Eph receptor tyrosine kinases and their ephrin ligands have been implicated in embryonic vascular development and in in vivo models of angiogenesis. Eph proteins may also regulate tumor neovascularization, but this role has not been previously investigated. To screen for Eph proteins expressed in tumor blood vessels, we used tumor xenografts grown in nude mice from MDA-MB-435 human breast cancer cells or KS1767 human Kaposi's sarcoma cells. By immunohistochemistry, the ephrin-A1 ligand and one of its receptors, EphA2, were detected throughout tumor vasculature. Double-labeling with anti-CD34 antibodies demonstrated that both ephrin-A1 and EphA2 were expressed in xenograft endothelial cells and also tumor cells. Furthermore, EphA2 was tyrosinephosphorylated in the xenograft tumors, indicating that it was activated, presumably by interacting with ephrin-A1. Ephrin-A1 and EphA2 were also detected in both the vasculature and tumor cells of surgically removed human cancers. In an in vitro angiogenesis model, a dominant negative form of EphA2 inhibited capillary tube-like formation by human umbilical vein endothelial cells (HUVECs), demonstrating a requirement for EphA receptor signaling. These data suggest that ephrin-A1 and EphA2 play a role in human cancers, at least in part by in¯uencing tumor neovascularization. Eph proteins may represent promising new targets for antiangiogenic cancer treatments. Oncogene (2000) 19, 6043 ± 6052.

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Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling. In contrast, mutation to the negatively charged glutamic acid disrupted SH2 domain binding without reducing EphB2 kinase activity. By using a panel of EphB2 mutants, we found that kinase activity is required for the changes in cell-matrix and cell - cell adhesion, cytoskeletal organization, and activation of mitogen-activated protein (MAP) kinases elicited by EphB2 in transiently transfected cells. Instead, the two juxtamembrane SH2 domain binding sites were dispensable for these effects. These results suggest that phosphorylation of tyrosines 605 and 611 is critical for EphB2-mediated cellular responses because it regulates EphB2 kinase activity.

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance ofa proper tissue organization during liver regeneration.

We show that diverse human tumors obtained directly from surgery or biopsy can grow at high frequency in vitro for long periods of time and still maintain many of their in vivo properties. The in vivo properties maintained in vitro include three-dimensional growth; maintenance of tissue organization and structure, including changes associated with oncogenic transformation; retention of differentiated function; tumorigenicity; and the growth of multiple types of cells from a single tumor.It has not been known whether human tumors can grow at high frequency for long periods of time in vitro and still maintain many oftheir critical in vivo properties. Tumor MATERIALS AND METHODSEstablishment of Human Tumor Explants in Culture. The specialized collagen gel (A.E.F., P. H. Gumerlock, and S. H. Hinrichs, unpublished data) is a commercial product of Health Design Industries (Rochester, NY) and is manufactured from pigskin. The material comes as dehydrated squares, which were removed from their sterile packages and placed in 60-mm plastic tissue-culture dishes containing Eagle's minimal essential medium (MEM) with 10% fetal bovine serum (Irvine Scientific), 0.1 mM nonessential amino acids, and the antibiotics gentamicin (100 ,g/ml) and cefotaxime (95 ,ug/ml). The gels were soaked in this medium with at least one fluid change before use.Immediately after surgery or biopsy, tumor sections in MEM with Hanks' salts and 10% fetal bovine serum were brought to the laboratory. Necrotic tissue was cut away, and the remaining healthy tumor tissue was minced with scissors into '4-mm3 bits. Five to ten of these tumor bits were placed on the collagen surface, where they tended to stick or become embedded in loose fiber structure. Medium was added until the upper part of the gel was reached but not covered. The cultures were refed twice a week.Fixation, Embedding, and Staining of Tumor Cells. Cells growing in the collagen gels were fixed in 10% formalin for at least 48 hr. The fixed material was washed for 1 hr in slowly running tap water to rinse out the formalin and then was processed through a series of changes of ethanol (70-100%), with each change lasting 1 hr. The material was then treated with xylene or chloroform and finally embedded in paraffin. After the paraffin hardened, the material was sectioned at 5Am and then dried for 10-15 min on a slide warmer. The next day the slides were stained with Gill's hematoxylin no. 2 and eosin and then mounted.Autoradiography of Tumor Cells. Cells were labeled metabolically, in the above-described medium used for establishment of tumor explants, with [methyl-3H]thymidine (1 ,uCi/ml, Ci/mmol, ICN; 1 Ci = 37 GBq) for 24 hr. After labeling, the medium was removed and the gels were washed three times for 5 min each with nonradioactive medium. Gels were then fixed, dehydrated, embedded in paraffin, and sectioned. The slides, after deparaffinization with xylene, were coated with Kodak NTB-2 emulsion (diluted 1:1 with water) at 40'C and stored in the dark at 40C for 1 week. The slides ...

Enoxaparin is commonly used to prevent venous thromboembolism (VTE) [1, 2] but has not been well-studied in patients with extreme obesity, a population at high risk for VTE. We prospectively compared three enoxaparin dosing regimens for the achievement of goal peak anti-Factor Xa levels in medically-ill patients (n=31) with extreme obesity (body mass index (BMI) ≥ 40 kg/m2). Patients were assigned to receive fixed-dose (FD) enoxaparin 40mg daily (QDay, n=11), weight-based, lower-dose (LD) enoxaparin 0.4 mg/kg QDay (n=9), or weight-based, higher-dose (HD) enoxaparin 0.5 mg/kg QDay(n=11). The average BMI and weight of the entire cohort was 62.1 kg/m2 (range 40.5-82.4) and 176 kg (range 115-256 kg) and did not differ between groups. Peak anti-Factor Xa levels were significantly higher in the HD group compared to either LD or FD groups. Patients in the HD group achieved target anti-Factor Xa levels more frequently than the LD and FD groups (p<0.05). Peak anti-Factor Xa levels did not correlate with age, weight, BMI, or creatinine clearance, demonstrating the predictability of weight-based enoxaparin dosing. There were no adverse events (e.g. bleeding, thrombosis, thrombocytopenia). To our knowledge, this is the first prospective comparative study demonstrating that in extremely obese, medically-ill patients enoxaparin 0.5 mg/kg QDay is superior to fixed-dose and lower-dose enoxaparin for the achievement of target anti-Factor Xa levels.

Venous thromboembolism (VTE) is a significant cause of morbidity and mortality in hospitalized patients. Where appropriate, evidence-based methods of prophylaxis are implemented and the burden of VTE can be reduced substantially. Obesity, including morbid obesity, is associated with a high risk of VTE and, unfortunately, fixed doses of US FDA-approved anticoagulant regimens, including unfractionated heparins, low-molecular-weight heparins and factor Xa inhibitors, may not provide optimal VTE prophylaxis in these patients. Although the data are still limited, a rapidly growing body of literature and cumulative evidence suggests that anticoagulant dose adjustments in morbidly obese patients may optimize pharmacodynamic activity and reduce VTE risk. With the prevalence of morbid obesity continuing to rise, more high-quality clinical data are needed to better understand the pathobiology of VTE in obesity and provide effective, yet safe, prevention strategies.

IMPORTANCE e-Cigarette, or vaping, product use-associated lung injury (EVALI) has caused more than 2800 illnesses and 68 deaths in the United States. Better characterization of this novel illness is needed to inform diagnosis and management. OBJECTIVE To describe the clinical features, bronchoscopic findings, imaging patterns, and outcomes of EVALI. DESIGN, SETTING, AND PARTICIPANTS This case series of 31 adult patients diagnosed with EVALI between June 24 and December 10, 2019, took place at an academic medical center in Salt Lake City, Utah. EXPOSURES e-Cigarette use, also known as vaping. MAIN OUTCOMES AND MEASURES Symptoms, laboratory findings, bronchoscopic results, imaging patterns, and clinical outcomes. RESULTS Data from 31 patients (median [interquartile range] age, 24 [21-31] years) were included in the study. Patients were primarily men (24 [77%]) and White individuals (27 [87%]) who used e-cigarette products containing tetrahydrocannabinol (THC) (29 [94%]). Patients presented with respiratory (30 [97%]), constitutional (28 [90%]), and gastrointestinal (28 [90%]) symptoms. Serum inflammatory markers were elevated in all patients. Bronchoscopy was performed in 23 of 28 inpatients (82%) and bronchoalveolar lavage (BAL) revealed the presence of lipid-laden macrophages (LLMs) in 22 of 24 cases (91%). BAL samples tested positive for Pneumocystis jirovecii (3 patients [13%]), rhinovirus (2 patients [8%]), human metapneumovirus and Aspergillus (1 patient each [4%]); all except human metapneumovirus were determined to be false-positives or clinically inconsequential. The exclusive or dominant computed tomography (CT) pattern was organizing pneumonia in 23 of 26 cases (89%). Patients received antibiotics (26 [84%]) and corticosteroids (24 [77%]), and all survived; 20 patients (65%) seen in follow-up showed marked improvement, but residual symptoms (13 [65%]), radiographic opacities (8 [40%]), and abnormal pulmonary function tests (8 of 18 [44%]) were common. CONCLUSIONS AND RELEVANCE In this case series, patients with EVALI characteristically presented with a flu-like illness with elevated inflammatory markers, LLMs on BAL samples, and an organizing pneumonia pattern on CT imaging. Bronchoscopic testing for infection had a high incidence of false-positive results. Patients had substantial residual abnormal results at early (continued) Key Points Question What are the typical clinical, radiographic, and bronchoscopic findings and clinical outcomes of e-cigarette, or vaping, product use-associated lung injury (EVALI)? Findings This case series of 31 patients found that EVALI typically presented as a flu-like illness with elevated inflammatory markers and an organizing pneumonia pattern on computed tomography imaging. Bronchoscopy showed lipid-laden macrophages and had a high rate of false-positive results for infection. Meaning The findings of this study suggest that EVALI has a characteristic clinical and radiographic presentation and that bronchoscopy has limited utility in its evaluation.