Clinical Features and Outcomes of Patients with Pancreaticobiliary Malignancies in Los Angeles County and Their Association with CA 19-9 Levels.

Although CA 19-9 is a commonly used tumor marker in the management of PBMs, the literature describing outcomes in patients with PBMs who have undetectable or low (hereinafter "low") CA 19-9 levels remains scarce. In this study, we sought to compare clinical features and outcomes in patients with PBMs and low CA 19-9 levels to those with normal and elevated CA 19-9 levels. METHODS: We retrospectively collected data on patients with biopsy-confirmed PBMs and stratified patients into categories based on their CA 19-9 level at diagnosis. Survival curves were estimated for patients in each of the three aforementioned CA 19-9 groups using the Kaplan-Meier method and compared using a Cox proportional hazards regression model. RESULTS: Of the 283 patients identified, 23 (8.1%) had low, 70 (24.7%) had normal, and 190 (67.1%) had elevated CA 19-9 levels. After controlling for sex, age, BMI, the presence of metastases at the time of diagnosis, and treatment with curative intent, the hazard ratio for death in the elevated CA 19-9 group compared to the low CA 19-9 group was 1.993 (95% CI 1.089-3.648; p = 0.025). CONCLUSION: The elevated CA 19-9 level compared to the low CA 19-9 level and the presence of metastases were associated with an increased hazard of death, while treatment with curative intent was associated with a decreased hazard of death.

Yield of Post-Acute Diverticulitis Colonoscopy for Ruling Out Colorectal Cancer.

Background and Aims: Colonoscopy is recommended post-acute diverticulitis (AD) to exclude underlying adenocarcinoma (CRC). However, post-AD colonoscopy utility remains controversial. We aimed to examine yield of post-AD colonoscopy in our majority-Hispanic patient population. Methods: Patients undergoing post-AD colonoscopy between 11/1/2015-7/31/2021 were identified from a prospectively maintained endoscopic database. AD cases without computed tomography confirmation were excluded. Pertinent data, including complicated vs uncomplicated AD, fecal immunochemical test (FIT) result post-AD/pre-colonoscopy, and number/type/location of non-advanced adenomas, advanced adenomas, and CRC, were abstracted. Analyses were conducted using two-sample Wilcoxon rank-sum and Fisher's exact tests. Results: 208 patients were included, of whom 62.0% had uncomplicated AD. Median age was 53, 54.3% were female, and 77.4% were Hispanic. Ninety non-advanced adenomas were detected in 45 patients (21.6%), in addition to advanced adenoma in eight patients (3.8%). Two patients (1.0%) had CRC, both of whom had complicated AD in the same location seen on imaging, and one of whom was FIT+ (the other had not undergone FIT). Patients with uncomplicated versus complicated AD had similarly low rates of advanced adenomas (4.7% vs. 2.5%, p=0.713). FIT data were available in 51 patients and positive in three (5.9%); non-advanced adenomas were found in all three FIT+ patients. No FIT- patient had an advanced adenoma or CRC. Conclusion: Colonoscopy post-AD is generally low yield, with CRC being rare and found only in those with complicated AD. Colonoscopy post-complicated AD appears advisable, whereas less invasive testing (e.g. FIT) may be considered post-uncomplicated AD to inform the need for colonoscopy.

Clinical comparison of tear film breakup time measurements in normal dogs using three different methods of fluorescein solution administration.

OBJECTIVE: To evaluate whether the method of fluorescein administration affects the results of tear film breakup time (TFBUT) measurement in normal dogs. ANIMALS STUDIED: Thirty-seven client and hospital staff owned dogs over 1 year of age with no known comorbidities or administration of systemic or topical ophthalmic medications. PROCEDURES: A prospective randomized three-way crossover study was conducted. All dogs received an abbreviated ophthalmic examination to rule out ocular surface disease. Using a 30-min washout interval period, each dog's right eye was received: (a) direct application of fluorescein stain strip with one drop of sterile eyewash, (b) direct application of fluorescein stain strip with two drops of sterile eyewash, or (c) application of one drop from a premade fluorescein solution (dilution of one strip in 0.3 mL sterile eyewash). Eyes were assessed using the cobalt blue filter of a slit lamp biomicroscope. TFBUT measurements were summarized as means ± standard deviation. The methods were compared using mixed model analysis of variance. All analyses were performed using sas version 9.4. RESULTS: Thirty-seven dogs met the inclusion criteria. Mean TFBUT ± standard deviation (SD) for the three described methods were: (a) 16.58s ± 6.9, (b) 15.98s ± 7.1, and (c) 16.43s ± 8.1. No differences between fluorescein stain application techniques were observed (p = .92). CONCLUSION: The technique of fluorescein solution administration did not affect TFBUT measurement in this population of healthy dogs.

Causes, outcomes, and owner satisfaction of dogs undergoing enucleation with orbital implant placement.

OBJECTIVE: To describe clinical and histopathologic pre-operative diagnoses as well as associated post-operative complications following orbital silicone implantation in dogs undergoing enucleation and evaluate owner satisfaction. ANIMALS STUDIED: One hundred and eighty-six dogs who underwent enucleation with orbital implant. PROCEDURES: Medical records from dogs that underwent enucleation with orbital implant performed at Virginia-Maryland Veterinary Teaching Hospital between 2007 and 2019 were reviewed. Owners were surveyed via telephone regarding client satisfaction. RESULTS: Enucleation followed by orbital implant placement occurred in 215 eyes of 186 dogs. The most common pre-operative diagnoses were glaucoma (68.8%), uveitis (17.7%), cataracts (15.8%), intraocular neoplasia (13.0%), and lens luxation (10.7%). The most common histopathologic diagnoses were retinal degeneration (46.5%), uveitis (39.5%), cataract (29.8%), retinal detachment (27.4%), and secondary glaucoma (26.5%). Fourteen eyes (6.5%) from ten dogs had post-operative complications reported including orbit cellulitis (n = 11), implant migration (n = 1), and implant extrusion (n = 1). Five of these dogs (50%) had concurrent diabetes mellitus. Median complication time from surgery was 41 days (range: 11-541 days). Ninety-five owner survey responses were completed with a median time of 6.3 years following surgery. Most owners, 85.3% (n = 81), were satisfied with the post-operative outcome. CONCLUSION: Enucleation with implantation of an orbital implant is a viable and safe method for irreversibly blind eyes. Diabetes mellitus may be a risk factor for the development of post-operative complications. Intraocular neoplasia was not associated with development of post-operative complications. Results of this study indicated high owner satisfaction rates for improving cosmetic appearance after enucleation in dogs.

Circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection.

UNLABELLED: Human parainfluenza viruses (HPIVs) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the hemagglutinin-neuraminidase receptor-binding protein (HN) and the fusion envelope glycoprotein (F), which together comprise the molecular fusion machinery; upon receptor engagement by HN, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo. IMPORTANCE: Acute respiratory infection is the leading cause of mortality in young children under 5 years of age, causing nearly 20% of childhood deaths worldwide each year. The paramyxoviruses, including human parainfluenza viruses (HPIVs), cause a large share of these illnesses. There are no vaccines or drugs for the HPIVs. Inhibiting entry of viruses into the human cell is a promising drug strategy that blocks the first step in infection. To develop antivirals that inhibit entry, it is critical to understand the first steps of infection. We found that clinical viruses isolated from patients have very different entry properties from those of the viruses generally studied in laboratories. The viral entry mechanism is less active and more sensitive to fusion inhibitory molecules. We propose that to interfere with viral infection, we test clinically circulating viruses in natural tissues, to develop antivirals against respiratory disease caused by HPIVs.

Interaction between the hemagglutinin-neuraminidase and fusion glycoproteins of human parainfluenza virus type III regulates viral growth in vivo.

UNLABELLED: Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain's HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN's altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE: Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN's altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.

Identification of a region in the stalk domain of the nipah virus receptor binding protein that is critical for fusion activation.

Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.

Adaptation of human parainfluenza virus to airway epithelium reveals fusion properties required for growth in host tissue.

UNLABELLED: Paramyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). We have previously shown that viruses with an HN/F pair that is highly fusogenic in monolayers of immortalized cells due to mutations in HN's secondary sialic acid binding site are growth impaired in differentiated human airway epithelium (HAE) cultures and in vivo. Here we have shown that adaptation of HPIV3 to growth in the lung is determined by specific features of HN and F that are different from those required for growth in cultured immortalized cells. An HPIV3 virus bearing a mutated HN (H552Q), which is fit and fusogenic in immortalized cells but unfit for growth in the lung, evolved into a less-fusogenic but viable virus in differentiated human airway epithelium. Stepwise evolution led to a progressive decrease in efficiency of fusion activation by the HN/F pair, with a mutation in F first decreasing the activation of F by HN and a mutation in HN's secondary sialic acid binding site decreasing fusion activation further and producing a stable virus. Adaptation of HPIV3 to successful growth in HAE is determined by specific features of HN and F that lead to a less easily activated fusion mechanism. IMPORTANCE: Human parainfluenza viruses (HPIVs) are paramyxoviruses that cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide, but there are currently no vaccines or antivirals available for treatment. Enveloped viruses must fuse their membrane with the target cell membrane in order to initiate infection. Parainfluenza virus fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. The receptor-binding hemagglutinin-neuraminidase (HN), upon receptor engagement, activates the fusion protein (F) to penetrate the target cell and mediate viral entry. In this study, we show that the precise balance of fusion activation properties of these two glycoproteins during entry is key for infection. In clinically relevant tissues, viruses evolve to acquire a set of fusion features that provide key clues about requirements for infection in human beings.

The second receptor binding site of the globular head of the Newcastle disease virus hemagglutinin-neuraminidase activates the stalk of multiple paramyxovirus receptor binding proteins to trigger fusion.

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN's globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN's site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation.

Mechanism of fusion triggering by human parainfluenza virus type III: communication between viral glycoproteins during entry.

Parainfluenza viruses enter host cells by fusing the viral and target cell membranes via concerted action of their two envelope glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion protein (F). Receptor-bound HN triggers F to undergo conformational changes that render it fusion-competent. To address the role of receptor engagement and to elucidate how HN and F interact during the fusion process, we used bimolecular fluorescence complementation to follow the dynamics of human parainfluenza virus type 3 (HPIV3) HN/F pairs in living cells. We show that HN and F associate before receptor engagement. HN drives the formation of HN-F clusters at the site of fusion, and alterations in HN-F interaction determine the fusogenicity of the glycoprotein pair. An interactive site, at the HN dimer interface modulates HN fusion activation property, which is critical for infection of the natural host. This first evidence for the sequence of initial events that lead to viral entry may indicate a new paradigm for understanding Paramyxovirus infection.

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

A recombinant sialidase fusion protein effectively inhibits human parainfluenza viral infection in vitro and in vivo.

BACKGROUND: The first step in infection by human parainfluenza viruses (HPIVs) is binding to the surface of respiratory epithelial cells via interaction between viral receptor-binding molecules and sialic acid-containing receptors. DAS181, a recombinant sialidase protein containing the catalytic domain of Actinomyces viscosus sialidase, removes cell surface sialic acid, and we proposed that it would inhibit HPIV infection. METHODS: Depletion of sialic acid receptors by DAS181 was evaluated by lectin-binding assays. Anti-HPIV activity in cultured cell lines and in human airway epithelium was assessed by the reduction in viral genomes and/or plaque forming units on treatment. In vivo efficacy of intranasally administered DAS181 was assessed using a cotton rat model. RESULTS: DAS181-mediated desialylation led to anti-HPIV activity in cell lines and human airway epithelium. Intranasal DAS181 in cotton rats, a model for human disease, significantly curtailed infection. CONCLUSIONS: Enzymatic removal of the sialic acid moiety of HPIV receptors inhibits infection with all tested HPIV strains, both in vitro and in cotton rats. Enzyme-mediated removal of sialic acid receptors represents a novel antiviral strategy for HPIV. The results of this study raise the possibility of a broad spectrum antiviral agent for influenza virus and HPIVs.

Human parainfluenza virus infection of the airway epithelium: viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity.

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN's receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN's F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN's F-triggering activity may have promise in vivo.