IV fibrocytesThe expression of CX26, CX30, CTGF, AQP1, CALDESMON, CAII, NKCC1, NaGJB3 geneCX31, also known as gap junction beta-3 protein (GJB3) or DFNA2B/DFNB91, is a gap junction protein, and in humans it is encoded by the CRYM gene. In humans, CRYM is expressed exclusively in the inner ear, as shown by a cDNA microarray analysis of gene expression24CRYM , also known as NADP-regulated thyroid-hormone-binding protein (THBP), is a crystallin structural protein encoded by the Drosophila gene grainyhead. In the common marmoset, GRHL2 expression was observed in the lateral wall spiral ligament, hair cells, supporting cells, and SGNs , a multi-subunit enzyme that mediates acidification of eukaryotic intracellular organelles. In contrast to the limited expression in the spiral limbus in mouseCOCH gene. In humans, the expression of COCH is restricted to the inner earCOCH  is coded by the To validate specificities of the antibodies used in this study, we performed immunostaining of the skin, the small intestine or the kidney of common marmoset . Antibod333435Hearing loss is the most frequent congenital sensory impairment. Approximately 1 out of every 1000 newborns suffers from deafnessWe chose six genes that cause congenital hereditary progressive hearing loss and we characterized their differential expression pattern between the common marmoset and rodents.GJB3 gene, which encodes CX31, cause DFNA2B/DFNB91. Hearing loss in DFNA2B has a progressive and down-sloping pattern. Remarkably, CX31 knockout mice do not suffer from hearing loss33Mutations in the CRYM gene cause progressive and down-sloping hearing loss24CRYM mutations cause hearing loss through disturbing the thyroid hormone-binding properties of the cochleaMutations in the GRHL2 gene cause DFNA28, resulting in progressive, down-sloping hearing lossGRHL2 is embryonic lethal, and no established hearing loss model is availableMutations in the DFNA5 cause progressive, down-sloping hearing impairmentIn vitro studies have shown that the transfection of mutant DFNA5 causes cell death, whereas transfection of wild-type DFNA5 does notDFNA5 do not suffer from deafnessDFNA5-induced hearing loss are thought to be gain-of-function mutations. In mice, it is expressed  in the stria vascularis and the greater epithelial ridge, as determined by RT-PCRMutations in ATP6B1 gene causes dRTA with hearing loss. In autosomal recessive dRTA, a substantial fraction of the patients have sensorineural hearing loss with a progressive, down-sloping patternMutation of the COCH gene cause DFNA9, which is associated with progressive hearing lossCOCH gene between mouse, humanMutations in the In this study, we analyzed six proteins causing progressive hereditary hearing loss, five of which  do not recapitulate human symptoms in the mouse model . Their ai.e., knockout) model may sometimes explain the symptoms. For example, deficiency of Connexin 30 in mice causes severe hearing impairment as observed in human patients of GJB6, which is caused by mutations in CX30Mouse mutants are a useful and powerful tool for examining the pathophysiologies of hereditary diseases, including deafness, but they are not always applicable. For missense mutations, a gain-of-function (including a dominant negative function) model may account for the symptoms, and, in these instances, a transgenic or knock-in approach is effective. For example, transgenic mice expressing a deafness-causing mutant of connexin 26, a mutant carrying the R75W mutation, have hearing impairments similar to those observed in human patientsHowever, these models are based on the assumption that the distributions of targeted gene products are same in humans and rodents, and we have found examples showing that this assumption is not always true by examining non-human primate cochleae. Considering the correlation of phenotypical difference and differential expression patterns, it may be helpful to examine the expression profiles of target genes in primates and rodents before creating a mouse mutant. The common marmoset model offers a useful platform to evaluate expression patterns in the primate because it avoids the difficulties of immunostaining human temporal bone, which is time-consuming and technically difficult.in vivo model will be helpful in the future. Knowledge of the inner ear has so far been largely dependent on experiments performed in rodent models , primarily because of well-established techniques in the biotechnology: The reagents are optimized to those species and the genetic modification is enabled. However, our results indicate that the difference in the expression patterns may lead to the phenotypical discrepancy between rodent models and human patients. We propose the common marmoset, a non-human primate that is easy to handle, as a promising option for bridging the species gap. Studies using genetically modified marmosets57Also, the difference in the life span of human and mouse may result in the failure of recapitulating human disease phenotypes in mice, especially for the bilateral progressive hearing loss or adult-onset hearing loss. In those diseases, the life-span of mouse may not be long enough considering the onset of the phenotypes. For these diseases, non-human primates, which have longer life span than mice, would be more appropriate. Another possibility that should be raised in the discussion of the discrepancies in phenotypes of human cases and mouse models is that of a human deafness gene being erroneously assigned as a deafness gene. Whether a mutation found in patients is pathogenic is essentially defined through an inductive process; thus, supporting experiments in disease models evaluating such mutation are of great importance. In this process, confirmations using human disease cells or non-human primate Fixed and decapitated cadaverous heads of the 3–6 years old common marmosets were kindly provided from Reona Kobayashi, Takahiro Kondo, Kimika Yoshino-Saito, and Seiji Shiozawa. Fixed small intestine and kidney samples were also kindly provided from them. The animal experiments were approved by the ethics committee of Keio University (number: 11006) and were in accordance with the guidelines of the National Institutes of Health, and the Ministry of Education, Culture, Sports, Science and Technology of Japan.Temporal bones from young adult marmosets were dissected, fixed, decalcified with Decalcifying Solution B  for 3–4 weeks, and embedded in Tissue-Tek O.C.T. compound  for cryosection. The 7 μm sections were used for immunohistochemistry.Small intestines, kidneys and facial skins from young adult marmosets were used for validations of antibodies. Those tissues were dissected and fixed with 4% PFA, and the specimens were embedded in Tissue-Tek O.C.T. compound for cryosection. The 7 μm sections were used for immunohistochemistry.Temporal bones from 3 weeks old mice (C57BL/6) were dissected, fixed, decalcified with Decalcifying Solution B for 3 days, and embedded in Tissue-Tek O.C.T. compound for cryosection. The 7 μm sections were used for immunohistochemistry. This animal experiments using mice were approved by the ethics committee of Keio University (number: 08020) and were in accordance with the guidelines of the National Institutes of Health, and the Ministry of Education, Culture, Sports, Science and Technology of Japan.After a brief wash with PBS, sections were heated (80 °C) in 10 mM citrate buffer (pH 6) for 1 h. After a brief wash, the sections were preblocked for 1 h at room temperature with 10% normal serum in PBS, incubated with primary antibodies at 4 °C overnight, and incubated with Alexa Fluor-conjugated secondary antibodies  for 60 min at room temperature. The nuclei were counterstained with Hoechst 33342. For validations of secondary antibodies immunohistochemistry was performed without primary antibody in an overnight incubation step.Primary antibodies were incubated with or without (control) peptide used for generating antigens provided from antibody suppliers. After overnight incubations following immunohistochemical procedures were performed with using these antibody as primary antibody.The primary antibodies used in this study are as follows: anti-SOX2 , anti-CX26 , anti-CX30 , anti-CX31 , anti-CTGF , anti-AQP1 , anti-CALDESMON , anti-CA II , anti-NKCC1 , anti-Na+/K+-ATPase α1 , anti-CALBINDIN , anti-ZO-1 , anti-CRYM , anti-MYOSIN 7a , anti-GRHL2 , anti-DFNA5 , anti-COCH , anti-β-TUBULIN III , anti-ATP6B1 , and anti-PERIPHERIN .For GRHL2, PrEST Antigen GRHL2  and for ATP6B1 .How to cite this article: Hosoya, M. et al. Distinct Expression Patterns Of Causative Genes Responsible For Hereditary Progressive Hearing Loss In Non-Human Primate Cochlea. Sci. Rep.6, 22250; doi: 10.1038/srep22250 (2016).


Hearing loss is the most common sensory deficit in humans, affecting 1 in 500 newborns. Due to its genetic heterogeneity, comprehensive diagnostic testing has not previously been completed in a large multiethnic cohort. To determine the aggregate contribution inheritance makes to non-syndromic hearing loss, we performed comprehensive clinical genetic testing with targeted genomic enrichment and massively parallel sequencing on 1119 sequentially accrued patients. No patient was excluded based on phenotype, inheritance or previous testing. Testing resulted in identification of the underlying genetic cause for hearing loss in 440 patients (39 %). Pathogenic variants were found in 49 genes and included missense variants (49 %), large copy number changes (18 %), small insertions and deletions (18 %), nonsense variants (8 %), splice-site alterations (6 %), and promoter variants (<1 %). The diagnostic rate varied considerably based on phenotype and was highest for patients with a positive family history of hearing loss or when the loss was congenital and symmetric. The spectrum of implicated genes showed wide ethnic variability. These findings support the more efficient utilization of medical resources through the development of evidence-based algorithms for the diagnosis of hearing loss.The online version of this article (doi:10.1007/s00439-016-1648-8) contains supplementary material, which is available to authorized users. Hearing loss is the most common sensory deficit in humans. It is diagnosed in 1 in 500 newborns and affects half of all octogenarians  for clinical genetic testing from January 2012 to September 2014. All genetic screenings were done on a custom-designed TGE + MPS panel called OtoSCOPE® v4  or v5  which targets 66 or 89 deafness-associated genes, respectively (Table S2) using custom-designed SureDesign capture technology . Each platform included all known NSHL and NSHL mimic genes at the time of design . Samples were analyzed in pools of 48 samples sequenced on an Illumina HiSeq  flow cell using 100-bp paired-end reads. If pre-determined quality control values were not met, the sample was rerun, as previously described  for sample preparation. The testing platform was either OtoSCOPEData were analyzed using a local installation of the open-source Galaxy software  and <0.0005, respectively] , minor allele frequency (MAF) from 1000 Genomes Project Database and the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project Exome Variant Server [thresholds for recessive and dominant NSHL were <0.005 .All provided clinical and phenotypic data were recorded. Diagnostic rates were compared using the Fisher exact test  or Chi-square test (comparing more than 2 groups), with 1119 unrelated patients were sequentially accrued during the study period. Relations were not included; otherwise, there were no exclusionary criteria. Patient demographics were binned into broad key categories: inheritance, onset, severity, laterality, physical exam and previous genetic testing  (Table S3). Of these patients, 101 (23 %) received a genetic diagnosis implicating an NSHL mimic, which included Usher syndrome (59 patients), Pendred syndrome (29 patients), Deafness-infertility syndrome , Alström syndrome (1 patient), autosomal dominant non-ocular Stickler syndrome (1 patient), branchiootorenal syndrome (BOR) (2 patients), MYH9-associated disease (1 patient), and Wolfram syndrome (1 patient) (Table S4).During the course of this study, the TGE + MPS platform was updated from v4 to v5 as part of our standard operating procedure, increasing the number of genes screened from 66 to 89. Of the 711 patients analyzed on v5, 11 patients carried causative variants in genes not included in v4, thus increasing the diagnostic rate by 2 % in all patients screened with V5 and accounting for 4 % of all positive diagnoses (11 of 263 positive diagnoses). Read metrics for V4 and V5 are shown in Table S5. Although patients sequenced with v5 had a lower average number of reads and lower average target coverage, the percentage of reads overlapping target was higher, as was the coverage at 1, 20, and 30×.Our analysis of 1119 patients identified 5900 variants, which we reported to healthcare providers. 14 % of variants were considered causally related to the hearing loss phenotype and reported as pathogenic or likely pathogenic; 4 % were previously reported pathogenic variants for recessive hearing loss, with a second variant not identified (carrier status); and 82 % of variants were reported as VUSs. The median number of reported variants was 4 (range = 0–14) and 5 (0–19) for v4 and v5, respectively .p < 0.05), while in patients with a family history of recessive hearing loss it was only 41 % (not significant—n.s.). In patients with no family history of hearing loss, the diagnostic rate was 37 % (p < 0.05).There was considerable phenotypic diversity that impacted the overall diagnostic rate of 39 % Fig. . In patip < 0.005 in both cases). Patients with bilateral hearing loss were significantly more likely to receive a diagnosis than patients with asymmetric or unilateral hearing loss . Patients with conductive or mixed hearing loss had a decreased likelihood of receiving a genetic diagnosis , but the difference was not significant.When age of onset is considered, patients with congenital hearing loss had a diagnostic rate of 44 %, which was significantly greater than the diagnostic rate in patients with childhood (29 %)- or adult (28 %)-onset hearing loss (p < 0.005), as compared to patients with NSHL . In patients with a clinical diagnosis of Usher or BOR syndromes, the diagnostic rate was 31 and 37 %, respectively. In none of the 15 patients with neurological findings  and hearing loss was a non-syndromic genetic cause for deafness identified (Table S6).Any kind of abnormality on physical exam decreased the likelihood of a genetic diagnosis using this panel . With congenital onset and symmetric hearing loss, the rate increased to 48 % (p < 0.005), and if the physical examination was normal, it increased further to 51 % (p < 0.005). The same trend was true for patients with family histories of dominant and recessive hearing loss—their diagnostic rates jumped to 67 and 55 %, respectively, when the hearing loss was congenital and symmetric and the physical examination was otherwise normal.Combining demographic characteristics provided a more realistic assessment of the diagnostic rate Figs. , 2. PatiFor adult-onset hearing loss, the diagnostic rate was 28 %, however, if the family history was positive, the diagnostic rate climbed to 50 %, and if the patient also had symmetric hearing loss, the diagnostic rate jumped again to 67 %.Only when the hearing loss was unilateral was there a marked negative impact on diagnostic rate (1 % of patients). This finding, when combined with any other characteristic, decreased diagnostic success Fig. .p < 0.005). In the cohort self-identified as Caucasian , the diagnostic rate was 38 %. However, in cohorts self-identified as Asian  and Middle Eastern , the diagnostic rate was 63 and 72 %, respectively (p < 0.005). The diagnostic rate was lowest in African Americans , at 26 %, p < 0.05 , STRC (16 %), SLC26A4, (7 %) and TECTA (5 %), although this list varied based on degree of hearing loss. For example, while variants in GJB2 were the most common cause of severe-to-profound hearing loss (20 %), STRC accounted for 30 % of diagnoses in persons with mild-to-moderate hearing loss, followed closely by GJB2 (25 %) and then TECTA (7 %). SLC26A4 pathogenic variants were identified in 7 % of patients with positive diagnoses; however, all of these patients had severe-to-profound hearing loss (10 % of severe-to-profound hearing loss).In total, 49 genes were causally implicated in hearing loss Table . HoweverSTRC-related deafness was just as likely to be diagnosed as GJB2-related deafness , but in Middle Eastern or Asian patients, GJB2 diagnoses were more common than STRC diagnoses . No African American patients were diagnosed with GJB2-related hearing loss . These genes contributed to 20 % of all 440 diagnoses, including one dominant diagnosis.The profile of causal variant type differed with inheritance pattern. Amongst all 440 diagnoses, 49 % were due to missense variants (Table S7); however, if the hearing loss was dominantly inherited, missense variants were diagnosed 85 % of the time, as compared to 46 % with recessive inheritance. Variants predicting null alleles were much more common with recessive diagnoses—CNVs, indels, nonsense variants, and splice variants made up 20, 19, 9, and 6 % of recessive and 2, 3, 5, and 5 % of dominant diagnoses. 146 CNV alleles in 9 different genes were identified as causative in 88 patients  at which identified genetic variants in each patient were discussed in the context of the patient-specific phenotype, a genetic cause of hearing loss was identified in 440 patients (39 %) (Table S3). Several smaller studies have reported similar diagnostic rates .EYA1, a well-recognized phenotype–genotype association  of the patients in this study self-identified as Caucasian and had a diagnostic rate of 38 %. In patients of Middle Eastern ethnicity, the diagnostic rate was higher (72 %), an increase that reflects the higher coefficient of inbreeding in this population  suggests that there is a ‘discovery gap’ to fill in these ethnic groups , which is in agreement with other reports , underscoring the requirement that all comprehensive genetic testing panels for hearing loss include CNV detection.Of variants with a MAF of <0.01, the largest majority were of unknown significance . In addition, however, we identified several known or likely pathogenic variants associated with ARNSHL in genes without a second causal variant. For example, 151 of the 679 patients, in whom a genetic diagnosis was not made, carried reported ARNSHL-causal variants without having a second variant in the coding sequence of that gene. This carrier rate of 22 % is roughly 8 times higher than that reported in hearing control populations and suggests that many of these patients have yet-to-be-identified non-coding mutations  captures this area of active study in an open-source, continuously updated, interpretational database that we maintain on all variant positions interrogated on the OtoSCOPE platform.In summary, we believe that comprehensive genetic testing is a foundational diagnostic test that allows healthcare providers to make evidence-based decisions in the evaluation of hearing loss thereby providing better and more cost-effective patient care Fig. ), mandatSupplementary material 1 (PDF 408 kb)Supplementary material 2 (PDF 211 kb)


Telocytes (TCs) are a peculiar type of interstitial cells with very long prolongations termed telopodes. TCs have previously been identified in different anatomic structures of the heart, and have also been isolated and cultured from heart tissues in vitro. TCs and fibroblasts, both located in the interstitial spaces of the heart, have different morphologies and functionality. However, other than microscopic observation, a reliable means to make differential diagnosis of cardiac TCs from fibroblasts remains unclear. In the present study, we isolated and cultured cardiac TCs and fibroblasts from heart tissues, and observed their different morphological features and immunophenotypes in primary culture. Morphologically, TCs had extremely long and thin telopodes with moniliform aspect, stretched away from cell bodies, while cell processes of fibroblasts were short, thick and cone shaped. Furthermore, cardiac TCs were positive for CD34/c-kit, CD34/vimentin, and CD34/PDGFR-β, while fibroblasts were only vimentin and PDGFR-β positive. In addition, TCs were also different from pericytes as TCs were CD34 positive and α-SMA weak positive while pericytes were CD34 negative but α-SMA positive. Besides that, we also showed cardiac TCs were homogenously positive for mesenchymal marker CD29 but negative for hematopoietic marker CD45, indicating that TCs could be a source of cardiac mesenchymal cells. The differences in morphological features and immunophenotypes between TCs and fibroblasts will provide more compelling evidence to differentiate cardiac TCs from fibroblasts. Telocytes (TCs), a distinctive type of interstitial cells, have firstly been identified by Popescu’s group . TCs areTo date, transmission electron microscopy (TEM) remains a golden standard method for identifying TCs, since not a single immunomarker can be considered specific for detecting TCs . HoweverThe aim of the present study was to observe different morphological features of cardiac TCs and fibroblasts in primary culture, and to determine their different immunophenotypes by using a set of double immunofluorescent staining for CD34/c-kit, CD34/vimentin, and CD34/PDGFR-β, which might provide more reliable means and evidence to discriminate between TCs and fibroblasts in heart tissues.ad libitum access to food and water. All animal experiments were conducted under the guidelines on the use and care of laboratory animals for biomedical research published by National Institutes of Health . This study was approved by the committee on the Ethics of Animal Experiments of Tongji Hospital (Permit Number: KYSB-2014–41). All surgery was perfomed under sodium pentobarbital anesthesia and all efforts have been made to mininmize suffering.Male C57BL/6 mice, weighed 25–30 g (10–12 weeks), were purchased from the animal research center of Fudan University. Mice were housed in a temperature-controlled room on a 12 h light/dark cycle, with 3 pieces, transferred into 50 mL centrifuge tube, and incubated on an orbital shaker at 37°C for 35 min with the collagenase solution as described above. After 25 mL ice-cold HBSS was added into the digest to inhibit collagenase activity, dispersed cells were separated from non-digested tissues by passing through a 40-μm-diameter cell strainer and collected with centrifugation at 1000 rpm for 5 min at 4°C. After washed once by centrifugation in HBSS, cells were resuspended with 10 mL DMEM/F12 supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin, and seeded into sterile culture dishes. Cells were then cultured in a humidified atmosphere of 5% CO2 at 37°C for 2 h to purify the cell suspension with TCs by allowing fibroblasts attachment. The unattached cells (containing TCs) were collected and cultured in DMEM/F12 supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin for 24 h and culture medium was changed. Cell cultures were examined using inverted biological microscope , and TCs and fibroblasts were photographed under 200× magnification at 48 h, 72 h and 96 h after seeded.After mice were anesthetized with 1% pentobarbital sodium, the hearts were isolated under sterile conditions and kept in Hank’s balanced salt solution , supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin  and 0.01 mM HEPES . After transported to the cell culture room and rinsed again with fresh HBSS, the hearts were transferred into a sterile culture dish containing DMEM/F12  supplemented with 0.25 mg/ml collagenase typeⅡ  and 0.01 mM HEPES. The hearts were then minced into 1 mm2 until soft. After DMEM containing 1% BSA and 100μM CaCl2 was added into the digest to inhibit collagenase activity, dispersed cells were separated by 100-μm-diameter cell strainer and collected with centrifugation at 1500 rpm for 5 min at 4°C. After washed with DMEM containing 1% BSA and 500μM CaCl2 and layered over 5ml of DMEM containing 6% BSA, cells were centrifugated at 800 rpm for 10 min to separat a clear upper layer from a darker layer. Cells from the latter were cultured in DMEM/F12 supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.Pericytes were isolated and cultured as previously described . BrieflyBMSCs were isolated and cultured as our previous described . BrieflyAfter washed with PBS for three times, cells were fixed in 4% paraformaldehyde for 30 min, washed with PBS, and permeabilized with 0.5% Triton X-100 for 30 min. Cells were then washed with PBS and blocked in 3% bovine serum albumin (BSA) for 1 h. After that, cells were incubated overnight at 4°C with rat monoclonal anti-CD34  and rabbit polyclonal anti-c-kit  primary antibodies diluted by 1:100 in 1% BSA. After washed with PBS for three times, cells were incubated with goat anti-rat FITC-labelled  and goat anti-rabbit rhodamine-labelled  secondary antibodies diluted by 1:200 in 3% BSA for 2 h, and then stained with DAPI . Finally, cells were kept in fresh PBS at 4°C in dark before observation. The images were taken under a magnification of 200× with fluorescent inverted microscope . Similar procedures were used for double immunofluorescent staining for CD34/vimentin  or CD34/PDGFR-β  or CD34/α-SMA .Cells were harvested by trypsinization and washed twice with cold PBS. TCs were stained with allophycocyanin-conjugated anti-mouse CD29  and CD45  while Rat-BMSCs were stained with FITC-conjugated anti-rat CD29  and CD45 . As negative controls, cell aliquots were incubated with PBS under the same conditions. After incubation away from light for 30 min at 4°C, cells were analyzed by MoFlo XDP (Beckman Coulter) and FlowJo software (Tree Star).Cardiac TCs in primary culture were purified from fibroblasts by using differential adhesion method. In the present study, fibroblast attachment occurred within 2 h after seeded, while TC attachment took 24 h. To observe the different morphological features of both cell types, cardiac TCs and fibroblasts were observed under light microscope and photographed at 48 h, 72 h and 96 h from the day of primary culture. As shown in To identify different immunophenotypes of cardiac TCs and fibroblasts, three different double immunofluorescent methods  were used. As shown in As shown in Cardiac TC and fibroblasts were isolated from heart tissues and their different morphological features were observed in primary culture. Meanwhile, we provided further evidence showing that cardiac TCs were positive for CD34/c-kit, CD34/vimentin, and CD34/PDGFR-β, while fibroblasts were only positive for vimentin and PDGFR-β, which clearly differentiated cardiac TCs from fibroblasts in primary culture.Isolation and culture of TCs from a given organ is of great interest to study the peculiar morphological features and functionality of TCs ,32,33,58Double immunolabeling is of great importance to make differential diagnosis of TCs from other cells in primary cultures or tissues ,13,59. IIt has been indicated that TCs could be a source of cardiac mesenchymal cells . To addrIn conclusion, the present study shows the different morphological features and immunophenotypes between cardiac TCs and fibroblasts in primary culture. Our results present here, as well as the comparison of gene profiles, microRNAs signatures and proteome features of these two cell types highly desirable to be determined in the future, will provide new lines of evidence to differentiate TCs from fibroblasts in heart tissues.


Liver fibrosis is a wound-healing response which engages a variety of cell types to encapsulate injury. Telocyte (TC), a novel type of interstitial cell, has been identified in a variety of tissues and organs including liver. TCs have been reported to be reduced in fibrotic areas after myocardial infarction, human interstitial wall's fibrotic remodelling caused either by ulcerative colitis or Crohn's disease, and skin of systemic sclerosis. However, the role of TCs in human liver fibrosis remains unclear. Liver samples from human liver biopsy were collected. All samples were stained with Masson's trichrome to determine fibrosis. TCs were identified by several immunofluorescence stainings including double labelling for CD34 and c-kit/CD117, or vimentin, or PDGF Receptor-α, or β. We found that hepatic TCs were significantly decreased by 27%–60% in human liver fibrosis, suggesting that loss of TCs might lead to the altered organization of extracellular matrix and loss the control of fibroblast/myofibroblast activity and favour the genesis of fibrosis. Adding TCs might help to develop effective and targeted antifibrotic therapies for human liver fibrosis. Liver fibrosis, primarily as a result of chronic damages to the liver such as chronic viral hepatitis and fatty liver diseases associated with obesity, is a worldwide burden Telocytes (TCs) are a novel type of interstitial cell identified in a variety of tissues and organs including liver Telocytes have been reported to be reduced in myocardial infarction, especially in fibrotic areas In this study, based on immunofluorescence methods including double labelling for CD34 and c-kit/CD117, or vimentin, or PDGF Receptor-α, or β, we showed that in human liver fibrosis, hepatic TCs were significantly decreased, suggesting that loss of TCs might lead to the altered organization of extracellular matrix and loss the control of fibroblast/myofibroblast activity and favour the genesis of fibrosis.Human liver fibrosis samples were obtained from liver biopsy examination in Division of Gastroenterology and Hepatology, Digestive Disease Institute, Tongji Hospital. As healthy control tissues, samples were from patients who underwent surgery because of neoplastic pathologies. Specimens were collected at least 10 cm from the margin of the tumour. Healthy specimens were carefully chosen to be sure no inflammatory or neoplastic infiltration occur. All samples were divided to healthy or fibrosis specimens based on histopathological examination. All the patients had signed a written informed consent form and the Institutional ethical committees approved this study. The investigation conforms to the principles that are outlined in the Declaration of Helsinki regarding the use of human tissues.Liver samples were fixed in 4% paraformaldehyde in PBS, and were embedded in paraffin, and sectioned at 5-μm thick. Haematoxylin–eosin and Masson's trichrome staining were performed with standard procedures to investigate liver histological and fibrotic changes.For investigate the changes in TCs in human liver fibrosis, double immunofluorescent staining for CD34/PDGFR-α or CD34/PDGFR-β or CD34/Vimentin or CD34/c-Kit was used as our previously reported t-test for independent samples. P-values that were less than 0.05 were considered to be statistically significant.All analyses were evaluated using SPSS 19.0 International Business Machines Corporation, Armonk, NY, USA. Data are expressed as mean ± SD and were analysed by using the Student's n + 4) or a fibrosis phase (n + 9).The histopathological changes in all liver samples in this study were determined by haematoxylin–eosin and Masson's trichrome staining. In healthy liver stage group, hepatocytes had a radial array with clear central veins without occurrence of inflammation, necrosis and fibrosis Fig.. In fibrAlthough transmission electron microscopy (TEM) examination is a golden standard for the identification of TCs, immunolabelling, especially double immunolabelling, remains a useful tool for discrimination between TCs and other cells, as well as for semi-quantitative data analysis. As we and many others have reported, here we used four different double labelling immunofluorescence methods to analyse the changes in TCs in human liver fibrosis, including double labelling for CD34 and PDGFR-α, CD34 and PDGFR-β, CD34 and vimentin, and CD34 and c-kit/CD117. Quantitative analysis of FigureThis study reports a novel finding that TCs are reduced in human liver fibrosis, mirroring the recent findings as described in the colonic wall in ulcerative colitis, the terminal ileum of patients affected by small bowel Crohn's disease, and skins, gastric wall, lung and myocardium in systemic sclerosis Fibrosis is a wound-healing response which engages a variety of cell types to encapsulate injury via either cell-to-cell contacts or shedding microvesicles or exosomes or paracrine secretion including microRNAs It remains to be determined whether the reduction in TCs precede the process of fibrosis or just a consequence of the fibrotic process. However, based on the literature, several relevant and potential roles could be proposed. First, the reduction in TCs in liver could participate in the abnormal 3D organization of the extracellular matrix as they are involved in intercellular signalling In conclusion, this study firstly demonstrated the decrease in TCs in human liver fibrosis based on a series of double immunoreactions. Considering the fact that intramyocardial transplantation of cardiac TCs could decreases MI and improves post-infarcted cardiac function


Borrelia burgdorferi ospC types from the southeastern U.S.A. supported the common belief that various ospC types are geographically restricted and host specific. Being widely distributed in the region, the southeastern population of B. burgdorferi is represented by a surprisingly small number of ospC types. Types B, G and H are dominant or common and are invasive, while scarce type L, restricted mostly to the southeastern U.S.A., is believed to rarely if ever cause human Lyme disease. OspC type B and L strains are represented in the region at the same rate, however their distribution among tick vectors and vertebrate hosts is unequal.Analysis of B. burgdorferi ospC type L strains to disseminate into host tissues. Mice were infected by subcutaneous injections of B. burgdorferi strains of various ospC types with different invasive capability. Spirochete levels were examined in ear, heart, bladder and joint tissues. Noninfected I. ricinus larvae were fed on infected mice until repletion. Infection rates were determined in molted nymphs. Infected nymphs were then fed on naïve mice, and spirochete transmission from infected nymphs to mice was confirmed.Direct diagnostics was used to analyze the ability of B. burgdorferi ospC type L strains from the southeastern U.S.A. have comparable potential to disseminate into host tissues as ospC types strains commonly associated with human Lyme disease in endemic European and North American regions. We found no difference in the invasive ability of ospC type B and L strains originated either from tick vectors or vertebrate hosts. Borrelia burgdorferi sensu lato (s.l.) species is characterized by its tick vectors, host spectrum, geographical distribution and, for the pathogenic species, its organotropism [B. burgdorferi strains, classified by ospC type, reveals the ratio between that type‘s frequency in vector ticks compared to human patients [It is known that each otropism . The relpatients .B. burgdorferi s.l. endemic area, despite prevailing dogma concerning LD in the United States [Ixodes scapularis  is the only vector of the spirochetes in the eastern U.S.; B. burgdorferi is antigenically and genetically uniform in North America in contrast to the situation in Europe; B. burgdorferi does not occur in wildlife in the southern U.S.A. and thus, humans in the Southeast could not acquire LD [I. scapularis-Peromyscus leucopus enzootic cycle in the northeastern U.S.A. is mirrored by the I. scapularis-P. gossypinus transmission cycle in the Southeast which is further enhanced by Ixodes affinis and Ixodes minor as vital maintenance vectors of Borrelia in areas where they occur, and Sigmodon hispidus and Neotoma floridana as additional major reservoir hosts [B. burgdorferi strains that cause LD in both areas will differ as well. Our previous research showed that of the 4 ospC types, B, G, H and N that have been detected in LD patients in the northeastern and midwestern U.S.A., 3 types, B, G, and H, at a lower rate, are widely distributed in the southeastern United States [OspC type B is associated with severe LD around the world [ospC types H and G are commonly detected in tissues at disseminated sites of LD patients from the northeastern and midwestern U.S.A. [B. burgdorferi ospC type L strains were not considered to have any impact in LD in North America in general and, in the southeastern U.S.A. particularly, because: i) ospC type L was considered to be restricted to Europe only; ii) ospC type L is recognized as very rare worldwide; and iii) ospC type L strains were previously detected in ticks only [Published data on prevalence of Lyme disease (LD) spirochetes in vector ticks and vertebrate hosts in the southeastern U.S.A. qualifies this region as d States . Brieflyquire LD ,4. It isir hosts ,5. As thhe world ,7, ospC n U.S.A. ,8. B. bucks only ,6–9.B. burgdorferi ospC type L strains are associated with the non-human biting tick I. affinis that feeds on the same vertebrate hosts as human-biting I. scapularis in the southeastern U.S.A. [Borrelia strains from I. affinis might be transmitted to humans [B. burgdorferi ospC types B and L strains are the most prevalent in the region. They are represented at the same rate overall, but their distribution among the tick vectors and the rodent hosts is unequal. While 75% of highly invasive ospC type B strains were isolated from rodents and 25% from ticks, in the case of ospC type L, host- and vector - originated strains represented 37.5% and 62.5%, respectively. For a long time it was believed that ospC type L strains are incapable of causing human LD [B. burgdorferi ospC type L strain (SLV-1) was cultured from a skin biopsy from a Slovenian patient with acrodermatitis chronica atrophicans (ACA), commonly associated with B. afzelii infection [I. ricinus nymphs collected in Switzerland (NE5222 and NE5266) and Slovakia (SKT-2) [ospC type L strain (SCW-9) was isolated from secondary sites of infection of cotton rat (Sigmodon hispidus) trapped in South Carolina, U.S.A. [ospC type L strains are one of the two most prevalent in this region [B. burgdorferi ospC type L strains to disseminate into vertebrate hosts and to compare their invasive potential with infective strains.Globally rare n U.S.A. . Howevero humans . B. burgnfection . Additiorain SCW- was isolrain SCW- was isolBorrelia strains were grown in BSK-H medium  according to the protocol described previously [ospC types defined earlier [ospC type B strain SLV-2 , cotton mouse (Peromyscus gossypinus) originated ospC type L strain SCCH-30 , I. ricinus nymphs originated ospC type L strain NE5222, ospC type B strain NE5264 and ospC type V strain NE5248  and I. affinis female originated ospC type B strain SCW-53 . Six weeks old female C3H/HeN mice  were infected by subcutaneous injections of 105 spirochetes in 100 μl of BSK-H medium per mouse (2 mice per strain). Presence of spirochetes in ear biopsies was determined at weekly intervals by PCR. Total DNA was extracted using a NucleoSpin Tissue Kit  according to the manufacturer’s protocol. Detection of spirochetes was performed by amplification of a 154 bp fragment of flagellin gene using primers FlaF1 (AAGCAAATTTAGGTGCTTTCCAA) and FlaR1 (GCAATCATTGCCATTGCAGA) [rd week after infection the presence of spirochetes was confirmed in all ear biopsy samples regardless of the ospC type of strain used for infection. I. ricinus larvae were obtained from the pathogen-free tick colony of the Institute of Parasitology (Czech Republic). At the 4th week after inoculation, non-infected larvae were fed on infected mice until repletion (100 larvae per mouse) and left to molt. Infection rates were determined in pools of ten molted nymphs using PCR described above. Nymphs were considered to be infected if >80% of them were PCR positive. Ten infected nymphs were then fed on naïve mice (5 mice per strain); spirochete transmission from infected nymph to mice was determined as described above. At week 6th, spirochete load was determined in positive biopsies from mouse ear, heart, bladder and joint by quantitative PCR using primers described above, the TaqMan FlaProbe1 (TGCTACAACCTCATCTGTCATTGTAGCATCTTTTATTTG) and a LightCycler 480 (Roche) [ospC genes from final mouse samples confirmed their identity to the ospC genes of B. burgdorferi strains used for initial infection [Low passage eviously ,12. Straeviously  and were earlier : human o (Roche) . SpirochB. burgdorferi ospC type B strains and ospC type L strains, previously found only in ticks  and L  strains were comparable in each tissue analyzed (Table ospC type B (NE5264) and L (NE5222) strains originated from human biting I. ricinus was almost equal in each tissue (Table ospC type B (SCW-53) originated from non-human biting I. affinis was more than 10 times higher in mouse joints than was the spirochete load of human-originated ospC type B (SLV-2) or human-biting I. ricinus originated (NE5264) strains. Our results confirm that tick originated strains NE5264 and NE5222 of ospC types B and L and host originated ospC type B strain SLV-2, show the same pattern of dissemination in all analyzed host tissues, with no preferential site of infection. Identical pattern of dissemination was revealed for non-human biting tick originated ospC type B strain SCW-53, and rodent originated ospC type L strain SCCH-30 , with joints as the preferable site of spirochete accumulation. Analysis of I. ricinus originated ospC type V strain NE5248 showed that, in contrast to types B and L, this ospC type preferably disseminates into bladder, not joints (Table PCR was used to confirm infection of laboratory mice with invasive ks Table . An ospCed Table . Dissemiue Table . HoweverB. burgdorferi ospC type L strains originated from human, rodent or hard tick were able to disseminate into laboratory mice as well as invasive ospC type B strains that are responsible for systemic disease in humans. Therefore, further study on the pathogenicity of B. burgdorferi ospC type L strains is warranted. The qPCR results in this study defined heart as a tissue with the lowest load of spirochetes, while the joints showed the highest load of borrelia in mice infected either with ospC type B or L strains (Table B. burgdorferi with Lyme arthritis [ospC type L strains to disseminate into vertebrate host tissues in the same manner as invasive ospC type B strains, known to be responsible for severe disease in humans worldwide, increases the possible disease risk to humans in the southeastern U.S.A., the region, where studied strains are widely distributed [ns Table . Our resrthritis ,16. The tributed .


P < 0.01) during follow-up. The incidence rate ratios (IRRs) of death were 2.5 times higher in ACPP patients than in comparisons (P < 0.01). The risk of death was particularly high in the first month after ACPP  and still high for ∼6 months . After adjusting for age, gender, selected comorbidities, geographic area, and monthly income, the hazard ratio of death for ACPP patients was still 2.4 times higher than for comparisons. Older age (≥35 years), male gender, diabetes mellitus, coronary artery disease, hypertension, stroke, mental disorder, and lower monthly income also predicted death. ACPP significantly increased long-term mortality. In addition to early follow-up after acute treatment, comorbidity control and socioeconomic assistance are needed for patients with ACPP.Acute anticholinesterase pesticide (organophosphate and carbamate) poisoning (ACPP) often produces severe complications, and sometimes death. We investigated the long-term mortality of patients with ACPP because it is not sufficiently understood. In this retrospective nationwide population-based cohort study, 818 patients with ACPP and 16,360 healthy comparisons from 1999 to 2010 were selected from Taiwan's National Health Insurance Research Database. They were followed until 2011. Ninety-four (11.5%) ACPP patients and 793 (4.9%) comparisons died ( Under high oxidative stress, cells typically undergo necrosis because of tissue damage, which can include subchronic and chronic toxicity and inflammation in various tissue types throughout the body.16 Some studies7 have been conducted but focus almost exclusively on predicting acute mortality and managing complications; however, the long-term prognosis of ACPP is still unclear. Therefore, we used Taiwan's National Health Insurance Research Database (NHIRD) to investigate, in a nationwide retrospective cohort study, the long-term mortality of patients with ACPP. We aimed to determine whether patients with ACPP have a higher mortality risk than do the general population because of the chronic toxicity of anticholinesterase pesticides and neurologic sequelae.Zunec et al17 The NHIRD contains all claims data from 1996 through 2011. This study used the Longitudinal Health Insurance Database 2000 (LHID2000), a subdataset of the NHIRD, which contains all claims data of 1 million  beneficiaries who were randomly selected in 2000. The age, gender, and healthcare costs between the LHID2000 dataset and all NHI enrollees are not significantly different.The Taiwan National Health Insurance (NHI) Program is a universal healthcare system that covers nearly 100% of the country's population.In this retrospective cohort study, we selected patients from the LHID2000 who had been newly diagnosed with ACPP (ICD-9 code 989.3) between January 1, 2002, and December 31, 2010 as the ACPP cohort. Members of the comparison cohort  were randomly selected from the LHID2000 by matching age, gender, and index date (when ACPP was first diagnosed in the database) with the ACPP cohort.We linked to the diagnostic codes through the NHIRD and collected data including demographics, comorbidities, survival status, and date of death. Comorbidities affecting mortality that may have presented before the index date were defined as follows: diabetes mellitus (DM) (ICD-9 code 250), coronary artery disease (CAD) (ICD-9 codes 410–414), stroke (ICD-9 codes 430–438), hypertension (HTN) (ICD-9 codes 401–405), and mental disease (ICD-9 codes 290–319). We considered these to be comorbidities if they occurred either in the inpatient setting or in 3 or more ambulatory care claims coded before the index date. Patients were followed from the index date to the date of death or the end of the database period. All citizens in Taiwan are required to participate in the NHI, and their enrollment must be withdrawn within 30 days postmortem. Therefore, patients recorded as deceased or disenrolled within 30 days of their discharge were presumed dead, and the discharge date was designated as the date of death. Figure This study was conducted according to the Declaration of Helsinki. The Institutional Review Board at the Chi-Mei Medical Center approved this study and waived the need for informed consents from patients because the dataset consists of deidentified data. This waiver does not affect the rights and welfare of the patients.2 tests for categorical variables and Student t test for continuous variables to compare the demographic characteristics and comorbidities18 and Poisson regression by calculating the incidence rate ratio (IRR) to compare the risk of death18 between ACPP cohort and Comparison cohort. The survival curves between 2 cohorts were compared by Kaplan–Meier analysis and the log-rank test.18 We used multivariate Cox proportional hazard regressions with adjustment for confounders18 to determine the effect of ACPP, age, gender, comorbidities, geographic region, and monthly income on the risk of death. We used SAS  for all the analyses in this study. Significance was set at P < 0.05 (2-tailed), except for the Poisson regression, in which a more conservative level of significance was set at 0.0033 (0.05/15) with Bonferroni correction because of the multiple comparisons performed.We used Pearson χWe recruited 818 patients with ACPP cohort and 16,360 age-, gender-, and index date-matched comparison cohort  for death during the follow-up period. After patient age, gender, comorbidities, geographic region, and monthly income had been adjusted for, ACPP  was still an independent predictor of mortality in all patients . Older people and men in Taiwan might have more opportunity than do others to use pesticide for suicide.12 Older age also predicted death for acute ACPP.23 The elderly are in poorer physiological condition and have altered toxicokinetics and toxicodynamics which predispose them to poor outcomes.23 However, younger and female patients may have fewer comorbidities; therefore, ACPP is one of the most likely factors that leads to death.The majority of patients with ACPP were older (≥35 years) and male Table ; however24 reported that nearly half of the patients with ACPP had a history of a mental disorder (43.2%) and were undergoing long-term treatment with antipsychotic and antidepressant medications (25.4%). Another study12 in Taiwan reported that intentional exposure from suicide attempt, occupational exposure, and accidental exposure were 64.72%, 15.82%, and 13.27% in the patients with ACPP, respectively. In the United States, unintentional ACPP exposure  rarely resulted in mortality.25 On the other hand, several clinical studies26 suggested that chronic exposure to organophosphate pesticides might be associated with mental disorders. Whatever the reason that these comorbidities predict mortality after ACPP, the present study's results indicate that we have to manage not only the poisoning in patients with ACPP, but also the patients’ comorbidities and socioeconomic problems.Comorbid DM, CAD, stroke, HTN, and mental disorder, and a monthly income <NT$ 15,840 predicted mortality. A recent hospital-based studyThis study has some limitations. First, The NHIRD did not indicate the severity of the ACPP, did not specify whether the pesticide was an organophosphate or carbamate, did not specify the exposure route, and gave no detailed personal information like smoking status, body mass index, treatment , or physical activity. All of these might be confounding factors. Second, misclassification of ACPP could not be completely avoided through using the ICD-9 codes in the claim records. It is possible that there were more cases of ACPP that were minor and treated, but not be correctly diagnosed. However, the consequence of such misclassification should be inclusion of ACPP patients in the comparison group and thus would tend to cause underestimation rather than overestimation of the relative risk. Therefore, our major conclusion that ACPP significantly increased long-term mortality should still be valid. Third, data on the cause of death, exposure types , and association between death and exposure types were not available in this study, and therefore further studies to clarify related issues. For example, patients caused by intentional or occupational exposures may have higher mortality than those caused by accidental exposure. Fourth, this study could not evaluate whether there was a difference in long-term mortality between organophosphate and carbamate. Further studies on this issue are warranted. Fifth, our findings might not be generalizable to other nations.This first nationwide population-based cohort study on ACPP showed that it not only caused acute mortality within 1 month but also increased long-term mortality during the first 6 months after poisoning. ACPP had a greater effect on younger and female patients. Comorbid DM, CAD, stroke, HTN, and mental disorder, and a low monthly income were also independent mortality predictors. In addition to acute treatment, early follow-up and secondary mortality prevention are needed for patients with ACPP.