Diagnostic per-lesion performance of a simulated gadoxetate disodium-enhanced abbreviated MRI protocol for hepatocellular carcinoma screening.

AIM: To evaluate the diagnostic per-lesion performance of a simulated gadoxetate disodium-enhanced abbreviated MRI (AMRI) in cirrhotic and chronic hepatitis B (CHB) patients for hepatocellular carcinoma (HCC) screening. MATERIALS AND METHODS: Seventy-nine consecutive patients at risk for HCC due to cirrhosis and/or CHB were included in this retrospective study. For each patient, the first gadoxetate disodium-enhanced MRI between 2008 through 2014 was analysed. Two independent readers read an anonymised abbreviated image set comprising axial T1-weighted (W) images with fat saturation in the hepatobiliary phase, 20 minutes or more after gadoxetate injection, and axial T2W single-shot fast spin echo images. Each observation >10 mm was scored as negative or suspicious for HCC. Inter-reader agreement was assessed. A composite reference standard was used to determine the per-lesion diagnostic performance for each reader. RESULTS: Inter-reader agreement was substantial (κ = 0.75). The final reference standard showed 27 HCCs in 13 patients (median 21 mm, range 11-100 mm). The two readers each correctly scored 23 as suspicious for HCC (sensitivity = 85.2%), scored a total of 27 and 32 observations as suspicious for HCC (positive predictive value [PPV] = 85.2% and 71.9%), and scored 83 and 78 observations or complete examinations as negative for HCC (negative predictive value [NPV] = 95.2% and 94.9%). CONCLUSIONS: The AMRI protocol provides higher per-lesion sensitivity and NPV than reported values for ultrasound, the current recommended technique for screening, and similar per-lesion sensitivity and PPV to reported values for complete dynamic contrast-enhanced MRI.

Complications of foot and ankle surgery in patients with diabetes.

Treatment of the foot and ankle in patients with diabetes often is fraught with complications, frequently multifactorial in nature. Because of the multidisciplinary approach to the patient with diabetes, it is imperative that the patient and all healthcare professionals who are treating the patient recognize the foot at risk, and the clinical hallmarks of Charcot neuroarthropathy. Failure to do so often leads to disastrous results, such as ulceration, destruction of normal foot architecture, and progressive deformity too severe to accommodate a brace, thereby necessitating surgical intervention. The surgical treatment of the foot in a patient with diabetes requires knowledge of the pathophysiology of the neuroarthropathic (Charcot) foot, so that the appropriate timing, extent of surgical intervention, and postoperative treatment in this unique population assures a higher success rate. One also must recognize associated factors that may be present in these patients, such as peripheral vascular compromise and poor nutritional status.

The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing.

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

Probing the interaction of dengue virus envelope protein with heparin: assessment of glycosaminoglycan-derived inhibitors.

A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.

Evidence for the expression of a second CD6 ligand by synovial fibroblasts.

OBJECTIVE: CD6, a cell surface glycoprotein expressed primarily on T cells, may function as a costimulatory molecule and may play a role in autoreactive immune responses. Recently, a CD6 ligand termed CD166 (previously known as activated leukocyte cell adhesion molecule [ALCAM]) has been identified and shown to be expressed on activated T cells, B cells, thymic epithelium, keratinocytes, and in rheumatoid arthritis synovial tissue. However, the results of functional studies have suggested the existence of a second CD6 ligand. The present study was undertaken to seek evidence for a second CD6 ligand on cultured synovial fibroblasts. METHODS: Flow cytometric and biochemical techniques were applied, using anti-CD166 monoclonal antibody (mAb) and a recombinant CD6 fusion protein, to determine whether cultured synovial fibroblasts and other cell types expressed a non-ALCAM CD6 ligand. RESULTS: CD14- fibroblastic synoviocytes showed greater binding of a recombinant CD6 fusion protein than of anti-ALCAM mAb. With interferon-gamma treatment of synovial fibroblasts, binding of both reagents increased, but this was more marked for binding of CD6 fusion protein. Exposure of synovial fibroblasts to other cytokines or to the superantigen staphylococcal enterotoxin A also regulated binding of CD6 fusion protein and anti-ALCAM mAb in a discordant manner. Immunoprecipitation of proteins from membrane extracts of synovial fibroblasts with a CD6-Ig fusion protein revealed a novel 130-kd band distinct from CD166; an identical molecule was also precipitated from membranes of HBL-100 tumor cells. CONCLUSION: Taken together with previous data regarding CD6 and CD166 function, the present findings strongly suggest the existence of a second CD6 ligand distinct from CD166, which can be expressed by synovial fibroblasts as well as other cells.

Imaging of osteochondral lesions of the talus.

A review of the literature reveals that all studies have been performed retrospectively. These studies have included limited numbers of low-grade lesions and no prospective, randomized, comparison studies have been performed to suggest the superiority of CT scanning over MR imaging. The following conclusions, however, can be made. Plain radiographs are useful in the initial evaluation of patients with acute or chronic complaints of ankle pain and swelling. These initial studies, however, may not identify all osteochondral lesions of the talus, particularly lower grade lesions. CT scanning can accurately identify and localize a lesion while defining its extent. It has been suggested that CT scanning can be used to assess whether bony healing has occurred at follow-up. MR imaging can also precisely identify, localize, and define an OLT with the advantage of assessing the integrity of the overlying cartilage. It can detect lower grade lesions with improved sensitivity and may aid in the differentiation of Stage II and Stage III lesions. Using the preceding observations, the following approach is recommended in the evaluation and work-up of an osteochondral lesion of the talus (Fig. 7). The patient who presents with ankle pain and swelling should have weight-bearing radiographs of the ankle obtained. If these films demonstrate an osteochondral lesion of the talus, staging of the lesion should be performed. In lesions that appear nondisplaced on plain radiography (low grade; stable), MR imaging is recommended so the clinician can evaluate the integrity of the overlying cartilage and assess the true stability of the lesion. In lesions that appear displaced on plain radiography (high grade; unstable), the CT scan is the preferred modality in order to provide accurate assessment of lesion size and location. It should be noted, however, that no study has prospectively [figure: see text] compared the efficacy of these two modalities in the evaluation of osteochondral lesions. If a symptomatic patient presents with negative plain films, then an initial period of immobilization using a cast or boot brace is recommended. This is followed by joint mobilization and range of motion exercises. If the patient remains symptomatic at the 4 to 6 week followup period, then an MR image should be performed. This study provides information regarding soft-tissue impingement, proliferative synovitis, and other bony and soft-tissue pathology. The authors have found that despite the results of bone scintigraphy, an MR image is invariably obtained. Because of this the authors do not recommend bone scintigraphy in the evaluation and diagnosis of OLT.

Intradermal anti-loxosceles Fab fragments attenuate dermonecrotic arachnidism.

OBJECTIVE: Bites from the brown recluse spider and other arachnids from the genus Loxosceles frequently induce necrotic skin lesions that can be recalcitrant to treatment and disfiguring. The authors used a rabbit model of dermonecrotic arachnidism to address the therapeutic efficacy of intradermal (id) polyclonal anti-Loxosceles Fab fragments (alphaLoxd Fab) raised against Loxosceles deserta spider venom. METHODS: Fab fragments were prepared by papain digestion and affinity chromatography from the IgG fraction of L. deserta antivenom raised in rabbits. Eighteen inbred New Zealand white rabbits were assigned to six groups of three. The rabbits received L. deserta venom (3 microg, id) injections into each flank. Cohorts of rabbits received single id injections (at one venom site/rabbit) of 30 microg alphaLoxd Fab at different times (T = 0, 1, 2, 4, 8, and 12 hours) after venom injection. In each rabbit the opposite flank was left untreated. As an additional control, one group of rabbits (T = 0) received nonspecific Fab (30 microg, id) in the opposite flank. Dermal lesions were quantified as a function of time through the use of a series of digital photographs and imaging software. In addition, myeloperoxidase (MPO) activity, a measure ofneutrophil accumulation, was determined in lesion biopsies. Lesion areas and MPO activities were analyzed by repeated-measures analysis of variance (ANOVA). RESULTS: Lesion areas and MPO activity were markedly reduced when alphaLoxd Fab was administered very early after venom injections. As the interval between venom inoculation and antivenom treatment increased, the therapeutic benefit of alphaLoxd Fab decreased. The final time tested that demonstrated therapeutic efficacy of alphaLoxd Fab was T = 4 hours. Lesion attenuation was no longer apparent when alphaLoxd Fab was given 8 hours post inoculation. CONCLUSIONS: Intradermal administration of alphaLoxd Fab attenuates Loxosceles-induced dermonecrotic lesion formation when given up to 4 hours after venom inoculation in this rabbit model.

Pseudoaneurysm of the lateral malleolar artery after an ankle sprain: case report and review of the literature.

A pseudoaneurysm of the peroneal artery or one of its branches is rare after trauma. The diagnosis is frequently delayed, resulting in substantial morbidity to the patient. The infrequent occurrence of this condition has resulted in a lack of diagnostic and treatment guidelines. However, when recognized, prompt attention involving either surgical ligation or embolization of the injured arterial branch is the most reliable method of treatment. Presented is a report of a patient who developed a lateral malleolar arterial pseudoaneurysm after an ankle sprain, which was treated successfully with aneurysmal excision and surgical ligation of the injured arterial branch. A review of the literature and treatment recommendations follow.

Midfoot fusion technique for neuroarthropathic feet: biomechanical analysis and rationale.

To test the hypothesis that a plate applied to the plantar (tension) side of the medial midfoot provides stronger fixation than midfoot fusion with screw fixation, we biomechanically compared the two constructs for midfoot fusion. We created a model of midfoot instability in eight matched pairs of cadaver legs by section of joint capsule, ligaments, and tendons about Lisfranc's joints, and then performed a load-to-failure study to compare the fixation provided by a plantarly applied third tubular plate with that by cortical screws. After an initial load deformation curve to 1000 N was obtained, specimens were cyclically loaded at 200 to 750 N for 3000 cycles and then loaded to failure (screw pullout, fracture, or deformation >3 mm). Comparing the plantar plate and midfoot fusion with screw fixation constructs, a plate applied to the plantar (tension) aspect of the medial midfoot provides a stronger, sturdier construct than does midfoot fusion with screw fixation.

Overlapping functions of E- and P-selectin in neutrophil recruitment during acute inflammation.

Selectin adhesion molecules mediate leukocyte rolling on activated endothelium, a prerequisite to leukocyte accumulation at sites of inflammation. The precise role of each selectin (E-, P-, and L-) in this process is unclear and may vary depending on the particular inflammatory stimulus, vascular bed, leukocyte subset, and species; most data suggest discrete functional roles for each selectin. To define the relative roles of E- and P-selectin in mediating neutrophil accumulation in acute dermal inflammation, mice genetically deficient in E-selectin, P-selectin, or both E- and P-selectin were injected intradermally with zymosan. Luminal endothelial expression of E- and P-selectin in response to zymosan was documented in wild-type mice by intravenous administration of fluorochrome-labeled anti-E- and anti-P-selectin antibodies. In mice deficient in E- or P-selectin, neutrophil accumulation was unchanged or only subtly reduced relative to wild-type control mice. In mice deficient in both E- and P-selectin, neutrophil accumulation was significantly reduced (87% at 4 hours and 79% at 8 hours). These data demonstrate that, in this model of acute inflammation, there is considerable overlap in the functions of E- and P-selectin; loss of both selectins was required to impair neutrophil accumulation.

Complement activation occurs on subendothelial extracellular matrix in vitro and is initiated by retraction or removal of overlying endothelial cells.

Vascular endothelium is continuously exposed to plasma complement, which could generate a potent proinflammatory signal if activated on the vascular wall. Normal endothelium, however, expresses an anti-inflammatory phenotype, which includes resistance to complement fixation. As activated endothelium converts to a proinflammatory phenotype, we investigated the effect of cytokines on endothelial susceptibility to complement fixation. Cytokine-treated HUVEC were exposed to human serum as a source of complement, and C3 deposition was quantified. IL-1beta and TNF-alpha in combination with IFN-gamma markedly increased endothelial C3 deposition; however, immunofluorescence microscopy revealed that the endothelial cells had retracted, and that bound C3 was concentrated not on cells but in areas of exposed subendothelial extracellular matrix (ECM). Studies with cell-free ECM indicated that complement activation required only ECM exposure and was independent of cellular activation. C3 deposition on ECM was reproduced by reconstituting the alternative pathway, which generated a stable C3 convertase on ECM, but not on endothelial cells. C3b and iC3b were identified on ECM exposed to purified alternative pathway components and serum, respectively. In conditions associated with endothelial disruption, exposure of subendothelial ECM could induce complement fixation and contribute to inflammation and vascular damage.

Posttraumatic posterior tibialis tendon insertional elongation with functional incompetency: a case report.

We present a case report and literature review of distal intrasubstance rupture of the posterior tibial tendon with progressive pes planovalgus secondary to tendon incompetence. Three months after a severe ankle sprain, a 25-year-old basketball player presented with ankle weakness and pain. Treatment by advancement of the posterior tibial tendon to the navicular and medial displacement osteotomy of the calcaneal tuberosity restored alignment, strength, and full function.

Decay-accelerating factor is a component of subendothelial extracellular matrix in vitro, and is augmented by activation of endothelial protein kinase C.

The vasculature is protected from complement activation by regulatory molecules expressed on endothelial cells. However, complement fixation also occurs on subendothelial extracellular matrix (ECM) in vitro, and is initiated simply by retraction or removal of overlying cells. To investigate mechanisms controlling vascular complement activation, we examined subendothelial ECM for the presence of complement regulatory proteins. Decay-accelerating factor (DAF) was found on both human umbilical vein endothelial cells (HUVEC) and in their ECM; in contrast, membrane cofactor protein was found only on cells. ECM and HUVEC DAF were distinguishable based on several properties. While HUVEC DAF is anchored to cell membranes by a phospholipase C-sensitive glycosylphosphatidylinositol linkage. DAF was removed from ECM only by proteolytic digestion. Cytokines (TNF-alpha, IL-1 beta, IL-4) increased HUVEC DAF expression, but had minimal effect on ECM DAF; in contrast, phorbol 12-myristate 13-acetate (PMA) and wheat germ agglutinin markedly increased DAF on both HUVEC and ECM. The effect of PMA was mediated by activation of protein kinase C. The complement regulatory potential of ECM DAF was assessed by evaluating the effect of DAF-neutralizing antibodies on C3 deposition on HUVEC ECM, as well as on HeLa cell ECM, which had a considerably higher DAF content. DAF blockade enhanced C3 deposition on HeLa ECM, but had no effect on HUVEC ECM. As ECM DAF is likely to be immobile, i.e. able to interact only with C3 convertases forming in the immediate vicinity, its ability to regulate complement activation may be particularly density dependent, and contingent on endothelial-dependent up-regulation.

Attenuation of interleukin-8 expression in C6-deficient rabbits after myocardial ischemia/reperfusion.

Neutrophil accumulation and activation of the complement system with subsequent deposition of the cytolytic membrane attack complex (MAC) have been implicated in the pathogenesis of myocardial ischemia/reperfusion injury. The MAC, when present in high concentrations, promotes target cell lysis. However, relatively little is known about the potential modulatory role of sublytic concentrations of the MAC on nucleated cell function in vivo. In vitro studies demonstrated that the MAC regulates cell function by promoting the expression of pro-inflammatory mediators, including adhesion molecules and pro-inflammatory cytokines. We examined, using C6-deficient and C6-sufficient rabbits, the regulatory role of the MAC in mediating IL-8 expression and subsequent neutrophil recruitment in the setting of myocardial ischemia/reperfusion injury. C6-deficient and C6-sufficient rabbits were subjected to 30 min of regional myocardial ischemia followed by a period of reperfusion. In addition to a significant reduction in myocardial infarct size in C6-deficient animals, analysis of myocardial tissue demonstrated a decrease in neutrophil influx into the infarcted region. The reduction in neutrophil influx correlated with the decreased expression of the neutrophil chemotactic cytokine IL-8, as determined by ELISA and immunohistochemical analysis. The results derived from this study provide evidence that the MAC has an important function in mediating the recruitment of neutrophils to the reperfused myocardium through the local induction of IL-8.

Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate.

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.

CD6 dependent interactions of T cells and keratinocytes: functional evidence for a second CD6 ligand on gamma-interferon activated keratinocytes.

The CD6 glycoprotein is expressed by T lymphocytes and is hypothesized to interact with one or more ligands expressed on antigen presenting cells (APCs). We show that CD6 mediates binding of the transformed CD4+ T cell line Hut 78 to gamma-interferon activated keratinocytes (KCs). A recombinant CD6-Ig fusion protein has been reported to bind to a CD6 ligand ALCAM, but this is the first demonstration that cell-cell adhesion of human T lymphocytes can be CD6 dependent. The known CD6 ligand ALCAM (CD166) is expressed on cultured KCs but does not appear to mediate KC-Hut 78 binding, suggesting the existence of additional CD6 ligands expressed on KCs. In functional studies using autologous KCs as APCs for tetanus toxoid specific T cell clones, KCs +/- gamma-interferon are unable to stimulate autologous T cells with recall antigen. Therefore interaction of T cell CD6 with CD6 ligands on KCs does not provide sufficient co-stimulation of primed T cells to support responses to nominal antigen.

Use of receptors immobilized on microspheres to identify ligand binding sites in tissue sections: detection of lymph node ligands for L-Selectin.

Particulate microspheres bearing immobilized probes can be used to identify ligands expressed by cells and require only brightfield microscopy for detection. There are distinct advantages to using microspheres to detect low affinity interactions; microspheres require no secondary amplification or detection procedures subsequent to the binding interaction, reducing opportunities for detachment of bound probe, and concentrating probes on microspheres may greatly increase binding avidity. Selectin leukocyte-endothelial adhesion molecules undergo low affinity binding to ligands, and these interactions may be difficult to detect with standard techniques. The aim of this study was to determine if immobilizing recombinant L-Selectin on microspheres would facilitate detection of specific tissue ligands. Microspheres were incubated with sections of rabbit peripheral lymph node in a modified Stamper-Woodruff assay, and binding was assessed by brightfield microscopy. L-Selectin-IgG microspheres bound to high endothelial venules, known to be sites of expression for L-Selectin ligands. Specificity was indicated by the lack of binding of microspheres coated with control protein, and inhibition of binding by antibody to L-Selectin and by competitive antagonists of L-Selectin ligand interactions.

Demonstration of binding of dengue virus envelope protein to target cells.

The nature of the initial interaction of dengue virus with target cells and the extent to which this interaction defines tropism are unknown. Infection of some cells may involve antidengue antibody-mediated immune adherence to cells bearing immunoglobulin Fc receptors; however, this mechanism does not explain primary infection or the infection of cells without Fc receptors. We hypothesized that dengue virus envelope protein mediates initial binding to target cells. To test this hypothesis, a recombinant chimeric form of dengue type 2 virus envelope protein was used as a probe to investigate binding to the surfaces of potential target cells. Envelope protein was expressed amino terminal to the heavy-chain constant region of human immunoglobulin G containing the Fc receptor binding motif; the binding mediated by envelope determinants was distinguishable from the binding mediated by immunoglobulin Fc determinants. We found that the recombinant chimera bound to Vero, CHO, endothelial, and glial cells through envelope protein determinants and to monocytes and U937 cells by Fc-Fc receptor interactions. The highest level of binding was to Vero cells; binding was dose and time dependent and saturable. Examination of partial-length recombinant envelope proteins indicated that the binding motif was expressed between amino acids 281 and 423. Recombinant envelope protein inhibited infection of Vero cells by dengue virus, indicating the functional significance of the interaction of envelope protein and target cells in infectivity. These results suggest that envelope protein binding to a non-Fc receptor could explain the cell and tissue tropism of primary dengue virus infection.

Cloning of the cDNA for rabbit L-selectin and expression of recombinant protein with a kinase target site to facilitate radiolabeling.

The cDNA encoding rabbit L-Selectin has been cloned from a cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 377 amino acids, including a signal peptide of 38 amino acids. Sequence analysis demonstrated extensive homology with L-Selectin's from other species. Recombinant rabbit L-Selectin protein was expressed in eukaryotic cells in a chimeric construct incorporating the entire extracellular portion of the protein coding region, a phosphokinase target site to allow high activity radiolabeling with 32P, and the constant region of the heavy chain of human IgG1 to facilitate purification and detection. Amino-terminal peptide sequencing of recombinant L-Selectin confirmed that the signal peptide had been removed at the expected site.