Comparison of Neuroinflammation Induced by Hyperphosphorylated Tau Protein Versus Ab42 in Alzheimer's Disease.

Both neurofibrillary tangles and senile plaques are associated with inflammation in Alzheimer's disease (AD). Their relative degree of induced neuroinflammation, however, is not well established. Mouse models of AD that expressed either human Aß42 (n = 7) or human hyperphosphorylated tau protein alone (n = 3), wild type (n = 10), and human AD samples (n = 29 with 18 controls) were studied. The benefit of using mouse models that possess only human tau or amyloid-b is that it allows for the individual evaluation of how each protein affects neuroinflammation, something not possible in human tissue. Three indicators of neuroinflammation were examined: TLRs/RIG1 expression, the density of astrocytes and microglial cells, and well-established mediators of neuroinflammation (IL6, TNFα, IL1ß, and CXCL10). There was a statistically significant increase in neuroinflammation with all three variables in the mouse models with human tau only as compared to human Aß42 only or wild-type mice (each at p < 0.0001). Only the Aß42 5xFAD mice (n = 4) showed statistically higher neuroinflammation versus wild type (p = 0.0030). The human AD tissues were segregated into Aß42 only or hyperphosphorylated tau protein with Aß42. The latter areas showed increased neuroinflammation with each of the three variables compared to the areas with only Aß42. Of the TLRs and RIG-1, TLR8 was significantly elevated in both the mouse model and human AD and only in areas with the abnormal tau protein. It is concluded that although Aß42 and hyperphosphorylated tau protein can each induce inflammation, the latter protein is associated with a much stronger neuroinflammatory response vis-a-vis a significantly greater activated microglial response.

Comprehensive Single-Cell Immune Profiling Defines the Patient Multiple Myeloma Microenvironment Following Oncolytic Virus Therapy in a Phase Ib Trial.

PURPOSE: Our preclinical studies showed that the oncolytic reovirus formulation pelareorep (PELA) has significant immunomodulatory anti-myeloma activity. We conducted an investigator-initiated clinical trial to evaluate PELA in combination with dexamethasone (Dex) and bortezomib (BZ) and define the tumor immune microenvironment (TiME) in patients with multiple myeloma treated with this regimen. PATIENTS AND METHODS: Patients with relapsed/refractory multiple myeloma (n = 14) were enrolled in a phase Ib clinical trial (ClinicalTrials.gov: NCT02514382) of three escalating PELA doses administered on Days 1, 2, 8, 9, 15, and 16. Patients received 40 mg Dex and 1.5 mg/m2 BZ on Days 1, 8, and 15. Cycles were repeated every 28 days. Pre- and posttreatment bone marrow specimens (IHC, n = 9; imaging mass cytometry, n = 6) and peripheral blood samples were collected for analysis (flow cytometry, n = 5; T-cell receptor clonality, n = 7; cytokine assay, n = 7). RESULTS: PELA/BZ/Dex was well-tolerated in all patients. Treatment-emergent toxicities were transient, and no dose-limiting toxicities occurred. Six (55%) of 11 response-evaluable patients showed decreased paraprotein. Treatment increased T and natural killer cell activation, inflammatory cytokine release, and programmed death-ligand 1 expression in bone marrow. Compared with nonresponders, responders had higher reovirus protein levels, increased cytotoxic T-cell infiltration posttreatment, cytotoxic T cells in significantly closer proximity to multiple myeloma cells, and larger populations of a novel immune-primed multiple myeloma phenotype (CD138+ IDO1+HLA-ABCHigh), indicating immunomodulation. CONCLUSIONS: PELA/BZ/Dex is well-tolerated and associated with anti-multiple myeloma activity in a subset of responding patients, characterized by immune reprogramming and TiME changes, warranting further investigation of PELA as an immunomodulator.

The differential expression of toll like receptors and RIG-1 correlates to the severity of infectious diseases.

The toll like receptors (TLRs) and RIG-1 are proteins involved in the initial reaction of the innate immune system to infectious diseases and, thus, can provide much information to the surgical pathologist in terms of the molecular dynamics of the infection. The TLRs (TLR1, 2, 3, 4, 7, 8) and RIG-1 distribution as determined by immunohistochemistry was examined in the following diseases: human papillomavirus (n = 30 including 15 squamous intraepithelial lesions (SIL), 5 cancers, and 10 controls); molluscum contagiosum (n = 8 including 4 controls), SARS-CoV2 (n = 52 including 20 mild, 5 fatal, and 27 controls) and reovirus infection as oncolytic therapy. Mild, regressing infection (molluscum contagiosum, mild SARS-CoV2 and low grade SIL) each showed the same pattern: marked up regulation of at least three of the TLRs/RIG-1 with decreased expression of none compared to the controls. Severe infection (fatal SARS-CoV2, and cervical cancer) each showed marked decrease expression in at least three of the TLRs/RIG-1. We recently documented an equivalent marked decrease expression of the TLRs/RIG-1 in the placenta in fatal in utero infections. The reoviral infected tissues showed an overall pattern of marked increase expression of TLRs/RIG-1, consistent with a strong anti-viral response. Thus, the in situ testing of infectious diseases by a panel of these early infectious disease recognition proteins may allow the surgical pathologist to predict the outcome of the disease which, in turn, may assist in the understanding of the role of the TLRs/RIG-1 in determining the fate of a given infectious process.

The rapid diagnosis of Mycoplasma pneumonia using in situ hybridization on clinical samples.

The microbiological etiology of seasonal upper respiratory illnesses in the United States is dominated by viruses, including influenza A, B, respiratory syncytial virus, and SARS-CoV2. Mycoplasma pneumonia, treatable with antibiotics, can also cause upper respiratory symptoms and is typically associated with about 15 % of cases. There is no clinical or radiologic finding diagnostic of Mycoplasma pneumonia infection and PCR-based testing is not routinely used in the clinical setting. Further, the bacteria grows slowly in culture and the diagnostic IgM response will take days after the onset of infection. Thus, a rapid diagnostic test for Mycobacterium pneumonia infection is needed. This study documented two cases of Mycoplasma pneumonia infection of the upper respiratory system using in situ hybridization in a series of over 20 patients who were being tested for SARS-CoV2 infection. The respiratory secretions were placed on a glass slide, fixed in 10 % buffered formalin, and then tested using a Mycoplasma pneumonia probe. The high bacterial number associated with acute infection allowed for straightforward detection by in situ hybridization in a few hours. Antibiotic therapy led to rapid resolution of the symptoms. This highlights the ability of standard in situ hybridization as a rapid diagnostic test for Mycoplasma pneumonia in the clinical setting.

The differential expression of toll like receptors and RIG-1 in the placenta of neonates with in utero infections.

Novel biomarkers of in utero infections are needed to help guide early therapy. The toll like receptors (TLRs) and retinoic acid-inducible gene 1 (RIG-1) are proteins involved in the initial reaction of the innate immune system to infectious diseases. This study tested the hypothesis that a panel of TLRs and RIG-1 in the placenta could serve as an early biomarker of in utero infections. The TLRs and RIG-1 expression as determined by immunohistochemistry was scored in 10 control placentas (normal delivery or neonatal damage from known non-infectious cause), 8 placentas from documented in utero bacterial infection, and 7 placentas from documented in utero viral infections blinded to the clinical information. The non-infected placentas showed the following profile: no expression (TLR1, TLR3, TLR4, TLR7, TLR8), moderate expression (TLR2), and strong expression (RIG-1). The bacterial and viral infection cases shared the following profile: no to mild expression (TLR 2, TLR7, and RIG1), moderate expression (TLR4), and strong expression (TLR1, TLR3, and TLR8). The histologic findings in the chorionic villi were equivalent in the infected cases and controls, underscoring the need for molecular testing by the surgical pathologist when in utero infection is suspected. The results suggest that a panel of TLRs/RIG-1 analyses can allow the pathologist and/or clinician to diagnose in utero infections soon after birth. Also, treatments to antagonize the effects of TLR1, 3, and 8 may help abrogate in utero neonatal damage.

The amplification of CNS damage in Alzheimer's disease due to SARS-CoV2 infection.

Pre-existing Alzheimer's disease is a risk factor for severe/fatal COVID-19 and infection by SARS-CoV2 virus has been associated with an increased incidence of un-masked Alzheimer's disease. The molecular basis whereby SARS-CoV2 may amplify Alzheimer's disease is not well understood. This study analyzed the molecular changes in autopsy brain tissues from people with pre-existing dementia who died of COVID-19 (n = 5) which was compared to equivalent tissues of people who died of COVID-19 with no history of dementia (n = 8), Alzheimer's disease pre-COVID-19 (n = 10) and aged matched controls (n = 10) in a blinded fashion. Immunohistochemistry analyses for hyperphosphorylated tau protein, α-synuclein, and ß-amyloid-42 confirmed the diagnoses of Alzheimer's disease (n = 4), and Lewy body dementia (n = 1) in the COVID-19 group. The brain tissues from patients who died of COVID-19 with no history of dementia showed a diffuse microangiopathy marked by endocytosis of spike subunit S1 and S2 in primarily CD31+ endothelia with strong co-localization with ACE2, Caspase-3, IL6, TNFα, and Complement component 6 that was not associated with SARS-CoV2 RNA. Microglial activation marked by increased TMEM119 and MCP1 protein expression closely paralleled the endocytosed spike protein. The COVID-19 tissues from people with no pre-existing dementia showed, compared to controls, 5-10× fold increases in expression of neuronal NOS and NMDAR2 as well as a marked decrease in the expression of proteins whose loss is associated with worsening Alzheimer's disease: MFSD2a, SHIP1, BCL6, BCL10, and BACH1. In COVID-19 tissues from people with dementia the widespread spike-induced microencephalitis with the concomitant microglial activation co-existed in the same areas where neurons had hyperphosphorylated tau protein suggesting that the already dysfunctional neurons were additionally stressed by the SARS-CoV2 induced microangiopathy. ACE2+ human brain endothelial cells treated with high dose (but not vaccine equivalent low dose) spike S1 protein demonstrated each of the molecular changes noted in the in vivo COVID-19 and COVID-19/Alzheimer's disease brain tissues. It is concluded that fatal COVID-19 induces a diffuse microencephalitis and microglial activation in the brain due to endocytosis of circulating viral spike protein that amplifies pre-existing dementia in at least two ways: 1) modulates the expression of proteins that may worsen Alzheimer's disease and 2) stresses the already dysfunctional neurons by causing an acute proinflammatory/hypercoagulable/hypoxic microenvironment in areas with abundant hyperphosphorylated tau protein and/or ßA-42.

The differential immune response in mild versus fatal SARS-CoV2 infection.

This study compared the immune response in mild versus fatal SARS-CoV2 infection. Forty nasopharyngeal swabs with either productive mild infection (n = 20) or negative for SARS-CoV2 (n = 20) were tested along with ten lung sections from people who died of COVID-19 which contained abundant SARS-CoV2 and ten controls. There was a 25-fold increase in the CD3+T cell numbers in the viral positive nasopharyngeal swabs compared to the controls (p < 0.001) and no change in the CD3+T cell count in the fatal COVID-19 lungs versus the controls. CD11b + and CD206+ macrophage counts were significantly higher in the mild versus fatal disease (p = 0.002). In situ analysis for SARS-CoV2 RNA found ten COVID-19 lung sections that had no/rare detectable virus and also lacked the microangiopathy typical of the viral positive sections. These viral negative lung tissues when compared to the viral positive lung samples showed a highly significant increase in CD3+ and CD8 T cells (p < 0.001), equivalent numbers of CD163+ cells, and significantly less PDL1, CD11b and CD206+ cells (p = 0.002). It is concluded that mild SARS-CoV2 infection is marked by a much stronger CD3/CD8 T cell, CD11b, and CD206 macrophage response than the fatal lung disease where viral RNA is abundant.

Histologic, viral, and molecular correlates of heart disease in fatal COVID-19.

Cardiac manifestations are common in severe COVID-19. This study compared the histologic, viral, and molecular findings in cardiac tissue in fatal COVID-19 (n = 11) and controls (n = 11). In situ hybridization (SARS-CoV2 RNA) and immunohistochemistry for viral proteins and the host response were quantified for the samples and compared with qRTPCR and Western blot data. Control hearts showed a high resident population of macrophages that had variable ACE2 expression. Cardiac ACE2 expression was 10× greater in the heart tissues of cases and controls with obesity or type II diabetes. Multifocal endothelial cell swelling and degeneration, perivascular edema plus microvascular thrombi were unique to the cases. SARS-CoV2 RNA and nucleocapsid protein were rarely detected in situ in any COVID-19 heart. However, in each case abundant SARS-CoV-2 spike protein was evident. Co-expression experiments showed that the spike protein localized mostly to the ACE2+ interstitial macrophages/pericytes that were activated as evidenced by increased IL6 and TNFα expression. Western blots confirmed the presence of the viral spike protein, but not the nucleocapsid protein, in the cardiac homogenates. The intercalated disc proteins connexin 43, the primary cardiac gap junction protein, and NaV1.5, the predominant cardiac sodium channel, each showed marked lateral migration in the myocytes in the cases, which would increase the risk of reentrant arrhythmias. It is concluded that the viral spike protein, endocytosed by macrophages/pericytes, can induce a myocarditis with the possibility of conduction dysfunction due to abnormal localization of key intercalated disc proteins.

Correction: MiR-34a/c-Dependent PDGFR-α/ß Downregulation Inhibits Tumorigenesis and Enhances TRAIL-Induced Apoptosis in Lung Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0067581.].

A Standardization Protocol for the In Situ Detection of SARS-CoV2 RNA and Proteins.

This manuscript details a stringent protocol for the in situ detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) RNA and 4 different viral proteins: envelope, spike, membrane, and nucleocapsid. Key aspects of the protocol are: (1) analysis of adjacent (serial) sections for viral RNA and at least 2 viral proteins; (2) cytologic alterations in the cells scored as virus positive based on an hematoxylin and eosin stain; (3) in situ demonstration of a host response in the cells scored as virus positive; (4) co-labeling experiments that show that the viral RNA and/or proteins co-localize with each other and the angiotensin converting enzyme 2 (ACE2) receptor; and (5) lack of signal in equivalent tissues obtained before the pandemic. Optimization conditions for the four viral proteins as well as the ACE2 receptor were each antigen retrieval in an EDTA solution which facilitates co-expression analyses. It is recommended not to use either electron microscopy or qRTPCR as methods to corroborate in situ SARS-CoV2 detection. This stringent protocol, that relies on sequentially labeled serial sections and can be completed in one working day, demonstrated the following: (1) infectious SARS-CoV2 is abundant in the lung in fatal coronavirus disease-2019 and is seen primarily in macrophages and endothelial cells; (2) circulating viral capsid proteins (spike, envelope, membrane without RNA) are evident in multiple organs including the skin and brain where it is endocytosed by ACE2+ cells and induce an endothelialitis; (3) both the infectious virus and circulating spike protein induce complement activation and cytologic changes in the viral positive cells.

The histologic and molecular correlates of liver disease in fatal COVID-19 including with alcohol use disorder.

Hepatic disease is common in severe COVID-19. This study compared the histologic/molecular findings in the liver in fatal COVID-19 (n = 9) and age-matched normal controls (n = 9); three of the fatal COVID-19 livers had pre-existing alcohol use disorder (AUD). Controls showed a high resident population of sinusoidal macrophages that had variable ACE2 expression. Histologic findings in the cases included periportal/lobular inflammation. SARS-CoV2 RNA and nucleocapsid protein were detected in situ in 2/9 COVID-19 livers in low amounts. In 9/9 cases, there was ample in situ SARS-CoV-2 spike protein that co-localized with viral matrix and envelope proteins. The number of cells positive for spike/100× field was significantly greater in the AUD/COVID-19 cases (mean 5.9) versus the non-AUD/COVID-19 cases (mean 0.4, p < 0.001) which was corroborated by Western blots. ACE2+ cells were 10× greater in AUD/COVID-19 livers versus the other COVID-19/control liver samples (p < 0.001). Co-expression experiments showed that the spike protein localized to the ACE2 positive macrophages and, in the AUD cases, hepatic stellate cells that were activated as evidenced by IL6 and TNFα expression. Injection of the S1, but not S2, subunit of spike in mice induced hepatic lobular inflammation in activated macrophages. It is concluded that endocytosed viral spike protein can induce hepatitis in fatal COVID-19. This spike induced hepatitis is more robust in the livers with pre-existing AUD which may relate to why patients with alcohol abuse are at higher risk of severe liver disease with SARS-CoV2 infection.

Endothelial cell damage is the central part of COVID-19 and a mouse model induced by injection of the S1 subunit of the spike protein.

Neurologic complications of symptomatic COVID-19 are common. Brain tissues from 13 autopsies of people who died of COVID-19 were examined. Cultured endothelial and neuronal cells were incubated with and wild type mice were injected IV with different spike subunits. In situ analyses were used to detect SARS-CoV-2 proteins and the host response. In 13/13 brains from fatal COVID-19, pseudovirions (spike, envelope, and membrane proteins without viral RNA) were present in the endothelia of microvessels ranging from 0 to 14 positive cells/200× field (mean 4.3). The pseudovirions strongly co-localized with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons demonstrated increased NMDAR2 and neuronal NOS plus decreased MFSD2a and SHIP1 proteins. Tail vein injection of the full length S1 spike subunit in mice led to neurologic signs (increased thirst, stressed behavior) not evident in those injected with the S2 subunit. The S1 subunit localized to the endothelia of microvessels in the mice brain and showed co-localization with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons showed increased neuronal NOS and decreased MFSD2a. It is concluded that ACE2+ endothelial damage is a central part of SARS-CoV2 pathology and may be induced by the spike protein alone. Thus, the diagnostic pathologist can use either hematoxylin and eosin stain or immunohistochemistry for caspase 3 and ACE2 to document the endothelial cell damage of COVID-19.

A Covid-19 Patient with Complement-Mediated Coagulopathy and Severe Thrombosis.

We report a patient with severe Covid-19-associated coagulopathy and type 2 diabetes mellitus who tested positive for antiphospholipid antibodies (aPL). Analysis of skin specimens suggested direct SARS-CoV-2 viral-induced and complement-mediated vascular injury and thrombosis, consistent with prior reports. Serial aPL testing demonstrated high levels of anticardiolipin antibodies (aCL) that declined to insignificant levels over a period of 5 weeks. SARS-CoV-2 RNA was detected in nasopharyngeal swab specimens on serial assays performed over the same 5-week period, though it was not detected thereafter. We hypothesize that SARS-CoV-2 viral-induced aPL contributed to severe Covid-19-associated coagulopathy in this patient.

Docked severe acute respiratory syndrome coronavirus 2 proteins within the cutaneous and subcutaneous microvasculature and their role in the pathogenesis of severe coronavirus disease 2019.

The purpose of this study was to examine the deltoid skin biopsy in twenty-three patients with coronavirus disease 2019 (COVID-19), most severely ill, for vascular complement deposition and correlate this with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA and protein localization and ACE2 expression. Deltoid skin microvascular complement screening has been applied to patients with various systemic complement-mediated microvascular syndromes, best exemplified by atypical hemolytic uremic syndrome. In 21 of 23 cases, substantial microvascular deposition of complement components was identified. The two patients without significant complement deposition included one patient with moderate disease and a severely ill patient who although on a ventilator for a day was discharged after 3 days. The dominant microvascular complement immunoreactant identified was the terminal membranolytic attack complex C5b-9. Microvascular complement deposition strongly colocalized in situ with the SARS-CoV-2 viral proteins including spike glycoproteins in the endothelial cells as well as the viral receptor ACE2 in lesional and nonlesional skin; viral RNA was not evident. Microvascular SARS-CoV-2 viral protein, complement, and ACE2 expression was most conspicuous in the subcutaneous fat. Although the samples from severely ill patients with COVID-19 were from grossly normal skin, light microscopically focal microvascular abnormalities were evident that included endothelial cell denudement, basement membrane zone reduplication, and small thrombi. It is concluded that complement activation is common in grossly normal skin, especially in the subcutaneous fat which may provide a link between severe disease and obesity, in people with severe COVID-19, and the strong colocalization with the ACE2 receptor and viral capsid proteins without viral RNA suggests that circulating viral proteins (ie, pseudovirions) may dock onto the endothelial of these microvessels and induce complement activation.

Cytologic and molecular correlates of SARS-CoV-2 infection of the nasopharynx.

Infection by SARS-CoV-2 commonly begins in the nasopharynx, and the cytologic and molecular correlates are not characterized. Fifty-eight cytologic preps (20 oral and 38 from the nasopharynx) were obtained from ten patients and analyzed in a blinded fashion for SARS-CoV-2 spike and envelope protein by immunohistochemistry and viral RNA by in situ hybridization. qRTPCR identified three positive cases and seven controls; the three cases reported mild symptoms that resolved in 2-3 days. Blinded analyses confirmed the presence of the SARS-CoV-2 spike and envelope proteins and viral RNA in the three cases and viral absence in the seven controls. A signal for the positive cases was evident in each nasopharyngeal and none of the oral samples. Viral RNA/proteins localized exclusively to glandular cells and was present in high copy number. Blinded analysis of the cytology documented that the glandular cells infected by SARS-CoV-2 showed marked degeneration with ciliocytophthoria; viral inclusions were not evident. Co-expression analysis showed viral infected cells had increased apoptosis, marked by strong expression of activated caspase 3. Weekly serial testing of two of the cases showed persistence of productive viral infection for up to 2 weeks after symptom onset. It is concluded that the target cell of SARS-CoV-2 in the head and neck region is the glandular cell of the nasal passages, that viral infection is lytic and associated with high copy number that facilitates viral spread. The method outlines a simple, rapid test for productive SARS-CoV-2 based on immunohistochemistry or in situ hybridization of the glandular cells from the nasopharynx.