Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue.

uPAR (CD87), the receptor for the urokinase-type plasminogen activator (uPA) facilitates tumor cell invasion and metastasis by focusing uPA proteolytic activity to the cell surface. As uPAR exists in various molecular forms, it is desirable to use well defined antibodies for analyses of uPAR antigen expression in human malignant tumors by immunological methods. Therefore, twelve monoclonal antibodies (MAbs) directed against uPAR were generated by using nonglycosylated, recombinant human uPAR (spanning amino acids 1 to 284), expressed in Escherichia coli, as the immunogen. The reaction pattern of these MAbs with the immunogen and a series of carboxyl-terminally truncated versions of uPAR demonstrated that at least six different epitopes of uPAR are recognized. All MAbs reacted under reducing conditions in immunoblot analyses with E. coli-expressed uPA and also with highly glycosylated, functionally intact, recombinant human uPAR expressed in Chinese hamster ovary (CHO) cells. Seven of the MAbs recognized CHO uPAR under nonreducing conditions as well. By flow cytofluorometric analyses, three of these MAbs were shown to bind to native human uPAR present on the cell surface of monocytoid U937 cells with MAb IIIF10 being the best. Saturation of uPAR with uPA on U937 cells completely blocked interaction of MAb IIIF10 with uPAR (mapped epitope, amino acids 52 to 60 of domain I of uPAR). In turn, preincubation of U937 cells with MAb IIIF10 efficiently reduced binding of uPA to uPAR, indicating that the epitope detected by MAb IIIF10 is located within or closely to the uPA-binding site of uPAR, and thus, this site may be a target to influence uPA/uPAR-mediated proteolysis in tumors. Binding of MAbs IID7 or IIIB11 (mapped epitope, amino acids 125 to 132 of domain II of uPAR) to uPAR is not affected when uPAR is occupied by uPA. As these MAbs reacted strongly with cellular uPAR antigen in formalin-fixed paraffin-embedded tumor sections, the domain-II-specific antibodies IID7 and IIIB11 may be useful for immunohistochemical studies of uPAR expression in tissue remodeling processes in tumor invasion. In conclusion, we have devised well defined and epitope-mapped MAbs to uPAR that are highly specific tools for detection and targeting of uPAR in tumor tissue.     

Uniparental disomy 7 in Silver--Russell syndrome and primordial growth retardation

Maternal uniparental disomy for the entire chromosome 7 hasso far been reported in three patients with intrauterine andpostnatal growth retardation. Two were detected because theywere homozygous for a cystic fibrosis mutation for which onlythe mother was heterozygous, and one because he was homozygousfor a rare COL1A2 mutation. We investigated 35 patients witheither the Silver-Russell syndrome or primordial growth retardationand their parents with PCR markers to search for uniparentaldisomy 7. Four of 35 patients were found to have maternal disomy,including three with isodisomy and one with heterodisomy. Thedata confirm the hypothetical localization of a maternally imprintedgene (or more than one such gene) on chromosome 7. It is suggestedto search for UPD 7 in families with an offspring with sporadicSilver-Russell syndrome or primordial growth retardation.     

Differentiation of C3b receptors on human lymphocytes, phagocytes, erythrocytes and renal glomerulus cells by monoclonal antibodies.

C3b receptor protein was purified form human erythrocytes by 2 M KBr solubilization and affinity chromatography on C3-coated sepharose. This material served as antigen for raising monoclonal antibodies. To investigate the distribution and antigenetic relationship between the receptors for C3b on human erythrocytes, lymphoid and phagocytic cells, as well as kidney cells three monoclonal antibodies were selected which inhibited the binding of EAC14 degrees 23b to complement receptor-bearing cells. This could be shown for human erythrocytes by inhibiting the immune adherence reaction, for tonsil lymphocytes, Raji cells, and guinea-pig spleen cells by inhibition of rosette formation of these cells with EAC14 degrees 23b, and for human renal glomeruli by blocking of the the adherence of EAC14 degrees 23b to kidney sections. In contrast, these monoclonal antibodies were not capable of inhibiting rosette formation of human granulocytes and monocytes with EAC14 degrees 23b. The antibodies only interfered with the rosette formation, of EAC14 degrees 23bi and EAC14 degrees 23d with Raji cells and tonsil lymphocytes-if at all-at high concentrations, whereas the rosette formation of Raji cells and tonsil lymphocytes with EAC14 degrees 23b was influenced by supernatants of the selected clones up to a dilution of 1:10(3) to 1:10(5).     

Human immunodeficiency virus 1 favors the persistence of infection by activating macrophages through TNF

Macrophages play a major role in HIV-1 persistence. In the present paper, we demonstrate that the absence of apoptosis in HIV-1-infected primary human monocyte-differentiated macrophages (MDM) correlates with an increase in anti-apoptotic (Bcl-2 and Bcl-x(L)) and a decrease in pro-apoptotic (Bax and Bad) proteins. This is associated with macrophage activation as shown by tumor necrosis factor (TNF) production and NF-kappaB activation upon infection. TNF production was shown to be involved in the upregulation of Bcl-2 and Bcl-x(L) because this increase was abolished by an anti-TNF anti-serum or an inhibitor of TNF synthesis. In parallel, inhibition of TNF production induced an increase in the number of apoptotic cells. Furthermore, using an inhibitor of NF-kappaB activation, we demonstrated that TNF-induced upregulation of Bcl-x(L) and Bcl-2 occurs, respectively, through a NF-kappaB-dependent and an NF-kappaB-independent pathway.     

Immunohistochemical study of the expression of MUC5AC and MUC6 in breast carcinomas and adjacent breast tissues

AIM: To study the protein expression patterns of MUC5AC and MUC6 in normal and diseased breast tissues and to compare their expression with that of a mucin (MUC1) normally expressed in mammary tissues. METHODS: Formalin fixed, paraffin wax embedded tissue from 69 cases of invasive breast carcinoma and surrounding breast tissue was studied immunohistochemically with monoclonal antibodies against MUC1 (SM3), MUCSAC (CLH2), and MUC6 (CLH6), using the avidin-biotin-peroxidase method. RESULTS: MUC5AC was detected in five of 68 cases of invasive carcinoma including one of three cases of pure colloid carcinoma. MUCSAC expression in the adjacent normal breast epithelium was present in one of 29 cases and in one of two cases of ductal carcinoma in situ. None of 15 cases of ductal hyperplasia without atypia was positive for MUCSAC. MUC6 was present in 15 of 65 cases of invasive carcinoma, in four of 29 cases of normal adjacent epithelium, two of 15 cases of ductal hyperplasia without atypia, and one of two cases of ductal carcinoma in situ. MUC1 immunoreactivity detected by the SM3 antibody was present in 50 of the 67 cases of invasive carcinoma, but expression was also detected in benign epithelium. All invasive carcinomas expressing MUCSAC were positive for MUC1 and four were positive for MUC6. No significant association was found between the expression of these mucins and tumour size, histological grade, node status, oestrogen receptor status, p53 positivity, or c-ErbB-2 overexpression. CONCLUSIONS: This study documents the expression of two different mucins (MUCSAC and MUC6) not described as being expressed by normal breast tissues in a minority of breast carcinomas, as well as in normal and hyperplastic epithelium. Although the role of mucins in malignant transformation and the progression of breast cancer is not well understood, in some cases, there is probably an upregulation of several genes that encode distinct mucin proteins.     

Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains.

A regulatory gene (dtxR) responsible for iron-dependent repression of the toxin (tox) and siderophore genes in Corynebacterium diphtheriae was cloned and characterized. A DNA fragment carrying dtxR repressed expression of a tox-lacZ gene fusion in Escherichia coli DH5 alpha in a high-iron environment but not under low-iron conditions. A protein with mobility corresponding to approximately 28 to 29 kDa was identified as the product of the dtxR gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A shuttle vector designated pCM2.6 was constructed which carries the origin of replication from C. diphtheriae plasmid pNG2 and confers resistance to chloramphenicol in E. coli and C. diphtheriae. DNA fragments carrying dtxR were cloned into pCM2.6, and the hybrid shuttle plasmids were transformed by electroporation into wild-type C. diphtheriae C7(beta) and the regulatory mutant C7(beta)hm723, which produces toxin and siderophore constitutively under high-iron conditions. Expression of the cloned dtxR determinant did not affect the phenotype of C. diphtheriae C7(beta). In C. diphtheriae C7(beta)hm723, expression of cloned dtxR restored full repression of siderophore production and partial repression of diphtheria toxin production during growth in a high-iron environment.     

Production of type-1 and type-2 cytokines by peripheral blood mononuclear cells of psoriatic patients.

We investigated the function of peripheral blood mononuclear cells (PBMC) in 16 patients with active psoriasis, in 15 patients with static psoriasis and in 27 healthy volunteers, by examining in vitro proliferation and antigen- and mitogen-stimulated production of interleukin-2 (IL-2) and IL-4. Plasma levels of the neuropeptide substance P were also determined. Defective alloantigen (ALLO)- and phytohaemagglutinin (PHA)-stimulated IL-2 production was detected in 42% and in 45% of psoriatic patients, respectively. The number of defective IL-2 responders was higher in static (60%) than in active (25%) psoriasis. The reduction of IL-2 responses in the former group was associated with an increase of IL-4 production. Thus PBMC of 66% of patients with static psoriasis but none of the patients with active psoriasis produced elevated amounts of PHA-stimulated IL-4. Variations of plasma substance P levels followed the same pattern of IL-4, being higher in static than in active psoriasis. These observations suggest a co-ordinated action of IL-4 and substance P as modulators of the clinical course of psoriasis. Our data show a possible correlation between the clinical evolution of psoriasis and the production of type-1 and type-2 cytokines, suggesting that the former may have a prominent role in the activation of psoriasis, while the latter may play a protective role.     

Coreceptor Switch of [MLV(SIVagm)] pseudotype vectors by V3-loop exchange

Retroviral vectors derived from murine leukemia virus (MLV) have been pseudotyped with a variant of the envelope glycoprotein (Env) of nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to result in [MLV(SIVagm-wt)] vector particles. The variant env gene encodes a full-length surface envelope glycoprotein (SU) and a C-terminally truncated transmembrane protein (TM). To change the coreceptor usage of this vector from CCR5 to CXCR4, which is predominant on human CD4-positive lymphocytes, the putative V3-loop of SIVagm SU was replaced by that of the T cell tropic HIV-1 variant BH10. The resulting [MLV(SIVagm-X4)] vectors were shown to specifically transduce CD4/CXCR4-positive cell lines, demonstrating the equivalent function in cell entry and choice of coreceptor usage of the V3-loops of SIVagm and HIV-1. These modified vectors were able to transduce primary human lymphocytes and were resistant to neutralization by sera from HIV-1-infected individuals. The [MLV(SIVagm-X4)] pseudotype vector generated is thus a promising candidate vector, e.g., for in vivo gene therapy of HIV-1 infection.     

Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.