Non‐organ‐specific autoimmunity in adult 47,XXY Klinefelter patients and higher‐grade X‐chromosome aneuploidies

Current literature regarding systemic autoimmune diseases in X‐chromosome aneuploidies is scarce and limited to case reports. Our aim was to evaluate the frequency of anti‐nuclear (ANAs), extractable nuclear (ENA), anti‐double‐stranded DNA (dsDNAs), anti‐smooth muscle (ASMAs) and anti‐mitochondrial (AMAs) antibodies in a large cohort of adults with Klinefelter’s syndrome (KS, 47,XXY) and rare higher‐grade sex chromosome aneuploidies (HGAs) for the first time. Sera from 138 X‐chromosome aneuploid patients [124 adult patients with 47,XXY KS and 14 patients with HGA (six children, eight adults)] and 50 age‐matched 46,XY controls were recruited from the Sapienza University of Rome (2007–17) and tested for ANAs, ENAs, anti‐dsDNAs, ASMAs and AMAs. Non‐organ‐specific immunoreactivity was found to be significantly higher in patients with 47,XXY KS (14%) than in the controls (2%, p = 0.002). Among all the antibodies investigated, only ANAs were observed significantly more frequently in patients with 47,XXY KS (12.1%) than in the controls (2%, p = 0.004). No anti‐dsDNA immunoreactivity was found. Stratifying by testosterone replacement therapy (TRT), non‐organ‐specific autoantibody frequencies were higher in TRT‐naive (p = 0.01) and TRT‐treated groups than in controls. No patients with HGA were found positive for the various autoantibodies. Non‐organ‐specific autoantibodies were significantly present in 47,XXY adult patients. Conversely, HGAs did not appear to be target of non‐organ‐specific immunoreactivity, suggesting that KS and HGAs should be considered as two distinct conditions. The classification and diagnosis of systemic autoimmune diseases is frequently difficult. To support a correct clinical evaluation of KS disease and to prevent eventual secondary irreversible immune‐mediated damages, we highlight the importance of screening for non‐organ‐specific autoimmunity in Klinefelter’s syndrome.     

Sensitivity of SARS‐CoV‐2 to different temperatures

This study was designed to investigate the sensitivity of SARS‐CoV‐2 to different temperatures, to provide basic data and a scientific basis for the control of COVID‐19 epidemic. The virus was dispersed in 1 mL basal DMEM medium at a final concentration of 10^3.2 TCID₅₀/mL and then incubated at 4, 22, 30, 35, 37, 38, 39 and 40°C for up to 5 days. The infectivity of residual virus was titrated using the Vero E6 cell line. The results showed that the virus remained viable for 5 days at 4°C, and for 1 day only at 22 and 30°C. We found that the infectivity of the virus was completely lost after less than 12 hours at 37, 38 and 39°C, while at 40°C, the inactivation time of the virus was rapidly reduced to 6 hours. We show that SARS‐CoV‐2 is sensitive to heat, is more stable at lower temperatures than higher temperature, remains viable for longer at lower temperatures, and loses viability rapidly at higher temperatures.     

Limbic‐predominant age‐related TDP‐43 encephalopathy neuropathologic change and microvascular pathologies in community‐dwelling older persons

Limbic‐predominant age‐related transactive response DNA‐binding protein 43 (TDP‐43) encephalopathy neuropathologic change (LATE‐NC) and microvascular pathologies, including microinfarcts, cerebral amyloid angiopathy (CAA), and arteriolosclerosis are common in old age. A relationship between LATE‐NC and arteriolosclerosis has been reported in some but not all studies. The objectives of this study were to investigate the frequency of co‐occurring LATE‐NC and microvascular pathologies and test the hypothesis that arteriolosclerosis, specifically, is related to LATE‐NC in brains from community‐dwelling older persons. Analyses included 749 deceased participants with completed data on LATE‐NC and microvascular pathology from 3 longitudinal clinical pathologic studies of aging. Given the specific interest in arteriolosclerosis, we expanded the examination of arteriolosclerosis to include not only the basal ganglia but also two additional white matter regions from anterior and posterior watershed territories. Ordinal logistic regression models examined the association of microvascular pathology with LATE‐NC. LATE‐NC was present in 409 (54.6%) decedents, of which 354 (86.5%) had one or multiple microvascular pathologies including 132 (32.3%) with moderate‐severe arteriolosclerosis in basal ganglia, 195 (47.6%) in anterior watershed, and 144 (35.2%) in posterior watershed; 170 (41.5%) with moderate‐severe CAA, and 150 (36.6%) with microinfarcts. In logistic regression models, only posterior watershed arteriolosclerosis, but not other regions of arteriolosclerosis was associated with a higher odds of more advanced LATE‐NC stages (Odds Ratio = 1.12; 95% Confidence Interval = 1.01–1.25) after controlling for demographics, AD, and other age‐related pathologies. Capillary CAA, but not the severity of CAA was associated with an increased odds of LATE‐NC burden (Odds Ratio = 1.71; 95% Confidence Interval = 1.13–2.58). Findings were unchanged in analyses controlling for APOE ε4, vascular risk factors, or vascular diseases. These findings suggest that LATE‐NC with microvascular pathology is a very common mixed pathology and small vessel disease pathology may contribute to LATE‐NC in the aging brain.     

N‐CAM Dysfunction and Unexpected Accumulation of PSA‐NCAM in Brain of Adult‐Onset Autosomal‐Dominant Leukodystrophy

Previously, myelin from cerebral white matter (CWM) of two subjects of a family with orthochromatic adult‐onset autosomal‐dominant leukodystrophy (ADLD) was disclosed to exhibit defective large isoform of myelin‐associated glycoprotein (L‐MAG) and patchy distribution only in the elder subject. L‐MAG and neural cell adhesion molecule (N‐CAM) (N‐CAM 180, 140, and 120) are structurally related and concur to myelin/axon interaction. In early developmental stages, in neurons and glia N‐CAM is converted into polysialylated (PSA)‐NCAM by two sialyltransferases sialyltransferase‐X (STX) and polysialyltransferase‐1 (PST). Notably, PSA‐NCAM disrupts N‐CAM adhesive properties and is nearly absent in the adult brain.Here, CWM extracts and myelin of the two subjects were searched for the expression pattern of the N‐CAM isoforms and PSA‐NCAM, and their CWM was evaluated for N‐CAM, STX and PST gene copy number and gene expression as mRNA. Biochemically, we disclosed that in CWM extracts and myelin from both subjects, PSA‐NCAM accumulates, N‐CAM 180 considerably increases, N‐CAM 140 is modestly modified and N‐CAM 120 remarkably decreases; duplication of genes encoding N‐CAM, STX and PST was not revealed, whereas PST mRNA was clearly increased. Immunohistochemically, in CWM of both subjects, we found an unusually diffuse accumulation of PSA‐NCAM without inflammation markers. PSA‐NCAM persistence, up‐regulated PST mRNA and previously uncovered defective L‐MAG may be early pathogenetic events in this ADLD form.     

Development of a high‐sensitivity ELISA detecting IgG,IgA and IgM antibodies to the SARS‐CoV‐2 spike glycoprotein in serum and saliva

Detecting antibody responses during and after SARS‐CoV‐2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non‐hospitalized SARS‐CoV‐2‐infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti‐spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti‐spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT‐PCR confirmed, non‐hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti‐spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS‐CoV‐2 infection.     

Comparison of the replication and neutralization of different SARS‐CoV‐2 Omicron subvariants in vitro

BackgroundNew Omicron subvariants are emerging rapidly from BA.1 to BA.4 and BA.5. Their pathogenicity has changed from that of wild‐type (WH‐09) and Omicron variants have over time become globally dominant. The spike proteins of BA.4 and BA.5 that serve as the target for vaccine‐induced neutralizing antibodies have also changed compared to the previous subvariants, which is likely to cause immune escape and the reduction of the protective effect of the vaccine. Our study addresses the above issues and provides a basis for formulating relevant prevention and control strategies.MethodsWe collected cellular supernatant and cell lysates and measured the viral titers, viral RNA loads, and E subgenomic RNA (E sgRNA) loads in different Omicron subvariants grown in Vero E6 cells, using WH‐09 and Delta variants as a reference. Additionally, we evaluated the in vitro neutralizing activity of different Omicron subvariants and compared it to the WH‐09 and Delta variants using macaque sera with different types of immunity.ResultsAs the SARS‐CoV‐2 evolved into Omicron BA.1, the replication ability in vitro began to decrease. Then with the emergence of new subvariants, the replication ability gradually recovered and became stable in the BA.4 and BA.5 subvariants. In WH‐09‐inactivated vaccine sera, geometric mean titers of neutralization antibodies against different Omicron subvariants declined by 3.7~15.4‐fold compared to those against WH‐09. In Delta‐inactivated vaccine sera, geometric mean titers of neutralization antibodies against Omicron subvariants declined by 3.1~7.4‐fold compared to those against Delta.ConclusionAccording to the findings of this research, the replication efficiency of all Omicron subvariants declined compared with WH‐09 and Delta variants, and was lower in BA.1 than in other Omicron subvariants. After two doses of inactivated (WH‐09 or Delta) vaccine, cross‐neutralizing activities against various Omicron subvariants were seen despite a decline in neutralizing titers.     

SARS‐CoV‐2 infection aggravates chronic comorbidities of cardiovascular diseases and diabetes in mice

BackgroundCardiovascular diseases (CVDs) and diabetes mellitus (DM) are top two chronic comorbidities that increase the severity and mortality of COVID‐19. However, how SARS‐CoV‐2 alters the progression of chronic diseases remain unclear.MethodsWe used adenovirus to deliver h‐ACE2 to lung to enable SARS‐CoV‐2 infection in mice. SARS‐CoV‐2’s impacts on pathogenesis of chronic diseases were studied through histopathological, virologic and molecular biology analysis.ResultsPre‐existing CVDs resulted in viral invasion, ROS elevation and activation of apoptosis pathways contribute myocardial injury during SARS‐CoV‐2 infection. Viral infection increased fasting blood glucose and reduced insulin response in DM model. Bone mineral density decreased shortly after infection, which associated with impaired PI3K/AKT/mTOR signaling.ConclusionWe established mouse models mimicked the complex pathological symptoms of COVID‐19 patients with chronic diseases. Pre‐existing diseases could impair the inflammatory responses to SARS‐CoV‐2 infection, which further aggravated the pre‐existing diseases. This work provided valuable information to better understand the interplay between the primary diseases and SARS‐CoV‐2 infection.     

ATP/IL‐33‐triggered hyperactivation of mast cells results in an amplified production of pro‐inflammatory cytokines and eicosanoids

IL‐33 and ATP are alarmins, which are released upon damage of cellular barriers or are actively secreted upon cell stress. Due to high‐density expression of the IL‐33 receptor T1/ST2 (IL‐33R), and the ATP receptor P2X7, mast cells (MCs) are one of the first highly sensitive sentinels recognizing released IL‐33 or ATP in damaged peripheral tissues. Whereas IL‐33 induces the MyD88‐dependent activation of the TAK1‐IKK2‐NF‐κB signalling, ATP induces the Ca²⁺‐dependent activation of NFAT. Thereby, each signal alone only induces a moderate production of pro‐inflammatory cytokines and lipid mediators (LMs). However, MCs, which simultaneously sense (co‐sensing) IL‐33 and ATP, display an enhanced and prolonged activation of the TAK1‐IKK2‐NF‐κB signalling pathway. This resulted in a massive production of pro‐inflammatory cytokines such as IL‐2, IL‐4, IL‐6 and GM‐CSF as well as of arachidonic acid‐derived cyclooxygenase (COX)‐mediated pro‐inflammatory prostaglandins (PGs) and thromboxanes (TXs), hallmarks of strong MC activation. Collectively, these data show that co‐sensing of ATP and IL‐33 results in hyperactivation of MCs, which resembles to MC activation induced by IgE‐mediated crosslinking of the FcεRI. Therefore, the IL‐33/IL‐33R and/or the ATP/P2X7 signalling axis are attractive targets for therapeutical intervention of diseases associated with the loss of integrity of cellular barriers such as allergic and infectious respiratory reactions.     

Interleukin‐4 protects mice against lethal influenza and Streptococcus pneumoniae co‐infected pneumonia

Streptococcus pneumoniae co‐infection post‐influenza is a major cause of mortality characterized by uncontrolled bacteria burden and excessive immune response during influenza pandemics. Interleukin (IL)‐4 is a canonical type II immune cytokine known for its wide range of biological activities on different cell types. It displays protective roles in numerous infectious diseases and immune‐related diseases, but its role in influenza and S. pneumoniae (influenza/S. pneumoniae) co‐infected pneumonia has not been reported. In our study, we used C57BL/6 wild‐type (WT) and IL‐4‐deficient (IL‐4^−/−) mice to establish co‐infection model with S. pneumoniae after influenza virus infection. Co‐infected IL‐4^−/− mice showed increased mortality and weight loss compared with WT mice. IL‐4 deficiency led to increased bacterial loads in lungs without altering influenza virus replication, suggesting a role of IL‐4 in decreasing post‐influenza susceptibility to S. pneumoniae co‐infection. Loss of IL‐4 also resulted in aggravated lung damage together with massive proinflammatory cytokine production and immune cell infiltration during co‐infection. Administration of recombinant IL‐4 rescued the survival and weight loss of IL‐4^−/− mice in lethal co‐infection. Additionally, IL‐4 deficiency led to more immune cell death in co‐infection. Gasdermin D (GSDMD) during co‐infection was induced in IL‐4^−/− mice that subsequently activated cell pyroptosis. Treatment of recombinant IL‐4 or inhibition of GSDMD activity by disulfiram decreased immune cell death and bacterial loads in lungs of IL‐4^−/− co‐infected mice. These results suggest that IL‐4 decreases post‐influenza susceptibility to S. pneumoniae co‐infection via suppressing GSDMD‐induced pyroptosis. Collectively, this study demonstrates the protective role of IL‐4 in influenza/S. pneumoniae co‐infected pneumonia.     

The detection of long‐lasting memory foot‐and‐mouth disease (FMD) virus serotype O‐specific CD4+ T cells from FMD‐vaccinated cattle by bovine major histocompatibility complex class II tetramer

Foot‐and‐mouth disease (FMD) is a highly contagious, economically devastating disease of cloven‐hooved animals. The development of long‐lasting effective FMD vaccines would greatly benefit the global FMD control programme. Deep analysis of adaptive immunity in cattle vaccinated against FMD is technically challenging due to the lack of species‐specific tools. In this study, we aimed to identify CD4⁺ T‐cell epitopes in the FMD virus (FMDV) capsid and to phenotype the CD4⁺ T cells that recognize them using bovine major histocompatibility complex (BoLA) class II tetramer. A BoLA class II tetramer based on the DRA/DRB3*020:02 allele and FMDV antigen‐stimulated PBMCs from bovine vaccinates were used to successfully identify four epitopes in the FMDV capsid, three of which have not been previously reported; two epitopes were identified in the structural protein VP1, one in VP3 and one in VP4. Specificity of the three novel epitopes was confirmed by proliferation assay. All epitope‐expanded T‐cell populations produced IFN‐γ in vitro, indicating a long‐lasting Th1 cell phenotype after FMD vaccination. VP3‐specific CD4⁺ T cells exhibited the highest frequency amongst the identified epitopes, comprising >0·004% of the CD4⁺ T‐cell population. CD45RO⁺CCR7⁺ defined central memory CD4⁺ T‐cell subpopulations were present in higher frequency in FMDV‐specific CD4⁺ T‐cell populations from FMD‐vaccinated cattle ex vivo. This indicates an important role in maintaining cell adaptive immunity after FMD vaccination. Notably, FMDV epitope‐loaded tetramers detected the presence of FMDV‐specific CD4⁺ T cells in bovine PBMC more than four years after vaccination. This work contributes to our understanding of vaccine efficacy.     

Role of miR‐21‐5p/FilGAP axis in estradiol alleviating the progression of monocrotaline‐induced pulmonary hypertension

BackgroundAberrant expression of microRNAs (miRNAs) has been associated with the pathogenesis of pulmonary hypertension (PH). It is, however, not clear whether miRNAs are involved in estrogen rescue of PH.MethodsFresh plasma samples were prepared from 12 idiopathic pulmonary arterial hypertension (IPAH) patients and 12 healthy controls undergoing right heart catheterization in Shanghai Pulmonary Hospital. From each sample, 5 μg of total RNA was tagged and hybridized on microRNA microarray chips. Monocrotaline‐induced PH (MCT‐PH) male rats were treated with 17β‐estradiol (E₂) or vehicle. Subgroups were cotreated with estrogen receptor (ER) antagonist or with antagonist of miRNA.ResultsMany circulating miRNAs, including miR‐21‐5p and miR‐574‐5p, were markedly expressed in patients and of interest in predicting mean pulmonary arterial pressure elevation in patients. The expression of miR‐21‐5p in the lungs was significantly upregulated in MCT‐PH rats compared with the controls. However, miR‐574‐5p showed no difference in the lungs of MCT‐PH rats and controls. miR‐21‐5p was selected for further analysis in rats as E₂ strongly regulated it. E₂ decreased miR‐21‐5p expression in the lungs of MCT‐PH rats by ERβ. E₂ reversed miR‐21‐5p target gene FilGAP downregulation in the lungs of MCT‐PH rats. The abnormal expression of RhoA, ROCK2, Rac1 and c‐Jun in the lungs of MCT‐PH rats was inhibited by E₂ and miR‐21‐5p antagonist.ConclusionsmiR‐21‐5p level was remarkably associated with PH severity in patients. Moreover, the miR‐21‐5p/FilGAP signaling pathway modulated the protective effect of E₂ on MCT‐PH through ERβ.     

Non‐structural protein 1‐specific antibodies directed against Zika virus in humans mediate antibody‐dependent cellular cytotoxicity

There is growing interest in understanding antibody (Ab) function beyond neutralization. The non‐structural protein 1 (NS1) of Zika virus (ZIKV) is an attractive candidate for an effective vaccine as Abs against NS1, unlike the envelope or premembrane, do not carry the risk of mediating antibody‐dependent enhancement. Our aim was to evaluate whether ZIKV NS1 Abs elicited following natural infection in humans can mediate antibody‐dependent cellular cytotoxicity (ADCC). We evaluated the isotype specificity of ZIKV‐specific Abs in immune sera and supernatants from stimulated immune PBMC and found that Abs against ZIKV NS1 and virus‐like particles were predominantly of the IgG1 isotype. Using a recently developed FluoroSpot assay, we found robust frequencies of NS1‐specific Ab‐secreting cells in PBMC of individuals who were naturally infected with ZIKV. We developed assays to measure both natural killer cell activation by flow cytometry and target cell lysis of ZIKV NS1‐expressing cells using an image cytometry assay in the presence of ZIKV NS1 Abs. Our data indicate efficient opsonization of ZIKV NS1‐expressing CEM‐NK^R cell lines using ZIKV‐immune but not ZIKV‐naïve sera, a prerequisite of ADCC. Furthermore, sera from immune donors were able to induce both NK cell degranulation and lysis of ZIKV NS1 CEM‐NK^R cells in vitro. Our data suggest that ADCC is a possible mechanism for ZIKV NS1 Abs to eliminate virally infected target cells.     

Human CD4+ T cells specific for dominant epitopes of SARS‐CoV‐2 Spike and Nucleocapsid proteins with therapeutic potential

Since December 2019, Coronavirus disease‐19 (COVID‐19) has spread rapidly throughout the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID‐19 is mediated by both short‐lived neutralizing antibodies and long‐lasting virus‐reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID‐19 patients with a potentially protective response to two major antigens of the SARS‐CoV‐2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus‐reactive CD4⁺ T cells, we were able to confirm a set of nine immunodominant epitopes and characterize T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR‐αβ sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR‐transgenic CD4⁺ T cell therapy of COVID‐19.