Human Papilloma Virus Vaccination Among Adolescents in a Community Clinic Before and After Intervention.

Human Papilloma Virus (HPV) is the most common sexually transmitted disease with over 14 million infections in 2008. Certain HPV types have been identified in up to 70% of cases of cervical and anal cancers. Despite being safe and effective, HPV vaccination rates remain low. Vaccination and demographic data was collected pre-and post-intervention. Among 13 thru 17-year-old cohort females were significantly more likely to be fully vaccinated. Assessment also found that patients insured by Medicaid were significantly more likely to be fully vaccinated than patients insured privately. Post-intervention vaccination rate is similar to baseline rates. There was non-significant improvement in HPV vaccination coverage after intervention. Male and privately insured patients of Creighton's Pediatric Clinic have lower HPV vaccination coverage than their counterparts. More direct efforts are needed in vaccination process and policy in the clinic to improve immunization against HPV among children and adolescents.

Composite lymphomas: Experience from a tertiary cancer center in Kerala, South India.

OBJECTIVES: Composite tumors are defined as tumors in which there are two different intermixed histologic types. Our objective was to study the clinical and pathologic features of five cases of composite lymphoma. MATERIALS AND METHODS: Our study included five patients of composite lymphoma diagnosed over a period of 5 years. Clinical presentation, hematological parameters including peripheral smear, bone marrow aspirate, and histopathological examination of lymph node including immunohistochemistry (IHC) were studied. Treatment and follow-up details were also noted. RESULTS: All the five cases were in the adult age group ranging from 44 to 72 years. All the cases were composite follicular lymphoma (FL) and mixed cellularity classical Hodgkin lymphoma (CHL). Diagnosis in all cases was suspected on morphology by identification of distinct neoplastic follicles in FL and classic Reed-Sternberg cells in CHL and confirmed by IHC. CONCLUSION: Although rare, composite lymphomas should be kept in mind. Careful histopathological examination of lymph node with identification of distinct morphological features along with IHC helps to arrive at the definitive diagnosis.

Therapeutic applications for ligands of retinoid receptors.

Synthetic retinoids, ligands for the RAR and RXR members of the steroid/thyroid superfamily of nuclear hormone receptors, are used for the treatment of psoriasis, acne, photoaging and cancer. Retinoid mechanisms of action for these conditions largely involve effects on epithelial differentiation and modulation of inflammation with some impact on the immune system. Retinoid medicinal chemistry in recent years has identified ligands highly specific for one of the three RAR subtypes (RAR-alpha) and for the RXR family of receptors, as well as antagonists for the RARs, RARalpha and the RXRs. Structure-activity relationships among the novel retinoid classes are reviewed along with potential therapeutic activities and side effects. RAR-alpha specific retinoids inhibit cancer cell growth but lack other retinoid toxicities, including skin irritation now ascribed to RAR-gama. RXR-specific retinoids lower blood glucose in animal models of type 2 diabetes albeit with a potential for mild hypothyroidism. Function-selective retinoids, especially a class of RAR antagonists called inverse agonists, have unexpected gene regulatory activity. Given the diverse properties and tissue distributions of the retinoid receptors, synthesis of additional classes of receptor-specific and function-selective ligands has the potential to produce novel therapeutic applications.

A cytokeratin 8-like protein with plasminogen-binding activity is present on the external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines.

Plasminogen binding to cell surfaces may be important for tumor invasion and other processes that involve cellular migration. In this investigation, the principal plasminogen-binding protein was identified in the plasma membrane fraction of rat hepatocytes. The protein had an apparent mass of 59 kDa, was insoluble in a spectrum of detergents, and was identical to cytokeratin 8 (CK 8) as determined by sequence analysis of nine amino acids at the N terminus of two cyanogen bromide fragments. The 59 kDa protein bound CK 8-specific antibody in western blot analyses. These studies demonstrate that CK 8 or a CK 8-like protein binds plasminogen. Given this newly determined and potentially important CK 8 function, immunofluorescence and immunoelectron microscopy studies were performed to determine whether CK 8 may be present on the external surfaces of unpermeabilized, viable hepatocytes. All of the cells in each preparation were immunopositive with two separate CK 8-specific antibodies. A punctate pattern of immunofluorescence was detected on the cell surface with approximately even intensity from cell to cell. By immunoelectron microscopy, CK 8 was preferentially associated with microvilli. In order to determine whether other epithelial cells express cell-surface CK 8, immunofluorescence and immunoelectron microscopy studies were performed with HepG2 hepatocellular carcinoma cells and with BT20 and MCF-7 breast carcinoma cells. The pattern of antigen expression was equivalent with each cell type and comparable to that observed with hepatocytes. These studies support the hypothesis that CK 8 is associated with the external cell surface where it may express important proteinase receptor function.

Plasmin cleaves betaglycan and releases a 60 kDa transforming growth factor-beta complex from the cell surface.

Plasmin regulates the activity and distribution of transforming growth factor beta (TGF-beta) and other growth factors. The purpose of the present investigation was to determine the effects of plasmin on cellular receptors for TGF-beta. AKR-2B fibroblasts were affinity-labelled with 125I-TGF-beta 1 and 125I-TGF-beta 2, demonstrating betaglycan, the type-I TGF-beta receptor and the type-II TGF-beta receptor. Treatment of TGF-beta-affinity-labelled cells with plasmin (10-100 nM) for 1 h profoundly and selectively decreased recovery of TGF-beta-betaglycan complex. The type-I and type-II receptors were not plasmin substrates. A radiolabelled complex with an apparent mass of 60 kDa was detected by SDS/PAGE in both the medium and cell extracts of plasmin-treated affinity-labelled cells. In order to demonstrate that plasmin cleavage of betaglycan did not require prior exposure of the betaglycan to cross-linking agent, AKR-2B cells were treated with plasmin first and then affinity-labelled. Markedly decreased TGF-beta binding to cellular betaglycan was observed. Although plasmin treatment of AKR-2B cells decreased overall binding of 125I-TGF-beta 1 and 125I-TGF-beta 2, the rate at which the cells degraded bound 125I-TGF-beta at 37 degrees C was not changed. AKR-2B cells treated with plasmin demonstrated slightly increased [3H]thymidine incorporation; the plasmin-treated cells retained their ability to respond to TGF-beta. Conditioned medium from plasmin-treated AKR-2B cells contained increased amounts of active TGF-beta as determined in Mv 1 Lu epithelial-cell-proliferation assays. Specific cleavage of betaglycan represents a novel mechanism whereby plasmin may regulate the assortment of receptors available for TGF-beta. In addition, plasmin may facilitate transfer of active TGF-beta between neighbouring cells by releasing the active growth factor from the cell surface.

Nerve growth factor-gamma activates soluble and receptor-bound single chain urokinase-type plasminogen activator.

Nerve growth factor-gamma (NGF-gamma) is a serine proteinase which reversibly associates with the well characterized neurotrophin NGF-beta. In this study, we demonstrated that NGF-gamma cleaves recombinant single chain urokinase-type plasminogen activator (scu-PA), converting the zymogen into a two-chain form (tcu-PA). The apparent masses of the two u-PA chains were 33 and 22 kDa, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). There was no evidence for secondary cleavage sites or further digestion of tcu-PA by NGF-gamma, even when conversion of scu-PA was complete. The NH2-terminal sequence of the 33-kDa band was Ile-Ile-Gly-Gly-Glu, indicating that NGF-gamma cleaved scu-PA at Lys158-Ile159, the plasmin cleavage site. Cleavage of scu-PA by NGF-gamma resulted in scu-PA activation. The kcat and Km for this reaction, as determined in a continuous assay with the tcu-PA-specific substrate L-pyroglutamyl-glycyl-arginine-p-nitroanilide hydrochloride (S-2444), were (4.1 +/- 0.6) x 10(-2) s-1 and 2.3 +/- 0.4 microM, respectively. The catalytic efficiency (kcat/Km) for scu-PA activation by NGF-gamma was 1.3 x 10(4) M-1 s-1, compared with 6.2 x 10(5) M-1 s-1 for the activation of scu-PA by plasmin. NGF-gamma-cleaved scu-PA which was bound to receptors on U937 monocytoid cells. The apparent masses of the resulting u-PA cleavage products were identical to those generated in solution as determined by SDS-PAGE. Cell-associated scu-PA was activated by NGF-gamma, as determined by the generation of activity against the tcu-PA-specific fluorogenic substrate, glutamyl-glycyl-arginine-7-amino-4-methyl coumarin. By activating scu-PA, NGF-gamma may initiate the u-PA-dependent cell-surface proteinase cascade and support NGF-beta activities which involve cellular migration and/or extracellular matrix remodeling.

Binding of anisoylated Lys-plasminogen streptokinase activator complex to cells in culture.

Anisoylated Lys-plasminogen streptokinase activator complex (APSAC) was purified from Eminase by chromatography on Superose-12. Purified APSAC did not significantly deacylate within 4 h at 4 degrees C in solution as determined by hydrolysis of D-Val-L-leu-L-lys-p-nitroanilide HCl (S-2251). At 37 degrees C, maximum amidase activity developed in 120 min; epsilon-amino-n-caproic acid (EACA) did not affect the apparent rate of APSAC deacylation but stabilized the streptokinase-plasmin(ogen) complex (SkPl) which formed. APSAC bound to C6 glioma cells and human umbilical vein endothelial cells (HUVECs) in culture. Binding as completely inhibited by EACA suggesting an essential role for the plasminogen kringle domains. Cell-associated APSAC deacylated to form active SkPl which hydrolyzed S-2251 and D-Val-Leu-Lys-7-amino-4-methyl coumarin. The rate of APSAC deacylation was increased when the APSAC was cell-associated. APSAC that was initially bound to C6 cells or HUVECs also activated 125I-plasminogen. This activity may have reflected cell-associated APSAC or APSAC but dissociated into solution. Plasmin was recovered bound to cells and in solution. These studies demonstrate that APSAC associates with cell-surfaces and retains activity. In the circulation, cell-surfaces may provide a significant pharmacologic compartment for intravenously administered APSAC.

Reaction of nerve growth factor gamma and 7S nerve growth factor complex with human and murine alpha 2-macroglobulin.

The kallikrein-like serine proteinase nerve growth factor gamma (NGF-gamma) reacted with the plasma proteinase inhibitor human alpha 2-macroglobulin (h alpha 2M). The h alpha 2M subunits were cleaved, the electrophoretic mobility of h alpha 2M in nondenaturing polyacrylamide gels was increased, and the intrinsic fluorescence of h alpha 2M was increased with a slight blue-shift. These changes are well-characterized components of the alpha 2M/proteinase reaction mechanism. In N alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) hydrolysis experiments, the catalytic efficiency (kcat/KM) of the h alpha 2M-NGF-gamma complex was decreased by 98.5% compared with free NGF-gamma. This decrease is unique since other alpha 2M-proteinase complexes retain significant amidase activity. For comparison, we determined that the catalytic efficiency of alpha 2M-trypsin is decreased by 58% compared with free trypsin under equivalent conditions. The rate of NGF-gamma inhibition by h alpha 2M was (1.0 +/- 0.1) x 10(4) M-1 s-1 as determined by BAPNA hydrolysis. A similar value was determined by monitoring the change in intrinsic fluorescence. NGF-gamma, which was bound within the intact 7S NGF complex, also reacted with h alpha 2M, albeit at a very slow rate. This reaction may have depended exclusively on slow reversible dissociation of NGF-gamma from the 7S complex. NGF-gamma was rapidly inhibited by murine alpha 2M (m alpha 2M).(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinogenolytic and fibrinolytic activity of cell-associated plasmin.

Binding of plasmin(ogen) to rat C6 glioma cells is saturable and kringle-domain dependent. This interaction was studied as a model of plasmin(ogen) receptor interactions in nucleated mammalian cells. Apparent 125I-plasmin dissociation from C6 cell binding sites was slow; however, the dissociation rate was increased when the solution contained diisopropyl phosphoryl-plasmin (0.3 microM), fibrinogen (0.16 or 0.8 mg/ml), 1.08 mM D-Val-L-Leu-L-Lys-p-nitroanilide-HCl (S-2251), or epsilon-amino-n-caproic acid (EACA, 5.0 mM). EACA promoted the most rapid dissociation of plasmin. C6 cell-associated plasmin and plasmin in solution demonstrated similar amidase activity. Only specifically bound plasmin (75% of total binding) was active against S-2251. Plasmin that was initially bound to C6 cells digested fibrinogen in a time- and plasmin concentration-dependent manner. alpha 2-Antiplasmin (alpha 2AP, 0.1 microM) completely inhibited fibrinogenolysis by plasmin that was initially C6- or human umbilical vein endothelial-cell associated. Since alpha 2AP reacts selectively with plasmin in solution (minimally with plasmin bound to cells), fibrinogen digestion by cell-associated plasmin probably occurred only after the plasmin dissociated into solution. Crosslinked fibrin clots were formed in uniform layers over C6 cells. If the cells were incubated with plasmin before addition of fibrinogen and thrombin, the clots were rapidly lysed. alpha 2AP incompletely inhibited fibrinolysis when added after fibrin polymerization (44% inhibition with 0.1 microM alpha 2AP). Fibrinolysis was completely inhibited when alpha 2AP was added before fibrin polymerization. These studies suggest that plasmin must first dissociate from cellular binding sites to mediate fibrinogenolysis or fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antifibrinolytic activities of alpha-N-acetyl-L-lysine methyl ester, epsilon-aminocaproic acid, and tranexamic acid. Importance of kringle interactions and active site inhibition.

alpha-N-acetyl-L-lysine methyl ester (NALME) is a lysine analogue that reportedly binds to low-affinity lysine binding sites in plasmin(ogen) and miniplasmin(ogen). In the studies presented here, we show that NALME has antifibrinolytic activity; however, unlike the therapeutic agents epsilon-amino-n-caproic acid (epsilon ACA) and tranexamic acid (TEA), the activity of NALME is based on inhibition of the plasmin active site. NALME (0.1-10 mM) significantly inhibited the amidase activity of plasmin, miniplasmin, and streptokinase-plasmin complex without affecting alpha-thrombin or tissue plasminogen activator. epsilon ACA and TEA (0.1-10 mM) did not affect the amidase activity of plasmin or miniplasmin. A kinetic analysis showed that NALME is a competitive inhibitor of D-Val-L-Lys-p-nitroanilide HCl (S-2251) hydrolysis by plasmin; NALME binding to plasmin completely prevented S-2251 binding. The Kl for the plasmin-NALME interaction was 0.4 mM. epsilon ACA and TEA inhibited fibrin monomer digestion by plasmin and miniplasmin without binding to the active site of either enzyme. This result suggests that epsilon ACA and TEA function as antifibrinolytics by disrupting the noncovalent association of fibrin monomer with a domain common to both plasmin and miniplasmin (probably kringle 5). NALME inhibited fibrin monomer digestion principally by decreasing amidase activity. NALME was the only lysine analogue that prevented fragment X formation; TEA and epsilon ACA primarily inhibited the formation of fragments Y and D. When plasmin was incubated simultaneously with alpha 2-antiplasmin and alpha 2-macroglobulin, epsilon ACA increased the fraction of plasmin reacting with alpha 2-macroglobulin; NALME had no effect on the plasmin distribution.(ABSTRACT TRUNCATED AT 250 WORDS)