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Consistent with the biochemistry of coronaviruses as well established over decades, SARS-CoV-2 makes its initial attachment to host cells through the binding of its spike protein (SP) to sialylated glycans (containing the monosaccharide sialic acid) on the cell surface. The virus can then slide over and enter via ACE2. SARS-CoV-2 SP attaches particularly tightly to the trillions of red blood cells (RBCs), platelets and endothelial cells in the human body, each cell very densely coated with sialic acid surface molecules but having no ACE2 or minimal ACE2. These interlaced attachments trigger the blood cell aggregation, microvascular occlusion and vascular damage that underlie the hypoxia, blood clotting and related morbidities of severe COVID-19. Notably, the two human betacoronaviruses that express a sialic acid-cleaving enzyme are benign, while the other three-SARS, SARS-CoV-2 and MERS-are virulent. RBC aggregation experimentally induced in several animal species using an injected polysaccharide caused most of the same morbidities of severe COVID-19. This glycan biochemistry is key to disentangling controversies that have arisen over the efficacy of certain generic COVID-19 treatment agents and the safety of SP-based COVID-19 vaccines. More broadly, disregard for the active physiological role of RBCs yields unreliable or erroneous reporting of pharmacokinetic parameters as routinely obtained for most drugs and other bioactive agents using detection in plasma, with whole-blood levels being up to 30-fold higher. Appreciation of the active role of RBCs can elucidate the microvascular underpinnings of other health conditions, including cardiovascular disease, and therapeutic opportunities to address them.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16 + T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16 + T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16 + T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16 + cytotoxic T cells. Proportions of activated CD16 + T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19., Competing Interests: Declaration of interests V.M.C. is named together with Euroimmun GmbH on a patent application filed recently regarding SARS-CoV-2 diagnostics via antibody testing. A.R.S. and H.E.M. are listed as inventors on a patent application by the DRFZ Berlin in the field of mass cytometry., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

COVID-19 presents with a wide range of clinical neurological manifestations. It has been recognized that SARS-CoV-2 infection affects both the central and peripheral nervous system, leading to smell and taste disturbances; acute ischemic and hemorrhagic cerebrovascular disease; encephalopathies and seizures; and causes most surviving patients to have long lasting neurological symptoms. Despite this, typical neuropathological features associated with the infection have still not been identified. Studies of post-mortem examinations of the cerebral cortex are obtained with difficulty due to laboratory safety concerns. In addition, they represent cases with different neurological symptoms, age or comorbidities, thus a larger number of brain autoptic data from multiple institutions would be crucial. Histopathological findings described here are aimed to increase the current knowledge on neuropathology of COVID-19 patients. We report post-mortem neuropathological findings of ten COVID-19 patients. A wide range of neuropathological lesions were seen. The cerebral cortex of all patients showed vascular changes, hyperemia of the meninges and perivascular inflammation in the cerebral parenchyma with hypoxic neuronal injury. Perivascular lymphocytic inflammation of predominantly CD8-positive T cells mixed with CD68-positive macrophages, targeting the disrupted vascular wall in the cerebral cortex, cerebellum and pons were seen. Our findings support recent reports highlighting a role of microvascular injury in COVID-19 neurological manifestations.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Clinical manifestations of coronavirus disease 2019 (COVID-19) in pregnant women are diverse, and little is known of the impact of the disease on placental physiology. Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has been detected in the human placenta, and its binding receptor ACE2 is present in a variety of placental cells, including endothelium. Here, we analyze the impact of COVID-19 in placental endothelium, studying by immunofluorescence the expression of von Willebrand factor (vWf), claudin-5, and vascular endothelial (VE) cadherin in the decidua and chorionic villi of placentas from women with mild and severe COVID-19 in comparison to healthy controls. Our results indicate that: (1) vWf expression increases in the endothelium of decidua and chorionic villi of placentas derived from women with COVID-19, being higher in severe cases; (2) Claudin-5 and VE-cadherin expression decrease in the decidua and chorionic villus of placentas from women with severe COVID-19 but not in those with mild disease. Placental histological analysis reveals thrombosis, infarcts, and vascular wall remodeling, confirming the deleterious effect of COVID-19 on placental vessels. Together, these results suggest that placentas from women with COVID-19 have a condition of leaky endothelium and thrombosis, which is sensitive to disease severity.

Corona virus disease 2019 (COVID-19) causes symptoms from multiple organs after infection by severe acute respiratory syndrome corona virus 2 (SARS CoV-2). They range from early, low blood oxygen levels (hypoxemia) without breathlessness ("silent hypoxia"), delirium, rashes, and loss of smell (anosmia), to persisting chest pain, muscle weakness and -pain, fatigue, confusion, memory problems and difficulty to concentrate ("brain fog"), mood changes, and unexpected onset of hypertension or diabetes. SARS CoV-2 affects the microcirculation, causing endothelial cell swelling and damage (endotheliitis), microscopic blood clots (microthrombosis), capillary congestion, and damage to pericytes that are integral to capillary integrity and barrier function, tissue repair (angiogenesis), and scar formation. Similar to other instances of critical illness, COVID-19 is also associated with elevated cytokine levels in the systemic circulation. This review examines how capillary damage and inflammation may contribute to these acute and persisting COVID-19 symptoms by interfering with blood and tissue oxygenation and with brain function. Undetectable by current diagnostic methods, capillary flow disturbances limit oxygen diffusion exchange in lungs and tissue and may therefore cause hypoxemia and tissue hypoxia. The review analyzes the combined effects of COVID-19-related capillary damage, pre-existing microvascular changes, and upstream vascular tone on tissue oxygenation in key organs. It identifies a vicious cycle, as infection- and hypoxia-related inflammation cause capillary function to deteriorate, which in turn accelerates hypoxia-related inflammation and tissue damage. Finally, the review addresses the effects of low oxygen and high cytokine levels in brain tissue on neurotransmitter synthesis and mood. Methods to assess capillary functions in human organs and therapeutic means to protect capillary functions and stimulate capillary bed repair may prove important for the individualized management of COVID-19 patients and targeted rehabilitation strategies., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The objective of this study was to elucidate the pathophysiology that underlies severe COVID-19 by assessing the histopathology and the in situ detection of infectious SARS-CoV-2 and viral capsid proteins along with the cellular target(s) and host response from twelve autopsies. There were three key findings: 1) high copy infectious virus was limited mostly to the alveolar macrophages and endothelial cells of the septal capillaries; 2) viral spike protein without viral RNA localized to ACE2+ endothelial cells in microvessels that were most abundant in the subcutaneous fat and brain; 3) although both infectious virus and docked viral spike protein was associated with complement activation, only the endocytosed pseudovirions induced a marked up-regulation of the key COVID-19 associated proteins IL6, TNF alpha, IL1 beta, p38, IL8, and caspase 3. Importantly, this microvasculitis was associated with characteristic findings on hematoxylin and eosin examination that included endothelial degeneration and resultant basement membrane zone disruption and reduplication. It is concluded that serious COVID-19 infection has two distinct mechanisms: 1) a microangiopathy of pulmonary capillaries associated with a high infectious viral load where endothelial cell death releases pseudovirions into the circulation, and 2) the pseudovirions dock on ACE2+ endothelial cells most prevalent in the skin/subcutaneous fat and brain that activates the complement pathway/coagulation cascade resulting in a systemic procoagulant state as well as the expression of cytokines that produce the cytokine storm. The data predicts a favorable response to therapies based on either removal of circulating viral proteins and/or blunting of the endothelial-induced response., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2) is a necessary step for SARS-CoV-2 infection permissiveness. In light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyze ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing β -cells. ACE2 is also highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human β -cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increase ACE2 expression in the β -cell line EndoC- β H1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct β -cell virus tropism., (Copyright © 2020 Fignani, Licata, Brusco, Nigi, Grieco, Marselli, Overbergh, Gysemans, Colli, Marchetti, Mathieu, Eizirik, Sebastiani and Dotta.)

The recently described severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people, with thousands of fatalities. It has prompted global efforts in research, with focus on the pathophysiology of coronavirus disease-19 (COVID-19), and a rapid surge of publications. COVID-19 has been associated with a myriad of clinical manifestations, including the lungs, heart, kidneys, central nervous system, gastrointestinal system, skin, and blood coagulation abnormalities. The endothelium plays a key role in organ dysfunction associated with severe infection, and current data suggest that it is also involved in SARS-CoV-2-induced sepsis. This critical review aimed to address a possible unifying mechanism underlying the diverse complications of COVID-19: microvascular dysfunction, with emphasis on the renin-angiotensin system. In addition, research perspectives are suggested in order to expand understanding of the pathophysiology of the infection., (Copyright © 2020 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.)

Early clinical evidence suggests that severe cases of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are frequently characterized by hyperinflammation, imbalance of renin-angiotensin-aldosterone system, and a particular form of vasculopathy, thrombotic microangiopathy, and intravascular coagulopathy. In this paper, we present an immunothrombosis model of COVID-19. We discuss the underlying pathogenesis and the interaction between multiple systems, resulting in propagation of immunothrombosis, which through investigation in the coming weeks, may lead to both an improved understanding of COVID-19 pathophysiology and identification of innovative and efficient therapeutic targets to reverse the otherwise unfavorable clinical outcome of many of these patients., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Vascular damage has been reported to contribute to atresia formation in several diseases including biliary atresia. This study focused on the extrahepatic biliary plexus in experimental biliary atresia. Newborn BALB/cAnNCrl-pups were infected with rhesus rotavirus within 24 hr after birth to induce experimental biliary atresia. The extrahepatic biliary plexus was examined by confocal microscopy on whole-mount preparations, scored by three independent researchers, and further evaluated at the subcellular level with transmission electron microscopy. Imaging results revealed a progressive destruction of the extrahepatic biliary vascular plexus in the course of experimental biliary atresia induced by rotavirus infection. Endothelial cell damage was already visible as cell swelling and necrosis in the first days after infection and a damaged microcirculation that rapidly deteriorated with progression of obliterative cholangiopathy, was observed in the infected mice as early as 72 hr after birth. In experimental biliary atresia, the destruction of the extrahepatic biliary vascular plexus starts already in the first days postinfection and clearly precedes the morphological symptoms of atresia. The deterioration of the vascular bed architecture continues with disease progression. Therefore, we conclude that the (ultra)structural changes in the extrahepatic biliary microvasculature occurring before the visible onset of atresia has a predictive diagnostic value and this impairment in blood supply to the extrahepatic bile duct may be an important contributing factor to the pathogenesis of acquired biliary atresia. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:818-824, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Diseases caused by dengue virus (DENV) are a major public health problem worldwide, considered one of the infections with more prevalence in tropical and subtropical zones of the world. Despite the intense research in the pathogenesis of DENV, this feature is not well understood. One of the main target cells for DENV infection is monocytes; these phagocytes can play a dual role, since they are essential to control viremia, but they also participate in the induction of tissue damage during DENV infection. Monocytes produce different pro-inflammatory cytokines and chemokines in response to infection, and also mediate endothelial damage. In peripheral blood, monocytes can be divided into three different subpopulations, namely classical, intermediate and non-classical, which differ in frequency, cytokine production, among others. Studies in the last years suggest that non-classical monocytes have higher affinity for microvasculature endothelium compared to other type of monocytes, which implies that they could be more involved in the increase of endothelial permeability observed during DENV infection. This review provides a general view of the role of monocytes and their subpopulations in DENV pathogenesis and its effect in viral replication. Finally, the potential contribution of these phagocytes in the alterations of endothelial permeability is discussed.

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB) to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC) lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59) persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs), without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α) and transiently induced, but failed to secrete, IFN-β and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7), IRF9, and IFN-stimulated genes (ISGs) 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV) persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread. IMPORTANCE ZIKV persists in patients, crossing placental and neuronal barriers, damaging neurons, and causing fetal microencephaly. We found that ZIKV persistently infects brain endothelial cells that normally protect neurons from viral exposure. hBMECs are not damaged by ZIKV infection and, analogous to persistent HCV infection, ZIKV constitutively induces and evades antiviral ISG and IFN responses to continuously replicate in hBMECs. As a result, hBMECs provide a protective niche for systemic ZIKV spread and a viral reservoir localized in the normally protective blood-brain barrier. Consistent with the spread of ZIKV into neuronal compartments, ZIKV was released basolaterally from hBMECs. Our findings define hBMEC responses that contribute to persistent ZIKV infection and potential targets for clearing ZIKV infections from hBMECs. These results further suggest roles for additional ZIKV-infected ECs to facilitate viral spread and persistence in the protected placental, retinal, and testicular compartments., (Copyright © 2017 Mladinich et al.)

The polymicrobial dysbiotic subgingival biofilm microbes associated with periodontal disease appear to contribute to developing pathologies in distal body sites, including the brain. This study examined oxidative stress, in the form of increased protein carbonylation and oxidative protein damage, in the tumor necrosis factor-α (TNF-α) transgenic mouse that models inflammatory TNF-α excess during bacterial infection; and in the apolipoprotein knockout (ApoE-/-) mouse brains, following Porphyromonas gingivalis gingival monoinfection. Following 2,4-dinitrophenylhydrazine derivatization, carbonyl groups were detected in frontal lobe brain tissue lysates by immunoblotting and immunohistochemical analysis of fixed tissue sections from the frontotemporal lobe and the hippocampus. Immunoblot analysis confirmed the presence of variable carbonyl content and oxidative protein damage in all lysates, with TNF-α transgenic blots exhibiting increased protein carbonyl content, with consistently prominent bands at 25 kDa (p = 0.0001), 43 kDa, and 68 kDa, over wild-type mice. Compared to sham-infected ApoE-/- mouse blots, P. gingivalis-infected brain tissue blots demonstrated the greatest detectable protein carbonyl content overall, with numerous prominent bands at 25 kDa (p = 0.001) and 43 kDa (p = 0.0001) and an exclusive band to this group between 30-43 kDa* (p = 0.0001). In addition, marked immunostaining was detected exclusively in the microvasculature in P. gingivalis-infected hippocampal tissue sections, compared to sham-infected, wild-type, and TNF-α transgenic mice. This study revealed that the hippocampal microvascular structure of P. gingivalis-infected ApoE-/- mice possesses elevated oxidative stress levels, resulting in the associated tight junction proteins being susceptible to increased oxidative/proteolytic degradation, leading to a loss of functional integrity.

Human brain microvascular endothelial cells (HBMECs), the major cell type in the blood-brain barrier (BBB), play a key role in maintaining brain homeostasis. However, their role in the BBB innate immunity against HIV invasion of the central nervous system (CNS) remains to be determined. Our early work showed that TLR3 signaling of HBMECs could produce the antiviral factors that inhibit HIV replication in macrophages. The present study examined whether exosomes from TLR3-activated HBMECs mediate the intercellular transfer of antiviral factors to macrophages. Primary human macrophages could take up exosomes from TLR3-activated HBMECs. HBMECs-derived exosomes contained multiple antiviral factors, including several key IFN-stimulated genes (ISGs; ISG15, ISG56, and Mx2) at mRNA and protein levels. The depletion of exosomes from TLR3-activated HBMECs culture supernatant diminished HBMECs-mediated anti-HIV activity in macrophages. In conclusion, we demonstrate that exosomes shed by HBMECs are able to transport the antiviral molecules to macrophages. This finding suggests the possibility that HIV nonpermissive BBB cells (HBMECs) can help to restore the antiviral state in HIV-infected macrophages, which may be a defense mechanism against HIV neuroinvasion., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV., Importance: Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Sepsis, also known as septicemia, is one of the 10 leading causes of death worldwide. The rising tide of sepsis due to bacterial, fungal and viral infections cannot be stemmed by current antimicrobial therapies and supportive measures. New paradigms for the mechanism and resolution of sepsis and consequences for sepsis survivors are emerging. Consistent with Benjamin Franklin's dictum 'an ounce of prevention is worth a pound of cure', sepsis can be prevented by vaccinations against pneumococci and meningococci. Recently, the NIH NHLBI Panel redefined sepsis as 'severe endothelial dysfunction syndrome in response to intravascular and extravascular infections causing reversible or irreversible injury to the microcirculation responsible for multiple organ failure'. Microvascular endothelial injury underlies sepsis-associated hypotension, edema, disseminated intravascular coagulation, acute respiratory distress syndrome and acute kidney injury. Microbial genome products trigger 'genome wars' in sepsis that reprogram the human genome and culminate in a 'genomic storm' in blood and vascular cells. Sepsis can be averted experimentally by endothelial cytoprotection through targeting nuclear signaling that mediates inflammation and deranged metabolism. Endothelial 'rheostats' (e.g. inhibitors of NF-κB, A20 protein, CRADD/RAIDD protein and microRNAs) regulate endothelial signaling. Physiologic 'extinguishers' (e.g. suppressor of cytokine signaling 3) can be replenished through intracellular protein therapy. Lipid mediators (e.g. resolvin D1) hasten sepsis resolution. As sepsis cases rose from 387 330 in 1996 to 1.1 million in 2011, and are estimated to reach 2 million by 2020 in the US, mortality due to sepsis approaches that of heart attacks and exceeds deaths from stroke. More preventive vaccines and therapeutic measures are urgently needed., (© 2015 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Hantavirus nephropathy (HVN) is an uncommon etiology of acute renal failure due to hantavirus infection. Pathological features suggestive of HVN historically reported are medullary interstitial hemorrhages in a background of acute interstitial nephritis (AIN). However, interstitial hemorrhages may be lacking because of medullary sampling error. This emphasizes that other pathological criteria may be of interest. We performed a retrospective clinicopathological study of 17 serologically proven HVN cases with renal biopsy from 2 nephrology centers in northern France. Histologic analysis was completed by immunohistochemistry with anti-CD3, anti-CD68, and anti-CD34 antibodies. Three control groups were not related to hantavirus infection: acute tubular necrosis (ATN) of ischemic or toxic etiology and AIN were used for comparison. Renal biopsy analysis showed that almost all HVN cases with medullary sampling (9/10) displayed interstitial hemorrhages, whereas focal hemorrhages were detected in 2 of the 7 "cortex-only" specimens. ATN was common, as it was present in 15 (88.2%) of 17 HVN cases. By contrast, interstitial inflammation was scarce with no inflammation or only slight inflammation, representing 15 (88.2%) of 17 cases. Moreover, HVN showed inflammation of renal microvessels with cortical peritubular capillaritis and medullary vasa recta inflammation; peritubular capillaritis was significantly higher in HVN after comparison with ischemic and toxic ATN controls (P = .0001 and P = .003, respectively), but not with AIN controls. Immunohistochemical studies highlighted the involvement of T cells and macrophages in renal microvascular inflammation related to HVN. Our study showed that microvascular inflammation, especially cortical peritubular capillaritis, and ATN are important histologic features of HVN., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To construct lentivirus siRNA vectors targeting ezrin (E), radixin (R) and moesin (M), transfect rat primary pulmonary microvascular endothelial cells (PMVECs) with the vectors and observe the effects of the transfection on E, R, M gene expressions and influenza virus replication., Methods: According to the principles of siRNA synthesis, siRNA sequences targeting E, R and M genes were synthesized and cloned into lentiviral plasmid GV248 to construct lentiviral siRNA vectors Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. Random Lenti-control-siRNA was also constructed targeting nonsense sequences. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Rat PMVECs were transfected with Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. The levels of ezrin, radixin and moesin mRNA and proteins were detected by real-time quantitative PCR and Western blotting, the distributions of ERM proteins were detected using confocal laser scanning microscope; After lentivirus-transfected PMVECs were infected with influenza virus, the effects of lentiviral vectors on influenza virus replication were measured by real-time quantitative PCR., Results: Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA and Lenti-control-siRNA were successfully constructed according to sequencing identification of recombinant plasmids. The infection efficacy of lentiviral siRNA vectors in rat PMVECs was above 80% at a multiplicity of infection (MOI) of 10. The transcription and expressions of ezrin, radixin and moesin in rat PMVECs were significantly inhibited by Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA respectively. Lentivirus control (containing no target genes) and Lenti-control-siRNA inhibited influenza virus replication in influenza virus-infected PMVECs, while Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA increased the levels of influenza virus mRNA in the infected PMVECs., Conclusion: Lentiviral siRNA vectors targeting E, R and M genes were constructed successfully and could inhibit E, R and M gene expressions specifically and effectively. Lentiviral vectors could inhibit influenza virus replication whereas lentiviral siRNA vectors targeting E, R and M facilitated influenza virus replication.

2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

Cytomegalovirus (CMV) infects a majority of the population worldwide. It has been implicated in cardiovascular disease, induces microvascular dysfunction, and synergizes with hypercholesterolemia to promote leukocyte and platelet recruitment in venules. Although platelets and platelet-associated P-selectin contribute to cardiovascular disease inflammation, their role in CMV-induced vascular responses is unknown. We assessed the role of platelets in CMV-induced microvascular dysfunction by depleting platelets and developing bone marrow chimeric mice deficient in platelet P-selectin. Wild-type and chimeric mice received mock or murine (m)CMV intraperitoneally. Five weeks later, some mice were switched to a high-cholesterol diet (HC) to investigate the synergism between mCMV and HC. Arteriolar vasodilation and recruitment of leukocytes and donor platelets in venules were measured at 11wk. mCMV with or without HC caused significant endothelial dysfunction in arterioles. Platelet depletion restored normal vasodilation in mCMV-HC but not mCMV-ND mice, whereas protection was seen in both groups for platelet P-selectin chimeras. Only mCMV + HC elevated leukocyte and platelet recruitment in venules. Leukocyte adhesion was reduced to mock levels by acute platelet depletion but was only partially decreased in platelet P-selectin chimeras. Platelets from mCMV-HC mice and, to a lesser extent, mCMV-ND but not mock-HC mice showed significant adhesion in mCMV-HC recipients. Our findings implicate a role for platelets, acting through P-selectin, in CMV-induced arteriolar dysfunction and suggest that the addition of HC leads to a platelet-dependent, inflammatory infiltrate that is only partly platelet P-selectin dependent. CMV appeared to have a stronger activating influence than HC on platelets and may represent an additional therapeutic target in vulnerable patients., (Copyright © 2014 the American Physiological Society.)

The severity of West Nile virus (WNV) infection in immunocompetent animals is highly strain dependent, ranging from avirulent to highly neuropathogenic. Here, we investigate the nature of this strain-specific restriction by analyzing the replication of avirulent (WNV-MAD78) and highly virulent (WNV-NY) strains in neurons, astrocytes, and microvascular endothelial cells, which comprise the neurovascular unit within the central nervous system (CNS). We demonstrate that WNV-MAD78 replicated in and traversed brain microvascular endothelial cells as efficiently as WNV-NY. Likewise, similar levels of replication were detected in neurons. Thus, WNV-MAD78's nonneuropathogenic phenotype is not due to an intrinsic inability to replicate in key target cells within the CNS. In contrast, replication of WNV-MAD78 was delayed and reduced compared to that of WNV-NY in astrocytes. The reduced susceptibility of astrocytes to WNV-MAD78 was due to a delay in viral genome replication and an interferon-independent reduction in cell-to-cell spread. Together, our data suggest that astrocytes regulate WNV spread within the CNS and therefore are an attractive target for ameliorating WNV-induced neuropathology.

Background: Our previous study showed an efficient targeting of islets of Langerhans by adenoviral injection via the celiac trunk. Unexpectedly, none of the endothelial cells was infected given the direct contact between adenoviruses and the capillary wall. The present study intended to provide an efficient approach for adenoviral targeting of the microcapillary endothelial cells in the pancreas., Methods: We prepared microspheres of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with a size comparable to the diameter of capillary (5-10 µm). Scanning electron microscopy was applied to verify that adenoviruses carrying a green fluorescence protein gene were complexed with PHBHHx-microspheres after 30 min of co-incubation. The complexes were then injected into the pancreas of mice via the celiac trunk., Results: Approximately 40% of endothelial cells in the pancreas were labeled 5 days after surgery. Islet cells were labeled occasionally, whereas labeling of the acinar and ductal tissues was barely detectable. Endothelium targeting was inefficient in other internal organs. Consistent with the reported superior tissue compatibility of PHBHHx, no discernable microspheres were found in all of the organs examined. Furthermore, splenocyte activation was dampened when adenoviruses were complexed with the microspheres., Conclusions: The present study has established an approach for efficient pancreatic capillary targeting by using microsphere-adenoviral complexes. This procedure could be invaluable for the treatment of capillary-related diseases., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background & Aims: Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS., Methods: We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication., Results: Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis., Conclusions: Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The central nervous system (CNS) is a major target of several important human and animal viral pathogens causing congenital infections. However, despite the importance of neuropathological outcomes, for humans in particular, the pathogenesis, including mode of neuro-invasion, remains unresolved for most congenital virus infections. Using a natural model of congenital infection with an RNA virus, bovine viral diarrhoea virus in pregnant cattle, we sought to delineate the timing and mode of virus neuro-invasion of and spread within the brain of foetuses following experimental respiratory tract infection of the dams at day 75 of pregnancy, a time of maximal risk of tissue pathology without foetal death. Virus antigen was first detected in the foetal brains 14 days postinfection of dams and was initially restricted to amoeboid microglial cells in the periventricular germinal layer. The appearance of these cells was preceded by or concurrent with vasculopathy in the same region. While the affected microvessels were negative for virus antigen, they expressed high levels of the type I interferon-stimulated protein ISG15 and eventually disappeared in parallel with the appearance of microcavitary lesions. Subsequently, the virus spread to neurons and other glial cells. Our findings suggest that the virus enters the CNS via infected microglial precursors, the amoeboid microglial cells, in a 'Trojan horse' mode of invasion and that the microcavitary lesions are associated with loss of periventricular microvasculature, perhaps as a consequence of high, unrestricted induction of interferon-regulated proteins., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

Nipah virus (NiV), a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC) in the viremic spread of the virus in the swine host. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8- cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166) highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain.

Background: Human Parvovirus B19 (PVB19) has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far., Methods and Findings: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP) color reporter gene in the non-structural segment 1 (NS1) of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304). The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber). NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05) and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05) compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05). The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR) analysis., Conclusions: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

Background: Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH., Methods: The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB., Results: HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB., Conclusion: In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.

Multiple sclerosis (MS) is an inflammatory neurological disease that is widely regarded as the outcome of complex interactions between a genetic predisposition and an environmental trigger. Epstein-Barr virus (EBV) has recently been associated with the onset of MS, yet understanding how it elicits autoimmunity remains elusive. Neuroinflammation, including the entry of autoreactive T cells, likely follows a breach of the blood-brain barrier (BBB) leading to CNS lesions in MS. We show that EBV can infect human BBB cells leading to increased production of pro-inflammatory mediators that result in immune cell adherence thus modeling a key step in MS pathogenesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease., Aim: To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease., Methods: We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces., Results: We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium., Conclusion: The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.

Expression of tight junction proteins between brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) is lost during development of human immunodeficiency virus (HIV) encephalitis (HIVE). Although many studies have focused on the strains of virus that induce neurological sequelae or on the macrophages/microglia that are associated with development of encephalitis, the molecular signaling pathways within the BMECs involved have yet to be resolved. We have previously shown that there is activation and disruption of an in vitro BBB model using lentivirus-infected CEMx174 cells. We and others have shown similar disruption in vivo. Therefore, it was of interest to determine if the presence of infected cells could disrupt intact cerebral microvessels immediately ex vivo, and if so, which signaling pathways were involved. The present data demonstrate that disruption of tight junctions between BMECs is mediated through activation of focal adhesion kinase (FAK) by phosphorylation at TYR-397. Inhibition of FAK activation is sufficient to prevent tight junction disruption. Thus, it may be possible to inhibit the development of HIVE by using inhibitors of FAK.

How neuroinflammatory activities affect signaling pathways leading to blood-brain barrier (BBB) injury during HIV/AIDS are currently unknown. Our previous work demonstrated that HIV-1 exposure activates pro-inflammatory genes in human brain microvascular endothelial cells (HBMEC) and showed that these genes are linked to the janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Here, we report that HIV-1 gp120 protein activated STAT1 and induced interleukin (IL)-6 and IL-8 secretion in HBMEC. IL-6, IL-8, and gp120 increased monocyte adhesion and migration across in vitro BBB models. The STAT1 inhibitor, fludarabine, prevented gp120-induced IL-6 and IL-8 secretion. Inhibitors of STAT1, mitogen activated protein kinase kinase (MEK) (PD98059), and phosphatidyl inositol 3 kinase (PI3K) (LY294002), blocked gp120-induced STAT1 activation and significantly diminished IL-8-, IL-6-, and gp120-induced monocyte adhesion and migration across in vitro BBB models. These data support the notion that STAT1 plays an important role in gp120-induced inflammation and BBB dysfunction associated with viral infection. Results also suggest crosstalk between STAT1, MEK, and PI3K pathways in gp120-induced BBB dysfunction. Inhibition of STAT1 activation could provide a unique therapeutic strategy to decrease neuroinflammation and BBB dysfunction in HIV/AIDS.