Immune checkpoint inhibitors (ICIs) are the treatment of choice for several cancers and can be associated with remarkable clinical benefit, but can also cause serious immune-related adverse events (irAEs). Management of rare and severe irAEs is challenged by an incomplete knowledge of their natural history and pathogenetic mechanisms. We report a case of fatal acute-onset gastro-intestinal (GI) hypomotility from myenteric plexus neuropathy following a single dose of ipilimumab plus nivolumab given for treatment of Merkel cell carcinoma (MCC).A 66-year-old man with recurrent metastatic MCC involving several organs  developed profound pharyngeal dysphagia and ileus that started 7 days after receiving a single administration of combination immune checkpoint blockade consisting of nivolumab (3 mg/kg) and low-dose ipilimumab (1 mg/kg). A swallowing study showed oropharyngeal dysfunction and aspiration. Imaging studies were consistent with diffuse intestinal paresis. An extensive work-up did not reveal obvious causes of these symptoms, and enteric plexopathy was suspected. Empiric immune suppressive therapy was initiated urgently. Despite an escalating immunosuppressive regimen that included high dose steroids, tacrolimus and therapeutic plasma exchange, no improvement in GI motility was seen and the patient suffered repeated episodes of aspiration. Seven weeks after the onset of GI hypomotility, the patient succumbed to sepsis from intestinal perforations. At autopsy, histologic specimens obtained from the entire GI tract (pharynx to rectum) showed near complete loss of ganglion cells within the myenteric and submucosal plexuses. An associated inflammatory infiltrate was not seen, suggesting a ‘burned out’ phase of illness. C4d complement deposition was found at the ganglionic sites, suggesting antibody-mediated pathogenesis. Remarkably, at sites of previously suspected Merkel cell metastases, no residual viable Merkel cell carcinoma was identified.GI-tract paresis due to myenteric neuritis is a rarely reported toxicity of ICIs. Because the window of reversibility is likely to be very brief, quick and decisive interventions are warranted. Subtle functional and anatomic perturbations of the myenteric nervous system from the use of ICIs may be more prevalent than realized and should be suspected and addressed in both clinical and investigational settings. Immune checkpoint inhibitors (ICIs) have been associated with unprecedented clinical success in patients with many cancer types and have ushered in a therapeutic revolution in cancer immunotherapy. Monoclonal antibodies of this class stimulate T cell function by blocking suppressive ‘checkpoint’ receptors such as cytotoxic T-lymphocyte antigen (CTLA)-4 (e.g. ipilimumab), programmed death (PD)-1  or its target ligand, PD-L1  .While ICIs have substantially improved cancer outcomes, inhibiting immune regulatory mechanisms can also result in unique toxicities, termed immune related adverse events (irAEs), which include, but are not limited to, colitis, hepatitis, dermatitis, pneumonitis and endocrinopathies such as hypophysitis , 4. The As experience with the ICIs grows, rare and sometimes fatal side effects are emerging, including myocarditis , asystolWe had previously reported the case of a patient with metastatic melanoma who developed severe constipation from myenteric ganglionitis following treatment with two doses of ipilimumab . We now clinicaltrials.gov; NCT02488759). The patient provided informed consent for treatment and for information sharing as part of a research protocol approved by the University of Washington/Fred Hutchinson Cancer Research Center IRB. He received dual immune checkpoint blockade with one dose of ipilimumab (1 mg/kg) plus nivolumab (3 mg/kg) administered on day 1.A 66-year-old Caucasian man presented with recurrent, metastatic MCC that involved several sites in liver, bones and disseminated lymphadenopathy. Primary MCC diagnosed 3 years before was located on the forearm without lymph node involvement and was treated with surgery and adjuvant radiation therapy. Serologic testing for antibodies to the Merkel cell polyomavirus T-antigen oncoprotein was negative . The patAt the time of treatment on day 1, he had no concerning neurologic, GI or other symptoms. Seven days after the administration of first dose of combination immunotherapy, the patient reported diffuse muscle aches, urinary retention, abdominal distention and a sensation of ‘gagging’ with swallowing attempts. He began taking low doses of oxycodone 5 mg every 4-6 h with partial relief. Four days later (day 11 post-immunotherapy), he was hospitalized due to worsening symptoms and a need for supportive care. A CT scan showed air-filled loops of small and large bowel and an increased stool burden within the right colon consistent with pseudo-obstruction without clear signs of mechanical obstruction. Diverticulosis was present without pericolonic fat stranding suggestive of diverticulitis. Despite methylnaltrexone (given for treatment of possible opioid induced constipation) and escalating doses of laxatives, no bowel movement was produced. Abdominal distention and constipation persisted, together with recurrent bouts of emesis. On day 13 post-immunotherapy, the patient developed hypoxic respiratory failure, attributed to an episode of aspiration, and required mechanical ventilation. A CT scan showed mildly distended fluid filled small bowel loops, moderate to large stool burden in the right colon and fecalization of the terminal ileum contents. Microbiological blood and urine cultures were sterile. The patient was treated with supportive care for 5 days after which extubation was successful. Video laryngoscopy showed a widely patent airway, with severe aspiration due to swallow delay/discoordination, weakness of base of tongue, hyolaryngeal excursion, moderate to severely weak pharyngeal constrictors and cricopharyngeal dysfunction. The patient was also noted to have nasal regurgitation due to uncoordinated velar elevation. A fluoroscopic study of oral contrast (gastrografin) showed absence of contrast progression to colon 48 h following administration.On day 22 post-immunotherapy, the patient’s respiratory status decompensated again and repeat intubation was required. A second CT scan of the abdomen showed partial regression of the MCC metastases in the liver and resolution of retroperitoneal lymphadenopathy. A fluid-filled esophagus and dilated loops of proximal small bowel, again without a transition point, was essentially unchanged from the prior scan. Placement of a nasogastric tube returned nearly 2 L of bilious and later feculent material. This clinical presentation of diffuse intestinal paresis and pharyngeal dysfunction following administration of immune checkpoint blockade suggested the possibility of immune-mediated neuropathy of the enteric nervous system or a similar irAE (based on our prior experience with a similar case published by our group) . Due to Evaluating whether this patient was suffering from an immune adverse effect of checkpoint immunotherapy on myenteric ganglia was challenging because the myenteric plexus, which lies within the muscularis propria, is evaluable only by a full-thickness biopsy. The feasibility of this endoscopic procedure was discussed extensively with the multidisciplinary team involved in the patient’s care, but was considered to pose a prohibitively high risk for perforation and poor wound healing. There was also concern that use of endoscopic cautery after biopsy could ignite bowel gases in the setting of obstipation. The patient’s three prior episodes of pulmonary decompensation resulted in increased risk for prolonged intubation following anesthesia. These considerations also placed the patient at prohibitively high risk for surgery. Thus, invasive diagnostic testing was not attempted. Serologic testing for antibodies to the acetylcholine receptor was negative. Additional serologic tests for anti-Hu antibodies as well as antibodies to P/Q type and N type calcium channels, neuronal (V-G) potassium channels, striated muscle, antineuronal nuclear antibodies, anti-glial nuclear antibodies, Purkinje cell cytoplasmic antibodies, amphiphysin antibodies and CRMP 5 were negative. The patient did not exhibit any arrhythmia during periods of cardiac monitoring.Following 1 week of treatment with high dose steroids, there was no apparent improvement in gut motility and the patient continued to have no bowel movement or passage of flatus. On day 30, the patient was started on tacrolimus with a target trough of 5–15 ng/mL. Enemas were attempted to induce defecation, but were unsuccessful. Plasmapheresis to remove potential pathogenic autoantibodies was initiated on day 45 post-treatment and repeated every other day for a total of 5 treatments. A venting gastrostomy tube was placed on day 49 due to worsening abdominal distention. On day 51 post-treatment, a CT scan showed focal inflammatory stranding and air involving the transverse colon compatible with diverticulitis with contained perforation. The known MCC tumors had regressed further .To investigate the possible pathogenetic involvement of the humoral immune system, we stained histologic sections of colon tissue for C4d. C4d is a serine protease in the classical complement cascade that can be generated and deposited during complement activation by the antibody complexes, resulting in formation of multi-molecular complexes with immune effector and cytolytic activity . We founImmunotherapy with ICIs improves cancer outcomes, but can be associated with varied side effects. In the case above, a single dose of nivolumab plus ipilimumab led to complete eradication of a patient’s widely metastatic MCC, but unfortunately was complicated by rapid and irreversible destruction of the patient’s myenteric ganglions that ultimately proved fatal. The case described above illustrates both the promises and pitfalls of the power of immune checkpoint blockade.The striking clinical findings in this case were a marked impairment in managing oral secretions, a lack of flatus/bowel movements, uncoordinated swallowing seen on video laryngoscopy, and radiologic findings of fluid-filled bowels with minimal transit of oral contrast. These symptoms began within 1 week after the initiation of immune checkpoint blockade and prior to the use of opiates and several of the other therapies, including the use of enemas, tacrolimus, methylnaltrexone, and prednisone, which were applied during his hospitalization and have been potentially associated with gastrointestinal perforation , 25.In the current case, no conspicuous inflammatory infiltrate was seen around the ganglia at the time of autopsy (58 days post-treatment). This absence of infiltrating inflammatory cells in the ganglionic region could be due to either a ‘burned-out’ phase of the illness wherein the target of the immune attack has already been destroyed, or due to intense escalating immune suppression, or both.The present case involved the entire length of the gastrointestinal tract (pharynx to rectum) both clinically and pathologically, whereas in the previously reported case, constipation had predominated and the patient did not report any complaints related to swallowing. Despite these differences in clinical presentation, we now performed a careful retrospective review of autopsy slides from the prior case that revealed lymphocytes infiltrating myenteric ganglia throughout the gastrointestinal tract (data not shown).Both cases were associated with quick-onset immune responses against the cancer, resulting in complete pathologic eradication of disseminated metastases. Even though the two patients had differing cancer types (melanoma and MCC), there remains the possibility of a shared antigen between the myenteric neurons and the cancer cells.The striking absence of bowel movements and atony of the gut in this patient suggested damage to the myenteric plexus, similar to a case described previously by our group , in whicTwo other instances of intestinal pseudo-obstruction associated with immune checkpoint inhibitor therapy have been recently reported, but the myenteric plexus was not evaluated in these cases. A 57-year-old man with poorly differentiated neuroendocrine tumor developed pseudo-obstruction after 11 doses of pembrolizumab . In thisThe full spectrum of neurologic complications from checkpoint inhibitors is unknown, but is likely to be broad and heterogeneous. Others have reported cases of Guillain-Barré-like demyelinating polyneuropathy induced by ipilimumab, but no autopsy was available . PatientThe focal activation of complement proteins surrounding the myenteric plexus suggests the presence of antibodies directed at antigens expressed on myenteric nervous system. While our patient tested negative for anti-Hu antibodies, the development of anti-Hu antibodies is one well described mechanism of paraneoplastic neuropathy associated with both small cell lung cancer  and MerkIn our patient, the paucity of inflammatory cells and absence of neurons within the gut at autopsy suggests that further intensification of the patient’s immunosuppression closer to the time of death would not have been beneficial. This observation strongly suggests careful consideration of early and intensive immunosuppression, soon after development of suggestive symptoms, ideally after rapid confirmation of a pathologic diagnosis. While immune suppression was escalated fairly rapidly in our case after day 22, the initial delay in beginning steroids and the late introduction of plasmapheresis (day 45) may have contributed to irreversible damage. Until the exact mechanisms of this unusual and severe toxicity become apparent, we suggest prompt institution of therapies targeting both cellular and humoral immunity, the latter probably being quite important given the finding of C4d deposition at the target site of myenteric plexus. Concurrent rather than sequential implementation of immune suppression should be considered. Analysis of pooled data from Phase 2 and 3 trials of immunotherapy with combined ipilimumab plus nivolumab shows that antitumor efficacy was similar between those patients who discontinued immunotherapy due to irAEs and those who did not, suggesting that early discontinuation of ICIs and institution of immunosuppression may not completely abrogate anti-cancer activity .Because of uncertainty regarding the effects of immune suppression on ICI-induced anti-tumor response, a prompt and definitive pathologic diagnosis of ganglionitis may be needed to justify aggressive immunosuppresion. In our case, the pathologic mechanism for the clinical hypomotility syndrome was suspected relatively early on, but still could not be confirmed prior to death because the process of obtaining a full thickness biopsy was evaluated as prohibitively high risk. Obtaining tissue for histologic diagnosis should be considered early in the clinical course with input from GI and surgery teams, especially when the clinical diagnosis is uncertain. Sigmoidoscopy to the splenic flexure with extensive biopsies is usually sufficient for the diagnosis of immune mediated colitis. In the absence of diarrhea an esophagogastroduodenoscopy and sigmoidoscopy or colonoscopy with biopsies may be useful in evaluating for enteritis and signs of intestinal paresis. Deep rectal biopsies can safely evaluate Meissner’s submucosal plexus for neuropathy. Full thickness biopsies, usually performed by a surgical team, involve the entire thickness of the colon, which carries additional risks. Surgical biopsy may be reserved for cases with an unclear diagnosis following the above evaluations. In addition to establishing histologic diagnosis, there is also a need for objectively monitoring functional improvement in gut motility, especially to guide management of immune suppressive therapies. Potential cases could be evaluated by CT scans and by monitoring contrast transit time as well as video fluoroscopic swallow studies and speech pathology evaluation. Rectal or esophageal manometry may be helpful in objectively tracking progress in gut motility.The rarity of reported myenteric plexopathy as a potential irAE highlights the need to disseminate experiences such as this one, so that early recognition may prompt intervention. While constipation is a frequently reported adverse reaction in trials of immunotherapy, this has not typically been regarded as an irAE. It is unclear if neuronal injury commonly contributes to immunotherapy-related constipation with a mild presentation , 38, or


Visual impairment occurred as an infrequent form of chemotherapeutic toxicity and was often underestimated despite of several reports. We described a case of acute unilateral visual impairment after one cycle of intravenous chemotherapy of a normal dose, aiming at raising attention to chemotherapy-induced ocular toxicity.The patient developed a progressive vision loss in the right eye during the chemotherapy. After one cycle of intravenous chemotherapy, her visual acuity decreased by 0.6 in the right eye (VOD = 0.4) compared to the previous value of 1.0 (VOD = 1.0). No evidence of ocular infiltration was observed from the cerebral magnetic resonance imaging (MRI). During her follow-up period, we documented the ophthalmologic examinations including visual acuity, visual field (VF), visual evoked potential (VEP), electroretinogram (ERG), fundus photograph (FP), fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). Ophthalmoscope examination and fundus photograph showed optic disc edema, fuzzy boundary and linear hemorrhages in her right eye. Fundus fluorescein angiography (FFA) revealed capillary underdevelopment at the nasal and superior temporal area of the optic disc in the early phase and capillary fluorescein leakage in the late phase. The result of VEP test suggested the impaired function of the optic nerve. Thus, a diagnosis of nonarteritic anterior ischemic optic neuropathy (NAION) was made by the ophthalmologist according to these results. The patient was prescribed prednisone combined with neuroprotective drugs, which did not work. After the cessation of chemotherapy, her impaired vision gradually recovered.This is the first reported case of acute visual impairment in a patient who underwent chemotherapy of a normal dose. It is indicated that while receiving benefits from chemotherapy, cancer patients simultaneously suffer from the risk of vision loss. In recent years, it has been reported that chemotherapy could induce irreversible or reversible visual loss due to an infrequent form of chemotherapeutic toxicity. For instance, a case that a male patient who had lung cancer suffered from acute unilateral blindness after 5 cycles of cisplatin/gemcitabine  which was repeated every four weeks. The patient complained of visual loss in the right eye two weeks after the first cycle of chemotherapy, and she received the second cycle of chemotherapy as planned.A 48-year-old woman developed tinnitus and enlarged cervical lymph nodes. Nasopharyngoscopy examination and biopsy confirmed the diagnosis of nasopharyngeal squamous cell carcinoma, thus, she received the anti-tumor therapy of a combination of docetaxel, cisplatin and fluorouracil . Her visual acuity had a decrease of 0.6 in the right eye (VOD = 0.4) compared to the original value VOD = 1.0), and the relative afferent pupillary defect (RAPD) was tested for the right eye. She had a history of high myopia and amblyopia in her left eye evidenced by the low visual acuity . The current vision impairment in her right eye therefore seriously affected her quality of life. Ophthalmoscope examination and fundus photograph showed optic disc edema, fuzzy boundary and linear hemorrhages in the right eye  . ImpaireDue to a sharp decrease of visual acuity, the patient was suggested to consult in an ophthalmology clinic for etiological diagnosis and potential therapy. The diagnoses of glaucoma, cataracts, macular degeneration and other eye diseases were excluded; the right optic disk edema was observed by funduscopic examination; and a diagnosis of NAION was made with etiology unknown. NAION is the most common cause of acute visual loss in people aged over 50, resulting from non-inflammatory small vessel ischemic damage to the anterior portion of the optic nerve. However, the cause and pathogenesis of this disorder remain unclear , 5. Neur2 daily as part of a chemotherapeutic salvage regimen for non-Hodgkin lymphoma, but received actual dose of 100 mg/m2 daily for 4 days inadvertently. Except for anorexia, nausea and tinnitus, he developed bilateral decreased vision immediately after the treatment. The ERG showed diminished a-wave and missed b-wave [Vision loss attributed to cisplatin, docetaxel and fluorine has been reported in several previous studies. Cisplatin-associated retinal toxicity including blurred vision, color vision defects, and electroretinographic (ERG) changes was dose-dependent or unique to high doses. There was a case that a patient with lung cancer who received five cycles of cisplatin/gemcitabine treatment for lung cancer was admitted to the emergency room unfortunately and complained of acute blindness in his left eye. His fundus examination was normal in both eyes, and the MRI of the left optic nerve and orbit did not reveal any relevant findings. A diagnosis of left retrobulbar optic neuritis was made eventually . There wd b-wave . A clinid b-wave . The resd b-wave . AnotherWhen patients suddenly develop vision loss while receiving chemotherapy, the possibility of chemotherapy-induced NAION should be considered. Most ophthalmic complications are reversible if recognized in an early phase. Besides, dosage reduction or agent cessation could rescue patients from vision loss. However, if the optic nerve or the retinal was involved, patients might develop irreversible vision loss. Induction chemotherapy plays a crucial role in the treatment of local advanced nasopharyngeal carcinoma, and presented here was one case of severe visual impairment induced by chemotherapy. Therefore, it is indicated that cancer patients take the risk of vision loss while benefiting from chemotherapy.Although it has been reported that intravenous chemotherapy of cisplatin and docetaxel could cause retinal toxicity, this is the first case demonstrating that intravenous administration of chemotherapy (TPF) for NPC could induce NAION and cause irreversible vision loss. This reported case suggests that when patients suddenly develop visual impairment after receiving chemotherapy, the possibility of ophthalmic complications should be considered. In addition, as soon as symptoms are recognized, the patient should be scheduled for further examinations in the ophthalmology clinic, and a cooperation between oncologists and ophthalmologists is necessary for subsequent treatment.


Leydig cell tumors (LCTs) are rare testicular tumors that may be a cause of precocious puberty in males. We present a 5-year-old boy with a five-month history of pubic hair appearance associated with an increase in penile length, scrotal hyperpigmentation, change in body odor, and bone age advanced by two years. His hormonal tests revealed the diagnosis of pseudo precocious puberty. Testicular ultrasound showed a unilateral right testicular enlargement. Surgery was performed to remove the mass. Histopathology confirmed the diagnosis of LCT. This case report highlights the importance of consideration of testicular tumors in boys with precocious puberty. Leydig cell tumors (LCTs) are a sex cord-stromal gonadal tumor which arise from Leydig cells . They arA 5-year-old boy was brought to the pediatric endocrinology clinic with a five-month history of pubic hair appearance associated with aggressiveness, increased penile length, and change in body odor. He was a product of full-term, normal vaginal delivery, with uneventful medical and surgical histories. There was no family history of endocrine tumors or precocious puberty. On examination, his vitals were unremarkable. Pubic hair and genitalia were Tanner stage II and the volume of each testis was 4 ml Figure . HormonaThis case demonstrates a rare instance of precocious pseudo puberty caused by an LCT. While testicular tumors in adults are a widely discussed topic, there is a significant shortage of data about prepubertal testicular tumors. Testicular tumors are rare in pediatric patients and account for only 1% of solid tumors among this age group . PrepubeTesticular tumors are subdivided into germ-cell tumors  and sex-cord stromal cell tumors  . LCTs wiPrecocious puberty in boys is defined as the presence of testicular enlargement, penile growth, or pubic hair before the age of nine. Causes of precocious puberty can be categorized into two distinctive entities; central, or “true” precocious puberty, which involves an increase in GnRH, and peripheral, or “pseudo” precocious puberty, which occurs in a setting of low levels of GnRH. Central precocious puberty involves premature activation of the hypothalamic-pituitary-gonadal axis, and interaction between the central nervous system and various endocrine glands yields a sequential pattern of development of secondary sexual characteristics which is most often accompanied by a growth spurt . In pseuA literature review revealed several similar cases of prepubertal boys with LCTs presenting with precocious pseudo puberty manifested by increased penile length and various signs of androgenization -20. MéndAlthough rare, LCT is an important cause of precocious pseudo puberty in children. Testicular ultrasound can aid in early diagnosis and treatment in order to decrease the psychological and somatic impact of precocious puberty on prepubertal children.Although rare, LCT is an important cause of precocious pseudo puberty in children. Testicular ultrasound can aid in early diagnosis and treatment in order to decrease the psychological and somatic impact of precocious puberty on prepubertal children.


Despite the availability of an effective prophylactic vaccine leading to sterilizing immunity, hepatitis B virus (HBV) is responsible for chronic liver disease in more than 250 million individuals, potentially leading to cirrhosis and hepatocellular carcinoma. Antiviral drugs able to completely suppress virus replication are indeed available but they are, by and large, unable to eradicate the virus. Several alternative new treatment approaches are currently being developed but none have so far captured the interest of clinicians for possible clinical development. A constant feature of chronic HBV infection is T-cell exhaustion resulting from persistent exposure to high antigen concentrations as shown by the high expression of programmed cell death protein 1 (PD-1) by HBV-specific CD8 T cells. One way of tackling this problem is to develop HBV-specific neutralizing antibodies that would clear excess envelope proteins from the circulation, allowing for nucleos(t)ide analogs or other antiviral drugs now in preclinical and early clinical development to take advantage of a reconstituted adaptive immunity. Several fully human monoclonal antibodies (mAb) have been developed from HBV-vaccinated and subjects convalescent from acute hepatitis B that show different properties and specificities. It is envisaged that such neutralizing mAb may be used as adjuvant treatment to reduce viral protein load, thus rescuing adaptive immunity in an effort to optimize the effect of antiviral drugs. Hepatitis B virus (HBV) is responsible for acute and chronic hepatitis, potentially leading to cirrhosis and hepatocellular carcinoma [reviewed in ]. HepatiB-cell responses also play a fundamental role in HBV infection. Antibodies specific for HBsAg are critical for the neutralization of free extracellular HBV, thus preventing viral entry into susceptible hepatocytes . HoweverCurrent evidence supports the view that a coordinated activation of both T and B cells is critical for eradication of infection. Of note, CD4+ T cells control CD8+ T cell activity and antibody production , and theHumoral immunity seems to play an important, but only partially understood role in the control of HBV infection . This isIn contrast with the wealth of studies on antibody responses to HBV proteins, there is limited information on the B-cell phenotypic and functional features during HBV infection, particularly during the chronic phase. Of note, and in contrast with evidence of exhausted T cells in the peripheral blood of patients with chronic HBV infection, bulk peripheral blood B cells express several activation markers and produce large amounts of immunoglobulin in response to innate and adaptive immunity signals, significantly more than B cells from patients with chronic HCV infection . In agrein vitro , was reported to control immune responses at the innate and the adaptive levels, and only a few studies have investigated the role of Bregs in chronic hepatitis B. Bregs classically suppress immune function through secretion of the inhibitory cytokine IL-10, which inhibits the production of pro-inflammatory cytokines and supports regulatory T cell differentiation. One study reported increased Breg cells in PBMC of patients with chronic HBV infection during spontaneous hepatic flares, demonstrating suppressive IL-10 mediated activity of Bregs on HBV-specific CD8 cells in vitro . Enhancein vitro . HoweverTupaia belangeri model of HBV infection in vivo, whereas others apparently did not or had a low neutralization titer . The anti-HBV humAbs were originally developed to obtain sustainable reagents for immunoprophylaxis of HBV re-infection in liver transplant recipients and other settings, such as treatment of accidental needle-sticks and in the prevention of vertical-perinatal HBV transmission. Anti-HBs antibodies are usually administered as an additional prophylactic measure while waiting for the appearance of neutralizing anti-HBs antibodies. However, it has not escaped our attention that another potentially important application may emerge in the future as an adjuvant treatment of chronic HBV infection.Human memory B cells and plasma cells are a major source of antibodies. Indeed, B cells undergo somatic mutations and selection by antigen and T-cell help in response to pathogens and persist for life as memory cells in a given individual, rapidly responding to booster immunization to yield a large number of plasma cells. Even though long-lived plasma cells are the main source of serum antibodies, these terminally differentiated cells are certainly not ideal for long-term culture. Several methods have been developed to isolate human monoclonal antibodies (humAbs). The original relatively simple approach consists of the isolation of single antigen-specific Epstein-Barr virus (EBV)-immortalized B cells from donors with high-titer specific immunoglobulin (Ig) after labor-intensive, time-consuming rounds of cloning steps . This apon titer . Intereson titer . The datAntibodies are usually effective in prophylaxis if given shortly after viral exposure, their efficiency declining after infection. However, highly potent and/or broadly cross-reactive human monoclonal antibodies may counteract viral escape mechanisms and open new avenues for intervention. For instance, potent HIV-1 envelope-specific broadly neutralizing antibodies (super-antibodies) have been identified that may have potential therapeutic applications . In chroCooperation between the humoral and cellular arms of adaptive immunity is required for efficient elimination of pathogens. To this end, antiviral humAbs can be exploited to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), which takes place via specific receptors recognizing the Fc fragment of IgGs (FcγRs) expressed on the membrane of several cells involved in innate and adaptive immune responses. The FcγR family includes several classes of receptors that show different affinity for the Fc fragment. One of these, the low affinity FcγRIII (CD16), is the most potent activating receptor of NK cells mediating ADCC . To evalThe findings discussed above suggest that humAbs targeting HBV envelope epitopes might indeed have a potential for combination therapies in chronically infected individuals. Phase I clinical trials are therefore eagerly awaited in this setting.MM wrote and conceived the manuscript. AC, DM, and SM contributed to conception and generated data. BO, SV, and SM revised the manuscript, read, and approved the submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months.ClinicalTrials.gov (NCT02342522) and is completed.We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated  to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group . No important unexpected adverse events or side effects of remote ischaemic conditioning were observed.Remote ischaemic conditioning does not improve clinical outcomes  at 12 months in patients with STEMI undergoing PPCI.British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden. Despite timely reperfusion with primary percutaneous coronary intervention (PPCI), morbidity and mortality following acute ST-elevation myocardial infarction (STEMI) remain substantial, and improvements in mortality and survival free of heart failure have plateaued.3In the clinical setting, the cardioprotective remote ischaemic conditioning stimulus can be applied using serial inflations and deflations of a pneumatic cuff placed on the upper arm or thigh to induce brief cycles of ischaemia and reperfusion.Evidence before this studyTo improve clinical outcomes in patients with ST-elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (PPCI), new treatments are needed to protect the heart against acute ischaemia–reperfusion injury and thus reduce myocardial infarct size and prevent heart failure. Remote ischaemic conditioning with transient ischaemia and reperfusion of the arm or leg as an adjunct to PPCI has been shown in several small randomised controlled trials to reduce myocardial infarct size and increase myocardial salvage in patients with STEMI when compared with use of PPCI alone. However, whether remote ischaemic conditioning can improve clinical outcomes such as mortality and heart failure has not been confirmed. A PubMed search undertaken up to the start date of the trial  using the terms “remote ischemic conditioning; acute myocardial infarction; mortality; and heart failure” revealed no randomised controlled trials investigating the effect of remote ischaemic conditioning on clinical outcomes in patients with STEMI treated by PPCI.Added value of this studyIn this large, sufficiently powered, multicentre, randomised controlled trial of 5401 patients with STEMI, we investigated whether remote ischaemic conditioning applied as an adjunct to PPCI could reduce the incidence of cardiac death or hospitalisation for heart failure within 12 months post-PPCI, when compared with PPCI alone. Our findings showed that remote ischaemic conditioning did not improve clinical outcomes in terms of cardiac death or hospitalisation for heart failure at this timepoint. Furthermore, no improvements in any of the secondary endpoints were found, including major adverse cardiovascular and cerebral events and myocardial infarct size evaluated by troponin T release. Furthermore, no effects of the intervention versus the control were seen among prespecified subgroup analyses by age, presence of diabetes, pre-PPCI coronary flow, ischaemia time, or infarct location.Implications of all the available evidenceThe CONDI-2/ERIC-PPCI trial has provided definitive evidence that remote ischaemic conditioning offers no benefits on either myocardial infarct size or clinical outcomes at 12 months in patients with STEMI treated with PPCI. Two follow-up randomised controlled trials and a prospective single-centre randomised controlled trial, published since the commencement of our trial, had suggested that remote ischaemic conditioning might improve clinical outcomes in patients with STEMI undergoing PPCI. The two follow-up trials were not prospectively designed or powered to detect a difference in clinical outcomes with remote ischaemic conditioning. The prospective single-centre trial might not have been sufficiently powered, and it had an extended follow-up period. Until now, remote ischaemic conditioning had been the most promising potential cardioprotective strategy for improving clinical outcomes following STEMI. Therefore, identification of novel cardioprotective targets and discovery of innovative approaches to cardioprotection are needed to improve clinical outcomes in patients with STEMI treated by PPCI. Such approaches might include combination multitarget therapy. Remote ischaemic conditioning might still be beneficial in other clinical settings of acute ischaemia–reperfusion injury, including renal transplantation, acute kidney injury, and stroke.We did an international, multicentre, single-blind, randomised controlled trial at 26 centres in the UK (the ERIC-PPCI component study), and at four hospitals in Denmark, two hospitals in Spain, and one hospital in Serbia (the CONDI-2 component study). The CONDI-2/ERIC-PPCI study received ethical approval from regional and national health service research ethics committees and was conducted in accordance with the principles of good clinical practice. In the CONDI-2 component study, all participants provided written informed consent before randomisation. In the ERIC-PPCI component study, all patients provided initial verbal assent before randomisation, which was followed by written informed consent. The London School of Hygiene & Tropical Medicine Clinical Trials Unit  coordinated the trial in collaboration with the Cardiology Trial Unit and Department of Clinical Epidemiology of Aarhus University Hospital . Details of the trial design have been reported previously,Patients with chest pain and suspected ST-segment elevation on electrocardiogram (ECG) were screened for possible inclusion. Patients were included if they were older than 18 years of age, had ST-segment elevation on ECG, and were eligible for PPCI. Exclusion criteria were previous coronary artery bypass graft surgery, myocardial infarct within the previous 30 days, left bundle branch block on ECG, treatment with therapeutic hypothermia, conditions precluding use of remote ischaemic conditioning , and life expectancy of less than 1 year due to a non-cardiac pathology.Sealed Envelope , and was stratified by centre with use of random permuted blocks (block sizes of 4 or 6). For CONDI-2, randomisation was done through a web-based clinical trial support system accessible 24 h a day  and stratified by centre using random permuted blocks . In both trials, access to the randomisation website and list were strictly controlled at each site and limited to unmasked study team members. Study team members collecting the data and assessing outcomes were masked to the treatment allocation.In both trials, patients were randomly allocated (1:1) to a remote ischaemic conditioning group or a control group by a designated study team member who was unmasked to treatment allocation. For ERIC-PPCI, randomisation was done via a secure website, An automated AutoRIC cuff device  was used to deliver the remote ischaemic conditioning protocol, which comprised four alternating cycles of cuff inflation for 5 min to 200 mm Hg and deflation for 5 min. At the UK sites, the control group received a sham simulated remote ischaemic conditioning intervention. At the sites in Denmark, Spain, and Serbia, the control group received standard therapy. The remote ischaemic conditioning and control protocols were implemented before PPCI and did not delay the onset of PPCI. Where ambulance transit times allowed, remote ischaemic conditioning was administered in the ambulance (in Denmark and Spain). Where ambulance transit times were shorter, remote ischaemic conditioning was delivered immediately upon arrival at the PPCI centre (in Serbia and the UK).Before PPCI, patients were treated with 300 mg aspirin orally or intravenously, 600 mg clopidogrel or 180 mg ticagrelor orally, and standard weight-adjusted heparin intravenously. The PPCI procedure followed guideline recommendations;The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months post-randomisation in the intention-to-treat population. Hospitalisation for heart failure included heart failure both during the index hospitalisation and during rehospitalisation for heart failure. Hospitalisation was defined as treatment occurring during a hospital admission. Heart failure was judged to be present on the basis of at least one of the following symptoms and signs: new or worsening dyspnoea, orthopnoea, or paroxysmal nocturnal dyspnoea; increasing fatigue or worsening exercise tolerance; ejection fraction less than 45% on echocardiography; new pulmonary oedema seen on chest x-ray in the absence of a non-cardiac cause; crepitations believed to be due to pulmonary oedema; and use of loop diuretics to treat presumed pulmonary congestion.Prespecified secondary endpoints, analysed in the intention-to-treat population, included combined cardiac death or hospitalisation for heart failure at 30 days; cardiac death and hospitalisation for heart failure  at 30 days and 12 months; major cardiovascular and cerebral adverse events  with 80% power and 5% significance would have required 4300 patients (2000 patients in ERIC-PPCI and 2300 in CONDI-2), allowing for 15% attrition. A blinded review of accruing data revealed that the estimated event rate was lower than anticipated and the sample size was, therefore, increased to 5400 patients (2800 patients in ERIC-PPCI and 2600 in CONDI-2).The primary and secondary time-to-event outcomes were compared between the remote ischaemic conditioning and control groups with use of a Cox regression model stratified by the two component studies on an intention-to-treat basis, and presented with Kaplan-Meier curves. To account for recurrent hospitalisation for heart failure and competing mortality risk, a negative binomial model was used to compare the total number of outcomes experienced by the two groups up to 12 months after randomisation. The proportion of patients with an implantable cardioverter-defibrillator, biventricular pacemaker, or single-chamber or dual-chamber pacemaker at 12 months was compared using a generalised linear model for a binary outcome with a log link.Prespecified analyses by subgroup  were done on the primary outcome. The subgroups were analysed by including an interaction term between treatment group and subgroup in the Cox regression model.Troponin T values were converted into a 48-h AUC summary measure for analysis, using multiple imputation by chained equations to account for missing data. As the distribution of the outcome variable was highly skewed, a log transformation was done. Differences were analysed by use of a linear regression model with the treatment effect presented as a ratio of the geometric means.We also did a per-protocol analysis of all primary and secondary outcomes limited to patients who had a confirmed STEMI, received the full intervention as specified in the protocol, and had a completed PPCI.Results were considered statistically significant if the two-sided p value was less than 0·05. Full details of the statistical methods are provided in the protocol and statistical analysis plan, which are available in ClinicalTrials.gov (number NCT02342522) and is a planned prespecified collaboration between the CONDI-2 and ERIC-PPCI studies  were included in the intention-to-treat analysis . The treThere was no evidence of difference between the control group (8·6% [n=220]) and the remote ischaemic conditioning group (9·4% [n=239]) with respect to the combined primary end point of cardiac death or hospitalisation for heart failure at 12 months  within 30 days, major cardiovascular and cerebral adverse events  after 30 days, or incidence of implantable cardioverter-defibrillator implantation within 12 months . NumbersThe AUC for high-sensitivity troponin T did not differ between the control group  and the remote ischaemic conditioning group  patients and transient pain or paraesthesia was reported for 147 (5·8%) patients. There were no withdrawals due to adverse events.In this large, appropriately powered, prospective, international, multicentre trial, we found no clinically meaningful beneficial effects of remote ischaemic conditioning administered as an adjunct to PPCI on clinical outcomes  at 12 months in patients with STEMI when compared with PPCI alone. Furthermore, remote ischaemic conditioning had no effect on major secondary endpoints, including myocardial infarct size assessed by cardiac biomarkers.These findings are in direct contrast to previously published clinical studies of remote ischaemic conditioning in patients with STEMI that reported increased myocardial salvage  and reductions in myocardial infarct size (assessed by cardiac biomarkers and cardiac imaging),12vs 11 [5%] in the control group) and hospitalisation for heart failure events (eight [3%] in the remote ischaemic conditioning group vs 17 [8%] in the control group) were small. An alternative explanation could be that the primary effect of remote ischaemic conditioning was on post-STEMI left ventricular remodelling rather than acute myocardial infarct size—although this mechanism would contradict experimental animal studies, which have clearly shown a beneficial effect of remote ischaemic conditioning on reducing acute myocardial infarct size. Notably, a clinical study investigating the cardioprotective effects of ischaemic postconditioning in patients with STEMI treated by PPCI showed no effect on myocardial infarct size, but still found reduced severity of adverse left ventricular remodelling at 1 year of follow-up, especially in patients with microvascular obstruction.Although myocardial infarct size is a known independent determinant of clinical outcomes post-PPCI in patients with STEMI, it is unclear whether a reduction in myocardial infarct size by a cardioprotective intervention applied as an adjunct to PPCI can be translated into improved clinical outcomes. The RIC-STEMI trial by Gaspar and colleaguesFollow-up (median 3·8 years) of participants in the initial CONDI-1 trialThe reasons for the failure of remote ischaemic conditioning to reduce myocardial infarct size and improve clinical outcomes in our study are not clear. One potential reason could relate to the remote ischaemic conditioning protocol itself. Although four 5-min cycles of upper arm cuff inflations and deflations applied before PPCI has been shown to increase the myocardial salvage indexThe timing of the remote ischaemic conditioning protocol in relation to reperfusion by PPCI might also be important. Previous clinical studies have shown that remote ischaemic conditioning is effective at reducing myocardial infarct size when administered before PPCI  due to infarcts in the left anterior descending artery.Remote ischaemic conditioning has shown mixed effects on outcomes in other clinical settings including cardiac surgery. Three large, prospective, multicentre, randomised controlled trials failed to find any beneficial effects of remote ischaemic conditioning on post-surgical clinical outcomes,A potential limitation of our study is the short follow-up time of 12 months post-STEMI for the primary outcome of cardiac death and hospitalisation for heart failure, which might have been too short to observe any effect of remote ischaemic conditioning on clinical outcomes.In summary, the findings from our trial provide conclusive evidence that remote ischaemic conditioning offers no benefits on either myocardial infarct size or clinical outcomes at 12 months in patients with STEMI treated by PPCI. Unfortunately, remote ischaemic conditioning had been the most promising cardioprotective strategy for improving clinical outcomes following STEMI, and few other prospective options exist. As such, further studies are needed to identify novel cardioprotective targets and innovative approaches to cardioprotection such as combination multitarget therapy.Requests for data collected for the study can be made to the co-corresponding authors and will be considered on an individual basis. Additional, related documents are immediately available  and can be requested from the co-corresponding authors.


The retina is a complex tissue with multiple cell layers that are highly ordered. Its sophisticated structure makes it especially sensitive to external or internal perturbations that exceed the homeostatic range. This necessitates the continuous surveillance of the retina for the detection of noxious stimuli. This task is mainly performed by microglia cells, the resident tissue macrophages which confer neuroprotection against transient pathophysiological insults. However, under sustained pathological stimuli, microglial inflammatory responses become dysregulated, often worsening disease pathology. In this review, we provide an overview of recent studies that depict microglial responses in diverse retinal pathologies that have degeneration and chronic immune reactions as key pathophysiological components. We also discuss innovative immunomodulatory therapy strategies that dampen the detrimental immunological responses to improve disease outcome. The retina is part of the central nervous system with more than 60 distinct retinal cell types, high level organization, and a evolutionarily conserved structure , 2. The As early as 1897, Spanish neuroscientist Ramón y Cajal began to appreciate that there were more cell types in the brain than just neurons and astrocytes , 12. In In the normal retina, microglia cells are located in the plexiform layers where they exhibit elaborate ramified processes responsible for immune surveillance of the retina . Noxious+ cells as early as 8-weeks post transplantation, and by the 6th month, all retinal myeloid cells were eGFP+  derived cells from eGFP-transgenic mice into irradiated normal adult mice . Retinalre eGFP+ . Using Cantation . Of noteantation , 18. Howantation . In contantation . The autantation . They arantation . Indeed,antation , 21.Therefore, to further analyse the contribution of circulating BM derived progenitors on the turnover of resident microglia in the CNS, a separate study employed parabiosis experiments . Parabio+ microglial progenitors throughout the CNS  early in development . MoreoveFollowing the development phase, microglia cells move to occupy the inner and outer plexiform layers where they adopt a quiescent phenotype characterized by very small somata and extensively ramified filopodia-like processes . They foCross-talk between microglia and other retinal cells is enabled by the assortment of cell surface molecules on the microglial cell membrane , 37. TheSialic acids, covalently linked to cellular membrane proteins and lipids, also contribute to the inhibition of the innate immune system in the CNS including the retina , 43. TheA further neuroimmune regulator is CX3CL1 , a constitutively expressed neuron derived chemokine which binds to its sole receptor CX3CR1 present in microglia and maintains them in a quiescent mode . Indeed,Microglia also exchange functionally significant signals with Müller cells both in health and disease states, and this bidirectional communication can act as mediator of neuron-microglia crosstalk [As a result of the heterocellular signaling and crosstalk in the retina, microglia cells are rendered extremely sensitive to slight changes in their environment, demanding efficient inhibitory mechanisms to maintain them in a quiescent neuroprotective state . RPE celA sustained chronic pro-inflammatory environment is an important common denominator of retinal degenerative diseases and neurological disorders that affect vision . NeuroinAMD, with a global prevalence of ~170 million people, refers to a progressive deterioration of the macula . It is aThere is substantial evidence to indicate the involvement of inflammation and dysregulated innate immunity in the pathogenesis of AMD , 75, 78.Consistently, enlarged amoeboid microglia have been found in the vicinity of RPE cells overlying drusen in retinal sections from dry AMD patients . In the Indeed, the role of microglial reactivity in severity of disease has been clearly demonstrated in several mouse models of AMD. However, it is important to note that conclusions drawn from these studies are limited in nature since rodents lack an anatomical macula. Nonetheless, these models effectively mimic distinct features of human pathology including accumulation of immune cells such as macrophages or microglia in the subretinal space, photoreceptor degeneration, choroidal neovascularization, funduscopically visible drusen-like lesions, and RPE degeneration . We willBright white light for example can be used to mimic the photoreceptor cell death and retinal degeneration witnessed in human AMD patients . IntenseAnother useful model for dry AMD is generated by immunizing mice with carboxyethylpyrrole (CEP)-adducted proteins . CEP is To study microglial immune responses in wet AMD, a laser induced bruch's membrane photocoagulation model is used . BrieflyHereditary degenerations of the human retina are a diverse group of clinically and genetically heterogeneous blinding diseases with more than 260 causal genes identified to date . InheritPde6b gene that codes for the β-subunit of cGMP phosphodiesterase (PDE), and mimics to a great extent the RP in human patients bearing mutations in the human homolog of the gene  mouse which harbors a missense mutation in the Pde6b gene but displays a much slower onset of retinal degeneration, a similar infiltration of microglia into the outer nuclear layer is observed at around P21 as the number of apoptotic rods increase  is a severe ocular complication of diabetes mellitus and is also the leading cause of blindness among the working age populations of industrialized regions . DR can Early indications of inflammation involvement in the pathogenesis of DR came from a study reporting a low incidence and high regression rate of DR when diabetic patients were treated with salicylates for rheumatoid arthritis complications . Since tIn human DR, hypertrophic and amoeboid microglia are present at different stages of the disease . In the Akita mouse model which harbors a spontaneous missense mutation in the insulin 2 gene, discrete pockets containing bloated microglia with shorter less branched processes can be observed in the diseased retina  can be found all over the retina and in the subretinal space following 12 months of hyperglycaemia (In the Goto-Kakizaki (GK) inbred rat model of type II diabetes, numerous Iba1lycaemia . The enhlycaemia . Alteratlycaemia . Notablylycaemia .Microglia reaction is also elicited in pharmacologically induced diabetes where toxic glucose analogs alloxan and streptozotocin (STZ) are used to preferentially destroy pancreatic beta cells and induce a state of insulin-dependent diabetes , 141. StGlaucoma refers to a distinctive group of optic neuropathies characterized by the progressive demise of retinal ganglion cells (RGCs) and their axons, thinning of the retinal nerve fiber layer and cupping of the optic disc . GlaucomConsistent with this hypothesis, aqueous humor from glaucoma patients have been shown in several studies to contain higher levels of pro-inflammatory cytokines and chemokines, suggesting that neuroinflammatory processes play a key role in glaucomatous neurodegeneration –156. MicMurine models of glaucoma where increased ocular hypertension occurs naturally or is experimentally induced using genetic manipulation or surgical procedures have been instrumental in understanding microglia's role in disease pathogenesis . Using DNitric oxide (NO) synthesized by microglia also plays a role in the pathophysiology of glaucoma , 169. Inin-vitro experiments conducted on primary glia cultures isolated from rat retina demonstrated that microglia, and not astrocytes were the primary sources of IL-6 following elevated pressure conditions  cap on their glycocalyx made up of α2-8-glycosidically linked N-acetylneuraminic acid residues and which are attached by means of Hildebrandt et al.  and SchnUnder pathological conditions such as elevated inflammation and oxidative stress, degradation of sialic acid caps occurs leaving neurons and other cells with a damaged glycocalyx –186. Dur1−42 with nanomolar concentrations of low molecular weight polySia with an average degree of polymerization 20 (polySia avDP20) significantly reduces pro-inflammatory gene transcription, inflammation-induced phagocytosis, and oxidative burst , formerly referred to as the peripheral benzodiazepine receptor (PBR), is a highly conserved 5α-helical transmembrane protein located on the outer mitochondrial membrane (OMM) . TSPO exin-vitro experiments in the lab, we have demonstrated that treatment of microglia cells with XBD173 induces pregnenolone synthesis, and that inhibition of this process with aminoglutethimide partially abolishes XBD173 therapeutic effects  belongs to the type I IFN family which are best known for their ability to induce a cellular antimicrobial state during a viral or bacterial infection , 217. IFWe used the laser-induced choroidal neovascularization (CNV) mouse model that mimics features of exudative AMD to test this hypothesis . Briefly2O2-induced apoptosis  and SOCS3 , 226. IFpoptosis . Notablypoptosis .Ifnb−/− mice exhibited striking reductions in Pi3K and Akt mRNA and protein levels  . GSK-3 ifunction . HoweverMinocycline is a highly lipophilic, semi-synthetic tetracycline derivative which is mainly used in the treatment of acne vulgaris and rheumatoid arthritis . Howeverrd10 mouse model of human RP, treatment with minocycline significantly reduced microglia-mediated photoreceptor apoptosis, improving retinal structure and function (Consistent with its anti-inflammatory properties, minocycline benefits during retinal pathophysiology have been confirmed in several experimental models. In a light-induced retinal degeneration mouse model, minocycline treatment was shown to significantly inhibit photoreceptor apoptosis and preserve retinal structure . Moreovefunction . In a rafunction . Similarfunction . In the function . Of notefunction .Minocycline protective effects have also been examined in the human retina . RetinitStudies using rodent models in the past decade have been instrumental in understanding the role of microglial responses in retinal pathologies. Ample evidence generated from these studies shows unequivocally that microglia reactivity and chronic inflammation is a common hallmark of various retinal pathologies, and that pharmacological targeting of overly reactive microglia ameliorates disease pathogenesis. However, why and how immunologic checkpoints become overwhelmed resulting in dysregulation of microglial inflammatory responses remains poorly understood. Therefore, prior to development of microglial-based therapeutic approaches, further studies are needed to understand the etiological factors and molecular mechanisms that propagate microglial responses during retinal degeneration. In addition, owing to the numerous overlapping insults that predispose retinal pathologies, it will be prudent to develop microglia based therapeutic approaches that will complement, or even synergize, other retinal therapeutic approaches.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ability to recover purposeful movement soon after debilitating neuromuscular injury is essential to animal survival. Various neural and mechanical mechanisms exist to preserve whole-limb kinematics despite exhibiting long-term deficits of individual joints following peripheral nerve injury. However, it is unclear whether functionally relevant whole-limb movement is acutely conserved following injury. Therefore, the objective of this longitudinal study of the injury response from four individual cats was to test the hypothesis that whole-limb length is conserved following localized nerve injury of ankle extensors in cats with intact nervous systems. The primary finding of our study was that whole-limb kinematics during walking was not immediately preserved following peripheral nerve injuries that paralyzed subsets of ankle extensor muscles. Instead, whole-limb kinematics recovered gradually over multiple weeks, despite having the mechanical capacity of injury-spared muscles across all joints to achieve immediate functional recovery. The time taken to achieve complete recovery of whole-limb kinematics is consistent with an underlying process that relies on neuromuscular adaptation. Importantly, the gradual recovery of ankle joint kinematics remained incomplete, discontinuing once whole-limb kinematics had fully recovered. These findings support the hypothesis that a whole-limb representation of healthy limb function guides a locomotor compensation strategy after neuromuscular injury that arrests progressive changes in the joint kinematics once whole-limb kinematics is regained. Summary: Longitudinal study of four cats documented recovery of locomotor kinematics in one hindlimb following nerve injury and muscle paralysis focused at the ankle joint. Whole-limb length was not conserved immediately, but recovered pre-injury levels gradually through kinematic adjustments distributed across joints. The ability to recover purposeful movement soon after debilitating neuromuscular injury is essential to animal survival. Various mechanisms exist to counteract impairments and conserve functionally relevant movement . For exaDespite the presence of various mechanisms to compensate for injury by exploiting biomechanical redundancy , it is uWithin 1 month after experimental denervation of a subset of ankle extensor muscles, cats and rats exhibit normal whole-limb length and orientation of the injured limb during walking, despite lingering kinematic irregularities at each of the joints . It is pClarifying the early development of kinematic compensation in awake, behaving cats should assist in identifying the underlying neuromuscular compensatory mechanisms. The finding that whole-limb kinematics is corrected immediately after denervating a subset of the primary muscles acting at a joint would indicate that existing neural and/or mechanical mechanisms can compensate to maintain whole-limb kinematics. Alternatively, whole-limb kinematics might not be immediately restored, suggesting that the underlying neuromuscular compensatory mechanisms involve processes of learning or plasticity that develop gradually. Thus, by knowing the early development of whole-limb recovery from injury, we might gain better understanding of the processes that enable animals to achieve purposeful and robust locomotion well in advance of time-consuming tissue regeneration.Therefore, the objective of the present study was to test the hypothesis that whole-limb length is conserved immediately following localized nerve injury of ankle extensors in cats with intact nervous systems. The kinematics of voluntary locomotion were examined in a convenience sampling of cats before and after unilateral denervation of one or another subset of the primary ankle extensor muscles that were utilized in other electrophysiological studies. Despite the variety of specific nerves injured, we were able to test the general hypothesis about limb-level versus joint-level kinematic changes after a localized injury. Results were uniform across individual cats, showing that whole-limb length was not conserved in the first day post-injury. Instead, limb length deviated considerably from pre-injury values for more than one week in every animal. Because recovery of whole-limb kinematics was not immediate, it seemed to rely on the development of a new motor strategy that falls outside the normal capacity of existing locomotor compensation mechanisms. We also found that recovery of whole-limb kinematics progressed gradually over a week or longer in parallel with recovery of ankle kinematics. Importantly, once whole-limb kinematics reached pre-injury values, ankle joint kinematics ceased to recover before reaching its pre-injury values. These findings support the hypothesis for a neuromuscular compensation strategy that prioritizes task-level function of the whole-limb over individual joints .Changes in kinematic parameters were evident throughout the injured hindlimb in the first 20 h after injury. Data presented in Whole-limb length for each of the four; was clearly shorter over virtually the entire stance phase of gait when compared with before and within the first 20 h after nerve injury, limb length was clearly shorter over virtually the entire stance phase for all four cats  and 2. BOn the first day after injury, the trajectory of whole-limb orientation deviated slightly from the pre-injury trajectory . A patteThe remaining two joint angles measured in this study, i.e. hip and knee joint angles, generally demonstrated increased stance-phase extension on the first day after nerve injury. The deficit in whole-limb length that was evident on post-injury day 1 extended several days longer as shown in Even at 3 weeks, however, ankle joint kinematics failed to return to the pre-injury range in contrast to whole-limb length, which recovered fully. Recovery of ankle angle, although correlated with whole-limb length, was distinguished by its failure to attain pre-injury levels. The curves in The present study is the first to show a failure to preserve whole-limb kinematics during walking in the first few days following peripheral nerve injuries that paralyzed subsets of ankle extensor muscles. Earlier reports on adult cats with similar injuries report that whole-limb length recovered fully by 1 month, despite the persistence of abnormal joint kinematics . While tSince there is no possibility for recovery of the denervated muscles, the gradual changes in ankle joint kinematics in the first few weeks following injury must be due to changes in the relative activations and mechanical contributions from the remaining intact ankle extensor muscles. As we only tested cats walking on a level surface at a moderate speed (0.7 m/s), we can assume that the intact muscles were well within their maximal force-producing capacity to restore normal joint and limb kinematics immediately after paralyzing a subset of ankle extensors. Prilutsky and colleagues  showed aDespite the muscular capacity for immediate restoration  recoveryNeural or mechanical adaptation is inferred as the underlying basis for the recovery of ankle kinematics that occurs slowly, but well in advance of any significant peripheral reinnervation of denervated  ankle extensors. Progressive recovery of ankle kinematics involves increased activation of injury-spared ankle extensor muscles . These aWhile peripheral nerve injury resulted in an immediate and significant change in whole-limb length, the observed deficits in limb orientation were much smaller. We expected a smaller effect on orientation, which is determined largely by the swing phase of gait and powered by flexor muscles which were uninjured in our study. Recovery of whole-limb kinematics was achieved after localized muscle paralysis by utilizing a new combination of component joint kinematics. Whole-limb length returned to normal through a two-step process involving immediate increases in extension primarily at the hip joint and variously at the knee joint, followed by a gradual yet incomplete return of subnormal extension at the ankle and MTP joints. Coordination across joints in recovery was clearly demonstrated by our observation that progressive reduction in ankle yield discontinued once whole-limb kinematics reached normal values. The arrest of ankle recovery that occurs when whole-limb kinematics reach full recovery suggests locomotor compensation after injury may be controlled through a hierarchical control structure , where gAlthough whole-limb kinematics may function as control targets of the central nervous system, other targets, such as limb force generated against the ground, may be equally pertinent. Humans are suggested to control limb force during locomotion . Recent Kinematic data are unavailable, either in this or previous reports to our knowledge, for cats sham controlled for nerve-injury surgery. We cannot rule out, therefore, the possibility that factors associated with the surgical procedure for cutting nerves influenced whole-limb recovery, which was first measured 20 h after surgery. Among factors potentially confounding recovery from nerve injury are surgical anesthesia and post-operative pain or discomfort at the surgical site. We ascribe minimal concern to anesthesia, since the isofluorane clearance from all tissues occurs in less than four hours . Pain atIn summary, our longitudinal study of the injury response from four individual cats documents the unexpected finding that whole-limb kinematics is not immediately conserved following localized nerve injury, regardless of affected muscles. These results provide important insight into what appears to be a progressive neuromuscular process of locomotor compensation. Our observations showing preferential recovery of limb kinematics over individual joint kinematics provide support for a hierarchical control strategy of compensation in legged locomotion.Kinematics data collected from cats engaged in separate studies of nerve injury in our lab made it possible to address this objective with existing data. Limb kinematics during treadmill walking were available for four adult cats studied over several days beginning soon after nerve injuries that paralyzed primary ankle extensor muscles to produce similar losses of ankle joint extensor torque. These features, described below in detail, met our study objective through a case study of longitudinal data from four individual animals.2) where they had ample space to walk, run, climb and play. Ambient temperature (24°C) and light-dark cycles (12 h) were controlled and dry food and water were provided ad libitum. All cats were studied before and after nerve injury as described below.All procedures were performed according to protocols approved by the Wright State University Laboratory Animal Care and Use Committee. Four purpose-bred (Class A) adult cats (mass 2.5–4.5 kg), two male and two female, were acquired from Liberty Research, Inc.  and housed separately by gender in large open-floor rooms , and tracheal intubation was used to deliver gaseous anesthesia (1.2-2.5% isoflurane in 100% oxygen) throughout surgery, and intravenous (IV) catheterization of the right cephalic vein for administration of fluid . Oxygen saturation, pulse rate, respiration rate, end-tidal COThe nerves supplying the medial gastrocnemius (MG), lateral gastrocnemius (LG), soleus (S) and plantaris (P) muscles were exposed through an incision (∼4 cm) overlying the popliteal fossa in the left leg. Transection of nerves to these muscles produced robust changes in ankle joint kinematics trajectory as described in earlier studies . In two The complete set of muscles acting to produce ankle extension in normal cats includes MG, LG, S, P and the flexor hallucis- and digitorum-longus (FHL and FDL). For each of these muscles in the adult cat, Lawrence and Nichols (1999) report maximal ankle torque produced at an ankle-joint angle of 110°. From these values, we estimate that for each of the two kinds of injury studied here, (xG and xLGSP), the total maximum ankle-joint torque produced in the sagittal plane by all injury-spared muscles combined was reduced to approximately 40% of normal (∼70/160 N-cm).Before surgery, all cats were trained by food reward to walk within a clear plastic enclosure (120 cm×120 cm×60 cm) mounted over the moving belt of a motorized treadmill . Kinematic measurements began when cats were trained to the point of completing at least 20 contiguous steps at the constant speed of 0.7 m/s, which was the speed most readily adopted by all four cats. Kinematic data were collected longitudinally for each of the four cats, beginning at least 1 week before nerve injury, and extending 5 days or longer afterwards (see Results). Data were collected up to the day before surgery (pre-injury control) and resumed within 20 h post-surgery, with each session lasting less than 1 hour, once per day. Without exception, cats gave no evidence of discomfort or disinterest in treadmill walking or food reward following nerve injury. Note that cats were housed in the recovery cage for the first 5 days after surgery to permit recovery of the skin incision before being returned to the colony room. The first recording session captured each cat's very first treadmill steps following surgery.For purposes of filming limb kinematics, colored-tape markers were temporarily placed on the skin at the superior iliac crest (IC), greater trochanter (hip), lateral malleolus of the fibula (ankle), fifth metatarsal-phalangeal joint (MTP) and the distal-most part of the fifth digit (toe) on the left leg . TreadmiThe spatial location of the markers was processed and analyzed offline using KineTracer software (Kissei), which allowed for assessment of hip, knee, ankle and MTP joint angles; i.e. the joint kinematics. Since skin movement errors are very high for the knee, its position was triangulated using the measured lengths of the femur and tibia (obtained post-mortem) and the positions of the hip and ankle markers . Once thWhole-limb and individual joint kinematic data collected from multiple steps per session of treadmill walking met criteria for using means and standard deviations as valid descriptive statistics. Statistical comparisons were made within individual cats from data obtained at different time points before and after nerve injury.


Haplopelma schmidti). The unusual pharmacology of DkTx is its high avidity for its receptor (TRPV1), a property that has been attributed to a bivalent mode-of-action. DkTx has subsequently proven a powerful tool for elucidating the structural basis for the function of the TRPV1 channel. Interestingly, all tandem repeat peptides functionally characterized to date share this high avidity to their respective binding targets, suggesting they comprise an unrecognized structural class of peptides with unique structural features that result in a characteristic set of pharmacological properties. In this article, we explore the prevalence of this emerging class of peptides, which we have named Secreted, Cysteine-rich REpeat Peptides, or “SCREPs.” To achieve this, we have employed data mining techniques to extract SCREP-like sequences from the UniProtKB database, yielding approximately sixty thousand candidates. These results indicate that SCREPs exist within a diverse range of species with greatly varying sizes and predicted fold types, and likely include peptides with novel structures and unique modes of action. We present our approach to mining this database for discovery of novel ion-channel modulators and discuss a number of “hits” as promising leads for further investigation. Our database of SCREPs thus constitutes a novel resource for biodiscovery and highlights the value of a data-driven approach to the identification of new bioactive pharmacological tools and therapeutic lead molecules.Peptide toxins isolated from animal venom secretions have proven to be useful pharmacological tools for probing the structure and function of a number of molecular receptors. Their molecular structures are stabilized by posttranslational formation of multiple disulfide bonds formed between sidechain thiols of cysteine residues, resulting in high thermal and chemical stability. Many of these peptides have been found to be potent modulators of ion channels, making them particularly influential in this field. Recently, several peptide toxins have been described that have an unusual tandem repeat organization, while also eliciting a unique pharmacological response toward ion channels. Most of these are two-domain peptide toxins from spider venoms, such as the double-knot toxin (DkTx), isolated from the Earth Tiger tarantula ( The existence of evolutionarily formed, two-domain toxins with bivalent activity, was first observed in Rhodniin; a highly specific serine protease inhibitor with two Kazal-type domains, isolated from prolixus . More reprolixus  and π-heprolixus , revealsThe protein “universe,” that is, the total space of all proteins from every species, consists of a vast and complex network of biomolecules. The rise of next-generation sequencing technologies has provided an excellent platform in which to explore this space. At first glance, the observable protein landscape appears disordered and random. However, upon further analysis, patterns of increasingly well-defined clusters are beginning to emerge. A growing number of detailed protein classifications now exist, with many groups branching from smaller central points; defined broadly by their structural similarities . Due to extracellular translocation. The purpose for this distinction is to help guide the identification of highly stable protein ligands that interact with extracellularly located receptors. Our ability to isolate and study these interactions may provide vital clues in the development of more sophisticated therapeutics needed for the treatment of complex pathophysiological disorders. Few disorders parallel in complexity to those associated with impaired ion-channel function; termed ‘channelopathies.’ Due to the fundamental physiological role of ion-channel activity, impairment can result in dramatic dysregulation of primary functions, affecting tissues such as cardiac muscle  N-terminally located, that determine its cellular and subcellular locations . There ec muscle , skeletac muscle , as wellc muscle  and reguc muscle . TherefoBoophilus microplus. Boophilin revealed a double Kunitz-type protease inhibitor architecture with a possible bi-functional role, inhibiting both circulating thrombin as well as the membrane bound thrombin activation intermediate, meizothrombin , contains two independently folded ICK domains connected by a linker , demonstrated great potential for neuroprotection from stroke induced cerebral ischemia within a rat model , a 75-residue peptide isolated from the Australian funnel-web spider  and TrEMBL; the automatically annotated and unreviewed sequence data (≈114 million entries in 2018-04)   was usedIn the initial stage of generating the SCREPs dataset it is important to avoid accidental exclusion of any true SCREP sequences. Ideally, the SCREPs identifying algorithm would be applied directly to the total UniProtKB database (≈114 million sequences as of May 2018), however, processing a dataset of this size is a computationally very intensive. Therefore, reducing the dataset size is necessary for making this task more feasible. To capture all potential SCREPs, appropriate filters to extract secreted proteins must be applied, the first of which is to exclude intra-cellular proteins. However, identifying proteins that are extracellularly secreted encounters many inherent difficulties. As previously mentioned, secreted proteins contain a signal sequence region, and the primary tool used to identify the presence of this region is SignalP . DespiteConversely, there is a need to consider sources of false negatives in the initial dataset. UniProt facilitates the search of sequences that have an identified signal region, which currently contains ≈8 million sequences. However, applying SPA to only sequences containing this signal region is not sufficient in capturing all SCREPs. This is due to the occurrence of uploaded protein sequences derived from native material wherein the signal region has undergone proteolytic cleavage prior to sequencing. Often in this situation, the uploaded sequences have been manually reviewed and annotated, allowing for a simple search of proteins with a “secreted” subcellular annotation, which currently results in ≈300,000 sequences. At the time of applying this data mining process, the signalP dataset contained 7.7 million sequences, and the total “secreted” dataset held 291,558 sequences. Combined, the starting platform in which SPA was applied contained just over 8 million protein sequences.The existing population of TR, multi-domain protein architectures is difficult to define, especially proteins with highly diverged sequences between domain repeats. Identifying the presence of TR proteins has proven to be a difficult task that is not easily accomplished; several different approaches have been presented with inherent strengths and weaknesses. Past attempts at estimating the existing population of TR proteins within the Swiss-Prot database identified ≈13–20% occurrence . Many biPresented here is the SCREPs processing algorithm (SPA); a custom-built in-house tool developed specifically to address the unique primary sequence features found within SCREPs. Prior to the application of the SPA tool, identification and removal of any signal sequence region that may exist within the protein is essential. Naturally, the overall aim of identifying repeating units applies only to the mature protein sequence. The first stage of the SPA initially processes each sequence to identify suitable SCREP candidates based upon simple sequence features. This involves restricting the input sequence size to between 20 and 500 aa’s in length; the minimum is set to avoid possible false identification of small non-domain repetitive elements, and the upper limit is established to avoid much larger proteins, e.g., transmembrane receptors. Additionally, to identify sequences that would contain at least two independently folded DRDs requires a minimum of 4 cysteine residues, i.e., the formation of at least one disulfide-bond per domain.n) of amino acids is counted from the N-terminus and used to divide the protein sequence into two segments. These two segments are used for pairwise sequence alignment by blastp . This approach revealed the existence of a bimodal distribution of cysteine density. Sequences with 2–3% cysteines contribute to over one third of the resulting SPA output Figure . The domAn additional step within the SCREPs refinement process is the identification and removal of sequence redundancy. To determine what level of redundancy may exist between sequences within the entire SCREPs population , we used cd-hit  to clustProteins within the refined SCREPs dataset, represent a great level of diversity with 166 unique taxonomic classes. A distribution of these major classes demonstrates the possible central role this novel protein architecture may play, bridging distantly related organisms between two distinct domains of life. A significant portion of SCREPs are clearly found within both bacteria (green) and eukaryotic organisms (blue) Figure . The curDifferent functional relationships are likely to exist between SCREPs within particular taxonomic groups, leading to clusters of specific biological roles throughout. So far, we have already observed such roles as protease inhibition and ion-channel modulation from venomous and parasitic species. However, there still appears to be a large amount of SCREPs observed within non-venomous and parasitic classes also, these are particularly apparent within the vertebrate classes Mammalia, Actinopterygii, and Aves Figure . FurtherGiven the apparent taxonomic diversity among SCREPs, we further investigated whether this level of diversity extends to the structural landscape as well. Due to the nature of this dataset, there is little to no structural information available for these proteins, therefore, the InterPro resource was used . The ideUsing InterProScan, 98.4% of the sequences within the refined SCREPs dataset were identified as having matching domain sequence features to at least one of the databases within the InterPro consortium. The resulting output of InterProScan after processing thousands of multi-domain protein sequences generates an immense amount of data to interpret and a manual approach toward such analysis is very limited. Using a custom, in-house developed tool to summarize domain distributions showed that SCREPs are by no means limited to the already identified Kazal and ICK domains. Surprisingly, these folds were not among the top 20 most abundant SCREP folds, which comprise a large diversity of structural domains Figure . PlottinWith an understanding of both the size and diversity of the SCREPs dataset, the question emerges of how this potentially useful resource may be fully utilized to identify functionally active peptides. To identify SCREPs that are likely to demonstrate ion-channel activity, it is necessary to narrow our focus toward smaller subpopulations of the database. To achieve this, we employed additional, widely available bioinformatics tools and resources to refine our database. We first used a dataset of toxins and proteins derived from venomous organisms (Tox-Prot) . The conA more sophisticated approach of interpretation is essential when attempting to accurately correlate any structure-activity-relationship, in particular, a cause for caution is necessary in avoiding inaccurate inferences. The Tox-Prot dataset downloaded for use in this comparative analysis contained a total of 6,722 protein sequences (February 2018), but consisted of approximately of this dataset contains sequence fragments that were excluded. Further refinement of this dataset by cd-hit to remove any redundant sequences reduced the original Tox-Prot dataset by ≈26%, resulting in a non-redundant and fragment-free dataset containing 4,976 venom protein and toxin sequences.e-value cut off of 10-6; a value set in order to identify alignments of moderate to high similarity. The resulting dataset of Tox-Prot SCREPs (TP-SCREPs) contained a considerable 9,925 alignments, matching 494 Tox-Prot sequences with 5,944 SCREPs. The BLAST results identified 25 SCREP sequences that were also present within the refined Tox-Prot dataset. Confirming the functionality of our approach, these include the previously mentioned SCREPs such as DkTx, CpTx-1, as well as Hi1a and its 3 additional variants; Hi1b-d. Within the remaining 25 SCREP sequences shared between datasets, the identification of a few putative SCREPs is observed, these include two spider derived peptides; Pn1a from Phoneutria nigriventer with an apparent double thyroglobulin type domain, U19-barytoxin-Tl1a, a double Kunitz type peptide isolated from Trittame loki, as well as two double WAP type snake derived peptides; Waprin-Phi1 and Waprin-Enh1, isolated from Philodryas olfersii and Pseudoferania polylepis, respectively.The first and probably most valuable relationship to observe is the presence of any significant pairwise alignments identified using BLAST. Local alignment of the mature sequences from both SCREPs and Tox-Prot, was performed using the standard blastp program with an Arachnida and Gastropoda classes. Surprisingly, the distribution of TP-SCREPs does not reflect the same bias toward these taxonomic groups . The ICIT dataset was generated from the Swiss-Prot database by keyword searching for the terms “toxin” or “venom” within all reviewed proteins of the Metazoan kingdom. Finally, a search to identify which of these sequences are associated with “ion channel impairing” functionality supported with experimental evidence, produced the final ICITs dataset containing 890 unique sequences. This dataset can be further divided based upon their associated ion-channel interactions, allowing for the possibility of guiding the selection of ion-channel specific homologous SCREP sequences. Like the refinement of all previous datasets, cd-hit was applied which generated a dataset of 836 non-redundant ICITs.e-value cut off at 10-6, and this resulted in a total of 3,391 significant local alignments (>35 bit score), with 2,034 SCREPs (ICIT-SCREPs) from 50 unique ICITs. Similar to the BLAST analysis between the Tox-Prot data, a few overlapping sequences were identified which revealed the presence of three SCREPs already within the ICIT dataset, including DkTx, Hi1a, and a precursor protein from the anemone Urticina grebelnyi  do not share functionality, the functional analysis of these 50 ICITs may still provide valuable insight toward the SCREPs that do. Naturally, further experimental investigation is required to conclusively determine which ICIT-SCREPs out of the 2,034 homologous sequences may share ion-channel activity. However, simple bioinformatics analysis is the first step toward identifying possible ion-channel modulating SCREPs, reducing the number of peptides required for any functional screening endeavors.2, as well as a few representatives from knottin-type scaffolds. These peptides display varying degrees of observed functionality toward their ion-channel targets in terms of potency, selectivity, and levels of supporting experimental evidence. In terms of their ion-channel targets, most of these toxins (51%) inhibit voltage gated potassium channels (KV), an even amount (10.2%) target both voltage gated calcium and sodium channels , similarly, an equal amount (6.1%) of toxins target both ryanodine receptors (RYR) and transient receptor cation channels of the vanilloid group (TRPV), and finally (8.2%) target acid sensing ion channels (ASICs). It is possible that the common occurrence of homologous ICITs found targeting the homotetrameric KV channels may indicate a potential bias toward SCREP interactions due to their multivalent ligand behavior. The nature of SCREPs with internally homologous domains may perhaps favor binding with molecules that display structural symmetry, enabling multivalent interactions to occur across identical subunits such as that observed within potassium channels , and Mollusca (4.1%). As previously mentioned, the predominant domain type within the ICIT-SCREPs dataset is the BPTI/Kunitz domain, and unsurprisingly this domain constitutes the majority (64%) of the homologous ICITs. A bias toward this domain type may possibly be due to either the method used in this approach, or simply just a direct reflection of the abundance of Kunitz domains within the SCREPs data. Several other shared domain types exist which include scorpion long chain toxins (cystine-stabilized a/β fold: CSaβ), cysteine-rich venom proteins (CRVP), PLAchannels .To more closely examine putative ion-channel modulating SCREPs, we selected high-similarity hits to three ICITs with high levels of experimental annotation that represented two different fold types and three different ion-channel targets Table . These hV1.1 inhibition [IC50 0.9 ± 0.1 nM;  . These two unstudied SCREP sequences represent exciting and new possible examples of bivalent ion-channel ligands.Similar to APEKTx1, APHC2 is another sea anemone-derived Kunitz peptide isolated from channels . The hig) Figure . AdditioPsalmopoeus cambridgei (The third ICIT alignment investigated was using PcTx-1 Figure . Isolatembridgei , this ismbridgei , and funmbridgei , 2006. Tmbridgei . As prevmbridgei , and theThe unique TR multidomain structure of SCREPs is likely to be responsible for their observed multivalent interactions. The investigation of SCREP ligands with multiple binding sites on symmetrical targets may lead to new insights on the role of bivalency in ion-channel pharmacology. Previous observations from DkTx and Hi1a, suggest that these features may enhance the selectivity of these ligands, capable of differentiating between ion-channel subtypes. Additionally, the combined interactions between multiple binding sites may result in increased levels of ligand avidity, with desirable pharmacological consequences. The formation of a tripartite complex between DkTx, TRPV1, as well as phospholipid interactions, have proven useful in the structural elucidation of this ligand receptor complex . AdditioThe data driven methods discussed above, provide a taxonomically unbiased approach toward the investigation of SCREPs. The practical application of our data mining and analysis pipeline, illustrates an excellent opportunity in which to diverge from repeating past methods used in bio-discovery efforts . By deviThe investigation of ion-channel activity depends greatly upon the use of functionally active molecular tools, the interaction of which provides valuable insights into these incredibly complex macromolecules. Animal toxins provide an excellent source of useful ion-channel ligands, and the discovery of novel bioactive ligands is a key component in the study of ion-channel pharmacology, necessary for the therapeutic development of new molecules aimed at treating an array of problematic channelopathies. SCREPs provide an exciting new source of structurally unique proteins with a high level of both taxonomic and structural diversity. Periodic sampling of the SCREP sequence space indicates an increase of approximately 21% of the total dataset size within just 12 months, suggesting this to be an expanding resource.Presented here, is a detailed analysis of SCREP sequences identified when using the data mining pipeline which we have developed. An effort toward providing a comprehensive description of the SCREPs data mining pipeline will hopefully supply the reader with the necessary details to further explore this novel resource. Despite several bioinformatic challenges that exist, we are still able to confidently identify many putative SCREP sequences that have great potential in displaying unique structure and pharmacology. The analysis of venom derived SCREPs provides an excellent approach in the identification of potentially bioactive peptides, likely to contribute to our understanding of the structural biology, function, and possibly even diversity of ion channels.MiM generated the data presented and performed the data analyses. MeM and MiM designed and developed the bioinformatic tools used with input from EU. EU and MeM conceived of and directed the research with input from MiM. MiM wrote the manuscript with input from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer IV and handling Editor declared their shared affiliation at the time of review.