Lung Ultrasound Findings and Endothelial Perturbation in a COVID-19 Low-Intensity Care Unit

Hypercoagulability and endothelial dysfunction related to inflammation have been clearly demonstrated in COVID-19. However, their influence on thromboembolism, lung alterations and mortality in low-intensity-care patients with COVID-19 is not completely clarified. Our aims were to evaluate the prevalence of deep vein thrombosis (DVT) with compressive ultrasound (CUS); to describe lung ultrasound (LUS) features; and to study coagulation, inflammatory and endothelial perturbation biomarkers in COVID-19 patients at low-intensity care unit admission. The predictive value of these biomarkers on mortality, need for oxygen support and duration of hospitalization was also evaluated. Of the 65 patients included, 8 were non-survivors. CUS was negative for DVT in all patients. LUS Soldati and Vetrugno scores were strongly correlated (rho = 0.95) with each other, and both significantly differed in patients who needed oxygen therapy vs. those who did not (Soldati p = 0.017; Vetrugno p = 0.023), with coalescent B lines as the most prevalent pattern in patients with a worse prognosis. Mean (SD) levels of thrombomodulin and VCAM-1 were higher in non-survivors than in survivors (7283.9 pg/mL (3961.9 pg/mL) vs. 4800.7 pg/mL (1771.0 pg/mL), p = 0.004 and 2299 ng/mL (730.35 ng/mL) vs. 1451 ng/mL (456.2 ng/mL), p < 0.001, respectively). Finally, in a multivariate analysis model adjusted for age, sex and Charlson score, VCAM-1 level increase was independently associated with death [OR 1.31 (1.06, 1.81; p = 0.036)]. In conclusion, in a cohort of mild COVID-19 patients, we found no DVT events despite the highly abnormal inflammatory, endothelial and coagulation parameters. The presence of lung alterations at admission could not predict outcome. The endothelial perturbation biomarker VCAM-1 emerged as a promising prognostic tool for mortality in COVID-19

Prevalence of disease and relationships between laboratory phenotype and bleeding severity in platelet primary secretion defects

BACKGROUND: The prevalence of platelet primary secretion defects (PSD) among patients with bleeding diathesis is unknown. Moreover, there is paucity of data on the determinants of bleeding severity in PSD patients. OBJECTIVE: To determine the prevalence of PSD in patients with clinical bleeding and to study the relationships between the type of platelet defect and bleeding severity. METHODS: Data on patients referred for bleeding to the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan (Italy) in the years between 2008 and 2012 were retrieved to study the prevalence of PSD. Demographic, clinical and laboratory information on 32 patients with a diagnosis of PSD was used to compare patients with or without associated medical conditions and to investigate whether or not the type and extension of platelet defects were associated with the bleeding severity score (crude and age-normalized) or with the age at first bleeding requiring medical attention. RESULTS: The estimated prevalence of PSD among 207 patients with bleeding diathesis and bleeding severity score above 4 was 18.8% (95% confidence interval [CI]: 14.1-24.7%). Patients without associated medical conditions had earlier age of first bleeding (18 vs 45 years; difference: -27 years; 95% CI: -46 to -9 years) and different platelet functional defect patterns (Fisher's exact test of the distribution of patterns, P\u200a=\u200a0.007) than patients with accompanying medical conditions. The type and extension of platelet defect was not associated with the severity of bleeding. CONCLUSIONS: PSD is found in approximately one fifth of patients with clinical bleeding. In patients with PSD, the type and extension of laboratory defect was not associated with bleeding severit

Mediterranean spotted fever and hearing impairment : a rare complication

Mediterranean spotted fever (MSF) is caused by Rickettsia conorii and transmitted by the brown dog tick Rhipicephalus sanguineus. It is prevalent in southern Europe, Africa and central Asia. The disease usually has a benign course and is characterized by fever, myalgia and a characteristic papular rash with an inoculation eschar ('tache noir') at the site of the tick bite. Severe forms of disease can have cardiac, neurologic or renal involvement. Nervous system complications are unusual and may develop in the early phase of disease or as a delayed complication. Neurological symptoms include headache and alterations of the level of consciousness, and some cases of meningoenchefalitis and Guillain-Barre syndrome have been also reported. Peripheral nerve involvement is reported only in a limited number of case reports. We describe a case of Rickettsia conorii that was complicated with hearing loss and did not respond to specific treatment. Hearing loss is a rare event, but clinicians should be aware of this complication

Surfactant protein D modulates HIV infection of both T-cells and dendritic cells

Surfactant Protein D (SP-D) is an oligomerized C-type lectin molecule with immunomodulatory properties and involvement in lung surfactant homeostasis in the respiratory tract. SP-D binds to the enveloped viruses, influenza A virus and respiratory syncytial virus and inhibits their replication in vitro and in vivo. SP-D has been shown to bind to HIV via the HIV envelope protein gp120 and inhibit infectivity in vitro. Here we show that SP-D binds to different strains of HIV (BaL and IIIB) and the binding occurs at both pH 7.4 and 5.0 resembling physiological relevant pH values found in the body and the female urogenital tract, respectively. The binding of SP-D to HIV particles and gp120 was inhibited by the presence of several hexoses with mannose found to be the strongest inhibitor. Competition studies showed that soluble CD4 and CVN did not interfere with the interaction between SP-D and gp120. However, soluble recombinant DC-SIGN was shown to inhibit the binding between SP-D and gp120. SP-D agglutinated HIV and gp120 in a calcium dependent manner. SP-D inhibited the infectivity of HIV strains at both pH values of 7.4 and 5.0 in a concentration dependent manner. The inhibition of the infectivity was abolished by the presence of mannose. SP-D enhanced the binding of HIV to immature monocyte derived dendritic cells (iMDDCs) and was also found to enhance HIV capture and transfer to the T-cell like line PM1. These results suggest that SP-D can bind to and inhibit direct infection of T-cells by HIV but also enhance the transfer of infectious HIV particles from DCs to T-cells in vivo

Inactivation of respiratory syncytial virus by zinc finger reactive compounds

<p>Abstract</p> <p>Background</p> <p>Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (<it>Pneumoviridae</it>), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins.</p> <p>Results</p> <p>Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats <it>S.hispidus </it>and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease.</p> <p>Conclusions</p> <p>This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.</p

Identification of HIV-1 Epitopes that Induce the Synthesis of a R5 HIV-1 Suppression Factor by Human CD4+ T Cells Isolated from HIV-1 Immunized Hu-PBL SCID Mice

We have previously reported that immunization of the severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood mononuclear cells (PBMC) (hu-PBL-SCID mice) with inactivated human immunodeficiency virus type-1 (HIV-1)-pulsed-autologous dendritic cells (HIV-DC) elicits HIV-1-reactive CD4+ T cells that produce an as yet to be defined novel soluble factor in vitro with anti-viral properties against CCR5 tropic (R5) HIV-1 infection. These findings led us to perform studies designed to identify the lineage of the cell that synthesizes such a factor in vitro and define the epitopes of HIV-1 protein that have specificity for the induction of such anti-viral factor. Results of our studies show that this property is a function of CD4+ but not CD8+ T cells. Human CD4+ T cells were thus recovered from the HIV-DC-immunized hu-PBL-SCID mice and were re-stimulated in vitro by co-culture for 2 days with autologous adherent PBMC as antigen presenting cells, APC previously pulsed with inactivated HIV in IL-2-containing medium to expand HIV-1-reactive CD4+ T cells. Aliquots of these re-stimulated CD4+ T cells were then co-cultured with similar APC's that were previously pulsed with 10 μg/ml of a panel of HIV peptides for an additional 2 days, and their culture supernatants were examined for the production of both the R5 HIV-1 suppression factor and IFN-Υ. The data presented herein show that the HIV-1 primed CD4+ T cells produced the R5 suppression factor in response to a wide variety of HIV-1 gag, env, pol, nef or vif peptides, depending on the donor of the CD4+ T cells. Simultaneous production of human interferon (IFN)-Υ was observed in some cases. These results indicate that human CD4+ T cells in PBMC of HIV-1 naive donors have a wide variety of HIV-1 epitope-specific CD4+ T cell precursors that are capable of producing the R5 HIV-1 suppression factor upon DC-based vaccination with whole inactivated HIV-1

Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-Dependent Variation in Quaternary Structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses

Topology of the C-Terminal Tail of HIV-1 gp41: Differential Exposure of the Kennedy Epitope on Cell and Viral Membranes

The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the “intracytoplasmic domain” based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical “Kennedy epitope” (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned

Cryoelectron Tomography of HIV-1 Envelope Spikes: Further Evidence for Tripod-Like Legs

A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a cryoelectron microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions. Perhaps most notable was the presence of three splayed legs projecting obliquely from the base of the spike head toward the viral membrane. Subsequently, a second 3D image of SIV spikes, also obtained by cryoET, was published by another group which featured a compact vertical stalk. We now report the cryoET analysis of HIV-1 virion-associated Env spikes using enhanced analytical cryoET procedures. More than 2,000 Env spike volumes were initially selected, aligned, and sorted into structural classes using algorithms that compensate for the “missing wedge” and do not impose any symmetry. The results show varying morphologies between structural classes: some classes showed trimers in the head domains; nearly all showed two or three legs, though unambiguous three-fold symmetry was not observed either in the heads or the legs. Subsequently, clearer evidence of trimeric head domains and three splayed legs emerged when head and leg volumes were independently aligned and classified. These data show that HIV-1, like SIV, also displays the tripod-like leg configuration, and, unexpectedly, shows considerable gp41 leg flexibility/heteromorphology. The tripod-like model for gp41 is consistent with, and helps explain, many of the unique biophysical and immunological features of this region