Epstein-Barr virus (EBV) provides survival factors to EBV+ diffuse large B-cell lymphoma (DLBCL) lines and modulates cytokine induced specific chemotaxis in EBV+ DLBCL.

Diffuse large B-cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non-Hodgkin lymphomas. Epstein-Barr virus (EBV) -positive DLBCL of the elderly is a newly recognized subtype that accounts for 8-10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL-derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin-4 and interleukin-21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4- or CCR7-mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant-negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV-positive lines. These results show that EBV plays an important role in EBV-positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV-carrying DLBCL cells and might have therapeutic implications.

Latency type-dependent modulation of Epstein-Barr virus-encoded latent membrane protein 1 expression by type I interferons in B cells.

We report that type I interferons (IFNs) upregulate latent membrane protein 1 (LMP-1) expression by direct activation of the ED-L1 promoter in several Epstein-Barr virus (EBV)-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulates LMP-1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs. Furthermore, our results may explain the expression of LMP-1 in memory B cells of systemic lupus erythematosus patients.

STAT6 signaling pathway activated by the cytokines IL-4 and IL-13 induces expression of the Epstein-Barr virus-encoded protein LMP-1 in absence of EBNA-2: implications for the type II EBV latent gene expression in Hodgkin lymphoma.

In line with the B-lymphotropic nature of Epstein-Barr virus (EBV), the virus is present in several types of B-cell lymphomas. EBV expresses a different set of latent genes in the associated tumors, such as EBV nuclear antigen 1 (EBNA-1) and latent membrane proteins (LMPs; type II latency) in classical Hodgkin lymphomas (HLs). We previously reported that exposure of in vitro EBV-converted, HL-derived cell line KMH2-EBV to CD40-ligand and interleukin-4 (IL-4) induced the expression of LMP-1. Here, we show that exposure to IL-4 or IL-13 alone induced LMP-1 in the absence of EBNA-2. Induction of LMP-1 by IL-4 and IL-13 was mediated by the signal transducer signal transducer and activator of transcription 6 (STAT6) and a newly defined high-affinity STAT6-binding site in the LMP-1 promoter. IL-4 induced LMP-1 also in Burkitt lymphoma-derived lines and in tonsillar B cells infected with the EBNA-2-deficient EBV strain P3HR-1. Furthermore, coculture of EBV-carrying Burkitt lymphoma cells with activated CD4(+) T cells resulted in the induction of LMP-1 in the absence of EBNA-2. Because Hodgkin/Reed-Sternberg cells are known to secrete IL-13, to have constitutively activated STAT6, and to be closely surrounded by CD4(+) T cells, these mechanisms may be involved in the expression of LMP-1 in EBV-positive chronic HLs.

IL-21 imposes a type II EBV gene expression on type III and type I B cells by the repression of C- and activation of LMP-1-promoter.

Epstein-Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4(+) T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies.

Monocytes enhance cell proliferation and LMP1 expression of nasal natural killer/T-cell lymphoma cells by cell contact-dependent interaction through membrane-bound IL-15.

Nasal natural killer (NK)/T-cell lymphoma (NNKTL) is an Epstein-Barr virus (EBV)-related malignancy with poor prognosis and has distinct histological features characterized by angiocentric and polymorphous lymphoreticular infiltrates including inflammatory cells such as granulocytes, monocytes, macrophages and lymphocytes. Here, we show that the monocytes enhance proliferation as well as LMP1 expression of NNKTL cells by cell contact-dependent interaction through membrane-bound interleukin (IL)-15. We used two EBV-positive NK-cell lines, SNK6 and KAI3, which originated from two patients-SNK6 from a patient with NNKTL and KAI3 from a patient with a severe mosquito allergy. We cocultured the cell lines with granulocytes or monocytes and examined whether proliferation, survival and LMP1 expression of the cells changed. Although cocultured granulocytes did not affect proliferation, survival or LMP1 expression of the cells, cocultured monocytes enhanced both proliferation and LMP1 expression in a dose-dependent manner. These phenomena were not seen when monocytes were placed in a separate chamber. Moreover, the monocyte-inducible proliferation and LMP1 expression were inhibited by treatment with an antibody against IL-15. Furthermore, production of interferon-gamma-inducible protein (IP)-10 were enhanced by coculture with monocytes and were inhibited by the antibody. Immunohistological studies confirmed that a number of infiltrating CD14-positive monocytes contacted CD56-positive lymphoma cells in all of 20 NNKTL tissues tested. These results suggest that monocytes enhance cell growth as well as LMP1 expression of NNKTL cells by cell contact-dependent interaction through membrane-bound IL-15. In the microenvironment of NNKTL tissue, a positive feedback loop of interaction between lymphoma cells and monocytes may be present and contribute to lymphoma progression.

Type I interferons directly down-regulate BCL-6 in primary and transformed germinal center B cells: differential regulation in B cell lines derived from endemic or sporadic Burkitt's lymphoma.

Type I interferons (IFN) exert multiple effects on both the innate and adaptive immune system in addition to their antiviral and antiproliferative activities. Little is known, however about the direct effects of type I IFNs on germinal center (GC) B cells, the central components of adaptive B cell responses. We used Burkitt's lymphoma (BL) lines, as a model system of normal human GC B cells, to examine the effect of type I IFNs on the expression of BCL-6, the major regulator of the GC reaction. We show that type I IFNs, but not IFNγ, IL-2 and TNFα rapidly down-regulate BCL-6 protein and mRNA expression, in cell lines derived from endemic, but not from sporadic BL. IFNα-induced down-regulation is specific for BCL-6, independent of Epstein-Barr virus and is not accompanied by IRF-4 up-regulation. IFNα-induced BCL-6 mRNA down-regulation does not require de novo protein synthesis and is specifically inhibited by piceatannol. The proteasome inhibitor MG132 non-specifically prevents, while inhibitors of alternate type I IFN signaling pathways do not inhibit IFNα-induced BCL-6 protein downregulation. We validate our results with showing that IFNα rapidly down-regulates BCL-6 mRNA in purified mouse normal GC B cells. Our results identify type I IFNs as the first group of cytokines that can down-regulate BCL-6 expression directly in GC B cells.

The proapoptotic function of SAP provides a clue to the clinical picture of X-linked lymphoproliferative disease.

Deletion or mutation of the SAP gene is associated with the X-linked lymphoproliferative disease (XLP) that is characterized by extreme sensitivity to Epstein-Barr virus (EBV). Primary infection of the affected individuals leads to serious, sometimes fatal infectious mononucleosis (IM) and proneness to lymphoma. Our present results revealed a proapoptotic function of SAP by which it contributes to the maintenance of T-cell homeostasis and to the elimination of potentially dangerous DNA-damaged cells. Therefore, the loss of this function could be responsible for the uncontrolled T-cell proliferation in fatal IM and for the generation of lymphomas. We show now the role of SAP in apoptosis in T and B lymphocyte-derived lines. Among the clones of T-ALL line, the ones with higher SAP levels succumbed more promptly to activation induced cell death (AICD). Importantly, introduction of SAP expression into lymphoblastoid cell lines (LCL) established from XLP patients led to elevated apoptotic response to DNA damage. Similar results were obtained in the osteosarcoma line, Saos-2. We have shown that the anti-apoptotic protein VCP (valosin-containing protein) binds to SAP, suggesting that it could be instrumental in the enhanced apoptotic response modulated by SAP.

Drug sensitivity patterns of HHV8 carrying body cavity lymphoma cell lines.

BACKGROUND: Primary effusion lymphoma (PEL) is a rare KSHV/HHV8-associated high-grade non-Hodgkin's lymphoma (NHL) of B-cell origin, characterized by serous effusions in body cavities. Most patients are HIV-infected men with severe immunosuppression and other HHV8-associated diseases such as Kaposi's sarcoma (KS). The prognosis for those infected is poor, with a median survival of less than 6 months in most cohorts. Sustained complete remission is rare. High-dose chemotherapy regimens are used to improve remission rate and survival. The aim of the present study was to compare the drug sensitivity pattern of the available primary effusion (body cavity based) lymphoma-derived cell lines in order to find additional, potentially effective drugs that are not included in current chemotherapy treatment protocols. METHODS: We have analyzed 11 cell lines against 27 frequently used cytostatic drugs in short term (3 days) survival assays using automated high throughput confocal microscopy. RESULTS: All cell lines showed a distinct, individual drug sensitivity pattern. Considering the in vitro used and clinically achieved drug concentration, Vinorelbine, Paclitaxel, Epirubicin and Daunorubicin were the most effective drugs. CONCLUSIONS: We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay-guided individualized therapy might be required to optimize therapeutic response.

Multi-center real-world comparison of the fully automated Idylla™ microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer.

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.

Fibronectin-mediated uptake of gelatin-coated latex particles by peritoneal macrophages.

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (Clg) to promote the uptake of 125I-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM). The uptake of g-Ltx* by PM was enhanced by Clg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake. Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37 degrees C after particle uptake removed less than 15% of the radioactivity incorporated by the monolayers. However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by greater than 75%. Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex. Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed Clg-dependent particle uptake. Phagocytosis of g-Ltx* by PM in the presence of Clg and heparin was confirmed by electron microscopy. Finally, g-Ltx* could also be effectively opsonized with Clg at 37 degrees C before their addition to the monolayers. These studies suggest that the recognition of g-Ltx* in the presence of Clg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements. Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells.

Contribution of fibrin stabilization to clot strength. Supplementation of factor XIII-deficient plasma with the purified zymogen.

The contribution of fibrin stabilization to clot strength, measured as the static elastic modulus, was evaluated in human plasma by two independent procedures. In the first approach, amine inhibitors of fibrin stabilization were examined for their effects on the rigidity of normal plasma clots. It is a unique property of these inhibitors that they do not interfere with the reversible aggregation of fibrin molecules, i.e., do not delay clotting time, but selectively prevent only the formation of gamma-glutamyl-epsilon-lysine protein-to-protein linkages. Though the compounds tested were of different chemical structures and potencies, a fivefold reduction in clot strength was obtained in each instance. This value of 20% of normal seems to correspond to the rigidity of the Factor XIII-deficient plasma clot because, as demonstrated by the second approach, when a plasma specimen that genetically lacked the fibrin stabilizing factor was supplemented by the addition of measured amounts of the purified zymogen, a fivefold increase in clot strength could be achieved. The described procedure of evaluating Factor XIII in terms of correcting the elastic modulus of a deficient plasma clot is considered an important assay for the functional competence of purified preparations of the zymogen for the purpose of therapeutic application.

A pathological inhibitor of fibrin cross-linking.

Lewis et al. recently reported on a patient who died of hemorrhages attributable to an acquired inhibitor of fibrin-stabilizing factor. They indicated that the inhibitor was associated with the immune globulins. Using the postmortem serum in the isolated fibrin cross-linking system, we have now further localized the site of inhibition in the scheme of blood coagulation. The interference occurs at the transpeptidation step catalyzed by the thrombin-activated fibrin-stabilizing factor. The patient's serum also uniquely delayed the clotting time of Homarus plasma, a test for specific inhibitors of transpeptidation. Since the inhibitor was effective in two such widely different systems, it probably is not an antibody, but falls into the category of cross-linking inhibitors which we have previously described (4, 5, 10, 12-17). While the exact nature of the inhibitor remains unknown, we raise the question whether some unusual metabolic transformation of isonicotinic acid hydrazide (with which the patient was treated and which itself we found to be a potent inhibitor fibrin cross-linking), in combination with a macromolecule, might not have given rise to an inhibitory compound.

Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.

Fibrinoligase, the fibrin cross-linking enzyme, transiently appearing during the course of coagulation in normal blood, was shown to catalyze the incorporation of a fluorescent amine, monodansylcadaverine [or N-(5-aminopentyl)-5-dimethylamino-1-naphthalene-sulfonamide] into casein. The reaction provided the basis of a sensitive fluorimetric method for measuring the activity of the enzyme (and also of similar other transpeptidases, such as transglutaminase). In tests involving plasma, certain difficulties had to be overcome which were mainly due to the fact that the enzyme itself does not occur in citrated plasma. Only its precursor (fibrin-stabilizing factor or factor XIII) is present, still requiring limited proteolytic activation by thrombin. Thus, in order to measure amine incorporation with plasma as a source of the factor, thrombin must be added. This necessitated a differential desensitization of the intrinsic fibrinogen so that the latter could not clot and could not thereby interfere with amine incorporation. Also, the thrombin-inactivating capacity of plasma had to be saturated to enable full conversion of the factor to the transpeptidase. Concentrations of casein, monodansylcadaverine, calcium, and hydrogen ions were chosen to permit almost maximal velocity of amine incorporation. A linear relationship with regard to plasma concentration could be obtained only under such conditions. No similar assay is presently available for quantitatively evaluating fibrin-stabilizing factor levels in plasma.The amine incorporation test was applied to a clinical case of hereditary total fibrin-stabilizing factor deficiency. The effect of transfusion therapy was studied, and some of the patient's relatives were examined. Whereas a paternal aunt and uncle gave values well within the normal range, a brother and the mother proved to be partially deficient and could be considered as heterozygous carriers. The father appeared to have a reduced level of fibrin-stabilizing factor, though not quite as low as the other two relatives. Two infusions (1 liter each) of fresh normal plasma, administered about 26 hr apart, brought levels in the patient's plasma close to those found in the mother and brother. The corrective power of the transfusions, however, rapidly declined within 5-6 days. Futility of the last transfusion could be ascribed to the appearance of a neutralizing antibody directed against the precursor stabilizing factor, a serious complication. General diagnostic versatility and potential of the quantitative amine incorporation assay with plasma is discussed.

Difference between type I autoimmune inhibitors of fibrin stabilization in two patients with severe hemorrhagic disorder.

Inhibitors of fibrin stabilization of apparently autoimmune origin, found in two severely bleeding unrelated patients (W. G. and G. A.), were compared with regard to their biological target specificities, potencies and immunological characteristics. Both interfered only with the activation of fibrin stabilizing factor (coagulation Factor XIII) and, while totally preventing the conversion of this zymogen to the functional transamidating enzyme, fibrinoligase (Factor XIII(a)), they showed very little inhibition toward the enzyme itself. Thus, according to the classification of Lorand concerning biological specificities, both can be characterized as Type I inhibitors of fibrin stabilization. Potencies of the two inhibitors were quite similar when measured in conjunction with the plasma zymogen, but they differed remarkably in tests with platelet Factor 13. The inhibitor of patient W. G. prevented the activation of the zymogen from platelets, but that of G. A. had no effect on the platelet factor. It may therefore be concluded that the inhibitor of W. G. is directed exclusively against the a subunit which is a common constituent of plasma as well as platelet factors. The inhibitor of G. A., however, must be targeted against determinants uniquely characteristic for the ab ensemble of the plasma zymogen including the b subunit. On the basis of this difference in target specificity, the inhibitor of W. G. is designated as Type I-1 and that of G. A. as Type I-2. The inhibitors of both patients were isolated as immunoglobulins, and neutralization tests revealed that the antibody of W. G. comprised mainly heavy chains of the IgG1 and light chains of the kappa class. The antibody of G. A. proved to be considerably more heterogeneous and contained IgG1 and IgG3 heavy chains as well as kappa- and lambda-light chains. The finding that the antibody of W. G. inhibited conversion of platelet Factor 13 and also its thrombinmodified form, but had no effect on the thrombin and Ca(2+)-activated factor, is an indication that antigenic determinants existing both on the native zymogen and on its hydrolytically modified form become buried in the Ca(2+)-dependent step of activation. This is clear evidence for the occurrence of a significant conformational change in the protein structure attendant to the process of unmasking of its enzymic activity.

Cytokine mediated induction of the major Epstein-Barr virus (EBV)-encoded transforming protein, LMP-1.

In the in vitro infected B-cells six EBV-encoded nuclear antigens (EBNA-1-6) and three latent membrane proteins (LMP-1, -2A, -2B) are expressed (type III latency). In addition, other restricted forms of latency occur in the EBV-carrying malignancies. In Burkitt lymphoma (BL) only EBNA-1 is expressed (type I), while in Hodgkin lymphoma (HL), T-, and NK-lymphoma, and nasopharyngeal carcinoma EBNA-1 and LMPs are expressed (type II). B-cells with these three expression patterns have been detected in healthy virus carriers. While in type III latency two viral transcriptional activators, EBNA-2 and -5, are responsible for LMP-1 expression, the mechanism that controls the expression of LMP-1 in type II latent cells is not known. In order to study the interaction of EBV- and HL-derived cells, we studied the in vitro EBV-converted subline of the KMH2 cells that express only EBNA-1 and LMP-2A. Interestingly, exposure of the KMH2-EBV cells to CD40-ligand and IL-4 induced LMP-1 expression, in the absence of EBNA-2. In BL cell lines lacking EBNA-2 another cytokine, IL-10, could induce LMP-1 expression. IL-10 induced LMP-1 also in tonsillar B-cells infected with the EBNA-2-deleted virus strain P3HR-1. Our results show that cytokines are responsible for the expression of LMP-1 in type II latent B-cells. These signals are available in the germinal center environment and in the granulation tissue of HLs. Based on these results we propose that LMP-1 expression is induced by extracellular signals and is not a constitutive characteristic of the EBV-carrying type II B-cells. Cytokine mediated induction of LMP-1 may also explain the heterogeneous expression of this viral gene seen in normal and malignant cells.

Cross-linking and proteolysis in Ca2(+)-treated lens homogenates.

It was previously shown (Lorand et al. (1985) Biochemistry 24, 1525) that treatment of lens homogenate with Ca2+ produces two sets of changes which are catalyzed by intrinsic enzymes of the lens and which can be readily seen by alterations in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of proteins. With the aid of differential inhibitors of the two reactions (e.g., dansylcadaverine and leupeptin) it was possible to distinguish the transglutaminase-dependent cross-linking of proteins from the proteolytic degradative phenomena. We have now shown that the proteins which are affected by the two processes can be compartmentalized differentially by centrifuging the lens homogenate after exposure to Ca2+. The dimeric and oligomeric beta-crystallin products of transglutaminase-mediated cross-linking are most clearly visible in the soluble supernatant, whereas the proteolytically susceptible proteins--possibly structural in nature, including vimentin--are predominantly present in the pellet. We have found a compound, 2-[3-(diallylamino)propionyl]benzothiophene, which, by virtue of acting as a noncompetitive inhibitor of transglutaminase as well as of calpains I and II, effectively blocked both the cross-linking seen in the supernatant and the proteolysis seen in the pellet fraction, though perhaps with somewhat different sensitivities.

Storage, ultrastructural targeting and function of toposomes and hyalin in sea urchin embryogenesis.

This study compares by immunogold labeling the ultrastructural localization of a hexameric 22S glycoprotein, called toposome, with that of hyalin in unfertilized eggs and cells of hatched sea urchin blastulae. Nearly all hyalin is present in the electron translucent compartment of the cortical granules and in the translucent non-cortical pigment granules. In the blastula both of these intracellular stores have vanished and hyalin now forms a broad band below the apical lamina. By contrast, in the egg toposomes are present on the surface, as well as stored in yolk granules and in the electron dense lamellar compartment of the cortical granules. In the hatched blastula, toposomes that have been modified by limited proteolysis in the yolk granules, are associated with the plasma membranes of all newly formed cells, while the toposomes originating from the cortical granules have been incorporated as unmodified 160 kDa polypeptides into an extracellular double layer enveloping the embryo on the outside of the hyaline layer. From evidence discussed in detail, we conclude that the extracellular toposomes rivet the apical lamina to the surface and underlying cytoskeleton of the microvilli, while the modified toposomes from the yolk granules are responsible for position specific intercellular adhesion as they are released to the surface of newly formed cells. We propose that all the material stored in yolk granules is utilized for the assembly of new membranes.

ß-catenin helices in the cytoplasm of sessile serrated adenoma/polyps and conventional colorectal adenomas.

Initiation and progression in conventional adenomas is triggered by deregulation of WNT/ß-catenin signaling. In the absence of WNT signal (off-state), ß-catenin prevents phosphorylation of GSK3ß, leading to aberrant nuclear accumulation in human tumors. It has been postulated that mutations in the ß-catenin gene are always associated with a morphologically-neoplastic course. While investigating the nuclear expression of ß-catenin in 170 colorectal biopsies, we observed a non-previously reported phenomenon, namely the presence of ß-catenin cytoplasmic helices in 29% (n=7) of 24 sessile serrated adenoma/polyps (SSA/P), in 24% (n=13) of 54 adenomas, in 8% (n=3) of 38 specimens with IBD, but in none (0/54) with normal mucosa. The earliest ß-catenin helices were found at the bottom of SSA/P glands (the domain of stem cells in the colorectal mucosa). It is submitted that ß-catenin helices might highlight a non-previously described cytoplasmic phenomenon evolving during the serrated-carcinoma pathway in SSA/P, and during the adenoma-carcinoma pathway in conventional adenomas.

Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology.

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.