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Acute respiratory failure and a systemic coagulopathy are critical aspects of the morbidity and mortality characterizing infection with severe acute respiratory distress syndrome-associated coronavirus-2, the etiologic agent of Coronavirus disease 2019 (COVID-19). We examined skin and lung tissues from 5 patients with severe COVID-19 characterized by respiratory failure (n= 5) and purpuric skin rash (n = 3). COVID-19 pneumonitis was predominantly a pauci-inflammatory septal capillary injury with significant septal capillary mural and luminal fibrin deposition and permeation of the interalveolar septa by neutrophils. No viral cytopathic changes were observed and the diffuse alveolar damage (DAD) with hyaline membranes, inflammation, and type II pneumocyte hyperplasia, hallmarks of classic acute respiratory distress syndrome, were not prominent. These pulmonary findings were accompanied by significant deposits of terminal complement components C5b-9 (membrane attack complex), C4d, and mannose binding lectin (MBL)-associated serine protease (MASP)2, in the microvasculature, consistent with sustained, systemic activation of the complement pathways. The purpuric skin lesions similarly showed a pauci-inflammatory thrombogenic vasculopathy, with deposition of C5b-9 and C4d in both grossly involved and normally-appearing skin. In addition, there was co-localization of COVID-19 spike glycoproteins with C4d and C5b-9 in the interalveolar septa and the cutaneous microvasculature of 2 cases examined. In conclusion, at least a subset of sustained, severe COVID-19 may define a type of catastrophic microvascular injury syndrome mediated by activation of complement pathways and an associated procoagulant state. It provides a foundation for further exploration of the pathophysiologic importance of complement in COVID-19, and could suggest targets for specific intervention. [ABSTRACT FROM AUTHOR]

Adjunctive immunohistochemistry tests for human papillomavirus (HPV) infection include p16 and Ki67 as well as the more recently discovered biomarkers importin-β, exportin-5, Mcl1, and PDL1. The purpose of this study was to compare the expression of these biomarkers in HPV infection due to the high-risk types such as HPVs 16, 18, 31, 33, 35, and 51 versus lesions that contain the low risk types HPV 2, 6 or 11. We studied 35 lesions with low risk HPV types (verruca vulgaris = 10 cases, condyloma acuminatum = 15 cases, CIN 1 with HPV 6/11 = 10 cases) and 25 CIN 1 or 2 lesions with a high-risk HPV type. The 25 high-risk positive CIN 1–2 cases had strong expression of the panel p16, Ki67, importin-β, exportin-5, Mcl1, and PDL1 where each protein localized to the cells in the parabasal aspect of the lesion. In comparison, neither p16, importin-β, exportin-5, Mcl1, nor PDL1 were increased in the epithelia of the lesions with the low risk HPV types; Ki67 showed variable expression. HPV viral capsid L1 protein and viral DNA were excellent markers of infection in the lesions with low risk types. Thus, p16, importin-β, exportin-5, Mcl1, and PDL1 are not only biomarkers of high-risk HPV infection but can also differentiate such lesions from those that contain low risk HPV types. Low risk HPV infections can be best differentiated from their mimics by viral L1 capsid detection and/or HPV DNA by in situ hybridization. • HPVs 2, 6/11 cannot induce oncogenesis (low risk types) • HPVs 16, 18, 31,33,35,51 and others can induce cancers (high risk types) • This paper describes biomarkers strongly induced only by high risk types • These biomarkers include p16, importin-β, exportin-5, PDL1, and MCL1 • Low risk HPV infections were best diagnosed by in situ analysis of DNA or L1 protein [ABSTRACT FROM AUTHOR]

The cause of epidermodysplasia verruciformis is infection by human papillomavirus, usually types 5 or 8, and it exhibits a high potential for malignant transformation. The diagnostic histologic features of epidermodysplasia verruciformis are not always present and can be mimicked by non-viral diseases. The purpose of this study was to interrogate such lesions for new potential biomarkers to aid in the diagnostic accuracy. HPV DNA was high copy and localized to the upper half of the lesion in cells with cytologic features that included perinuclear halos, blue-grey cytoplasm, and hyper/parakeratosis. Serial section analyses demonstrated that there was increased expression of importin-β, exportin-5, Mcl1, p16, Ki67 and PDL1 in 13/13 epidermodysplasia verruciformis lesions. Each of these proteins localized primarily to the less differentiated cells in the parabasal aspect of the lesion. Only Ki67 and exportin-5 were expressed in the normal epithelia, though much less so, in 13/13 aged matched controls. It is concluded that the host response to HPV 5/8 infection in epidermodysplasia verruciformis includes the up regulation of several proteins including p16, Ki67, importin-β, exportin-5, Mcl1, and PDL1. Thus, these proteins may serve as new biomarkers of this disease that can aid in cases that are equivocal for epidermodysplasia verruciformis on histologic examination. • Epidermodysplasia verruciformis (EDV) is caused by HPV infection. • HPV types including 5, 8, and others can cause EDV. • In situ testing to diagnose EDV must include all possible types. • This paper describes various biomarkers strongly induced by HPV in EDV. • These biomarkers include Ki67, p16, importin-β, exportin-5, PDL1, and MCL1. [ABSTRACT FROM AUTHOR]

Abstract Importin-β, exportin-5, p16, Ki-67, Mcl1, PDL1, and cFLIP are each over-expressed in the majority of CIN 1 lesions. These biomarkers, plus HPV E6/E7 RNA, were analyzed in carcinoma-in-situ (CIS), microinvasive, and squamous cell carcinoma (SCC) of the uterine cervix and cervical carcinoma cell lines. Only p16 and Ki-67 continued to be over-expressed in CIS, with a concomitant marked increase in E6/E7 RNA. There was a highly significant increase in PDL1 expression and decrease in Ki-67 (each p < 0.001) in microinvasive cancer compared to CIS whereas p16 and E6/E7 remained stable. As the lesion progressed to SCC, p16 and E6/E7 RNA remained strongly overexpressed with a concomitant over expression of importin-β and Ki67. HPV positive Caski cells showed significant elevations of p16, importin-β, exportin-5 and PDL1 compared to the HPV negative cervical cancer cell line C33A, consistent with viral induction of these biomarkers. The data suggest that PDL1 may be a useful biomarker to differentiate CIS from microinvasive cancer and, thus, anti-PDL1 therapy may inhibit the progression of CIS to the invasive stage. Highlights • p16 and Ki67 are the standard biomarkers of CIN 1 with high risk types. • Importin-β, exportin-5, Mcl1, PDL1, and cFLIP are also biomarkers of high risk CIN 1. • Except for p16 and Ki67, these biomarkers are much reduced in CIN 3. • Microinvasive cervical cancer is marked by marked increase in PDL1 expression. [ABSTRACT FROM AUTHOR]

Abstract Acute human papillomavirus (HPV) infection of the cervix (cervical intraepithelial neoplasia, CIN) is marked by high copy episomal viral DNA and L1/L2 capsid protein expression (productive infection) in the cells towards the surface that facilitate sexual viral transmission. Viral DNA is low copy and not associated with viral capsid protein expression in the less differentiated lower part of the CIN (nonproductive infection). The purpose of this study was to examine the host response in these two areas. Serial section and co-localization analyses demonstrated that in 29/33 (88%) of cases the NF-κB pathway was activated and localized to the suprabasal nonproductively infected cells in the CIN lesions. There was a concomitant increased expression of importin-β, exportin-5, Mcl1, p16, Ki67 and cFLIP in 32/33 (96%) of CIN lesions that likewise localized primarily to the nonproductively infected cells. Only Ki67 and exportin-5 were expressed, though much less so, in the adjacent, normal squamous epithelia. The viral proteins E1^E4 and L1 were localized to productively infected cells whereas E6/E7 protein/RNA was rarely present in early CIN. It is concluded that the host viral response to acute cervical HPV infection includes strong increased expression of proteins besides p16 and Ki67. These include importin-β, exportin-5, Mcl1, and cFLIP in cells with low copy and relatively quiescent viral DNA that, in turn, may serve as new biomarkers of this disease. Highlights • Early CIN is marked by strong productive infection in koilocytes towards the mucosal surface. • In early CIN the suprabasal zone contains relatively quiescent low copy HPV DNA. • The suprbasal low viral copy zone contains several biomarkers of early HPV infection. • These biomarkers include, besides p16 and Ki67, Mcl1, cFLIP, importin-β, and exportin-5. [ABSTRACT FROM AUTHOR]

Abstract Spinal cord paralysis is relatively common after surgical repair of thoraco-abdominal aortic aneurysm (TAAA) and its etiology is unknown. The present study was designed to examine the histopathology of the disease and investigate whether miR-155 ablation would reduce spinal cord ischemic damage and delayed hindlimb paralysis induced by aortic cross-clamping (ACC) in our mouse model. The loss of locomotor function in ACC-paralyzed mice correlated with the presence of extensive gray matter damage and central cord edema, with minimal white matter histopathology. qRTPCR and Western blotting showed that the spinal cords of wild-type ACC mice that escaped paralysis showed lower miR-155 expression and higher levels of transcripts encoding Mfsd2a, which is implicated in the maintenance of blood-brain barrier integrity. In situ based testing demonstrated that increased miR-155 detection in neurons was highly correlated with the gray matter damage and the loss of one of its targets, Mfsd2a, could serve as a good biomarker of the endothelial cell damage. In vitro, we demonstrated that miR-155 targeted Mfsd2a in endothelial cells and motoneurons and increased endothelial cell permeability. Finally, miR-155 ablation slowed the progression of central cord edema, and reduced the incidence of paralysis by 40%. In sum, the surgical pathology findings clearly indicated that the epicenter of the ischemic-induced paralysis was the gray matter and that endothelial cell damage correlated to Mfsd2a loss is a good biomarker of the disease. MiR-155 targeting therefore offers new therapeutic opportunity for edema caused by traumatic spinal cord injury and diagnostic pathologists, by using immunohistochemistry, can clarify if this mechanism also is important in other ischemic diseases of the CNS, including stroke. Highlights • Ischemic spinal cord injury is a relatively common cause of paralysis after aortic aneurysm repair. • The histologic changes suggest gray matter disease. • In situ testing showed marked increase of miR-155 in motor neurons and endothelial cells. • Immunohistochemistry showed a concomitant loss of Mfsd2a in these cells. • Reduction of miR-155 much abated the spinal cord damage and in situ molecular correlates. [ABSTRACT FROM AUTHOR]

This work focused on immunohistochemistry markers of acute viral infections. Viral infected cells were detected by in situ based methods (reovirus, rabies virus) or cytologic changes (human papillomavirus, molloscum contagiosum virus, herpes simplex virus). Two proteins involved in nuclear trafficking, importin-β and exportin-5, were detected in the infected cells for each virus and not in the control tissues. A wide variety of other proteins, including caspase-3, and bcl-2 family members (bcl2, bclX, MCL1, BAK, BAX, BIM, BAD) showed wide variations in expression among the different viral infections. Specificity of the importin-β and exportin-5 signals varied greatly with different commercially available peroxidase conjugates. It is concluded that immunohistochemistry detection of importin-β and exportin-5 may be useful markers of acute viral infection, which suggests that increased nuclear trafficking may be an important concomitant of viral proliferation. [ABSTRACT FROM AUTHOR]

This study examined the molecular correlates of Down's dementia. qRTPCR for chromosome 21 microRNAs was correlated with in situ hybridization, immunohistochemistry for microRNA targets, mRNAs located on chromosome 21, and neurofibrillary tangles in human and the Ts65 dn mouse Down's model. qRTPCR for the microRNAs on the triplicated chromosome showed miR-155 dominance in brain tissues (14.3 fold increase, human and 24.2 fold increase, mouse model) that co-expressed with hyperphosphorylated tau protein. miR-155 was not elevated in Alzheimer's disease or neonates with Downs' syndrome. Chromosome 21 genes APP/BA-42, DYRK1a and BACH1 were not correlated to pathologic changes in Down's dementia. Validated CNS targets of miR-155 that were present in controls and Alzheimer's disease but lacking in Down's dementia brains included BACH1, CoREST1, bcl6, BIM, bcl10, cyclin D, and SAPK4. It is concluded that Down's dementia strongly correlated with overexpression of chromosome 21 microRNA 155 with concomitant reduction of multiple CNS-functional targets. This study highlights the need for anatomic pathologists to determine the specific and diverse pathways cells may take to form neurofibrillary tangles in the different dementias. [ABSTRACT FROM AUTHOR]

Acute reoviral infection has been extensively studied given the virus's propensity to target malignant cells and activate caspase-3 mediated apoptosis. Reovirus infection of malignant N1E-115 mouse neuroblastoma cells led to significant increased expression of importin-β and exportin-5 mRNAs (qRTPCR) and proteins (immunohistochemistry) which was partially blocked by small interfering LNA oligomers directed against the reoviral genome. Co-expression analysis showed that the N1E-115 cells that contained reoviral capsid protein had accumulated importin-β and exportin-5, as well as activated caspase 3. Reoviral oncolysis using a syngeneic mouse model of multiple myeloma similarly induced a significant increase in importin-β and exportin-5 proteins that were co-expressed with reoviral capsid protein and caspase-3. Apoptotic proteins (BAD, BIM, PUMA, NOXA, BAK, BAX) were increased with infection and co-localized with reoviral capsid protein. Surprisingly the anti-apoptotic MCL1 and bcl2 were also increased and co-localized with the capsid protein suggesting that it was the balance of pro-apoptotic molecules that correlated with activation of caspase-3. In summary, productive reoviral infection is strongly correlated with elevated importin-β and exportin-5 levels which may serve as biomarkers of the disease in clinical specimens. [ABSTRACT FROM AUTHOR]

Understanding the metabolic profile of neurons with the hyperphosphorylated tau protein characteristic of Alzheimer's disease is essential to unraveling new potential therapies and diagnostics for the surgical pathologist. We stratified 75 brain tissues from Alzheimer's disease into hyperphosphorylated tau positive or negative and did co-expression analyses and qRTPCR for importin-β and exportin-5 plus several bcl2 family members and compared the data to controls, Down's dementia and Parkinson's disease. There was a significant increase in the expression of importin-β and exportin-5 in Alzheimer's disease relative to the three other categories (each p value < 0.0001) where each protein co-localized with hyperphosphorylated tau. Both apoptotic and anti-apoptotic proteins were each significantly increased in Alzheimer's disease relative to the three other groups. Neurons with hyperphosphorylated tau in Alzheimer's disease have the profile of metabolically active cells including increased exportin-5 and importin-β mRNA and proteins which indicates that immunohistochemistry testing of these proteins may aid the surgical pathologist in making a definitive diagnosis. [ABSTRACT FROM AUTHOR]

Interdigitating dendritic cell sarcoma (IDS) is a rare form of hematologic malignancy associated with an aggressive clinical course. Only 4 prior cases have been described as originating in the skin. We encountered two male patients ages 47 and 61 years of age who presented with solitary cutaneous neoplasms diagnosed as IDS. Histologic exam showed a coalescing nested and multinodular proliferation of large pleomorphic epithelioid cells. In one case an initial diagnosis of melanoma was rendered. A recurrence 8 months later was then interpreted as a primary cutaneous IDS. This patient died of widespread metastatic disease within 2 years from his initial surgery. The other patient has recently undergone wide excision and radiation without any recurrence or metastatic disease during this short follow up time period. Both patients had a tumor exhibiting the same phenotypic profile comprising leukocyte common antigen, SOX10, S100, CD68, and CD163 positivity. In reviewing the 4 other reported cases, there is a similar older male predominance (mean age of 58 years) although women affected were significantly younger (mean age of 28 years); there was a predilection for the proximal extremities and the face. Patients treated with excision only developed recurrent disease with one patient subsequently dying of metastatic disease. Primary cutaneous IDS is a highly aggressive hematologic malignancy that has many overlapping features with poorly differentiated epithelioid and spindle cell melanoma including SOX10 staining. An aggressive treatment protocol at the beginning could optimize patient survival. [ABSTRACT FROM AUTHOR]

The absence of any histologic correlate for Alzheimer's disease despite its commonness and severe clinical sequelae may offers clues to its etiology. Recent evidence strongly suggests that the central event of this disease is the hyperphosphorylation of neuronal tau protein and not the beta amyloid precipitates. In each case, essential and soluble neuronal proteins derivatives form insoluble aggregates that can readily be detected by immunohistochemistry using antibodies specific for the misfolded proteins. Immunohistochemistry also demonstrates that neurons with hyperphosphorylated tau protein are viable. Experimental evidence using neuronal cell cultures suggests that the affected neurons in Alzheimer's disease may have undergone molecular changes that include accumulation of anti-apopotic proteins MCL1 and cFLIP that do not allow the cell to undergo programmed cell death but, rather, to “immortalize” and thus accumulate hyperphosphorylated tau protein in the neuronal cell body and beta amyloid in downstream dendrites. We describe a simplified protocol to demonstrate such changes based on tagged LNA modified microRNA/antimicroRNA oligomers and cell cultures. Co-expression showed that the tagged antimiR-512 strongly localized with the markedly up-regulated proteins MCL1 and cFLIP with concomitant accumulation of hyperphosphorylated tau protein. The data underscore to the anatomic pathologist that the diagnosis of Alzheimer's disease is best accomplished by simple immunohistochemistry tests correlated to the clinical history and the key role pathologists can play in understanding the cause of the disease. [ABSTRACT FROM AUTHOR]

Lichen sclerosus (LS) is a cutaneous disease of unknown etiology that often involves the vulva or foreskin but also can affect extragenital sites. Regardless of the anatomic site, the histomorphology and presumably pathogenesis are similar. Perhaps a clue to the pathophysiology of LS lies in its frequent association with morphea, specifically, when occurring in an extragenital context. In our experience a striking feature evident in established lichen sclerosis (LS) is one of superficial vascular drop out whereby residual vessels exhibited endothelial cell necrosis and microvascular basement membrane zone thickening, the latter reflective of antecedent episodes of microvascular injury. We sought to understand the pathophysiology that underlies the distinct vascular changes and in doing so, shed light on the pathogenesis of LS. We examined 44 cases of LS over a period of 2019 to 2021. We were able to obtain past medical histories in 34 of the 44 cases. Regarding pathological assessment, the predominant focus was on microvascular changes. We assessed the role of C5b-9 mediated vascular injury in the pathogenesis of the vasculopathy and enhanced type I interferon signaling in vessels given the morphologic semblance to the select interferonopathy syndromes, namely fibrosing dermatomyositis and Kohlmeier Degos disease. We examined the expression of CMV DNA and protein based on prior observations in an earlier study that isolated early protein expression in the microvasculature in the setting of LS and scleroderma. From a clinical perspective, the most striking association was an older age at the time of diagnosis (mean age of 62 years and median age of 61.5 years) and the presence of vascular comorbidities of diabetes, hypertension, and hyperlipidemia in almost 80% of cases. All cases showed significant microvascular changes in the superficial corium with the most frequent findings being those of significant basement membrane zone reduplication and vascular drop out. A number of cases showed prominent microvascular deposits of C5b-9 in the zone of hyalinizing fibrosis or subjacent to the discernible table of fibroplasia in the absence of enhanced type I interferon signaling. In no case were there viral cytopathic changes associated with CMV affecting the endothelium. The studies that encode CMV DNA or protein did not show a significant role for CMV reactivation in endothelium in the majority of the studied cases. It is concluded that the pathophysiology of LS includes a microvascular injury syndrome within the papillary dermis. The mechanism of endothelial cell injury is complement mediated at least in part and could reflect an adaptive immune response targeting endothelium indicative of classic complement pathway activation when coexisting with morphea or occurring in younger individuals. A non-immune based endothelial dysfunction and complement mediated injury unrelated to antibody driven classic complement pathway activation are more likely pathogenetically in the setting of certain diseases like diabetes mellitus and hypertension. Vascular drop out can be explained by the diminished endothelial progenitor pool needed to repopulate the damaged microvessels in certain settings like hypertension and diabetes. • Lichen Sclerosus • Microvascular Injury • C5b-9 • Complement • Diabetes [ABSTRACT FROM AUTHOR]

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 TLRs and RIG-1 are among the earliest proteins to respond to an infection. • The TLRs and RIG-1 were compared in mild versus severe infections. • Mild infections are marked by marked increases in the TLRs/RIG-1. • Severe infections are marked by marked decreases in some TLRs/RIG-1. • The surgical pathologist is an a unique position to make these in situ observations. [ABSTRACT FROM AUTHOR]

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. • Many different infectious agents can cause upper respiratory signs and symptoms. • Rapid testing for Mycoplasma pneumonia infection is not readily available. • Mycoplasma pneumonia is highly responsive to antibiotic therapy. • Mycoplasma pneumona produces very high copy DNA, making it easy to detect in situ. • A rapid in situ hybridization test confirmed Mycoplasma pneumonia infections in this study. [ABSTRACT FROM AUTHOR]

Monocytes are critical components of the innate immune system and they can differentiate into dendritic cells (DCs). Cutaneous neoplasms of dendritic cell origin are uncommon and mostly represented by histiocytic lesions derived primarily from Langerhans cells. The myeloid DC (mDC) while recognized in the immunology literature does not have a well-defined neoplastic cutaneous counterpart. Eleven patients with a diagnosis of cutaneous mDC dyscrasia were evaluated. Routine hematoxylin and eosin stain were performed followed by selective phenotypic studies. The patients were older without a gender predilection and exhibited an asymptomatic papular skin rash with a waxing and waning course. The biopsies demonstrated a dermal based monomorphic small mononuclear cell infiltrate. The cells expressed CD14, CD11c, HLA-DR, as well as granzyme and lysozyme that defines terminally differentiated monocyte/dendritic cells. Expression of BDCA-3 (CD141) by the tumor cells indicated that they were myeloid dendritic cells (mDC2). Each patient had a prior or subsequent diagnosis of an abnormal bone marrow biopsy that included myelodysplastic syndrome, myelofibrosis, chronic myelomonocytic leukemia, and acute myelogenous leukemia. We propose the term cutaneous mDC cell dyscrasia for distinctive infiltrates of differentiated mDCs reflective of underlying myeloproliferative disease. The clinical course is variable and can be indolent although it is strongly correlated with myelodysplastic syndrome that included leukemia. [ABSTRACT FROM AUTHOR]

False-positive results in diagnostic immunopathology can lead to unnecessary treatments. The purpose of this study was to do a side-by-side comparison of 10 different antibodies commonly used in the clinical laboratory altering only the horseradish peroxidase (HRP) conjugate. The automated Leica BOND-MAX platform was used to study serial sections from 203 tissues including controls compared in a blinded fashion using the HRP conjugates from Leica (Refine HRP), Ventana Medical Systems (Ultraview HRP), and Enzo Life Sciences (Polyview HRP). False-positive results, defined as signal from cases known to not contain the target, were noted in 23 (13%) of 171 cases with the Leica HRP, 62 (36%) of 171 cases with the Ventana HRP, and no cases with the Enzo HRP. Each data set was performed simultaneously allocating 1 tray for each of the 3 different HRP conjugates. HER2/neu analysis from triple-negative breast cancers were scored as positive by immunohistochemistry in 6 (24%) of 25 cases using either the Refine or Ultraview HRP and in 0 of 25 cases with the Enzo conjugate. It is concluded that false-positive results in a wide spectrum of diagnostic immunopathology tests can occur from 13% to 36% of cases with commonly used commercial assays based on the HRP conjugate. [ABSTRACT FROM AUTHOR]

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. • Pattern recognition receptors in the placenta show a unique pattern with in utero infections • TLRs 1, 3, and 8 are markedly increased whereas TLR2 and RIG-1 markedly decreased in the placenta with in utero infections • These new data may assist in the diagnoses of in utero infections right after birth • The histologic findings in the control versus infected placenta tissue (chorionic villi) were equivalent, underscoring need for molecular testing • The histology in the control versus infected placenta tissue were equivalent, underscoring need for molecular testing [ABSTRACT FROM AUTHOR]

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 β-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 βA-42. • Microvascular damage is a central part of CNS COVID-19. • Endocytosis of the viral spike protein is highly correlated with this damage. • The microvascular damage is superimposed on the plaques and tangles of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

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. • Mild infection showed a much stronger T cell response versus fatal SARS-CoV2 infection • Mild infection had a much stronger selective macrophage response (CD11b, CD206) than fatal viral infection • Fatal COVID-19 lung without in situ virus had a strong T cell response suggesting the latter was muted by the virus • H&E findings differentiated the in situ virus negative (organizing pneumonia) from the virus positive fatal lung COVID-19 tissues [ABSTRACT FROM AUTHOR]

The discovery of the human papillomavirus (HPV) vaccine illustrates the power of in situ–based pathologic analysis in better understanding and curing diseases. The 2 available HPV vaccines have markedly reduced the incidence of cervical intraepithelial neoplasias, genital warts, and cervical cancer throughout the world. Concerns about HPV vaccine safety have led some physicians, health care officials, and parents to refuse providing the recommended vaccination to the target population. The aims of the study were to discuss the discovery of HPV vaccine and review scientific data related to measurable outcomes from the use of HPV vaccines. The strong type-specific immunity against HPV in humans has been known for more than 25 years. Multiple studies confirm the positive risk benefit of HPV vaccination with minimal documented adverse effects. The most common adverse effect, injection site pain, occurred in about 10% of girls and was less than the rate reported for other vaccines. Use of HPV vaccine should be expanded into more diverse populations, mainly in low-resource settings. [ABSTRACT FROM AUTHOR]

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 Na V 1.5, 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. • Cardiac disease in fatal COVID-19 is associated with viral spike protein, but not the infectious virus. • The viral spike protein is endocytosed in interstitial macrophages and induces a myocarditis. • The histologic findings show perivascular edema, endothelial cell damage, and microthrombi. [ABSTRACT FROM AUTHOR]

An 11-year-old female presented with multiple oral lesions for several months. Histopathological findings suggested focal epithelial hyperplasia (FEH), also known as Heck disease. FEH is strongly associated with Human papillomavirus (HPV), especially genotypes 13 and 32. An oral swab of a mucosal lesion was subsequently obtained for cytology, immunohistochemistry and in situ hybridization. In addition, in situ hybridization and immunohistochemistry were also performed retrospectively on the biopsy specimen for correlation. The cytology specimen showed squamous cells with enlarged, slightly atypical nuclei and rare perinuclear halos. The histology findings included papillomatosis with acanthosis, mild nuclear atypia and focal perinuclear halos. The immunohistochemistry for the consensus HPV L1 capsid protein was found in both the cytology and biopsy specimens indicating that the lesion was HPV-related. High viral copy numbers of HPV 13 were detected by in situ hybridization in both the cytology and histology specimens. Although histologic features of FEH have been well characterized in the literature, to our knowledge, this is the first case to describe in FEH with adjunct immunohistochemistry and in situ hybridization results. Furthermore, these findings assisted in our diagnosis since the patient's clinical presentation was a diagnostic challenge with smooth dome-shaped papules instead of the typically described flat-topped verrucous lesions seen in FEH. In summary, our case reveals that there is a high concordance between the HPV 13 detection in the cytology and histology of FEH, and that performing cytology in addition to histology can be used to optimize diagnostic evaluation towards appropriate patient care. • Patient with an unusual clinical presentation of FEH confirmed on histology and cytology. • HPV 13 was detected in both the biopsy and cytology specimens by in situ hybridization. • Presence of productive HPV infection confirmed by consensus antibody directed against the viral L1 capsid protein. • First report to reveal correlation between the HPV 13 detection in the cytology and histology of FEH. [ABSTRACT FROM AUTHOR]

Abstract: An increasing body of evidence highlights an intriguing interaction between microRNAs and transcriptional factors involved in determining cell fate, including the well known “genome guardian” p53. Here we show that miR-205, oncosuppressive microRNA lost in breast cancer, is directly transactivated by oncosuppressor p53. Moreover, evaluating miR-205 expression in a panel of cell lines belonging to the highly aggressive triple negative breast cancer (TNBC) subtype, which still lacks an effective targeted therapy and characterized by an extremely undifferentiated and mesenchymal phenotype, we demonstrated that this microRNA is critically down-expressed compared to a normal-like cell line. Re-expression of miR-205 where absent strongly reduces cell proliferation, cell cycle progression and clonogenic potential in vitro, and inhibits tumor growth in vivo, and this tumor suppressor activity is at least partially exerted through targeting of E2F1, master regulator of cell cycle progression, and LAMC1, component of extracellular matrix involved in cell adhesion, proliferation and migration. [Copyright &y& Elsevier]

Summary: A 59-year-old previously healthy man had flulike symptoms of fever and diarrhea for a week, which worsened despite treatment with antibiotics. After admission, his medical condition rapidly deteriorated with renal failure, heart failure, and a marked increase of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase. The patient died of a cardiac arrhythmia 3 days after the admission. The autopsy showed diffuse myocarditis with a granulocytic and monocytic infiltrate, necrotizing arteritis of the coronary arteries, and fulminant hepatitis, with microvesicular steatosis and necrosis. Cell-free serum showed high copies of human herpesvirus 6 B variant DNA by polymerase chain reaction. Human herpesvirus 6 B was identified in the heart, liver, lung, and spleen by immunohistochemistry. No parvovirus B19 was evident in the heart by immunohistochemistry. Human herpesvirus 6 is increasingly found in association with myocarditis in immunocompromised patients; however, histopathologic features and the clinical severity of this disease have not yet been clearly defined. Only 4 to 5 cases of human herpesvirus 6 fulminant myocarditis have been reported, all in young children or immunosuppressed patients. To the best of our knowledge, this is the first case in the English literature of human herpesvirus 6 fulminant myocarditis and hepatitis in an immunocompetent adult with a fatal outcome. In addition, several pathologic features of our case have not been previously reported. [Copyright &y& Elsevier]

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. • Viral spike protein is more abundant in the liver in fatal COVID-19 than viral RNA. • The viral spike protein is especially high with pre-existing alcohol use disorder. • The viral spike protein induces a pro-inflammatory hepatitis. • The study highlights the key role that anatomic pathologists have in dissecting the mechanisms of COVID-19. [ABSTRACT FROM AUTHOR]

Abstract: A 30-year-old palomino Standardbred horse was seen with a persistent penile squamous cell carcinoma. After failure using multiple treatments, surgical removal was successfully carried out under local anesthesia using Mohs micrographically controlled surgery. Subsequent examination of the malignant tissue identified bovine papillomavirus exclusively within the nuclei and cytoplasm of tumor cells. The use of Mohs surgery would appear to be useful as an effective, precisely controlled treatment for managing genital squamous cell carcinoma in equine subjects. The presence of papillomavirus within tumor cells would suggest the possibility of causation by an oncogenic papilloma virus and would indicate that careful monitoring of the surgical site should be performed. [Copyright &y& Elsevier]

Background: The hamartoma nevus sebaceus (NS) presents at birth or early childhood as a yellowish plaque characterized histologically by variable acanthosis, papillomatosis, sebaceus hyperplasia, and proliferations of adnexal structures. Clinically apparent human papillomavirus (HPV) infection is also recognized by acanthosis and papillomatosis. Objective: We sought to determine the prevalence and physical state of HPV DNA in NS. Methods: DNA was retrieved from 44 formalin-fixed, paraffin-embedded samples of NS (22 with secondary tumors [eg, trichoblastoma, verruca, syringocystadenoma papilliferum] and two epidermal nevi [EN]). Nested polymerase chain reaction with multiple degenerate consensus and type-specific primers and direct sequencing of polymerase chain reaction products was performed. For selected cases, in situ hybridization using probes specific for HPV 5 and 8 and for high-risk genital-mucosal HPV types was performed. Results: HPV DNA was detected in 82% of NS and both EN, and consisted of genital-mucosal HPV types in 52% (HPV 6, 16, and 33) and a diverse variety of epidermodysplasia verruciformis–associated HPV types in 61%, including well-known epidermodysplasia verruciformis HPV types (5, 8, 15, 20, 22, 24, 36, 37, 38, and 80) and putatively novel epidermodysplasia verruciformis HPV types (DL285, DL287, DL436, and alb-1, -2, -3, -5, -6, -7, -8, -10, -11, -12, and -13). HPV coinfection was frequent, found in 48% (two HPV genotypes in 35% and 3 in 13%). Of NS and EN, 42% had HPV genotypes associated with cancer (ie, HPV 5, 8, 16, 20, 33, and 38); the two most commonly identified HPV types where HPV 16 (39%) and HPV 38 (18%). No differences were detected comparing frequency of HPV DNA detected with respect to age or presence of a secondary tumor. Histologically, all NS and EN showed HPV-associated cytopathic effects (ie, perinuclear halos, altered keratohyaline granules). By in situ hybridization, 64% (18/28) were positive, showing a low-intensity, punctate nuclear signal in epidermal and adnexal keratinocytes, indicating viral integration and low viral genome copy number. Limitations: Absence of adjacent, uninvolved normal-appearing skin control samples. Conclusion: HPV DNA is prevalent in NS, and HPV 16, the most frequently detected genotype, appears to be integrated into the host genome. Whether HPV represents a commensal infection caused by localized cutaneous predisposition, or is an essential factor in the pathogenesis of NS is unknown. The high frequency of oncogenic HPV types implicates maternal transmission of HPV and infection of an ectodermal stem cell leading to an epigenetic mosaic and altered skin development manifested along Blaschko''s lines. [Copyright &y& Elsevier]

Summary: Molecular in situ–based assays are a useful adjunct to the diagnosis of viral infections by the surgical and cytopathologist. In some cases, the viral nucleic acids and/or proteins are abundant and easily detected by in situ hybridization or immunohistochemistry. In other cases, such as the one integrated provirus typical of latent retroviral localization, in situ polymerase chain reaction amplification is required to localize the virus in the intact cell. Direct correlation of viral localization and the histologic changes will demonstrate in many cases that routine histopathology often does not provide sufficient information to determine what specific cells are infected and/or the number of infected cells in a given biopsy. By combining this information with, for example, cytokine localization, one can elucidate much about the pathophysiology of the viral infection that cannot be afforded by polymerase chain reaction–based methods alone. [Copyright &y& Elsevier]