We report for the first time a way to predict the 2-dimensional extension of an internal limiting membrane (ILM) defect by detecting the area with dissociated optic nerve fiber layer (DONFL)-like spots in the preoperative optical coherence tomography (OCT) en-face images.Case 1 was a 67-year-old man with metamorphopsia and decreased vision in his right eye. His best-corrected visual acuity (BCVA) was 20/100, with a pterygium, a moderate nuclear cataract, and an epiretinal membrane (ERM). Case 2 was a 73-year-old man with metamorphopsia and decreased vision in his left eye. His BCVA was 20/25, with a moderate nuclear cataract and an ERM. Both patients underwent simultaneous cataract surgery and pars plana vitrectomy with ERM and ILM peeling. Brilliant Blue G staining, performed before ERM and ILM peeling, revealed an unstained area. A careful evaluation of the area showed that it was not covered by either the ERM or ILM. A postoperative evaluation of the preoperative OCT images obtained from these cases showed DONFL-like low-brightness spots in the ILM defect area on the OCT en-face images.OCT en-face images may indicate the area of the ILM defect. To avoid iatrogenic damage to the retinal nerve fiber layer by touching/pinching it with forceps, detecting areas with DONFL-like spots in the preoperative OCT en-face images may be useful to predict an ILM defect. Pars plana vitrectomy (PPV) with membrane peeling is performed to treat a symptomatic epiretinal membrane (ERM). Some surgeons also perform internal limiting membrane (ILM) peeling, which can damage the inner retinal layers and the physiology of the Müller cells, because the ILM is comprised of the basal membrane and the end-feet of the Müller cells , 2. HoweWhen performing ILM peeling, it is common to use stains such as indocyanine green, trypan blue, or Brilliant Blue G (BBG) –6. ERMs We present two cases in which areas with dissociated optic nerve fiber layer (DONFL)-like low-brightness spots were detected on the optical coherence tomography (OCT) en-face images and a defect of the ILM in the area was confirmed during the ERM surgery. Both patients provided written informed consent for publication of patient-related materials.A 67-year-old man presented with metamorphopsia and decreased vision in his right eye. The best-corrected visual acuity (BCVA) was 20/100, and slit-lamp examination revealed a pterygium and a moderate nuclear cataract. An idiopathic ERM was detected on fundus photography and OCT  had already occurred, which was confirmed by spraying triamcinolone acetonide during core vitrectomy. BBG was sprayed before the ERM and ILM peeling and the surgeon waited about 1 min with the BBG covering the macula. When the surgeon removed the BBG, an unstained area was identified (Fig. A 73-year-old man presented with metamorphopsia and decreased vision in his left eye. The BCVA was 20/25, and slit-lamp examination revealed a moderate nuclear cataract. An idiopathic ERM was detected on fundus photography and OCT (Fig. Similar to case 1, the PVD had already occurred and an area that was unstained by BBG before ERM and ILM peeling was found Fig. c. The abWe described two cases in which areas with DONFL-like spots were detected on the OCT en-face images, and an ILM defect in those areas was confirmed during the ERM surgery. To the best of our knowledge, this is the first report to predict the 2-dimensional extension of an ILM defect by detecting an area with DONFL-like spots in the preoperative OCT en-face images.Previous studies have reported that tearing and folding of the ILM associated with an ERM occur in 8.6 to 12.03% of cases , 8. MoreIn the current cases, a DONFL-like low-brightness spots area was observed in the area of the ILM defect. In the previous reports, the edge of the ILM defect was observed as a hyporeflective band on near-infrared fundus images and the prominent ILM findings were referred to as a “spaghetti sign” on OCT B-scans , 9. It iA preoperative ILM defect is a potential pathogenesis of an ERM. Foos hypothesized that an initial process of ERM formation might be glial cell migration from the retina by microbreaks in the ILM –12. The Tadayoni et al. first noticed the unusual appearance of the optic nerve fiber layer after membrane peeling and referred to this appearance as the “DONFL” . The DONThe DONFL has been reported to occur in only 54 to 62% of patients who underwent ILM peeling , 20. ConIn conclusion, we present two cases in which areas with DONFL-like spots were detected on the preoperative OCT en-face images, and defects in the ILM in those areas were confirmed during the ERM surgery. To avoid iatrogenic damage to the RNFL by touching/pinching it with forceps, predicting an ILM defect by detecting areas with DONFL-like spots in the preoperative OCT en-face images may be useful.


PRKCA, DUSP6, BMP4) and several other genes known from neuronal disorders . Moreover, associations of gene body methylation alterations with corresponding gene expression changes revealed that especially the three signaling pathway genes JAK3, MECOM, and TNXB differ strongly in their expression between patient-matched intra- and extracranial metastases. Our analysis contributes to an in-depth characterization of DNA-methylation differences between patient-matched intra- and extracranial melanoma metastases and may provide a basis for future experimental studies to identify targets for new therapeutic approaches.Melanomas frequently metastasize to distant organs and especially intracranial metastases still represent a major clinical challenge. Epigenetic reprogramming of intracranial metastases is thought to be involved in therapy failure, but so far only little is known about patient-specific DNA-methylation differences between intra- and extracranial melanoma metastases. Hierarchical clustering of the methylomes of 24 patient-matched intra- and extracranial melanoma metastases pairs revealed that intra- and extracranial metastases of individual patients were more similar to each other than to metastases in the same tissue from other patients. Therefore, a personalized analysis of each metastases pair was done by a Hidden Markov Model to classify methylation levels of individual CpGs as decreased, unchanged or increased in the intra- compared to the extracranial metastasis. The predicted DNA-methylation alterations were highly patient-specific differing in the number and methylation states of altered CpGs. Nevertheless, four important general observations were made: (i) intracranial metastases of most patients mainly showed a reduction of DNA-methylation, (ii) cytokine signaling was most frequently affected by differential methylation in individual metastases pairs, but also MAPK, PI3K/Akt and ECM signaling were often altered, (iii) frequently affected genes were mainly involved in signaling, growth, adhesion or apoptosis, and (iv) an enrichment of functional terms related to channel and transporter activities supports previous findings for a brain-like phenotype. In addition, the derived set of 17 signaling pathway genes that distinguished intra- from extracranial metastases in more than 50% of patients included well-known oncogenes (e.g.  Melanoma development is triggered by DNA mutations in melanocytes, which are mainly induced by solar ultraviolet radiation4. Especially BRAF mutations are frequently involved in the development of melanomas by enhancing cell proliferation through an uncontrolled activation of the MAPK signaling pathway7. Melanomas also frequently metastasize to other organs like lymph node, lung, liver or brain8. In fact, intracranial metastases are observed in about 50% of metastatic melanoma patients9 and without treatment affected patients only survive on average about four months10.Melanomas represent the most aggressive form of skin cancer and their occurrence has increased over the past few years12. These approved therapies significantly improve the survival of patients with extracranial metastases15. However, for intracranial metastases, the response duration of BRAF/MEK inhibitors is limited to a few months and the efficacy of immune checkpoint inhibitors is substantially reduced in symptomatic patients20. Thus, intracranial metastases are the leading cause of death for melanoma patients21. Therefore, more detailed knowledge about molecular differences between intra- and extracranial metastases is required to provide a better basis for the development of therapeutic strategies for intracranial metastases.There are several treatment options for metastatic melanoma including BRAF and MEK inhibitors or immune checkpoint inhibitors24. Several studies analyzed the gene mutation landscape with different results. Recently, genes  have been predicted to differ in their mutational status between intra- and extracranial metastases6, whereas another older study had reported an unchanged mutational landscape between both types of metastases22. Moreover, intra- and extracranial metastases have also been compared at the gene expression level25 and at the level of DNA copy number alterations22. Additionally, a study that utilized a melanoma brain metastasis model described a neuron-like melanoma phenotype suggesting that the brain micro-environment plays an important role for the adaptation of metastatic melanoma cells to the brain26. A more general view of metastases in the brain micro-environment was obtained by a recent single cell transcriptome study that discovered two general archetypes of human brain metastases: a proliferative and an inflammatory archetype, which are shaped by tumor-immune interactions27.Over the last years, several studies were done to better understand differences between intra- and extracranial metastases at the molecular level. Three studies identified an upregulation of the PI3K/Akt pathway in intra- compared to extracranial metastases30. Changes of DNA-methylation in promoter regions are well-known to directly alter the expression of affected genes31, but also DNA-methylation changes in other genomic regions like enhancers or gene bodies can influence the expression of genes34. Two studies with melanoma metastases from different patients have revealed a strong heterogeneity between DNA-methylation profiles of intra- and extracranial metastases36. It is very likely that a large proportion of this epigenetic heterogeneity can be attributed to the fact that metastases from different patients had been compared. To account for this inter-patient heterogeneity, a comparative analysis of genome-wide DNA-methylation profiles of patient-matched intra- and extracranial metastases pairs could help to better characterize DNA-methylation differences to improve the understanding of molecular mechanisms that may contribute to therapy resistance. The potential of such an approach has recently been demonstrated for other omics layers by comparing transcriptomic25 and genomic profiles6 of patient-matched intra- and extracranial metastases pairs. In addition, paired analyses of DNA-methylation profiles of cell lines of metastatic and primary melanomas identified driver genes that contribute to metastases formation38.In addition, epigenetic alterations have been reported to play an important role for metastases formationHere, we analyze DNA-methylation profiles of patient-matched melanoma metastases pairs from intra- and extracranial metastases. To account for the common developmental history of the patient-matched metastases, a Hidden Markov Model (HMM) approach is considered for the personalized analysis of the genome-wide DNA-methylation profiles of patient-specific metastases pairs. The predicted methylation differences are further analyzed in the context of known functional genomic elements, cancer-relevant signaling pathways, and clinical meta-information. Further, the most frequently altered genes across all patients were intensively analyzed based on gene ontologies, pathway information and literature studies to derive a set of potential key pathway candidate genes that could be involved in the manifestation of differences between intra- and extracranial melanoma metastases. Our study contributes to a better characterization of DNA-methylation differences between intra- and extracranial metastases and may help to select potential molecular targets for the development of novel therapeutic strategies for intracranial metastases.Our data set represents genome-wide methylation profiles of patient-matched intra- and extracranial melanoma metastases of 14 patients . Therefore, we utilized a specifically adapted three-state standard first-order HMM for the prediction of methylation alterations in individual metastases pairs40  and known transcription co/factors   enrichment analysis was performed for the candidate gene set Fig. D. Intere38 were analyzed for their methylation behavior in our patient-matched intra- and extracranial metastases. For the study by Vizoso et al.37, 2410 of 2620 CpGs that differed in methylation between metastatic and primary melanoma cell lines were shared with our methylome data set  tended to show an increased methylation across all metastases pairs, whereas the other CpGs showed much stronger methylation differences between both clusters.The initial candidate gene set was further reduced to the 17 included signaling pathway genes that were altered in the same direction in at least 8 of 14 patients to enable a focused analysis of the selected cytokine, ECM, MAPK, and PI3K/Akt signaling pathway genes. Corresponding methylation changes between intra- and extracranial metastases pairs are shown in Fig. PRKCA, CACNG8, RASGRF1, CACNA2D3), (ii) genes involved in cell growth , (iii) genes involved in cell death , and (iv) genes involved in the regulation of cell adhesion . Further, strong literature connections to cancer were found for ten candidate genes. Most of these genes were associated with cancers of metabolic systems  followed by breast cancer , cancers of the central nervous system (PRKCA), or leukemia . Some of the candidate genes are also known to play a role in neuronal diseases like Alzheimer’s disease (EIF4B), schizophrenia (CACNG8) or Parkinson’s disease (TNR).In addition, the 17 signaling pathway candidate genes were considered for an in-depth annotation analysis and literature search to summarize potential functions and connections to cancer and other diseases Table . OverallCACNA2D3, DUSP6, EIF4B, GDF15, IGF1R, PRKCA, RASGRF1, SGK1), (ii) the other three genes with decreased gene body methylation showed a trend towards decreased expression in intra- compared to extracranial metastases , and (iii) both genes with increased gene body methylation  showed a trend towards decreased expression in intra- compared to extracranial metastases. Further, a ranking of these 13 genes according to their strengths of expression differences between patient-matched intra- and extracranial metastases showed that especially JAK3, MECOM, and TNXB differed strongly in their expression between intra- and extracranial metastases . Thus, this validation study indicates that gene body methylation alterations could be functionally linked to expression changes of affected genes and may thereby contribute to differences between intra- and extracranial melanoma metastases.The identified 17 signaling pathway genes that best distinguished intra- from extracranial metastases predominantly showed alterations of gene body methylation Fig. , Table 267, we have focused on an in-depth analysis of genome-wide methylomes of patient-matched pairs of intra- and extracranial melanoma metastases. The initial hierarchical clustering analysis of these methylomes clearly showed that the patient-specific origin of the individual metastases had a stronger influence on the methylome than the organ in which a metastasis was found. This was also confirmed by an additional hierarchical clustering of patient-matched metastases pairs from patients for which samples of different histological regions of patient-specific metastases were available. Such an impact of the patient-specific ancestry of metastases has also recently been reported for gene expression profiles of melanoma metastases25. To account for this, we used a well-established three-state HMM approach40 to perform a patient-specific analysis of each individual metastases pair. This enabled us to predict the most likely underlying biologically plausible methylation status for each CpG of each individual metastases pair.The development of intracranial melanoma metastases is a crucial factor for survival of melanoma patients. Therefore, the identification of molecular differences between intra- and extracranial metastases is a very important step towards a better characterization of molecular alterations that may contribute to treatment resistance of intracranial melanoma metastases. Since epigenetic alterations could potentially be involved in the establishment and manifestation of molecular differences between intra- and extracranial metastases70.We observed a general trend towards decreased methylation in intra- compared to extracranial melanoma metastases at the level of individual CpGs and at the level of affected genes. This loss of methylation was found for most patient-matched pairs, but several others also showed a more balanced behavior between losses and gains of methylation or a clear increase in methylation in intracranial metastases. Thus, the potential role of methylome alterations in the development of melanoma metastases seems to be very complex varying from patient to patient. Still, the more frequently observed global reduction of methylation levels in intracranial metastases may represent a specific adaptation mechanism of those metastases to the specific micro-environment of the brain. Such an adaptation would also be supported by the general fact that a loss of methylation can play important roles in tumor formation and progression by influencing genomic stability and gene expression35. Due to the strong patient heterogeneity and the small number of patients, it is not very surprising that no associations of the predicted methylome alterations could be found in relation to patient survival or treatment. Since the patient-specific metastases pairs within the cohort were selected by focusing on patients whose metastases occurred in comparable time frames, it is only possible to say that from relatively few up to many methylation alterations  may occur in similar time frames between the extra- and intracranial metastasis. Another important observation was that the most discriminative CpGs that distinguished intra- from extracranial metastases tended to show largely homogeneous methylation alterations for treated and untreated patients suggesting that they could represent more general marker candidates independent of the treatment status.As suggested by the hierarchical clustering and as further identified by the HMM-based predictions of the methylation states of individual CpGs, the methylomes of the individual metastases were very patient-specific. Additional comparisons of the HMM-based predictions of methylome alterations of multiple metastases pairs from the same patient to metastases pairs of other patients clearly confirmed this patient-specific alterations of the methylomes. This observation is in good accordance with findings for methylomes of melanoma brain metastases71 and may thereby influence cancer development72. At the level of cancer-relevant signaling pathways, mainly genes of MAPK, PI3K/Akt, ECM and cytokine signaling were frequently affected by a loss of methylation in intracranial metastases. MAPK and PI3K/Akt signaling, which are involved in the regulation of cell proliferation and survival, are already known for their important roles in the regulation of proliferation of melanoma brain metastases23. Both pathways are targeted by drugs such as BRAF and PI3K inhibitors for the treatment of metastatic melanoma76. The cytokine signaling pathway plays an important role in immune responses and is further known to be involved in tumor progression78. Interestingly, 13 of all 24 metastases pairs showed an enrichment of decreased methylation alterations of cytokine pathway genes indicating that this pathway could be involved in the manifestation of differences between patient-specific intra- and extracranial metastases. However, cytokine pathway genes were not significantly enriched among the candidate genes that shared the same methylation state in at least 50% of patients. Thus, different cytokine pathway genes are affected by methylation changes in individual patients.Further, a functional annotation analysis of the predicted methylome alterations showed several interesting patterns across the patient-matched metastases pairs. Differential methylation was observed on a genome-wide scale and functional genomic elements like promoters, enhancers and gene bodies were significantly affected. Additionally, a loss of methylation in intracranial metastases was revealed for transposon families, most dominantly for long terminal repeats (LTR) transposons (12 of 24 pairs). Such a hypomethylation can trigger a reactivation of transposons, which could contribute to additional DNA mutations79. Further, some of these individual gene candidates also showed frequent associations with neurodegenerative diseases. This might indicate shared adaptations to the brain microenvironment.Considering the candidate gene set of genes that were observed to have the same methylation status in at least 50% of patients, genes were often associated with neuron-type ontology terms like synapses and voltage-gated channels. Since the analyzed regions of the metastases were marked by an experienced pathologist, only a minimal contamination by normal brain cells is expected, which cannot explain the observed strong neuron-type associations. Thus, the methylation alterations in intracranial metastases may have contributed to a brain-like phenotype, supporting similar previous findingsBMP4 and MECOM. BMP4 is a growth-factor that also plays an essential role in neurogenesis80. The observed increased gene body methylation of BMP4 in intra- compared to extracranial metastases was associated with decreased expression in intracranial metastases, which may contribute to differences in cell growth and proliferation between intra- and extracranial metastases. MECOM is a well-known oncogene involved in cell proliferation and apoptosis and reported to be overexpressed in e.g. leukemia81, ovarian cancer82, and glioblastoma83. Again, the observed increased gene body methylation of MECOM was associated with decreased expression in intracranial metastases, which may contribute to differences in metastasis manifestation and progression between intra- and extracranial metastases. All other CpGs differed in their methylation between both clusters. The larger cluster showed mostly decreased methylation of CpGs in intra- compared to extracranial metastases, whereas the smaller clustered showed a more heterogeneous methylation behavior of the individual CpGs including increased and decreased methylation in intracranial metastases. In addition, most of the gene-associated CpGs (88.9%) were located within the gene body region, whereas only 6.7% were associated with the gene promoter region. Thus, alterations of gene body methylation patterns seems to be an important feature to distinguish intra- and extracranial metastases pairs. In addition to our observed and other potentially existing alterations of gene expression levels of affected genes34, such gene body alterations may also have an impact on splicing84. However, it is important to note that the observed difference in the proportions of gene body and promoter methylation alterations is also influenced by the fact that the underlying microarray represents more CpGs within gene body regions (56.6%) than in promoter regions (13.7%). Nevertheless, our in-depth gene annotation analyses of the most frequently altered signaling pathway genes suggest that intra- and extracranial metastases potentially differ in cell-cell signaling, cell adhesion, cell growth, and cell death. Some of the identified genes may therefore also contribute to known differences in treatment efficiencies for intra- and extracranial metastases.In addition, the candidate gene set was reduced to known cancer signaling pathway genes to enable an in-depth analysis of the resulting 17 candidate genes Table . Corresp37 showed that the genes that were associated with these regions can also be altered in their methylation in patient-matched pairs of intra- and extracranial metastases. Especially the top-ranking candidate gene TBC1D16 found by Vizoso et al.37 also showed decreased methylation in patient-matched intra- compared to extracranial metastases pairs in more than 70% of patients. Thus, the loss of methylation of TBC1D16 in metastatic melanoma cell lines that has been reported to trigger a metastatic cascade37 might also be involved in the manifestation of differences between intra- and extracranial metastases.Further, an analysis of genomic regions that have previously been reported to be altered in methylation between matched metastatic and primary melanoma cell linesCACNA2D3, DUSP6, EIF4B, GDF15, IGF1R, PRKCA, RASGRF1, SGK1), but there were also three genes with decreased gene body methylation that were associated with decreased expression in intra- compared to extracranial metastases . Both genes with increased gene body methylation were associated with decreased expression in intra- compared to extracranial metastases . Thus, gene body methylation can potentially influence the expression of affected genes, but potential relationships between methylation and expression alterations were complex and gene-specific. However, TNXB, MECOM, and JAK3 were the three top-ranking genes with altered gene body methylation that significantly differed in their expression between patient-matched intra- and extracranial metastases. Since these three genes are known to be involved in the regulation of cell adhesion, cell death, or immune responses  in combination with normalization by the R function preprocessFunnorm utilizing the sex of patients. The CpGs that were represented by probes on the microarray were further filtered for polymorphic and off-target probes based on data from86. LogGenome-wide DNA-methylation profiles of paired intra- and extracranial melanoma metastases from one of our prior projects were used to determine patient-specific methylome alterations (Gene Expression Omnibus (GEO): GSE203152). The considered part of this data set comprised methylation profiles of 37 metastases samples from 14 melanoma patients. Each of these patients had an intracranial and an extracranial melanoma metastasis during the course of the disease. For seven patients, multiple samples from histologically different regions of the same metastasis were considered. Detailed information about the different metastases types and clinical data are summarized in Table 39. Manhattan distance was chosen as distance metric between individual samples due to the high dimensionality of the data87 in combination with Ward’s minimum variance linkage method (ward.D2)88 for clustering. The pvclust approach was used with 10,000 bootstrapping repetitions in order to analyze the stability of the clustering. This stability was quantified using the approximate unbiased p value . This value ranges from 0 to 100, where a larger value indicates greater stability. The same bootstrap approach was also considered to evaluate the robustness of the hierarchical clusterings of the patient-specific metastases pairs to determine similarities and differences of multiple metastases pairs of different histological regions of the same patient or between patients  state “−” models CpGs with decreased methylation in the intracranial compared to the corresponding extracranial metastasis, (ii) state “89. State-posterior decoding was used to assign each CpG in a patient-specific logTo obtain a logentclass1pt{minima90. CpG-island annotations were obtained from the R package IlluminaHuman Methylation EPIC Cando85. Enhancer annotations were obtained by the R package annotatr utilizing annotations from FANTOM5. Signaling and metabolic pathway genes and cancer-related annotations of genes were taken from91. Transposon annotations were obtained from UCSC genome browser92. Statistical significance for enrichment of increased and decreased methylated CpGs in a specific functional category was tested using a one-sided Fisher’s exact test (R function fisher.test). All categories with a FDR-adjusted p value (R function p.adjust93) below 0.05 were considered to be significantly enriched.Each CpG was annotated according to the overlap of its genomic location with the location of functional genomic features. Promoter and gene annotations were obtained from the IlluminaHuman Methylation EPIC arrays regulation features resourceClinical meta-information about time between patient-matched metastases, treatment and survival were available for most patients . In addition, an in-depth annotation and literature analysis was done for the 17 annotated signaling pathway genes of the gene candidates using Uniprot66, GeneCards65 and Pubmed (https://pubmed.ncbi.nlm.nih.gov/) to obtain more detailed insights into potentially existing differences between intra- and extracranial metastases.To identify gene candidates that were frequently affected by DNA-methylation changes, all CpGs within gene-specific promoters, enhancers and gene bodies were ranked across all patients based on their numbers of predicted decreased or increased methylation by the HMM . This resulted in three genes with FDR-adjusted p values less than 0.05  for which an additional direct comparison of the patient-matched intra- and extracranial expression levels was done by a paired t-test. This additional analysis accounted equally for each of the eight patients by considering average intra- or extracranial gene expression levels for patients for which measurements of multiple regions of the same metastasis were available .Gene expression data for 13 of 17 selected signaling pathway candidate genes with altered methylation were available from a companion project to analyze if and how DNA-methylation alterations influence the expression of these genes. This validation data set comprised processed patient-matched RNA-seq gene expression measurements of intra- and extracranial melanoma metastases that were available for 8 of 14 patients of our DNA-methylation cohort . The studies were conducted in accordance with the Declaration of Helsinki. All experiments were performed in accordance with relevant named guidelines and regulations. Informed consent for the usage of the tumor material was obtained from all included patients.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.Supplementary Information 13.Supplementary Information 14.Supplementary Information 15.Supplementary Information 16.


Opioid use disorder (OUD) is a chronic disease requiring long-term treatment and is associated with opioid overdose and increased risk of mortality. However, existing randomized clinical trials focused on short-term treatment engagement and detoxification rather than overdose or mortality risk due to limited follow-up time and ethical considerations. We used a hypothetical trial framework to conduct a retrospective cohort study to assess the effectiveness of time-varying buprenorphine-naloxone on opioid overdose and death. We identified 58,835 insured adult patients with OUD diagnosis in the US, 2010–2017. We fit a marginal structural model using inverse probability weighting methods to account for measured baseline and time-varying confounders, as well as selection bias due to possibly differential loss-to-follow-up. We found that receipt of buprenorphine-naloxone was associated with reduced risk of opioid overdose (hazard ratio (HR) = 0.66, 95% confidence interval (CI): 0.49, 0.91), death , and overdose or death . The E-value for death was 7.8, which was larger than the upper 95% CI of the association between each measured baseline variable and all-cause death, which implies that the unmeasured confounding itself may not explain away the estimated effect of treatment on the endpoint of all-cause mortality. This syThe benefit of BUP-NX treatment for patients with opioid use disorder is supported by evidence from clinical trials. One randomized clinical trial compared BUP-NX versus clonidine on retention and treatment success (negative urine sample test of illicit opioids) at the end of a 14-day-follow-up . The res2.Randomized clinical trials thus far have provided evidence supporting the short-term efficacy of BUP-NX on detoxification. However, opioid use disorder is a chronic disease requiring long-term treatment and is associated with opioid overdose and increased risk of mortality ,12. The When a randomized trial is not feasible or ethical, conducting a retrospective cohort study using routinely-collected data to estimate the effectiveness of the treatment is an alternative. In the light of the real-world evidence, Hernán et al. developed a framework to improve the design and conduct of retrospective studies . This fr3.® Data Mart  from 1 January 2010 to 31 December 2017. Major protocol elements of the hypothetical randomized trial and the rationale, design, and method for the retrospective cohort study emulating the conceptual trial are provided in We first conceptualized a hypothetical trial to address the research question, regardless its feasibility in the real world. Then, we designed a retrospective cohort study to emulate the hypothetical trial as closely as possible using an administrative claims data: the Optum’s de-identified Clinformatics The first eligible OUD diagnosis in the database was defined as the start of study follow-up (time zero) for that patient. The one-year period before time zero was defined as the baseline period. Patients were followed for up to two years after their OUD diagnosis. The follow-up ended when the outcome occurred, the patient was lost-to-follow-up, or the patient reached the end of the database (31 December 2017), whichever happened first. All patients were required to be continuously enrolled in the database during the baseline period and at least 18 years old at time zero with at least 30 days of follow-up after baseline to allow for ascertainment of outcomes. Patients who had received buprenorphine-naloxone or naltrexone, or who had visited methadone clinics during the baseline period were excluded. Patients who died during the baseline period were also excluded. Patients missing their state of residence or gender information were excluded . No impuSeveral existing studies have shown that patients are at a high risk of overdose and death during the first four-week after treatment cessation ,18 and iWe identified the following covariates that were known or suspected risk factors for the outcomes: age, gender, type of insurance, region, type of insurance product, history of opioid overdose, chronic pain, depression, alcohol use disorder, opioid use, and Charlson comorbidity index (CCI)  20–22].–22.20–22Patient baseline characteristics were summarized overall at baseline. Descriptive statistics were calculated as means with standard deviations (SD) for continuous variables and percentages for categorical variables. The outcomes were analyzed with time-to-event statistical methodology and relative comparisons were estimated as hazard ratios (HR). We first fit an unadjusted Cox model with the BUP-NX exposure and time-to-event outcomes. The exposure was parameterized as a time-varying indicator of receipt of any BUP-NX in the 30-day interval ,27. We fWe designed a series sensitivity analyses to assess the model fit, robustness of the results regarding study design, and vulnerability to potential unmeasured confounding. We checked the exposure and censoring weight distributions. Then, we evaluated an alternative loss-to-follow-up definition established by Lesko et al. . This al4.We successfully identified 58,835 adult patients diagnosed with opioid use disorder from 2010 to 2017. Approximately half of the patients were male (49%) and the mean age of the analytical sample was 51 years (SD = 17.6). Among all patients, 55% were enrolled in commercial insurance and the rest were enrolled in Medicare Advantage. Most patients used a health maintenance organization or point of service as their insurance product (69%). During the one-year baseline period, the overall disease burden measured by the CCI was 1 (SD = 1.64), which means that on average patients had one out of the seventy comorbidities included in CCI. During the baseline period, a small proportion of patients had a documented history of opioid overdose (4%) and 15% had documented alcohol use disorder. Almost half of the patients had depression (47%) and more than two-thirds of patients had chronic pain conditions (79%) during the baseline period .The total person-time for opioid overdose, death, and composite endpoint were 63,910 person-years, 65,180 person-years, and 63,881 person-years, respectively. During the study follow-up out of a total of 58,835 patients, 1908 (3.2%) patients had at least one opioid overdose, 1051 (1.8%) patients died, and 2874 (4.9%) patients experienced the composite endpoint of either death or opioid overdose during the follow-up. There were 5722 patients who had received at least one BUP-NX dispensing before having an opioid overdose, or were lost to follow-up (whichever came first). For the other two endpoints (death and composite), there were 5835 and 5720 patients who had at least one BUP-NX dispensing before overdose, death, or were lost to follow-up (whichever came first). We focused on discussion on results from the MSM that accounted for both measured baseline and time-varying confounding and possibly differential loss to follow-up . PatientThe mean of the product of the two weights  was close to 1, which suggests no evidence of model misspecification or positivity assumption violations  15]. Th. Th15]. 5.In this retrospective cohort study, we evaluated the effectiveness of time-varying buprenorphine-naloxone dispensing on reducing subsequent opioid overdose and/or death through an application of causal inference methods with a trial emulation framework using routinely-collected health data. The data source is a large national administrative claims database from 2010 to 2017 and included 58,835 patients diagnosed with OUD in the United States. A marginal structural model was applied to analyze the time-to-event outcomes and conduct inference in an observational setting. With adjustment for measured baseline and time-varying variables and possible selection bias due to loss to follow-up, we found that receiving BUP-NX during the two-year follow-up could decrease the hazard of opioid overdose and/or death among patients who were diagnosed with OUD, as compared to not receiving BUP-NX during follow-up.Medication treatment retention or sustained medication treatment has been shown to be one of the most important protective factors associated with a lower risk of opioid overdose. Furthermore, the risk of overdose after treatment discontinuation is high regardless of the treatment duration before discontinuation . Chang eOur study provides new insights into OUD treatment by considering important improvements in study design and statistical methodology. Conditioning on treatment initiation leads to a depletion of potential patients who comprise an important part of the target population of all insured adult patients diagnosed with OUD. This could distort the study sample from the target patient population of interest, rendering questions among the entire patient population with OUD difficult to address. Consequently, the finding otherwise would be limited to patients who had both the disease and had received a certain type and duration of treatment. The eligibility criteria to identify the target patient population and definition of the start of follow-up in this study were carefully designed based on the concept of trial emulation for an ideal randomized study to address this research question . In a reFrom a statistical method perspective, the time-dependent Cox model can be subject to bias when the time-dependent confounders are also affected by previous exposure, and g-methods are needed to properly adjust for time-varying confounding ,32. We aWe conducted a sensitivity analysis for the definition of lost to follow-up . SupposeTo conduct causal inference based on information from an observational study, the conditional exchangeability assumption must hold . This assumption cannot be tested with the observed data , howeverThis study has several limitations. Giving the nature of observational studies without a randomly assigned intervention, there could be unknown and/or unmeasured confounding. In our study, there is no information about OUD severity, illicit substance use, community naloxone distribution, and socioeconomic status variables , which could also be important confounders. The results also depend on the model being correctly specified. There could be measurement error using diagnosis codes in claims data . The endIn summary, we designed a retrospective cohort study using administrative claims data to evaluate buprenorphine-naloxone dispensing on health outcomes, including overdose. With an application of a marginal structural model under the conceptual trial emulation framework, our study provides evidence of the effectiveness of buprenorphine-naloxone treatment against opioid overdose and/or death. The findings of this study should be interpreted in light of its known limitations, including a retrospective design and possibly unmeasured important confounders and OUD disease severity. Future retrospective study with electronic health records that include more detailed information on confounders and prospective cohort studies will provide further insights into treatment of opioid use disorder.Supplemental File


Symptom checkers have been widely used during the COVID-19 pandemic to alleviate strain on health systems and offer patients a 24-7 self-service triage option. Although studies suggest that users may positively perceive web-based symptom checkers, no studies have quantified user feedback after use of an electronic health record–integrated COVID-19 symptom checker with self-scheduling functionality.In this paper, we aimed to understand user experience, user satisfaction, and user-reported alternatives to the use of a COVID-19 symptom checker with self-triage and self-scheduling functionality.We launched a patient-portal–based self-triage and self-scheduling tool in March 2020 for patients with COVID-19 symptoms, exposures, or questions. We made an optional, anonymous Qualtrics survey available to patients immediately after they completed the symptom checker.Between December 16, 2021, and March 28, 2022, there were 395 unique responses to the survey. Overall, the respondents reported high satisfaction across all demographics, with a median rating of 8 out of 10 and 288/395 (47.6%) of the respondents giving a rating of 9 or 10 out of 10. User satisfaction scores were not associated with any demographic factors. The most common user-reported alternatives had the web-based tool not been available were calling the COVID-19 telephone hotline and sending a patient-portal message to their physician for advice. The ability to schedule a test online was the most important symptom checker feature for the respondents. The most common categories of user feedback were regarding other COVID-19 services , policies, or procedures, and requesting additional features or functionality.This analysis suggests that COVID-19 symptom checkers with self-triage and self-scheduling functionality may have high overall user satisfaction, regardless of user demographics. By allowing users to self-triage and self-schedule tests and visits, tools such as this may prevent unnecessary calls and messages to clinicians. Individual feedback suggested that the user experience for this type of tool is highly dependent on the organization's operational workflows for COVID-19 testing and care. This study provides insight for the implementation and improvement of COVID-19 symptom checkers to ensure high user satisfaction. During the COVID-19 pandemic, symptom checkers have become an essential tool for providing patients with on-demand access to triage recommendations . These tSymptoms checkers are widely considered to be popular with patients because they provide them with 24-7 access to health information, risk assessments, and in some cases, test and appointment scheduling. A handful of studies have reported moderately high user satisfaction ratings for COVID-19 symptom checkers. One study of 296 patients who were predominantly health care workers reported 56% found their institution’s internal web-based symptom checker tool useful . SymptomWhile these studies suggest that users may positively perceive web-based symptom checkers, they are insufficient to understand the patient and tool characteristics that contribute to user experience. Furthermore, no studies have quantified user feedback after the use of an electronic health record (EHR)–integrated COVID-19 symptom checker with self-scheduling functionality. Symptom checkers that are EHR-integrated and offer self-scheduling may have higher user satisfaction because they decrease the time it takes for patients to be scheduled for necessary appointments or tests .As symptom checkers become more ubiquitous for COVID-19 and other use cases, it is important to understand user perceptions and to know which features make them attractive. This type of user feedback can inform product development and improvement for these symptom checkers and any other digital health tools. In addition, it is important to understand whether there are demographic differences that drive user perceptions of the tool, as these may impact health disparities.At the University of California, San Francisco (UCSF), in March 2020, we launched one of the first COVID-19 symptom checkers with self-triage and self-scheduling capabilities. We designed a survey to collect feedback and user experience upon use. This is the first known study to conduct user research on an EHR-integrated symptom checker with self-scheduling functionality. Our primary aims in this analysis are to examine patterns in user experience and user satisfaction by demographic characteristics, determine what patients view as alternatives to symptom checker use, and gather actionable feedback for symptom checker improvements.UCSF Health is a large academic health system providing approximately 1.7 million outpatient visits annually. The UCSF primary care practices serve approximately 90,000 empaneled patients. As of January 2022, approximately 95% of adult primary care patients were enrolled in UCSF’s EHR-tethered patient portal.In early March 2020, UCSF established a COVID-19 telephone hotline, which became the primary telephone intake point for all UCSF patient and employee inquiries regarding COVID-19, including general questions, exposures, symptom assessments, and test scheduling requests.The UCSF COVID-19 Symptom Checker was developed as an EHR-tethered portal-based self-service option for patients with symptoms of or exposure to COVID-19 or those who are requesting a COVID-19 test. After answering a series of branched logic questions about exposures, symptoms, and comorbidities, patients are directed to the appropriate disposition based on their predicted risk level. The triage algorithm used in this tool was identical to the one used on the telephone hotline. UCSF uses a commercially available EHR from Epic Systems. In early March 2020, we used our EHR vendor’s configuration tools to design, configure, and deploy our UCSF COVID-19 Symptom Checker, which launched on March 12, 2020 . PatientIf appropriate based on their responses, patients could self-schedule SARS-CoV-2 RNA tests, video visits, or in-person appointments directly through the tool, as described previously . When paThe optional, anonymous user feedback survey for the UCSF COVID-19 Symptom Checker was built on Qualtrics and consisted of 12 total questions . The surWe designed the survey to be lightweight and easy to complete. Questions were a combination of slider-style rating questions, multiple-choice, select-all-that-apply, and optional open-ended questions. Only the first question, in which patients were asked about their overall rating of the tool, was required. The patients were then asked to assess their agreement on Likert scale with statements describing their experience and about how they would have sought care had the tool not been available. Additionally, the participants were asked about the most important features to them and were able to comment on any technical difficulties experienced. The respondents were asked optional demographic questions on age, gender, ethnicity, and race.For this analysis, we included all adults who responded to the Qualtrics feedback survey from December 16, 2021, until March 28, 2022. A subanalysis of the peak of the omicron surge  and its effect on patient satisfaction and experience was conducted. We defined the peak as the period during which the tool averaged over 200 unique users a day.This study was approved by the UCSF Institutional Review Board (20-30903).t test for continuous variables. Visualizations were created using the ggplot2 library. A multivariate linear regression analysis was performed with a primary outcome of user overall rating and the demographic questions of the survey. For the purposes of multivariate analysis, respondent ethnicity was stratified as non-Hispanic and Hispanic, and race was stratified as White and non-White. To assess the collinearity of covariates, variable inflation factors were calculated with a cutoff of <10. Moreover, P<.05 was considered significant.Data were exported from the Qualtrics survey and analyzed using R 3.5.1 (The R Foundation). The participants’ longitude and latitude at the time of response based on Qualtrics estimation using respondent IP addresses were matched to respective census block tract and area deprivation index national percentile as a proxy for socioeconomic status . DescripConstructive open-ended responses were assigned 1 of 10 categories by a physician reviewer who was familiar with the ambulatory COVID-19 care structure. We did not categorize responses that were purely complementary or that did not offer specific feedback.From December 16, 2021, until March 28, 2022, there were 395 total responses to the experience survey . During When asked about the most important feature of the tool, over half of the users  cited the ability to schedule their COVID-19 test online. The second most popular feature was 24-7 access to triage advice if they had COVID-19 symptoms or exposure .There were 288 responses during the omicron surge from December 16, 2021, to January 28, 2022. The median 1-10 rating during that time was 8 (IQR 4-10), and 139 (48.2%) users gave the tool a rating of 9 or 10. There was no difference in overall rating for responses during the Omicron surge compared with before or after the Omicron surge (Patient demographics of the survey respondents are summarized in Respondents gave a wide variety of answers when asked what they would have done if they did not have access to the web-based tool . The mosThe most common categories of open-ended feedback were as follows: (1) requested changes to other COVID-19 services , policies, or procedures; (2) request for additional tool functionality; and (3) lack of appointment availability . This feThis study is the first, to our knowledge, to conduct user research on an EHR-integrated COVID-19 symptom checker with self-scheduling functionality. In this analysis, users generally perceived the symptom checker positively and usually reported that the tool improved their care experience. The most popular features were the ability to self-schedule a COVID-19 test online and 24-7 access to triage advice. Constructive or negative feedback on the tool was often a reflection of the larger ecosystem of care delivery for COVID-19, particularly during times of high demand, rather than related to the digital tool itself. This points toward the need for systems to develop robust, high-quality services in conjunction with usable and functioning digital health tools to aid in access.This analysis supports existing literature ,11,13 thThis survey was completely optional, and we observed a low response rate, so there may be a risk for nonresponse bias. However, recent research suggests that the trends observed may accurately represent attitudes of the population despite low response rate ,18. FurtThis analysis may be useful to health systems that are trying to weigh the benefits of developing or integrating a COVID-19 symptom checker or similar tool with potential costs. In addition to the other established benefits, high patient satisfaction may make investment in COVID-19 symptom checkers worthwhile. Second, this tool may help to prevent front-line staff and physician burnout  by decreSeveral of the qualitative trends we observed may be useful to health systems and developers for the design and improvement of symptom checkers and other patient self-service tools. First, the most common category of written feedback was pertaining to the COVID-19 services, policies, and procedures, as well as appointment availability at the health system, rather than an intrinsic aspect of the tool. For that reason, it is essential that health systems first optimize their operational workflows and rightsize their capacity for tests and visits prior to or in conjunction with implementing such a tool. Second, users frequently requested software features that were beyond the current capabilities of the platform we used. Developers of these tools must therefore weigh the benefit of more nimble tools with user-friendly features  with the cost of their development and integration into the EHR. Third, users rarely reported technical challenges, suggesting usability of simple patient-portal–embedded tools. Fourth, the most popular feature of the symptom checker was the ability to self-schedule a COVID-19 test online, suggesting that EHR-integrated tools with the ability to offer self-scheduling of tests, screening exam, or imaging may be perceived more positively by patients compared with those without such features. Finally, our experience from reviewing continuous user feedback reinforced the notion that embedding a simple user feedback survey into a digital tool is a helpful way to promote iterative development.COVID-19 symptom checkers have effectively aided health systems in handling high volumes of triage and scheduling requests during the COVID-19 pandemic. We report high user satisfaction and user experience across demographic groups. Furthermore, patient-reported alternatives to the use of this tool suggest it may have saved thousands of phone calls and patient messages. COVID-19 symptom checkers are likely to remain in use for the near future in a diverse array of settings, and an examination of characteristics of use provides insight to improve the patient experience.


Colchicine acts upstream in the cytokines cascade by inhibiting the nod-like receptor protein 3 (NLRP3) inflammasome while interleukin 6 (IL-6) receptor antagonists, such as tocilizumab, block the end result of the cytokines cascade. Hence, adding colchicine to tocilizumab with the aim of blocking the early and end products of the cytokines cascade, might reduce the risk of developing cytokine storm.We aim to conduct an open-label randomized controlled trial to evaluate the efficacy and safety of adding colchicine to tocilizumab among patients with severe COVID-19 pneumonia to reduce the rate of invasive mechanical ventilation and mortality. We will include patients with severe COVID-19 pneumonia who received tocilizumab according to our local guidelines. Enrolled patients will be then randomized in 1:1 to colchicine versus no colchicine. Patients will be followed up for 30 days. The primary outcome is the rate of invasive mechanical ventilation and will be determined using Cox proportional hazard model.Given colchicine’s ease of use, low cost, good safety profile, and having different anti-inflammatory mechanism of action than other IL-6 blockade, colchicine might serve as a potential anti-inflammatory agent among patients with severe COVID-19 pneumonia. This study will provide valuable insights on the use of colchicine in severe COVID-19 when added to IL-6 antagonists.The Medical Research Center and Institutional Review Board at Hamad Medical Corporation in Qatar approved the study protocol (MRC-01-21-299). Results of the analysis will be submitted for publication in a peer-reviewed journal. The mechanisms by which COVID-19 induces ARDS is currently thought to be due to the activation of the innate immune system leading to hypercytokinemia and hence cytokine storm.The novel coronavirus infection (COVID-19) significantly contributes to increased mortality in many countries, with a continuously increasing number of infected cases worldwide.,5 SARS-CoV-2 acts on the macrophages to release the nod-like receptor protein 3 (NLRP3) inflammasome, leading to the conversion of pro-interleukin (IL)-1 and pro-IL-18 to their active forms.,6 The production of IL-1 leads to interleukin 6 (IL-6) synthesis which is a cytokine that induces C reactive protein (CRP) and has been considered a major proinflammatory agent in the COVID-19 cytokine storm.–10 Consequently, a few biological therapies that have been studied and used for the treatment of COVID-19 to target specific cytokines augmented by the disease, including anakinra for IL-1, and tocilizumab for IL-6. Early in the COVID-19 pandemic, the therapeutic efficacy of IL-6 antagonists was evaluated and showed conflicting results.–15COVID-19 disease course can be divided into three phases; early infection phase, where the virus penetrates host cells in the lung parenchyma; pulmonary phase, in which the viral propagation causes lung tissue injury and hence the host immune response is activated; and the inflammatory cascade, which is triggered by the viral RNA and it is comprised of inflammasome activation that drives the cytokine storm.,17 Colchicine has been shown to interfere with the activation of the NLRP3 inflammasome, reducing IL-1 production, which in turn prevents IL-6 release and neutrophils and macrophages activation.–20 A few studies have evaluated the use of colchicine in hospitalized patients with COVID-19. The GRECO-19 trial was the first open-label randomized trial that evaluated the efficacy of colchicine versus usual care in non-critically ill hospitalized patients. The study included 105 patients and found a significant reduction in the primary outcome of a two-point deterioration on World Health Organization disease severity scale. Additionally, in a single center cohort study in Italy, the use of colchicine in 122 hospitalized patients with COVID-19 compared with 140 patients who received hydroxychloroquine and/or intravenous dexamethasone; and/or lopinavir/ritonavir, resulted in a significantly better survival rate at 21 days . The RECOVERY trial of colchicine in hospitalized COVID-19 patients found that colchicine was not associated with mortality or morbidity benefit in comparison to usual care. Moreover, there are a few ongoing trials in different countries evaluating the use of colchicine in COVID-19 in comparison to standard care, while excluding the concurrent use of IL-6 antagonists: .Colchicine is one of the oldest anti-inflammatory therapies that is approved for treating and preventing acute gout attacks and familial Mediterranean fever.Given the controversy around the therapeutic alternatives for COVID-19 pneumonia, adding colchicine to tocilizumab with the aim of blocking the early and end products of the cytokines cascade, might reduce the risk of developing cytokine storm. Therefore, we aim to conduct an open-label randomized controlled trial to evaluate the efficacy and safety of adding colchicine to tocilizumab among patients with severe COVID-19 pneumonia.This is a single center open-label randomized controlled trial aims to evaluate the efficacy and safety of using colchicine versus no colchicine among patients with severe COVID-19 pneumonia who received tocilizumab and admitted with severe COVID-19 pneumonia to COVID-19 tertiary facilities at Hamad Medical Corporation (HMC) in Qatar, including Hazm Mebaireek General Hospital (HMGH), Cuban Hospital (CH), and Communicable Disease Center (CDC). The study will cover the following objectives; primary: evaluate the efficacy of colchicine versus no colchicine in terms of the need of invasive mechanical ventilation among patients with severe COVID-19 pneumonia who received tocilizumab; secondary: assess the impact of colchicine on in-hospital mortality, duration of mechanical ventilation, and intensive care unit (ICU) length of stay among patients with severe COVID-19 pneumonia who received tocilizumab, evaluate the safety of using colchicine among patients with severe COVID-19 pneumonia who received tocilizumab.The study will include patients admitted to HMC COVID-19 facilities, including HMGH, CH, and CDC with a confirmed diagnosis of severe COVID-19 pneumonia.Patients will be considered eligible to participate in the study, if they meet the following criteria:Alert and conscious adults with the age of >18 years old2 < 94% on room air at sea level, respiratory frequency > 30 breaths/minutes, or lung infiltrates >50%Severe COVID-19 pneumonia defined as SpOReceived tocilizumab within 10 days prior to enrollmentTocilizumab is co-administered with a systemic corticosteroidAgree to sign the written informed consentPatients will be ineligible to be recruited in case they have any of the following:Severe COVID-19 pneumonia requiring invasive mechanical ventilationCreatinine clearance calculated using Cockcroft-Gault equation <30 mL/minEnd stage renal disease on hemodialysisAlanine transaminase (ALT) and/or aspartate transaminase (AST) > 5 upper limit of normalPregnancyLactationConcurrent use of a strong CYP3A4 inhibitor , a moderate CYP3A4 inhibitor  or a P-gp Inhibitor COVID-19 patients who meet the eligibility criteria of the study will be randomized using web-based randomization to receive colchicine (intervention arm) versus receiving no colchicine (control arm) in 1:1 ratio. Allocation numbers will be generated using the randomization website, and stratification will be balanced by gender  and age , resulting in 6 strata.Participants allocated in the intervention arm will receive colchicine using the following dosing schedule:1. Day 1: Colchicine 1 mg and 0.5 mg after 1 hour.2. Day 2:• Weight >70 kg: 0.5 mg twice daily for 14 days, or discharge, whichever happens first• Weight <70 kg: 0.5 mg once daily for 14 days, or discharge, whichever happens firstIn case of concomitant use of azithromycin:1. Day 1: Colchicine 1mg2. Day 2:• Weight >70 kg: 0.5 mg twice daily for 14 days, or discharge, whichever happens first• Weight < 70 kg: 0.5 mg once daily for 14 days, or discharge, whichever happens firstThroughout the 14-day course, in case of elevation of liver enzymes (ALT/AST) by more than 5 upper limit of normal and/or decrease in creatinine clearance to <30 mL/min, colchicine will be stopped, and outcomes will be censored.During the 30-day follow up period, the participants will be monitored for the following outcomes.Rate of invasive mechanical ventilationTime to invasive mechanical ventilationDuration of invasive mechanical ventilation30-dayin-hospital mortalityICU length of stayHospital length of stayAdverse drug reaction (ADR) due to colchicineColchicine discontinuation ratePatients will be followed up until discharge or for 30 days, whichever comes first as shown in Figure  Assuming the rate of invasive mechanical ventilation is about 50% in the control arm, a minimum sample size of 190 patients will provide 80% power to detect a relative risk reduction of approximately 40% and absolute risk reduction of 20% in the intervention group. To account for 20% dropout rate, the estimated sample size would be 230 patients randomized in 1:1 ratio (115: 115).The rate of invasive mechanical ventilation was about 50% among COVID-19 patients in Qatar in 2020.For the baseline characteristics, descriptive statistics in the form of mean and standard deviations or median and interquartile range will be calculated for interval variables and percentages for categorical variables. Chi-square or Fisher exact tests will be used for categorical variables to compare the two groups. Student t test will be applied for normally distributed variables and Mann–Whitney U test for non-normally distributed interval variables.P value <.05 will indicate statistical significance.For the primary outcome of rate of invasive mechanical ventilation, it will be reported as percentages, and difference between the two arms will be evaluated using Chi-square or Fisher exact tests, as appropriate, and presented using absolute risk reduction and relative risk reduction. Additionally, Cox proportional hazard regression analysis  will be used to determine the association between colchicine use and time-to-primary outcome, with the adjustment of baseline imbalances between the two groups, if any. The results are to be presented as hazard ratio (HR), with a 95 % CI and P value <.05 will indicate statistical significance. Statistical analyses will be performed using SPSS 24.0 statistical package.Secondary outcomes, including 30-day in-hospital mortality, ADR due to colchicine, and colchicine discontinuation rate will be reported as percentages, and difference between the two arms will be evaluated using Chi-square or Fisher exact tests, as appropriate, and presented using absolute risk reduction and relative risk reduction. Remaining outcomes, including time to invasive mechanical ventilation, duration of invasive mechanical ventilation, ICU length of stay, and hospital length of stay will be reported as continuous data in mean days or median days according to data distribution, and difference between the two arms will be evaluated using Student t test for normally distributed variables and Mann–Whitney U test for non-normally distributed variables. All reported p-values will be two-sided, and Colchicine use, like any other medication, can cause adverse drug events, and the most common adverse drug events reported with colchicine use are gastrointestinal: diarrhea, nausea, and vomiting.:Other side effects that have been reported with colchicine use (with unknown frequency), includeNeurological: sensory motor neuropathyDermatological: alopecia, maculopapular rash, purpura, rashHematological: leukopenia, granulocytopenia, thrombocytopenia, pancytopenia, aplastic anemiaHepatobiliary: elevated AST, elevated ALTMusculoskeletal: myopathy, elevated creatine kinase, myotonia, muscle weakness, muscle pain, rhabdomyolysisReproductive: azoospermia, oligospermiaIn view of strict safety precautions followed worldwide and in Qatar to limit the spread of COVD-19, investigators will not be able to assess the development of ADRs by direct contact with the patients. Therefore, the following steps will be followed:After enrollment, investigators will seek safety information regarding the development of possible ADRs by contacting over the phone the managing teams of the enrolled patients during the follow-upperiod on daily basis.Monitoring the enrolled participants for safety outcomes includes monitoring the development of the adverse drug events mentioned above, and if the study participant develops an adverse event due to colchicine, an adverse drug event form will be filled in the patient’s profile electronically within 24 hours of event, and it will be recorded in the data collection sheet.The study principal investigator will be notified by the study investigator who identified the ADR to update the ADR report that will be reviewed by the data safety monitoring board (DSMB).Severity of the ADR will be assessed using NCI Common Terminology Criteria for Adverse Events (CTCAE), v4.0.Severe ADRs will reported by the principal investigator to the IRB.Medical judgment will be used to determine the relationship, considering all relevant factors, including pattern of reaction, and the decision to stop colchicine will depend on the severity of the adverse reaction. In case of severe adverse events (Grade 3–5) due to colchicine, the medication will be stopped.The DSMB, an independent committee from the study investigators, will comprise 2 established clinical investigators, and 1 biostatistician who are independent from study investigators. The primary role of this board will be the continuous independent review of the study reports developed by the study principal investigator, concerning: study progress; effectiveness and safety reports, with the conduction of the formal interim analysis.P value for the final analysis will be conducted at .049. Cox regression will be used for the efficacy primary endpoint and Chi square test will be used for the safety endpoint (colchicine induced ADR). The stopping rule for futility will be based on conditional power. Discontinuation of the study because of futility will be considered at the time of the interim analysis if the accumulating data suggest a very low likelihood (<1%) of a statistically significantly lower primary end point event rate for colchicine arm.An interim analysis for efficacy and safety will be conducted after recruiting 50% of the panned sample size. The DSMB will employ the O’Brien-Fleming alpha spending function for stopping the study for efficacy and safety. The O’Brien-Fleming stopping rule will be used at an alpha of 0.005, and the  Later in 2021, the REMAP-CAP trial evaluated the use of a single injection of an IL-6 receptor blocker, tocilizumab or sarilumab, compared to placebo among 800 patients in need of respiratory or blood-pressure support or both and found that IL-6 antagonist resulted in an in-hospital mortality of 27%, as compared with 36% in the control group. The RECOVERY trial which included 4116 hospitalized patients with COVID-19 pneumonia demonstrated that tocilizumab significantly reduced all-cause mortality at 1 month in comparison to usual care . On the contrary, the COVACTA trial evaluated the use of tocilizumab versus placebo among 452 patients with severe COVID-19 pneumonia and it showed that the mortality rate was 19.7% in the tocilizumab group versus 19.4% in the control group.It remains controversial whether blocking IL-6 alone is effective. A meta-analysis of seven retrospective observational studies using tocilizumab in patients with severe COVID-19 found that tocilizumab resulted in lower all-cause mortality compared to placebo without achieving statistical significance. Furthermore, the risk of ICU admission and the need for mechanical ventilation were similar between the 2 groups.,17 This specifically makes it a potential anti-inflammatory therapy for COVID-19. Colchicine differs from IL-6 receptor blockers by having pleotropic mechanisms of action, acting upstream of the cascade of cytokines, and being an oral agent.Colchicine, which is an old anti-inflammatory drug, targets multiple cytokines in the inflammatory cascade of the cytokine storm induced by COVID-19. Therefore, adding colchicine to IL-6 antagonists with the aim of blocking the early and end products of the cytokines cascade, might reduce the risk of developing cytokine storm and therefore the need for invasive mechanical ventilation and eventually death.Given colchicine ease of use, low cost, good safety profile, having different anti-inflammatory mechanism of action than other IL-6 blockade, and the controversy of an effective therapy for COVID-19 so far, colchicine might serve as a potential anti-inflammatory agent among patients with severe COVID-19 pneumonia. Colchicine acts upstream in the cytokines cascade by inhibiting the NLRP3 inflammasome while IL-6 receptor antagonists block the end result of the cytokines cascade. The latter might be the reason behind the controversies surrounding tocilizumab efficacy in treating COVID-19 as it has been thought that the ongoing replication of the virus overwhelms the drug capacity to control the disease burden.The main strength of our study is that it will evaluate the use of colchicine in COVID-19 pneumonia in a novel setting, where it will be used on top of tocilizumab, which is to the best of our knowledge is innovative in the literature of the pharmacological options for COVID-19.To sum up, our study is an open-label randomized trial that will provide valuable insights on the use of colchicine in severe COVID-19 when added to IL-6 antagonists as it aims to evaluate the efficacy and safety of adding colchicine to tocilizumab among patients with severe COVID-19 pneumonia.This study will be carried out according to the research regulations of the Medical Research Center and Institutional Review Board at HMC in Qatar; it obtained the approval in August, 2021 (MRC-01-21-299). Results of the analysis will be submitted for publication in a peer-reviewed journal and international conferences.AR, AM, SMA, MAA, SA, and MN: conceived the study.AR, and AA: designed the analysis planAR, MN, and AHA wrote the first draft of the protocol.All authors reviewed and agreed on the final version of the manuscript.


Human cytomegalovirus (HCMV) remains an important cause of transplant-related morbidity and mortality. The incidence of HCMV recurrence in the donor seronegative (D-)/recipient seropositive (R+) group is significantly higher than in other serostatus combinations as a result of a lack of pre-existing HCMV-specific memory T-lymphocytes in the donor, coupled with the eradication of the recipient's cellular immunity due to the conditioning regimen.We describe the case of an 8-year-old βE-thalassemic girl from Bangladesh who was seropositive for human cytomegalovirus (HCMV) and underwent hematopoietic stem cell transplantation from a HLA-matched, unrelated, HCMV-seronegative donor. Despite administering antiviral prophylaxis with commercial pooled anti-HCMV immunoglobulin (Ig) from day +1, the post-transplant course was complicated by prompt viral reactivation, and foscarnet therapy was initiated. The virus was refractory to treatment, leading rapidly to complete bone marrow failure, and targeted immunotherapy was proposed as a second-line therapy. Hypothesizing that the patient and her relatives may have been exposed to similar HCMV strains, we selected the patient's mother, who presented a high HCMV antibody titer, as the donor of virus strain-specific anti-HCMV Ig and T-lymphocytes. Complete viral clearance was achieved after two transfusions of the mother's plasma. Subsequently, the patient underwent a haploidentical rescue transplant, promptly reaching full hematological recovery.These findings suggest that treatment with virus strain-specific Ig may offer a new therapeutic option for critically ill patients. •Human cytomegalovirus (HCMV) remains an important cause of transplant-related morbidity and mortality.•Reactivation of multiple viral strains is particularly challenging to eradicate. .•Virus strain-specific Ig is an inexpensive, non-toxic therapy to treat HCMV reactivation and improve transplant outcomes. .•The efficacy of this approach as a means of prevention has only been demonstrated previously in mouse models. SeveralWe describe a case of a pediatric HSCT recipient who had complications of HCMV recurrence and was treated successfully with targeted immunological therapy, using haploidentical donor-derived immunoglobulin (Ig).2An 8-year-old girl was diagnosed at the age of two in her country of origin (Bangladesh) as βE-thalassemic. She was treated with monthly blood transfusions and weekly iron chelation with deferoxamine, plus supplementation with folic acid. A suboptimal regimen of chelation therapy led to severe systemic siderosis, and her family travelled to Italy to undergo HSCT. Since a human leukocyte antigen (HLA)-matched related donor was unavailable at that time, an HLA-matched (10/10) unrelated donor was identified. The patient was HCMV-seropositive, whereas the donor was HCMV-seronegative, thus posing a high-risk serological combination (D-/R+) for post-transplant HCMV infection and associated complications.8 total nuclear cells/kg recipient weight, and the vitality of the cells contained in the graft was 96.9%. Tacrolimus and mycophenolate mofetil were used for graft-versus-host disease (GVHD) prophylaxis. Anti-HCMV prophylaxis commenced at day +1 with commercial pooled anti-HCMV Ig  at a dose of 100 U/kg twice a week.The conditioning regimen consisted of fludarabine, busulfan, thiotepa, and anti-thymocyte globulin (ATG). The infused donor's bone marrow contained 3.79 × 10The initial post-transplant period was uneventful. The first occurrence of HCMV viremia  was detected at day +14, and antiviral therapy with foscarnet was started immediately. At day +17, neutrophil engraftment was achieved, but not the platelet and the erythrocyte counterparts. At day +20, the patient presented fever without clinical symptoms and without an increase of C-reactive protein. Afterwards, a full blood count revealed a progressive drop in leukocytes and platelets, whereas HCMV viremia continued to increase despite antiviral therapy. The patient also developed a severe, HCMV-related hemorrhagic cystitis, which required frequent platelet transfusions, initially once daily for 5 days, and thereafter twice daily for 7 days.6 CD34 cells/kg of mother's graft. GVHD prophylaxis was performed with post-transplant cyclophosphamide, tacrolimus, and mycophenolate mofetil. The engraftment was rapid, with a neutrophil count of >500/μL achieved at day +15, and the final platelets and packed red blood cells were transfused at day +20. Since HCMV viremia remained negative during the entire post-transplant period, we discarded the use of the mother's virus-specific T-lymphocytes. The patient was discharged at day +27 in perfect clinical condition, with continuation of antiviral valganciclovir prophylaxis. Immunosuppressive treatment and antiviral prophylaxis were discontinued six months later, and the patient remains in perfect health 2 years after the second transplant.As the infection appeared unresponsive to foscarnet, ganciclovir treatment was started at day +25. This switch in antiviral therapy followed an increase in viral load, reaching a maximum titer of 5100 viral genome copies/mL at day +30. Simultaneously, the clinical condition of the patient continued to deteriorate, and the leukocyte count decreased drastically. Therefore, the diagnosis of secondary graft failure due to HCMV infection was established. Because of the failure of anti-HCMV treatment, ganciclovir was withdrawn, and foscarnet therapy was resumed. The possible explanations for treatment failure include the presence of antiviral resistance mutations or an ineffective activity of the Ig administered. As the first option seemed improbable because resistance mutations usually arise later than 6 weeks after HSCT, we opted to address the latter option. Therefore, we considered using antibodies (Abs) that were more likely to inhibit the HCMV strains causing the infection in the patient. Although the diversity of circulating HCMV strains is known to be extensive , we hypoAY446894.2), and non-synonymous variants known to confer resistance were identified. No resistance mutations were detected.Therapeutic interventions, HCMV load, and white blood cell count during the entire episode are summarized in 3Various factors are implicated in the outcome of HCMV infection in transplant patients, the first of which relates to the HCMV serological status of donor and recipient. The serological status of the first donor may have played an important role in this case, as it has been reported that using an HCMV-seronegative unrelated HSCT donor (D-) for a seropositive recipient (R+) results in reduced overall survival after myeloablative conditioning. In contrast, an HCMV seropositive donor is associated with improved event-free survival and reduced non-relapse mortality . Thus, tIn relation to viral factors, the clinical course of the patient may have been influenced by two main factors: the presence of multiple HCMV strains or the presence of mutations conferring antiviral resistance. Infection with multiple HCMV strains is common after transplantation , and is In the present study, the initial failure of antiviral therapy could not be explained by the development of resistance mutations in the HCMV strains present in the patient. This finding is not surprising, as detection of resistance mutations is unusual during the first 6 weeks of antiviral therapy, whereas the sample analysed in this case was collected only 13 days after initiation of foscarnet treatment .In regard to the initial failure of the immunotherapy with commercial pooled Ig, this may have been due to the lack of particular Abs with neutralizing capacity against the HCMV strains present in the patient. Assuming that the donors providing the Ig had been exposed to strains circulating in Europe, the Abs may have been more suited for neutralizing HCMV strains commonly circulating in that region. However, it is possible that the strains present in the patient, who originated from Bangladesh, may have differed immunologically from strains in other regions of the world. The extent to which this is true is unknown, as current knowledge of HCMV genome variability is derived almost entirely from strains circulating in a few European countries. Therefore, it would be useful to assess the neutralizing capacity of commercial pooled Ig in order to avoid potential therapeutic failure, especially in patients from non-European countries. The clinical problem in this case was addressed successfully by using the Ab-rich plasma of the patient's mother in a kind of personalized immunotherapy, on the premise that both mother and daughter may have been exposed to the same pool of viral strains. In this HCMV-positive recipient with a severe viral reactivation, Ig obtained from a household donor afforded an efficacious treatment that led to complete viral clearance.To the best of our knowledge, this is the first report of the successful use of HCMV strain-specific Ig therapy in an HSCT recipient. The efficacy of this approach as a means of prevention has been demonstrated previously only in mouse models involving murine cytomegalovirus (MCMV) reactivation after bone marrow transplantation . In thisOur case highlights the importance of monitoring HSCT recipients comprehensively, longitudinally and with high sensitivity for viral factors, including the presence of multiple strains and resistance mutations. Importantly, it also provides an affordable targeted immunological therapy in patients from countries with constrained access to alternative therapies.4HSCT is a complex and specific setting in which a recipient may rapidly develop viral reactivation and consequent life-threatening complications. Ab-rich plasma may represent a fast and feasible therapeutic option to overcome viral activity and allow successful engraftment of the donor's bone marrow.No specific funding was received.The authors have obtained consent from the parents of the patient to publish individual patient data.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Orthodontically induced inflammatory root resorption (OIIRR) is considered an undesired and inevitable complication induced by orthodontic forces. This inflammatory mechanism is regulated by immune cells that precede orthodontic tooth movement (OTM) and can influence the severity of OIIRR. The process of OIIRR is based on an immune response. On some occasions, the immune system attacks the dentition by inflammatory processes during orthodontic treatment. Studies on the involvement of the PD-1/PD-L1 immune checkpoint have demonstrated its role in evading immune responses, aiming to identify possible novel therapeutic approaches for periodontitis. In the field of orthodontics, the important question arises of whether PD-L1 has a role in the development of OIIRR to amplify the amount of resorption. We hypothesize that blocking of the PD-L1 immune checkpoint could be a suitable procedure to reduce the process of OIIRR during orthodontic tooth movement. This review attempts to shed light on the regulation of immune mechanisms and inflammatory responses that could influence the pathogenesis of OIIRR and to acquire knowledge about the role of PD-L1 in the immunomodulation involved in OIIRR. Possible clinical outcomes will be discussed in relation to PD-L1 expression and immunologic changes throughout the resorption process. Orthodontic tooth movement (OTM) is induced by mechanical orthodontic forces (compressive and tensile strain) and stimulated by the profound remodeling that occurs in the alveolar bone and periodontal ligament (PDL) . On the Within the periodontium, the dentine of the root is covered with a thin layer of cementum. During OTM, local over-compression of the PDL may induce a hyalinization of the cementum, resulting in simultaneous cementum resorption in line with the removal of hyalinized tissue. This pathological process that causes substance loss from mineralized cementum and dentine is termed “orthodontically induced inflammatory root resorption” (OIIRR) . OIIRR oDuring OTM, specific immunocompetent cells migrate to the periodontal ligament. Among the PDL resident cells, immune cells such as neutrophiles, antigen-presenting cells (APCs: dendritic cells and macrophages) , naturalThe programmed cell death-ligand 1 (PD-L1) is a type-I transmembrane protein with an extracellular domain at its N-terminus that functions as an immune checkpoint. It suppresses the immune response by interacting with the programmed death cell receptor (PD)-1 that is found to be expressed in T cells , B cellsIn this article, we review current evidence regarding immunology in OIIRR and discuss the potential immunomodulatory properties of PD-L1 in the context of OIIRR. Furthermore, the possible clinical implication of PD-L1 in OIIRR prevention and therapy will be addressed.OIIRR occurs on almost all tooth roots exposed to orthodontic forces. It takes place in approximately 80% of orthodontically treated patients  and is iIn 2002, Brezniak et al. proposed the more descriptive and accurate term of orthodontic force-induced root resorption in light of the actual histologic process and termed it OIIRR . The patThe cause and risks of OIIRR are complex, but it is believed that sterile inflammatory processes include different components: mechanical forces (magnitudes), types of mechanical forces , direction and duration of force, tooth root morphology, distance of root apical movement, surrounding matrix, PDL, cementum, and certain biological messengers ,38. The The key resorptive cells involved in OIIRR of cementum include mono-nucleated macrophage-like cells, osteoclasts, dentinoclasts, and odontoclasts, the latter of which are extremely similar in the morphologic and functional aspects to those of osteoclasts, albeit slightly smaller  Figure .Endocytotic vesicles containing liberated apatite crystals are found in odontoclasts, implying that demineralization in the resorptive microenvironments is not completely comparable with osteoclasts . ActivatThe immunological system response accompanied with inflammatory responses during OTM and OIIRR relies on the cooperative activities between innate and adaptive immunity . MonocytIn addition, multinucleated TRAP-positive giant cells participate in hyalinized tissue removal and the adjacent root structure resorption . TRAP-poDecompression alters the resorption process and repair of the cementum process is initiated once the applied orthodontic forces have been discontinued ,50. PhysThis process usually takes at least 6–8 weeks to become radiographically visible . HoweverIn response to orthodontic forces, an inflammatory process is involved in the occurrence of cementum-repairing activities . InflammThe underlying immune process of OIIRR is still not completely understood. The hyaline necrosis of the periodontium causes damage to the cementum, thus the dentine matrix is exposed . Once thIn the initial phase of orthodontic force application (within 3 days), the first infiltrating immune cells that reside in mouse PDL mainly consist of neutrophils, monocytes, lymphocytes, and APCs ,54. BesiTwo distinct in vitro phenotypes of macrophages exist: the classically activated phenotype  and the alternatively activated phenotype  . Both ar+ and iNOS+ M1 macrophages [Under prolonged duration of orthodontic force application, OIIRR was found to be concomitant with an increased infiltration of CD68rophages . The sevrophages . The macThe NK cells participate in the innate immune regulation by activating signaling pathways related to OIIRR . The up-+ DCs were expressed around the hyalinized tissue between dentine and cellular cementum [The DCs are normal resident cells in the PDL and mainly play roles as APCs to react with T and B cells. Upon orthodontic force application, increased numbers of CD11bcementum , indicat+ T cells [The involvement of T cells  was also T cells . Low lev T cells , further T cells .+ B lymphocytes in PDL [B cells are already primed for orthodontic forces . In conjs in PDL  in respoCytokines and chemokines are substances released by immune cells for the communication of signals between immune and non-immune cells  Figure . It has Porphyromonas gingivalis  and its components, PD-L1 was up-regulated in cells belonging to the oral masticatory mucosae such as primary human gingival keratinocytes [Concerning the progress of OIIRR, recent studies have tried to associate its initialization with a specific antigen present in the PDL that might alter the immunologic response, PD-L1 . It is winocytes  and humainocytes , with a inocytes . PD-L1 hinocytes , human oinocytes , human Pinocytes , and huminocytes .+ T cell-mediated local inflammatory reaction. This suggests that they have protective properties against excessive tissue damage [+ and CD8+ T lymphocytes from periodontitis patients compared with healthy individuals [The PD-L1-expressing keratinocytes promote the regulation of a CD4e damage . The PD-e damage . Oral PDe damage . Both PDividuals . Recentlividuals . Moreoveividuals . These n2 enhances the PD-L1 expression in cementoblasts. P. gingivalis peptidoglycan also up-regulate the PD-L1 expression in cementoblasts and may induce tolerogenic signaling to T cells. When cementoblasts were subjected to a modified compression, they expressed stable PD-L1, which in turn may facilitate their antigenic escape from immune surveillance. In addition, it was revealed that HIF-1α plays an pivotal role in the PD-L1 modulation [Under physiological conditions, PD-L1 has been detected in different in vivo and in vitro models in various periodontal cell types. It has been demonstrated to be up-regulated upon immune activation conditions, such as inflammations . In recedulation . Anotherdulation . Based odulation  (Figure Regarding the regulation of the T cell immune response, the functional profile of PD-L1 in a human inflammatory environment has previously been reviewed. It is widely accepted that T cells are required in immune response and that they play essential roles during OTM and OIIRR . Shelby As the literature states, a PD-L1 checkpoint is functional in tumor-associated macrophages  and is rAs OIIRR is linked to interactions between an inflamed microenvironment and the immune response of the host, we proposed for the first time that the host immune system can be modulated to affect the tooth root resorption and repair process during OIIRR. One of the possible mechanisms is to target the PD-L1 molecule. The PD-L1 inhibitors that target PD-1 have been shown to improve the outcome of melanoma survival . This inFuture in vitro studies should be performed with primary human cementoblasts as well as in animal models to verify the mechanisms. The link between PD-L1 expression and orthodontic forces needs to be evaluated by comparing how the checkpoint is expressed on T lymphocytes from patients with OIIRR to those from healthy people.However, it should be noted that the use of PD-L1-targeted drugs to control OIIRR in orthodontics poses too great a systemic risk in the future. Given the importance of PD-1 in suppressing self-reactive T cell-mediated immune responses, immunosuppression caused by the PD-1/PD-L1 immune checkpoint inhibitors can result in the infiltration of T cells into organs all over the body, causing an immune response . Some ofTo date, there has not been a published study about its use in orthodontics. Moreover, the major question remains about the dosage of the drugs regarding their application in dentistry. If clinicians do not account for these variations and collect data about adverse events, we will never gain knowledge about the real incidences of treatment-related side effects in relation to PD-1/PD-L1 inhibitors in dentistry.Furthermore, especially in orthodontics, it is also essential to understand the economic impact of this influential, but expensive therapy. Until now, the cost-effectiveness of PD-1/PD-L1 immune checkpoint inhibitors has not yet been systematically evaluated.Root resorption, especially OIIRR, is linked to the interactions between inflammation and the host immune response. There is little evidence about how immune cells and non-immune cells in the periodontium contribute to OIIRR and how they interact with PD-L1. The establishment of the role of PD-L1 in an orthodontically induced microenvironment represents a significant emerging step toward unraveling the recognition mechanisms for immune cells and pathogenic components concerning inflammatory conditions during OIIRR. Thus, transgenic/gene knockout technologies should be conducted to elucidate cellular and molecular immunologic processes and mechanisms induced by orthodontic forces. The precise pathway mechanisms and the immune reactions behind the role of PD-L1 in OIIRR should be revealed in future work. With this knowledge, it is the goal to provide novel therapeutic approaches to prevent or treat OIIRR.