Diabetic individuals are considered a vulnerable population during the COVID-19 Pandemic, and several studies noted worse outcomes, including death, among those who get infected. Diabetic emergencies, such as ketoacidosis (DKA), are common and potentially life-threatening conditions in uncontrolled patients. While the pathophysiological background of the relationship between COVID-19 and DKA is not fully understood, early reports available so far indicate that patients with pre-existing diabetes who get infected with the SARS-CoV 2 virus are at higher risk of DKA. It was also suggested that DKA is a poor prognostic sign for infected patients, these being at higher risk of developing worse forms of COVID-19 disease and having high mortality. Therefore, healthcare personnel dealing with such patients face a considerable challenge, as the correct and safe emergency management of such cases is far from established. This article aimed to conduct a study that reviews the current published data available about patients with DKA and COVID-19. Diabetic ketoacidosis (DKA) is a well-known medical emergency occurring in uncontrolled diabetic patients, responsible for significant mortality and morbidity, therefore highlighting the vital importance of early diagnosis and treatment in managing these patients. Although more frequently seen in patients with Type 1 diabetes, considering the metabolic disorder background of absolute insulin deficiency, diabetic ketoacidosis is not uncommon in Type 2 diabetes patients. Prompt action is required whenever clinical suspicion is present ,4.The novel-coronavirus that causes severe acute respiratory syndrome 2 (SARS-2) disease COVID-19) emerged from Wuhan, China, in December 2019, leading to a global pandemic, with more than 44 million cases and over 1.1 million deaths confirmed as of 28.10.2020 ,6. Despi emerged Furthermore, lack of physical activity because of lockdowns imposed by the government in many countries might be another negative factor for diabetic patients, as sedentarism plays a significant role in the progression of disease severity and metabolic imbalance ,10.Considering all these aspects, both the healthcare personnel dealing with diabetic patients and those on the front-line of the pandemic caring for COVID-19 patients should be aware of the challenges in managing diabetic patients during these times, paying careful attention to acute complications such as DKA or HHS. Also, new patients presenting with DKA should be carefully screened for COVID-19, which could be a possible infectious triggering factor of the metabolic imbalance ,12.There little data available regarding the pathophysiological relationship between diabetic ketoacidosis and COVID-19 and the impact of the pandemic on the evolution of acute diabetic complications.The study aimed to review current evidence about the epidemiology, pathophysiological mechanisms, and diagnostic and management challenges of DKA during the COVID-19 pandemic.A literature search was performed using “COVID-19”, “diabetes”, and “diabetic ketoacidosis” as keywords through the “PubMed” and “Google-Scholar” search engines.The first published studies about the evolution and prognosis of diabetic patients with COVID-19 originate from China. It was reported that COVID-19 patients who had diabetes had higher hospital admission rates and developed worse forms of pneumonia than infected patients without previous medical conditions. Another study reported that diabetes was the second most frequent comorbidity after hypertension in a sample of 140 patients diagnosed with Covid-19 and hospitalised in Wuhan . These oBesides the previously discussed, more commonly studied pathophysiological relationships between COVID-19 and diabetes, another important cause of concern when dealing with diabetic patients during the COVID-19 pandemic lies in managing acute hyperglycaemic complications DKA. DKA is the most common acute metabolic complication of diabetes caused by a relative or absolute insulin deficiency, which leads to reduced glucose uptake and utilisation by cells and excessive lipolysis. This further causes uncontrolled production of ketone bodies and a state of acidosis. Although DKA is an inflammatory state, it is commonly triggered and associated with an underlying acute illness, such as an infectious disease . RecentlMore retrospective studies, case reports and a series of cases have observed that DKA is not an uncommon occurrence among hospitalised COVID-19 patients, which could be one of the causes of the higher mortality among these patients. In a Chinese study, almost half of the Covid-19 hospitalised patients died; all had medium and severe forms of Covid 19, with diabetes as a comorbidity and a 39% mortality in the studied cohort .The first reports of DKA in COVID-19 patients outside China were by Croft A et al. 2020), who published the evolution of five new-onset DKA in a series of patients with Type 2 diabetes hospitalised for COVID-19. The authors highlighted the unusual severity of DKA in these cases of previously well-controlled diabetic patients, suggesting the significant negative impact of the novel coronavirus infection on insulin regulation and metabolic balance 20, who p,22, someIn cross-sectional UK study on 218 patients admitted with laboratory-confirmed COVID-19, pre-existing Type-2 diabetes was found in 61 patients (28%) and Type-1 diabetes in 6 (2.8%) patients. In this cohort, four patients (1.8%) presented DKA during the hospital stay, three of them having pre-existing Type-2 diabetes, and one was a newly diagnosed case based on high glycated haemoglobin C (HbA1C) and glucose levels . A studyBesides DKA, another life-threatening acute metabolic complication of diabetes is HHS. Although the two conditions can be seen distinctively, both can sometimes overlap, especially in elderly, uncontrolled Type 2 diabetes patients ,27. As tA retrospective study reported six cases with both DK and HHS and positive SARS-CoV 2 tests admitted to an intensive care unit in New York, USA . All sixThe exact underlying mechanisms of how SARS-CoV 2 infection could accelerate DKA development, or how DKA contributes to a more severe evolution of the COVID-19 disease, leading to worse clinical outcomes and higher mortality, are still a subject of debate. The novel SARS-CoV 2 coronavirus belongs to a family of coronaviruses that have previously caused two significant outbreaks, one in the 2002-2004 SARS-1 outbreak and another, but with a higher mortality rate of MERS in 2012 ,30. SeveAngiotensin 1-7 has an antagonist role compared to Angiotensin 2 by stimulating insulin production and vasodilatation ,34,35. AThis mechanism might explain the possible risk of COVID-19 patients developing DKA and metabolic deterioration. Further studies are needed to understand the complex role of the COVID-19 infection on pancreatic endocrine function and its consequences. Increased insulin resistance during COVID-19 infection might also be triggered by the frequently used medication to treat these patients, especially corticosteroids, but also remdesivir, lopinavir/ritonavir darunavir/cobicistat and interferon-b1. Cortisol, a potent endogenous stress hormone, is a known glucose elevating agent, states of hypercortisolism being frequently a cause for the metabolic deterioration of diabetic patients. Therefore, it could be hypothesised that cortisol, together with other stress hormones like catecholamines, might be involved in the vicious cycle of diabetic patients with COVID-19 during the hyper-inflammatory stress response phase , 36. AnoFurther studies are needed to evaluate the role of other possible markers of disease severity, like pro-inflammatory cytokines or Vascular Endothelial Growth Factor (VEGF), which is known to be a predictor of poor outcome in ARDS . Some stAs recommended by the latest guidelines, the key components of DKA management include fluid and potassium replacement, intravenous insulin therapy and close monitoring of serum ketones, electrolytes, as well as acid-base status. It is recommended that patients continue long-acting analogue insulin therapy alongside intravenous infusions to prevent rebound hyperglycaemia after the intravenous treatment has ceased ,46.Management of DKA during the COVID-19 pandemic poses a significant challenge, as there is no current consensus or guidelines for this issue. In the case of compliant and clinically stable patients, where home testing of ketones is available, early treatment and prevention of this condition could be, in theory, done at home with self-hydration with oral fluids and following the therapy instructions by the healthcare team . This coA known helpful strategy for managing uncomplicated and mild DKA that could also be used during the COVID-19 pandemic consists of the utilisation of subcutaneous insulin therapy; this would reduce the need for prolonged direct contact with healthcare personnel and nursing time. A. Cochrane review evaluated five randomised clinical trials concluded that subcutaneous rapid-acting insulin therapy is not inferior to intravenous insulin therapy for the treatment of mild DKA . HoweverThe pro-inflammatory state of COVID-19 could explain this and the disturbance of the Angiotensin 1-7 counter-regulation of Angiotensin 2, leading to a higher degree of insulin resistance and possibly the accompanying organ damage involving the pancreatic islet cells. Careful attention should be paid to the concomitant use of corticosteroids and vasopressors in critically ill patients, as these could significantly influence the need for insulin. Therefore, a rapid adjustment method of insulin delivery to address fluctuations in glucose levels is vital for critically ill COVID-19 patients with DKA ,51. AnotThe nutrition of COVID-19 patients in ICU units is another matter of debate. There are no specific guidelines or data regarding the optimal nutritional support of COVID-19 diabetic patients. General recommendations for critical COVID-19 patients indicate that gastric enteral nutrition should be performed as early as possible after admission and should be preferred over parenteral nutrition. If gastric enteral nutrition is not possible or sufficient, parenteral nutrition (PN) can be prescribed using case-by-case decision-making. Risks of overfeeding and hyperglycaemia are to be taken into consideration, especially in diabetic patients. Liver function should also be closely monitored in all patients receiving gastric enteral nutrition or parenteral nutrition .Another significant challenge in managing these patients lies in the administration of intravenous fluids. Although restoring fluid balance is a crucial aspect of treating ketosis, the lung involvement of COVID-19 infection could be worsened by increased extravascular lung water that would lead to severe hypoxia and potentially dangerous outcomes . Therefoand pH. Patients with severe acidosis (pH < 7.1) should be treated more aggressively regarding fluid replacement than those with milder acidosis. The conservative approach to fluid replacement, although protecting the lungs, could lead to impaired clearance of ketones causing prolonged and worse ketonemia ,59.As DKA leads to a state of acidosis, patients frequently resort to respiratory-compensation tachypnoea; this state may be impaired in COVID-19 infected individuals.Probably the most troublesome complication in all COVID-19 patients is acute hypoxemic respiratory failure . The manThe use of a high-flow nasal cannula is a viable option for avoiding or delaying intubation, providing, as it does, adequate pre-oxygenation before the need for intubation and mechanical ventilation .The principles of respiratory support in COVID-19 respiratory failure are similar to those for ARDS caused by other diseases: low tidal volumes and driving pressures and adjusting the positive end-expiratory pressure depending on each patient’s requirements to protect the lungs. The addition of a severe metabolic complication like DKA makes the management and decision-strategy in the intensive-care unit handling of these patients even more problematic, as there is a lack of consensus and data regarding these issues , 62.Diabetic ketoacidosis management during the COVID-19 pandemic poses a significant challenge. Patients with pre-existing diabetes are considered a vulnerable group to SARS-CoV-2 related complications, and the virus could potentially lead to a worsening of glycaemic control and acute complications like DKA. Mortality in COVID-19 infected diabetic patients is high, resulting in DKA producing even worse clinical outcomes and prognosis. Close and continuous monitoring is mandatory for DKA patients with Covid-19, making it challenging for healthcare facilities to ensure a safe environment for both personnel and patients through enforcing strict protective and contact-limiting measures. Management of DKA patients with COVID-19 could be handled by aggressive intravenous insulin therapy and fluid restoration in some critically ill patients while taking a conservative approach to fluid administration to avoid excessive extravascular lung volumes that could worsen the respiratory function. Further research is needed to understand the pathogenesis of DKA during COVID-19 infection and to provide reliable guidelines on how to manage these patients.


Clinicians may wish to query not only individual but also neighborhood-level health when considering cognition.Living in neighborhoods with lower incomes, lower education/occupational levels, and/or higher crime increases one’s risk of developing chronic health problems including cardiovascular disease risk factors and stroke. These cardiovascular health problems are known to contribute to cognitive decline and dementia. The purpose of this study was to determine the association of neighborhood socioeconomic resources and crime-related psychosocial hazards on stroke risk and cognition, hypothesizing that cardiovascular health would mediate any relationship between the neighborhood-level environment and cognition. The study evaluated 121 non-demented Chicago-area adults  for cardiovascular health problems using the Framingham Stroke Risk Profile 10-year risk of stroke (FSRP-10). The cognitive domains that were tested included memory, executive functioning, and attention/information processing. Neighborhood socioeconomic resources were quantified at the census tract level ; crime-related psychosocial hazards were quantified at the point level. Structural equation modeling (SEM) did not show that the FSRP-10 mediated the relationship between neighborhood characteristics and domain-specific cognition. The SEM results did suggest that higher crime rates were associated with a higher FSRP-10 ( Increasingly, studies show that neighborhood-level factors of the social and built environment are associated with a broad range of individual-level outcomes among older adults . For exaNeighborhood-level socioeconomic disadvantage based not only on lower income, education, and/or occupation levels but also on psychosocial hazards including social disorganization and public safety concerns are also associated with increased rates of cognitive decline and lower cognitive performance more generally, even after controlling for individual-level socioeconomic status (SES) . FurtherThe aim of the present cross-sectional study was to determine the relationships between neighborhood-level socioeconomic disadvantages and individual-level health and cognition in non-demented community-dwelling older adults. Based on the literature linking neighborhood socioeconomic disadvantage ,5,11,12 This study was funded by the National Institute on Aging to investigate cardiovascular disease risk factors, neighborhood ‘health’ factors, cognition, and brain aging. The study was approved by the University of Illinois at Chicago Institutional Review Board (UIC IRB #2012-0142) as well as the Rush University Medical Center IRB (#16102101). It was conducted in accordance with the Declaration of Helsinki with written informed consent obtained from all participants.Described in detail elsewhere ,15,16, iFollowing successful completion of the telephone screening, eligible individuals were scheduled for a more detailed in-person screening that included the Mini-Mental State Examination (MMSE)  and the One-hundred and twenty-one participants met all inclusion and exclusion criteria and were enrolled in the study. We excluded 10 participants who were administered Spanish versions of the cognitive measures given concerns about comparability of select test measures used in the current analyses, and 6 individuals who either lacked information on key variables in our analyses or evidenced incidental findings on MRI. Thus, 106 participants contributed to the current analyses.n = 21), participants were asked to provide their immediately prior address and duration of residence at that location for geocoding.Participants provided their current address and duration of residence at their current address. If the stated duration of residence was less than 5 years (Geographic information systems (GIS) coding was used to analyze participants’ addresses. Data on neighborhood-level income, education, and employment were collected at the census tract level from the American Community Survey (2010 to 2014). Neighborhood-level crime data were collected at the point level (using a 1-mile buffer) from the Chicago Police Department’s Citizen Law Enforcement Analysis and Reporting (CLEAR) database accessed in 2015. Given that available GIS data were not in perfect one-to-one correspondence with all study visit years, we time lagged GIS data to 2 years prior to a participant’s year of study visit to maximize our sample. Thus, the resulting GIS data represented the socioeconomic environment captured 2 years prior to a participant’s study visit.In accordance with methods previously outlined , a standCrime variables representing per capita rates of homicide, robbery, and assault were quantified and combined using a similar PCA procedure as that described above. This allowed for the construction of a composite score that accounted for individual standardized weighted coefficients of these 3 variables . Higher Participants received a medical history interview and physical examination conducted by trained staff and a registered nurse, respectively, from the UIC Clinical Research Center (CRC). This evaluation included two seated blood pressure measurements separated by 5 min, anthropometrics including height and weight, and a confirmed 12 h fasting blood draw for health-related variables such as glucose and hemoglobin A1c. An electrocardiogram and medication review were also performed. This evaluation allowed for an assessment of the revised Framingham Stroke Risk Profile score 10-year risk of stroke (FSRP-10) . The FSRParticipants underwent a comprehensive neuropsychological assessment conducted by trained research assistants. We statistically grouped select cognitive test measures shown to be particularly vulnerable to increased cardiovascular disease and stroke risk in older adults . As detThe LMR domain was based on total recall across five consecutive learning trials, long delay free recall, and a recognition discriminability index from The California Verbal Learning Test-II (CVLT-II) . The AIPX2) statistic and several practical fit indices were utilized to evaluate the model, including the root mean square error of approximation (RMSEA), the comparative fit index (CFI), and the Tucker–Lewis index (TLI). While the chi-squared test is sensitive to sample size bias, it is considered an adequate metric for samples between 75 and 200 with suggested cutoff values greater than p = 0.05 representing better fit [Descriptive characteristics of our sample and initial bivariate correlations of key variables of interest were conducted using SPSS Version 27. Structural equation modeling (SEM) was employed using Mplus (Version 8) to evaluate whether stroke risk mediates relationships between neighborhood socioeconomic characteristics and cognition. Specifically, we tested three models examining stroke risk as a mediator of any association between the neighborhood-level PCA scores of socioeconomics and crime with cognition, i.e., LMR, AIP, and EF composite scores (separately). In order to assess the extent to which the model fit the data, the chi-squared , and obtained an average of 16 years of education. Additional participant characteristics are outlined in As may be seen from p < 0.001. Thus, lower neighborhood-level socioeconomic resources were associated with higher neighborhood-level crime. The entire correlation matrix is outlined in A bivariate Pearson correlation matrix was created using the following variables of interest: neighborhood-level socioeconomic- and crime-related composites, as well as individual-level stroke risk (FSRP-10) and cognition . Of the correlations between the two neighborhood-level composites and individual-level stroke and cognitive status variables, only the relationship between the socioeconomic and crime composite scores were significant, r(105) = −0.65, Initial models testing whether stroke risk mediated the relation between predictors (neighborhood-level socioeconomic resources and crime) and outcome variables  run with all paths freely estimated demonstrated that the direct effects between predictor and outcome variables were nonsignificant (data not shown). Thus, these direct effects were constrained to zero for final models outlined below.X2  = 1.81, p = 0.41; RMSEA < 0.01; CFI = 1; TLI = 1.25), FSRP-10 was not significantly associated with the LMR composite score, β(106) = −0.004, p = 0.86.While model fit was adequate for using LMR  = 2.62, p = 0.27; RMSEA = 0.05; CFI = 0.91; TLI = 0.78); despite TLI results, FSRP-10 was significantly correlated with AIP (β(105) = 0.04, p = 0.03). Specifically, higher 10-year stroke risk was associated with higher values (equating to worse performance) on attention/information processing tasks. Thus, we felt that the overall model fit was acceptable. Additionally, neighborhood socioeconomic characteristics, in terms of socioeconomic resources (β(105) = 2.38, p = 0.04) as well as crime-related psychosocial hazards (β(105) = 1.26, p = 0.02), were significantly correlated with FSRP-10. The final model is described graphically in All but one model fit statistic was adequate for the AIP model  = 3.53, p = 0.17; RMSEA = 0.09; CFI = 0.74; TLI = 0.36) and was, therefore, not considered further.The EF model had poor overall fit  on attention/information processing tasks. Similar investigations of other cognitive domains including learning and memory as well as executive functioning did not suggest a similar results profile. Thus, while stroke risk may not formally mediate the relationship between neighborhood-level characteristics and domain-specific and individual-level cognitive skills, higher crime rates were associated with stroke risk and a higher stroke risk was associated with reduced cognitive performance after accounting for other neighborhood socioeconomic characteristics. Furthermore, our study suggests that where one lives is associated with how one lives both physically and, in turn, cognitively, particularly as it relates to attention and information processing.The results of this study contribute to the literature in several ways. First, this study represents a growing body of literature that aims to draw connections between neighborhood-level factors and individual-level health outcomes ,27, exteWhile the underlying biological mediators of the associations revealed in this study cannot be gleaned directly from the work presented, suggestions for future investigations incorporating biological biomarkers may be found in the literature. For example, the literature suggests that chronic exposure to stressful environments has a negative impact on health, including an increased risk for developing cardiovascular disease risk factors such as hypertension, diabetes, and coronary heart disease, all of which are associated with increased risk for stroke and other adverse outcomes . An undeThe assumption of chronic exposure to stressful environments may not, however, apply to all neighborhood-level characteristics of the environment in our structural equation models. Specifically, the significant association between our measure of socioeconomic resources and stroke risk was in opposition to our hypotheses as well as much of the literature. Thus, our association between lower socioeconomic resources and lower stroke risk contrasts previous work suggesting that living in more socioeconomically impoverished neighborhoods has an adverse effect on individual-level health ,29. PostThe strengths of this paper include the fact that this is amongst the first study to examine the mediators between neighborhood-level socioeconomic environments including crime-related psychosocial hazards and cognition. Additional strengths include our analytic sample consisting of a diverse cohort of community-dwelling older adults without dementia and our focus on stroke risk, not prevalent or incident disease as related to neighborhood context and cognition. Additionally, we employed sophisticated statistical methodology and publicly available data on neighborhood-level health to understand the interplay between neighborhood- and individual-level health on cognition in older adults. Nonetheless, our work should be considered within the context of its limitations.Noted limitations of our work include the cross-sectional nature of this study which does not allow for an understanding of causality. While our requirement that participants provide an address that denoted at least a five-year duration of exposure to their neighborhood environment does not counter our cross-sectional design, when combined with our findings that individuals are likely to live in socioeconomically similar regions throughout their life ,32, the This study demonstrated that higher amounts of neighborhood-level crime negatively associated with stroke risk and, in turn, this stroke risk associated with attention and information processing. While work is ongoing to clarify the role of socioeconomic resources, more specifically neighborhood-level educational attainment, on individual-level physical and cognitive health outcomes, our findings with neighborhood-level crime have clinical practice implications. Specifically, the neighborhood represents an important context that should be considered by clinicians as part of the diagnostic interview and case conceptualization process. While it may not be common for clinicians to specifically ask about the neighborhood in which their patients live, doing so may provide a wealth of information about daily, chronic stressors that have implications for symptom presentation and possibly even long-term cognition.


Laser induced maculopathy includes retinal photoreceptor disruption, macular hole, macular hemorrhage, and rarely choroidal neovascularization (CNV). Here we report a case of laser induced CNV that was treated by intravitreal anti-vascular endothelial growth factor (VEGF) injection and resulted in visual improvement and CNV resolution during 1-year follow up. In addition, the case of laser induced CNV treated with intravitreal anti-VEGF injections are reviewed for the first time in literature.A 7-year-old boy presented to our department with blurred vision in his right eye for 2 months. The symptom immediately happened after the boy staring at the laser beam for a few seconds. Examination of ocular fundus with slit lamp showed yellowish lesion in macula in his right eye.CNV was confirmed by fundus examinations, including color fundus photograph, spectral domain optical coherence tomography, fluorescein angiography, and spectral domain optical coherence tomography angiography.After the diagnosis of laser induced CNV, intravitreal ranibizumab  injection was performed.After 1 injection of intravitreal ranibizumab, the best corrected visual acuity improved from 20/50 to 30/50 and CNV gradually regressed during 1-year follow up.For young patients with laser induced CNV, intravitreal anti-VEGF injections may be helpful in visual improvement and CNV regression. Moreover, age seems to be a significant factor thus we propose that old animals may be more appropriate for laser induced CNV animal models of age-related macular degeneration. Laser induced maculopathy has been reported to involve retinal photoreceptor disruption, macular hole, macular hemorrhage and CNV.–6 Patients with laser induced CNV typically present with blurred vision, central scotomas, and metamorphopsia.,8 The symptoms and the prognosis largely depend on the power and wavelength of the laser and the duration of the exposure.Laser instruments have been applied in many aspects of human activity including industry, military and medicine and entertainment, which has led to more and more accidental eye injuries. Intravitreal injection of antivascular endothelial growth factor (VEGF) is another treatment because of its antiangiogenetic and anti-inflammatory function.One of the treatments for laser induced CNV is corticosteroids, which may reduce inflammation caused by the laser, however, the role of steroids remains a debated topic due to variable outcomes.We herein reported a 7-year-old boy who suffered from accidentally laser induced CNV. After 1 intravitreal injection of ranibizumab , the CNV gradually regressed during 1-year follow up. Moreover, the cases of laser induced CNV which were treated with intravitreal anti-VEGF injections are reviewed for the first time in literature and that prompts us to think further about the rationality of laser induced CNV as an animal model of neovascular age-related macular degeneration (AMD).2A 7-year-old Chinese boy presented to our hospital in October 2019 with blurred vision in the right eye (OD) for 2 months. Symptoms occurred immediately after the laser beam of a laser pointer shotting directly into his right eye for a few seconds. The laser pointer is “a class III laser product, wavelength: 650 nm, the maximum output power: less than 100mW” according to the sticker on it. The boy was found to suffer from fundus lesions by a local ophthalmologist and was advised to turn to our department for further diagnosis and treatment. The child was given full-term birth and had no significant past medical history or family history.On presentation, his best corrected visual acuity (BCVA) was 20/50 OD and 20/20 in the left eye (OS). The intraocular pressures and the anterior segments were normal in both eyes. Funduscopic examination demonstrated a yellowish-white lesion in the macula OD Fig. A. SpectrA diagnosis of laser induced CNV was made. After detailed informed consent from the patient's parents, an intravitreal injection of 0.5 mg/0.05 mL ranibizumab was performed under general anesthesia. At 1-month follow-up visit, the BCVA improved to 20/40 OD. Compared with the SD-OCTA findings before injection , which could not legally be promoted as a laser pointer. Thus, the tragedy immediately happened after the boy staring at the laser beam for a few seconds.The incidence of laser induced eye injures has been on a rise because the wide use of laser in many spheres of human activity such as industry, reported a 12-year-old boy and Tofolean IT et al reported a 31-year-old woman whose BCVA improved to 20/20 after only 1 anti-VEGF injection while another 33-year-old patient regained 20/20 after 3 intravitreal injections. Besides, the study groups of Tran K, Forshaw TRJ, Chang CY and Veronese C reported either complete resolution of CNV or visual improvement in young patients (aged between 12 and 26 years old). It appears that anti-VEGF treatment is quite useful for young patients who suffer from laser induced CNV. However, another case reported that a 55-year-old male dermatologist was diagnosed CNV secondary to laser exposure and after 2 intravitreal injections an increase in the size of the CNV with extensive arborization was observed by OCTA. Although there was only 1 case of the elderly patient with laser induced CNV whose anti-VEGF therapeutic effect was poorer than that of young patients, it should be noted that age may be an important factor of anti-VEGF therapeutic effect. CNV is 1 feature in neovascular age-related macular degeneration, which is modeled in animals by laser photocoagulation. However, researchers tend to not take the ages of animals into consideration. Espinosa-Heidmann et al have conducted a research on the impact of age on the severity of neovascularization in a mouse model of laser-induced CNV and they found that age was associated with more severe CNV, defined as larger surface area, greater vascularity, and greater cellularity. Therefore, we suspect that aged animals should be applied to duplicate animal model for AMD, although further studies are required to study the underlying molecular mechanisms on this.After diagnosis of laser induced CNV, intravitreal ranibizumab was performed. The CNV was observed to regress rapidly in the first month after injection and regress gradually during the 1 year follow up, while the BCVA improved from 20/50 to 30/50. We reviewed the published articles which reported laser induced CNV with treatment of intravitreal anti-VEGF injections. Xu K et alAlthough the BCVA of our patient restored to 30/50 after 1 anti-VEGF injection, laser exposure left a permanent eye injury on the boy. Apparently, accidental eye injury due to laser exposure has become a serious public health issue which requires more education to prevent this catastrophe.In summary, we report a 7-year-old boy with laser induced CNV whose BCVA improved from 20/50 to 30/50 1 year after an intravitreal injection of ranibizumab and CNV gradually regressed. In addition, it is the first time in literature to review the cases of laser induced CNV with intravitreal anti-VEGF injections performed and we propose to duplicate CNV animal models for AMD with old animals, which needs further studies to investigate.Conceptualization: Caixin Li, Peirong Lu.Data curation: Caixin Li, Jianqing Li, Xinzhu Chen.Funding acquisition: Peirong Lu.Investigation: Caixin Li, Jianqing Li, Xinzhu Chen.Project administration: Caixin Li, Peirong Lu.Resources: Peirong Lu.Supervision: Caixin Li, Peirong Lu.Visualization: Caixin Li, Jianqing Li.Writing – original draft: Caixin Li, Jianqing Li.Writing – review & editing: Caixin Li, Jianqing Li, Xinzhu Chen, Peirong Lu.


Antiretroviral therapy (ART) has led to dramatic improvements in survival for people living with HIV, but is unable to cure infection, or induce viral control off therapy. Designing intervention trials with novel agents with the potential to confer a period of HIV remission without ART remains a key scientific and community goal. We detail the rationale, design, and outcomes of a randomised, placebo-controlled trial of two HIV-specific long-acting broadly neutralising antibodies (bNAbs): 3BNC117-LS and 10-1074-LS, which target CD4 binding site and V3 loop respectively, on post-treatment viral control.n = 36); in arm B, participants receive placebo infusions followed by ATI. The primary endpoint will be time to viral rebound within 36 weeks after ATI. Upon viral rebound, the participant and researcher are unblinded. Participants in arm A recommence ART and complete the study. Participants in arm B are invited to restart ART and enroll into Stage 2 where they will receive open-label LS bNAbs, followed by a second ATI 24 weeks after. Secondary and exploratory endpoints include adverse events, time to undetectable viraemia after restarting ART, immunological markers, HIV proviral DNA, serum bNAb concentrations in blood, bNAb resistance at viral rebound, and quality of life measures.RIO is a randomised, placebo-controlled, double-blinded prospective phase II study. Eligible individuals will have started ART within 3 months of primary HIV infection and have viral sequences that appear to be sensitive to both bNAbs. It will randomise 72 eligible participants 1:1 to the following arms via a two-stage design. In Stage 1, arm A participants are given dual long-acting (LS-variants) bNAbs infusions, followed by intensively monitored Analytical Treatment Interruption (ATI) (The two-stage design was determined in collaboration with community involvement. This design allows all participants the option to receive bNAbs. It also tests the hypothesis that bNAbs may drive sustained HIV control beyond the duration of detectable bNAb concentrations. Community representatives were involved at all stages. This included the two-stage design, discussion on the criteria to restart ART, frequency of monitoring visits off ART, and reducing the risk of onward transmission to HIV-negative partners. It also included responding to the challenges of COVID-19.Clinical.trials.gov and EudraCT and has approval from UK Ethics and MHRA.The protocol is registered on The online version contains supplementary material available at 10.1186/s13063-022-06151-w. The order of the items has been modified to group similar items  has dramatically improved survival for people living with HIV, preventing both disease progression and onward transmission. ART alone cannot cure HIV infection due to viral persistence in an inaccessible reservoir of latently infected cells . Also, denv protein have shown promise in addressing these issues. Neutralising antibodies prevent pathogens from infecting host cells, by blocking virus entry through disrupting virus-receptor interaction, antibody-dependent cell-mediated cytotoxicity (ADCC), and effector cell-derived soluble factors inhibiting viral spread . Mild local and systemic reactions may be observed, and participants will have a weekly medical review including regular health screening questions, for the first 8 weeks after receipt of the infusions, and subsequently twice monthly. Infusion of antibodies carries a potential risk of thromboembolism, and coagulation screening tests will be performed at screening. Individuals at increased risk of thromboembolic disease will not be eligible for inclusion in the study.In the absence of a surrogate marker of HIV cure, the only measure of post-treatment viral control is to interrupt antiretroviral therapy . There aThere is limited data on the impact of HIV on both SARS-CoV-2 acquisition; recent evidence suggests that people living with HIV may have worse outcomes and mortality of severe COVID-19 , 32. It During the trial, all participants enrolled would have received at least one dose of the SARS-CoV-2 vaccine under the national vaccination programme. They would also be offered PPE and SARS-CoV-2 screening measures as described above, to mitigate the risk of SARS-CoV-2 acquisition during the trial.This randomised phase II study will determine whether a combination of dual long-acting bNAb may result in sustained HIV suppression in the absence of ART amongst individuals with immune recovery treated since primary HIV infection.The study design allows the testing of two hypotheses, the first being that combination long-acting bNAbs will result in a sustained and quantifiable period of viral control following ATI compared with placebo amongst a specifically selected group of individuals who started ART in primary HIV infection.The second hypothesis that bNAbs may drive sustained HIV control beyond the duration of detectable bNAb concentrations, through processes mediated by the host innate immunity. This is based on evidence that HIV may be controlled in the absence of antiretroviral therapy, in individuals often termed ‘elite controllers’, and this control is mediated by highly potent HIV-1-specific CD8 T cells . In macaAt the time of writing, the first participant was consented in May 2021 and recruitment is expected to complete in 2022. The current version of the protocol is 4.0, dated 5th March 2021.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


The emergency department (ED) offers an important opportunity to identify patients with opioid use disorder (OUD) and initiate treatment. However, post-ED follow-up is challenging, and novel approaches to enhance care transitions are urgently needed. Outcomes following ED visits have traditionally focused on overdose, treatment engagement, and mortality with an absence of patient reported outcomes (PROs), for example patient ability to schedule follow-up OUD treatment appointments or pick up a prescription medication, that may better inform evaluation of treatment pathways and near-term outcomes after acute events. In the context of increasing novel secure mobile health  platforms, we explored the feasibility and acceptability of electronically collecting PROs from ED patients with non-medical opioid use to enhance care in the ED and transitions of care.ED patients with non-medical opioid use or opioid overdose who endorsed willingness and ability to complete electronic surveys after discharge were enrolled from a tertiary, urban academic ED. Participants were enrolled in an mHealth platform, shared electronic health records with researchers, and completed electronic surveys of PROs at baseline, three- and thirty-days post discharge from the hospital, including questions about ability to schedule a follow-up appointment, pick up a prescription medication and overdose risk behaviors. Primary outcomes were measures of feasibility and acceptability of electronic PRO collection among ED patients with non-medical opioid use.Among 1,808 patients assessed for eligibility between June-December 2019, 101 of 130 (78%) eligible adult patients consented to participate. Ninety-six (95%) of 101 patients completed registration in the mHealth platform, and 77/96 (80%) were successful in sharing their electronic health data. Completion rates for the baseline, three-day and thirty-day surveys were 97% (93/96), 49% (47/96) and 42% (40/96). Implementation challenges included short engagement window during ED visit, limited access to smartphones/computers, insufficient battery life of participant phone to access email and password, forgotten emails and passwords, multi-step verification processes for account set-up, and complaints about hospital care, most of which were effectively addressed by study personnel.ED patients with OUD were willing to share electronic health information and PROs, although implementation challenges were common, and more than half of participants were lost-to-follow-up after hospital discharge at 30 days. Efforts to streamline communication and remove barriers to engagement are needed to improve the collection of PROs and pathways of care in ED patients with OUD.Clinical Trial Registration ClinicalTrials.gov (NCT03985163). Date of Registration: June 10, 2019, Retrospectively registered . https://clinicaltrials.gov/ct2/show/record/NCT03985163 The US has seen rapidly increasing opioid-associated morbidity and mortality, with increased rates of fatal and non-fatal overdose and opioid-related utilization of inpatient and emergency department (ED) care –4. ED viMobile health  technology, defined by the National Institutes for Health as “the use of mobile and wireless devices to improve health outcomes, healthcare services and health research,” has significant potential in enhancing follow up among patients with non-medical opioid use and OUD . AdditioWe conducted an observational study of ED patients with non-medical opioid use. Participants were enrolled from the ED at Yale New Haven Hospital (YNHH), a 1,450-bed teaching hospital located in New Haven, Connecticut with a catchment area of 350,000 that includes a diverse ethnic and cultural mix; non-Latino white (48%), non-Latino Black (30%), Latino (18%), Other (2.5%) and Asian (< 1%) and is the most nationally representative community in terms of socioeconomics and education . The YNHThe study enrolled patients presenting to the YNHH ED for opioid overdose or those screening positive for OUD or non-medical opioid use, based on the NIDA Quick Screen, a brief 30-day substance use screener . EligiblAfter providing informed consent, participants enrolled in an mHealth platform, Hugo, and were asked to 1) access and share EHR data with the study and (2) complete electronic questionnaires at enrollment, 3-day and 30-days post discharge from the hospital accessible by an electronically delivered link by text or email. A detailed study flow is illustrated in Fig.  access aHugo is a sync-for-science platform that allows patients to access personal data from multiple health systems, share data with researchers, and complete surveys for researchers using personal email and/or mobile devices . To enroBased on patient preference, surveys were delivered by email and/or text using a secure link and could be completed on any computer or smart device. No questions were mandatory, and participants were permitted to skip questions they preferred not to answer. Surveys were considered “complete” if they had ≥ 1 answer, and participants clicked through the entire survey and received a gift card. Participants received a minimum of four email, text or phone reminders as needed and were given up to 30 days to complete each survey, after which they were considered lost to follow-up (LTFU). The baseline, 3-day and 30-day surveys included 12, 33–42 and 44–56 questions total, based on skip logic. Participants received a $10 electronic gift card for each survey completion, which was delivered automatically through a third-party vendor (Tremendous). Participants were given the option to receive an electronic gift card by text or email instantly or provide an address to have the gift card mailed. Electronic gift cards could be redeemed online only, while mailed gift cards were redeemable at brick and mortar stores.PRO selection was guided by our interdisciplinary steering committee that included technical experts and representatives from National Institute on Drug Abuse, National Library of Medicine, Centers for Disease Control and Prevention, U.S. Food and Drug Administration, U.S. Department of Health and Human Services, academic researchers, and a community member with personal experience with OUD. Survey questions represent a mix of investigator generated questions designed to collect outcomes that could inform ED quality improvement for which validated surveys were not available (i.e. difficulty scheduling a follow-up appointment or filling a medication) and validated structured instruments designed to collect PROs (PROMIS surveys). The baseline survey consisted of demographics, DSM-5 criteria for OUD, and history of overdose and treatment for OUD, while the 3-day and 30-day surveys included recent treatment and prescription history, an overdose risk behavior survey as well as standardized patient reported outcomes including PROMIS Global-10, PROMIS Severity of Substance Use (Past 30 days) and Treatment Effectiveness Assessment (TEA) . The PROAs EDs often have staff responsible for making follow-up patient calls regarding late resulting tests  referrals, or patient satisfaction, this step was designed to test the feasibility of a pragmatic strategy to integrate PRO feedback to enhance patient outcomes using an existing resource at many EDs. Following the 3-day questionnaire only, a phone call was triggered if the patient reported challenges picking up a prescription or scheduling a follow-up appointment for OUD treatment. During the phone call, a trained RA provided information and resources about OUD treatment, as appropriate. Challenges were captured qualitatively based on interviews and feedback from study RAs.Our feasibility outcomes included the proportion of eligible patients who were willing to enroll in the study overall and the ability to link patient’s EHR to the mHealth platform. Acceptability outcomes include response rates to the baseline, 3-day and 30-day surveys and the ability to reach patients by telephone when a PRO triggered a follow-up telephone call.Screening, enrollment, and follow-up data were recorded by study RAs in case report forms using the Qualtrics XM Platform. Survey data were collected and stored by Hugo and were extracted and transferred via the Hugo platform by researchers. At the end of the study, Hugo staff shared a comprehensive analytic dataset including EHR data for all participants via secure transfer. Using a unique identifier to link participants, all data were merged in Microsoft Excel and analysis was performed using SAS version 9.4. For analysis purposes, “completers” included participants completing two or more surveys and “non-completers” completed no surveys or baseline only, as nearly all baseline surveys (97%) were completed during enrollment in the ED and do not accurately capture engagement post-discharge from the hospital. As a pilot feasibility study, no power analyses were used.As depicted in the CONSORT diagram Fig. , 1,808 pNinety-six out of 101 patients registered an account with Hugo, of which 80% (n = 77) shared electronic health data. Among patients registered with Hugo, 81 opted to receive surveys by email only, and 15 opted to receive surveys by email and text. Reasons for failed Hugo registration included technological challenges  (n = 4) and time constraints (n = 1). Reasons for failed EHR linkage among 15 Hugo users included challenges activating an account , difficulty accessing existing account (account locks after three failed login attempts), and trouble linking MyChart with Hugo (password or connectivity issues with Hugo). Forty-four (44%) patients needed help establishing an email account prior to registering with Hugo, 35 (35%) needed help accessing their existing email account, 77 (77%) patients needed assistance creating an account in the online EHR portal, MyChart, and 14 (14%) needed help accessing their existing MyChart account. Compared to participants who did not require assistance creating an email account, those who required assistance were significantly less likely to complete the 3-day and 30-day follow-up survey , 49% (47/96) and 42% (40/96), and details about survey completion are described in Table In this pilot study utilizing a novel mHealth technology within the unique ED setting, we found that patients with non-medical opioid use were willing to share electronic health information and provide PRO data related to non-medical opioid use and the navigation of post-ED care. We found that collecting PROs through a mHealth platform was feasible and acceptable to ED patients with non-medical opioid use. Several key implementation challenges were identified, but notably, despite public and media attention to data security, patient concern about linking electronic health data across platforms was not a prominent challenge or noted limitation. Patients were willing to use mHealth technology to provide PROs relating to details of ED visit and SUD referral, ability to pick up prescribed medications, ability to schedule follow-up appointments and additional measures relating to quality of life and substance use. Additionally, ED patients with non-medical opioid use may be more amenable to providing PRO data related to ED care and to identify assistance needed with transitions of care when collected through a mHealth platform due to reduced stigma. Therefore, collection of PROs through an mHealth platform in ED patients with non-medical opioid use offers a potential strategy to gather patient-focused data and to inform a feedback loop to help facilitate assistance with prescription medications or follow-up appointments that would be feasible within usual ED clinical workflows and outside of a research setting.Overall, 95% of patients were able to complete their registration in the mHealth platform, and 80% were successful in sharing their electronic health data. Several challenges were identified within this pilot, including logistical, technical and patient-specific barriers, most of which have been identified in other non ED-based mHealth PRO surveys . We idenImportantly, lack of access to telephone for the vast majority of ED patients with non-medical opioid use was not a barrier to participation, which is consistent with a prior study finding that an estimated 95% of patients in our ED report having cell phone access, and that smartphone access remains high among patients with substance use disorders . ConsistCompletion rates for the baseline, three-day and thirty-day surveys in our study were 97%, 49%, and 42% respectively. While the three-day and thirty-day response rates are low, they are not entirely inconsistent with other ED and hospital-based follow-up surveys. One survey of patients admitted with acute traumatic injury found that 19/26 participants completed the survey with a three month follow-up rate of 38% . AnotherIn future studies follow-up rates may be enhanced without significantly increasing follow-up attempts by “testing” participant follow-up phone numbers in the ED, collecting participants preferred time of day for follow-up calls and by collecting patients preferred social media method of contact if applicable (e.g. Facebook). Beyond the frequency of follow-up attempts, prior work has suggested that multi-modal approaches to follow-up may be more successful and implementation of PRO collection within this study may have also resulted in greater follow-up at the risk of limiting generalizability given our goal of evaluating feasibility of integration into a standard clinical workflow .There are several limitations associated with this study. Participant concerns around the feasibility and acceptability of sharing EHR and PROs may be mitigated by the presence of in-person RAs or by incentive for study compensation. Individuals who were willing to participate in the study may not be generalizable to all ED patients with non-medical opioid use. Additionally, the development of an effective collaboration with our emergency psychiatry colleagues designed to enhance enrollment of ED patients who had been evaluated and cleared by psychiatry prior to discharge led to an overrepresentation of patients who were enrolled after a psychiatric evaluation, as patients were in the ED longer and therefore easier to capture for enrollment.ED patients with OUD were willing to share electronic health information and PROs, although implementation challenges were common, and about half of participants were lost-to-follow-up within 30 days of hospital discharge within a real-world, mHealth application with modest follow-up effort. Future work to streamline integration and remove barriers to engagement is needed to improve the collection of PROs and to advance patient centered care initiatives for patients with OUD.


Immunotherapy involving immune checkpoint inhibitors (ICIs) for enhancing immune system activation is promising for tumor management. However, the patients’ responses to ICIs are different. Here, we applied a non-negative matrix factorization algorithm to establish a robust immune molecular classification system for colorectal cancer (CRC). We obtained data of 1503 CRC patients . In the training cohort, 42.8% of patients who exhibited significantly higher immunocyte infiltration and enrichment of immune response-associated signatures were subdivided into immune classes. Within the immune class, 53.1% of patients were associated with a worse overall prognosis and belonged to the immune-suppressed subclass, characterized by the activation of stroma-related signatures, genes, immune-suppressive cells, and signaling. The remaining immune class patients belonged to the immune-activated subclass, which was associated with a better prognosis and response to anti-PD-1 therapy. Immune-related subtypes were associated with different copy number alterations, tumor-infiltrating lymphocyte enrichment, PD-1/PD-L1 expression, mutation landscape, and cancer stemness. These results were validated in patients with microsatellite instable CRC. We described a novel immune-related class of CRC, which may be used for selecting candidate patients with CRC for immunotherapy and tailoring optimal immunotherapeutic treatment. Early screening, surgical procedure advancements, and multidisciplinary systemic therapies have led to a decline in CRC mortality rates, and have helped achieve a 5-year survival rate of 90% in early-stage patients5. However, the 5-year survival rate is less than 15% among patients diagnosed with metastasis, and 25% of patients newly diagnosed with CRC are expected to present with metastases at the time of diagnosis7. Moreover, the incidence of early-onset CRC, which is defined as CRC occurring in individuals < 50 years of age, continues to increase considerably9. CRC is highly heterogeneous, resulting in varied prognoses even among patients exhibiting the same tumor stage, which poses challenges in the clinical management of the patients. The heterogeneous nature of CRC may be associated with differences in genetic mutations, the tumor microenvironment (TME), gut microbiota profile, and metabolism characteristics12. Therefore, the classification of CRC at the molecular level is urgently needed to improve risk stratification and adopt precision therapies for CRC prevention and management.Colorectal cancer (CRC) is the third most common malignancy reported worldwide and is the second leading cause of cancer-related deaths16. Application of monoclonal antibodies to inhibit immune checkpoints has resulted in the achievement of promising and durable responses in multiple cancers20. However, immune checkpoint inhibitors (ICIs) have shown effectiveness in only a few patients with CRC, and only those with the microsatellite instability (MSI) phenotype but not those with the microsatellite-stable (MSS) phenotype respond to PD-1 blockade 22. The MSI phenotype is represented by enrichment of tumor-infiltrating lymphocytes (TILs) and production of neoantigens, which facilitate immune activation. Moreover, accumulating evidence has shown that tumor mutation burden (TMB), PD-1/PD-L1 expression, and TILs correlate with ICI response25. However, a lack of thorough understanding of TME complexity has hindered the application of anti-tumor immune therapies. For example, immunotherapies that repress tumor-associated macrophages have been applied in the clinical setting, but minimal response has been observed with monotherapy26. Therefore, comprehensive profiling of the immune molecular characteristics of CRC is warranted.Tumor evolution is a consequence of complex interactions between tumor cells and the TME, including immunocytes and stromal cells. Immunotherapy has transformed the traditional paradigm of clinical cancer therapy and exhibits potent anti-tumor efficacy in various cancer types27, interpretation of multimodal data28, and omics analyses29. NMF can be used in the identification of cancer molecular subtypes based on gene expression profiles through the identification of exemplar genes33. In this study, we used NMF to virtually microdissect the molecular patterns that are closely associated with immune activation in CRC and validated the resulting molecular classification in three independent cohorts.Owing to technical advancements, several computational methods are available for dissecting tumor heterogeneity. Non-negative matrix factorization (NMF) analysis is a virtual microdissection approach that has been widely applied in numerous research fields, including image representationhttp://xena.ucsc.edu/) and used as the training cohort. Only patients diagnosed with adenocarcinoma (n = 488) were selected for the subsequent analysis. Three additional CRC datasets with the required clinical information were obtained from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) and used as an external validation cohort. The three cohorts—GSE39582, GSE14333, and GSE17538—comprised 1015 patients.A total of 1503 patients with CRC, whose gene expression profiles and clinicopathological characteristics were available, were selected; the relevant data were obtained from public databases. The Cancer Genome Atlas-Colon Cancer (TCGA-COAD) and The Cancer Genome Atlas-Rectal Cancer (TCGA-READ) datasets were downloaded from UCSC XENA (We used the NMF package (v0.23.0) to perform microdissection of the mRNA expression profiles in the TCGA-COAD and TCGA-READ datasets. After testing, it was found that K = 10 was the number of factors that could be applied for good segmentation of TCGA data. To extract data on immune-related NMF factors, ESTIMATE was used to analyze the immune enrichment score in TCGA samples. Statistical analysis revealed that among all NMF factor groups, patients whose data were decomposed into the seventh factor had a higher immune enrichment score; hence, the seventh factor was defined as an “immune factor.”According to the difference between the load value of the seventh factor and the maximum load of other factors, all genes were sorted from high to low, and the genes with the top 150 weights were considered to be the key factors distinguishing immune and non-immune classes. This classification was achieved through the utilization of the NMF Consensus command of the NMF package with top 150 genes, which was further refined using the multidimensional scaling (MDS) random forest method of the randomForest (v4.6-16) package. The clusterProfiler (v3.14.3) package was used to analyze the function of the 150 immune-related genes.padj < 0.05 and absolute value of log2 fold change (FC) > 1. To determine the gene sets and pathways enriched in the immune and non-immune groups, the clusterProfiler (v3.14.3) package was used to conduct Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway functional enrichment analysis of the differentially expressed genes (DEGs); simultaneously, all genes were enriched through GSEA using the fgsea (v1.12.0) package.Nearest template prediction (NTP)  and the single-sample Gene Set Enrichment Analysis (ssGSEA) method in the Gene Set Variation Analysis (GSVA) (v1.34.0) package were used to evaluate gene expression signatures and enrichment of molecular pathways that represented different inflammatory states or immune cells. Through NTP of stroma activation, the immune class data were further bifurcated into immune-suppressed and immune-activated subtypes. DESeq2 software was used to select genes that were significantly differentially expressed between the immune and non-immune groups with We screened the top 150 dysregulated genes between the immune and non-immune classes. Similar to the training cohort, the NMF and the ESITIMAT algorithms were adopted to separate the validation cohort samples into immune and non-immune classes based on the expression of the selected 150 genes. Furthermore, NTP was used to further divide the immune group into immune-suppressed and immune-activated subtypes.To predict the responses to ICI therapy in patients with CRC, tumor immune dysfunction and exclusion (TIDE) and SubMap analysis (GenePattern module “SubMap”) were used. SubMap is an algorithm used to assess similarities in gene expression between previously defined immunophenotypes and responders or non-responders to anti-PD-1 treatments in the MD Anderson melanoma cohort.https://gdac.broadinstitute.org). A statistical comparison was then conducted to determine the difference between the immune and non-immune groups in amplification or deletion events at the arm and focal levels. Previously, Rooney et al. calculated the neoantigen number of TCGA tumors, from which the neoantigen number of TCGA-COAD and TCGA-READ could be obtained34. Furthermore, Saltz et al. estimated the abundance of TILs based on the images of hematoxylin and eosin (H&E)-stained sections of 13 TCGA tumor types, which included data on CRC35. Mutation data were collected from TCGA (https://tcga-data.nci.nih.gov), and the TMB was evaluated using the maftools (v2.6.05) package. To obtain data on the significantly mutated cancer genes (p < 0.01), we used the MutSigCV (v1.41) package to analyze the mutation data. Independent tests were then performed to further determine the mutations with significant differences among the different groups (p < 0.05). Finally, the mutation landscape oncoprint was generated using maftools. The mRNA stemness index (mRNAsi) was analyzed through one-class logistic regression, which can help predict stemness in poorly differentiated tumors.Copy number alterations (CNAs) were calculated using GISTIC2.0 from GDAC Firehose . Kaplan–Meier analysis and the log-rank test were conducted to compare patients’ survival between different immunophenotypes.Unless otherwise noted, all analyses were conducted using R software (v3.6.1), and the significance level was set at 36 generated using the ESTIMATE algorithm, the seventh of the 10 expression patterns (NMF factors) was found to be enriched in patients with high immune enrichment scores and exhibited a significantly higher immune enrichment score average than most of the remaining patterns  of solid tumors by conducting an extensive immunogenomic analysis of over 10,000 tumors derived from 33 distinct cancer types. We observed a significant enrichment of C2 , and a significant decrease in C1 , in the immune class compared with those in the non-immune class  and a significant decrease in CMS2  in the immune class compared with those in the non-immune class  of the patients in the immune class that lacked stromal enrichment, whereas the remaining 53.1% (111/209) had a relatively higher stromal enrichment scores , TGF-β response signatures of T cells (T cells-TBRs), late TGF-β, and extracellular matrix cytokines (C-ECM) were all significantly enriched in the stromal-activated class than those observed in the remainder of the immune class  cells, MDSCs, and macrophages signatures were significantly upregulated in the immune-suppressed subtype , and myeloid-derived suppressor cells (MDSCs) are regarded as immune-suppressive cellsTo validate the recurrence of our established immune-related subtypes, we performed NMF analysis and activated stromal signature-based separation in three CRC cohorts  and disease-free survival (DFS) outcomes, the immune-suppressed subclass showed the poorest prognosis, and the non-immune class exhibited moderate prognosis outcomes in both training and validation cohorts Fig. a. We per45, which is an effective computational method for predicting response to ICIs, was also used to evaluate the ability of our molecular classification to predict ICI response. We observed that the ratio of ICI response in the immune-activated subclass was higher than that in the other two groups, whereas the immune-suppressed subclass showed the worst ICI responses  patients were allocated to the immune-activated subclass, which exhibited higher immune cell infiltration and immune activation signaling, implying these patients were potential responders to ICI therapy Fig. . We reca49. However, the immune landscape in CRC remains poorly understood. A thorough investigation of different immune and stromal components in the TME may help understand complex tumor heterogeneity, develop more precise therapies, and refine current immunotherapeutic strategies. Particularly, establishment of an immune-related classification may provide data on more accurate subtypes of patients with CRC to help clinicians select more effective therapies. In this study, we presented a comprehensive immune-related classification of CRC based on the NMF algorithm according to CRC expression profiles. Using the top 150 exemplar genes of the immune pattern for consensus clustering, three immune-related molecular groups were identified, namely immune-activated, immune-suppressed, and non-immune. Molecular classification associated with different immune signaling pathways and immune cell types was established and validated in three independent cohorts. Notably, the immune-related classes correlated with patient prognosis and response to ICI therapy, and this finding might be used to tailor clinical immunotherapy strategies for patients with CRC.Accumulating evidence has demonstrated the essential role of the TME in cancer progression50. Emerging evidence has suggested NMF to be a promising method for classifying tumor molecular subtypes51. Using the NMF algorithm, we identified an immune pattern in the training cohort. Through consensus clustering of the exemplar genes of that immune pattern, CRC could be distinguished as immune and non-immune classes. Of the 488 patients in the training cohort, 42.8% were grouped under the immune class, which showed higher enrichment of infiltrating immune cells, cytolytic activity, and IFN signaling than the non-immune class. Effective immunotherapy is often limited by the immunosuppressive TME, which includes immunosuppressive cells and cytokines. Calon et al.52 revealed a pro-metastatic program induced upon secretion of IL11 by TGF-β-stimulated CAFs in the tumor niche. Accumulating evidence has suggested that the C-ECM level is upregulated by activated CAFs to trigger the recruitment of immunosuppressive cells53. To investigate the different components in the CRC TME, we further dissected the gene expression profiles of the immune class and divided them into immune-activated and immune-suppressed subclasses based on a stromal-activated signature calculated using the NTP method. Interestingly, 46.9% of the patients in the immune class were assigned to the immune-activated subclasses, which showed lower enrichment of the stromal-related signatures and immunosuppressive cells—such as TGF-β, C-ECM, PD-1, Treg, and MDSC signatures—than that observed in the immune-suppressed subclass. This immune molecular classification was also validated in three independent cohorts. For example, the immune-activated subclass accounted for 40.5% of the patients, whereas the immune-suppressed subclass accounted for 59.5% of the patients in the immune class of the GSE17538 cohort. These results indicate that NMF may be used as a helpful classifier for stratifying immune molecular subtypes of patients with CRC.The NMF algorithm, a multiplicative update algorithm, can be used to dissect substantial volumes of data using a limited number of components54. As a smaller number of patients respond to ICI therapy, identification of effective biomarkers for predicting patient suitability to immunotherapy is warranted. Recently, emerged immunological and non-invasive methods, such as liquid biopsy using peripheral T cell receptor repertoire of PD-1+ CD8+ lymphocytes, provide a non-invasive approach for selecting patients with metastatic CRC who could benefit from ICI treatment55. Meanwhile, with the advances in computational methods, such as SubMap and TIDE, a novel algorithm was established to predict ICI therapy response. SubMap analysis is conducted using an algorithm to evaluate the similarity in gene expression profiles between patients in different molecular subtypes and in patients with metastatic melanoma treated with ICIs56. Notably, here, the results of the SubMap analysis showed that patients in the immune-activated subtypes exhibited similar gene expression profiles to those of patients with melanoma who responded to anti-PD-1 immunotherapy, which suggested that patients in the immune-activated subclass could respond to PD-1. Moreover, survival analysis shows that patients in the immune class, especially in the immune-activated subclass, presented with better OS and DFS. These results may have clinical implications in terms of formulating prognosis and treatment decisions.Currently, treatment paradigms have been shifting from targeting the tumor cell compartment to focusing on the development of strategies for regulating the TME57. Lower CNAs were linked to the elevated infiltration of immunocytes and cytokines, which implied a higher likelihood of successful ICI therapy58. We analyzed the relationship between the established molecular subtypes and genetic features. We observed that CNA amplification was significantly lower in the immune class than that in the non-immune class. The levels of other canonical biomarkers that could help predict response to ICI, such as TIL and PD-1/PD-L1 expression, were significantly higher in the immune class, which confirmed patients in the immune class could potentially benefit from immunotherapy.Genomic studies have revealed that CNAs, TMB, and neoantigens demonstrate a predictive value for ascertaining susceptibility to immunotherapiesHerein, we proposed a novel immune molecular classifier based on gene expression profiles analyzed using the NMF algorithm. Patients in the immune-activated subclass present with favorable prognosis and may respond positively to anti-PD-1 immunotherapy. This study provides new insights into tumor heterogeneity using an integrated and multifaceted approach to enhance the discovery of clinically important molecular subtypes that respond to immunotherapy.Supplementary Information.