Long noncoding RNAs (lncRNAs) play important roles in tumorigenesis and progression of different cancers and they have been potential biomarkers for cancer diagnosis and prognosis. As the most common endocrine malignancy, precise diagnosis and prognosis of papillary thyroid cancer (PTC) is of great clinical significance. Here, we aim to identify new hub lncRNAs for marking PTC and constructed prognostics signatures based on lncRNA- miRNA-mRNA competing endogenous RNAs (ceRNA) network to predict overall survival (OS) and disease-free survival (DFS) respectively. Five reliable hub lncRNAs were identified by integrating differential genes of four Gene Expression Omnibus (GEO) gene chips using the RobustRankAggreg (RRA) method. Based on differential analyses and interaction prediction, a lncRNA-mRNA co-expression network and a lncRNA-miRNA-mRNA ceRNA network were established. Then a comprehensive function characterization of the five hub lncRNAs was performed, including validation dataset testing, receiver operating characteristic (ROC) curve analysis, and functional analysis on two networks. All results suggest that these five hub lncRNAs could be potential biomarkers for marking PTC. The ceRNA network was used to identify RNAs which were associated with PTC prognosis. Two prognostic signatures were developed using univariate and step-wise multivariate Cox regression analyses and both of them were independent prognostic indicators for PTC OS and DFS. Tumor microenvironment difference analysis between high and low-risk patients showed that dendritic cells activated and macrophages M0 may be a possible target for immunotherapy of PTC. In addition, disclosing the potential drugs that may reverse the expression of hub genes may improve the prognosis of patients with PTC. Here, connectivity map (CMap) analysis indicates that three bioactive chemicals  are promising therapeutic agents for PTC. So, the paper presents a comprehensive study on diagnosis, prognosis, and potential drug screening for PTC based on the five hub lncRNAs identified by us. Thyroid cancer is the most common endocrine malignancy and the incidence has been rapidly increasing in the past 4 decades . As the As we all know, human cancers are frequently correlated with the change of transcription pattern and the transcriptome is not only restricted to protein-coding RNAs but also refers to the multiple noncoding members . The bioMeanwhile, cancer biomarkers need to have a strong specificity for a particular disease condition and lncRNAs have been emerging as crucial players in the control of gene expression . PreviouStudies have indicated that lncRNAs could play important roles as ceRNAs in certain cancers, such as breast cancer, colorectal cancer, pancreatic cancer, and so on . They alIn this study, we integrated the data from four GEO databases with the most PTC samples and the Cancer Genome Atlas (TCGA) so as to screen crucial lncRNAs. Five hub lncRNAs were achieved by robust rank aggregation (RRA) method for data integration of different databases. To comprehensively validate five hub genes, their expression difference analysis and the receiver operating characteristic (ROC) diagnostic analysis were performed based on four GEO datasets, TCGA and Gene Expression Profiling Interactive Analysis (GEPIA) database, respectively. Meanwhile, lncRNA-mRNA co-expression network and lncRNA-miRNA-mRNA ceRNA network were also constructed. Functional analysis on mRNAs involved in the two networks along with the deep-literature exploring five hub lncRNAs and these mRNAs all indicate that they are all involved in cancer-related functions. So, the five hub lncRNAs give promising potentiality for diagnosing PTC.We also established two prognostic risk models for PTC OS and DFS, namely PTC-mi1m4 and PTC-m3, respectively, by screening all genes in ceRNA network. To elucidate the potential pathogenesis of PTC, Gene Oncology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Disease Ontology (DO) enrichment analyses were performed. The proportions of 22 immune cells of PTC were analyzed to estimate the tumor microenvironment in patients with PTC. Among them, two immune cells were demonstrated to be associated with the prognosis of PTC and they may be the potential target of immunotherapy.Finally, connectivity map (CMap) analysis was performed based on five prognosis-related mRNAs to screen potential bioactive chemicals. Three promising drugs were predicted as compounds that play vital roles in PTC-related biological processes and may provide potential treatment of PTC.http://www.ncbi.nlm.nih.gov/geo/query/acc.cdi), four gene chips with the most sample pairs were selected, including GSE29265 with 20 pairs of normal and PTC samples, GSE3678 with seven pairs, GSE3467 with nine pairs, and GSE33630 with 49 PTC samples and 45 normal samples. Furthermore, the RNA-Seq counts data of 501 PTC and 58 normal tissues were downloaded from TCGA data center . Meanwhile, we obtained the clinical information of 496 PTC patients from cBioPortal for Cancer Genomics . After deleting PTC samples without either expression data or clinical information, 490 eligible PTC, and 58 normal tissues remained for the construction of PTC OS prediction model. Since 14 of 490 PTC samples lack clinical information about PTC DFS, the remaining 476 samples were used for DFS prediction.In this study, based on the same sequencing platform of Affymetrix Human Genome U133 Plus 2.0 Array from GEO database  in R package “edgeR.”p < 0.05 were selected as differentially expressed ones. However, different differential lncRNAs were extracted from different gene chips respectively. Here, in order to achieve more valid and representative differential lncRNAs as well as to remove the bath effect, RRA method in R package was employed to integrate the differentially expressed gene lists resulting from differential expression analysis of four individual datasets. The RRA method can detect genes that are ranked consistently better than expected and then assign a significance score for each gene. The significance scores provide a rigorous way to keep only the statistically relevant genes in the final list so that genes identified by this method will be robust, convincing, and significant .To establish the lncRNA-mRNA co-expression network, the Pearson correlation analysis was performed between expression levels of hub lncRNAs and differential mRNAs in TCGA so as to select co-expressed mRNAs that are correlated with hub lncRNAs with the coefficient value of |Cor|>0.5 and http://starbase.sysu.edu.cn) was used to predict lncRNA-miRNA interactions. Those predicted miRNAs only proved to be differentially expressed by TCGA data are regarded as those which were used to construct the ceRNA network. mRNAs targeted by those miRNAs interacting with hub lncRNAs were predicted using miRTarbase (http://mirtarbase.cuhk.edu.cn/), miRDB (http://www.mirdb.org/), and TargetScan 7.2 (http://www.targetscan.org/vert_72/). Similarly, only those predicted target mRNAs that also differentially expressed TCGA can be involved in the ceRNA network. Finally, the lncRNA-miRNA-mRNA ceRNA network was established and visualized using Cytoscape .For the purpose of constructing lncRNA-miRNA-mRNA ceRNA network, starBase v2.0  and Kaplan-Meier survival curve analyses were performed by R package of “survivalROC,” “survival,” and “survminer.” Area under curve (AUC) value from the ROC curve and concordance index (C index) were calculated to determine the prognosis accuracy of the two signatures.Using the other clinicopathological factors associated with PTC patients’ OS or DFS time as confounding variables, clinical characteristics including age, gender , and stage  were also analyzed using univariate and multivariate Cox regression. This stratified analysis was conducted to determine whether the prognostic signature is independent of these clinical factors.http://rdrr.io/github/singha53/amritr/src/R/supportFunc_cibersort.R). The sums of immune cells of each PTC patient were equal to 1. The Wilcoxon test was used to test the prominent difference of immune cells’ proportions between high and low-risk groups that was divided according to OS-associated signature and DFS-associated signature respectively. The Pearson correlation coefficient was calculated to study the correlations between 22 immune cells and key genes involved in two risk models with the cutoff values of |Cor|>0.2 and p < 0.05. So, the distinctive immune cells were identified that not only show significant differences between high and low-risk groups but are correlated with the expression levels of genes. Finally, univariate and multivariate Cox regression analyses were used to further identify those which may be associated with OS or DFS of patients.In order to evaluate the proportions of all 22 immune cells in PTC tissues, CIBERSORT methods were used based on the gene expression profile by running CIBERSORT script from the website (http://broadinstitute.org/cmap) was used to predict the effect of drugs on the particular gene expression patterns in tumors. In order to study functional connections between the key genes associated with OS and DFS of PTC patients and bioactive chemicals, the up-regulated and down-regulated tags from the key genes were uploaded into the CMap online tool. How closely a compound is connected to the uploaded signature depends on the connectivity score with a range from −1 to 1. A positive connectivity score indicates that the compound promotes the query gene expression, whereas a negative connectivity score indicates that the compound represses the query gene expression.The CMap online tool  based on the same sequencing platform were selected in this study. In total, eight differentially expressed lncRNAs were recognized in GSE29265 gene chip, including three lncRNAs with higher expression and five lncRNAs with lower expression . A totalThe five hub lncRNAs are SLC26A4-AS1, RNF157-AS1, NR2F1-AS1, ST7-AS1, and MIR31HG. Among them, RNA expressions of NR2F1-AS1 and MIR31HG in PTC tissues were significantly up-regulated compared with normal tissues, while expressions of the other three genes were significantly down-regulated in all four GEO datasets . In ordeIn order to further verify the potentiality of five hub lncRNAs for marking PTC, the diagnostic performance of these five hub lncRNAs were demonstrated by ROC curve analysis based on four GEO datasets and TCGA, as shown in Finally, a deep literature-exploring was implemented and all five hub genes have been confirmed as having important roles in PTC and other cancers. The overexpression of SLC26A4-AS1 could decrease cell migration, invasion, and proliferation, and thus had anti-oncogenic effects in PTC . But NR2p < 0.05, the interactions between five lncRNAs and 2716 differential mRNAs in TCGA were researched. A total of 647 mRNAs were significantly related to the five hub lncRNAs, so the lncRNA-mRNA co-expression network was constructed. The network graph is shown in Further, functions of the co-expressed mRNAs with hub lncRNAs were investigated. By Pearson correlation analysis with the cutoff values of |Cor| > 0.5 and Firstly, mRNAs involved in lncRNA-mRNA co-expression network were divided in to common or specific ones. If mRNAs are related with two or more lncRNAs, they are defined as common and those that only connect with one lncRNA are specific mRNAs. As displayed in To construct lncRNA-miRNA-mRNA ceRNA network, the starBase v2.0 was used to predict the target miRNAs of five hub lncRNAs and 713 were identified. Then, 17 target miRNAs were determined by intersecting 167 differentially expressed miRNAs in TCGA and 713 predicted miRNAs. Consequently, miRDB, miRTarBase, and TargetScan 7.2 were used to predict probable target mRNAs of the above 17 miRNAs and extracted the intersections from the three online analysis tools. By overlapping the predicted mRNAs to 2716 differential derived from TCGA, 68 target mRNAs that may exert critical functions in PTC were discovered.Based on the achieved lncRNA-miRNA pairs and miRNA-mRNA pairs, the lncRNA-miRNA-mRNA ceRNA network was constructed . The potp-values of five hub lncRNAs are all much higher than 0.05, as shown in Initially, the five hub lncRNAs were used to establish the prognosis model. However, the univariate Cox analysis results of five hub lncRNAs prove that the p < 0.05 as the criteria. As a result, nine mRNAs including TMEM184A, SRCIN1, PI4K2A, FADS6, ITGA3, KRT80, ADM, TOB1, and DCBLD2 were found to be correlated with PTC DFS. On the other hand, four miRNAs and nine mRNAs, namely hsa-miR-1305, hsa-miR-4501, hsa-miR-3652, hsa-miR-665, PASS2, SCD, THBS2, ID4, FHL2, MEX3A, DSEL, DCBLD2, and TMEM184A, were significantly associated with PTC OS. Then, in order to further screen out an optimal combination from these genes, stepwise multivariate Cox regression analysis was conducted and subsequently two predictive signatures named PTC-mi1m4  and PTC-m3  were obtained for PTC OS and DFS respectively.To identify the potential RNAs with prognostic characteristics, univariate Cox proportional hazards regression analysis was performed for five lncRNAs, 17 miRNAs, and 68 mRNA expression data and those related to patient OS or DFS were selected by using in vitro . The time-dependent ROC analysis shows that the AUC values for predicting 5-years and 10-years OS rates are 0.781 and 0.823respectively with C index of 0.775  is of practical significance for cancer diagnosis and treatments. The 22 immune cells form the major non-tumor constituents of tumor tissues, and can perturb the tumor signal and have an important role in cancer biology . We knowThen the differences of immune cells’ proportions between high and low-risk groups divided according to OS-associated signature and DFS-associated signature were further estimated by using Wilcoxon test and displayed in In general, the proportion of macrophages M0 is higher in high-risk patients either based on OS-associated signature or DFS-associated signature, which may indicate that macrophages M0 would be unfavorable to the prognosis of PTC, since it has been demonstrated by the study of Xie et al. that macrophages M0 as well as dendritic cells activated and Tregs were observed to play a tumor-promoting role in PTC .Discovering novel effective drugs may improve the prognosis of patients with PTC. In our two signatures, seven feature mRNAs related to the prognosis of PTC were achieved. It is expected that drugs targeted to them may be of great potential in the therapy of PTC. Except two without GPL96 probe ID, the remaining five mRNAs including PAPSS2, TOB1, ID4, SCD, and DCBLD2 were uploaded into the CMap web tool as down-regulated tags and up-regulated tags respectively to screen the compounds that can reverse the expression of these five hub genes. A negative connectivity score indicates that the compound represses the query gene expression. So, the top three bioactive compounds with connectivity scores close to -1 were determined as the potential therapeutic agents for PTC. The chemical structures of three compounds are shown in Among the three compounds, it has been reported that pioglitazone may be the potential drug in patients with PAX8-PPARγfusion protein (PPFP) thyroid cancer and thyroid cancer. It can also promote apoptosis in human glioblastoma LN-18 cells . BenseraAlthough the survival rate of PTC is relatively high, its recurrence rate is also high. Accurate diagnosis, prognosis for PTC patients, and discovering more potential drugs are of great significance in PTC clinical practice. lncRNAs have been indicated as an important biomarker for different cancers, such as colorectal cancer, gastric cancer, ovarian cancer, and so on . MoreoveFirstly, five hub lncRNAs were identified by integrating four gene chips  from GEO using differential expression analysis combined with RRA approach. The five lncRNAs of SLC26A4-AS1, NR2F1-AS1, MIR31HG, ST7-AS1, and RNF157-AS1 then underwent comprehensive validation tests. Significant expression difference could be observed between tumor and normal tissues in four GEO datasets, TCGA, and GEPIA databases, Moreover, ROC curve analysis shows that these five hub genes yield excellent diagnostic efficiency between tumor and normal tissues based on all four GEO datasets and TCGA and almost all AUC values higher than 0.8 in all five datasets. Actually, previous researchers have identified lncRNAs as prognostic markers of PTC, such as TTTY10 , LINC002In addition, lncRNA-mRNA co-expression network analysis shows that the common co-expressed mRNAs of the five hub lncRNAs are mainly involved in the cancer-related biological processes or pathways, which can indicate to some extent that these hub lncRNAs play crucial roles in PTC and other cancers. Finally, by deep literature-exploring, all of the five lncRNA genes have been confirmed as having important roles in cancers. All the above analysis proves that they would be potential biomarkers for PTC diagnosis.However, the five hub lncRNA genes give poor correlation with the survival prognosis of PTC patients by univariate Cox regression analysis. So based on this, we aim to investigate the prognosis features from their interacting miRNAs and target mRNAs, since much more prognostic signatures have been constructed using miRNAs and mRNAs in cancers, such as gastric cancer, endometrial carcinoma, and so on . Among 7The immune cells are an essential part in the tumor microenvironment and the effects of them on therapy is simulative or impedimental. Meanwhile, the activation status of immune cells may be different in different cancer tumors . TherefoDisclosing the potential drugs that may reverse the expression of hub genes may improve the prognosis of patients with PTC. Therefore, we performed CMap analysis on the five feature mRNAs derived from two risk models to screen the potential compounds for the therapy of PTC. Three compounds  were identified. Through literature-searching, all three bioactive compounds were shown to regulate PTC-related biological processed or pathways by targeting to the five feature mRNAs, but the practical applicability of those drugs should be experimentally confirmed in future researches.


Primary Epstein–Barr virus infection in pediatric patients with inflammatory bowel disease during immunomodulation with thiopurines has been associated with increased risk for malignancies or hemophagocytic lymphohistiocytosis. We determined Epstein–Barr virus (EBV) seroprevalence at inflammatory bowel disease (IBD) diagnosis and seroconversion during follow-up in a large single center cohort of children with IBD. EBV serology results and patient characteristics were retrospectively retrieved from the hospital documentation system. EBV seronegative patients at IBD diagnosis were prospectively retested. We report on IBD patients with symptomatic active EBV infection and a complicated disease course, and those diagnosed with malignancy with respect to EBV status and drug exposure. Of 402 patients, 194 (48%) had available EBV serology results at time of IBD diagnosis at a median of 12 years (IQR 9–14 years). Thereof, 102 (53%) were EBV-positive. Of 92 EBV-negative patients, 66 were retested and 17% showed a seroconversion at a mean follow-up time of 4.3 years (SD 3 years). Three children treated with azathioprine experienced acute clinically relevant EBV infection 2, 2.5, and 4 years after IBD diagnosis, two developed signs of hemophagocytic lymphohistiocytosis. Three cases of malignancy occurred in the cohort, though none seemed to be triggered by EBV. In conclusion, almost 50% of pediatric IBD patients were EBV-naïve following diagnosis and may be at increased risk to develop severe EBV infection during immunosuppressive therapy, potentially associated with complications such as hemophagocytic lymphohistiocytosis or malignancy. Since 0.0007) . In Epst 0.0007) . In this 0.0007) . In a pr 0.0007) .EBV is a human herpesvirus that has been linked to numerous malignancies including endemic (African) Burkitt’s lymphoma, post-transplantation lymphoproliferative disease, Hodgkin’s and B-cell lymphomas, and nasopharyngeal and gastric carcinoma ,11. The It is relevant to understand the dynamics of EBV infection in children with IBD in different populations and how this translates into clinical practice. As the risk for complications seems higher in children with primary EBV-infection during therapy with thiopurines, we hypothesize that EBV-naïve children at IBD diagnosis are at higher risk to develop clinically relevant EBV infection or associated morbidities. The aim of this study is to determine the seroprevalence of EBV at the time of IBD diagnosis, the consecutive seroconversion, and EBV-related complications .The cohort consisted of children and adolescents diagnosed with Crohn’s Disease (CD), ulcerative colitis (UC), or IBD-unclassified (IBD-U) before the age of 18 years at our institution between 2003 and 2019. Medical charts were reviewed to identify patient characteristics, EBV status at diagnosis and during follow-up, and received therapies. Special focus was paid to patients with primary EBV infection who showed signs of HLH or on those developing any malignancy during the time period. Detailed case reports are also given. Since 2011, immunological testing was performed as a part of the diagnostic work up in newly diagnosed pediatric patients in our institution. This includes immunoglobulin concentrations , antibody response to common vaccines , lymphocyte subsets , and a dihydrorhodamine flow cytometric test to exclude chronic granulomatous disease (CGD). In cases of very early onset (VEO)-IBD or atypical clinical course, such as HLH, whole exome sequencing was performed within research projects to detect known and unknown monogenetic disorders mimicking IBD. EBV-naïve subjects at IBD diagnosis and during follow-up were prospectively invited for EBV serology testing. Written informed consents of patients and/or caregivers were obtained. The retrospective and the prospective parts of the study were approved by the Ethics Committee of the Ludwig Maximilian University Munich (No. 18-603).EBV serology testing was comprised of testing for anti-viral capsid antigen (VCA)-IgM, anti-VCA-IgG, anti-Epstein–Barr nuclear antigen (EBNA)-IgG, and anti-Epstein–Barr early antigen (EA)-IgG. EBV serology was tested in blood samples based on enzyme-linked immunosorbent assay (ELISA) technique , 2004–2018 NovaLisa, Novatec (anti-VCA-IgM), 2006–2018 virion/serion ELISA , since 2018 DiaSorin LIAISON . Pipetting was performed by Tecan EvoLyzer  and processing by Siemens BEP III , Emil-von-Behring-Str. 76, 35041 Marburg, Germany) since 2004. It was counted as a positive serology if anti-VCA-IgG or anti-VCA-IgM were positive, whereby constellations, including a positive anti-VCA-IgM (without a positive anti-EBNA-IgG), were suggestive of an acute or shortly passed primary EBV infection.In certain cases, when serological testing was suggestive of primary EBV infection, an in-house quantitative real time polymerase chain reaction (PCR) from blood samples was performed to detect and determine the viral load.All virological testing  was carried out in an accredited clinical diagnostic laboratory.p values of ≤0.05 were considered statistically significant.Descriptive statistics with frequency, percentage, and a 95% confidence interval (95% CI) were reported for all patients in the cohort stratified in EBV status. The distribution of EBV status was compared in different strata of nominal variables by using the Chi-square test. Fisher’s exact test was performed where applicable. A univariate logistic regression analysis was performed to determine the association of a considered factor with EBV status. Estimated risks  and 95% CI were reported. In total, 402 patients fulfilled the inclusion criteria , thereofp = 0.963) nor gender (p = 0.735) were associated with EBV status. EBV seronegative children were significantly younger  than EBV-positive ones , with a positive EBV serology in 102 (53%) of them . All 102p = 0.073).Of the 92 children with documented negative EBV serology at diagnosis, 66 (72%) were retested after a mean follow-up time of 4.3 (SD 3.0) years and 11 of 66 (17%) patients had seroconverted . SeroconOf the 11 children with seroconversion, 3 showed an acute symptomatic EBV infection. A 7-year-old boy with IBD-U acutely presented with fever, reduced general condition, cervical lymphadenopathies, and hepatosplenomegaly, while being treated with azathioprine and 5-ASA for four years. Laboratory tests showed pancytopenia, elevated liver enzymes, increased ferritin , and high soluble interleukin-2-receptor levels . HLH was considered as five out of eight criteria  were metA 17-year-old girl presented six years after her CD diagnosis with acute onset of weakness and headaches while being treated with combotherapy with azathioprine for 2.5 years and infliximab for 1.5 years. Laboratory tests showed leucopenia, thrombocytopenia, elevated liver enzymes, and mildly elevated ferritin (554 ng/mL). Azathioprine dose was lowered from 100 mg to 50 mg (1 mg/kg) per day. Anti-VCA-IgG and anti-VCA-IgM were positive but EBV PCR came back negative. The laboratory findings normalized within four weeks and the azathioprine dose was increased to 100 mg per day without further complications. The patient remained in clinical and endoscopic remission until she was transferred to adult care at the age of 21 years.The third patient with acute clinical EBV infection and later diagnosis of XIAP deficiency has been reported previously ,19. In bDuring the 16-year period, three patients of the cohort were diagnosed with malignancy. A 17-year-old female patient presented with weight loss, night sweats, a cough, and fatigue three years after diagnosis of extensive CD. A CT scan revealed a stage four intrapulmonary lymphoma. At that time, she was treated with combotherapy with azathioprine and infliximab for two years. EBV serology was negative at IBD and at Hodgkin’s lymphoma diagnosis. She was treated by oncology, presented a relapse one year later, but stayed in remission since then.An 18-year-old male patient diagnosed with CD at the age of 6 years, developed a testicular non-seminomatous germ cell tumor six months after referral to adult care. At the age of 13 years, routine EBV serology revealed positive anti-EA-IgG serology without anti-VCA-IgM or anti-VCA-IgG, while EBV PCR was negative. Follow-up serology four years later showed neither anti-EA-IgG or anti-VCA-IgG positivity; however, at the time of referral to adult care, anti-VCA-IgG was positive for the first time. He was in clinical remission, was treated with infliximab for the last six years, and was thiopurine naïve. Anti-TNFα was paused, and orchiectomy and adjuvant chemotherapy were performed.The third patient with malignancy was diagnosed with UC and primary sclerosing cholangitis (PSC) at 12 years of age. Annual routine colonoscopy disclosed an adenocarcinoma in the colon by 19 years of age; EBV serology was negative one year earlier. Over the disease course, she was treated with azathioprine, 5-ASA, ursodeoxycholic acid, as well as some courses of prednisolone and adalimumab. This commenced at the age of 15 years, but switched to vedolizumab because of the lack of response 2.5 years later. She was then transferred to the adult program for colectomy.In this cohort of 402 pediatric patients with IBD, 48% of children were tested for EBV infection at the time of diagnosis and a further 3% at any timepoint during follow-up. Of those with available results at IBD diagnosis, serology indicated passed infection in 53% with older age being the only associated risk factor. Of initially EBV-naïve patients, 17% showed seroconversion when tested as part of this study at a mean follow-up time of 4.3 years. The only drug associated with seroconversion was azathioprine with a 3.7 times higher risk in exposed to non-exposed patients.Our results confirm previous studies from New York with a low EBV seropositivity in early teenagers with IBD  while yoPrimary EBV infection before 10 years of age is usually asymptomatic or with symptoms of a common cold. In adolescents, the primary infection is more often symptomatic and known as infectious mononucleosis . In our Another important aspect is the higher chance in pediatric CD patients to have unrecognized XIAP deficiency as in one patient of our cohort. With the onset of CD at the age of 8 years and an almost therapy-refractory course with severe perianal disease, it took two years until diagnostic testing for XIAP deficiency was initiated during an episode of HLH possibly triggered by EBV infection. Early functional and genetic testing for XIAP deficiency, especially in children who are difficult to treat with CD and VEO-IBD, might be an additional strategy to anticipate, and possibly prevent severe morbidity related to EBV infection ,27.In our cohort of 402 pediatric IBD patients, three patients developed a malignancy, but none seemed to be triggered by EBV. One patient, who received combination therapy of azathioprine and anti-TNFα, developed a Hodgkin lymphoma independent of EBV infection. It is known that patients treated with thiopurines are at a four-to-six-fold increased risk to develop a lymphoproliferative disorder, not all are related to EBV but associated with thiopurine treatment ,4,5. AnoCurrent ECCO and ESPGHAN guidelines for the management of UC and CD recommend thiopurines as therapeutic option for maintaining remission for CD and in moderate to severe UC, either as mono-therapy or as concomitant therapy to infliximab ,30. EspeThe strengths of this study are that we prospectively assessed the EBV seroconversion rates in a relatively large cohort with half of the children and adolescents being EBV-negative at the time of IBD diagnosis.As a limitation, no serial follow-up testing is available, except in those who were tested for clinically suspected acute infection. Therefore, the exact age at the time of seroconversion remains unknown in children without clinical signs for mononucleosis. Since the overall number of patients in our study is relatively large, our cohort is too small to calculate the absolute risk ratios for EBV-associated comorbidities, such as HLH or malignancies. In addition, neither functional testing or genetic testing, especially for XIAP gene mutations , were brAbout 50% of pediatric IBD patients were EBV-naïve at diagnosis and 30% of pediatric IBD patients prior to their transfer to adult care were subject to increased risk to develop severe EBV infection during immunosuppressive therapy. Our data confirm the risk of developing HLH triggered by an acute EBV infection in children with IBD treated with thiopurines. This support the concept of testing for EBV status in children and young adults with IBD prior to starting maintenance therapy. If thiopurines are used in EBV n-ïve pediatric patients, parents and patients should be educated to contact the caring IBD physician in case of symptoms suggestive of mononucleosis to be investigated and closely monitored for signs of HLH. Functional or genetic testing for XIAP deficiency should be offered to male patients with EBV-related complications and those with therapy refractory severe CD-like disease.


Testing men for HIV during their partner’s pregnancy can guide couples-based HIV prevention and treatment, but testing rates remain low. We investigated a combination approach, using evidence-based strategies, to increase HIV testing in male partners of HIV-positive and HIV-negative pregnant women.ClinicalTrials.gov (NCT04124536) and all enrolment and follow-up has been completed.We did two parallel, unmasked randomised trials, enrolling pregnant women who had an HIV-positive test result documented in their antenatal record  and women who had an HIV-negative test result documented in their antenatal record  from an antenatal setting in Lusaka, Zambia. Women in both trials were randomly assigned (1:1) to the intervention or control groups using permuted block randomisation. The control groups received partner notification services only, including an adapted version for women who were HIV-negative; the intervention groups additionally received targeted education on the use of oral HIV self-test kits for their partners, along with up to five oral HIV self-test kits. At the 30 day follow-up we collected information from pregnant women about their primary male partner’s HIV testing in the previous 30 days at health-care facilities, at home, or at any other facility. Our primary outcome was reported male partner testing at a health facility within 30 days following randomisation using a complete-case approach. Women also reported male partner HIV testing of any kind (including self-testing at home) that occurred within 30 days. Randomisation groups were compared via probability difference with a corresponding Wald-based 95% CI. The trial is registered at vs 15 [28%] of 53, estimated probability difference −21·9% [95% CI −35·9 to −7·9%]) and trial 2 . However, reported male partner HIV testing of any kind was higher in the intervention group than in the control group in trial 1  and trial 2  due to increased use of HIV self-testing. Overall, 14 male partners tested HIV-positive. Across the two trials, three cases of intimate partner violence were reported (two in the control groups and one in the intervention groups).From Oct 28, 2019, to May 26, 2020, 116 women who were HIV-positive  and 210 women who were HIV-negative  were enrolled and randomly assigned to study groups. Retention at 30 days was 100 (86%) in trial 1 and 200 (95%) in trial 2. Women in the intervention group were less likely to report facility-based male partner HIV testing in trial 1 (3 [6%] of 47 Our combination approach increased overall HIV testing in male partners of pregnant women but reduced the proportion of men who sought follow-up facility-based testing. This combination approach might reduce linkages to health care, including for HIV prevention, and should be considered in the design of comprehensive HIV programmes.National Institutes of Health. Although the uptake of male partner HIV testing has remained low in many programme settings,4 several new interventions show promise, including assisted partner notification services, home-based testing, and HIV self-testing.6 However, these individual approaches alone might not be enough to broaden coverage to meet the ambitious HIV testing targets set by the global HIV/AIDS community. The combination of different evidence-based strategies could further enhance HIV testing uptake but, to date, there are little data to support combination strategies.Important global achievements have been made in the prevention of mother-to-child (PMTCT) HIV transmission. In sub-Saharan Africa, where the burden of HIV is greatest, involving male partners in health services improves antenatal care attendance, PMTCT programme uptake, and infant survival.7 Using this framework in the setting of comprehensive HIV services, mathematical modelling done by our group showed that small to moderate increases in male partner HIV testing might lead to substantial reductions in horizontal and vertical HIV transmission.8 Public health strategies, including index testing and active case finding,9 typically focus on pregnant women who test HIV-positive to efficiently identify male partners with undiagnosed HIV. However, such approaches can lead to missed opportunities because pregnant women who initially test HIV-negative, but remain at elevated risk for acquiring HIV, are overlooked. Universal health services for the male partners of all pregnant women  can increase acceptability, reduce stigma, and broaden overall reach and effect.We developed a couples-based framework for HIV prevention and treatment in antenatal settings.11Here, we report the primary findings of two parallel randomised trials designed to address gaps in male partner HIV testing: one in HIV-positive pregnant women and one in HIV-negative pregnant women. Our integrated intervention included two strategies recommended by WHO: assisted partner notification services  and secondary distribution of HIV self-test kits .We did two parallel, unmasked, individual-level, randomised controlled trials. Participating women were recruited at the Chipata Level 1 Hospital, a government health facility in Zambia’s capital city of Lusaka. Serving a catchment population of over 100 000, this busy facility has an antenatal HIV prevalence of 16%. Eligible participants met the following criteria: 18 years or older, pregnant and seeking antenatal care at the time of enrolment, documented HIV status in their antenatal record, at least one current male sex partner, willingness to provide her contact information, ability and willingness to provide informed consent, and willingness to adhere to study procedures. Women who had already tested for HIV with their partner  during the current pregnancy were excluded, as were those who expressed concerns about intimate partner violence or social harms at screening. Eligible women were offered enrolment into one of two trials according to their HIV status. In trial 1, we enrolled pregnant women who had an HIV-positive test result in their antenatal record; in trial 2, we enrolled women who had an HIV-negative test result in their antenatal record within the past 3 months during the current pregnancy. In each trial, participants were randomly assigned to either the control or intervention group.All study participants were fully informed of the study procedures and provided informed written consent. The study protocol was approved by research ethics committees or institutional review boards at the University of Zambia  and the University of North Carolina . We also obtained in-country approvals from the Zambia National Health Research Authority and the Lusaka District Health Office before the study began.12Women in both trials were randomly assigned (1:1) to the intervention or control groups using permuted block randomisation. The random allocation sequences with block sizes of 2, 4, and 6 for both trials were generated by the study biostatistician (KRM) using SAS version 9.4 and were kept confidential from the study team during study conduct. Randomisation assignments were placed in sealed, opaque numbered envelopes by personnel in Lusaka who were not directly involved with participant enrolment. At the enrolment visit, study staff would open the next envelope in sequence. These allocations were documented and monitored for quality assurance according to standard practice.13 These options are already provided to pregnant women living with HIV in Zambia, in a package that is generally referred to as assisted partner notification services (shortened to partner notification services in the remainder of this report).14 In line with our status-neutral approach, we also adapted this strategy for HIV-negative pregnant women; we offered the same four options for male partner testing, but tailored the counselling messages to focus on HIV prevention in the context of the woman’s HIV-negative status.Our team worked with community partners and hospital staff to provide information about the study and facilitate recruitment. In a prescreening stage, pregnant women who were interested in the study were first asked if they had been tested for HIV with their primary male partners during the current pregnancy. For women who had not undergone couple HIV testing with their partner in the current pregnancy, a full screening questionnaire was completed. All participants provided written informed consent in either English, Bemba, or Nyanja. At enrolment, participants answered questions about their sociodemographic characteristics, obstetrical history, and sexual health. They also provided information about individual and primary male partner HIV testing history, current HIV treatment and prevention measures, and history of intimate partner violence. Regardless of study group, participants in both trials were offered four options for male partner HIV testing: client self-referral , provider contract referral , provider referral , and dual referral .13 did not recommend confirmatory testing for non-reactive HIV self-test kits in low-risk individuals.14 However, in this study, facility-based HIV testing was viewed as an important entry point to HIV services for male partners and a key outcome of interest.In trial 1 (women who were HIV-positive), all participants informed the study staff of the partner notification approach they had previously selected as part of routine antenatal care. Women in trial 2 (women who were HIV-negative) were educated about the similar partner notification options and asked to select their preferred approach, which was then implemented by our study staff. In both trials, participants randomly allocated to the intervention groups additionally received targeted education on the use of oral HIV self-test kits for their partners (and for themselves if desired). Written instructions for the testing procedures were provided in English, Bemba, and Nyanja, using materials approved by the Zambia Ministry of Health. Up to five oral HIV self-test kits  per participant were offered as part of the intervention. HIV testing at a health facility by a healthcare provider was recommended to all study participants regardless of study group allocation. This included confirmatory HIV testing at a healthcare facility for those who first tested at their home or in the community, regardless of their HIV test result. This advice differed slightly from local HIV self-testing guidelines, which, similar to WHO recommendations at the time,15 We also asked about social harms related to trial participation, with questions adapted from previous studies in Malawi.16 Women who reported intimate partner violence or social harms at their follow-up visit were counselled. As appropriate, our staff provided referrals to the gender-based violence command centre, located on hospital grounds. Information about other adverse events were collected via interviews and medical record reviews.All participants were scheduled to return for a follow-up visit approximately 30 days from enrolment. At the follow-up visit, we collected information from pregnant women about primary male partner HIV testing, including the date, modality, and venue. We screened for intimate partner violence through a nine-item questionnaire previously adapted from the Kenya Demographic and Health Survey.With the emergence of COVID-19, we made modifications to our follow-up protocols from March, 2020 onward. Although our main outcome remained the same , we extended the window period for data collection. To promote physical distancing and to limit in-person contact, we also offered telephone interviews at follow-up for those unable to travel safely to the study clinic.17 to measure male partner HIV testing outcomes, we relied on the female participant’s report. To be included in the numerator for either of the study outcomes, the reported testing event had to occur within 30 days of randomisation. Of those women whose main partner had been tested for HIV, we reported the results of the HIV test. We also described incident social harms, intimate partner violence, and other serious adverse events.Our main outcome was the proportion of primary male partners reported to have tested for HIV at a health facility within 30 days of participant enrolment. Specifying testing at a health-care facility emphasises the importance of HIV testing by trained personnel and the need for engaging with HIV services. The short window  reflects the urgency of male partner HIV testing in the context of PMTCT, particularly for settings like Zambia where antenatal care typically begins later in pregnancy. A prespecified outcome was the proportion of primary male partners reported to have HIV testing of any kind (including HIV self-testing in the household and other community-based venues) within 30 days of randomisation. This outcome was added on May 1, 2020, before study completion. Similar to other studies,vs 45%) in facility-based HIV testing rates with our intervention strategy. An enrolment target of 116 HIV-positive pregnant women (58 women per group) provided approximately 80% power to detect this anticipated difference. In trial 2 (women who were HIV-negative), we estimated that 10% of male partners in the control group would receive facility-based HIV testing. With the addition of HIV self-testing (as provided to the intervention group), we expected a 15 percentage point increase in facility-based partner HIV testing rates . Based on these assumptions, our sample size of 210 HIV-negative pregnant women (105 per group) provided approximately 80% power. Differences in anticipated baseline HIV testing and effect size for trials 1 and 2 reflect our underlying assumptions about facility-based testing. Male partners of women who were HIV-positive might be more likely to seek facility-based testing to confirm results and to engage with treatment or prevention services as needed, because of their higher risk for HIV, for example, than male partners of women who were HIV-negative. Conversely, because male partners of women who were HIV-negative are more likely to test HIV-negative themselves, we reasoned that they could be less likely to return for facility-based HIV testing than male partners of women who were HIV-positive.18This study was designed a priori to assess acceptability, feasibility, and early effectiveness of partner HIV testing strategies. We calculated our sample size on the basis of large effect sizes in partner HIV testing outcomes. For each trial, we used a type I error rate of α=0·05 and two-sided CIs with no adjustment for multiplicity and anticipated 5% missing data and attrition for power calculations. In trial 1 (women who were HIV-positive), we assumed that 20% of male partners in the control group would be tested for HIV. We anticipated a 25 percentage point increase  and its corresponding Wald-based 95% CI. Using the same approach, we also estimated a probability difference to compare reported male partner HIV testing of any kind within 30 days of randomisation between study groups. We did a prespecified sensitivity analysis to estimate a probability difference (intervention–control) adjusted for partner age, age difference between the partner and study participant, travel time to the health facility, and partner HIV testing history by applying an augmented inverse probability weighted doubly robust method, with corresponding bias-corrected percentile bootstrapped 95% CIs.21 In these sensitivity analyses, all randomly assigned participants were included in the analysis. Best case bounds were constructed by assuming that all women missing outcome data in the intervention groups had a partner who tested within 30 days of randomisation and that every woman missing outcome data in the control groups did not have a partner who tested within 30 days of randomisation. The opposite assumptions were made when estimating the worst case bounds. Additionally, multiple imputation of missing partner HIV testing outcomes was done using fully conditional specification (discriminant function method) with 50 imputed datasets, separately for trials 1 and 2. Each imputation model included partner testing outcomes, randomisation group, the four baseline covariates in our adjusted analyses, and two-way interactions between randomisation group and each baseline covariate. Rubin’s rule was used to combine the results from the 50 imputed datasets.Women with no follow-up, or who presented after the visit window, were considered missing and excluded from the denominator for complete-case analyses. In prespecified sensitivity analyses we evaluated the effect of missing data by non-parametrically estimating best case and worst case bounds around our effect estimates.ClinicalTrials.gov (NCT04124536).An affirmative response to any question on either the social harms or intimate partner violence instruments administered at follow-up was counted as an incident social harm or intimate partner violence event. The number of women who reported social harms and intimate partner violence was summarised separately by randomisation group within each trial. All analyses were done using Windows SAS version 9.4. The trial is registered at The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of this paper.From Oct 28, 2019, to May 26, 2020, 426 pregnant women receiving antenatal care at the study site were approached about the study; 97 (23%) reported testing with their male partners during the current pregnancy and were excluded from further evaluation. The remaining 329 (77%) were assessed for full eligibility. Three HIV-negative women were found to be younger than 18 years and were also excluded. Of the remaining 326 women, 116 (36%) women who were HIV-positive were enrolled and randomly assigned in trial 1 ; 210 women who were HIV-negative were enrolled and randomly assigned in trial 2 . The last study visit was completed on July 3, 2020. Overall, retention was high: 100 (86%) of 116 women who were HIV-positive and 200 (95%) of 210 women who were HIV-negative completed follow-up . Key demReported male partner HIV testing methods, stratified by trial and randomisation group, are shown in vs 36%; estimated probability difference 40·7% [95% CI 23·0–58·4]). A similar, but smaller, difference was noted in the women who were HIV-negative: male partner HIV testing of any kind was more frequently reported in the intervention group than the control group  reported that their male partners had been tested for HIV. Of these, 13 (68%) of 19 male partners in the control group and 28 (78%) of 36 male partners in the intervention group were reported to be HIV-negative in HIV serodiscordant relationships. 13 (24%) of the 55 male partners had an HIV-positive result, of whom eight were reported to be taking ART. In trial 2, ten of 210 HIV-negative women enrolled did not report the primary outcome, and among the 200 who did, 134 (67%) reported that their male partners had been tested for HIV. The vast majority of these male partners tested HIV-negative: 52 (96%) of 54 male partners in the control group and all 80 (100%) in the intervention group. In the control group, one male partner received an indeterminate result and one received a positive HIV result; the latter received HIV testing at a health-care facility and was reported to be taking antiretroviral therapy. These findings are detailed in Social harms were infrequently observed throughout the study. One HIV-positive woman (control) reported intimate partner violence, male partner abandonment, and emotional and legal harm arising from study participation. Two other women who were HIV-positive (one intervention and one control) reported intimate partner violence, but did not link these events to their participation in this study. No women in trial 2 reported social harms or intimate partner violence. One maternal death occurred over the course of the study: a participant in trial 1 who had been allocated to the intervention group. She was diagnosed with meningoencephalitis due to advanced HIV disease, a condition deemed unrelated to study participation.We compared strategies for HIV testing of male partners of pregnant women who are HIV-positive and pregnant women who are HIV-negative in antenatal settings: partner notification plus secondary distribution of HIV self-test kits (intervention) versus partner notification alone (control). Our integrated intervention was associated with decreases in facility-based HIV testing and increases in HIV testing of any kind (driven by self-testing) in male partners of all women irrespective of their HIV status. Such combination strategies can help to expand male partner HIV testing out of antenatal settings. However, stronger supporting services might be needed to link specific groups  to health services.11 However, from a research perspective, it provides novel insights into male partner HIV testing behaviours. As HIV programmes in Lusaka, Zambia continue to evolve and incorporate an increasing number of facets of differentiated and decentralised care, other outcomes might become increasingly programmatically relevant in the future.To reach our primary endpoint, male partners of pregnant women had to undergo HIV testing at a health facility. We designated this as our primary outcome for several reasons. First, facility-based HIV testing services present important opportunities for education, couples-based counselling, and engagement with long-term care. For men who test positive for HIV using a self-test kit, the need for confirmatory HIV testing is evident. At the time of our study, HIV testing services also provided one of the few entry points into nascent HIV prevention programmes, including those for HIV pre-exposure prophylaxis. As such, we considered facility-based HIV testing as a reasonable proxy for engagement with comprehensive HIV services. Second, from a study design perspective, we sought a standard outcome that could be used across both trials, irrespective of the male partner’s HIV test result. Because WHO recommends facility-based HIV testing for key groups , we applied the same standard for all male partners. Finally, we recognise that this strict primary outcome might not fully align with the latest WHO guidelines for low-risk individuals who test negative for HIV using a self-test kit.23 expanded across our hospital catchment area. To better contextualise our findings within this changing programmatic landscape, we added a complementary outcome  in prespecified analyses. By considering both facility-based male partner HIV testing versus male partner HIV testing of any kind, our analysis provides a nuanced view about the potential effect of adding HIV self-testing. On one hand, when HIV self-testing is offered in addition to partner notification, the proportion of male partners who undergo HIV testing increases substantially. On the other hand, relocating HIV testing outside of the health-care infrastructure can create lost opportunities to engage with existing HIV services. This latter explanation is evident in our results, in which women in the intervention groups were less likely to report that their male partners accessed facility-based HIV testing services than those in the control groups. Although features of our study design could have contributed to these findings, including the short follow-up window of 30 days, our results appear consistent with other recent studies.24Over the course of study implementation, new approaches for HIV testing, including HIV self-testing and community-based testing,25 however, as evident from our results, gaps might remain. In the context of HIV prevention, such linkages could present further challenges. A proportion of male partners who test HIV-negative could still be at elevated risk for acquiring HIV: of the 55 women who were HIV-positive whose partner received HIV testing in our study, for example, 41 (75%) stated that their partners had tested HIV-negative; a surprisingly high rate of HIV serodiscordancy. Because many national programmes do not recommend facility-based, confirmatory HIV testing when HIV self-test kits are non-reactive (including in Zambia14), alternative venues might be needed to link men to comprehensive prevention services, including HIV pre-exposure prophylaxis.Our findings suggest that adding HIV self-testing to partner notification services can expand the coverage of male partner HIV testing and help to identify those in immediate need of HIV prevention or treatment. To fully realise this potential, however, stronger linkages to care are needed between communities and health facilities. For individuals living with HIV, efforts to promote linkages between HIV diagnosis and treatment have garnered increasing attention;26 Nevertheless, strategies relying on the secondary distribution of HIV self-test kits can place an undue burden on pregnant and breastfeeding women.27 Given the challenges inherent to HIV status disclosure, resources are needed to minimise intimate partner violence and social harms, and actively support those who face such issues.27We observed a low incidence of reported intimate partner violence and social harms. This could be attributed in part to our eligibility criteria for the study, which excluded those at high risk of partner violence or social harms. However, our findings did not differ by study group and are consistent with other studies of HIV self-testing of male partners.28 male partners might have tested without the index participant’s knowledge. It is also possible that participants responded in ways they felt were socially desirable in the health-care setting. Second, allocation to study groups was not masked. Participants were aware of their study group assignment and this could have influenced their subsequent health behaviours. Third, because of the importance of male partner engagement throughout pregnancy, we used a short window  for our outcome measures. It is possible that an increased uptake of male partner HIV testing could have been reported if we had extended the partner testing window beyond 30 days. Fourth, with the emergence of the COVID-19 pandemic in March 2020, public health measures implemented by the Zambian Government limited our ability to trace participants for missed visits. We did numerous sensitivity analyses to account for follow-up losses and these results were largely in agreement with our complete-case approach. Hospital-level mitigation strategies  could have also reduced access to HIV testing services, but rates of reported facility-based HIV testing did not substantially change over this period. Fifth, the partner notification component, which was part of both the control and intervention groups, included four different options, in line with WHO recommendations and Zambia HIV guidelines. Across both trials, the overwhelming majority of participants selected client selfreferral, which is a passive approach. In settings in which other options are preferred or more accessible, such comparisons might yield different results. Finally, this report focuses on the comparative outcomes in male partner HIV testing. Further analyses are underway to describe the feasibility and acceptability of the intervention, as well as its effect on couples HIV counselling and testing.Our study has numerous strengths, including its randomised design and broad eligibility criteria. Although we did separate trials for HIV-positive and HIV-negative pregnant women, our pooled standardised analysis showed consistent population-level effects when accounting for HIV prevalence. We also note several limitations. First, we relied on participant self-report for our main outcomes and this could result in recall and reporting biases. With growing access to community-based HIV testing,29 such integrated strategies can play an important role, and optimised linkages are needed to ensure that all men who undergo HIV testing receive timely access to comprehensive HIV prevention and treatment.In summary, our results show both the opportunities and the challenges inherent to this integrated strategy for male partner HIV testing. HIV self-testing and partner notification services increased male partner testing but reduced the proportion of men seeking facility-based HIV testing. As national HIV programmes seek to meet the ambitious 95–95–95 goals set by UNAIDS for 2030,1


RPE65-related LCA pivotal trials, which culminated in the first Food and Drug Administration-approved and European Medicines Agency-approved ocular gene therapy, have paved the way for a new era of genetic treatments in ophthalmology. At present, multiple clinical trials are available worldwide applying different techniques, aiming to achieve better outcomes and include more genes and variants. Genetic therapy is not only implementing gene supplementation by the use of adeno-associated viral vectors, but also clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing and post-transcriptional regulation through antisense oligonucleotides. Pharmacological approaches intending to decrease photoreceptor degeneration by supplementing 11-cis-retinal and cell therapy’s aim to replace the retinal pigment epithelium, providing a trophic and metabolic retinal structure, are also under investigation. Furthermore, optoelectric devices and optogenetics are also an option for patients with residual visual pathway. After more than 10 years since the first patient with LCA received gene therapy, we also discuss future challenges, such as the overlap between different techniques and the long-term durability of efficacy. The next 5 years are likely to be key to whether genetic therapies will achieve their full promise, and whether stem cell/cellular therapies will break through into clinical trial evaluation.Leber congenital amaurosis (LCA) is a severe congenital/early-onset retinal dystrophy. Given its monogenic nature and the immunological and anatomical privileges of the eye, LCA has been particularly targeted by cutting-edge research. In this review, we describe the current management of LCA, and highlight the clinical trials that are on-going and planned.  Leber congenital amaurosis (LCA) represents one of the severest diagnoses a family can receive regarding a child’s eyesight. It is characterised by early-onset visual impairment, nystagmus or roving eyes and severe photoreceptor dysfunction.RPE65, GUCY2D, NMNAT1, CEP290, AIPL1, and RDH12.RDH12, CRX, CRB1 and NMNAT1 can present with early-onset macular atrophy,CEP290 may be associated with other ciliopathy manifestations,RPE65 may show absent or decreased autofluorescence.The diagnosis of LCA is clinical; by ophthalmological evaluation and electrophysiology. However, genetic testing is critical to confirm the specific diagnosis and is key towards the administration of genotype-specific treatment, inclusion in clinical trials, prognostication, as well as for family planning and tailored screening of systemic conditions.Due to the severe visual impairment caused by LCA/EOSRD, the well-established genetic basis, availability of animal models and well-characterised clinical features, translational research has been particularly active. In this review, we aim to provide an update of the treatments that are currently available and those that are in clinical trial .The current management for most patients with LCA/EOSRD remains symptomatic and supportive, with treatment of possible associated complications Several clinical trials are currently available for individuals with inherited retinal degenerations (IRDs), including LCA/EOSRD, using different approaches. The optimal design of these studies is challenging. IRDs are rare, and therefore gathering large cohorts can be complicated; which is often addressed with multiple trial sites and consequently high costs. Also, determining the eligibility criteria and ideal therapeutic window may not be straightforward. Age is not always correlated with the severity of disease; the rate of degeneration differs significantly between individuals. Structural and functional retinal parameters are usually set as eligibility criteria, often based on natural history data. Robust prospective data take time to be gathered and there is rarely a consensus between different trials. The clinical trials currently available use different techniques as follows.Given the relative immune privilege of the eye and its favourable anatomical characteristics, ophthalmology has been one of the areas in which genetic therapy has thrived,Gene supplementation is based on the delivery of DNA to the cells’ nuclei via a viral vector.RPE65, OMIM 180069) is an enzyme that catalyses a critical step in the visual cycle, within the RPE.RPE65 cause 5%–10% of all LCA/EOSRD cases and these patients show severe, early-onset night blindness.RPE65 have been in development since 2007.Retinal pigment epithelium-specific 65 kDa  containing RPE65 complementary DNA, at two dose levels (NCT00643747).11 vg/mL) and all of them showed a statistically significant improvement in vision-guided mobility (maze navigation at different lux levels), retinal sensitivity (Octopus static perimetry), visual acuity, contrast sensitivity and reading speed, 6 months after the injection.Bainbridge et al conducted a phase I/II trial in which a subretinal injection of rAAV2-CBSB-hRPE65 was performed in 15 young adults (NCT00481546).Concomitantly, Hawswirth RPE65 that reached phase III. The latter involved 20 patients aged 3 and older, who had bilateral treatment with an AAV2 vector, with a 6 to 18 day interval between eyes.RPE65 and remaining viable retinal cells. An ophthalmologist has to evaluate the child to assess their fitness to participate and refer them to one of the four treatment centres in the UK (https://retinauk.org.uk/information-support/luxturna/). This will lead to more patients and longer follow-up, which will enable a better understanding of the durability of treatment response. The longest follow-up published so far has been 3 years, where the efficacy appeared relatively well maintained.Concurrently, Spark Therapeutics was developing Voretigene neparvovec . Luxturna is the first and only gene therapy for an IRD approved by the Food and Drug Administration .et al conducted a phase I/II trial where they treated twelve patients with rAAV2-CB-hRPE65 (NCT00749957). The participants were divided into two groups and received two different doses.Weleber GUCY2D account for 10%–20% of cases worldwide.GUCY2D (OMIM 600179) encodes the enzyme guanylate cyclase 2D, which synthesises cGMP, the intracellular messenger of photoreceptor stimulation.GUCY2D-LCA is characterised by severe congenital onset, profoundly reduced visual acuity, nystagmus, light sensitivity and absent/markedly reduced electroretinogram responses. However, it has been noted that these patients tend to show relatively preserved central photoreceptor structure until late in the disease.GUCY2D-LCA particularly suitable for interventional gene therapy trials.Biallelic variants in et al have developed SAR439483, using a modified AAV capsid (Y733F), a subretinal gene therapy aiming to restore expression of guanylate cyclase 2D and thereby preserve the structure of rods and cones.Boye AIPL1 generally do not have residual outer retinal structure beyond the age of 4 years.AIPL1-LCA, in infants and young children with remaining outer retinal layers at the central macula. This consists of a unilateral subretinal injection of an AAV8 vector, and is taking place at Great Ormond Street Hospital and Moorfields Eye Hospital, London, UK.Aryl hydrocarbon receptor-interacting protein-like 1 -LCA is a rare cause of LCA (1%–2% of cases).Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated enzyme (Cas) play a role in prokaryotic adaptive immunity.Another method to suppress the expression of a damaging variant is through the use of small molecules designed to interact with pre-mRNA, known as antisense oligonucleotides (AON). Through this bonding, these molecules can modulate the pre-mRNA splicing , or cause the degradation of certain transcripts.CEP290 have been associated with LCA, Joubert, Senior-Loken and Meckel syndromes.This gene (OMIM 610142) encodes a centrosomal protein of 290 kDa, a key component of cilia.CEP290-related LCA (NCT03140969) is targeting those carrying the variant c.2991+1655A>G in a homozygous or compound heterozygous state.The first interventional phase I/II clinical trial treating individuals with Staphylococcus aureus Cas9 and CEP290-specific guide RNAs.The first CRISPR-mediated retinal gene therapy clinical trial (NCT03872479) using EDIT-101 is ongoing. This phase I/II study started in 2019 and aims to enrol 18 participants harbouring the c.2991+1655A>G variant. The study consists of a subretinal injection of an AAV5 vector containing RPE65-related and LRAT (OMIM 604863)-related LCA/EOSRD, all-trans-retinal does not get converted back to 11-cis-retinal, interfering with the visual cycle, and ultimately leading to retinal degeneration and visual loss.cis-retinal may prevent (or slow) photoreceptor degeneration.Visual cycle modulators have been under development for the last 10 years, with the aim of either decreasing the accumulation of various retinoid derivatives or to supply deficient compounds.OMIM 6048-related cis-retinyl acetate orally (QLT091001), in patients with LRAT and RPE65-related retinal degeneration. The first study involved a daily dose of 40 mg/m2 for 7 days, was completed in 2013 and included 18 participants.2) for 7 days, with a minimum of 3 weeks in between treatment courses.RPE65-related autosomal dominant retinitis pigmentosa . The latter study showed an improvement in visual field in three out of five patients and in BCVA in one patient.A few studies  have tested the effect of supplementing 9-Patient-derived somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), retinal precursor cells and specialised cells such as photoreceptors or RPE.et al reported improved integration and functionality of the RPE in patches, when compared with RPE cells in suspension. These cell therapies are now being attempted for the first time in patients with RP and LCA; including plans for MERTK-associated LCA.Subretinal transplantation of human embryonic stem cell derived RPE cells has been undertaken in individuals with age-related macular degeneration (AMD) and Stargardt macular dystrophy.An optoelectronic device is able to transform an electrical signal to an optical one, and vice versa.Argus II  is the first retinal prosthetic device that became FDA approved, in 2013. It remains the only FDA approved retinal prosthesis and has been widely used, with over 350 implanted patients worldwide.The field of optogenetics aims to repurpose second-order and third-order neurons of the visual pathway (bipolar and ganglion cells),Due to favourable preclinical data, there are currently two trials involving optogenetics’ techniques in patients with advanced RP. We believe that patients with LCA may be eligible for these trials as well, given that they meet the requirement of severely decreased visual acuity and retinal degeneration. NCT02556736 is a phase I/IIa, open label, dose-escalation study launched in 2015 at multiple locations in the USA. It has fully recruited 12 adults who have received an intravitreal injection of the drug RST-001, a gene therapy encoding the protein channelrhodopsin-2.www.clinicaltrials.gov, accessed on October 2020).Undoubtfully, tremendous progress has been made since the first description of LCA by Theodore Leber 150 years ago. In depth multimodal evaluation and genotypic characterisation have made this condition attractive for pioneering research that could improve patients’ (and their families) quality of life, and advance the frontiers of medicine. Outstanding research has led to this condition being the only IRD with an FDA-approved gene therapy, and with 29 registered clinical trials, 19 of which are interventional  that over the next 5 years, clinical trials for LCA/EOSRD will continue to increase in number, with more treatments being approved. More genes and more treatment approaches are in the pipeline and will continue their development to a clinical trial and, hopefully, an approved drug in the next few years. There are many challenges, including establishing these therapies to be cost effective and scalable—including if more genetic therapies could be delivered intravitreally compared with subretinally, with all the inherent benefits. In addition, should durability wane, it will need to be determined if and when to retreat individuals that have already undergone gene therapy. A further future area of investigation will be the pros and cons of a patient receiving different treatments both in the same or in the contralateral eye, at the same or subsequent timepoints. As more therapies are developed, research exploring their synergic (or antagonistic) effect(s) will need to be undertaken.


Overall, 104 patients required mechanical ventilation, 35 needed dialysis, and 53 died during their hospitalization. In multivariable analyses, OPN levels ≥140.66 ng/mL (third tertile) were associated with a 3.5 × (95%CI 1.44–8.27) increase in the odds of death, and 4.9 × (95%CI 2.48–9.80) increase in the odds of requiring mechanical ventilation. There was no association between OPN and need for RRT. Finally, OPN levels in the upper tertile turned out as an independent prognostic factor of event-free survival with respect to the composite endpoint. Conclusion: Higher OPN levels are associated with increased odds of death and mechanical ventilation in patients with COVID-19, however, their utility in triage is questionable.Background: Severe coronavirus disease 2019 (COVID-19) is the result of a hyper-inflammatory reaction to the severe acute respiratory syndrome coronavirus 2. The biomarkers of inflammation have been used to risk-stratify patients with COVID-19. Osteopontin (OPN) is an integrin-binding glyco-phosphoprotein involved in the modulation of leukocyte activation; its levels are associated with worse outcomes in patients with sepsis. Whether OPN levels predict outcomes in COVID-19 is unknown. Methods: We measured OPN levels in serum of 341 hospitalized COVID-19 patients collected within 48 h from admission. We characterized the determinants of OPN levels and examined their association with in-hospital outcomes; notably death, need for mechanical ventilation, and need for renal replacement therapy (RRT) and as a composite outcome. The risk discrimination ability of OPN was compared with other inflammatory biomarkers. Results: Patients with COVID-19  had significantly higher levels of serum OPN compared to healthy volunteers (96.63 vs. 16.56 ng/mL,  COVID-19 represents a hyperinflammatory condition characterized by an excessive immune activation and an overshooting release of cytokines often leading to multi-organ failure ,3. HowevOsteopontin (OPN) is a 44 to 66 kDa integrin-binding extracellular matrix glyco-phosphoprotein involved in inflammatory response ,8 and arWe hypothesize that circulating levels of OPN are elevated in patients with COVID-19 and associated with worse in-hospital outcomes. To test our hypothesis, we leveraged the International Study of Inflammation in COVID-19 (ISIC) to measure serum OPN levels in a subset of its patients and examined their association with in-hospital death and need for mechanical ventilation or renal replacement therapy.We measured OPN levels in serum samples collected within 48 h of admission of patients hospitalized for COVID-19 enrolled in ISIC. The International Study of Inflammation in COVID-19 is an ongoing multi-center observational study that aims to characterize the role of inflammation in COVID-19 through the measurement of various blood-based biomarkers . Patientn = 217); Attikon University Hospital in Athens, Greece (n = 89); University Hospital of Dusseldorf, Germany (n = 28); Aachen University Hospital, Germany (n = 7). Participants in this ancillary study were randomly selected from a subset of ISIC patients who were hospitalized during the first COVID-19 surge from 1 February to 1 May 2020 after excluding those mechanically ventilated within 48 h of presentation. For the purpose of this study, 341 participants of ISIC with available serum samples from the following centers were included: University of Michigan in Ann Arbor, MI USA , both as a composite outcome and individually. Circulating OPN concentrations were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) that detects both full-length and cleaved human OPN according to the manufacturer’s instructions . C-reactive protein, ferritin, D-dimer and procalcitonin levels were measured at the request of the clinical team by the central laboratory of the respective institution of enrollment.n = 113], 55.81–140.65 ng/mL [n = 114], and ≥140.66 ng/mL [n = 114]), and present continuous variables as means (±standard deviation [SD]) or as median (25th to 75th interquartile range) for normally and non-normally distributed data, respectively. Categorical variables are presented as proportions (%). We used ANOVA, Kruskal–Wallis, and Chi-square tests to compare characteristics and outcomes between OPN tertiles. Spearman-rank was used to assess correlations between OPN and C-reactive protein, D-dimer, ferritin and procalcitonin biomarkers. We first report demographic and clinical characteristics stratified by OPN tertiles , diabetes mellitus, hypertension, coronary artery disease, and heart failure in the model. To evaluate the association between OPN and outcomes, we log-transformed OPN levels  and used binary logistic regression modeling including the following clinical characteristics: age, sex, race, body-mass index, smoking, eGFR on admission, diabetes mellitus, hypertension, congestive heart failure. We report adjusted odds ratios for each OPN tertile, with the lowest tertile serving as the reference group. We also performed a time-to-event analysis using Cox regression modeling using the composite outcome of death, mechanical ventilation and need for RRT. p-values ≤ 0.05 as statistically significant, and performed the analyses using SPSS 24 .Lastly, we compared the ability of OPN to that of C-reactive protein in discriminating risk of the composite outcome by computing areas under the curves (AUC) and comparing the AUCs using the DeLong’s test. We provided OPN’s test characteristics  for predicting the composite outcome using the highest tertile as a cut-off. We considered two-tailed 2 (SD: 7.58). The composite endpoint of need for mechanical ventilation (n = 104) or need for renal replacement therapy (n = 35) or death (n = 53) was met by a total of 192 patients (56.3%). A total of 341 COVID-19 patients from ISIC met the inclusion criteria for this study. The mean age of the study population was 61 years (SD: 16). Of the total number of patients, 61.9% were male and 27.0% were black. The mean BMI of the study cohort was 30.79 kg/mn = 40) and COVID-19 patients. OPN levels measured within 48 h of hospital admission were significantly higher in COVID-19 patients compared to healthy controls , procalcitonin , ferritin , and d-dimers .We first compared circulating levels of OPN between healthy controls  or renal replacement therapy (RRT) at some point during treatment, we investigated the predictive function of OPN concentrations upon hospital admission for these clinical endpoints. We observed a significant increase in MV rates from the lowest (15.9%) to the highest OPN tertile 50.1%, . OPN levCRP: 0.725, OPN/CRP: 0.728, The prediction of a combined composite endpoint  might represent a clinically more relevant tool to identify patients at high risk for complicated clinical course. We noted a stepwise increase in the incidence of the composite endpoint amongst patients within higher OPN tertiles . In detap = 0.027, We finally performed time-to-event analyses for the composite endpoint. Kaplan–Meier curve analysis revealed a significantly better event-free survival for COVID-19 patients who presented with initial OPN levels within the lower tertile . PatientThe COVID-19 pandemic poses an unprecedented challenge to even well-resourced healthcare systems. The unexpectedly high number of patients requiring hospitalization or even intensive care therapy resulted in overtaxing of medical systems and the inability to treat all patients optimally. The clinical course of SARS-CoV-2 infections is highly variable and can be only poorly predicted by classical clinical markers. However, an early risk stratification of COVID-19 patients upon hospital admission is essential not only to provide high-risk patients with the best possible medical treatment but also to optimally allocate the available medical resources and to prevent a health care system overload. In the present study, we report that circulating levels of OPN, an integrin-binding extracellular matrix glycol-phosphoprotein, are elevated in patients with COVID-19 upon hospital admission. We show that concentrations of OPN directly reflect disease severity and represent an independent risk factor for a more severe clinical course. As such, patients with high OPN levels at hospital admission were at higher need for mechanical ventilation and had a significantly impaired survival. Thus, this work supports the development of novel markers to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.Several clinical, medical, societal, and economic factors have recently been identified as major determinants of severe clinical courses and mortality in COVID-19 patients ,13. To np = 0.055) turned out as factors associated with circulating OPN levels. On the other hand, it was recently reported that specific antibodies directed against OPN are produced in the context of diseases associated with systemic inflammation  and might neutralize the effect of elevated OPN levels [OPN is a multifaceted molecule involved in the inflammatory response: it modulates the recruitment of monocytes-macrophages and regulates cytokine production in macrophages, dendritic cells, and T-cells. OPN has been classified as T-helper 1 cytokine and thus believed to exacerbate inflammation in several chronic inflammatory diseases, including lung diseases. In line to our findings, the working groups of Varim et al. described elevated levels of OPN in patients with COVID-19 and suggested a relationship between serum osteopontin levels and the disease severity . OPN is N levels . Thus, iThe present study has several strengths. First, the multinational study design at four study sites in the USA and central Europe. Second, the high degree of standardization in sample collection and analyses argues for the high reliability of our results. However, we do acknowledge some limitations, with the high number of patients with incomplete sets of biomarker measures being the most important one. This is due to the fact that first routine clinical markers were performed following standards of the local investigators and second the availability of OPN measurements was limited by the ability of local centers to collect samples within 2 days by the study team and often small residual volumes. However, we want to emphasize that the “missingness” of these data appeared to be random, limiting the bias introduced by missing data. Furthermore, there was an important difference in enrollment across centers, however, sensitivity analyses did not show differences in the association between biomarker and outcomes according to center. In summary, our data unraveled a previously unrecognized role of circulating OPN as a novel predictive and prognostic biomarker that enables the identification of COVID-19 patients at risk of experiencing a more severe course of disease. This easily accessible risk stratification tool might help to guide treatment decisions and facilitate an optimal distribution of medical resources during this ongoing pandemic when implemented in a panel of other of routinely accessed markers systemic inflammation and/or infection, which are widely available and analyzed in almost all patients suspected for COVID-19.


N-acetyllactosamine present in N-linked and O-linked glycoproteins and that seem to be implicated in inflammatory and immune responses as well as fibrotic mechanisms. This preliminary study investigated serum galectins as clinical biomarkers in lung transplant patients with chronic lung allograft dysfunction (CLAD), phenotype bronchiolitis obliterans syndrome (BOS).Galectins are proteins that bind β-galactosides such as Nineteen lung transplant patients  were enrolled in the study. Peripheral blood concentrations of galectins-1, 3 and 9 were determined with commercial ELISA kits.p = 0.0394). In logistic regression analysis, testing BOS group as dependent variable with Gal-1 and 3 as independent variables, area under the receiver operating characteristics (AUROC) curve was 98.9% . With the stable LTX group as dependent variable and Gal-1, 3 and 9 as independent variables, AUROC was 92.6% . In stable patients were observed an inverse correlation of Gal-3 with DLCO% and KCO%, and between Gal-9 and KCO%.Galectin-1 concentrations were higher in BOS than in stable LTX patients (Galectins-1, 3 and 9 are possible clinical biomarkers in lung transplant patients with diagnostic and prognostic meaning. These molecules may be directly implicated in the pathological mechanisms of BOS. The hypothesis that they could be new therapeutic targets in BOS patients is intriguing and also worth exploring. Lung transplant (LTX) is a lifesaving treatment option for patients with end-stage lung diseases, including emphysema, cystic fibrosis, pulmonary fibrosis and pulmonary arterial hypertension, unresponsive to maximal medical therapy or for whom no effective medical or other surgical alternative exists –4. Long-N-acetyllactosamine, present in N-linked and O-linked glycoproteins. Galectin structure features one or two carbohydrate recognition domains, on the basis of which they are classified as prototype galectins , chimera galectins  and tandem-repeat galectins . Galectins have numerous biological functions: they participate in cross-talk and maintenance of cell architecture, facilitating interactions between cells and components of the extracellular matrix  monitored at the Siena Regional Referral Centre for Sarcoidosis and other Interstitial Lung Diseases were enrolled in the study. Nine patients  were considered stable (CLAD-free), whereas the other ten patients  had CLAD (BOS phenotype). A healthy control group (HC) was also included  value. The baseline value was computed as the mean of the best two postoperative FEV1 measurements (taken > 3 weeks apart). BOS phenotype was assumed in case of a fall in FEV1 ≥ 20% (compared with baseline) in presence of an obstruction pattern at spirometry (FEV1/FVC < 0.7) without persistent radiologic pulmonary opacities  , Gal-3  and Gal-9  of controls were in line with those indicated by the kit manufacturer .The data did not show a normal distribution. The Mann–Whitney test was used for comparison of two variables, while non-parametric one-way ANOVA  and the Dunn test were used for multiple comparisons. The Chi-squared test was used for categorical variables, as appropriate. According to the presence of BOS phenotype, our population were divided in CLAD-free and CLAD-BOS group.https://biit.cs.ut.ee/clustvis).We also performed a logistic regression with HC as dependent variable against CLAD-BOS (or CLAD-free) to assess the potential of the serum galectins for differential diagnosis between the groups. Sensitivity, specificity, and positive and negative predicted values  were calculated for cut-offs of the different variables. Correlations between variables were studied by Spearman correlation and linear regression. A p-value less than 0.05 was considered statistically significant. Statistical analysis and graphic representation of the data was performed with GraphPad Prism 8.0 software. Unsupervised and supervised Principal Component Analysis (PCA) was performed by ClustVis . Galectin-1 concentrations were higher in CLAD-BOS than in CLAD-free patients (p = 0.0394); Gal-9 concentrations were higher in the CLAD-free group than in the HC group (p = 0.0231)     curve of 96.5%   Table . No sign8) Table .Table 2MBOS is the most frequent presenting phenotype of CLAD. It is characterized by bronchiole thickening and obstruction due to injury and inflammation of epithelial cells and small airway subepithelial structures, leading to fibroproliferation and aberrant tissue repair . Since pGal-1 has been demonstrated to play a negative prognostic role in several conditions, including cancer and fibrotic processes , 24. In In the present study, higher serum concentrations of Gal-3 were observed in CLAD patients with BOS than in healthy controls, although its levels in BOS patients did not differ significantly with respect to those of CLAD-free patients. Galectin-3 concentrations were correlated with DLCO% and KCO% in CLAD-free patients. Elevated Gal-3 concentrations are known to be correlated with various inflammatory and fibrotic conditions, including idiopathic pulmonary fibrosis, cardiovascular disease, autoimmune diseases, cancer and osteoarthritis, but no data is yet available on lung transplant patients , 36. GalOur limited number of patients does not allow us a safe interpretation of this result; however, the possible association with some pre-CLAD lung pathological process within the graft is interesting and need further exploration.Galectin-9 was first identified as a chemoattractant and activation factor of eosinophils in vitro and in vivo . It moduThe limits of the present study were its monocentric and retrospective nature and small statistical sample. However, as an exploratory study into the role of galectins in lung transplant patients, it could pave the way for new research into their clinical and pathogenic participation in BOS. No specific hypothesis on their mechanistic role in lung transplantation can be made at this point; however, our study shines a first spotlight on galectins in chronic lung allograft dysfunction suggesting their potentiality as marker for homeostatic mechanisms of the graft that worth to investigate.In conclusion, this is the first study to evaluate galectins concentrations in serum of LTX recipients. Our results reveal overexpression of these molecules in CLAD patients with BOS phenotype correlated with several respiratory function parameters. Galectins-1, 3 and 9 are possible clinical diagnostic and prognostic biomarkers, worthy of further study in larger populations in order to clarify their role in lung transplant. These molecules may be directly implicated in the pathological mechanisms of BOS. The hypothesis that they could be new therapeutic targets in BOS patients is intriguing and also worth exploring.