CD62L is required on effector cells for local interactions in the CNS to cause myelin damage in experimental allergic encephalomyelitis.

Adhesion molecules are believed to facilitate infiltration of leukocytes into the CNS of mice with experimental allergic encephalomyelitis (EAE). The role of the adhesion molecule CD62L (L-selectin) in the immunopathology of EAE is not known. To study this, we crossed CD62L-deficient mice with myelin basic protein-specific TCR (MBP-TCR) transgenic mice. CD62L-deficient MBP-TCR transgenic mice failed to develop antigen-induced EAE, and, despite the presence of leukocyte infiltration, damage to myelin in the CNS was not seen. EAE could, however, be induced in CD62L-deficient mice upon adoptive transfer of wild-type macrophages. Our results suggest that CD62L is not required for activation of autoimmune CD4 T cells but is important for the final destructive function of effector cells in the CNS and support a novel mechanism whereby CD62L expressed on effector cells is important in mediating myelin damage.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.

Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis.

The mechanism of CD40 ligand (CD40L)-mediated in vivo activation of CD4(+) T cells was examined by investigation of the development of experimental allergic encephalomyelitis (EAE) in CD40L-deficient mice that carried a transgenic T cell receptor specific for myelin basic protein. These mice failed to develop EAE after priming with antigen, and CD4(+) T cells remained quiescent and produced no interferon-gamma (IFN-gamma). T cells were primed to make IFN-gamma and induce EAE by providing these mice with B7.1(+) antigen-presenting cells (APCs). Thus, CD40L is required to induce costimulatory activity on APCs for in vivo activation of CD4(+) T cells to produce IFN-gamma and to evoke autoimmunity.

Inhibition of angiogenesis by the matrix metalloprotease inhibitor N-[2R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]-L-tryptophan methylamide.

The inhibitor N-[2R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]-L- tryptophan methylamide specifically blocks several matrix metalloproteases, enzymes which are thought to be involved in angiogenesis. An extract of Walker 256 carcinoma in Hydron pellets implanted in the corneas of Sprague-Dawley rats was used to stimulate angiogenesis from the vessels of the limbus. Angiogenesis was graded visually as the distance penetrated into the cornea and the number of vessels generated. The vessel area was also measured by image analysis using Image 1 software. Continuous i.v. administration of N-[2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]- L-tryptophan methylamide at 32 mg/kg/day (n = 17) via syringe pump reduced vessel number [25.06 +/- 5.9 (SEM) compared to 65.33 +/- 9.0] and vessel area (26.14 +/- 3.2 mm2 compared with 40.96 +/- 4.6 mm2), but not distance penetrated, compared to vehicle-treated control eyes after 6 days. These results confirm the suspected role for matrix metalloproteases in angiogenesis and suggest that inhibitors of these enzymes may be angiostatic agents.

Neu proto-oncogene amplification and expression in ovarian adenocarcinoma cell lines.

In this communication, the authors summarize their characterization of eight ovarian adenocarcinoma-derived cell lines for level of neu gene amplification, expression of neu transcripts and protein, and intraperitoneal tumorigenicity in nude mice. Two of the eight cell lines in our study (SKOV3 and YAOVBIX1) exhibited five- to ninefold neu DNA sequence amplification, accompanied by up to 200-fold overexpression of transcripts and protein (p185). Both of these cell lines expressed a major approximately 7.5 kb neu-complementary transcript not previously reported in other neu-positive tumor cell lines. One pair of cell lines (YAOVBIX1 and YAOVBIX3), isolated from a single ovarian carcinoma patient's ascites sample differed dramatically in regard to level of neu gene amplification and expression. Immunohistochemical staining of the primary ovarian tumor from which these two lines were derived demonstrated populations of both neu-positive and neu-negative malignant epithelial cells. Seven of the eight ovarian carcinoma lines produced intra-abdominal tumors after intraperitoneal injection into nude mice, irrespective of level of neu gene expression. This study demonstrates tumor cell heterogeneity with regard to neu gene amplification and expression in an ovarian adenocarcinoma, reveals the overexpression of novel neu-complementary transcripts in two independently isolated ovarian adenocarcinoma cell lines, and suggests that neu gene expression is not required for intraperitoneal tumorigenicity of ovarian carcinoma xenografts in a nude mouse model system.

[Significance, sources of error and limits of protein diagnosis in urine. I. Diagnostic methods]. / Aussagen, Fehlerquellen und Grenzen der Proteindiagnostik im Urin. I. Diagnostische Methoden.

Determination of proteins in the urine requires standardized collection and storage of urine. To quantify total protein and to separate single proteins electrophoretically unconcentrated urine should be used. Protein dye-binding methods, beta 2-microglobulin essay and polyacrylamide gel techniques can be recommended for routine urinalysis. However, the analytical limits and pitfalls of the methods must be considered. The application of the selectivity concept of proteinuria is restricted to patients with nephrotic syndrome.

Ultrastructural morphology and domain structure of a unique collagenous component of basement membranes.

A disulfide cross-linked collagenous fragment (7 S) has been isolated by pepsin solubilization from several tissues rich in basement membranes including bovine lung, human placenta, and the murine EHS tumor. Examination of this material by the rotary shadowing technique indicates that these fragments are similar to but not identical with the 7S collagen described recently [Risteli, J., Bächinger, H.P., Engel, J., Furthmayr, H., & Timpl, R. (1980) Eur. J. Biochem. 108, 239-250]. The central rodlike portion of the particles was found to be similar in length; however, the peripheral four arms of 7S particles from bovine and murine sources are 10 nm longer in comparison to those from human sources. In addition, about 5-7% of all the particles contain a fifth arm. Specific antibodies to bovine 7 S cross-react with murine 7 S but only to a rather limited extent with human 7 S. These antibodies react with antigenic sites located at the ends of the peripheral arms of the fragment as visualized directly with rotary shadowing techniques. The data are consistent with a structural difference in type IV collagens from bovine, human, and mouse which leads to pepsin cleavage at different sites in a particular noncollagenous region adjacent to 7 S. However, since bovine 7S antibodies cross-react with human and murine tissues by immunofluorescence despite the lack of complete serological cross-reactivity, it is suggested that type IV collagens from all three species have some degree of homology in this region.

Methods in laboratory investigation. Monoclonal antibodies to type IV collagen: probes for the study of structure and function of basement membranes.

Type IV collagen is one of the main constituents of basement membranes, yet it is unknown whether the structural framework at different sites is assembled from one unique type of molecule or whether different type IV collagen molecules exist. To study the composition, chemical identity, and organization of this protein in different organs we have prepared monoclonal antibodies to a type IV collagen preparation from human placenta. Swiss Webster mice were hyperimmunized, and splenic cells were fused with the three different myeloma cell lines SP2/0, NS1, and U1. Type IV collagen-specific hybrids were selected and cloned by limiting dilution and on hard agar. Monoclonal antibodies secreted by two clones were extensively characterized by ELISA-inhibition assay, immunoprecipitation, rotary shadowing, and immunofluorescence techniques. Unlike conventionally raised antibodies in rabbits, both monoclonal antibody reagents show species-specific binding exclusively to native type IV collagen from human placenta but not to a similar preparation from calf lung or to other types of collagen. After heat denaturation of the antigen binding was no longer observed. The M3F7 antibody-binding site is located within the triple helical domain of the type IV molecule, approximately 900 A removed from the amino terminal end as visualized by a metal shadow casting technique. The monoclonal antibody M3F7 precipitates material from pepsin-derived and radiolabeled type IV collagen, and analysis of the polypeptide chains in the immunoprecipitate by sodium dodecyl sulfate polyacrylamide gel electrophoresis suggests that two major fragments are contained in the precipitate, which yield polypeptides of about 100 and 50 kilodaltons. After rotary shadowing of antigen-antibody mixtures native collagen fragments of two different size classes that bind antibody are visualized. One fragment is approximately 1500 A in length, and the other measures about 2700 to 3000 A. The localization of the antigenic site on these fragments suggests that both are generated by pepsin cleavage at a site about 900 A removed from the amino terminal end. In immunofluorescence experiments the monoclonal antibodies stained all basement membranes in kidney, lung, placenta, or skin, suggesting that at least the type IV collagen molecule recognized by these monoclonal antibodies is shared by a variety of vascular and epithelial basement membranes.