Prebreeding Immunization of Beef Cows

A number of infectious agents are potential threats to the fetus of a pregnant cow and may result in abortion. These agents include Leptospira sp., Campylobacter fetus and viruses such as infectious bovine rhinotracheitis (IBR) and bovine virus diarrhea (BVD). Maintenance in the cow of a high level of immunity to these agents during pregnancy can insure protection of the fetus. In particular, vaccines against BVD and IBR viruses can establish protective immunity throughout gestation. An appropriate vaccination regimen prior to breeding is required to establish this protective immunity. This can be achieved with a single dose of certain modified live virus vaccines, but those vaccines must be administered at least 30 days prior to breeding to avoid interference with conception. We have evaluated an oil-adjuvanted inactivated virus vaccine in cattle with differing immunological histories. Two doses of the vaccine administered 30 days apart to serologically negative animals induced appreciable levels of BVD and IBR anti-viral antibodies with persisting titers throughout gestation. In other experiments a single dose of the vaccine was administered to: (1) animals given two doses of the vaccine several months earlier, (2) animals previously vaccinated with other inactivated virus vaccines, or (3) animals previously vaccinated with modified live virus vaccine. The vaccine consistently induced marked anamnestic responses in these animals. Not only did mean titers rise, but a vast majority of individual animals responded. This contrasts with efforts to boost titers with modified live virus vaccines where the effect may be erratic among animals. The safety and efficacy of selected inactivated viral vaccines argues for their use in prebreeding immunization of beef cows.

Investigation of the Potential Effects of Epizootic Hemorrhagic Disease Virus on Iowa Cattle

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus, causes significant mortality in white-tailed deer and can also cause disease in cattle. Objectives of this preliminary investigation were 1) to survey cattle at auction markets to determine the prevalence of anti-EHDV antibodies in Iowa cattle, 2) to determine EHDV seroprevalence in herds in which clinical EHD had been diagnosed, and 3) to determine whether EHDV is associated with stillbirths and/or congenital anomalies in calves. There was a 15% seroprevalence in auction market cattle; positive cattle were from southern, central, and western Iowa. Herds in which clinical EHD had been diagnosed had >60% seroprevalence. Viremia was detected in both clinically affected and unaffected cattle during an EHD outbreak. EHDV exposure was not consistently associated with congenital anomalies. Although additional surveillance is warranted, EHDV is unlikely to have a significant effect on the reproductive health of Iowa cattle.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Decreasing risk of hepatitis a infection in león, nicaragua: evidence from cross-sectional and longitudinal seroepidemiology studies

BACKGROUND AND OBJECTIVES Nicaragua is highly endemic for hepatitis A. We aimed to provide an estimate of the change in the age-specific risk of hepatitis A virus (HAV) infection based on serological data from cross-sectional and longitudinal samples collected in León, Nicaragua, in 1995/96 (n = 979) and 2003 (n = 494). METHODS The observed age-specific prevalence of anti-HAV antibodies was correlated to the age-specific risk of infection by calculating the probability of freedom from infection at a specific age. RESULTS The proportion of seropositive children aged 1.5 to 6 years was 42% in 2003 compared to 67% in 1995/96. Estimated annual risk of infection for a 3-year old child was 30% (95% CI: 27.0%, 33.1%) in 1995 and 15.5% (95% CI: 12.4%, 19.0%) in 2003. There was good agreement between estimates based on cross-sectional and longitudinal data. The age-specific geometric mean of the quantified anti-HAV antibody levels assessed in 2003 was highest at age 4 and decreased steadily up to age 40. CONCLUSIONS The substantially lower risk of HAV infection in 2003 than in 1995 for young children indicates a beginning transition from high to intermediate endemicity in León, Nicaragua. Consecutive age-stratified serosurveys are useful to assess changes in risk of infection following public health interventions. The decreasing age-specific GMC of anti-HAV antibodies during adulthood in a country with endemic HAV indirectly suggests that ongoing HAV exposure in the community has marginal boosting effect on antibody levels once protective immunity has been established by natural infection.

Biologika – Genese und Charakteristika immunvermittelter Nebenwirkungen

Die Therapie immunvermittelter und maligner Erkrankungen befindet sich im Umbruch. Zunehmend kommen gezielt mit molekularen Zellstrukturen interagierende therapeutische Proteine, sogenannte Biologika oder Biopharmaka, zum Einsatz. Biologika sind in der Regel Antikörper oder Fusionsproteine und werden gentechnologisch nach dem Vorbild körpereigener Strukturen in zellulären Expressionssystemen, wie zum Beispiel Hamster-Ovarzellen, hergestellt und in hochreiner Form verabreicht. Biologika haben mit den klassischen kleinmolekularen Medikamenten, den sogenannten Synthetika, sowohl in der Wirkungsweise wie auch hinsichtlich der Nebenwirkungen nur noch wenig gemein. Der zunehmende Einsatz von Biologika im klinischen Alltag stellt eine Herausforderung für die anwendenden Ärzte dar, die nebst den neuen Wirkmechanismen auch den Umgang mit komplexen Nebenwirkungsmustern von direkt mit dem Immunsystem interagierenden Substanzen kennen und behandeln lernen müssen. Akute Infusionreaktionen (infusion-related reaction, IRR) stellen mit einer Inzidenz von 0,1 – 3% die häufigste Nebenwirkung dar. Interessanterweise handelt es sich in den meisten Fällen nicht um IgE-vermittelte Reaktionen und lassen sogar eine Reexposition zu. Während regulatorische Behörden und die Pharmaindustrie sich vor allem auf anti-drug-antibodies (ADA)-vermittelte Nebenwirkungen konzentrieren, wollen wir hier einen Überblick über das Nebenwirkungsspektrum und mögliche Abklärungsalgorithmen geben.

Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

The sequence of addition of IL-3 and IgE-dependent/IgE-independent stimuli regulates RANKL expression in human basophils

Background: Receptor Activator of Nuclear Factor kappaB Ligand (RANKL), a member of the TNF superfamily, contributes to the imbalance of bone resorption and immunoregulation in rheumatoid arthritis. In mice, collagen induced arthritis was exacerbated by IL-3 and anti-IgER antibodies, two mediators activating basophils that are known as effector cells of allergy. Interestingly, our unpublished microarray data revealed that IL-3 induces RANKL mRNA in human basophils. Here we further investigate under which conditions human basophils express surface and/or soluble RANKL. Methods: One part of purified human basophils was co-stimulated with IL-3 and either IgE-dependent or IgE-independent stimuli. The other part of purified basophils was first primed with IL-3 and subsequently triggered with IgE-dependent or IgE-independent stimuli. Expression of surface and soluble RANKL were detected by flow cytometry, ELISA and real-time PCR. Results: By flow cytometry we show that IL-3 induces de novo expression of surface RANKL on human basophils in a time and dose dependent manner. Co-stimulation of basophils with IL-3 and an IgE-dependent stimulus reduces IL-3-induced expression of surface RANKL in a dose dependent manner while IgE-independent stimuli have no effect. In contrast, both IgE-dependent and IgE-independent stimuli enhance expression of surface and soluble RANKL in basophils that were first primed with IL-3 and then triggered. Real-time PCR analysis shows that surface hRANKL1 and soluble hRANKL3 are induced by IL-3 and reduced by co-stimulation with IL-3 and an IgE-dependent stimulus and thus confirms our flow cytometry data. Conclusion: RANKL expression in human basophils is not only dependent on IL-3 and IgE-dependent/IgE-independent stimuli but also on the sequence of their addition to cell culture. Based on our data, we suggest that basophils might have previously unidentified functions in bone resorption or immunoregulation via RANKL.

Importance of the collagen adhesin ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis.

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFDeltaace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFDeltaace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.

Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis.

Enterococcus faecium is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating E. faecium virulence determinants. Our previous studies showed that some clinical E. faecium isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic acm deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Deltaacm::cat is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P < 0.0001) and for valve colonization 1 and 3 hours after infection (P or=50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that acm transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in E. faecium collagen adherence by affinity-purified anti-Acm antibodies from E. faecium endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

Persistence of circulating ADAMTS13-specific immune complexes in patients with acquired thrombotic thrombocytopenic purpura

Anti-ADAMTS13 autoantibodies are the main cause of acquired thrombotic thrombocytopenic purpura. Binding of these antibodies to ADAMTS13 eventually results in the formation of antigen-antibody immune complexes. Circulating ADAMTS13-specific immune complexes have been described in acquired thrombotic thrombocytopenic purpura patients, however, the prevalence and persistence of these immune complexes over time has hitherto remained elusive. Here, we analyzed a large cohort of patients with acquired thrombotic thrombocytopenic purpura for the presence of free and complexed anti-ADAMTS13 antibodies. In the acute phase (n=68), 100% of patients had free IgG antibodies and 97% had ADAMTS13-specific immune complexes. In remission (n=28), 75% of patients had free antibodies (mainly IgG) and 93% had ADAMTS13-specific immune complexes. Free antibodies were mainly of subclasses IgG1 and IgG4, whereas IgG4 was by far the most prevalent in ADAMTS13-specific immune complexes. Comparison of ADAMTS13 inhibitor and anti-ADAMTS13 IgG (total and subclasses) antibody titers in acute phase and in remission samples showed a statistically significant decrease in all parameters in remission. Although non-significant, a trend towards reduced or undetectable titers in remission was also observed for ADAMTS13-specific immune complexes of subclasses IgG1, IgG2 and IgG3. For IgG4, no such trend was discernible; IgG4 immune complexes persisted over years, even in patients who had been treated with rituximab and who showed no features suggesting relapse.

The Role of Acidic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor 4 in High Grade Serous Carcinoma

Background: High grade serous carcinoma whether ovarian, tubal or primary peritoneal, continues to be the most lethal gynecologic malignancy in the USA. Although combination chemotherapy and aggressive surgical resection has improved survival in the past decade the majority of patients still succumb to chemo-resistant disease recurrence. It has recently been reported that amplification of 5q31-5q35.3 is associated with poor prognosis in patients with high grade serous ovarian carcinoma. Although the amplicon contains over 50 genes, it is notable for the presence of several members of the fibroblast growth factor signaling axis. In particular acidic fibroblast growth factor (FGF1) has been demonstrated to be one of the driving genes in mediating the observed prognostic effect of the amplicon in ovarian cancer patients. This study seeks to further validate the prognostic value of fibroblast growth receptor 4 (FGFR4), another candidate gene of the FGF/FGFR axis located in the same amplicon. The emphasis will be delineating the role the FGF1/FGFR4 signaling axis plays in high grade serous ovarian carcinoma; and test the feasibility of targeting the FGF1/FGFR4 axis therapeutically. Materials and Methods: Spearman and Pearson correlation studies on data generated from array CGH and transcriptome profiling analyses on 51 microdissected tumor samples were used to identify genes located on chromosome 5q31-35.3 that showed significant correlation between DNA and mRNA copy numbers. Significant correlation between FGF1 and FGFR4 DNA copy numbers was further validated by qPCR analysis on DNA isolated from 51 microdissected tumor samples. Immunolocalization and quantification of FGFR4 expression were performed on paraffin embedded tissue samples from 183 cases of high-grade serous ovarian carcinoma. The expression was then correlated with clinical data to assess impact on survival. The expression of FGF1 and FGFR4 in vitro was quantified by real-time PCR and western blotting in six high-grade serous ovarian carcinoma cell lines and compared to those in human ovarian surface epithelial cells to identify overexpression. The effect of FGF1 on these cell lines after serum starvation was quantified for in vitro cellular proliferation, migration/invasion, chemoresistance and survival utilizing a combination of commercially available colorimetric, fluorometric and electrical impedance assays. FGFR4 expression was then transiently silenced via siRNA transfection and the effects on response to FGF1, cellular proliferation, and migration were quantified. To identify relevant cellular pathways involved, responsive cell lines were transduced with different transcription response elements using the Cignal-Lenti reporter system and treated with FGF1 with and without transient FGFR4 knock down. This was followed by western blot confirmation for the relevant phosphoproteins. Anti-FGF1 antibodies and FGFR trap proteins were used to attempt inhibition of FGF mediated phenotypic changes and relevant signaling in vitro. Orthotopic intraperitoneal tumors were established in nude mice using serous cell lines that have been previously transfected with luciferase expressing constructs. The mice were then treated with FGFR trap protein. Tumor progression was then followed via bioluminescent imaging. The FGFR4 gene from 52 clinical samples was sequenced to screen for mutations. Results: FGFR4 DNA and mRNA copy numbers were significantly correlated and FGFR4 DNA copy number was significantly correlated with that of FGF1. Survival of patients with high FGFR4 expressing tumors was significantly shorter that those with low expression(median survival 28 vs 55 month p< 0.001) In a multivariate cox regression model FGFR expression significantly increased risk of death (HR 2.1, p<0.001). FGFR4 expression was significantly higher in all cell lines tested compared to HOSE, OVCA432 cell line in particular had very high expression suggesting amplification. FGF1 was also particularly overexpressed in OVCA432. FGF1 significantly increased cell survival after serum deprivation in all cell lines. Transient knock down of FGFR4 caused significant reduction in cell migration and proliferation in vitro and significantly decreased the proliferative effects of FGF1 in vitro. FGFR1, FGFR4 traps and anti-FGF1 antibodies did not show activity in vitro. OVCA432 transfected with the cignal lenti reporter system revealed significant activation of MAPK, NFkB and WNT pathways, western blotting confirmed the results. Reverse phase protein array (RPPA) analysis also showed activation of MAPK, AKT, WNT pathways and down regulation of E Cadherin. FGFR trap protein significantly reduced tumor growth in vivo in an orthotopic mouse model. Conclusions: Overexpression and amplification of several members of the FGF signaling axis present on the amplicon 5q31-35.3 is a negative prognostic indicator in high grade serous ovarian carcinoma and may drive poor survival associated with that amplicon. Activation of The FGF signaling pathway leads to downstream activation of MAPK, AKT, WNT and NFkB pathways leading to a more aggressive cancer phenotype with increased tumor growth, evasion of apoptosis and increased migration and invasion. Inhibition of FGF pathway in vivo via FGFR trap protein leads to significantly decreased tumor growth in an orthotopic mouse model.

The interaction of P160 BCR with BCR-ABL gene products unique to Philadelphia chromosome leukemias

The BCR gene is involved in the pathogenesis of Philadelphia chromosome-positive (Ph$\sp1$) leukemias. Typically, the 5$\sp\prime$ portion of BCR on chromosome 22 becomes fused to a 5$\sp\prime$ truncated ABL gene from chromosome 9 resulting in a chimeric BCR-ABL gene. To investigate the role of the BCR gene product, a number of BCR peptide sequences were used to generate anti-BCR antibodies for detection of BCR and BCR-ABL proteins. Since both BCR and ABL proteins have kinase activity, the anti-BCR antibodies were tested for their ability to immunoprecipitate BCR and BCR-ABL proteins from cellular lysates by use of an immunokinase assay. Antisera directed towards the C-terminal portions of P160 BCR, sequences not present in BCR-ABL proteins, were capable of co-immunoprecipitating P210 BCR-ABL from the Ph$\sp1$- positive cell line K562. Re-immunoprecipitation studies following complete denaturation showed that C-terminal BCR antisera specifically recognized P160 BCR but not P210 BCR-ABL. These and other results indicated the presence of a P160 BCR/P210 BCR-ABL protein complex in K562 cells. Experiments performed with Ph$\sp1$-positive ALL cells and uncultured Ph$\sp1$-positive patient white blood cells established the general presence of BCR/BCR-ABL protein complexes in BCR-ABL expressing cells. However, two cell lines derived from Ph$\sp1$-positive patients lacked P160 BCR/P210 BCR-ABL complexes. Lysates from one of these cell lines mixed with lysates from a cell line that expresses only P160 BCR failed to generate BCR/BCR-ABL protein complexes in vitro indicating that P160 BCR and P210 BCR-ABL do not simply oligomerize.^ Two-dimensional tryptic maps were performed on both BCR and BCR-ABL proteins labeled in vitro with $\sp{32}$P. These maps indicate that the autophosphorylation sites in BCR-ABL proteins are primarily located within BCR exon 1 sequences in both P210 and P185 BCR-ABL, and that P160 BCR is phosphorylated in trans in similar sites by the activated ABL kinase of both BCR-ABL proteins. These results provide strong evidence that P160 BCR serves as a target for the BCR-ABL oncoprotein.^ K562 cells, induced to terminally differentiate with the tumor promoter TPA, show a loss of P210 BCR-ABL kinase activity 12-18 hours after addition of TPA. This loss coincides with the loss of activity in P160 BCR/P210 BCR-ABL complexes but not with the loss of the P210 BCR-ABL, suggesting the existence of an inactive form of P210 BCR-ABL. However, a degraded BCR-ABL protein served as the kinase active form preferentially sequestered within the remaining BCR/BCR-ABL protein complex.^ The results described in this thesis form the basis for a model for BCR-ABL induced leukemias which is presented and discussed. ^

Function and formation of $\beta$1,4-galactosyltransferase-specific adhesions during growth and differentiation of F9 embryonal carcinoma cells

Galactosyltransferase (GalTase) is localized in the Golgi, where it functions in oligosaccharide synthesis, as well as on the cell surface where it serves as a cell adhesion molecule. GalTase-specific adhesions are functional in a number of important biological events, including F9 embryonal carcinoma (EC) cell adhesions. GalTase-based adhesions are formed by recognition and binding to terminal N-acetylglucosamine (GlcNAc) residues on its glycoprotein counterpart on adjacent cell surfaces. The object of this work has been to investigate the formation and function of GalTase-specific adhesions during F9 cell growth and differentiation. We initially investigated GalTase synthesis during differentiation and found that the increase in GalTase activity was specific for the Golgi compartment; surface GalTase levels remained constant during differentiation. These data indicated that the increase in cell adhesions expected with increased cell-matrix interaction in differentiated F9 cells is not the consequence of increased surface GalTase expression and, more interestingly, that the two pools of GalTase are under differential regulation. Synthesis and recognition of the consociate glycoprotein component was next investigated. Surface GalTase recognized several surface glycoproteins in a pattern that changes with differentiation. Uvomorulin, lysosome-associated membrane protein-1 (LAMP-1), and laminin were recognized by surface GalTase and are, therefore, potential components in GalTase-specific adhesions. Furthermore, these interactions were aberrant in an adhesion-defective F9 cell line that results, at least in part, from abnormal oligosaccharide synthesis. The function played by surface GalTase in growth and induction of differentiation was examined. Inhibition of surface GalTase function by a panel of reagents inhibited F9 cell growth. GalTase expression at both the transcription and protein levels were differentially regulated during the cell cycle, with surface expression greatest in the G1 phase. Disruption of GalTase adhesion by exposure to anti-GalTase antibodies during this period resulted in extension of the G2 phase, a result similar to that seen with agents known to inhibit growth and induce differentiation. Finally, other studies have suggested that a subset of cell adhesion molecules have the capability to induce differentiation in EC cells systems. We have determined in F9 cells that dissociating GalTase adhesion by galactosylation of and release of the consociate glycoproteins induces differentiation, as defined by increased laminin synthesis. The ability to induce differentiation by surface galactosylation was greatest in cells grown in cultures promoting cell-cell adhesions, relative to cultures with minimal cell-cell interactions. ^

The molecular cloning and characterization of human heparanase cDNA and the immunochemical localization of heparanase in metastatic melanomas

Heparanase, an endo-$\beta$-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a M$\sb{\rm r}\sim 97,000$ protein upon SDS-polyacrylamide gel electrophoresis of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, metastatic human A375-SM and mouse B16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse organs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells, but not in adjacent normal tissues. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells, but not in adjacent connective tissues.^ Monoclonal antibodies directed against murine heparanase were developed and characterized. Monoclonal antibody 10E5, an IgM, precipitated and inhibitated the enzymatic activity of heparanase. A 2.6 kb cDNA was isolated from a human melanoma $\lambda$gt11 cDNA library using the monoclonal antibody 10E5. Heparan sulfate cleavage activity was detected in the lysogen lysates from E. Coli Y1089 infected with the $\lambda$gt11 cDNA and this activity was inhibited in the presence of 10-fold excess of heparin, a potent inhibitor of heparanase. The nucleotide sequence of the cDNA was determined and insignificant homology was found with the gene sequences currently known. The cDNA hybridized to a 3.2-3.4 kb mRNA in human A375 melanoma, WI-38 fibroblast, and THP-1 leukemia cells using Northern blots.^ Heparanase expression was examined using Western and Northern blots. In comparison to human A375-P melanoma cells, the quantity of 97,000 protein recognized by the polyclonal anti-heparanase antibodies doubled in the metastatic variant A375-SM cells and the quantity of 3.2-3.4 kb mRNA doubled in A375MetMix, a metastatic variant similar to A375-SM cells. In B16 murine melanoma cell, the intensity of the 97,000 protein increased more than 2 times comparing with B16-F1 cells. The extent in the increase of the protein and the mRNA levels is comparable to the change of heparanase activity observed in those cells.^ In summary, the studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; (c) heparanase antigens are localized in invasive and metastatic murine and human melanomas in vivo, but not in adjacent normal tissues; (d) heparanase molecule appeared to be differentially expressed at the transcriptional as well as at the translational level; and (e) the size of human heparanase mRNA is 3.2-3.4 kilobase. ^

Immunochemical interactions of peptides with site-directed polyclonal immunoglobulin G

The objective of this study was to investigate the immunochemical nature of the polyclonal immune response to the 14mer peptide TINKEDDESPGLYG and to identify interactions among antibodies to more than one epitope. Two groups of rabbits were immunized with the 14mer peptide and a Keyhole Limpet hemocyanin (KLH) carrier, but with KLH attached either to the 14mer's N- or C-terminus. Two approximate epitopes were mapped by an antibody-capture enzyme-linked immunosorbent assay method using antiserum obtained when KLH was oriented on the C-terminus of the 14mer. A precise mapping of the epitopes performed with inhibition enzyme immunoassays (iEIAs) resulted in an N-terminal 6mer epitope TINKED and a C-terminal 10mer epitope EDDESPGLYG. The epitopes overlapped by two amino acids. IEIAs and iEIAs incorporating antibody-blocking peptides indicated that the two anti-epitope antibody fractions did not interfere with one anothers' epitope binding. It was postulated that the anti-TINKED and anti-EDDESPGLYG antibody fractions individually bind their respective hydrophobic epitope "core" region at the N- or C-terminal of peptide TINKEDDESPGLYG, while sharing the two hydrophilic overlap amino acids. This antibody "lap joint" binding interaction can be accomplished by each of the anti-epitope antibodies binding an opposite side of the epitope overlap region in the shallow periphery of its binding site. ^