Gaucher disease (GD) is an autosomal recessive inborn error of metabolism, belonging to the group of lysosomal storage diseases (LSDs). GD is caused by a defect in lysosomal glucocerebrosidase, responsible for glucosylceramide breakdown into glucose and ceramide. Because of this dysfunction, glucosylceramide progressively accumulates in the liver, spleen, bone marrow, bones, and in other tissues and organs, also causing anemia, hepatosplenomegaly, thrombocytopenia, and bone symptoms. Depending on neurological symptoms, GD is classified into three main types. Treatment options for LSDs, including enzyme replacement therapy, hematopoietic stem cell transplantation, small molecular weight pharmacologic chaperones, and, for some LSDs, gene therapy, are increasingly available. For this reason, many efforts are aimed at implementing newborn screening for LSDs since early detection accompanied by a prompt intervention has been demonstrated to be essential for reducing morbidity and mortality and for improved clinical outcomes. Herein, we report two siblings of preschool age, presenting with hepatosplenomegaly and thrombocytopenia. The initial suspicion of GD based on the clinical picture was further supported by biochemical confirmation, through newborn screening workflow, including first- and second-level testing on the same dried blood spot samples, and finally by molecular testing. Gaucher disease (GD) is a lysosomal storage disease (LSD) affecting the recycling of cellular glycolipids. Glucocerebroside is ordinarily degraded to glucose and lipid components, while it accumulates within the lysosomes of cells in GD. The incidence of GD in the general population ranges between 0.4 and 5.8 per 100,000 births, with a prevalence of 0.7 to 1.8 per 100,000  and 1 inDespite attempts to categorize the three types of GD, they comprise a continuum from severely affected collodion babies through those with acute and chronic neuronopathic forms, to patients with non-neuronopathic GD manifesting with skeletal and visceral involvement or Parkinson disease, to older adults with mild or no clinical manifestations ,7.GD results from deficiency of lysosomal glucocerebrosidase  ,7, a glyThe deficiency of GBA leads to the accumulation of glucocerebroside and other glycolipids within the lysosomes of macrophages . The tisMacrophages stuffed with glycolipids accumulate in many tissues, including those of the liver, spleen, bone, and bone marrow. However, pathologic lipid accumulation in macrophages accounts for less than 2 percent of the additional tissue mass in the liver and spleen . The infThe pathologic processes also occurring within bone are different: decreased mineral density, marrow infiltration, and infarction of bone. Although uncertain, the mechanisms leading to decreased bone mineral density may involve a failure to achieve peak bone mass, abnormal osteoclast regulation, or overproduction of cytokines by activated macrophages . In an iWe describe the presenting features, diagnostic approach, and initial response to therapy in two siblings with GD. In particular, the present study reports on the familial case of two siblings of preschool age presenting with hepatosplenomegaly and cytopenia, leading to referral of case 1 for suspected acute leukemia. After ruling out leukemia, GD became the clinical diagnosis, to be supported by biochemical confirmation, through newborn screening workflow, revealing the significantly low glucocerebrosidase activity and the strong accumulation of glucosyl-sphingosine on the same dried blood spot samples, and finally by molecular testing.Healthy parents of Moroccan origin, reporting themselves as unrelated; the mother was gravida 5, para 5. No evidence of familial diseases.The index case is a 22-month male, born at term after a regular pregnancy, with an uneventful neonatal period. He did well until he was 20 months old, when abdominal distension was noted, followed by progressive asthenia. Thus, he was first seen at the local hospital where severe anemia was documented. The child was referred under the suspicion of acute leukemia.On admission, he was pale, with normal skin and no evidence of hemorrhagic lesions. He had a weight of 11 Kg (25–50th centile); length of 84 cm (10–25th centile); and head circumference of 43 cm (<5th centile). His respiratory activity was normal, while the abdomen was prominent with marked hepatosplenomegaly. No lymphadenomegaly and no bone pain were apparent. The child was barely able to walk, but his neurological examination was normal. The main presenting features are summarized in In order to rule out leukemia or other hemo-proliferative disorders, bone marrow aspirate was performed: while leukemic metaplasia was not present, large macrophages stuffed with storage material were detected at morphological examination .Thus, within a couple of hours, leukemia was ruled out and the diagnosis of possible GD was made; confirmation by biochemical study was requested on the same day. Following confirmed diagnosis in the index case, we asked detailed information of the remaining four siblings. The older, 41-month sister, (case 2) was reported as well doing. Yet, on physical examination, she was pale, with normal skin and no evidence of hemorrhagic lesions. She had a weight of 14.9 kg (54th centile); height of 92 cm (11th centile); and head circumference of 50 cm (78th centile). Her respiratory activity was normal, while the abdomen was prominent with hepatosplenomegaly. No lymphadenomegaly, no bone pain, and normal neurological examination were reported. Her main presenting features are summarized in Newborn screening (NBS) for LSDs is performed as a first-tier test by measurement of lysosomal enzymatic activities in dried blood spot (DBS) samples. More precisely, there are two currently available methodologies used for determination of enzymatic activities: tandem mass spectrometry (MS/MS) and digital microfluidics fluorimetry (DMF-F). Multiplexing of the LSD enzymes are guaranteed by both platforms; the MS/MS has a higher analytical precision than DMF-F . DBS samHerein, the entire diagnostic workflow for NBS was applied on the DBS samples from pediatric patients with clinical diagnosis of GD.Case 1. First DBS sample, collected at 22 months of age, underwent primary screening, revealing low GBA activity . Through workflow, reduced enzymatic activity indicated second-tier testing on DBS, which confirmed the clinical diagnosis of GD with the specific biomarker LysoGb1 = 662.63 nM (cut-off < 31.1 nM). Case 2. First DBS sample from the older sister, collected at 41 months of age, underwent primary screening, and showed low GBA activity . Accordingly, the second-tier testing on DBS confirmed the clinical diagnosis of GD with LysoGb1 = 616.49 (cut-off < 31.1 nM). A familial study was carried out in all available members  for the phenotypic profile by quantifying both GBA activity and LysoGb1 levels, which exhibited normal values. GBA1, associated with GD (GBA1 gene (NM_ 000157.4) evidenced the pathogenic variant c.1448T>C (p.Leu483Pro). The c.1448T>C variant causes the thymine nucleotide to be changed into a Cytosine nucleotide at position 1448 in exon 10 of the GBA1 gene, causing the substitution of the amino acid Leucine by the amino acid Proline at codon 483 of the encoded protein (p.Leu483Pro). The molecular testing revealed the pathogenic familial mutation on both alleles for the symptomatic siblings. The familial mutation was found in monoallelic (heterozygous) form in both asymptomatic parents and the two older siblings, aged 18 and 13 years, respectively, also asymptomatic. One asymptomatic older sibling was not available for the diagnostic study. Sanger sequencing is described in full detail in the GBA1 gene in both GD cases as well as in the healthy carrier collaterals.The entire family was invited for mutation analysis of the gene  with GD . Sanger Following confirmation of the diagnosis of GD for both siblings, disease staging for assessment of possible bone lesions and CNS involvement was carried out with negative results. Based on the combination of clinical features, i.e., hepatosplenomegaly and cytopenia, with no evidence of bone or CNS involvement, these children were recognized as belonging to Type 1 , the non-neuronopathic variant, with prevalent involvement of the liver, spleen, bone, and hematological system.The enzyme replacement therapy (ERT) by i.v. imiglucerase (72 U/kg every other week) was started for the little boy (case 1). Soon thereafter, ERT by i.v. imiglucerase (66 U/kg every other week) was also started for his older sister (case 2). Treatment was very well tolerated and is still ongoing, after 12 and 7 doses administered, respectively, both at the dose of 60 U/kg. Clinical response was soon evident in both kids, with progressive reduction of the abdominal circumference and regression of cytopenia. The parents reported that the young boy (case 1), who was barely able to stand and walk without help at the time of the diagnosis, progressively improved his motor ability up to being able to run. Both patients had a reduction of liver and spleen size during ERT: liver diameter reduced in case 1 from 13 to 9.5 cm and in case 2 from 11 to 9 cm. Spleen size reduced as well: case 1 had a spleen length of 16 cm at the diagnosis, while at last follow-up the spleen dimensions were 11 × 3.7 × 3.5 cm; case 2 had an initial spleen length of 15 cm, while at last follow-up the spleen dimensions were 11 × 4.2 × 2.8 cm.p-value = 0.0170) and case 2  were found. In consideration of the presenting features at diagnosis and the improvement in clinical evidence during ERT for case 1, the correlation between the abdominal circumference and LysoGb1 levels was also investigated, showing a strong correlation between the two factors   in case 1  . DetailsThe present study describes a sibling pair of preschool age, diagnosed with GD based on the clinical manifestation of splenomegaly and cytopenia. Bone marrow aspiration, performed to rule out possible acute leukemia, clearly showed at morphological evaluation typical storage cells, which oriented the diagnosis. Children with splenomegaly and cytopenia should be rapidly addressed with a diagnostic work-up for GD. In the recent GAU-PED study, Pession et al. report that 14 out of 154 patients considered at high risk for GD because of cytopenia and organomegaly were diagnosed with confirmed GD . Based on the combination of clinical features, i.e., hepatosplenomegaly and cytopenia, with no evidence of bone or CNS involvement, these children were recognized as belonging to Type 1 , the non-neuronopathic variant, with prevalent involvement of the liver, spleen, bone, and hematological system.GBA gene on chromosome 1q21 [Interestingly, the parents did not realize that the slightly older sister was also affected, despite a very similar phenotype, so that the diagnosis was made only upon our specific request to check the remaining family members. As described above, GD is an autosomal recessive inborn error of metabolism resulting from biallelic mutations in the ome 1q21  and causome 1q21 . This lyome 1q21 ,19. Accuome 1q21 . Thus, following clinical diagnosis, the non-routinary biochemical work-up through the NBS workflow was started to document the defect in GBA activity and then confirmation was achieved by mutation analysis . Even thAfter definitive diagnosis of GD1, and after both of the siblings had started ERT, a follow-up was conducted by closely monitoring different clinical and biochemical parameters, as a multiparameter evaluation of the response to therapy. The careful follow-up highlighted the potential value of LysoGb1: indeed, LysoGb1 level decrease was well correlated with clinical improvement during ERT. This evidence suggests that DBS-based LysoGb1 determination is a highly valid and noninvasive approach for therapeutic monitoring of GD patients, of importance in the evaluation of ERT efficacy ,25,26.While LysoGb1 as a biomarker of GD disease has already been recognized, we have shown here that its values is well correlated with the improvement of clinical parameters during treatment. Above all, we have shown that it is both possible and convenient to monitor LysoGb1 via the analysis of DBS rather than venipuncture, a much-preferred option by patients, especially those of pediatric age .Through family history we investigated possible parental consanguinity, which was strongly denied. Yet, careful evaluation of their geographical origin showed that they originated from little villages sharing an obligate route, which suggested a likely common origin. Haplotype analysis confirmed that the parents not only share the same pathogenic mutation, but also share the entire haplotype, thus confirming their very likely consanguinity.It is well recognized that GD is one of the most prevalent of the LSDs, a large group of inherited metabolic diseases that globally present a combined frequency of ~1:5000 live births, even if the real prevalence might be higher than estimated when considering undiagnosed patients . MoreoveThe c.1448T>C variant found in this family is one of the four most common pathogenic variants for GD accounting for about 90% of the pathogenic variants; the frequency of this homozygous genotype in the affected patients’ population is 6% .GBA gene and its closely linked pseudogene, were found. Based on their findings, mutation 1226 was associated with a mild clinical phenotype, while mutation 1448 was associated with a more severe phenotype. In the present case, homozygosity for the so-called 1448 mutation, now defined as c.1448T>C (p.Leu483Pro), confirms its pathogenic role in causing a “chronic” phenotype of GD1, which may be included in type B, with no evidence of bone or CNS involvement during the first two or three years of life [In their pioneer genotype–phenotype study of GD1, Zimran et al. had already detected four mutations in the DNA of 47 unrelated patients with type GD1 . Two of  of life . This va of life . In addi of life ,35,36,37 of life .In a recent report on the outcome of ERT (60 U/kg imiglucerase every other week) in a 2-year-old child with GD at long term follow-up , hemogloIn a recent report of a telephone survey on 124 patients with GD1 who received GD1-specific therapy, 40% of patients reported that GD restricted their education/job and fun activities and were concerned about being emotional and financial burdens on others. Concerns regarding the risk of bone disease and Parkinson disease were also high (60%). Adolescent and adult patients with GD1 may have personal problems and concerns not captured by traditional outcome parameters but are potentially affecting their quality of life . To thisThe present study described the cases of two pre-school aged siblings, presenting with hepatosplenomegaly and cytopenia. The initial clinical picture addressed the suspicion of GD, further supported by biochemical confirmation, through newborn screening workflow, including first- (GBA activity determination) and second-level testing (LysoGb1 quantification) on the same DBS sample, and finally by molecular testing. The pathogenic variant c.1448T>C found in this family is very common in GD, although only 6% of cases bear this variant in homozygosity. Of potential interest, we report the use of DBS samples in the workup of biochemical confirmation and follow-up. Indeed, wider availability of ERT, together with the relevance of an early intervention, raises the interest in using NBS workflow in the diagnosis and follow-up of LSDs, in pediatric as well as in adult age . The useIn conclusion, with the present clinical cases, we further support the idea of stabilizing and recognizing the use of DBS samples, which guarantee an easy self-sampling and noninvasive sample collection procedure ,25,27, t


IntroductionMalignant melanoma (MM) is potentially a fatal type of skin cancer and a major health concern for the Caucasian population. It is a heterogeneous disease with a wide spectrum of manifestations. Therefore, in this study, we evaluated the clinicopathological characteristics of MM.MethodsWe retrospectively studied the clinicopathological characteristics of MM in 167 biopsy-proven cases of MM reported between January 2020 and December 2021 at Kings Mill Hospital, Sutton-in-Ashfield, United Kingdom. Clinical data such as the age, sex, and anatomical site of the lesion were obtained from the clinical referral forms. Biopsies of the lesions were performed, and the specimens collected were sent to the laboratory for histopathological study and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation evaluation. Formalin-fixed paraffin-embedded blocks (FFPE) were prepared, sectioned, and stained with hematoxylin and eosin for histological examination.Results2. The lower limb was the most common site of involvement (27.5%), followed by the thorax (25.1%). The most common histological subtype was superficial spreading melanoma (SSM) (77.8%), followed by nodular melanoma (14.4%). The in situ component was present in 95.8% of cases; a majority (92.2%) of the cases showed vertical growth phase, 71.9% of cases were at Clark’s level IV of invasion, regression was noted in 70.7% of cases, ulceration was present in 21.6% of cases, and microsatellites were present in 3% of cases. Perineural invasion was present in 3% of cases, and lymphovascular invasion (LVI) was present in 4.2% of cases. BRAF mutation testing was performed on 36 cases, out of which 20 cases (55.6%) showed BRAF mutation. Acral lentiginous melanoma and nodular melanoma were most likely to show ulceration . SSM and lentigo maligna melanoma were more likely to be associated with regression.A total of 167 cases of MM were included in the study. The age range was 23-96 years, with the median age at diagnosis found to be 66 years; males were more commonly affected (52.1%). The median Breslow thickness was 1.20 mm. The median mitotic activity was 1.0/mmConclusionThe study demonstrated that MM is prevalent among the elderly population with male predominance; SSM was found to be the most common subtype. The study further demonstrated various clinicopathological features of MM and its association with histological subtypes. Malignant melanoma (MM) is a neoplasm of melanocytic cells and most commonly affects the skin. However, it may involve mucosal surfaces such as the oral cavity, uveal tract of the eye, gastrointestinal mucosa, and leptomeninges . It is tMMs have two phases of growth: a horizontal  growth phase that only spreads through the epidermis and a vertical phase that extends into the dermis . MM is aMM displays a wide spectrum of clinicohistological manifestations. Hence, the identification and reporting of the disease is extremely important for the commencement of the specific treatment for better outcomes. Therefore, demographic details, clinical characteristics, and diagnostic measures are essential not only for histopathological reports but also for implying significant clinical concern in treatment options, which can only be achieved by regular organized studies. Therefore, this study aimed to determine the clinicopathological profile of MM and its clinical correlation in a United Kingdom (UK) population.We conducted a retrospective cross-sectional study at Kings Mill Hospital, Sutton-in-Ashfield, UK. A total of 167 cases of MM that were reported between January 2020 and December 2021 at our institute were enrolled in the study. All biopsy-proven cases of MM were included in the study. Clinicopathological data of the cases included in the study reported during the study period were retrieved from the institutional archive. Clinical data were collected from the clinical referral forms, which included the patient’s age, gender, and the site of the lesion. Cases with missing clinical data or tissue block and benign cases were excluded from the study. Institutional Review Board approval was not needed as it was a retrospective study. The clinical audit project was approved by Sherwood Forest Hospitals NHS Foundation Trust (Project Code: Path/CA/2021-22/04).Out of 167 cases, excisional biopsy was performed in 156, punch biopsy was performed in six, incisional biopsy was performed in three, and specimens were collected by the curette and shaving method in two cases. All specimens obtained were fixed with 10% neutralized formalin and were sent to the laboratory for histopathological evaluation. For histological examination, tissue blocks were prepared by removing water from the tissue by alcohol dehydration, which were then treated with xylene to clear off the alcohol from the tissue sample and then embedded in the paraffin wax at 56°C, after which they were cut into 4-5 µm sections. The sections are placed on a slide treated with L-lysine, dried, and sequentially treated with xylene, alcohol, and water, and were subsequently stained with hematoxylin and eosin, and were studied under a microscope by a senior histopathologist. The histological subtype, melanoma in situ, microsatellite lesions, ulceration, regression, tumor-infiltrating lymphocytes (TIL), growth phase, perineural invasion (PNI), lymphovascular invasion (LVI), and Clark’s level of invasion were observed. Breslow thickness, i.e., the distance between the granular layer of the epithelium and the deepest part of the tumor was measured using an ocular micrometer, and the mitotic rate was estimated by calculating the number of mitotic figures in the tumor cells within 10 high-power fields (HPF).The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation was detected by the amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) method. The 4-5 µm thick sections of formalin-fixed paraffin-embedded (FFPE) tissue were deparaffinized and treated with proteinase K at 56°C for digestion. Using the ZR Genomic DNA™ I kit , genomic DNA extraction was performed as per the guidelines. Quantification of the extracted DNA was performed using a NanoDrop 1000 Spectrophotometer V3.8 . PCR was performed in a final volume of 25 μl containing 1x buffer, 2 mM magnesium chloride (MgCl2), 400 nM primer FO (forward outer), 200 nM primer RO (reverse outer), and Fiwt, 800 nM primer Rimut, HotStarTaq DNA Polymerase  1 unit, 200 μM each deoxynucleoside triphosphate (dNTP), and 30 ng genomic DNA template. PCR amplification was performed. The product of PCR was analyzed with 2% agarose gel.Data analysisData analysis was performed using the IBM SPSS Statistics for Windows, Version 26.0 . The median for age, Breslow thickness, mitotic activity, peripheral margins, and deep margins were calculated, whereas frequencies and percentages for all other clinicopathological variables were evaluated. A p-value of < 0.05 was considered significant. Fisher’s exact tests were applied to determine the association of various clinicopathological features concerning the histological subtypes.A total of 167 cases of MM were included in the study. The median age in our study was 66±23 years , with NM (14.4%) being the second most common subtype. In our study, most cases (92.2%) demonstrated a radical growth phase, with most cases at Clark level IV of invasion, and the median mitotic activity was 1.0 mm². In most cases, an in situ lesion was present. Regression was noted in most cases (70.3%). Ulceration, microsatellite lesion, and TIL showed less frequency in our cases. Similarly, PNI and LVI were less frequently noted in our study.Panda et al. conducted a study on clinicopathological characteristics of MM in 182 cases, and similar to our study, they corroborated that MM most commonly occurs in the sixth decade of life, with males predominating . In mostBreslow thickness of the tumor is an important prognostic factor; tumors of less than 0.76 mm have the least risk for metastasis, whereas tumors 1.5 mm thick are at high risk for metastasis. Similarly, ulceration is the second most important prognostic factor; tumors with increased thickness are more likely to be ulcerated and are associated with a worse prognosis. The mitotic rate is an important predictor of survival when analyzed in the vertical growth phase . In our Furthermore, we found that SSM and ALM most commonly involved the lower limb, whereas LMM mostly occurred on the head and neck region and NM mostly involved the upper limb. The in situ lesion was more likely to be associated with SSM, LMM, and ALM. Ulceration was more likely to occur in cases of ALM and NM, and regression was noted in cases of SSM, LMM, and NM. PNI and LVI were more likely to be present in cases of NM, and TIL was found to be more likely to be present in cases of SSM. A study was conducted by El Sharouni et al. on 48,361 cases of MM ; similarBRAF mutations are reported to be present in approximately 50% of the cases of MM and are of therapeutic importance as they show a positive response to treatment with BRAF inhibitors . In our Molecular/gene expression profiling has provided new insights into the understanding of MM. Based on genomic profiling, four distinct genomic subtypes of MM are identified, these include BRAF-mutant, NF1-loss, NRAS-mutant, and triple wild-type (TWT). The later subtype (TWT) lacks the characteristic BRAF, RAS, and NF1 molecular alterations. It has also been shown that these molecular subtypes don’t correspond to histological subtypes or locations; however, acral and mucosal melanomas are more likely to be TWT . HoweverLimitationsThe study had a few limitations. The study was conducted in a single center and with a limited sample size. Moreover, as this was a retrospective study, follow-up of the patient was not determined. Also, risk factor stratification and therapeutic intervention studies were not conducted. Therefore, we propose that further prospective multicenter studies be conducted to understand the correlation of various clinicopathological characteristics with different subtypes of MM. Moreover, apart from BRAF, molecular testing for RAS and NF1 was not conducted in our study and BRAF was tested in a limited sample. In accordance with the studies done previously on MM, we found that MM was more prevalent among the elderly population and the risk of developing MM increases with increasing age, with male predominance. SSM was found to be the most common subtype. The lower limb was the most common site of occurrence. Moreover, we found that the SSM and LMM were more likely to be associated with favorable histological features (regression), whereas NM and ALM showed an association with poor histological parameters (ulceration).


Flares were observed in 10.3% of patients after the end of the vaccination cycle and 12.8% after the third dose. Being vaccinated for influenza was inversely associated with the onset of AEs after the second dose, at both univariable (p = 0.013) and multivariable analysis (p = 0.027). This result could allow identification of a predictive factor of vaccine tolerance, if confirmed in larger patient populations. A higher disease activity at baseline was not associated with a higher incidence of AEs or disease flares. Effectiveness was excellent after the second dose, with only 1/78 (1.3%) mild breakthrough infection (BI) and worsened after the third dose, with 9/47 (19.2%) BI (p < 0.002), as a probable expression of the higher capacity of the Omicron variants to escape vaccine recognition.Data on the risk of adverse events (AEs) and disease flares in autoimmune rheumatic diseases (ARDs) after the third dose of COVID-19 vaccine are scarce. The aim of this multicenter, prospective study is to analyze the clinical and immunological safety of BNT162b2 vaccine in a cohort of rheumatoid arthritis (RA) patients followed-up from the first vaccine cycle to the third dose. The vaccine showed an overall good safety profile with no patient reporting serious AEs, and a low percentage of total AEs at both doses (40/78 (51.3%) and 13/47 (27.7%) patients after the second and third dose, respectively ( At the beginning of 2021, a vaccine campaign against COronaVIrus Disease 2019 (COVID-19) started worldwide, and patients affected by autoimmune rheumatic diseases (ARDs) were included among the priority target groups ,4.Indeed, patients with ARDs are known to have a higher risk of serious infections and a slightly higher risk of complications from COVID-19 than the general population ,5,6,7.Currently, several authors evaluated the immunogenicity of COVID-19 vaccine (mainly BNT162b2 vaccine) in ARD patients through the analysis of humoral and cellular immunity. Overall, these studies showed that the great majority of ARD patients develop anti-receptor-binding domain (RBD) antibodies, with a lower proportion presenting also a specific T-cell response ,9,10. ThInflammatory arthritis, and, in particular, Rheumatoid Arthritis (RA), is characterized by spontaneous and unpredictable episodes of immunological and clinical worsening, defined flare ups. They may frequently be induced by infections, which have even been implicated in the etiology of the disease and may trigger a strong inflammatory reaction able to reactivate the disease . Thus, tWe carried out, therefore, a multicenter, prospective study to analyze the clinical and immunological safety of BNT162b2 vaccine, in a cohort of RA patients, in remission and with low-moderate disease activity, followed-up from the first vaccine cycle to the first booster dose.The protocol was approved by the Ethical Committee of INMI-Lazzaro Spallanzani-IRCCS . All participants signed a written informed consent.This is a prospective, real-life, multicenter study investigating the safety of mRNA vaccine  in a cohort of RA patients during first vaccine cycle and third dose administration (first booster).Patients with a diagnosis of RA according to the 2010 criteria of the European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) , under iWe conducted post hoc power calculations at a two-sided 5% significance level to evaluate the ability of the study to detect the difference observed between two groups. Moreover, to address this issue, in logistic regression odds ratios with their 95% confidence interval (95% CI) were also reported as estimate of the magnitude of effects that are consistent with the observed data.At T0 (time of the first dose of vaccination), several demographic and clinical data including body mass index (BMI), smoking habits, comorbidities, flu vaccination, ongoing immunosuppressive therapy, disease duration and disease activity, were collected . In part− DAS28CRP to infer for possible disease flare-ups. Flare-up was defined as ΔDAS28 ≥ 1.2 or ≥0.6, if final DAS28 > 3.2 . The dur− AEs collected as early local and systemic (reactogenicity), if occurred within 7 days from vaccination: pain, redness or swelling at the site of injection; generalized muscle or joint pain, headache, fever, chills, fatigue or vomiting. AEs of special interest, reported by specifying the organ/system involved. It has to be underlined that most systemic AEs, excepting for anaphylactic reactions, are closely evocative of flares; thus, they may hardly be discriminated from them. In this study, however, it was preliminarily established that systemic AEs were considered flares only if they were able to increase the DAS28CRP as described above, whereas, in case of mild symptomatology, they were categorized as AEs. AEs were also classified as non-serious or serious ;− Antinuclear antibodies (ANA) and rheumatoid factor (RF) in a subgroup of patients by using indirect immunofluorescence on hep-2 cells, and nephelometry, respectively;− Occurrence of breakthrough infections (BI) At T1 (2–4 weeks from the second vaccine dose) and T2 , the following parameters were assessed:The patients interrupted methotrexate and JAK-inhibitors intake for one week before and after, and CTLA-Ig one week before, each vaccine dose as suggested by ACR recommendations .2 test or Exact Fisher test, as appropriate. To evaluate the association of demographic and clinical characteristics at baseline and the occurrence of any adverse event after the second vaccine dose and after booster dose, univariable and multivariable logistic regression analyses were performed. In multivariable logistic regression, variables with p < 0.05 at univariable analysis were included. Statistical tests were considered significant at p < 0.05. Data were analyzed using Stata .Numerical variables were expressed as median and interquartile range (IQR) and compared among groups by means of the Mann–Whitney test. Categorical variables were expressed by numbers and percentages and compared among groups by Yates corrected, two tails, χIn total, 78 RA patients were assessed at the time of the first vaccine dose and followed-up after the second vaccine dose; however, only 47 of them were followed-up after the third dose. Demographic, clinical, and life-style factors of the patients are specified in Thirty-two percent of patients were receiving a combination of cs and bDMARDs. Finally, 43.6% were treated with low-dose corticosteroids  in combination to cs and bDMARDs.p < 0.002). However, it has to be underlined that only 19/40 (47.5%) patients who had AEs after the second dose received the third dose, versus 28/38 (74%) of those who did not suffer any AEs at T1 (p < 0.004). Only seven patients (9%) had AEs at both doses. Pain at the injection site (28.2%) was the most frequent AE at T1, followed by systemic AEs: fatigue (23.1%), arthralgia (19.2%), fever (15.4%), headache (11.5%) and myalgia (10.3%). At T2, a reduction in total AEs, that was significant for pain at injection site (p < 0.007) and arthralgia (p < 0.004), was observed (p = 0.039) with the occurrence of AEs, whereas receiving other bDMARDs and being vaccinated for seasonal influenza were inversely associated  with the occurrence of AEs at univariable analysis; however, only vaccination for seasonal influenza maintained a significant difference (p = 0.027) when adjusted in the multivariable analysis (AEs were observed in 40/78 (51.3%) and 13/47 (27.7%) patients at T1 and T2, respectively  and 6 at T2 (12.8%). Flares occurred during the first week after vaccination, and were generally mild and not clinically relevant. Indeed, although DAS28CRP slightly increased at T1 and T2 , it remaOngoing glucocorticoid therapy was the only baseline parameter significantly associated with the occurrence of flares; in particular, we did not find associations between having a higher disease activity at baseline and the onset of flares . FurtherAccording to the post hoc power analyses performed on the sample available to study outcomes after the second dose, only flu vaccination showed a quite good power, 73.5%, in detecting differences in proportions of AEs, and treatment with glucocorticoids a power of 77.7% in detecting difference in proportions of flares. Six out of thirty patients (20%) were positive for low-titer ANA (≤1:160) at baseline.A slight, not significant, increase in ANA titer (from 1:80 to 1:160) was found in three patients after the second dose. However, the titer remained stable after the third dose and ANA were not associated with the onset of disease flares or AEs. No new ANA appearance was found. Twenty-four out of thirty patients (80%) were positive for RF at baseline and no modifications have been observed at T1 and T2.p = 0.001). All infections were mild with the exception of one that was moderate; however, no patient required hospitalization  patients presented a breakthrough infection (BI), versus 9 in 47 (19.2%) after the third dose, which all occurred during the first month of 2022  ,39,40,44Here, the safety of anti-Severe Acute Respiratory Syndrome-CoronaVirus (SARS-CoV)-2 BNT162b2 vaccine after the first cycle and the third dose in a cohort of RA patients was longitudinally analyzed. The vaccine showed an overall good safety profile with no patient reporting SAEs, and a percentage of total AEs similar to that reported in the general population in large trials  that doeThe relatively low rate of AEs observed in our population also confirms data from previous studies in ARD patients after the first vaccine cycle. Tang et al. have recently systematically reviewed these data from 47 selected studies on a total of 4433 ARD patients after the first and second dose. The rate of SAEs was very low (0.04%). Local pain was the most common AE, followed by fatigue, arthro-myalgia and headache . The larWe observed 10.3% of flares after the end of the vaccination cycle and 12.8% after the third dose. These data are in agreement with previous studies which reported a general low incidence of disease flares in ARD patients with most being mild/moderate . In partVery few studies addressed vaccine safety after the third dose. Assawasaksakul et al. have prospectively evaluated a small population of systemic lupus erythematosus (SLE) and RA patients. They found no SAEs, and the three most common AEs were injection site pain, fatigue and fever . Even inHowever, analyzing the rate of AEs in patients affected by autoimmune arthritis is a challenge for clinicians. Indeed, it should be taken into account that the large majority of systemic AEs to vaccines  can resemble a disease flare and vice versa. As reported above, we tried to discriminate between AEs and flares, through the DAS28CRP, a universally accepted index which integrates both subjective  and objective (swollen joints and CRP) variables. However, it is impossible to exclude that some of the systemic AEs are not true AEs, rather mild disease flares, under the threshold to be detected by DAS28CRP modifications. The clinical safety was parallel to immunological safety, as inferred by slight, not significant, ANA titer modifications, without new autoantibody appearance, thus confirming the original result of Blank et al. in inflammatory arthritis after BNT162b2 vaccine  and our Both vaccine components, antigens and adjuvants, might induce autoimmunity onset and flares by different mechanisms, such as molecular mimicry, bystander activation and polyclonal activation ,16. BNT1The effectiveness of BNT162b2 COVID-19 vaccine in preventing infection was excellent at T1, with breakthrough mild COVID-19 infection occurring in only 1/78 (1.3%). After the third dose, the rate of BI worsened, with 9/47 infected patients , probably due to the spreading of the Omicron variant of concern, which mostly escapes recognition by original vaccine  and becaThis hypothesis is corroborated by the overall good immunogenicity of the original SARS-CoV-2 strain, emerged by our previous studies conducted on a part of these patients. Indeed, we already showed that the antibody specific response was present in almost all subjects after the first cycle and was significantly strengthened after the third dose of BNT162b2 mRNA vaccine ,43.However, recently, a very promising treatment for COVID-19 has been set up, including the chimeric antigen receptor (CAR) T-cells, which may be useful and effective not only in oncology, but even in autoimmune and viral diseases .The main limits of this study are the relatively low number of recruited patients and the lack of control groups, represented by non-vaccinated RA patients and vaccinated normal controls, that may affect the robustness of these results. Moreover, considering the multicentric real-life design of the study, reporting or investigator bias cannot be excluded, thus reducing the generalizability of results.Conversely, some strengths of the study are its prospective design, the presence of a homogeneous disease cohort of RA patients, followed-up from the first cycle to the third dose, the evaluation of disease activity by clinical examination and the analysis of immunological safety, which allow the reliability of data be increased. In conclusion, the current preliminary study confirms a good safety profile of the BNT162b2 COVID-19 vaccine, at both the first cycle and the third dose, in RA patients under immunosuppressive therapy. No SAEs occurred, and the rate of AEs and disease flares was low.For the first time, to the best of our knowledge, evidence was provided that being vaccinated for influenza was inversely associated with the onset of AEs after the second dose, thus allowing identification of a predictive factor of vaccine tolerance, should this result be confirmed in larger patient populations. A higher disease activity at baseline was not associated with a higher incidence of AEs or disease flares. The reduced vaccine effectiveness at T2 is a probable expression of the higher capacity of the Omicron variants to escape vaccine recognition. A future larger, controlled, study will allow to obtain definitive data on the mRNA COVID-19 vaccine safety in remitted and low-moderate disease activity RA patients.


Purpose: An intraocular hemorrhage is an adverse event that can lead to visual acuity impairment. Antithrombotic therapy with antiplatelet agents and anticoagulants may increase intraocular hemorrhage. However, since their frequency is low, studies on the risk of intraocular hemorrhage with these drugs, especially under combination therapy, are limited. This study aimed to investigate the occurrence of intraocular hemorrhages under monotherapy and combination therapy with antiplatelets and anticoagulants by analyzing a large pharmacovigilance database.Methods: Intraocular hemorrhage signals with oral antiplatelets and anticoagulants were evaluated by calculating reporting odds ratios and information components using the Japan Adverse Drug Reactions Report database from April 2004 to March 2022. In addition, differences in signals between younger and elderly patients, affecting factors, and time-to-onset from initial antiplatelet and anticoagulant treatments were analyzed.Results: Aspirin, clopidogrel, warfarin, apixaban, and rivaroxaban, but not ticagrelor, ticlopidine, prasugrel, dabigatran, and edoxaban showed intraocular hemorrhage signals under monotherapy. In combination therapy, dual therapy  and triple therapy (aspirin + P2Y12 inhibitors + warfarin) resulted in intraocular hemorrhage signals. Intraocular hemorrhage signals were observed in younger patients receiving monotherapy with aspirin and in elderly patients receiving monotherapy and combination therapy with warfarin. Affecting factors were diabetes mellitus in patients with prasugrel, use of medications for intravitreal injections, and posterior sub-Tenon injections with some antiplatelets and anticoagulants. The median period of intraocular hemorrhage occurrence after starting monotherapy with aspirin, clopidogrel, warfarin, or rivaroxaban was within 90 days.Conclusion: In addition to monotherapy with several antiplatelets and anticoagulants, combination therapy using aspirin, P2Y12 inhibitors, and warfarin has the potential risk of intraocular hemorrhage. Particular attention should be paid to the occurrence of intraocular hemorrhages in younger patients taking aspirin, in elderly patients taking warfarin, and within the first 90 days of antiplatelet and anticoagulant use. Additionally, APs and ACs are often used in combination therapy and monotherapy (The use of antiplatelets (APs), such as aspirin and P2Y disease –6. For e disease . However disease . Thus, iotherapy . AnalyziIn recent years, pharmacovigilance approaches for drug-associated adverse event (AE) signal detection using large databases, such as the Japanese Adverse Drug Event Report (JADER) based on spontaneous AE reports, have evolved . Drug-asIt has been reported that warfarin and some DOACs under monotherapy showed signals of choroidal hemorrhage, retinal hemorrhage, and vitreous hemorrhage using Vigibase, a pharmacovigilance database obtained from the World Health Organization . HoweverIn this study, we investigated the signals of IOHs during monotherapy or combination therapy using JADER database. In addition, we investigated differences in IOHs signals in terms of age, affecting factors for occurrence of IOHs, and time to onset of IOHs in patients taking oral APs and ACs.via the “oral” route were selected to evaluate the signals for IOHs in patients who received APs and ACs orally. The “demo” table was linked to the “drug,” “reac,” and “hist” tables using the patient identification number of each case. After data cleaning, 688,467 patients were included in this study  in the Medical Dictionary for Regulatory Activities . Seventeen PTs were determined as IOHs by two ophthalmologists from PTs grouped into the high level term (HLT) and Ocular haemorrhagic disorders (HLT code 10064464). IOHs because of external or physical reasons were excluded .This study used ROR and IC to evaluate the signal detection of IOHs, as previously reported ,10. The ROR equations:IC Equations:The calculations were performed using Microsoft 365 (Microsoft Corporation). The signals for IOHs were positive when the lower limit of the 95% CI of the ROR exceeded 1, and that of the IC exceeded 0.p values <0.05 were considered statistically significant. Statistical analysis was performed using EZR  as a graphical user interface for R version 4.1.2 . EZR is a modified version of R Commander  were calculated using JMP 13.0 . We evaluated APs and ACs in >10 patients reporting IOHs between the day of the initial dose and the day of the first occurrence of IOHs.The RORs and ICs of APs and ACs for IOHs under monotherapy are shown in 12 inhibitors: ROR, 3.55 [95%CI 2.74 to 4.60] and IC, 1.72 [95% CI 1.34 to 2.10]; with warfarin: ROR, 3.12 [95% CI 2.10 to 4.63] and IC, 1.51 [95% CI 0.93 to 2.08]; with DOACs: ROR, 3.64 [95% CI 2.15 to 6.18] and IC, 1.61 [95% CI 0.86 to 2.37]). P2Y12 inhibitors showed IOHs signals in combination with warfarin , but not DOACs . In combination therapy with three types of antithrombotic agents (triple therapy): DAPT with warfarin or DOACs, an IOHs signal was detected in combination with warfarin , but not with DOACs .The RORs and ICs of APs and ACs in combination therapy are shown in 12 inhibitors with warfarin  showed IOHs signals in the elderly group. In the combination therapy of DAPT with warfarin, the IOHs signal was detected in the elderly group .The RORs and ICs of APs and ACs that showed IOHs signals under monotherapy or combination therapy in the younger/elderly groups are shown in p = 0.006). IVI and PSTAI increased the frequency of IOHs in most APs and ACs with their presence.To investigate factors affecting the occurrence of IOHs under monotherapy or combination therapy of APs and ACs, we assessed the effect of comorbidity of hypertension, diabetes mellitus, and use of medications for IVI and PSTAI on the occurrence of IOHs . MultivaIn the time-to-onset analysis, two APs (aspirin and clopidogrel) monotherapy and three ACs  monotherapy with >10 reports of IOH were included in the evaluation. The median period [IQR] to the onset of IOHs in patients with APs and ACs was as follows: aspirin (72.5 [30.3–369.5] days), clopidogrel (79.5 [12.3–443.8] days), warfarin (29.0 [14.0–367.0] days), apixaban (174.0 [34.0–381.5] days), and rivaroxaban (90.0 [21.8–339.5] days). The time-to-onset of IOHs for combination therapy could not be evaluated because no more than 10 patients reported IOHs between initial administration and the date of the first occurrence of IOHs .12 inhibitors, warfarin, and DOACs. It has been reported that monotherapy of aspirin, and combination therapy taking more than 1 aspirin/clopidogrel/warfarin are associated with an increased risk of the IOHs occurrence in patients with neovascular age-related macular degeneration who had no previous intraretinal hemorrhage and no history of trauma, posterior segment surgery (12 inhibitors (prasugrel or ticagrelor) did not increase the risk of IOHs compared to aspirin and other P2Y12 inhibitor (clopidogrel). However, their study did not assess whether DAPT increases the risk of IOHs (12 inhibitors (DAPT), warfarin, or DOACs, alerting us to the risk of IOHs during aspirin monotherapy and combination therapy. In P2Y12 inhibitors monotherapy, clopidogrel but not other P2Y12 inhibitors, such as prasugrel, showed IOHs signals. In contrast, a retrospective study reported that the prevalence of spontaneous IOHs on prasugrel was higher than that on clopidogrel , a trend that differed from that observed with apixaban monotherapy. In fact, the combination therapy of apixaban with P2Y12 inhibitors was associated with a lower risk of thrombolysis in myocardial infarction major and minor bleeding, trial-defined primary bleeding events, and intracranial hemorrhage compared with the combination therapy of P2Y12 inhibitors with warfarin in meta-analysis (The signals of IOHs under monotherapy and combination therapy by APs and ACs, signal differences in ages, affecting factors, and their time-to-onset were analyzed using the JADER pharmacovigilance database. IOHs signals were shown in both monotherapies with aspirin and combination therapy of aspirin with P2Y surgery . Sun et  of IOHs . In addipidogrel . In our pidogrel , apixabapidogrel ; rivaroxpidogrel . Contrarpidogrel . Their manalysis , and theIn the age-based subgroup analysis, IOHs signals were observed in elderly patients receiving warfarin monotherapy and combination therapy. These results are consistent with reports that spontaneous IOHs occurrence was higher in elderly patients receiving warfarin treatment  and combHypertension, diabetes mellitus, IVI, and PSTAI are known to affect the occurrence of IOHs –25. In oIn the time-to-onset analysis, the median period of IOHs occurrence after starting monotherapy with APs and ACs, excluding apixaban, was within 90 days, and the 75-percentile period of IOHs occurrence with APs and ACs was approximately 360 days. The recommended period of DAPT after coronary artery diseases, such as acute coronary syndromes and percutaneous coronary intervention, is 1–12 months, depending on the pathophysiology, thrombotic risk, and bleeding risk of the target patients, followed by monotherapy with APs or ACs . We haveOur study had several limitations. First, spontaneous reporting systems, such as JADER, are passive reporting systems, and there are many biases, including under-reporting, over-reporting, and confounding by comorbidities. In addition to hypertension, diabetes mellitus, IVI, and PSTAI, intraocular surgery such as vitrectomy is considered a risk factor for IOHs. Because the JADER database does not include information on the presence of surgical history, the influence of intraocular surgery cannot be ruled out. Second, the influence of AP and ACs doses cannot be ruled out. For example, aspirin exhibits antiplatelet effects when administered at low doses; however, the limited information on drug doses in JADER makes accurate assessment difficult. Third, the number of cases of ticagrelor and prasugrel use and the number of evaluable cases in the time-to-onset analysis was small. Finally, we defined an IOHs signal as one with a significant difference in RORs and ICs to avoid false positive detection. Future investigations with more case-accumulated databases are needed to assess the IOHs signal and time-to-onset.In conclusion, the potential risk of IOHs increases during monotherapy with aspirin, clopidogrel, warfarin, apixaban, and rivaroxaban and during combination therapy with most APs and ACs, such as DAPT. In addition, aspirin use in younger patients, warfarin use in the elderly, and within the first 90 days of APs and ACs use should be considered for the occurrence of IOHs. Our results may help internists, ophthalmologists, pharmacists, and other medical staff predict and manage the occurrence of IOH under APs and ACs monotherapy and combination therapy.


Diabetes is a common chronic metabolic disease, and its incidence continues to increase year after year. Diabetic patients mainly die from various complications, with the most common being diabetic cardiomyopathy. However, the detection rate of diabetic cardiomyopathy is low in clinical practice, and targeted treatment is lacking. Recently, a large number of studies have confirmed that myocardial cell death in diabetic cardiomyopathy involves pyroptosis, apoptosis, necrosis, ferroptosis, necroptosis, cuproptosis, cellular burial, and other processes. Most importantly, numerous animal studies have shown that the onset and progression of diabetic cardiomyopathy can be mitigated by inhibiting these regulatory cell death processes, such as by utilizing inhibitors, chelators, or genetic manipulation. Therefore, we review the role of ferroptosis, necroptosis, and cuproptosis, three novel forms of cell death in diabetic cardiomyopathy, searching for possible targets, and analyzing the corresponding therapeutic approaches to these targets. The number of people in 2021 is 7.4 million more than that in 2019, an increase of 16%, which highlights the prevalence of diabetes worldwide. The international diabetes federation (IDF) estimates that this number will reach 783 million by 2045, a 46% increase that is more than double the projected population growth of 20% over the same period, with one-in-eight adults likely to have the disease (Diabetic cardiomyopathy (DCM), a disease common to most diabetic patients, is based on changes in the cardiac structure and systolic and diastolic functions. When patients have cardiac insufficiency, other clear causes, such as hypertension and structural or ischemic heart disease can be diagnosed . HoweverIn general, the intervention involves conventional sugar control measures such as diet and hypoglycemic drugs, in the early stage. When the disease progresses to the stage of heart failure, the corresponding treatment is given according to the current guidelines for heart failure . HoweverIn fact, the major risk factors for the development of diabetic cardiomyopathy are the progressive death of cardiomyocytes, changes in endothelial cells , and car2.2+-related dysfunction have been identified, as well as changes in substrate metabolism, insulin signal transduction, gene regulation, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, neurohumoral activation, and cardiac cell death  directly leads to ferroptosis . Spontan2.1.1.2.1.1.1.In human cells, Acetyl-CoA is a global currency that mediates carbon trading among the metabolic pathways, including glycolysis, the tricarboxylic acid cycle, amino acid metabolism, gluconeogenesis, and fatty acid synthesis . Acetyl-2.1.1.2.2·−), H2O2, and hydroxyl radical (HO·), which are derived from the four-electron reduction reaction of oxygen in the mitochondrial electron transport chain H pathway (P)H-dependent CoQ10 oxidoreductase, NAD(P)H can be used to reduce the oxidized ubiquinone. It is known that the reduced form of ubiquinone can capture the free radicals that mediate lipid peroxidation and limit the occurrence of lipid peroxidation. Thus, FSP1 can inhibit the occurrence of ferroptosis (P) H axis may play an important role in the occurrence of ferroptosis caused by lipid peroxidation.Similar to the regulation of iron, the presence of PUFA-PL-OOH in cells does not directly lead to ferroptosis. In fact, PUFA-PL-OOH can accumulate under the action of iron bivalent, resulting in cell death through damage to cell membranes or the production of lipid-derived electrophilic small molecules . This ac pathway . Ferropt pathway . FSP1 wa pathway . Recentl pathway , 65. FSProptosis . All in In a study conducted by Mao C, the effect of a GPX4 inhibitor on ferroptosis in tumor cells was reversed by dihydroorotate dehydrogenase (DHODH), and this reversal was more significant in tumor cells with a low expression of GPX4, suggesting that DHODH may be the third mechanism of inhibiting ferroptosis . The ant2.1.2.As mentioned earlier, the system Xc-/GSH/GPX4 axis is an important pathway for ferroptosis. Many drugs function in the system Xc-/GSH/GPX4 axis, such as the classic Erastin, when ferroptosis was originally associated with this compound. Erastin inactivates GPX4 by inhibiting the system Xc- and reducing the production of GSH, promoting ferroptosis –72. SkipThe accumulation of advanced glycation end products (AGEs) caused by hyperglycemia increases lipid peroxidation and the ferroptosis of cardiomyocytes , which tIncreasing evidence shows that ferroptosis is involved in the onset and progression of DCM, and ferroptosis also affects the pathological consequences of cardiac function –82. Thes2.2.Initially, necroptosis was not considered a new form of cell death. It was morphologically similar to necrosis and shared an upstream pathway with apoptosis. Later, researchers gradually distinguished the differences between them, which led to the emergence of necroptosis . As a we2.2.1.Necroptosis is a unique cell death mode in vertebrate cells, which is the second line of defense established by cells to resist pathogens. When apoptosis fails, necroptosis becomes a fail-safe mechanism. Abnormal regulation of necroptosis is associated with these diseases. The expression and activity of necroptosis signaling proteins are low in cancers, such as breast cancer  and ovarNature reported that caspase-8 induces and inhibits cell death at the same time. It induces apoptosis through death receptors such as TNFR1, and inhibits necroptosis through lysis and the inactivation of RIPK1 and RIPK3  activation through RIPK3 defects. Moreover, alleviating necroptosis in DCM mice demonstrates the existence of severe necroptosis in DCM mice  deficiency aggravated hyperglycemia-induced mitochondrial damage, increased ROS accumulation, promoted necrosis, and possibly activated NLRP3 inflammatory. Ultimately, these processes aggravated DCM in mice . RecentlDCM mice .It is certainly not enough to simply state a correlation. Irbesartan is a common clinical ARB drug. Existing studies have explored how irbesartan acts. Irbesartan has a cardioprotective effect on diabetic rats, and its mechanism may be related to the inhibition of the RIP1-RIP3-MLKL pathway, which alleviates necroptosis in cells. When it comes to the RIP1-RIP3 complex, AMPK inhibits the formation of this complex . Based o2.3.Similar to iron, copper is an indispensable trace element in all organisms, but it can exhibit cytotoxicity if the concentration of copper ions in cells exceeds the threshold for maintaining homeostasis , 105. In2.3.1.α-ketoglutaric acid and the consumption of succinic acid in cells, indicating that the loss of protein lipid acylation function blocks the progression of the tricarboxylic acid cycle (TCA)  . FDX1 anle (TCA)   .2.3.2.DCM is characterized by myocardial remodeling, including myocardial fibrosis and hypertrophy. As mentioned above, cuproptosis mainly occurs in the mitochondria, which means that cuproptosis affects the course of DCM by destroying mitochondrial structure and function . AbnormaCurrently, many studies have shown that an increasing copper level can reduce myocardial hypertrophy –119, butAs a newly proposed form of cell death, the current research on cuproptosis still focuses on its intervention in cancer therapy, which destroys the energy system of cancer cells by providing excessive copper to cancer tissues through targeting, leading to the death of cancer cells , 125, an3.Based on the above review, novel regulatory cell death mechanisms are involved in the cell death and progression of DCM, and it is worth noting that inhibition of any form of cell death can significantly restore damaged myocardial cell function or structure in DCM , 126–131As mentioned above, the AMPK pathway is also an important pyroptosis pathway . PyroptoOf particular interest to us is what these similarities represent. Is there an overlap between some forms of death? If there is overlap, are these overlapping processes an opportunity for the onset of DCM? Shedding light on these issues can help to better determine targets and target diseases. Metabolic disorders caused by diabetes are triggers for many forms of cell death. However, as mentioned above, there is currently no specific clinical treatment plan for DCM, and even treatment after heart failure only follows the guidelines for heart failure. If we can further study these forms of death, we may be able to solve this problem. After all, the incidence of DM is high and rising year by year, and cardiovascular diseases are also the main cause of death and disability at present.


Background: It is of utmost importance to identify populations with an elevated risk for COVID-19 and the factors influencing its outcomes. The present study aimed to investigate factors affecting mortality and length of stay (LOS) among COVID-19 patients in the hospitals of East Azerbaijan province, Iran, during 15 months of this pandemic.Study Design: The present study followed a retrospective cohort design.Methods: This retrospective study was conducted using data in the integrated syndromic surveillance system (ISSS) on patients admitted to the hospitals from February 21, 2020, to April 11, 2021. The association between variables of interest and death, as well as LOS, was investigated via multiple logistic regression and multiple linear regression analyses.Results: In total, 24 293 inpatients with a mean age of 54.0 ± 19.4 years were included in this study. About 15% of them lost their lives, whose mean age was 69.0 ± 14.6 years, significantly higher than the recovered ones (P < 0.001). Factors, such as above 49 years of age (P < 0.001), male gender , and having chronic diseases , were correlated with patient mortality. In addition, having chronic diseases  was associated with higher LOS in hospitals.Conclusion: In conclusion, older patients were at a higher risk of mortality and prolonged hospitalization. Furthermore, patients’ underlying diseases could cause a severe form of COVID-19, which can lead to death and increase patients’ LOS. Factors affecting the severity of this condition and the resulting mortality include male gender, ≥ 50 years of age, kidney disease, cerebrovascular disease, cardiovascular disease, respiratory disease, diabetes, hypertension, cancer, and obesity.5 Most COVID-19 cases have mild to moderate symptoms One significant aspect of epidemiological studies is to investigate the relationship between patients’ demographic characteristics, clinical signs and symptoms, and several underlying diseases, by which the severity of the disease and its outcomes, which could vary from patient to patient, can be determined, and then, to find the effects of these characteristics on disease outcomes. Moreover, knowing the temporal and spatial trends of the disease and its epidemiological aspects in different regions is one of the necessities for legislative decision-making to control and manage it. Therefore, using large population-based data, this study, as the first study in East Azerbaijan province, Iran, aimed to document the COVID-19 pandemic and investigate factors affecting mortality and length of stay (LOS) among patients in the hospitals of this province, during 15 months of this pandemic. This descriptive-analytical study was conducted on COVID-19 patients admitted to the hospitals affiliated with Tabriz University of Medical Sciences (TBZMED), East Azerbaijan Province, Iran, from February 21, 2020, to April 11, 2021. In total, 24,293 patients who tested positive for COVID-19 by reverse transcriptase-polymerase chain reaction (RT-PCR) and were hospitalized in TBZMED hospitals were recruited for the study. The data were obtained from the integrated syndromic surveillance system (ISSS) of the Vice Chancellor’s Office for Health at TBZMED. The ISSS is a national electronic platform under the supervision of the Center for Communicable Disease Management of the Ministry of Health and Medical Education of Iran, which collects data on target syndromes from public and private healthcare providers. The main purpose of the ISSS is to quickly detect outbreaks of infectious diseases, particularly emerging ones, such as COVID-19. Data on COVID-19 suspected patients have been recorded as Severe Acute Respiratory Infections syndrome. All suspected cases were subjected to COVID-19 RT-PCR testing and confirmation. Incomplete data and patients without RT-PCR test results were excluded. Demographic characteristics, clinical signs and symptoms, COVID-19 RT-PCR test results, underlying diseases , and disease outcomes  were retrieved from the ISSS. Additionally, data quality was evaluated for non-duplication and completeness.t test, and analysis of variance (ANOVA) were utilized in univariate analysis. In addition, multiple logistic regression and multiple linear regression analyses were conducted in multivariate analysis. All statistical analyses were performed by the SPSS software (version 24). Descriptive statistics were used to describe the data. Chi-square test,  The mean age of patients was 54.0 ± 19.4 years. Totally, 12,603 cases (51.9%) were male with a mean age of 54.6 ± 19.3 years, and 11 690 cases (48.1%) were female with a mean age of 53.5 ± 19.4 years. Among all the hospitalized cases, 8,653 patients (35.6%) had at least one underlying disease. The most common complications of the disease among the inpatients were ground-glass opacity , bilateral pulmonary infiltration , and primary viral pneumonia . During the study period, 3663 patients (15.1%) lost their lives due to COVID-19. The mean age of the dead cases was 69.0 ± 14.6 years, among whom 2027 (55.3%) were male with a mean age of 68.7 ± 15.0 years, and 1636 were female with a mean age of 69.5 ± 14.11 years. The total case fatality rate (CFR) in this study was estimated to be 15.1% (minimum = 6.15% in April 2021 and maximum = 18.3% in December 2020). It was 16.1% for males and 14.0% for females . The association between various factors and COVID-19 mortality was explored using multiple logistic regression analysis. As shown in  The median LOS was three days for patients . It was also three days for women (interquartile range [IQR] = 6) and three days for men (IQR = 7). Patients in the age group of 65-80 years experienced the highest LOS with a median of five days (IQR = 6). Moreover, the median LOS was five days in dead patients (IQR = 8) and three days (IQR = 6) for the recovered ones . The present study aimed to investigate the epidemiological characteristics of COVID-19 and factors affecting mortality and LOS in COVID-19 patients admitted to hospitals affiliated with TBZMED, Tabriz, Iran. It was found that patients with older age, male gender, and underlying conditions had significantly higher mortality due to COVID-19. Additionally, the LOS was significantly higher for patients with older age and a positive history of underlying conditions.6 and 8.1%.7 A systematic review that analyzed 39 studies calculated a CFR of 13%.8 In another systematic review involving 33 studies and 13,398 patients, CFR was estimated as 17.1%.9 These different fatality rates can be attributed to the ability of the healthcare system to respond to the pandemic, the predominant type of virus in the community at the time of the study, and the denominator of the different fractions in calculating the CFR of the disease. According to the results of the present study, CFR was 15.1% among hospitalized patients, and it was higher in men than women. Other studies have estimated the CFR of the COVID-19 disease in Iran as 13.5%13 The high CFR in old age was also attributed to the point that most older people were suffering from several underlying diseases and were at a higher risk for death if they had become ill.14 It was also found that higher age is the most influential factor in increasing the risk of COVID-19 mortality; therefore, the risk of mortality was the highest for patients above 80 years. Accordingly, 75% of all COVID-19 deaths were for those above 60 years. Numerous studies have further shown that old age is one of the essential predictors of patient mortality due to COVID-19.17 which may be due to biological and socio-behavioral factors, as well as the difference in medication efficacy, comorbidities, immune system response, and sex-related hormonal differences.17 In this study, male patients experienced a higher risk of mortality due to COVID-19, and about 52% of deaths occurred in men. Several studies on COVID-19 patients indicated that males experienced higher mortality than females,20 Based on the findings of this study, higher age and underlying diseases were related to prolonged hospitalization. The median LOS in this study was three days for both male and female patients; however, patients in the age group of 65-80 years had the highest LOS at hospitals. Furthermore, the LOS in dead patients was about 1.6 times longer than that in the cases recovered and discharged from the hospitals. These results were similar to previous findings. Considering that the ISSS data was used in this study, similar to other surveillance systems’ data, the present findings may have been affected by some errors in data collection, registration, and reporting, which cloud not be detected despite quality control efforts. Therefore, the results should be interpreted with caution.Case fatality rate (CFR) was calculated to be 15.1% among COVID-19 hospitalized patients. The CFR is higher in male patients than the females. Aging and underlying diseases are major risk factors for prolonged hospitalization and death among COVID-19 patients. More than half of COVID-19 hospitalized patients have at least one underlying disease.  It was concluded that aging and underlying diseases were the most influential factors in COVID-19 mortality, as well as LOS at hospitals, among inpatient cases. More than half of the dead cases experienced at least one underlying disease, and the older ones had the highest LOS at hospitals. The CFR was higher in hospitalized men than women in all age groups. High-risk populations were also identified in terms of patient mortality and LOS at hospitals. The findings could be used in implementing the necessary prevention and treatment measures for high-risk groups to reduce COVID-19 mortality in hospitalized patients. Moreover, they could be utilized by health authorities to intensify their attempts in terms of educating high-risk groups to have more compliance with prevention measures and maintain the risk perception of the population at the desired level to voluntarily support healthy behaviors. The authors hereby extend their gratitude to all healthcare workers contributing to this study, especially those involved in collecting patient information. The authors declare that they have no competing interests. This study was approved by the Ethics Committee of the Tabriz University of Medical Sciences, Tabriz, Iran, with the code of ethics as IR.TBZMED.REC.1400.007. The study was supported by funding from the Centers for Tabriz University of Medical Sciences. The funding covered data collection, analysis, and report writing.


Osteogenesis imperfecta (OI) is a rare congenital bone dysplasia generally caused by a mutation of one of the type I collagen genes and characterized by low bone mass, numerous fractures, and bone deformities. The collagen organization and osteocyte lacuna arrangement were investigated in the long bones of 17-week-old wildtype  and osteogenesis imperfecta mice  that is a validated model of severe human OI in order to assess their possible role in bone fragility. Fractures were counted after in vivo scanning at weeks 5, 11, and 17. Humerus, femur, and tibia diaphyses from both groups were analyzed ex vivo with pQCT, polarized and ordinary light histology, and Nano-CT. The fractures observed in the OIM were more numerous in the humerus and femur than in the tibia, whereas the quantitative bone parameters were altered in different ways among these bones. Collagen fiber organization appeared disrupted, with a lower birefringence in OIM than WT bones, whereas the osteocyte lacunae were more numerous, more spherical, and not aligned in a lamellar pattern. These modifications, which are typical of immature and less mechanically competent bone, attest to the reciprocal alteration of collagen matrix and osteocyte lacuna organization in the OIM, thereby contributing to bone fragility. Osteogenesis imperfecta (OI) is a rare genetic disease of connective tissues, whose prevalence is 1/10,000 to 1/20,000 births. In most cases, because of an abnormal production of type I collagen, patients present with short stature, fractures, bone deformities, scoliosis, pain, and disability. Extra-skeletal manifestations can also be observed, such as blue sclera, dentinogenesis imperfecta, hearing loss, etc. .COL1A1 and COL1A2 genes is responsible for about 80% of OI cases [Type I collagen, the main protein of bone matrix, is a heterotrimer composed of two alpha-1 chains and one alpha-2. A mutation of one of their respective OI cases . Such muOI cases .The bone matrix comprises two primary components: the organic phase, predominantly composed of 90% type I collagen, and the mineral phase, consisting of hydroxyapatite crystals. The three-dimensional structure of mature bone tissue depends on the lamellar organization of the collagen matrix, which is essential for the proper arrangement of hydroxyapatite crystals and overall bone matrix quality .2, which regulate osteoclast and osteoblast activity [The bone matrix is apposited by osteoblasts, differentiated from mesenchymal stem cells, which become osteocytes in the bone matrix after passing successive stages, namely osteoblast, osteoid osteocyte, mineralizing osteocyte, and finally mature osteocyte. In the mineralized matrix, osteocytes are embedded inside individual lacunae and are interconnected with each other via dendrites into canaliculi , leadingactivity .Osteocytes are oriented with a specific polarity, depending on the direction of collagen apposition, mineral formation, and loads . They coPLUS ; thereafter, the humerus, femur, and tibia were analyzed ex vivo with peripheral Quantitative Computed Tomography (pQCT) to assess bone mineral density and quantitative cortical bone parameters, microscopic birefringence to evaluate the collagen organization, as well as histology and Nano-CT to characterize the OL in cortical bone.The aim of the present study is to characterize the organization of extracellular matrix and osteocyte lacunae (OL) in long bones of 17-week-old wildtype (WT) and osteogenesis imperfecta mice (OIM) known to display type III osteogenesis imperfecta features [p < 0.0001), as highlighted by their weight at 5, 11, and 17 weeks (p = 0.4). Coherently, the femur and the tibia were significantly shorter in the OIM than in WT mice, whereas no significant difference was observed in the length of the humerus between both groups  and 54.59% (p < 0.01) lower gray value than WT ones.p < 0.001). The elliptic rapport, i.e., minor axis/major axis ratio, was closer to 1 in OIM than WT (p < 0.001), and the osteocyte lacuna roundness showed the same trend with a higher roundness in OIM (p < 0.0001).Quantitative analyses of longitudinal sections through the femur stained with Masson’s trichrome showed modifications of OL parameters in OIM bones B–D. OsteAs visible in p < 0.01) (p < 0.001)  in the OIM group (−11.74%) than the WT one ( < 0.01) A. The qu< 0.001) B. The vo< 0.001) C,D.This study highlighted the combined disorganization of collagen fibers and OL in the long bones of OIM, a murine model of type III OI. Furthermore, the usual bone mass parameters showed different variations between the humerus, femur, and tibia, as was the case of the fracture number.The general OI features of the OIM were attested by clinometric data, fracture assessment, and long bone densitometry (pQCT).As observed in previous studies, OIM were consistently lighter than their WT counterparts throughout the experiment , which iLike Cardinal et al.  and BargThe number of fractures included the humerus, femur, and tibia, as well as the fractures or deformation of the pelvis, calcaneum, ulna, and fibula. Vertebral fractures, not easily distinguishable, were not included. The use of 3D scans instead of 2D X-ray images made it possible to observe multiple fractures on the same bone, such as fractures of the femoral neck and diaphysis or the deltoid tuberosity and humeral diaphysis. These fractures were not always healed between consecutive scans. Our 3D methodology can also explain the higher numbers obtained, as compared with the data of other authors at 4 to 6 weeks of life, i.e., 1.2 to 1.8 fractures per mouse  and at tDensitometric pQCT data reflected the fragility of these OIM long bones in different ways. Indeed, BMD was significantly lower only in the OIM humerus than WT one, whereas cross-sectional area (CSA) and cortical bone area were significantly lower in the OIM femur and tibia than WT ones. These data do not correspond to the low BMD previously obtained in the femur and the tibia of OIM  and coulThe lower CSA and cortical bone area of OIM femur and tibia led to a significantly lower Stress–Strain Index (SSI) than that of WT bones, which indirectly assessed their fragility, as it was illustrated with 3-point bending test in previous studies ,22. The These variations in morpho-densitometric data of the cortical bone among the long bones of OIM draw attention to the need to evaluate different skeletal sites, including trabecular ones, in order to further characterize pathological and therapeutic changes. They also suggest the contribution of other non-quantitative factors in bone fragility, factors more directly linked to collagen abnormality and the disruption of osteoblastic lineage cells.The lower birefringence of OIM collagen fibers observed with polarized light microscopy attested to a loss of lamellar bone organization compared to WT cortical bone. In equine metatarsal bone, mechanical properties were shown to vary according to the orientation of lamellar bone, assessed by collagen fiber birefringence . The lowAs studied in equine, ovine, and murine bone, the arrangement of the osteocyte network is closely interdependent with the organization of the extracellular matrix . ConsistThis qualitative observation was further confirmed by the significantly higher density of OL in the OIM femur and humerus than in WT ones, quantified in 2D histology and 3D NanoCT, respectively. Similarly, a higher relative surface of osteocytes was reported in the bones of OI patients ,28,29 anAlthough we could not use Nano-CT data to analyze 3D lacunar shape because of the resolution (voxel size 1.667 µm), OL analyzed in the histological sections of femurs showed a significantly more pronounced elliptic shape and a higher roundness ratio in OIM than WT mice, in agreement with the more spherical OL observed by Carriero et al.  in OIM bSince the arrangement of type I collagen and the alignment of the osteocyte network in the bone matrix are closely interdependent, any mutation of the genes encoding collagen is expected to disrupt the organization of the osteocyte network, which has to be taken into account when investigating collagenopathy.Homozygous OIM/OIMB6C3Fe (strain a/a-Col1a2 OIM/J ) and homPLUS  under sevoflurane inhalation anesthesia at 5, 11, and 17 weeks of age. At 17 weeks, mice were euthanized with isoflurane. The right humerus, femur, and tibia were dissected, and bones showing fractures on scans were excluded from the study. The preserved bones were measured with a vernier caliper and fixed in formaldehyde. Because of no significative differences between males and females, the data were pooled in each group without gender distinction.After genotyping, mice were included in the osteogenesis imperfecta group  or wildtype one . Mice were weighed weekly between 5 and 17 weeks of age and scanned with 3D in vivo scan NanoSPECT/CTScans performed at 5, 11, and 17 weeks of age allowed to identify and count the fractures. Bone deformation, fracture, and callus formation were considered fractures. A deformation observed after an earlier fracture was not counted as a new one. Since the tail was not systematically radiographed, fractures of the tail were not counted.3 for cortical bone and 280 mg/cm3 for general bone parameters. Bone Mineral Density (BMD), Cross-Sectional Area (CSA), Cortical Bone Area (Cort. area), and Stress–Strain Index  were blinded and analyzed. For each parameter analyzed, the mean value consisted of the mean value of the three slices of each bone. The bones with fractures were not analyzed.The right humerus, femur, and tibia were analyzed ex vivo using a pQCT Research SA+ scanner equipped with the XCT 5.4 built-in software . Three parallel transverse slices through the midshaft were acquired with a distance of 0.03 mm between each other. Voxel size was 0.07 mm × 0.7 mm × 0.7 mm, and the threshold was 570 mg/cmRight femurs without fracture were decalcified, embedded in paraffin, and sliced into longitudinal and transverse sections. Three unstained longitudinal  and transverse slices  of each bone were observed under polarized light with Axioplan microscope  with 1.25 mm objective lens. After digitalization, collagen birefringence was assessed with ImageJ/Fiji software (v1.54f). The images were 8-bit transformed, and the birefringence of cortical bone was measured as the mean of the grey levels between 0 (black) and 255 (white). Two longitudinal slices of each bone were stained with Masson’s trichrome, observed under ordinary light microscopy, and scanned with an SCN400 slide scanner  at 20× magnification. OL were visually differentiated from vascular lacunae by their specific staining and were manually counted in cortical bone. The scans were 8-bit transformed, binarized via Fiji/BoneJ (v1.54f), and OL were hand-selected. Lacunae were analyzed with the ‘elliptic fit’ function. The ratio of the minor axis of the ellipse to the major axis was calculated. A value of 1 indicates that the two axes have the same length and, therefore, that the osteocyte lacuna is close to a circle. The roundness function was also used, which is the ratio of the perimeter of a perfect circle to the object’s actual perimeter. It represents how close the object is to the shape of a perfect circle. A roundness value close to 1 indicates a more circular shape.3 as defined by Carriero et al. [Humeri without fracture were scanned under Nano-CT scan , and 3000 images were taken in mid-diaphysis with a voxel size of 1.5 × 1.5 × 1.5 µm. The source, equipped with a tungsten target, was operated at 85 kV and 70 μA with an exposure time of 750 ms. An aluminum filter of 0.2 mm was applied to reduce beam hardening. All microCT datasets were reconstructed with the Datos|x software version 2.7.0  and exported as XY slices (.tiff). After reconstruction, all datasets were normalized to one sample to harmonize all gray levels. The process of normalization was conducted using a Matlab Script 7.0  ,33. A tot-test or corresponding non-parametric tests such as the Mann–Whitney test and Student’s t-test. Normality was verified with the Shapiro–Wilk normality test, Kolgomorov–Smirnov normality test, and QQ Plot. Results are expressed as mean ± SD or SEM. Data were considered significant if p < 0.05.Statistics were performed with GraphPad6.0 using a This study highlighted qualitative and quantitative alteration of bone tissue in OIM long bones, such as reduced collagen birefringence and disorganized osteocyte lacunae, which could contribute to bone fragility in addition to low BMD and/or impaired geometric data. These observations draw attention to the osteoblastic/osteocytic cell-matrix interactions that influence each other. Further studies would consider these parameters in correlation with mechanical testing, bone matrix composition, osteocyte–dendritic network, and osteocyte viability and activity.


Chronic schizophrenia is significantly influenced by negative symptoms, with several known contributors to secondary negative symptoms. However, the impact of these factors and negative symptoms on social functioning warrants further exploration.We assessed the clinical symptoms, antipsychotic adverse reactions, and social functioning of 283 hospitalized patients with chronic schizophrenia using various standardized interviews and scales. We conducted multiple regression and mediation analyses to elucidate the impact of secondary factors on negative symptoms, and the relationship among these “secondary factors,” negative symptoms, and social functioning.Our findings identified depressive symptoms, extrapyramidal symptoms, and positive symptoms as significant contributors to secondary negative symptoms. We found that negative symptoms play a notable mediating role in the effect of depressive and positive symptoms on social functioning. However, the relationship between positive symptoms, negative symptoms, and social functioning proved to be intricate.Our findings propose that negative symptoms act as pivotal mediators in the correlation between “secondary factors” (including the depressive symptoms and positive symptoms) and social functioning. The treatment of chronic schizophrenia necessitates focusing on key factors such as depressive and positive symptoms, which might significantly contribute to the development of secondary negative symptoms. Further research is essential to clarify the complex relationship among positive symptoms, negative symptoms, and social functioning in schizophrenia. PrimarySocial functioning, defined as the capacity to comprehend and partake in social activities, thus facilitating effective and regular involvement in social life , constitThe main objective of this study is 2-fold. Firstly, we aimed to investigate several potential “secondary factors” that might lead to negative symptoms in chronic schizophrenia. Secondly, by employing mediation analysis, the relationship among (1) negative symptoms, (2) secondary factors , and (3) social functioning were explored. For this study, our hypothesis suggests that the “secondary factors” has an impact on social functioning by a mediator of the severity of negative symptoms.2.2.1.The present study employed a cross-sectional design and conducted a sampling survey at the one of the Mental Health Centers of Beijing from March 1, 2018, to September 30, 2018. A total of 283 hospitalized patients between the ages of 18 and 60, who had been diagnosed with schizophrenia by two psychiatrists according to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), were recruited as participants. Eligible participants had a course of schizophrenia lasting more than 5 years and had been hospitalized for at least 5 years.2.2.Inclusion criteria of this study were as followed: (1) diagnosed with schizophrenia by the DSM-5 diagnostic criteria; (2) having a disease duration of more than 5 years; (3) hospitalized for longer than 5 years; (4) aged between 18 and 60; (5) had not have any changes in antipsychotic medication for at least 6 weeks; and (6) voluntary participation with informed consent. Exclusion criteria were: (1) having comorbid mental disorders; (2) having brain diseases or serious/unstable physical illnesses; (3) being pregnant or lactating; (4) having drug or alcohol abuse/dependence; (5) having received modified electroconvulsive therapy within the past 3 months; and (6) were unable to complete all surveys. Informed consent obtained from all patients and their guardians. The study protocol was approved by the Ethics Committee of the one Mental Health Center of Beijing (protocol number: 109).2.3.2.3.1.The positive and negative syndrome scale (PANSS) is a widely used tool designed to evaluate the severity of schizophrenia symptoms . The PAN2.3.2.The scale for assessment of negative symptoms (SANS) is a well-established instrument in evaluating negative symptoms across five domains, including affective flattening, alogia, avolition apathy, anhedonia-asociality, and attention. The scale consists of 25 items, which are rated on a six-point Likert scale ranging from 0 to 5 points, with higher scores indicating greater impairment . The ChiFactor analysis of the general psychopathological rating scales found that some items in SANS did not belong to the category of negative symptoms, including attentional impairment , inappropriate affect (SANS item 6), and poverty of content of speech (SANS item 10) , 8. Ther2.3.3.The scale for the assessment of positive symptoms (SAPS) scale is comprised of 34 items that evaluate positive symptoms of schizophrenia across four domains: hallucinations, delusions, bizarre behavior, and positive formal thought disorder . Each it2.3.4.The Simpson–Angus scale (SAS) is a validated sensitive tool to evaluate extrapyramidal side effects caused by neuroleptic drugs , includi2.3.5.The Calgary depression scale for schizophrenia (CDSS) was developed to evaluate depressive symptoms in individuals with schizophrenia. It consists of nine items, each rated on a Likert scale of 0–3, with higher scores indicate more severe symptoms .2.3.6.The Global Assessment of Functioning (GAF) is a widely used scale in both research and clinical practice for assessing the overall functioning of patients with schizophrenia, focusing on psychological, social, and occupational well-being . The totFour professional psychiatrists who had undergone training and consistency evaluation (the Intraclass Correlation Coefficient is 0.90) were recruited to evaluate the schizophrenia scale on site, and the survey results were collected. Each eligible patient was assessed by two psychiatrists on the same day. The first psychiatrist evaluated the patients and collected data on their sociodemographic information, clinical data, and psychiatric history through a questionnaire. The second psychiatrist administered all six scales to the patient.2.4.p < 0.05.Data analysis was performed using SPSS for Windows 23.0. Descriptive statistics were used to summarize continuous variables in terms of mean and standard deviation. Firstly, multiple linear regression was used to explore the factors that may significantly impact negative symptoms, with the total SANS score as the dependent variable and the common factors of secondary negative symptoms as independent variables. Secondly, we utilized a linear regression model to separately explore: (1) The prediction of social function as evaluated by Global Assessment of Functioning (GAF), by the extrapyramidal symptoms evaluated by SAS, depressive symptoms evaluated by CDSS, and positive symptoms evaluated by SAPS; (2) The combined prediction of these three factors  for negative symptoms; (3) The prediction of social function (GAF) by negative symptoms, evaluated by Scale for the Assessment of Negative Symptoms (SANS), after excluding items not related to negative symptoms. Furthermore, the impact of each negative symptom factor on social functioning was further studied using multiple linear regression, with the GAF score as the dependent variable and the SANS score on the five subscales as independent variables. Finally, mediation analysis was carried out using packages in R (Mediation)  to inves3.3.1.A total of 283 long-term hospitalized patients with chronic schizophrenia participated in this study, with an age range of 29–60 and an average age of 43.83 ± 8.19. The mean age of onset was 24.57 ± 8.042, the mean course of disease was 8.92 ± 5.075 years, and the mean length of hospital stay was 6.30 ± 4.535 years. Participants exhibited significant negative symptoms (mean SANS score of 19.548 ± 10.541), and relatively mild positive (mean SAPS score of 3.15 ± 4.466) and depressive symptoms (mean CDSS score of 1.70 ± 2.00). Moreover, their social functioning was impaired, as evidenced by a mean GAF score of 68.16 ± 7.345 see .3.2.p < 0.001). Moreover, the scores from the CDSS (p = 0.001), SAS (p = 0.007), and the SAPS (p = 0.048) significantly predict the SANS score. However, only the CDSS (p = 0.019) and SAPS (p = 0.045) scores significantly predict the GAF score  and social functioning, and partially mediated the relationship between positive symptoms  and social functioning  and Clinical Assessment Interview for Negative Symptoms (CAINS). These updated scales resonate more with current conceptualizations of negative symptoms and enable a clear distinction between primary and secondary negative symptoms. Secondly, our study lacked data pertaining to the temporal relationship between the onset of negative symptoms and the commencement and augmentation of antipsychotic treatment. As such, we were unable to verify whether all extrapyramidal symptoms we noted were indeed secondary to antipsychotic treatment. Thirdly, considering that not all aspects of function might be impacted by negative symptoms, future research could potentially adopt rating scales that offer a clear separation of functional aspects. This could provide a more nuanced assessment of patients’ social functioning. Fourthly, the relatively small sample size in our study may limit the extrapolation of our findings. To validate our results, larger-scale studies are recommended. Finally, to further unravel the temporal dynamics of negative symptoms and their contributory factors, it would be beneficial for future research to include longitudinal data.5.This study indicates that factors depressive symptoms and positive symptoms may play significant roles in the evolution of secondary negative symptoms in schizophrenia. Our findings propose that negative symptoms act as pivotal mediators in the correlation between “secondary factors” (including the depressive symptoms and positive symptoms) and social functioning. Therefore, in managing chronic schizophrenia, the targeted treatment of depressive symptoms, as well as the positive symptoms could represent a crucial strategy for enhancing social functioning by addressing secondary negative symptoms.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The study protocol was approved by the Ethics Committee of one Mental Health Center in Beijing (protocol number: 109). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.The concept for this article was proposed by YL, and NH, WL, HD, JS, HY, JC, WH, HW, XZ, PZ, SH, YC, and TF collaborated to develop the protocol, establish the search strategy, and define the inclusion and exclusion criteria. Data collection was conducted by NH, WL, HD, JS, HY, JC, WH, HW, PZ, SH, XZ, and YC, while YL performed the statistical analyses, interpreted the data, and created the figures and tables. NH and TF drafted the initial version of the report with critical feedback from YL and YC. NH, WL, HD, JS, HY, JC, WH, HW, XZ, PZ, SH, YC, TF, and YL participated in the interpretation of the findings, and revision of the report, and gave their final approval of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China under Grant Nos. 81901345, 82001445, and 82171538, the Natural Science Foundation of Beijing Municipality under Grant Nos. 7212035, and the Beijing Municipal Administration of Hospitals’ Youth Program under Grant No. QML20232006, QML20232003, and QML20211203.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.