Altered Antigen Expression Research Articles

Lyme borreliosis – an update

Lyme borreliosis is the most common tick-borne, infectious disease in the northern hemisphere. Disease manifestations in the United States and Europe vary as a result of geographic distribution of different species within the genospecies Borrelia burgdorferi sensu lato, which in turn are host-specific. Certain toxigenic B. burgdorferi strains cause early disseminated disease. The ability of Borrelial organisms to break down the extracellular matrix also promotes dissemination. B. burgdorferi are eliminated by complement-mediated lysis and by T and B cell activity of the specific immune response. Yet, B. burgdorferi can evade humoral immunity by means of type of protective mechanism by which it adheres to the proteoglycan decorin in the joints and skin. A further factor in the persistence of the pathogen is altered antigen expression. Re-infection usually occurs with a different strain, although repeated infection with the same strain is also possible after a certain period of latency. New developments in serologic testing include the use of recombinant native antigen as well as antigens produced in vivo such as VlsE (variable major protein-like sequence, expressed) or decorin-binding protein A. Diagnosis continues to be complicated by seropositivity of healthy individuals, the persistence of antibodies after therapy, and a lacking humoral immune response in patients with erythema migrans.

Flow cytometric expression of common antigens CD18/CD45 in blood from dogs with lymphoid malignancies: A semi-quantitative study

Flow cytometry is useful to study lymphoid malignancies since it allows both immunophenotyping of neoplastic cells and quantification of antigen expression. CD18 and CD45 are commonly exposed membrane antigens with different levels of expression on blood leukocyte and neoplastic cells. The aim of this retrospective study was to semi-quantitatively evaluate the expression of CD18 and CD45 in dogs with different lymphoid malignancies with blood involvement and to compare results with those from healthy dogs and dogs with reactive diseases. Blood samples from 13 dogs with precursor lymphoid malignancies, 20 with mature neoplasms (either chronic lymphocytic leukaemia or lymphoma), of different immunophenotypes, were compared with 24 healthy dogs and 12 dogs with different reactive diseases. The median fluorescence intensity (MFI) for CD18 and CD45 was recorded on lymphoid and granulocytic populations using dual colour flow cytometry, and the ratio between MFI for lymphoid and granulocytic populations (L/N ratio) was calculated to compare the results obtained in different sessions using an internal control (granulocyte fluorescence intensity). Significant decreases in the L/N ratio were detected in neoplastic samples for both CD18 (either precursors or mature versus controls) and CD45 (either precursors or mature versus control), while using MFI only slight differences were detectable in CD45 between precursors and controls. Neoplastic cells often exhibited lower expression of the L/N ratio for CD18, and mainly for CD45, most likely due to a less mature pattern than normal cells and/or to an aberrant quantitative expression of surface antigen. Moreover, more than 50% of neoplastic lymphoid cells exhibited L/N ratios that were not within the values observed in controls for at least one antigen. Altered L/N ratios, in particular decreases of CD45, were mainly observed in precursor neoplasms and in T-cell neoplasms. Detection of altered expression of common antigens, and in particular a L/N ratio for CD45 lower than a value of 103% may be useful as a confirmation of pseudo-clonality thus helping in differentiating reactive and neoplastic lymphocyte expansions.

Preoperative prediction of postoperative edema and effusion in pediatric cardiac surgery by altered antigen expression patterns on granulocytes and monocytes.

Postoperative edema and effusion (POEE) following cardiopulmonary bypass (CPB) surgery in children retards recovery and may aggravate postpericardiotomy (PPS), capillary leak syndrome (CLS), or multiorgan failure (MOF). Compared with complication-free children, POEE affected children have different preoperative serum levels of circulating cytokines and adhesion molecules. These levels may be used preoperatively to assess POEE, but their determination is time consuming, costly, and a substantial blood volume is required. Altered serum levels of cytokines and adhesion molecules also may be reflected in altered antigen expression on circulating blood leukocytes. The predictive potential of flow cytometric (FCM) leukocyte immunophenotyping was explored as a sensitive and fast method that required small blood samples. Blood samples taken 24 h preoperatively from 49 patients (3-18 years old) were stained with monoclonal antibodies for adhesion molecules (ICAM-1, LFA-1, Mac-1) or constitutive/activation markers (CD4, CD14, CD16, CD25, CD54, CD69, HLA-DR) and measured on a microbead calibrated FCM. Neutrophils, monocytes, and eosinophils from POEE patients express higher preoperative levels of LFA-1, monocytes, HLA-DR, and other activation markers (all P < 0.03). Over 89% of the patients were classified correctly by using two discriminant analysis methods (sensitivity, >76%; specificity, >86%; positive prediction, >80%; negative prediction, >83%). Granulocytes and monocytes of postoperative POEE patients exhibit significant preoperative immune activation, suggesting an increased risk for patients with atopic/allergic predisposition. Surgical trauma and CPB cause additional immune activation, leading to POEE by a summative response. Most patients at risk for POEE can be identified preoperatively by using data pattern analysis on FCM-derived parameters.

Expression of MUC1 and MUC2 and carbohydrate antigen Tn change during malignant transformation of biliary papillomatosis.

Biliary papillomatosis is characterized by papillary proliferations of biliary lining cells without invasion or metastasis. The neoplastic character and biological behaviour of this disease remain still speculative. These issues were examined in this study. Mucin core protein MUC1, MUC2, MUC3, MUC5AC and carbohydrate antigens (T, Tn and sialosyl Tn) were immunohistochemically examined, using 11 lesions of biliary papillomatosis from seven patients, and five lesions of biliary papillomatosis with foci of carcinoma from four patients. Five cases of papillary intrahepatic cholangiocarcinoma and 12 histologically normal livers were used as a control. Patients with biliary papillomatosis alone or with carcinoma were middle-aged or elderly (five men and six women). Microscopically, biliary papillomatosis showed a villous, papillo-tubular, papillary, or papillo-villous pattern with a thin fibrovascular core. Cytologically, they were classifiable into biliary epithelial or pyloric gland-like type. The former was frequent in the cases associated with carcinoma. Expression of MUC1, Tn antigen and sialosyl Tn antigen was frequent and marked in biliary papillomatosis alone and with carcinoma and also intrahepatic papillary carcinoma. In addition, marked expression of MUC1 and Tn antigen were rather frequent in biliary papillomatosis with carcinoma and intrahepatic biliary papillary carcinoma compared with biliary papillomatosis. MUC2 was rather frequent and marked in biliary papillomatosis alone compared to other two disease groups. Focal expression of MUC5AC and MUC2 was rather frequent and infrequent irrespective of disease group, respectively. Focal expression of T antigen was frequent in papillary ICC. Biliary papillomatosis could undergo overt malignant transformation along with altered phenotypic expression of MUC proteins and mucin carbohydrate antigens.

Three-color flow cytometry in the diagnosis of malignant lymphoma based on the comparative cell morphology of lymphoma cells and reactive lymphocytes.

This study examines the identification of unusual cell populations highly associated with lymphoma cells (UCP-L) in diagnostic biopsy specimens using three-color flow cytometry (3-FCM). Patterns of surface antigen expression were used to compare the morphology of distinct lymphoid cell populations present in biopsy specimens and determine the presence or absence of UCP-L. UCP-L were identified by their larger size as compared to admixed reactive lymphocytes, and the method is based on the concept that neoplastic lymphoma cells are larger than reactive lymphocytes. The comparison of relative cell sizes was determined by overlaying forward scatter histograms by multicolor gating using PAINT-A-GATE software. In order for separate gates to be set on UCP-L and reactive cell populations, UCP-L had to fulfill one or more immunophenotypic criteria. These included: (1) belonging to a subset of B cell antigen-positive cells showing restricted expression of kappa or lambda light chains; (2) belonging to a subset of CD4-positive cells having dim or absent expression of CD45RA; (3) showing alterations in antigen expression (loss, dimmer or brighter); or (4) expressing an immunophenotype that is present on only rare cell populations or is absent from reactive lymph nodes. The immunophenotypic profiles of the respective cell populations were demonstrated by cubic representations to assess more easily the co-expression of three antigens. The common morphology of UCP-L as defined by forward and side scatter grams was consistent with a 'lymphoid appearance' except in several cases of HTLV-I-positive T cell lymphoma and gammadelta T cell lymphoma. The immunophenotypic profiles of UCP-L were confirmed to correspond to the presumptive lymphoma cell population by use of a live gating procedure on the large cells, which eliminated interference by reactive cells or necrotic tissue fragments. Using this method, we identified UCP-L in 208 of 293 (71%) consecutive cases of non-Hodgkin's lymphomas, while no UCP-L were seen in 72 cases of non-specific hyperplasia of lymph nodes. Twenty-seven cases could not properly be examined about the existence of UCP-L because of massive necrosis, extensive fibrosis or strong non-specific staining reactions of unknown cause. When those cases were eliminated from the analysis, 80% of non-Hodgkin's lymphoma were found to contain UCP-L. In B cell lymphoma, the incidence of UCP-L in nodal lymphomas (80%) was much higher than in extranodal lymphomas (47%). Only one of 21 cases of Hodgkin's lymphoma was found to have UCP-L. The 3-FCM procedure was validated by the combined use of immunohistochemistry, morphologic examination, cytogenetic and antigen receptor gene rearrangement analysis by Southern blot hybridization. Our findings indicate that detection of UCP-L by 3-FCM is a reliable method to distinguish non-Hodgkin lymphomas from reactive hyperplasias in the majority of cases, even when the reactive cell population predominates over the malignant cell population.

Xenoantigens and Xenoantibodies: Their Modification

The hyperacute rejection of pig organs by primates, including humans, results from antibody-mediated complement activation. Protection from complement injury still leads to delayed rejection, which results from other mechanisms that may also be dependent on the presence of antibody. Anti-pig antibodies are directed largely, if not entirely, against alpha-galactose (alpha Gal) epitopes on pig vascular endothelium. Prevention of antibody-antigen binding is being attempted by methods that (1) alter antigen expression on the donor organ or (2) deplete the potential recipient of anti-alpha Gal antibody. To date, most progress has been made in depleting antibody by extracorporeal immunoadsorption using immunoaffinity columns of synthetic alpha Gal oligosaccharides. A pig organ transplanted into a recipient baboon depleted of antibody functions for several days--in contrast to minutes to hours in unmodified baboons. The return of antibody, however, results in rejection of the graft. Pharmacologic immunosuppressive agents are relatively ineffective for prolonged suppression of anti-alpha Gal production. Total-body irradiation and splenectomy are proving more successful. Studies are continuing with the aim of achieving a state of B cell tolerance toward alpha Gal epitopes.

The basement membrane zone in lichen sclerosus: an immunohistochemical study

The alteration in expression of basement membrane zone (BMZ) components in lichen sclerosus was investigated by immunohistochemical staining of skin biopsies from seven patients with histologically confirmed disease compared with controls. Monoclonal antibodies and polyclonal sera directed against proteins of the hemidesmosomes, anchoring fibrils, lamina lucida, lamina densa and BMZ collagens were used. Characteristic histological appearances at the dermo-epidermal junction were reflected in widespread alterations in antigen expression in the epidermal basement membrane and the papillary dermis. Expression of the proteins which constitute the structural scaffold (collagen IV and VII) were increased in lichen sclerosus. Expression of hemidesmosomal proteins which mediate adhesion and cell to matrix interaction (alpha 6 beta 4 and bullous pemphigoid antigen) and expression of anchoring filament components were markedly reduced, suggesting that the epidermal cells are exposed to selective damage.

Plasmodium falciparum:Altered Expression of Erythrocyte Membrane-Associated Antigens during Antigenic Variation

The O and R antigenic variants of thePlasmodium falciparumPalo Alto strain present differences in the morphology of the infected red blood cell membrane, in their adhesion properties, surface immunofluorescence, and agglutination specificities and importantly, induce a variant-specific protection after a primary infection inSaimiri sciureusmonkeys. To identify potential targets of variant-specific immunity, we have compared the antigenic makeup of both variants by immunoblot. O-specific monkey sera generated similar profiles on both parasite types, while R-specific sera showed a consistent difference on a high-molecular-mass undefined antigen. Distinct antibody specificities were eluted from the surface of O- or R-infected erythrocytes, generating variant-specific agglutination, surface immunofluorescence, and immuno-blot profiles. An antiserum raised to Pf60.1, predicted to cross-react with the cytoplasmic domain of PfEMP1, reacted with specific, SDS-soluble antigens in both variants. Antigens associated with the membrane of the infected red blood cells were further investigated using several specific antisera. The 85-kDaHRP1gene product was more abundant in O than in R parasites, while the reverse was observed for the PfEMP3 protein. These data indicate that O and R parasites differ in the expression of several antigens associated with the membrane of the infected red blood cell.

Taenia multiceps (Cestoda): Ia antigen expression and prostaglandin secretion by parasite-modified, murine peritoneal macrophages.

Taenia multiceps secretions modify accessory cell activity in macrophages. The present experiments were designed to elucidate the cellular mechanisms involved. While normal, murine peritoneal macrophages amplified mitogen-activated T-cell proliferation, macrophages modified by exposure to parasite secretions inhibited this proliferation. The modified behaviour was shown by glutaraldehyde-fixed as well as living macrophages, and modification was inducible by FPLC fraction 24 of coenurus fluid and was associated with an expanded population of 1a- macrophages. Secretory products of parasite-activated macrophages also inhibited T-cell proliferation, and secretion was prevented by indomethacin. The measurement of modified accessory activity was not influenced by the concentration of tritiated thymidine in lymphocyte proliferation assays. Consequently there is no evidence that the reported events are affected by macrophage-derived, cold thymidine secretion. It is concluded that T. multiceps si able to manipulate macrophage accessory function by mechanisms which involve altered histocompatibility antigen expression and the secretion of prostaglandin.

Increased Efficacy of CSA Following Rearterialization in the Rat OLTX Model

Many investigators presently use a rat orthotopic liver transplant (OLTX) model without rearterialization of the graft. Rearterialization has been demonstrated to have a variable effect on the rejection response in various strain combinations. However, there are little data on the effects in a model with immunosuppression. The influence of rearterialization on the efficacy of cyclosporine (CSA) in such a model was examined, with the hypothesis that rearterialization may alter the rejection response and efficacy of cyclosporine. OLTX was performed between adult male D Agouti rats and Lewis rats. Rearterialization was performed between the recipient and donor celiac axis, and CSA was delivered at 1 mg/kg/day by continuous infusion for 14 days postoperatively. Treatment groups consisted of no rearterialization/no CSA, rearterialization/no CSA, no rearterialization/CSA, and rearterialization/CSA. Survival time and histology of liver grafts were measured. Rearterialization itself did not prolong survival in this strain combination (median survival no rearterialization/no CSA is 11 days versus median survival rearterialization/no CSA is 10 days). The addition of CSA at this dose without rearterialization also did not prolong survival (median survival no rearterialization/CSA is 15.5 days). The combination of CSA with rearterialization did prolong survival significantly (median survival rearterialization/CSA is 22 days; P < 0.05 versus the other three groups). The mechanism of this increased efficacy is unknown, but may involve altered MHC antigen expression, altered metabolism of CSA, decreased toxicity of CSA, or decreased nonspecific inflammation in the rearterialized grafts. These data indicate that the type of model used has an impact on survival results, and a rearterialized model should be further studied to determine whether it is more representative of the clinical situation.

Immunohistochemical analysis of adhesion molecules in the micro-environment of portal tracts in relation to aberrant expression of PDC-E2 and HLA-DR on the bile ducts in primary biliary cirrhosis.

We have examined immunohistochemically the expression of adhesion molecules in the micro-environment of portal tracts and their relationship to the expression of the pyruvate dehydrogenase E2 complex (PDC-E2) and HLA-DR in liver biopsy specimens. Ten cases of primary biliary cirrhosis (PBC) and 19 controls were examined, including four cases of extrahepatic biliary obstruction, six of chronic viral hepatitis, and nine normal livers. In PBC, the damaged small bile ducts demonstrated an increased expression of PDC-E2 and an aberrant expression of HLA-DR; about half of these damaged bile ducts also expressed intercellular adhesion molecules (ICAM)-1 and a few expressed vascular adhesion molecule (VCAM)-1. In addition, lymphocyte function-associated antigen (LFA)-1 and very late antigen (VLA)-4 were expressed on infiltrating lymphocytes around these bile ducts. In contrast, in control livers, these alterations in antigen expression on the bile ducts were either not observed or were only focal and weak, when present. These findings suggest that ICAM-1/LFA-1 and also VCAM-1/VLA-4 linkages between the damaged bile ducts and lymphocytes may facilitate antigen-specific reactions such as the presentation of antigens, possibly PDC-E2, to the periductal lymphocytes in PBC. ICAM-1, VCAM-1, and E-selectin were strongly expressed on the endothelial cells of some vessels in the portal tracts in PBC, suggesting the facilitation of the recruitment of lymphocytes around the bile ducts of PBC. VCAM-1, a member of the immunoglobulin superfamily, has not hitherto been reported on bile ducts.

Progressive differentiation of human sebocytes in vitro is characterized by increasing cell size and altering antigen expression and is regulated by culture duration and retinoids

Increasing cell size, lipid accumulation, and altered antigen expression are features of sebaceous differentiation in vivo. Enhanced lipid synthesis with progressive differentiation is also present in cultured human sebocytes. This study was conducted to investigate the evolution of cell size and antigen expression of human sebocytes with progressive differentiation in vitro. Subconfluent human sebocyte cultures were examined for sebocyte differentiation evaluated on cytocentrifuge preparations by light microscopy and classified in stages according to morphological criteria described for sebocytes in vivo. Rates of 5.1 +/- 2.2% undifferentiated sebocytes, 29.2 +/- 4.9% early differentiated, 20.7 +/- 4.1% advanced differentiated, 37.6 +/- 6.4% fully differentiated, and 5.9 +/- 1.9% mature sebocytes were calculated in secondary cultures. The size of cultured sebocytes measured by computer-assisted planimetry significantly increased with progressive differentiation up to 4-5.5 times. The low rates of mature sebocytes and the only moderate increase of their size with progressive differentiation indicate an incomplete terminal differentiation in vitro. Sebocytes were subsequently stained with a series of monoclonal antibodies (mAb) to determine antigen expression using the alkaline phosphatase anti-alkaline phosphatase technique. The number of sebocytes labeled with the anti-keratin mAb CK8.12 and KL1, and the mAb 34D11 (82 kD protein) increased with progressive differentiation; significant differences were found after comparing early and advanced differentiated sebocytes. Sebocytes were positively stained with the anti-keratin mAb 6B10 (K 4), RPN1162 (K 7), CK13 (K 13), RPN1165 (K 19), CK8.60, and the mAb 115F5 (MAM-6c), OM-1 (sebaceous gland antigen), and 24F10 (basic polypeptides) only at late-stage differentiation. The expression of keratins 4, 7, 13, and 19 was confirmed by gel electrophoresis and immunoblotting. The data obtained were used to study the effects of the duration of cultivation and of the retinoids isotretinoin and tretinoin on sebocyte differentiation in vitro. Subcultivation of sebocytes upregulated, and treatment with isotretinoin but not with tretinoin downregulated labeling with mAb which recognize indicating progressive and late-stage differentiation.

High expresion rate of Tn antigen in metastatic lesions of uterine cervical cancers

The significance of altered expression of MN blood group antigens was examined by studies on the expressions of Thomsen-Friedenreich antigen (T antigen) and Tn antigen in primary and metastatic lesions of 29 human uterine cervical cancers. These antigens were measured by the avidin-biotin-peroxidase (ABC) method with peanut agglutinin (PNA) lectin for T antigen and Vicia villosa agglutinin (VVA) lectin for Tn antigen. Proportion of cancer cells expressing Tn antigen was higher in the metastatic lesions than in the primary tumors in 10 of the 29 cases, less in the metastasis than in the primary tumor in one case, and similar in the primary and metastatic lesions in the other 18 cases. Reaction for Tn antigen was positive in 24 (82.8%) of the 29 metastases, and in 17 (58.5%) of the 29 primary lesions. Thus, the rate of Tn antigen expression was significantly higher in the metastases than in the primary lesions ( P < 0.05). On the other hand, there was no significant difference between the immunoreactivities of T antigen in metastases and primary tumors. These findings support our previous suggestion that expression of Tn antigen is closely related to the metastasis to regional lymph nodes and may reflect an important role of this carbohydrate in the process of metastasis of cervical cancer.

Expression of carbohydrate antigens in human pulmonary adenocarcinoma.

The altered and anomalous expression of carbohydrate antigens (CA) occur in patients with lung carcinomas. To investigate the relationship between prognosis and immunohistochemical findings of CA in serum and pulmonary adenocarcinomas (PA), PA specimens surgically resected from 102 patients were studied with the use of A, B, H, Lewis(Le)a, Le(b), Le(x), Le(y), sialyl Le(a) (FH-7), sialyl Le(x-i) (SLX) (FH-6), sialosyl Tn (TKH-2), and CA 19-9. The Lewis blood group types were determined by the dot blotting method on patients' serum using monoclonal anti-Le(a), Le(b), Le(x), and Le(y). Formalin-fixed paraffin-embedded PA sections were stained using the avidin-biotin-peroxidase complex methods. There was no correlation between the patients' postoperative survival and tissue expression of CA, although survival correlated with the clinical stages, histologic differentiation of PA, and serum SLX in Stage 4. The higher the positive rate of both Le(b) and Le(y), the better their differentiation in PA specimens. Regardless of blood group type, PA showed high positive reactions for FH-6 (88%) and Le(y) (87%). The serum concentration of CA 19-9 correlated with immunohistochemical reactions of tumors (Spearman correlation coefficient = 0.33, P = 0.02). The expression of Le(a) in Lewis (a-b+) phenotype patients and Type 2 CA, such as Le(y) and FH-6, is characteristic in PA. The findings suggest that CA tissue expression may not reflect postoperative survival length of patients with PA, but biological aberrant fucosylation or sialylation as well as morphologic alteration may occur in neoplastic cells.

12-O-Tetradecanoylphorbol-13-Acetate Not Only Modulates Proliferation Rates, But Also Alters Antigen Expression and LAK-Cell Susceptibility of Normal Human Melanocytes In Vitro

For serial cultivation of normal human melanocytes media supplemented with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) are largely employed. By using a culture medium that permits cultivation of melanocytes without TPA, the effects of TPA on melanocyte proliferation, phenotype, and susceptibility to lymphokine-activated killer cells were studied. Addition of 50 ng/ml TPA to the medium induced rapid dendrite formation and increased the cell proliferation rate by 16-63% in mitogen-rich media (four of seven cultures, p < 0.01), and by 237% in mitogen-reduced media (p < 0.001). Furthermore, several phenotypic changes indicating early stages of melanocyte transformation were induced by 50 ng/ml TPA. These included increased expression of melanoma progression-associated antigens such as A.1.43 and A.10.33, upregulation of nerve-growth factor receptor as well as of the melanocyte-activation marker HMB-45 and of histocompatibility class I antigens. In contrast, the expression of the differentiation marker K.1.2 and of intercellular adhesion molecule-1 was decreased in TPA-treated cultures. Most of these changes persisted even after removal of TPA from the culture medium (> or = 2 weeks). Staurosporine, a protein kinase C inhibitor, modulated melanocyte-antigen expression similar to TPA, suggesting that protein kinase C downmodulation rather than activation by TPA is involved. In addition to the antigenic alterations, the susceptibility of TPA-treated melanocytes to lymphokine-activated killer cell cytotoxicity decreased by 40% (p < 0.01), possibly due to their altered surface antigen expression. The presented data reveal that the tumor promoter TPA hitherto used as a supplement of melanocyte culture media induces profound phenotypic and functional changes of the cultured cells, indicating incipient transformation of normal human melanocytes in vitro.

Myelo-monocytoid immunophenotypes of B-cell chronic lymphocytic leukemia cells.

Leukemia cells from ten patients with B-cell chronic lymphocytic leukemia (B-CLL) and one with B-CLL of mixed cell type (B-CLL/PL) were analyzed to delineate the cellular characteristics, especially the myelomonocytic nature. Most of the surface phenotypes were consistent with the previous reports. However, the frequency of positivity for CD22 and CD25 was much higher in the examined cases of B-CLL than in Western reports. B-CLL cells frequently possessed antigens which are mainly expressed in myelomonocytic cells: 8/10 for CD11b and 4/10 for CD11c, although the presence of CD14 was not apparent (0/10). In addition, colony stimulating factor-1 receptors (CSF-1R) were expressed in 6 of the 10 B-CLL cases. The reactivity with antigens such as CD14, CD11c, and CSF-R was markedly enhanced by in vitro incubation. Alterations in antigen expression and in in vitro survival by growth factors for myelomonocytic lineage were observed in certain cases. These data imply that not only are B-CLL cells phenotypically closely related to myelomonocytic lineage but that they may also be influenced by growth factors for that lineage.

Altered antigen expression of microglia in the aged rodent CNS.

Microglia, the resident macrophages of the central nervous system, are characterised by a highly specialized morphology and unusual antigenic phenotype. Microglia appear to be downregulated by their microenvironment when compared to other tissue macrophages. We have studied the microglia in brains of healthy, aged rats with a panel of monoclonal antibodies. We have found that microglia in the brains of these aged rats express antigens that are downregulated or absent from microglia of juvenile rats. The stimuli which give rise to this upregulated phenotype are not known. Age related changes in the phenotype of microglia should be taken into account when considering the possible role of microglia in neuropathological conditions.

Synergism of interleukin 6 and 1 alpha,25-dihydroxyvitamin D3 in induction of myeloid differentiation of human leukemic cell lines.

Interleukin 6 (IL-6) is a multifunctional cytokine with an important role in immunity. We analyzed the effect of recombinant human IL-6 in combination with 1 alpha,25-dihydroxyvitamin D3 (Vit. D3) on differentiation of the human myeloid leukemic cell lines U937 and HL-60 with respect to alterations in antigen expression and functional activity. Of a panel of antigens analyzed, only CD11b (the alpha chain of CR3), and CD14 (a cell surface protein recognizing the lipopolysaccharide-binding protein-lipopolysaccharide complex) had significantly increased expression. Expression of ICAM-1 (CD54), a ligand for LFA-1, was also found to be enhanced with a concomitant increase of ICAM-1 mRNA levels. Enhanced nonspecific esterase levels and induction of respiratory burst activity confirmed that cell differentiation was induced. Furthermore, IL-6 and Vit. D3 had a profound effect on functional activities, as shown by enhancement of rosetting between sheep erythrocytes, sensitized with C3bi (EAC), and either U937 or HL-60 cells. Also, phorbol myristate acetate-induced homotypic adhesion of U937, which is ICAM-1 dependent, was markedly induced by these agents. These results indicate an important role of IL-6 and Vit. D3 in myeloid cell function and development.

Immune-mediated cell separation methods.

230 Introduction The diversity among cells of the body can be revealed in vitro by physical methods such as lightand electron-microscopy, size and density separations, active and physical adhesion to a solid phase and electrophoretic methods. Recently, much progress has been made in our understanding of the functions of cells by using antigedantibody interactions to perform negative and positive selections for isolation of discrete populations and sub-populations. Such immune-mediated techniques heavily exploit the specificity of monoclonal antibody technology and benefit from the use of biological probes. Usually, probes are antibodies, often monoclonal, directed specifically to cellular antigens such as cell-membrane proteins. Three main approaches can be identified: (a) antibodies to cell-surface phenotypic markers are used to identify cell populations in situ such as in immunohistology, flow cytometry and rosetting, (b) antibody-mediated cytotoxicity which is usually a negative selection of a viable cell population and (c) antibody linked to a solid phase which can in turn be selected by techniques such as centrifugation (Staphylococcus aweus), column chromatography (Sepharose) or magnetism (Dynabeads, Magnabeads). These techniques are subject to usual technical problems such as contamination, variation in viability of the cells isolated and yield, so careful monitoring of the isolate and control of the procedures is essential. When using samples from ‘normal’ individuals regard must be paid to age and state of health since cell profiles vary. In patients, even more care is needed since disease is manifested by changes in tissues. In instances such as leukaemias, cell populations shift dramatically and may be severely reduced (e.g. acute myeloid leukaemia) or present in greatly increased numbers (e.g. chronic lymphocytic leukaemia). The application of immune-mediated cellseparation techniques in medical diagnostics and therapy varies from diagnosis of tumours by identification of altered antigen expression using im-

Alterations in antigen expression in superficial bladder cancer.

Bladder cancer can be viewed as a prototype for carcinogen-induced neoplasia. This has been demonstrated experimentally in a variety of systems and in man through epidemiological studies of occupational exposure to putative carcinogens. The natural history of this neoplasm demonstrates recurrence in time and space, i.e., multifocal disease. This clinical scenario is precisely what would be expected if a target tissue, e.g., urothelium, was continuously exposed to a weak carcinogen. The detection of gross disease is clinically easy. However, the ability to intervene at early stages and monitor the success of this treatment requires the definition of early markers for bladder cancer. Integrins are a family of cell surface proteins, many of which function as receptors for extracellular matrix components. Normal epithelial cells express the integrin alpha 6 beta 4 in association with an anchoring structure known as the hemidesmosome. Urothelium expresses alpha 6 beta 4 on the basal layer of cells similar to the distribution seen on other epithelial surfaces. Even early stages of bladder cancer demonstrate an alteration in the expression of this integrin. Low-stage bladder tumors express alpha 6 beta 4 diffusely throughout the tumor as well as at the invading margin. Altered expression of alpha 6 beta 4 may be an early marker for bladder cancer which may contribute to an invasive phenotype. A second potential marker is detected by DD23, an IgG1 murine monoclonal antibody triggered by the immunization of a BALB/c mouse with a fresh human bladder tumor specimen. The antigen detected by DD23 is not present on normal urothelial specimens.(ABSTRACT TRUNCATED AT 250 WORDS)