Tumor recognition of peanut agglutinin-immobilized fluorescent nanospheres in biopsied human tissues.

We are investigating an imaging agent for early detection of colorectal cancer. The agent, named the nanobeacon, is coumarin 6-encapsulated polystyrene nanospheres whose surfaces are covered with poly(N-vinylacetamide) and peanut agglutinin that reduces non-specific interactions with the normal mucosa and exhibits high affinity for terminal sugars of the Thomsen-Friedenreich antigen, which is expressed cancer-specifically on the mucosa, respectively. We expect that cancer can be diagnosed by detecting illumination of intracolonically administered nanobeacon on the mucosal surface. In the present study, biopsied human tissues were used to evaluate the potential use of the nanobeacon in the clinic. Prior to the clinical study, diagnostic capabilities of the nanobeacon for detection of colorectal cancer were validated using 20 production batches whose characteristics were fine-tuned chemically for the purpose. Ex vivo imaging studies on 66 normal and 69 cancer tissues removed from the colons of normal and orthotopic mouse models of human colorectal cancer, respectively, demonstrated that the nanobeacon detected colorectal cancer with excellent capabilities whose rates of true and false positives were 91% and 5%, respectively. In the clinical study, normal and tumor tissues on the large intestinal mucosa were biopsied endoscopically from 11 patients with colorectal tumors. Histological evaluation revealed that 9 patients suffered from cancer and the rest had adenoma. Mean fluorescence intensities of tumor tissues treated with the nanobeacon were significantly higher than those of the corresponding normal tissues. Correlation of magnitude relation of the intensity in individuals was observed in cancer patients with a high probability (89%); however, the probability reduced to 50% in adenoma patients. There was a reasonable likelihood for diagnosis of colorectal cancer by the nanobeacon applied to the mucosa of the large intestine.

Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy.

Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 10(6) M(-1)·s(-1) at 0.43 µM PnA to (1.1 ± 0.1) × 10(5) M(-1)·s(-1) at 12 µM PnA. CTB-GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 10(9) M(-1)·s(-1) at 0.5 nM CTB to (3.5 ± 0.2) × 10(6) M(-1)·s(-1) at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 µM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 10(9) M(-1) and (2.8 ± 0.5) × 10(13) M(-1), respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB-GM1 demonstrates positive ligand-ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein-ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

Simultaneous determination of concanavalin A and peanut agglutinin by dual-color quantum dots.

In this work, we designed a novel detection strategy to realize simultaneous determination of multiplex lectin by labeling glucosamine (G1) and galactosamine (G2) with different-colored semiconductor quantum dots (QDs). On the basis of the agglutination of the aminosugar-labeled QDs induced by the exclusive binding between the lectin and sugar on the QDs surfaces, the fluorescence emission of the QDs supernatant after centrifugation decreased with relevant lectin concentration [i.e., when concanavalin A (Con A) exists alone], only green color fluorescence emission from QDs-G1 supernatant decreased, so it is peanut agglutinin (PNA) and red color fluorescence emission from QDs-G2. Moreover, since QDs can be simultaneously excited with multiple fluorescence colors and have a larger Stokes shift than organic fluorophores, when both Con A and PNA are present in the sample, both of the green and red color fluorescence emission from QDs-G1 and QDs-G2 supernatant would decrease, thus realizing the simultaneous determination of Con A and PNA. The detection limits of Con A and PNA are 0.30 and 0.18 nM (3σ), respectively. Furthermore, the present detection method not only can determine the protein/lectins by fluorescence spectral method but also can realize visualization detection by UV lamp illumination. To the best of our knowledge, this is the first report of such analytical method in multiple and simultaneous lectin detection.

Design of a covalently bonded glycosphingolipid microarray.

Glycosphingolipids (GSLs) are well known ubiquitous constituents of all eukaryotic cell membranes, yet their normal biological functions are not fully understood. As with other glycoconjugates and saccharides, solid phase display on microarrays potentially provides an effective platform for in vitro study of their functional interactions. However, with few exceptions, the most widely used microarray platforms display only the glycan moiety of GSLs, which not only ignores potential modulating effects of the lipid aglycone, but inherently limits the scope of application, excluding, for example, the major classes of plant and fungal GSLs. In this work, a prototype "universal" GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-G(M1), with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-G(M2)). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays that incorporate GSLs for serodiagnosis is discussed.

Localization of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction.

One of the essential properties of mammalian, including sperm, plasma membranes is a stable transversal lipid asymmetry with the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), typically in the inner, cytoplasmic leaflet. The maintenance of this nonrandom lipid distribution is important for the homeostasis of the cell. To clarify the relevance of lipid asymmetry to sperm function, we have studied the localization of PS in boar sperm cell membranes. By using labeled annexin V as a marker for PS and propidium iodide (PI) as a stain for nonviable cells in conjunction with different methods (flow cytometry, fluorescence and electron microscopy), we have assessed the surface exposure of PS in viable cells during sperm genesis, that is, before and during capacitation as well as after acrosome reaction. An approach was set up to address also the presence of PS in the outer acrosome membrane. The results show that PS is localized in the cytoplasmic leaflet of the plasma membrane as well as on the outer acrosome membrane. Our results further indicate the cytoplasmic localization of PS in the postacrosomal region. During capacitation and acrosome reaction of spermatozoa, PS does not become exposed on the outer surface of the viable cells. Only in a subpopulation of PI-positive sperm cells does PS became accessible upon capacitation. The stable cytoplasmic localization of PS in the plasma membrane, as well as in the outer acrosome membrane, is assumed to be essential for a proper genesis of sperm cells during capacitation and acrosome reaction.

Age-related alterations in the carbohydrate residue composition of the cell surface in the unexposed normal human epidermis.

BACKGROUND: The results of the very few histochemical studies that have been performed so far on the lectin-binding profile of normal human epidermis are mostly controversial; thus, the carbohydrate residue composition of the cell surface in the latter still remains in dispute and the possible alterations in the epidermal lectin-binding profile are unknown. OBJECTIVE: To investigate the influence of age on the carbohydrate residue composition of the cell surface in unexposed normal human epidermis by means of lectin histochemistry. METHODS: Biopsy specimens obtained from the sun-protected (unexposed) buttock skin, divided into 5 age groups of 18 subjects each, were fixed in buffered formalin (10%) and embedded in paraffin. 4-mum sections were processed for histochemistry using a panel of six biotinylated lectins. RESULTS: In the unexposed normal human epidermis the concentration and distribution of cell surface beta-D-galactose, D-galactose-beta-(1,3 N-acetylo-D-galactosamine), beta(1,4 D-N-acetylo-D-glucosamine) and alpha-D-N-acetylo-D-galactosamine were almost identical in all age groups, whereas those of alpha-D-mannose, alpha-D-glucose and alpha-L-fucose revealed significant age-related differences. CONCLUSION: These findings may be due to an age-related decline in synthesis and/or transport of monosaccharides from the cytoplasm to the surface of epidermal cells. Thus, the corresponding lectins concanavalin A and Ulex europaeus agglutinin-I can only be used in comparative histochemical studies of the carbohydrate residue composition of the cell surface in the normal and pathological epidermis of individuals of the same age, whereas Ricinus communis agglutinin-I, peanut agglutinin, wheat germ agglutinin and Dolichos biflorus agglutinin, whose binding to carbohydrates is not affected by aging can be used in histochemical studies of carbohydrate residue composition of the cell surface in the normal and pathological epidermis in human subjects of any age.

Loss of PNA staining in mouse aortico-pulmonary septum is associated with mesenchymal cell apoptosis.

Massive apoptosis of mesenchymal cells in the septum of the aortico-pulmonary trunk was found in mouse fetuses at stage 14.5dpc. It was associated with the appearance of cavities in the mesenchymal tissue, presumably due to cell loss, a strong reduction in the extent of lectin PNA staining, and the induction of metallothioneins in specialized mesenchymal cells. Cell loss was spatially restricted to an inner area of the septum and was due to a distinct apoptotic pattern of cells, different from that in the heart wall. These events led to a rapid reduction of the aortico-pulmonary septum as occurs during the late stages of heart morphogenesis. It coincided with the migration of other cell types that invaded the cell-depleted septum, and contributed to the histiogenesis of the mature heart.

Altered regulation of Fc gamma RII on aged follicular dendritic cells correlates with immunoreceptor tyrosine-based inhibition motif signaling in B cells and reduced germinal center formation.

Aging is associated with reduced trapping of Ag in the form of in immune complexes (ICs) by follicular dendritic cells (FDCs). We postulated that this defect was due to altered regulation of IC trapping receptors. The level of FDC-M1, complement receptors 1 and 2, FcgammaRII, and FDC-M2 on FDCs was immunohistochemically quantitated in draining lymph nodes of actively immunized mice for 10 days after Ag challenge. Initially, FDC FcgammaRII levels were similar but by day 3 a drastic reduction in FDC-FcgammaRII expression was apparent in old mice. FDC-M2 labeling, reflecting IC trapping, was also reduced and correlated with a dramatic reduction in germinal center (GC) B cells as indicated by reduced GC size and number. Nevertheless, labeling of FDC reticula with FDC-M1 and anti-complement receptors 1 and 2 was preserved, indicating that FDCs were present. FDCs in active GCs normally express high levels of FcRs that are thought to bind Fc portions of Abs in ICs and minimize their binding to FcRs on B cells. Thus, cross-linking of B cell receptor and FcR via IC is minimized, thereby reducing signaling via the immunoreceptor tyrosine-based inhibition motif. Old FDCs taken at day 3, when they lack FcgammaRII, were incapable of preventing immunoreceptor tyrosine-based inhibition motif signaling in wild-type B cells but old FDCs stimulated B cells from FcgammaRIIB(-/-) mice to produce near normal levels of specific Ab. The present data support the concept that FcR are regulated abnormally on old FDCs. This abnormality correlates with a reduced IC retention and with a reduced capacity of FDCs to present ICs in a way that will activate GC B cells.

The suppressive effect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease.

Bacteroides, a predominant commensal bacteria in the gut, are thought to be responsible for the development of inflammatory bowel disease (IBD). In the present study, we examined whether or not bifidobacteria suppress B. vulgatus, a representative pathogenic Bacteroides species, in both the coculture system and the gnotobiotic murine model. As a result, Bifidobacterium infantis 1222 highly inhibited the growth of B. vulgatus in the coculture and also significantly suppressed the systemic antibody response raised by B. vulgatus colonizing the gut in gnotobiotic mice. Colonization of the mice by B. vulgatus increased the number of Peyer's patch (PP) cells bearing PNA (peanut agglutinin)+/anti-kappa+ phenotype, which represents plasma cell-like B cells. Moreover, treatment of those B. vulgatus-implanted mice with B. infantis 1222 abrogated such increase in the number of PNA+/anti-kappa+ cells. These results thus suggested that B. infantis 1222 protected the gut epithelial layer including the PP from being invaded by Bacteroides, thereby suppressing the systemic antibody response raised by Bacteroides.

Low-grade tubular-mucinous renal neoplasms: morphologic, immunohistochemical, and genetic features.

The current classification system of renal tumors is based on morphologic criteria, as supported by genetic findings. We present a group of previously unclassified tumors with similar morphologic and genetic features, suggesting a new entity within renal neoplasms. Seven renal tumors from five patients (ages 31-67 years) were analyzed. All cases were stained with periodic acid-Schiff, Hale's colloidal iron (HCI), and Alcian blue (AB) at pH 2.5/1.0 with and without hyaluronidase (HA) digestion. Immunohistochemical (IHC) stains were performed for CK8, CK18, CK19, vimentin, villin, Tamm-Horsfall protein (THP), renal cell carcinoma marker (RCC), epithelial membrane antigen (EMA), ulex europaeus agglutinin (UEA-1), soy bean agglutinin (SBA), peanut agglutinin (PNA), and MIB-1. Comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) studies were performed on all cases. All tumors showed circumscribed growth, a tubular growth pattern with focal solid areas, no significant nuclear atypia and absence of necrosis, desmoplasia, or inflammation. Abundant extracellular mucin was present. Immunohistochemistry stains support collecting duct origin (EMA+, PNA+, SBA+/-, CK 8/18/19+, vimentin+/-, UEA-1-, RCC-, villin-, THP-). The proliferative rate was low (<1%). CGH showed multiple consistent chromosomal losses (-1,-4, -6, -8, -9, -13, -14, -15, -22). Clinical outcome was favorable, with recurrences but no known distant metastases or death of disease. These findings are distinct from all previously classified renal neoplasms. Our data suggest the presence of a unique tumor entity within tumors of probable collecting duct origin: tubular-mucinous renal tumors of low malignant potential.

Placental vessel adaptation during gestation and to high altitude: changes in diameter and perivascular cell coverage.

The objective of this study was to determine how human placental vascular structures change during gestation and whether this would be altered by external factors such as reduced ambient oxygen. To achieve this, several experiments were carried out: Vessel profile diameter was measured and the presence of perivascular cells (pericytes or smooth muscle cells) noted. This was carried out in normal human first trimester and term placentae, and in term placentae obtained from high altitude and an ethnically matched lowland population. In addition, to characterize endothelial cells in human placenta a panel of endothelial markers anti-CD 105, CD31, CD34, Von Willebrand factor (vWF), Ulex europaeus agglutinin 1 (UEA I), Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Bandeieraea simplicifolia agglutinin 1 (BS 1) was used. The proportion of vessels associated with perivascular cells rises during gestation from 37 per cent in the first trimester to 63 per cent at term (P<0.0001) and vessels with perivascular cells have a larger median diameter at term. In placentae obtained at high altitude, the vessels are dilated and are less frequently associated with perivascular cells. The absence of perivascular cells may allow remodelling of capillaries and this is likely to be physiological important in the first trimester but also under physiological or pathological stress.

Quantitative determination of dietary lectin activities by enzyme-linked immunosorbent assay using specific glycoproteins immobilized on microtiter plates.

An immunoenzymatic method for the quantitative determination of dietary lectin activities employing immobilized glycoproteins was studied. Lectins from wheat germ (WGA), peanut (PNA), and jack bean (ConA) were added to microtiter plates coated with ovalbumin or asialofetuin and quantified by enzyme-linked immunosorbent assay (ELISA) with lectin-specific antibodies. ELISA responses for lectin activity were dose-dependent in the concentration range 30-1000 ng/mL for WGA and 80-1000 ng/mL for both PNA and ConA. Inhibition assays carried out with different saccharides confirmed that the binding of lectins to immobilized glycoproteins was specific. The proposed method is specific and sensitive, allowing the quantitative determination of lectin activities on raw samples by simple dilution of the extracts. Examples of application to wheat germ and roasted peanut extracts are reported.

Fate of the acrosome in ooplasm in pigs after IVF and ICSI.

BACKGROUND: ICSI bypasses the sperm-oolemma interactions that, in normal fertilization, depend on completion of the acrosome reaction. Morphological changes in the acrosomes of sperm in the ooplasm were therefore examined following IVF and ICSI using pig gametes. METHODS: In-vitro-matured porcine oocytes were used for ICSI or IVF. Oocytes were then stained with fluorescein isothiocyanate-conjugated peanut agglutinin lectin (FITC-PNA), which specifically labels the outer acrosomal membrane of boar sperm and the cortical granules (CG) in porcine oocytes. This was followed by observation under a confocal laser scanning microscope. RESULTS: In ICSI, PNA showed the presence of disintegrated acrosomes that classified into four categories. Heterogeneous chromatin decondensation was observed in the sperm with intact/disintegrated acrosome, whereas acrosomes were barely detected in oocytes which had formed a male pronucleus. Both in ICSI and IVF, PNA-positive tails were concomitantly observed with one type of disintegrated acrosome, which was considered to be acrosome-reacted. The disappearance of CG in activated oocytes after ICSI was similar to that after IVF. CONCLUSIONS: The PNA-binding properties of sperm head components introduced into the ooplasm during ICSI are different from those after IVF. The delay of sperm chromatin decondensation is associated with that of acrosomal disassembly. Acrosomes appear to disintegrate in the ooplasm whether or not the acrosome reaction has taken place. Oocytes undergoing ICSI appear normally activated in terms of meiotic resumption and CG exocytosis.

Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells.

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.

Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation.

This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) molecular markers linked to genes controlling semen freezability can be identified using amplified restriction fragment length polymorphism (AFLP) technology. Five ejaculates were collected from each of 129 boars. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and five straws (0.5 ml) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells, motility characteristics (computer-aided semen analysis; CASA), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (positive for fluorescein-labeled peanut agglutinin; PNA). Consistent between-boar variability was detected for postthaw sperm motility (P < 0.01), membrane integrity (P < 0.01), acrosome integrity (P < 0.01), and all CASA characteristics (P < 0.05). There was no significant difference between ejaculates (P > 0.05) or straws (P > 0.05) for any viability assessment. Multivariate pattern analysis of the viability data set highlighted three groups of boars producing spermatozoa with poor, average, and good postthaw recovery (42, 63, and 24 boars, respectively). DNA from Large White boars (n = 22) previously classified as good and poor freezers was screened for AFLP markers. Twenty-eight polymerase chain reaction primer combinations generated 2182 restriction fragment bands, of which 421 were polymorphic. Sixteen candidate genetic markers (P < 0.005) were identified by comparing the AFLP profile with semen freezability using logistic regression analysis. These findings support the hypothesis that there is a genetic basis for variation in postthaw semen quality between individuals, and that AFLP technology may be able to identify molecular markers linked to genes influencing this variation.

Collecting duct carcinoma mixed with common renal cell carcinoma: analysis of morphological characteristics using lectin histochemistry.

Recently, cases of collecting duct carcinoma have been reported which were thought to have arisen from the renal collecting ducts or distal tubuli. We present here a rare case of collecting duct carcinoma mixed with common (conventional) renal cell carcinoma. A 51-year-old man underwent right nephrectomy under the diagnosis of renal tumor. Histochemically, markers for the collecting ducts/distal tubuli, such as peanut agglutinin (PNA) and soybean agglutinin (SBA), were identified in the collecting duct carcinoma as well as on several luminar surfaces of the common renal cell carcinoma. Based on the results of histological, histochemical and chromosomal examinations, we speculated on the histogenesis of this collecting duct carcinoma.

Role of follicular dendritic cells in the apoptosis of germinal center B cells.

Follicular dendritic cells (FDCs) provide the most obvious source of antigens, which are essential for the differentiation of GC B cells. It has been reported that most proliferating B cells in germinal centers undergo apoptosis. Quantitative histology shows macrophages with apoptotic debris throughout the germinal center, the highest frequency of these cells being found in the dense FDC network. Based on these findings, we hypothesized that FDC may be involved in an apoptotic pathway of the germinal center B cells. To prove this hypothesis, we performed double immunohistochemical analysis using anti-FDC mAb and peanut agglutinin (PNA), with their respective TUNEL kits. Collated data showed that a great proportion of the apoptotic cells, most of which were positive for PNA, were in close contact with FDC, which indicated an interaction between FDC and B cells in the apoptotic pathway. Further studies using double immunohistochemical staining and FACS analyses demonstrated the expression of Fas-ligand (FasL) in a subset of the FDC. These results suggest that FDC may play a role in the apoptosis of germinal center B cells via Fas-FasL interaction.

Lectin binding patterns in nonsensory regions of rat cochlea during postnatal development.

The distribution of glycoconjugates was examined in the nonsensory regions of the rat cochlea during postnatal development using biotin-conjugated lectins. Temporal bones of rats at postnatal d 1 and at wk 2, 4 and 6 were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for paraffin wax embedding. The dewaxed sections were incubated with 7 biotinylated lectins, followed by avidin-biotin-peroxidase complex. A different staining pattern was observed in the stria vascularis, spiral ligament and spiral limbus in the age groups examined. The staining intensity varied between lectins and the reaction product exhibited limited disparity. The staining intensity for WGA increased with age in all the 3 nonsensory regions. The staining patterns for the other lectins differed in the various nonsensory regions examined indicating tissue specificity. The limited variations in the lectin binding patterns after 2nd wk of postnatal life also indicate that the changes in the carbohydrate moieties are established during the fetal period of cochlear development and limited changes take place during postnatal maturation of the nonsensory regions.

The use of immunostaining for bcl-2 and CD34 and the lectin peanut agglutinin in differentiating between basal cell carcinomas and trichoepitheliomas.

Fifteen unequivocal basal cell carcinomas (BCC) and ten unequivocal trichoepitheliomas (TE) were studied using the lectin peanut agglutinin (PNA), and the monoclonal antibodies Q bend 10 and bcl-2 oncoprotein directed against the antigens CD34 and bcl-2, respectively, to see whether these markers could be used to differentiate between the two tumors. Ten percent of TE demonstrated a continuous band-like peritumorous staining with PNA and 80% demonstrated a discontinuous band-like peritumorous staining with PNA, with the comparable figures for BCC being 40% and 20%, respectively. In addition, 40% of BCC showed focal areas of pemphigus-like staining in contrast with only 10% of TE. Using the antibody directed against bcl-2, TE demonstrated weak staining mainly confined to the basal layer of tumor cells in 20% of cases and staining of the cells throughout the tumor in 30% of cases. Similarly, BCC also showed staining of the basal layer of tumor cells in 7% of specimens and staining of cells throughout the tumor mass in 40% of specimens studied. Finally, with the antibody Q bend 10 directed against CD34, staining of the immediate peritumoral spindle-shaped cells was observed in 20% of TE compared with 7% of BCC. Despite reports in the literature, we found that none of these three markers can be reliably used to differentiate between TE and BCC.

Somatic hypermutation of an artificial test substrate within an Ig kappa transgene.

We have characterized a novel substrate for somatic hypermutation, confirming that non-Ig sequences can be targeted for mutation and demonstrating that this substrate allows for the rapid assay for mutations. An artificial sequence containing alternating EcoRV and PvuII sites (EPS) was inserted into the Vkappa167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is amplified using flanking transgene primers, and the PCR product is subsequently digested with either EcoRV or PvuII. A mutation is seen as the appearance of a larger fragment, indicating a base change in a restriction enzyme site. The original transgene, Vkappa167/EPS, showed evidence of a low level of mutation in both splenic hybridomas and Peyer's patch-derived or immunized splenic B220+ cells with high peanut agglutinin levels. Two derivative lines of Vkappa167/EPS were made, Vkappa167/POX and Vkappa167/PEPS. While none of the Vkappa167/POX transgenic lines demonstrated mutation, the Vkappa167/PEPS transgene was highly mutated in B220+ splenic B cells with high peanut agglutinin levels at a frequency similar to that of endogenous Ig genes. An analysis of splenic RNA from the unimmunized transgenic mice indicated that the levels of stable message in splenic B cells could not be correlated with the mutation seen in GC B cells. The mutable Vkappa167/PEPS transgenic line is a unique tool to study somatic hypermutation in vivo.