Background. Recent clinical trials suggest that nonoperativemanagement (NOM) of patients with acute, uncomplicated appendicitis is anacceptable alternative to surgery. However, limited data exist comparing thelong-term cost-effectiveness of nonoperative treatment strategies.Design. We constructed a Markov model comparing thecost-effectiveness of three treatment strategies for uncomplicated appendicitis:1) laparoscopic appendectomy, 2) inpatient NOM, and 3) outpatient NOM. The modelassessed lifetime costs and outcomes from a third-party payer perspective. Thepreferred strategy was the one yielding the greatest utility without exceeding a$50,000 willingness-to-pay threshold. Results. Outpatient NOM cost$233,700 over a lifetime; laparoscopic appendectomy cost $2500 more whileinpatient NOM cost $7300 more. Outpatient NOM generated 24.9270 quality-adjustedlife-years (QALYs), while laparoscopic appendectomy and inpatient NOM yielded0.0709 and 0.0005 additional QALYs, respectively. Laparoscopic appendectomy wascost-effective compared with outpatient NOM ; inpatient NOM was dominated by laparoscopicappendectomy. In one-way sensitivity analyses, the preferred strategy changedwhen varying perioperative mortality, probability of appendiceal malignancy orrecurrent appendicitis after NOM, probability of a complicated recurrence, andappendectomy cost. A two-way sensitivity analysis showed that the rates of NOMfailure and appendicitis recurrence described in randomized trials exceeded thevalues required for NOM to be preferred. Limitations. There arelimited NOM data to generate long-term model probabilities. Health stateutilities were often drawn from single studies and may significantly influencemodel outcomes. Conclusion. Laparoscopic appendectomy is acost-effective treatment for acute uncomplicated appendicitis over a lifetimetime horizon. Inpatient NOM was never the preferred strategy in the scenariosconsidered here. These results emphasize the importance of considering long-termcosts and outcomes when evaluating NOM. While usually safe and effective, appendectomy is not totally benign:surgery incurs perioperative risk and has been associated with short- and long-termcomplications, with reported rates ranging from 2% to 23%.13 The necessaryobservation period to assess these long-term outcomes is impractical for prospective trials,14 and certainly exceeds individual surgeons’ typical episodes of care. However,accurate assessment of the cost-effectiveness of NOM warrants consideration of thelong-term costs of appendicitis-related care, including the lifetime potential forrecurrence, ongoing risk for appendiceal malignancy, or potential postoperativecomplications such as adhesive bowel obstructions. Two prior cost-effectiveness analysescompared NOM with appendectomy in the adult and pediatric populations,16 but these analyses were completedover 1-year time horizons and did not model long-term costs.Proponents of nonoperative management (NOM) contend that it is less expensive thanappendectomy, as it minimizes operative costs and may reduce hospitalization days.Trials have estimated the costs of NOM versus surgical management for the indexhospitalization, but have not assessed long-term costs of recurrence or othercomplications.To determine the cost-effectiveness of NOM versus operative management of acuteuncomplicated appendicitis, we conducted an analysis comparing two NOM strategiesagainst laparoscopic appendectomy. A novel aspect of our analysis is the modeling ofboth short- and long-term costs over a reference patient’s lifetime to study theenduring effects of NOM and surgery. This analysis is intended to inform surgeons andthird-party payers regarding long-term economic implications when comparing NOM andlaparoscopic appendectomy as first-line therapies for uncomplicated appendicitis.This computer simulation modeling study was deemed exempt from human subject reviewbecause it uses only publicly available, deidentified, and aggregate datasources.We first defined the reference case as a healthy 20-year-old male in the UnitedStates presenting to the emergency room with imaging-confirmed uncomplicatedappendicitis. Uncomplicated appendicitis was defined as inflammation of theappendix in the absence of abscess, phlegmon, perforation, fecalith on imaging,or diffuse peritonitis on initial clinical exam.11 Two NOM arms were includedas we wished to consider both the most typical nonoperative treatment strategyas well as the potentially least resource-invasive, as recent pilot studies haveshown comparable outcomes between inpatient and outpatient management strategies.17 Appendectomy was considered the reference treatment, as it is the currentstandard of care.We constructed a decision model using decision analysis software . We used literature review and consultation with experts tobuild a decision tree comparing three management strategies: 1) laparoscopicappendectomy, 2) inpatient NOM with 3-day hospitalization for intravenousantibiotics (piperacillin-tazobactam) followed by completion course of 7 days ofoutpatient oral antibiotics (amoxicillin-clavulanate), and 3) outpatient NOMwith 7 days of oral outpatient antibiotics only (amoxicillin-clavulanate) . For eac19 and NOMmay result in a missed opportunity to treat an early, confined appendicealtumor. Recent data suggest that appendiceal tumors are incidentally discoveredin around 1% of appendectomies.2022 It is thus reasonable toconsider the potential downstream effects of leaving an unidentified tumor insitu following NOM over the rest of a patient’s life.Inpatient failure of NOM was defined as symptom progression during the indexhospital admission. Outpatient failure was defined as symptom progression orreturn of symptoms after discharge from the index hospitalization within 30 daysof initial presentation. Treatment failures occurring after 30 days wereconsidered to be recurrent appendicitis. Complicated failure or recurrence wasdefined as perforation with associated abscess. We considered short-termperioperative complications including ileus, surgical site infection, anestheticcomplications, and postoperative hemorrhage. We considered long-termcomplications including trocar-site hernia and adhesive small bowel obstruction.Finally, we accounted for the possibility that appendicitis may be the firstpresentation of an otherwise unrecognized appendiceal tumor,We also conducted additional scenario analyses to explore how much variousfactors would influence the assessment of NOM. These included the following: 1)if an older patient underwent NOM (if the base case was instead an average 40-or 65-year-old), 2) if patients who were managed nonoperatively never developedappendiceal cancer , 3) if patients who were managed nonoperatively neverfailed or recurred, and 4) if all failures or recurrences were uncomplicated.23 Age-appropriate background mortality and costs were considered in themodel.25 We used Markov modeling with a cycle length of 1 month toexamine probabilities of long-term events over the expected lifespan of thepatient. After initial chance nodes accounted for events and costs occurring inthe first 30 days, Markov nodes incorporating half-cycle correction were used.Incremental cost-effectiveness ratios were compared with a $50,000 per QALY gained willingness-to-pay threshold.26 For the strategies considered, we examined the incrementalcost-effectiveness ratios (ICERs)—defined as the incremental cost of a givenstrategy divided by its incremental benefit compared with the next bestalternative. Future costs and life-years were discounted at an annual rate of 3%.27The time horizon for the model was the expected lifetime of the reference case,derived from US life tables.28 and expert opinion from clinicians. Base case values were felt byclinicians to represent the best available estimates from the literature.Probabilities were obtained from literature review ; these w29 We summed the physician work, practice expense, and malpractice relativevalue units (RVUs) and multiplied by a conversion factor of 35.8887 (per CMSdata) for the Current Procedural Terminology (CPT) codes relevant to our study. Hospitalization costs were obtained from the CMS final rulefor fiscal year 2017,30 and were calculated by multiplying the conversion factor by the sum ofthe RVUs for 1 admit day (CPT 99222), 1 discharge day (CPT 99239), and anyintervening hospital days (CPT 99231). Average length of hospitalization perprocedure was obtained from literature review. We multiplied the sum of thelabor ($3420), non-labor ($2096), and capital cost rates ($447) by theappropriate DRG (diagnosis-related group) multiplier. The costs of emergencydepartment evaluation and follow-up clinic visits were obtained from the CMSHospital Outpatient Prospective Payment System.31 The sum of hospital charges and physician charges for a level 4 visit(CPT 99284) was added to the cost of imaging; assuming that half of the patientsreceived an abdominal computed tomography scan and half received an ultrasound.Medication costs were obtained from the Federal Supply Schedule.32 The average wholesale price per medication was multiplied by 0.64 toobtain the net final price paid by Medicaid for each medication . The Medicaid final price includes distribution anddispensing costs. Costs used in our analysis are summarized in Costs were considered from the perspective of a third-party payer. Proceduralcosts were calculated using the 2017 Centers for Medicare and Medicaid Services(CMS) physician fee schedule.33Utilities related to hospitalizations, surgical or procedural interventions, andrecovery from appendicitis or appendectomy were drawn from published literatureas available. Previously published studies assessing age-related backgrounddecline in health-related quality of life was used to estimate the baselineutility of the reference case.3437 We assumed that all NOMfailures or recurrences underwent laparoscopic appendectomy . All patients with complicated failures orrecurrences were assumed to undergo percutaneous drain placement byinterventional radiology followed by interval appendectomy. We assumed that 70%of patients returning with an appendiceal malignancy presented with a carcinoidtumor >2 cm and underwent right hemicolectomy, and the remaining 30%presented with Stage III appendiceal adenocarcinoma and underwent righthemicolectomy followed by 12 cycles of FOLFOX chemotherapy.3840 We purposefully assumedthat patients would present with advanced-stage malignancies due to a delay incancer diagnosis related to NOM. As a small percentage of patients undergoingappendectomy may be found to have malignancies at that time, we assumed thatthese patients would be cured by appendectomy alone and would not requirefurther treatment. We felt this assumption was reasonable, as we defined ourreference case as having confirmed appendicitis without concerning imagingfindings correlated with diagnosis of later-stage malignancy duringappendectomy.We assumed that all appendectomies were completed laparoscopically, in line withmodern case series describing very low rates of conversion to open appendectomyfor uncomplicated appendicitis.We first conducted one-way deterministic sensitivity analyses to evaluate how thedecision might change across the plausible range of each model parameter givenits uncertainty while holding all others at their base case values. We usedsensitivity analyses to assess the importance in changes of utilities for healthstates individually and in combination, while preserving the preference orderingof health states. Baseline values were varied over the ranges displayed in 41 The uncertainty for normal distributions was calculated using apreviously defined method.42 Values were repeatedly sampled from their appropriate uncertaintydistributions 10,000 times.We then conducted Monte Carlo probabilistic sensitivity analyses using betadistributions for input probabilities and utility weights and normaldistributions for defined procedural costs. Univariate utility distributionswere then correlated to establish joint uncertainty distributions exploitingordinal preferences over health states, in order to avoid bias and improveprobabilistic sensitivity analyses.Authors’ funding sources had no role in the study.When considered over the expected lifetime of the base case patient, outpatientNOM proved the least costly and generated the fewest QALYs and life-years . BothlaWe first assessed whether the cost-effectiveness of NOM was sensitive to the ageof the reference patient considered. When the age of the reference case wasincreased to either age 40 or age 65, laparoscopic appendectomy remained themost effective strategy and was cost-effective at age 40 but not at age 65.When we considered the situation where the risk of developing appendiceal cancerafter NOM was zero , appendectomy’s healthbenefits relative to NOM gained by removing the appendix were attenuated . Hence,When we considered the scenario where all NOM was successful , the costs of both NOM strategiesdecreased compared with the base case . OutpatiFinally, we considered the situation where all failures and recurrences wereuncomplicated and proceeded directly to appendectomy instead of requiringinitial percutaneous drainage followed by interval appendectomy. The cost ofboth NOM strategies decreased relative to the base case and outpatient NOMremained the least costly. QALYs and life-years increased for both NOMstrategies; appendectomy continued to generate more life-years and QALYs. Inthis scenario, laparoscopic appendectomy was no longer cost-effective comparedwith outpatient NOM . Inpatient NOM was dominated bylaparoscopic appendectomy.Model results were sensitive to changes in the following parameters in one-waysensitivity analyses: perioperative mortality rates during appendectomy, theprobability of developing recurrent appendicitis and the probability that therecurrence be complicated, the probability of developing an appendicealmalignancy after NOM, and the cost of laparoscopic appendectomy. When theprobability of perioperative mortality during laparoscopic appendectomy was>0.26%, outpatient NOM dominated laparoscopic appendectomy. When theprobability of developing recurrent appendicitis in the 5 years following NOMwas >26%, laparoscopic appendectomy dominated. Laparoscopic appendectomy alsodominated when the probability of a complicated appendicitis recurrence was>4.6%. Laparoscopic appendectomy dominated outpatient NOM when the long-termrisk of developing an appendiceal malignancy after NOM was <0.48%. Varyingthe cost of laparoscopic appendectomy also changed the preferred strategy: whengreater than $10,173 , outpatient NOM was preferred overlaparoscopic appendectomy. In no sensitivity analysis that we considered wasinpatient NOM the cost-effective strategy.We conducted a two-way sensitivity analysis when the probabilities of short-termNOM failure and long-term appendicitis recurrence were varied concurrently.In probabilistic sensitivity analyses, laparoscopic appendectomy was thepreferred strategy in 62% of 10,000 simulated cases when the willingness-to-paythreshold was set at $50,000 per QALY, while outpatient NOM was preferred in theother 38%. A cost-effectiveness acceptability curve is included as Our analysis finds that laparoscopic appendectomy is the most effective managementstrategy for acute uncomplicated appendicitis when the lifetime effects ofappendicitis-related treatment are considered. It is cost-effective compared withoutpatient NOM with an ICER of $32,300 per QALY. Inpatient NOM is the costlieststrategy and was less effective than laparoscopic appendectomy.16 A costanalysis of the APPAC trial comparing appendectomy to NOM concluded that NOMincurred fewer costs over the duration of the trial.13 In contrast, our model suggests that laparoscopic appendectomy is acost-effective treatment option despite the additional costs associated with surgerywhen the lifetime implications of appendicitis-related treatment are considered.Conversely, inpatient NOM as conventionally defined by prospective trials was notcost-effective over a lifetime horizon. This demonstrates the importance ofcollecting long-term data from registries and prospective trials assessing NOM.Prospective trials of short duration and individual surgeons’ personal experienceswill not account for long-term outcomes and may make NOM appear overlyfavorable.These findings are important as few other studies have modeled or assessed thelong-term health and economic consequences of NOM for uncomplicated appendicitis.Prior cost-effectiveness analyses assessing NOM were limited to a 1-year timehorizon and suggested that NOM dominated laparoscopic appendectomy.When considered over a patient’s lifetime, the differences in costs and QALYs betweenappendectomy and NOM were relatively small. This likely reflects the overallexcellent outcomes following appendicitis and low rates of long-term complicationsfollowing either management strategy. However, the sensitivity and scenario analysespresented here are informative in determining important factors in the long-termcost-effectiveness of appendicitis treatment. Appendectomy cost and perioperativemortality proved influential in one-way sensitivity analyses, suggesting thatminimizing operative costs and mortality are important targets in real-lifepractice. Model outcomes were insensitive to changes in age until age 65, suggestingthat these results are applicable to a broad adult population.4 Although rare, it is thus important to consider the possibility of futuremalignancy when considering NOM.2022The probabilities of developing and dying from appendiceal malignancies were stronglyinfluential on treatment preferences in this analysis, despite their relativelyrarity. Appendectomy was preferred over outpatient NOM when the probability offinding an incidental appendiceal malignancy at index appendicitis presentation wasgreater than 0.59%. Recent data suggest a 1.1% probability of incidentallydiscovering an appendiceal malignancy at index appendicitis presentation forpatients aged 18 to 65, and a 2.7% probability for ages >65.512 The thresholds of NOM failureand appendicitis recurrence resulting in dominance by laparoscopic appendectomy arenotably lower than those reported in prior cost-effectiveness analyses conductedover a 1-year time horizon, which found that NOM was not the preferred strategy whenshort- and long-term failure rates ranged from 32% to 45%.16 These results suggest thattreatment failure rates of large-scale pending prospective trials should be analyzedclosely as they bear significant financial implications for the cost-effectivenessof NOM, and that careful tracking of long-term recurrence rates is important.As expected, rates of NOM treatment failure and appendicitis recurrence wereinfluential on model outcomes and the preferred strategy. Two-way sensitivityanalyses revealed that the threshold rates of treatment failure and appendicitisrecurrence resulting in dominance of outpatient NOM over laparoscopic appendectomywere at the low end of recurrence rates seen in randomized trials, where treatmentfailure rates have reached as high as 38%.6 Although not yet common clinical practice, recent pilot studies have movedtoward assessing fully outpatient treatment strategies, consistent with the leastresource-invasive strategy presented here.17 Clinicians must be aware that NOM protocols that result in hospitalizationslonger than hospitalizations typical for laparoscopic appendectomies  are unlikely to prove cost-effective on a population level.Notably, inpatient NOM as defined in this analysis was never cost-effective or thepreferred strategy compared with outpatient NOM or laparoscopic appendectomy. Thedefinition of inpatient NOM was modeled to be consistent with the largest randomizedtrial to date: a 3-day admission for intravenous antibiotics followed by 7 days ofcompletion oral antibiotics.44; thus, cost estimates and their effects on the model may not begeneralizable to other settings. Parameters used to model outpatient NOM were drawnfrom prospective trials that included an all-oral antibiotic regimen, as no majorprospective trials to date have tested a fully outpatient NOM strategy. For thepurposes of studying the costs associated with outpatient treatment, we assumed thatoral antibiotics given in hospital would yield similar results to oral antibioticstaken at home. However, this may be unrealistic as patients may derive additionalbenefit from hospitalization alone, making outpatient management appear overlyeffective in this analysis.We recognize the limitations associated with this study, particularly the relativelylimited prospective data used to generate model parameters. This is especially truefor parameters involving long-term probabilities, including the long-term risk ofappendicitis recurrence after NOM and long-term risks of postoperativecomplications. Every effort was made to preferentially use model parameters drawnfrom high-quality randomized data, only supplementing with retrospective data andexpert clinician opinion if no other parameter estimates were available. Healthstate utilities used as model parameters were limited in availability and drawn fromsingle studies, and thus may significantly influence model outcomes. Health carecosts in the United States are known to exceed costs in other developednations4547 These results may not beapplicable to low-resource settings where preoperative diagnostic imaging is lessfrequently utilized and rates of negative appendectomy are higher.Finally, the model is built on the assumption that patients have imaging-confirmedappendicitis, consistent with modern surgical practice. Rates of negativeappendectomy have decreased significantly secondary to the high diagnostic accuracyof modern diagnostic imaging.In this cost-effectiveness analysis of treatment strategies for acute uncomplicatedappendicitis, laparoscopic appendectomy is the most effective management strategyfor acute uncomplicated appendicitis when considered over a lifetime horizon and iscost-effective compared with outpatient NOM. In cases where there is a desire not toperform surgery, outpatient NOM should be considered, as inpatient NOM as previouslydefined in randomized trials is costlier and is not cost-effective.


Bilateral salpingo-oophorectomy (BSO) is increasingly employed as a risk-reducing strategy for epithelial ovarian cancer (EOC). We report the third case of a patient developing primary peritoneal cancer two decades after a bilateral salpingo-oophorectomy. This 66-year-old female underwent a hysterectomy for pelvic pain at age 28 and a subsequent bilateral salpingo-oophorectomy (BSO) at age of 45 for a pelvic mass. Presenting with a 6-month history of increasing abdominal girth, decreased energy, and a reduction in appetite, she was consented for a unilateral salpingo-oophorectomy, omentectomy, and cytoreductive surgery. Pathology specimens revealed a high grade metastatic papillary serous carcinoma consistent with a primary gynecologic origin. It is unlikely that an occult malignancy was missed at the time of pathologic assessment following her previous BSO; therefore it provides evidence that primary peritoneal cancers can arise through the malignant transformation of benign endosalpingiosis. In North America, ovarian cancer has the highest case fatality rate amongst gynecological malignancies. In Canada each year, 1750 women will die from ovarian cancer , 3.A risk-reducing strategy employed for women with a strong family history of ovarian carcinoma/BRCA mutation carriers is prophylactic bilateral salpingo-oophorectomy (BSO). In addition, BSO at the time of hysterectomy for benign conditions has been suggested to reduce the risk of EOC, particularly in postmenopausal women . FurtherA 66-year-old female presented to the gynecology oncology clinic with a 6-month history of increasing abdominal girth, decreased energy, and a reduction in appetite. She reported a twenty pound weight gain over the preceding two months.The patient reported a previous hysterectomy at age 28 for pelvic pain and then a bilateral salpingo-oophorectomy at age 45 for a pelvic mass. Past medical history was significant for atrial fibrillation, type 2 diabetes mellitus, hypertension, and gastro-esophageal reflux disease. Her family history was significant for a maternal aunt with a diagnosis of breast cancer at age 58 and subsequent ovarian cancer at age 90.A diagnostic paracentesis was performed preoperatively and returned positive for an adenocarcinoma. An abdominal CT scan revealed a query 4.8 x 2.1 cm left ovarian cyst and omental cake. CA-125 was elevated at 278 U/mL . On examination, BMI was 38. Auscultation of the heart and lungs was normal. Abdominal examination showed shifting dullness. Rectovaginal examination revealed ascites, but no pelvic mass or nodularity in the cul-de-sac.With a presumed diagnosis of ovarian cancer, the patient was presented with two options, neoadjuvant chemotherapy, or primary cytoreductive surgery. After a discussion of risks and benefits of each approach, the patient consented for primary cytoreduction.A laparotomy was performed, and upon entering the peritoneal cavity, diffuse inflammation of the peritoneum and 12L of ascites were noted. Extensive carcinomatosis involving most peritoneal surfaces was identified with involvement of the ascending colon. Preoperatively we had queried the possibility of some residual ovary; however no ovary could be identified within the peritoneal cavity, nor with dissection into the retroperitoneum. A supracolic omentectomy, peritoneal stripping of both upper abdomen and pelvis, and right sided hemicolectomy with primary reanastomosis were performed.In the postoperative period, the patient developed rapid atrial fibrillation and became persistently hypotensive despite aggressive fluid resuscitation. She was brought to the intensive care unit for resuscitation, but continued to decline. At the patient's request, treatment was withdrawn; she died shortly thereafter.Pathology specimens revealed a high grade metastatic papillary serous carcinoma consistent with a primary gynecologic origin. The patient's previous surgical and pathology reports were obtained. The operative note from 1993 described that the patient had undergone a laparotomy with removal of both fallopian tubes and ovaries. Final pathology from that surgery confirmed removal of a normal left ovary and tube and the removal of a right ovary (containing a serous cyst) and a normal right fallopian tube. No borderline or malignant changes were identified.To our knowledge this is only the third case of a patient developing primary peritoneal cancer two decades after a BSO. A previous report by Casey et al. described a 55-year BRCA1 mutation carrier who developed primary peritoneal cancer 24.3 years following prophylactic salpingo-oophorectomy. Investigation of this patient's ovaries revealed no malignant or borderline changes . Piver dFor women with a BRCA mutation, the literature quotes a consistent risk of 3-4% risk of primary peritoneal cancer following prophylactic salpingo-oophorectomy over twenty years, with the vast majority of cases occurring within 3-4 years –8. The rOf the two prior cases presenting with primary peritoneal cancer two decades after prophylactic BSO, one patient was a known BRCA1 mutation carrier and the other patient had a significant family history for ovarian cancer. Unfortunately, the mutation status of our patient was unknown, as the patient passed away before any further treatment or genetic testing could be offered. The patient is a possible mutation carrier as there was a known family history of ovarian and breast cancer in a second degree relative. Furthermore, a somatic BRCA mutation at the tumoral level is a possibility.Regarding the pathogenesis of high grade serous tumors (HGST) of pelvic origin, a model gaining wide acceptance proposes that these tumors originate in fallopian tube epithelium (FTE) as premalignant growths dubbed STICs  which undergo malignant transformation and, due to their location, extend to nearby ovary and surrounding pelvic peritoneum , 10. SupConversely, in about half the cases of pelvic HGST, there are no concurrent fallopian tube neoplastic changes. This has led to the proposal that both low and high grade serous carcinomas can arise from benign fallopian tube epithelial (FTE) inclusions in the ovary  or on peritoneal surfaces  which undergo malignant transformation independently of changes in the fallopian tube . These iIn our case, the interval between BSO and the later development of primary peritoneal cancer was 21 years, making it unlikely that an occult malignancy was missed at the time of pathologic assessment following her BSO at the age of 45. We reviewed the pathology report from her original surgery, and it confirmed removal of both ovaries and fallopian tubes. Assuming no residual ovary/fallopian tube remained, the malignancy likely arose from benign peritoneal FTE inclusions  that had been sloughed from the patient's fallopian tubes before these were removed, subsequently undergoing malignant transformation many years later. It therefore provides evidence that primary peritoneal cancers can arise through the second mechanism described above, through the malignant transformation of benign endosalpingiosis. It further demonstrates that fallopian tube STICs are not a necessary intermediary step in the pathogenesis of primary peritoneal carcinomas.This case demonstrates a rare presentation of primary peritoneal cancer presenting twenty-one years after bilateral salpingo-oophorectomy.


Passive administration of broadly neutralizing antibodies (bNAbs) capable of recognizing a broad range of viral strains to non-human primates has led to protection from infection with chimeric SIV/HIV virus (SHIV). This data suggests that generating protective antibody responses could be an effective strategy for an HIV vaccine. However, classic vaccine approaches have failed so far to induce such protective antibodies in HIV vaccine trials. HIV-specific bNAbs identified in natural infection show high levels of somatic hypermutations, demonstrating that they underwent extensive affinity maturation. It is likely that to gain ability to recognize diverse viral strains, vaccine-induced humoral responses will also require complex, iterative maturation. T follicular helper cells (Tfh) are a specialized CD4+ T cell subset that provides help to B cells in the germinal center for the generation of high-affinity and long-lasting humoral responses. It is therefore probable that the quality and quantity of Tfh responses upon vaccination will impact development of bNAbs. Here, we review studies that advanced our understanding of Tfh differentiation, function and regulation. We discuss correlates of Tfh responses and bNAb development in natural HIV infection. Finally, we highlight recent strategies to optimize Tfh responses upon vaccination and their impact on prophylactic HIV vaccine research. Most successful vaccines  work by inducing long-lasting neutralizing antibody responses that prevent infection of target cells . CurrentEnv is the only viral protein expressed on the surface of free, mature HIV virions. Broadly neutralizing antibodies (bNAbs) are able to recognize a variety of different HIV strains by targeting conserved regions of the HIV envelope protein. Passive administration of bNAbs has been shown to prevent infection in non-human primate (NHP) models ,4,5. In High quality and long-lived humoral immune responses require help from a specialized CD4+ T cell subset called T follicular helper cells (Tfh) . Tfh celTfh cells are a specialized CD4+ T helper subset, characterized by the expression of CXCR5, the ligand for the chemokine CXCL13, which allows their migration into the GC of secondary lymphoid organs ,11. TherTfh differentiation is a multifactorial and multistep process , process it and present it to GC Tfh via MHCII. During this process, B cells compete for limited Tfh help: High affinity B cells, which were able to capture and therefore present more antigen compared to B cells with lower affinity, are more likely to receive Tfh signals ,53. SeleTfh help in the GC occurs via direct cell-cell interactions with B cells and secretion of cytokines. Some important mediators for B cell survival, proliferation, and differentiation are summarized in GC Tfh cells have been shown to form a pool of memory cells upon antigen clearance in both mice and humans. Memory Tfh cells localize together with antigen-specific memory B cells in the draining lymph node (LN) for the rapid induction of humoral responses upon re-exposure to antigen . A subsecTfh represent a heterogeneous population that can be classified into multiple subsets based on polarization and activation status. Differential expression of the chemokine receptors CXCR3 and CCR6 allows the distinction of Th1-like (CXCR3+CCR6-), Th1Th17-like (CXCR3+CCR6+), Th17-like (CXCR3−CCR6+), and Th2-like (CXCR3−CCR6-) cTfh subsets. These cTfh subsets express transcription factors and can produce cytokines upon stimulation that are typically associated with Th1, Th2, Th17, and Th1Th17 CD4+ subsets . Using tTfh subsets based on the expression of CXCR3 and CCR6 can also be identified in the LN of macaques . HoweverGiven the important role of Tfh cells for the generation of high-affinity antibody responses, it is not surprising that absence of or impaired Tfh responses hamper the generation of protective antibodies after infection or vaccination . HoweverTfh cells are characterized by the high expression of multiple co-inhibitory receptors including PD-1, TIGIT or CD200 ,76,77. WIn addition, GC responses are controlled by the recently identified T follicular regulatory cells (Tfr) ,82,83. TTfh cell are highly permissive to HIV or SIV infection ,87,88, yAlthough the increase of Tfh cell frequencies in lymphoid tissue is not mirrored in peripheral blood of HIV-infected individuals, cTfh cells present qualitative defects in viremic and ART-treated subjects compared to uninfected controls, with a decreased ability to induce B cell differentiation and antibody production in vitro ,96. ThesIn untreated HIV infection, bNAbs typically develop after a few years of chronic antigen exposure. Because of immune escape of the autologous strain, these humoral responses are not associated with viral control . These fCorrelates between Tfh responses and development of bNAbs in humans have been largely restricted to the analysis of blood as access to lymphoid tissue is limited. Differences in the cTfh response between HIV+ subjects with high or low levels of neutralizing antibody activity were detected with a high frequency of CXCR3-PD-1+ cTfh being associated with the development of bNAbs ,98. DuriHowever, most studies mentioned rely on the analysis of the bulk Tfh population. Further studies are necessary to specifically investigate HIV-specific Tfh responses and the correlation of phenotype and function with protective antibody development, which might differ from observations made on total Tfh cells. Such investigations have been hampered by the limited tools available to study T cell antigen specificity independently of cytokine production. As Tfh show only limited cytokine production upon activation, they are more likely to be missed with standard assays such as intracellular cytokine staining or enzyme-linked immunospot (ELISPOT). Activation-induced marker (AIM) assays, recently developed by our group and others ,103,104,Classical vaccination strategies, while successful for the induction of protective antibodies against various pathogens, have failed to induce protective and long-lasting humoral responses against HIV. Given the importance of the quantity and quality of Tfh cells for the development of effective antibody responses in natural HIV infection several groups sought to determine how Tfh responses could be induced or modulated to improve humoral immunity  as well as activate innate immune cells via binding to specific receptors  agonists). Based on their different effects, specific adjuvants therefore guide distinctive types of generated immune responses. Alum is the most widely used adjuvant for vaccinations  and has shown great success in vaccinations against for example hepatitis A and B, human papilloma virus, diphtheria, or tetanus . Howeverhttps://clinicaltrials.gov/ct2/show/NCT02968849?term=HVTN702&rank=1, accessed 27 May 2018). This study is based on the RV144 trial (ALVAC/AIDSVAX), which demonstrated partial antibody-mediated protection [Given the success of MF59 to enhance protective antibody titers in vaccines against other pathogens, it is currently used in some HIV vaccine efficacy trials, such as the HVTN702 study (ALVAC + gp120), conducted in South Africa  injection as this vaccination route demonstrated only minimal adverse effects . HoweverAnother factor is the differential distribution of DCs between tissue compartments. As mentioned before, DCs regulate CD4+ T cell differentiation. In a recent study, Krishnaswamy et al. identified migratory CD11b+ type 2 DCs as superior inducers of Tfh generation in mice due to their enhanced homing potential to the T-B border in secondary lymphoid organs . These CSince HIV is mainly transmitted across mucosal surfaces, an effective HIV vaccine likely depends on the generation of humoral responses in the mucosa. Mucosal antibody responses can be induced by systemic immunization routes, however, the exact mechanisms and how to specifically induce them via s.c., i.m., or i.d. immunization are not known . MucosalAs mentioned earlier, Tfh differentiation is promoted by prolonged DC-T interaction and Tfh maintenance is dependent on persistent antigen availability in the GC, suggesting that Tfh differentiation could be regulated by the amount of antigen given during vaccination. Indeed, GC Tfh and B cell responses are elevated when mice were immunized with higher doses of antigen . SimilarAn alternative strategy to enhancing the dose of the single vaccine infections is the prolonged administration of antigen. Single injection of a vaccine antigen might not recapitulate the sustained availability of antigen in the GC during the natural infection of replicating pathogens. This might be especially important for the generation of HIV-specific bNAbs as they require extensive SHM acquired during multiple rounds of GC reactions . To maniContinuous antigen delivery can also be achieved using immunization with mRNAs encoding for the antigen of interest (reviewed in ). SingleTfr cells represent an interesting target for inhibition to enhance GC Tfh and B cell function. Besides influencing Tfr/Tfh ratio at the differentiation level using certain TLR-agonists as mentioned before, Tfr cell function was directly targeted in mice. CTLA-4 blockade or knockout led to a decreased suppressive capacity of Tfr cells and augmented Tfh cell frequency and function, GC responses, and antigen-specific antibody responses upon immunization ,119. HowCurrent HIV-related vaccine studies in NHP animal models demonstrate that several new vaccination strategies alone or in combination are able to enhance GC Tfh and B cell responses, which will serve as basic concept for future studies. These strategies aim to enhance GC Tfh responses by broadly augmenting Tfh frequency, yet the question remains whether more targeted approaches and the induction of specifically efficient Tfh cells might be favourable for the generation of bNAbs upon vaccination. For this, a better understanding of Tfh function in the GC is indispensable.This review focuses on the development of strategies for a prophylactic HIV vaccine. However, enhancing antiviral responses also plays a role in HIV strategies of a functional cure that aims at inducing control of any residual viral replication by the host’s own immune system in the absence of ART. Administration of bNAbs in ART-treated HIV+ subjects delayed viral rebound after analytic treatment interruption until viral resistance mutations occurred , suggestTfh responses have been shown to be involved in the generation of protective antibody responses in various infectious diseases and after vaccination. Induction of more potent Tfh responses represents an interesting strategy for vaccines that show limited efficacy, including HIV. Understanding Tfh biology has allowed developing alternative strategies that aim to induce more efficient Tfh responses, which have shown some success in NHP studies. However, further efforts are needed to determine whether these strategies can be successfully applied to HIV vaccine trials in humans.


AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotic cells and organisms. As such, AMPK has attracted enormous interest in various disciplines. Accordingly, the current Special Issue “AMP-Activated Protein Kinase Signalling” is a present-day reflection of the field covering a wide area of research. Although widely conserved throughout evolution and expressed ubiquitously, the functions of AMPK in different tissues and cell types may vary to some extent. Therefore, it is worthwhile to focus on cellular functions of various different origins, or tissues and organs, as well as their interplay in the context of the whole organism. This Special Issue includes research articles and reviews addressing AMPK regulation and function in all biological organization levels in health and disease.Starting from the AMPK molecule, Yan et al. summarize the knowledge derived from crystal structures and provide expert insight into the molecular mechanisms of kinase activity modulation by adenine nucleotides . As pres2+/Calmodulin-dependent protein kinase kinase 2 (CaMKK2) are firmly established as physiological upstream kinases of AMPK. In addition, transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) has also been reported as AMPK upstream kinase, but did not receive full attention as discussed in detail [Apart from the allosteric mode of regulation, AMPK is part of a kinase cascade. Upstream regulation of AMPK involves one of several kinases capable of phosphorylating AMPK at Thr-172 in the α-subunit. Both liver kinase B1 (LKB1) and CaAs reviewed by Janzen et al., in skeletal muscle, AMPK activity is regulated by glycogen content . GlycogePrevious studies also reported that AMPK activation improved insulin sensitivity in endothelial cells for modulation of vascular homeostasis. Strembitska et al. investigate insulin-stimulated Akt phosphorylation in response to the AMPK activators AICAR, 991, and A-769662, the latter two chemicals targeting a different part of the AMPK molecule, the ADaM  site . However2+-induced assembly of tight junctions.Hypertension and kidney disease can be a consequence of suboptimal early-life conditions, that is, by renal programming. Tain and Hsu bring forward the argument that AMPK activators could be applied for renal reprogramming as a protection against disease development . Glosse Over the past decade, AMPK has emerged as a key player in the regulation of whole-body energy homeostasis. AMPK regulates food intake and integrates energy metabolism with several hormones, such as leptin, adiponectin, ghrelin, and insulin . In thisApart from acute effects on the activity of enzymes or localization of proteins, AMPK has also been shown to change gene expression patterns for long-term adaptation involving regulation of transcription factors and chromatin remodelling. Gongol et al. describe AMPK as a key player in epigenetic regulation and discuss the consequent physiological and pathophysiological implications . AMPK isLong-term memory depends on the induction of immediate early genes (IEGs). Didier et al. report that AMPK controls the expression of IEGs upon synaptic activation via the cAMP-dependent protein kinase (PKA)/cAMP response element binding (CREB) signalling pathway . AlthougWhile many studies focused their attention on the tumour-suppressor effect of AMPK activation, there is now growing evidence that AMPK plays a dual role in cancer, that is, inhibiting growth but enhancing survival. Adding to this discussion, Zhang et al. show that loss of AMPKα2 impairs sonic hedgehog medulloblastoma tumorigenesis . Silwal O-methyltransferase (COMT). Human reproduction represents a less mature field of AMPK research. Martin-Hidalgo et al. review the known cellular roles of AMPK in spermatozoa . The argIn summary, the current Special Issue provides a representative cross-section of AMPK research and topical reviews. Thanks to the authors submitting their precious work and insights that are presented in this Special Issue, our understanding of AMPK structure, function, and regulation has further progressed. Additionally, it turns out that AMPK biology is more complex than most of us originally anticipated, leading many of the contributing authors to highlight the fact that we still lack information and need to address new questions in subsequent studies. For example, the molecular structure of AMPK, although studied in great detail, does not provide information on the dynamic movements that are inherent to an allosteric enzyme. Moreover, “AMPK” is a heterogeneous mixture of twelve different heterotrimeric complexes  without considering splice variants. The concept emerges that isoforms of AMPK localized at different subcellular compartments may respond to specific cues and regulate only a subset of cellular processes that are now collectively attributed to AMPK. Indeed, AMPK isoform selectivity to specific substrates may arise from a compartmentalized AMPK signalling, rather than from distinct intrinsic kinase substrate specificity. Hence, the spatiotemporal regulation of individual AMPK complexes in various tissues and metabolic conditions awaits further clarification. Furthermore, the development of AMPK activating drugs is constantly progressing behind the scenes, holding more promise than ever for the possible treatment of human disease. AMPK research does not stand still. The knowledge about AMPK accordingly will steadily increase. Besides, the variety of research topics relating to AMPK may continue to evolve. As we are already working on the next edition, we encourage the reader to consider submission of their upcoming AMPK-focused work to the successor Special Issue entitled “AMP-Activated Protein Kinase Signalling 2.0”.


Classical plant breeding was extremely successful in generating high yielding crop varieties. Yet, in modern crops, the long domestication process has impoverished the genetic diversity available for breeding. This is limiting further improvements of elite germplasm by classical approaches. The CRISPR/Cas system now enables promising new opportunities to create genetic diversity for breeding in an unprecedented way. Due to its multiplexing ability, multiple targets can be modified simultaneously in an efficient way, enabling immediate pyramiding of multiple beneficial traits into an elite background within one generation. By targeting regulatory elements, a selectable range of transcriptional alleles can be generated, enabling precise fine-tuning of desirable traits. In addition, by targeting homologues of so-called domestication genes within one generation, it is now possible to catapult neglected, semi-domesticated and wild plants quickly into the focus of mainstream agriculture. This further enables the use of the enormous genetic diversity present in wild species or uncultured varieties of crops as a source of allele-mining, widely expanding the crop germplasm pool. For 10,000 years, humans have utilized the genetic diversity generated from spontaneous mutations and recombination for the selection of improved crops. These traditional breeding approaches have been extremely successful in delivering elite crop varieties with high yields and other enhanced traits, and even today, they remain the corner stone of plant breeding. In recent times, these classical breeding approaches could be accelerated by increasing selection efficiency using marker-assisted selection . HoweverAlready for classical breeding, the induction of DNA double-strand breaks (DSBs) by gamma irradiation was used to achieve genetic variability. The repair of these DSBs occurs in the large majority of cases by non-homologous end joining (NHEJ), which is error-prone . It resuSeveral recent studies have demonstrated the potential of CRISPR/Cas to generate a broad range of allelic diversity at specific loci.Shen et al. succeeded in editing eight yield or quality relevant genes in rice simultaneously . DespiteGRAIN SIZE3, GS3) and grain number  were edited in five different widely cultivated rice varieties. Loss-of-function mutations in these QTLs were described to enhance yield [GS3/Gn1a double mutants and GS3 single mutants to be isolated. Surprisingly, seven of the ten novel genotypes had decreased grain yield compared to the WT, indicating strong dependency of the editing outcome on genetic background and highlighting the utility of genetic diversity across different backgrounds.Another recent study showed that editing the same QTLs (Quantitative Trait Loci) can have different outcomes depending on the genetic background . Two QTLce yield , 39. TheGS3 and Gn1a, in addition to GRAIN WIDTH and WEIGHT 2 (GW2). All combinations of biallelic or homozygous single, double and triple mutants were obtained. The triple mutants showed increases in the yield related traits panicle length, flower number per panicle as well as grain length, width and weight. Unlike the study from Shen et al. [Zhou et al. achieved simultaneous editing of three yield related QTLs in elite rice backgrounds . They tan et al. , the resMATRILINEAL), induced generation of clonal diploid seeds from hybrids that stably preserved heterozygosity.The multiplexing capability of CRISPR combined with its high efficiency in rice could recently be harnessed to create a system enabling the clonal reproduction from F1 hybrids, thus preserving the favourable high degree of heterozygosity . SimultaCamelina sativa is dominated by polyunsaturated fatty acids and the development of new varieties rich in monounsaturated fatty acids is desirable. By targeting all three homeologs of the CsFAD2 (Fatty Acid Desaturase 2) gene involved in fatty acid metabolism, a diverse set of genetic combinations with single, double and triple knockouts could be generated. The obtained lines varied strongly in their lipid profiles, with monounsaturated fatty acid levels in the oil ranging from 10%, like in wild type, up to 62% in homozygous triple mutants. As complete mutants with the strongest change in oil profile showed growth defects, the large mutant diversity could then be used for genetic fine-tuning of the trait, combining improved oil profile without growth defect.As highlighted by another recent study, the polyploid nature of many crops can be a valuable source of genetic diversity . The oilCis-regulatory elements are noncoding DNA sequences that contain binding sites for transcription factors or other molecules influencing transcription, the most common examples being promoters and enhancers. Promoters are generally bound by a common set of conserved transcription factors. In contrast, enhancers are much more variable. They can be located remote from the regulated gene and not only upstream but also downstream and even in introns . FurtherSEPALLATA4 and FRUITFULL. The improved inflorescence architecture increased fruit number and weight as well as yield without a concomitant reduction in sugar content. Importantly, optimal inflorescence architecture could only be realized by a moderate increase in branching, which was dependent on alleles supporting reduced expression, one of them being in a heterozygous state. In contrast, combining CRISPR/Cas-mediated complete KO alleles in a homozygous state resulted in excessively branched inflorescences that produced infertile flowers. However, by targeting Cis-regulatory elements of above-mentioned genes with CRISPR, they generated a range of new alleles supporting varying expression levels for optimization of inflorescence architecture. The authors also identified a further promising Cis-regulatory element as editing target, LIN, which is another tomato SEPALLATA4 homolog. Alleles conveying reduced LIN expression might enable subtle increases in flower production. The fact that rice carries a homolog of LIN that controls panicle architecture and grain production [In this regard, the Lippman lab at CSHL has recently achieved pioneering breakthroughs. First, they achieved optimization of inflorescence architecture in tomato by generating new weak transcriptional alleles . They imoduction  suggestsFollowing this, the same group further developed this approach to a generally applicable genetic scheme for rapid generation and evaluation of novel transcriptional alleles . In thisFine-tuning of gene expression can also be achieved by targeting upstream ORFs (uORFs), short protein-coding elements located in the 5’UTR of an mRNA, upstream of the main ORF. Usually, uORFs act as post-transcriptional inhibitors of translation of the downstream pORF. They are quite widespread, in plants, around 30–40% of genes exhibit uORFs . Now, thThere are over 300,000 plant species. Less than 200 are used commercially, and only 3 species, wheat, rice and maize, provide most of the energy for human consumption , 54. FurSolanum pimpinellifolium, which exhibits a high degree of stress-tolerance [Zsögön et al. report de-novo domestication of the ancestral tomato relative olerance . Much ofSP; SELF PRUNING), flowering time  and fruit size , in all four accessions . SP is a flowering repressor and weak alleles provide a compact determinate growth that enables simple mechanized harvesting. However, the effect from CRISPR generated null alleles of Ppr-sp was too strong, limiting fruit production similar to the null sp allele in tomato, where a weak transcriptional allele is optimal. SP5G was identified recently as an important domestication gene since null alleles eliminate day length sensitivity in tomato and other crops [Ppr-sp5g mutants did not show a useful effect. However, the mutants showed moderate shoot termination resulting in higher fruit amount along each shoot. The Physalis orthologue of CLV1 was chosen as target for its involvement in the CLAVATA-WUSCHEL meristem size pathway influencing fruit size. Weak transcriptional CLV3 alleles mediate enlarged fruits in many crops, whereas clv3 null alleles mediate excessive and disorganized fruit production. Since CLV1 acts as one of several redundant CLV3 receptors, clv1 null alleles might mimic weak transcriptional CLV3 alleles. Indeed, the resulting Ppr-clv1 mutants showed a 24% increase in fruit mass.More recently, rapid improvement of domestication traits hinting at de-novo domestication were undertaken in an orphan crop of the Solanaceae family, protocol . Initialer crops . ConcernOpposed to traditional breeding approaches, improving crops by genome editing requires a much higher degree of genomic and bioinformatic knowledge, as it depends on functionally characterised candidate genes. But an increasing number of genes underlying QTLs is identified  and the There are many traits that can be improved by simple knock-out mutations in the coding sequence of genes, but other traits require edits in regulatory sequences to generate new transcriptional alleles for fine-tuning of gene expression. To unlock the potential of changes in regulatory parts of the genome for dosage effects, the genetic scheme developed by the Lippman lab  seems hiTaVRN1-A1 promoter in wheat protoplasts, which is involved in the regulation of vernalization. By deep-sequencing they identified a variety of mutations in all three targeted regulatory elements. Base-editing can also be used for elimination of specific splicing isoforms by inducing G to A conversions in the respective 5′ splice sites. This way, specific splicing events and the corresponding mature mRNA forms can be eliminated [The base editing system enables precise C-to-T or A-to-G editing in a specified sequence range by fusion of Cas9 nickase with cytidine or adenine deaminase , 65. Reciminated .The EvolvR system provides another elegant way by which site-specific genetic diversity can be generated . It reliZea mays ssp. parviglumis), wild emmer wheat (Triticum dicoccoides) and common wild rice (Oryza rufipogon) could be re-domesticated to generate novel varieties that retain lost traits. What is still limiting de-novo domestication efforts is the availability of efficient transformation procedures and genomic knowledge. However, the latter limitation will be overcome soon as an increasing number of wild species and minor crops are being sequenced. Finally, it should be noted that the trend of ever increasing homogeneization in modern agriculture might be suboptimal considering our changing climate [Finally, CRISPR mediated de-novo domestication provides another new exciting possibility. On the one hand, this enables exploiting wild relatives of crops as a valuable source of allele mining, which could widely expand the crop germplasm pool. This should prove to be very useful considering the genetic impoverishment of many crops and the resistance of wild plants against a broad range of stresses . On the  climate . EffortsUnfortunately, in many areas the development of new crop varieties by genome editing is hampered by strict GMO  regulation, especially those areas adhering to a process rather than a product based regulatory framework, such as the European Union, where the authorization of new varieties developed by genome editing techniques is subjected to time- and cost intensive admission procedures. The recent ruling of the European Court of Justice decreed that targeted mutagenesis using genome editing tools is subject to the strict GMO legislation, even if the product is completely free of any transgene (ECJ 2018). This constitutes a considerable barrier to innovation and progress in these areas. In order to derive all the benefits from the new genome editing techniques and restore innovation, a switch to a product based regulatory framework is urgently needed in Europe. Fortunately, most other countries are not facing such an impediment to innovation, leaving no doubt that on a global scale CRISPR/Cas will continue to revolutionize plant breeding.The genetic bottlenecks imposed on our modern crops by the long domestication process have removed most of the genetic diversity available for breeding, which makes further improvement of elite varieties by traditional breeding technology a cumbersome process. CRISPR/Cas based new breeding tools including multiplex editing, fine-tuning of gene expression and de-novo domestication now provide plant breeders with exciting new opportunities to generate genetic diversity for breeding in an unprecedented way.


Tourism represents an important opportunity to provide sustainable funding for many ecosystems, including marine systems. Tourism that is reliant on aggregating predator species in a specific area using food provisioning raises questions about the long-term ecological impacts to the ecosystem at large? Here, using opportunistically collected video footage, we document that 61 different species of fish across 16 families are consuming tuna flesh at two separate shark dive tourism operations in the Republic of Fiji. Of these fish, we have resolved 55 to species level. Notably, 35 (63%) of the identified species we observed consuming tuna flesh were from ostensibly non-piscivorous fishes, including four Acanthuridae species, a group primarily recognized as browsers or grazers of algae and epibenthic detritus. Our results indicate that shark diving is having a direct impact on species other than sharks and that many species are facultatively expanding their trophic niches to accommodate the hyperabundance of resources provided by ecotourism. Tourism has been long suggested as a mechanism for supporting sustainable use of protected ecosystems, particularly in marine areas . One forCalanus pacificus; [Orcinus orca; [Lepus americanus) have been observed scavenging on mammalian and avian prey [Sylvilagus floridanus) have been recorded scavenging avian prey [Although functional groups are a useful concept in ecology, the rigidly defined dietary boundaries for different functional groups do not adequately acknowledge the potential for plasticity that many species exhibit under certain circumstances. For example, in response to community and/or population level processes, some species opportunistically expand their expected trophic envelopes ,8. In macificus; ) to orcaus orca; ). In terian prey  while eaian prey .Castor canadensis) feed on chinook salmon (Oncorhynchus tshawytscha) carcasses during spawning aggregations [Functional groups may also blur during periods of resource hyperabundance. On land, Alaskan beaver  predictable in time, (2) short-lived, and (3) annual in periodicity. Food provisioning through tourism diverges from natural hyperabudnace in its periodicity, with food provisioning often occurring on a weekly or even daily basis.Carcharhinus leucas) from tuna heads represented only a minor component of their diet [In some ways, commercial shark feeding mimics natural exploitation of hyperabundant resources  as pelageir diet , a bioeneir diet . Thus atWhile the commercial draw of shark feeding is the sharks themselves, the ecological impact of food provisioning on the diet and behavior of the lower trophic-level fishes found within the shark feeding sites remains an open area of investigation. Here, we take a first step at resolving this question by reporting the diversity of fishes that were observed directly feeding upon human-facilitated trophic supplements at two shark-based ecotourism feeding areas in the Republic of Fiji. In doing so, we present evidence for the direct trophic supplementation by a commercial tourism operation of coral reef fishes across multiple functional groups and demonstrate that many of these fishes facultatively shift their diet despite ostensibly being consumers. Our work suggests that the resource subsidies created by shark-diving could percolate throughout the reef fish community and that these fish are drawing upon latent behavioral plasticity to expand their traditional trophic levels in response to anthropogenic hyperabundance occurring on daily time scales.We opportunistically collected observational data in Beqa Lagoon , off the island of Viti Levu in the Republic of Fiji. The area is a no-take marine protected area (MPA) where two companies, Beqa Adventure Divers (BAD) and AquaTrek, have established shark-based diving programs within the MPA.Thunnus obesus and Katsuwonus pelamis) to sharks, mostly bull sharks (Carcharhinus leucas) with occasional tiger  and sickle-fin lemon (Negaprion acutidens) sharks. The two organizations differ in that AquaTrek practices the dissemination of chum as an attractant before and during feeding events, while BAD exclusively hand feeds sharks. The sharks then consume fish heads while swimming past the customers, often passing within about 2 m from the tourists. Trained dive masters are stationed above the tourists and across the feeding arena to maintain safety measures. As sharks consume the heads, they create small flakes of tuna suspended in the water column providing a locally hyperabundant, temporally predictable, resource for other fish who swarm around and behind the shark .Because these observations were made as part of commercial dives, there were no additional permissions necessary. Additionally, there were no permits required to collect observational data on any endangered or protected species present. The dives take place six days a week, year-round, at a coral rubble area approximately 20 m deep. Each trip consists of two tank dives for about 10–15 tourists each. The main attraction of these dives is the hand feeding of tuna heads , white-tipped (Triaenodon obesus) and tawny nurse (Nebrius ferrugineus) sharks. The non-shark component of the community at both sites is mostly consistent and is composed of a suite of Indo-Pacific fishes commonly seen in Fiji (see http://fijisharkdive.com/conservation/shark-reef-fish-list/ for a list of species).Aquatrek has incorporated non-selective feeding methods and therefore feeds a broader spectrum of sharks and other predatory fish species, such as grouper, with limited discretion . AquaTreOur data consisted of opportunistically filmed shark feeding events that took place during July of 2016. During this time, one diver (MGM) recorded 29 minutes of video footage across three dives at AquaTrek, and 9 minutes of video footage across two dives at BAD, which was supplemented by 7 min of footage from 2012 shot by JAD. For each recording, we identified the species observed participating in feeding events. Video footage was reviewed after in-person observations of facultative consumption of food scraps by non-shark species. All fish species present and active at feeding events were identified to the lowest possible taxonomic level. We used an on-line database, FishBase  to queryTable 1). The sizes of the fish consuming tuna cover a wide spectrum, ranging from small  to massive , suggesting that larger sharks are not competitively excluding smaller fishes the way congeners are excluded at some sites [We observed a total of 61 different species of fish from 18 families directly consuming tuna pieces in the water column. Because of the schooling nature of many of these species, it was not feasible to calculate the number of individual observations. Of the 61 observed species, 55 were identified to the species level; of those 55, 35 (63.6%) were species reported as non-piscivorous or scavengers, with 20 (36%) being primarily fish eaters, 14 (25%) being listed as planktivores and 4 (7%) being listed as primary algae or detritus eaters  typically feed on zooplankton [A. sexfasciatus chase after scraps of flesh when sharks were fed tuna heads. Similarly, multiple members of the planktivorous Caeseionidae were found consuming small particles of tuna despite their diets usually consisting of pelagic crustaceans [Many of the species we observed already exhibit some dietary flexibility, particularly in light of temporally or spatially ephemeral food resources, suggesting that there is an inherent behavioral template that is being expanded upon within dive sites. For example, the planktivorous damselfish plankton ,23. Howeplankton . We freqstaceans .Chaetodon mertensii, C. kleinii and C. auriga), were able to expand their diets. Notably, these three fish on occasion demonstrate a more generalist nature, consuming and successfully digesting a number of food sources outside of their typical scleractinian invertebrates [In addition, fish that typically focus on benthic invertebrates, including the coral-eating Chaetodonitdae  normally feed on small benthic invertebrates living interstitially within coral heads [Thalassoma lunare (Labridae) will seasonally switch to piscine prey, consuming a large number of fish recruits [Other mobile benthic invertebrate predators that we observed feeding on tuna have demonstrated dietary flexibility in the wild. al heads  but can al heads . Similarrecruits , 30; somrecruits .Ctenochaetus striatus has very fine, comb-like teeth (the genus name translates to “bristle tooth”) and a gut microflora that effectively digests algae and benthic detritus [C. striatus and Acanthurus olivaceus by Choat et al. [Other species of fish observed consuming tuna in the water were more surprising given their physiological or behavioral limits. For example, the Acanthurid, detritus , 32. Hert et al.  suggesteLabroides bicolor Labridae), a roving obligate cleaner which consumes both ectoparasites and fish mucus [Lastly, the cleaner wrasse . The niche variation hypothesis  suggestsOur work represents an important first step in understanding how the ecology of coral reefs is impacted by tourism. Stable isotope studies have suggested that food provisioning at Beqa shark dive sites could meet daily nutritional diets for some bull sharks  Whether Our data were collected opportunistically, are observational by nature, and were limited by the dangers inherent in conducting field research in the presence of large, actively feeding sharks. As all observations were made from a safe recording distance, we were unable to definitively quantify the full extent to which allochthonous subsidies are percolating throughout these reef systems. It is possible that our results underestimate the scope of this trickle-down ecology and that small, cryptic fishes such as blennies, gobies, and other benthic dwellers may also be receiving supplementation, either directly or indirectly.per se that supplemental food is a significant component to the overall energy budget of these species. Future investigations using stable isotopes coupled with bite calculations should be carried out to determine if the excess food from shark provisioning is a major component of, or an ephemeral supplement to, their daily dietary budget. The continued proliferation of shark-based tourism and the ecological importance of this trophic supplementation increase the relevance of this topic. Moreover, these observations contribute to a larger body of research considering the ecological impacts of resource pulses, behavior flexibility, and the dynamics of functional groups within the Anthropocene.Our work here highlights the potential ecological impact of anthropogenically derived food supplementation on the daily energy budget of lower trophic level reef fish. In doing so, our research opens up several potentially interesting lines of further investigation. For example, what are the behavioral impacts of typically bottom-dwelling fish now feeding higher in the water column and the concordant potential shifts territoriality within and among species? For sex-changing species, resource availability helps drive mating strategy  and thusS1 FigAcanthurus xanthopterus, which were observed consuming the tuna flesh. The tuna head then drew attention from several piscivorous Lutjanus bohar. At 4 seconds a large Carcharhinus leucas frightens off other fish, and consumed the tuna head whole. Throughout the video the fish are surrounded by a school of planktivorous Abudefduf sexfasciatus.The tuna entered the water column from the top of the image, where it was immediately surrounded by several nominally herbivorous (GIF)Click here for additional data file.S2 FigC. lucas after it takes the tuna head, followed by another aggregation of smaller fish trying to get into the tuna holding container.Note the aggregation following the (MOV)Click here for additional data file.S3 FigRemora remora) and tangs (Acanturus sp.) break away to feed on a tuna head, while a snaller (Lutanus sp.) looks for an opening.Once opened several remora ((MOV)Click here for additional data file.


The inherent complexity in the immune landscape of pediatric rheumatic disease necessitates a holistic system approach. Uncertainty in the mechanistic workings and etiological driving forces presents difficulty in personalized treatments. The development and progression of immunomics are well suited to deal with this complexity. Immunomics encompasses a spectrum of biological processes that entail genomics, transcriptomics, epigenomics, proteomics, and cytomics. In this review, we will discuss how various high dimensional technologies in immunomics have helped to grow a wealth of data that provide salient clues and biological insights into the pathogenesis of autoimmunity. Interfaced with critical unresolved clinical questions and unmet medical needs, these platforms have helped to identify candidate immune targets, refine patient stratification, and understand treatment response or resistance. Yet the unprecedented growth in data has presented both opportunities and challenges. Researchers are now facing huge heterogeneous data sets from different origins that need to be integrated and exploited for further data mining. We believe that the utilization and integration of these platforms will help unravel the complexities and expedite both discovery and validation of clinical targets. Unraveling the etiology of pediatric rheumatic diseases exposes the complex heterogeneity inherent within the networks of immune pathophysiology. This mechanistic complexity underscores the challenge and uncertainty in precise disease characterization or sub-stratification. One illustrative example is the International League of Associations for Rheumatology (ILAR) classification of juvenile idiopathic arthritis (JIA) which serves to discriminate seven categories through a combination of clinical presentations, family history, serum, and genetic markers . Yet recImmunomics can be understood as the application of high dimensional technologies that aim to harvest information across a spectrum of biological processes, encompassing (a) genomics, (b) transcriptomics, (c) epigenomics, (d) proteomics, and (e) cytomics . This hoIn this review, we will discuss how conventional high dimensional immunomics platforms, and recent emerging technologies in single cell immune profiling (mass cytometry and single cell transcriptomics) have been deployed in pediatric rheumatic diseases. The resultant explosion of biological information entails corresponding challenges in bioinformatics analysis and public data sharing platforms, and how these issues are addressed will be examined. We believe the incorporation and integration of immunomics platforms in the research community will serve to illuminate and expedite both discovery and clinical validations.Genetic susceptibility is a cardinal aspect of pediatric rheumatic diseases, and comprehension of how individual genetic variants influence pathogenesis will subsequently guide prognostication and disease management. However, the challenge with pediatric rheumatic diseases is underpinned by its heterogeneity in disease susceptibility, clinical presentations as well as treatment outcomes. Genomics is well-placed to address this quandary by (a) identifying candidate genetic susceptibility loci through an unsupervised genome wide interrogation and (b) by streamlining disease classification into more homogenous subtypes, so that disease pathways may be elucidated and therapeutic selection more personalized. Another facet of immunological heterogeneity pertains to the inter-individual differences in immune repertoire present in T cell receptors (TCR). Advancements in TCR repertoire sequencing will add another dimension in the understanding of why certain individuals develop autoimmune diseases and maintain disease persistence. Major insights have since been gleaned from genetic studies across multiple pediatric rheumatic diseases, thereby augmenting our understanding of these diseases.Genome-wide association studies (GWAS) are hypothesis-free studies in which a dense array of genetic markers, achieving significant representation in the genomic sequence, are instructive for a trait of interest . A typicVTCN1, implicated in immune attenuation through B/T lymphocytes, as a novel JIA susceptibility locus in a 2009 study involving 279 JIA cases in the discovery cohort and 321 JIA cases in the validation cohort   . Since tHLA-DRB1*11 was uncovered as a strong systemic JIA (sJIA) risk factor following a meta-analysis of 9 independent case-control populations consisting 982 patients and 431 healthy children  and 14 novel loci reaching genome-wide significance (p < 1 × 10−6) were uncovered for the first time (PTPN22 regions for susceptibility to idiopathic inflammatory myopathies (IIM), of which JDM is a major subtype  loci  of 2.3, and HLA-DRB1*04 with a pooled OR of 1.9 from a meta-analysis of prior candidate gene studies of MHC II in JIA  loci . This isI in JIA . The impNEBL (OR 32.22) and TUBA3C (OR 21.03), both associated with cardiac muscle and tubulin respectively, were recently identified as risk factors for coronary artery lesions  in female JIA patients  and 17 n lesions . While t lesions . Future T-cell receptor (TCR) sequencing is targeted toward the complementary determining region 3 (CDR3) loops, where most of the diversity in these heterodimeric cell-surface receptors is contained. The CDR3 regions are formed by random rearrangements between noncontiguous variables, diversity and joining (VDJ) gene segments in the β-chain locus, and between analogous variable and joining (VJ) gene segments in the α-chain locus . This prTCR sequencing has helped to reaffirm trafficking of CD4 subsets shared between the autoimmune synovial microenvironment and the systemic circulation in JIA patients , 23. CirIn JIA patients non-responsive to conventional DMARDs or biologics therapy, immune reconstitution through autologous hematopoietic stem cell transplantation (HSCT) may be one of the remaining options . Two illThe transcriptome is the entire composite set of transcripts, both coding and non-coding, usually retrieved from a pre-selected subset of cells at a particular instance. This selective combination of transcripts, or the expression profile, gives another layer of biological insight pertaining to gene function, interaction and regulatory networks, which otherwise may not be apparent from the entire genetic sequence. From microarrays that capture limited ranges of known messenger RNAs (mRNAs) to the high-throughput next-generation sequencing (NGS) that can interrogate massive amounts of RNA in a genome-wide fashion, transcriptomics has greatly complemented genetic studies by identifying gene expression signatures for diagnostic discrimination or for shedding light on disease mechanisms , Table 2Microarrays quantitatively measure mRNA levels for thousands of genes in a biological sample, by relying on collections of oligonucleotide probes that capture cDNA or antisense RNA under high stringency conditions. Immobilized in defined positions on a solid matrix, labeled single-stranded nucleic acid fragments can be hybridized to these probes, and the amount of hybridization detected for a particular probe is proportional to the number of complementary fragments in the sample. Advances over the past decade have led to arrays for analysis of gene regulation , genome methylation signatures and even individual exons to assess alternative splicing. Due to their significantly greater dynamic range than reverse transcription polymerase chain reaction (RT-PCR) assays, microarrays are hence more adaptable to genome-wide high-throughput studies integral to decrypting the complex genetic networks in pediatric rheumatic disease .Since the 2000s, transcriptomic profiling of peripheral blood cells via microarrays has resulted in major discoveries in processes driving pediatric rheumatic diseases. In 2003, several groups independently identified the type I interferon signature in both pediatric and adult SLE patients –40. In pThe late 2000s saw several studies that sought to define disease-specific signatures as well as the biological basis behind various clinical phenotypes, especially active disease versus clinical remission. A 2007 study found 286 genes that were significantly up-regulated in peripheral blood mononuclear cells (PBMCs) isolated from active sJIA patients, and this signature was proposed to be disease-specific as most of the candidate genes did not overlap with those identified for other inflammatory diseases including RF-negative polyarticular JIA, KD and pediatric SLE , 46–48. Recent work has looked into whole blood gene expression profiles to determine diagnostic biomarkers as well as predictors of treatment response, in spite of the inherent “noise” from the composite signatures of multiple cellular subsets. The relative ease of collecting whole blood as opposed to fractionated cell subsets and the holistic examination of cross-talk between all innate and adaptive immune cells in disease pathogenesis make whole blood nevertheless an attractive candidate. Using whole blood microarray gene expression data obtained from the Trial of Early Aggressive Therapy in JIA , network approaches utilizing functional co-expressing gene modules unveiled extensive re-ordering of gene expression networks in polyarticular JIA patients following initiation of therapy. In particular, distinctions existed between responders and non-responders on how these networks evolved . A folloThere have also been contemporary microarray studies outlining the role of microRNAs (miRNAs) and individual exons in the pathophysiology of pediatric rheumatic diseases. miRNAs are short non-coding RNA molecules that downregulate specific mRNA transcripts either by translational repression or mRNA cleavage , and theRNA sequencing (RNA-seq) with NGS is the direct ultra-high-throughput sequencing of cDNA derived from transcripts in the sample, and it has several considerable advantages over microarrays: (a) detection of transcripts is free from probe-specific hybridization thus avoiding the need for a priori knowledge of targets, (b) broader dynamic range, (c) lower background signal, (d) increased sensitivity and reproducibility. As such, RNA-seq is able to efficiently measure genome-wide RNA abundance, detect novel and/or allele-specific transcripts and pinpoint alternative splice variants associated with pediatric rheumatic disease in an unbiased fashion. With that, RNA-seq is better poised as a discovery platform for holistic deep expression analysis as compared with microarray systems which is typically customized for specific questions .AIM2, IL18RAP, NLRC4, and IL-18 expression remains dysregulated at a lower intermediate level even in clinically inactive state as compared with healthy individuals. Another study sought to delineate the role of natural killer (NK) cells in sJIA by performing comparative RNA sequencing analysis of these FACS sorted NK cells from a cohort of 6 active sJIA patients and 6 healthy controls  in active disease. In conjunction with an altered plasma cytokine profile enriched in species that promote inflammation and NK cell survival, this thereby implicates the inflammatory milieu characteristic of sJIA in shaping the biologic behavior of NK cells and their consequent function in disease pathogenesis. Moving forward, future work can hence aim to better define RNA signatures in cells of innate immunity in sJIA patients that is stratified in relation to therapy response.Recent comprehensive RNA-seq transcriptome analyses, particularly in combination with fluorescence-activated cell sorting (FACS) or magnetic sorted cells have aided in the identification and characterization of dysregulated pathways in disease implicated cellular subsets. As sJIA involves prominent innate immune activation and lacks significant involvement of autoreactive T cells or autoantibodies , recent controls . Proinfl+ synovial T cells of patients with active disease  demonstrated enhanced expression of autophagy genes compared with PBMCs of patients and healthy controls  and carnitine palmitoyltransferase 1A (CPT1A) within the fatty acid synthesis pathway  when neutrophils from children with untreated RF-negative polyarticular JIA were compared with those from children with cystic fibrosis. Differential exon usage genes and long non-coding RNA (lncRNA) expression were also identified between the disease phenotypes. Interestingly, the prior study on sJIA reported a dissimilar neutrophil gene expression signature that lacked significant upregulation of IL-8 and IFNγ. Though there is a need to evaluate these findings in larger cohorts, both studies further contribute to the promise of potential neutrophil biomarkers for diagnosis and prognosis across JIA subtypes, given the proposed adaptability in neutrophil transcriptomes under specific biological contexts.While the role of innate immunity in non-systemic JIA subtypes is less clear, emerging evidence has hypothesized the importance of neutrophils in linking both arms of the immune system in disease pathogenesis. Notably, prior microarray analyses reflected differences in neutrophil expression profile that correlated with disease phenotypes in RF-negative polyarticular JIA . The samThere have been studies in non-systemic JIA subtypes that chose instead to work with unfractionated heterogeneous cell populations , though differing outcomes were noted in biomarker identification. Analysis of gene expression patterns in PBMCs of patients with polyarticular JIA at different treatment stages  as well as with healthy controls surprisingly failed to define molecular signatures that would assist in disease staging  or in diagnosis  . In retrEpigenomics broadly entails the hereditary and phenotypic traits that can alter function at the genome level without a direct change in the genetic sequence . These eDNA methylation of gene promoters, specifically at regions of CpG dinucleotides, is usually associated with reduced gene expression . Numerou+ T cells from healthy controls and JIA subjects. The Illumina platform was used to compare more than 25,000 CpGs sites, and analysis found significant decreased methylation at the IL-32 gene.Technologies to examine DNA methylation have undergone an increase in their capacity to examine unique CpG sites upon the creation of arrays in the late 1990s. For instance, current DNA methylation array platforms such as the Illumina Infinium HumanMethylation450 (M450K) BeadChip are now able to target more than 450,000 methylation sites, giving us a genomic wide view of epigenetic disruptions. This epigenomic view through CpG arrays is illustrated in a study on how differential T-cell DNA methylation may impact JIA . Before + T cell DNA methylation in oligoarticular JIA was conducted  of JIA. However, independently in another study, the CD4+ T cell DNA methylome of both polyarticular and extended oligoarticular JIA patients prior to and after withdrawal of antiTNFα therapy was investigated , typically known to be expressed across several cell types . The CpGpatients , 87. Thesponsive . This siChIP is an immunoprecipitation technique that enables analysis of a spectrum of protein-DNA interactions, including transcription initiation factors on promoters or silencers on regulatory sites as well as the specific localization of defined histone modifications . This isin-silico, solely based on the DNA sequence  and Roadmap Epigenomics projects. DNA-binding proteins such as enhancers and silencers cannot be predicted accurately sequence . ChIP-sesequence  can faci+ cells  is cross referenced against a mass spectra database through a software designed for the mass spectrometer configuration.Mass spectrometry (MS) became the predominant technique for examining proteins  at a proMS platforms are now adept at distilling candidate protein targets and increasingly being deployed to characterize proteomic profiles of pediatric rheumatic diseases . One stuIn JIA, MS has been employed to differentiate clinical subtypes. With the aid of MALDI-TOF-MS, Finnegan et al.  studied Apart from distinguishing disease subtypes, proteomics profiles have been exploited for biomarker discovery. Surface-enhanced laser desorption/ionization time-of flight (SELDI-TOF) MS was used in one study  of pediaThe enzyme-linked immunosorbent assay (ELISA) is commonly used for profiling of selected liquid analytes, in particular pertaining to immunological response. It uses an enzyme immunoassay (EIA), i.e., an enzyme reaction with its substrate, to detect the presence of a target antigen using specific antibodies. In pediatric rheumatic diseases, this technology has been actively used to profile cytokines or mediators involved in the disease process . For exaCytokine profiles screened through MIA kits, could potentially be used to monitor disease activity, determine treatment response and play a role in the prediction of disease flares. Despite the relatively low-throughput in screening potential compared to mass spectrometry, the convenience and robustness in validating and deploying ELISA diagnostic kits in hospital labs, explains their utility and widespread usage.Cytomics aims to understand complex cellular landscapes and systems at the single cell level by integrating molecular techniques  with digital spectra acquisition. Dissection of complex immune cellular phenotypes can augment our knowledge of how disease mechanisms operate. For instance, analysis of alterations in lymphoid and myeloid cells, allow for identification of immune cell subpopulations that are disease-specific  and may in vivo kinase assays can provide better information on signaling pathways that are crucial to understanding cellular processes and responses to receptor triggering.Flow cytometry is the key platform utilized in the field of cytomics. Since its introduction more than half a century ago, fluorescence-based flow cytometry has been extensively used for functional analysis and characterization of immune cells subsets . Technol+ T cells that are phenotypically similar to CD4+ T cells from the synovial micro-environment of JIA patients  provides the transcriptome of individual cells , which bVarious scRNA-seq protocols have been published over the past few years, and they may be classified based on how single cells are captured and how RNA levels from a single cell is quantified. Flow-based and microfluidic technologies have been commonly used to isolate single cells: the former is ideal for selecting specific cell subsets using fluorescently-tagged monoclonal antibodies bound to specific surface markers, while the latter  offers precise fluid control with an intricate system of valves and switches to isolate cells of interest. A modified version of conventional microfluidics technology, microdroplet-based microfluidics  allows a cell to be encapsulated in a droplet with a bead containing a unique barcode that will be attached to all downstream reads. As such, all droplets can be sequenced together in a high-throughput manner and reads accurately assigned to individual cells of origin. RNA quantification is achieved either by full-length or tag-based sequencing: while tag-based protocols only profile one end of each RNA and are hence thought to offer poorer read mappability; they are more amenable to highly parallel multiplexing and often incorporate the use of 4 to 10 bp long unique molecular identifiers that greatly reduce amplification biases .The advances in scRNA-seq hardware have also spurred parallel advancements in computational handling of increasingly complex data output, which demands generic bioinformatics tools previously employed in bulk sequencing data analysis to be tailored to specific challenges in the single-cell setting. For instance, the intrinsic stochasticity amongst single cells and technical variability from transcriptome sampling result in the violation of assumptions upon which most normalization methods are based , and instead introduce artifacts that bias downstream analyses . In respThe successes of scRNA-seq in cancer biology offer many lessons due to significant parallels with pediatric rheumatic diseases, including cell type heterogeneity, complex interactions between pathogenic cells and their microenvironment as well as immune dysregulation. For example, detailed modeling of transcriptional kinetics in individual tumor cells has facilitated the study of cancer evolution. In various cancers such as those of the intestinal and hematopoietic lineages, scRNA-seq has helped to characterize the layers of tumor hierarchies to uncover cell of origins as well as previously unknown populations that may be pathologically relevant . scRNA-s−THY1+, CD34−THY1+, CD34+) identified after integrating bulk and single-cell transcriptomics. Following subsequent histological and functional assays, the CD34−THY1+ fibroblasts appeared to play the strongest role in promoting synovial swelling and inflammation. scRNA-seq has also helped to delineate SLE pathogenesis and disease complications, though studies were conducted on adult patients. For instance, transcriptomic analyses of human renal and skin biopsy samples from adult SLE patients derived a signature composed of interferon-inducible genes in renal tubular cells that correlated with clinical parameters of lupus nephritis  and adult SLE. As synovial fibroblasts play important roles in initiating and driving RA by contributing to the proinflammatory milieu and promoting osteoclast function, scRNA-seq was used as part of a toolkit to define the molecular identity of the pathogenic fibroblasts . Compariephritis . Interesephritis , tools aThe mass cytometer or CyToF (cytometry time of flight) is essentially a mass spectrometer platform designed specifically to interrogate at the single cell resolution , examiniMass cytometry was performed on PBMCs from treatment-native JDM patients and healthy controls in an attempt to understand cellular signaling . In combThe same group also studied signaling abnormalities in polyarticular JIA and found that treatment-naïve patients displayed enhanced IFN-γ signaling in CD4 T cells and monocytes . Naïve Cex vivo with healthy donor's blood. This study shows the utility of mass cytometry in studying immune modifications in pediatric SLE, which may give us insights into disease pathogenesis.Pediatric SLE patients were also studied to evaluate the presence of immune dysregulation via mass cytometry. Different studies have offered conflicting information on the involvement of specific immune cell subsets in the pathogenesis of SLE. Some studies have showed that the circulating regulatory T cells are decreased while others have shown that numbers are the same while suppression of immune response is reduced , 133. Tht-distributed stochastic neighbor embedding algorithm (t-SNE)  presented unique challenges in deciphering and analysis; that saw the gradual acceptance of nonlinear dimensionality reduction algorithms such as  (t-SNE) , uniform (t-SNE)  or refin (t-SNE) . Applicahttp://www.immport.org/)   by the Nrt.org/) .Recently, the same authors demonstrated how the ImmPort data can be utilized in a massive framework through the implementation of the 10,000 Immunomes Project (10KIP) . The proThe ImmPort and the spin-off 10KIP provide an exemplary demonstration how the challenges associated with integrating diverse immunomics data can be surmounted. Despite these efforts, the authors have cited several limitations that persisted . FirstlyThe era of immunomics provides unprecedented access to platforms that encompass a wide array of capabilities to interrogate the complexity of pediatric rheumatic diseases. We have shown how various groups have tapped on these technologies to peer into the elaborate networks of immune cell subsets and related pathways, which have in turn given us important clues to pathological mechanisms. The resulting explosion of biological data has further presented the challenge of how to best integrate and assimilate such large amounts of data into a coherent narrative.Nevertheless, the need to improve stratification and personalization of existing therapeutic regimens as well as to provide new treatments necessitate continued in-depth research into the immune profile of pediatric rheumatic diseases. This would demand appreciation of each immunomics platform's strengths and limitations to design complementary approaches for addressing important questions. Starting from a biological sample , deep immune phenotyping can be first performed in an unsupervised manner , so as to obtain immune signatures of diverse cell subsets. To streamline high dimensional biological information regarding the sampled cells, computational algorithms can be put in place for dimensionality reduction and functional annotation to derive relevant immune signatures. Subsequently, populations of interest can be sorted for targeted downstream analyses  reiteratively and accumulated data may then be functionally validated against clinical correlates. This proposed framework permits initial unbiased interrogation of the biological sample at the single cell level that is not possible with conventional technologies, and target cell subsets can then be evaluated individually or in bulk at different levels of gene expression . All in all, judicious use of immunomics platforms will unequivocally identify unique cellular signatures which compose the key to unraveling the mysteries of autoimmune disease.In addition, it is imperative to maintain close interaction among researchers, clinicians, bioinformaticians, and technologists alike for continued evolution within the immunomics field, which will definitely provide exciting opportunities for all.No datasets were generated or analyzed for this study.ST, KY, JL, JY, and TA contributed to the writing and conceptualization of the article. JL, TA, and SA helped in the revision of the manuscript.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.