Expression of multiple functional chemokine receptors and monocyte chemoattractant protein-1 in human neurons.

Functional chemokine receptors and chemokines are expressed by glial cells within the CNS, though relatively little is known about the patterns of neuronal chemokine receptor expression and function. We developed monoclonal antibodies to the CCR1, CCR2, CCR3, CCR6, CXCR2, CXCR3 and CXCR4 chemokine receptors to study their expression in human fetal neurons cultured from brain tissue as well as the clonally derived NT2.N human neuronal cell line (NTera 2/cl.D1). Specific monoclonal antibody labeling demonstrated expression of CCR2, CXCR2, CXCR3 and CXCR4 on neurons from both sources. Co-labeling studies revealed strong expression of CXCR3 and CXCR4 on both dendritic and axonal processes, with a weaker expression of CXCR2 and CCR2. Reverse transcriptase-polymerase chain reaction analysis of pure NT2.N neurons confirmed RNA expression for CCR2, CXCR2, CXCR3 and CXCR4. No changes in the neuronal labeling pattern of chemokine receptor expression were noted when NT2.N neurons were grown on a supporting layer of astrocytes, again consistent with similar patterns seen in primary human fetal brain cultures. Analysis of single-cell calcium transients revealed a robust response to stromal derived factor-1alpha (CXCR4) and melanocyte growth-stimulating activity (CXCR2), and variable response to monocyte chemoattractant protein-1 (CCR2) or interferon-gamma inducible protein-10 (CXCR3). Finally, we detected the release of monocyte chemoattractant protein-1 from pure cultures of NT2.N neurons, but not undifferentiated NT2 cells. These data indicate that individual neurons may not only co-express multiple functional chemokine receptors, but also that neurons themselves produce chemokines which may influence cellular function within the central nervous system.

N-formylkansosaminyl-(1----3)-2-O-methyl-D-rhamnopyranose: the type-specific determinant of serovariant 14 of the Mycobacterium avium complex.

Significant occurrences of pulmonary and disseminated infections due to "atypical" mycobacteria have again focused attention on the Mycobacterium avium serocomplex. An examination of the major surface glycopeptidolipid antigen of one of the more prominent and one of the first described serovariants, the Boone strain (serovariant 14), has shown that it is characterized by a unique terminal disaccharide unit, N-formylkansosaminyl-(1----3)-2-O-methyl-D-rhamnopyranose. The structure of the entire oligoglycosyl hapten is N-formylkansosaminyl-(1----3)-2-O-methyl-alpha-D-rhamnopyranosyl-( 1----3)-2-O- methyl-alpha-L-fucopyranosyl-(1----3)-alpha-L-rhamnopyranosyl-(1----2)-6 -deoxy- L-talose. We had previously described a 4,6-dideoxy-2-O-methyl-3-C-methyl-4-(2-methoxy propanamido)-L-mannopyranose (an N-acylkansosamine) as the characteristic sugar of the trehalose-containing lipo-oligosaccharide antigens of strains of Mycobacterium kansasii. Thus, a derivative of this sugar is now found to occur in other mycobacteria as part of other antigens. The other distinguishing feature of the structure is the presence of D-rhamnopyranose, which occurs only sparingly in Nature.

Intra-abdominal desmoplastic small round cell tumor: immunohistochemical evidence for up-regulation of autocrine and paracrine growth factors.

Desmoplastic small round cell tumors (DSRCT) are highly aggressive tumors typically involving the serosal surfaces of the peritoneum. Patients often present with abdominal pain, an abdominal mass, ascites or signs of intestinal obstruction. Cytogenetic and molecular studies have identified a characteristic t(11;22)(p13;q12) translocation within the tumor cells. The fused gene product apparently aligns the NH2-terminal domain (NTD) of the EWS gene to the zinc finger DNA-binding domain of the WT1 gene. This product could lead to loss of the tumor suppressor effect of the WT1 gene as well as to an increase in EWS driven expression of growth factors normally repressed by WT1. We investigated this latter possibility by performing immunohistochemical studies on formalin fixed tissue from 10 cases of DSRCT and five Wilms' tumors using-antibodies to insulin-like growth factor (IGF)-II, the latency associated peptide of transforming growth factor (TGF)-beta 1, platelet-derived growth factor (PDGF)-AB chain and PDGF-alpha receptor, respectively. In general, tumor cells were strongly positive for these growth factors in DSRCT, while stromal cells were negative for IGF-II and positive for the other growth factors in parallel with the tumor cells. Wilms' tumor cells were essentially negative for PDGF-AB chains, but positive for IGF-II, and the latency associated peptide of TGF-beta 1 and variably positive for PDGF-alpha receptor. These findings support the proposed molecular mechanism of tumorigenesis for DSRCT and may help explain this tumor's poor prognosis.

Intra-abdominal desmoplastic small round cell tumor: immunohistochemical evidence for up-regulation of autocrine and paracrine growth factors.

Desmoplastic small round cell tumors (DSRCT) are highly aggressive tumors typically involving the serosal surfaces of the peritoneum. Patients often present with abdominal pain, an abdominal mass, ascites or signs of intestinal obstruction. Cytogenetic and molecular studies have identified a characteristic t(11;22)(p13;q12) translocation within the tumor cells. The fused gene product apparently aligns the NH2-terminal domain (NTD) of the EWS gene to the zinc finger DNA-binding domain of the WT1 gene. This product could lead to loss of the tumor suppressor effect of the WT1 gene as well as to an increase in EWS driven expression of growth factors normally repressed by WT1. We investigated this latter possibility by performing immunohistochemical studies on formalin fixed tissue from 10 cases of DSRCT and five Wilms' tumors using antibodies to insulin-like growth factor (IGF)-II, the latency associated peptide of transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-AB chain and PDGF-alpha receptor, respectively. In general, tumor cells were strongly positive for these growth factors in DSRCT, while stromal cells were negative for IGF-II and positive for the other growth factors in parallel with the tumor cells. Wilms' tumor cells were essentially negative for PDGF-AB chains, but positive for IGF-II, and the latency associated peptide of TGF-beta1 and variably positive for PDGF-alpha receptor. These findings support the proposed molecular mechanism of tumorigenesis for DSRCT and may help explain this tumor's poor prognosis.

Interleukin-6-associated laboratory parameters and immunohistochemistry in symptomatic stage A and B nodular sclerosing Hodgkin's disease in children.

Interleukin (IL)-6-associated laboratory parameters obtained at diagnosis on 17 children with histologically confirmed nodular sclerosing Hodgkin's disease (NSHD) are reported. When these patients were grouped as either symptomatic stage A or B, they were found to be similar in extent of disease, age, and gender. However, statistically significant differences between these two groups were observed for the means of the following IL-6-associated laboratory parameters: hematocrit (p = 0.019), platelet count (p = 0.009), serum albumin (p = 0.001), and ferritin (p = 0.037) concentrations. Moreover, trend analysis of abnormalcy revealed an increasing frequency of anemia, thrombocytosis, hypoalbuminemia, and hyperferritinemia between stage A and B patients and, when available, febrile controls (p values = 0.0012, 0.0009, 0.0406, and 0.0011, respectively). Correspondingly, IL-6 immunohistochemistry performed on archival material from representative cases in each group showed greater overall reactivity in specimens from stage B patients. A variety of cells accounted for this positivity for IL-6 antigen including Reed-Sternberg cells and their variants, lacunar cells, dendritic interdigitating cells, endothelial cells, fibroblasts, and vascular smooth muscle cells. In summary, greater and more frequent abnormalities in IL-6-associated laboratory parameters and increased immunohistochemical reactivity for IL-6 antigen coincide with the presence of fever in helping to identify children with clinical stage B NSHD.

Structure and antigenicity of the phosphorylated lipopolysaccharide antigens from the leprosy and tubercle bacilli.

A family of major arabinose- and mannose-containing phosphorylated lipopolysaccharides was isolated from Mycobacterium leprae and Mycobacterium tuberculosis. The only antigenic member of the family, lipoarabinomannan (LAM)-B, was purified by anion exchange and gel filtration chromatography in detergent and recovered in large quantities (15 mg/g of bacteria). It yielded a broad diffuse band on polyacrylamide gel electrophoresis but appeared homogeneous by this criterion and gel filtration. Besides arabinose and mannose, it contained glycerol and a polyol phosphate and was acylated by lactate, succinate, palmitate, and 10-methyloctadecanoate. The phosphate was released by alkalinolysis and identified by thin layer chromatography and gas chromatography-mass spectrometry as myoinositol 1-phosphate. Thus, the group-specific "arabinomannan" of the genus Mycobacterium in the native state is acylated, contains the substituents of phosphatidylinositol, and is apparently membrane associated. LAM-B is one of the dominant immunogens of the leprosy bacillus reacting readily with antibodies from lepromatous leprosy patients and monoclonal antibodies in plate and nitrocellulose enzyme-linked immunosorbent assay and on electrophoretic immunoblots. It is immunologically cross-reactive with a like product from M. tuberculosis. LAM-B is clearly the pervasive "glycoprotein" antigen of the leprosy bacillus and may be the long sought lipoteichoic acid-like polymer of Mycobacterium with a role in cell wall physiology, macrophage recognition, and perhaps an involvement in cross-protective immunity.

Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan.

Each of more than 30 monoclonal antibodies that had been raised against Mycobacterium leprae and previously classified as reactive with carbohydrate was shown to be directed against lipoarabinomannan, a prominent, highly pervasive, myo-inositol-phosphate-containing, cross-reactive antigen within the leprosy bacillus. Some of the antibodies preferentially bound to the lipopolysaccharide of M. leprae rather than to that of Mycobacterium tuberculosis, suggesting the presence of distinguishing structural features. The presence of alkali-labile inositol 1-phosphate in the lipopolysaccharide from M. tuberculosis and its apparent absence from the M. leprae product may account for the difference.

Generation of monoclonal antibodies to the specific sugar epitopes of Mycobacterium avium complex serovars.

Monoclonal antibodies have been generated to the unique distal sugar epitopes on the oligosaccharide haptens of the glycopeptidolipid antigens of clinically prominent members of the Mycobacterium avium serocomplex. Thus, antibodies are described that recognize the distal O-acetyl-alpha-L-rhamnopyranosyl residue of the specific glycopeptidolipid of M. avium serovar 1, the 4-O-acetyl-2,3-di-O-methyl-alpha-L-fucopyranose of serovar 2, the 4-O-methyl-alpha-L-rhamnopyranosyl-(1----4)-2-O-methyl-alpha-L- fucopyranosyl unit of serovar 4, the 4,6-(1'-carboxyethylidene)-3-O-methyl-beta-D-glucopyranosyl unit of serovar 8 [and the 4,6-(1'-carboxyethylidene)-beta-D-glucopyranosyl residue of serovar 21], and the 4-O-acetyl-2,3-di-O-methyl-alpha-L-fucopyranosyl-(1----4)-beta-D- glucuronopyranosyl unit of serovar 9. Epitope definition was arrived at through use of the pure, chemically defined glycopeptidolipid antigens and neoglycoproteins containing the chemically synthesized distal sugars of some select serovars. These monoclonal antibodies combined with the already published information on the structure of the antigen determinants and the tools used to arrive at these structures provide powerful means for fundamental studies on the role of these antigens in immunopathogenesis and for the precise mapping of the epidemiology of opportunistic infections caused by M. avium.

Quantification of active caspase 3 in apoptotic cells.

We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying relative amounts of active caspase 3 in apoptotic cells. Covalent modification of caspase 3 active sites with a biotinylated inhibitor differentiates active from latent caspases. Capture on an ELISA plate with an antibody specific for caspase 3 makes the assay specific for caspase 3. Detection is with horseradish peroxidase (HRP)-conjugated streptavidin that binds to the biotinylated inhibitor covalently bound to caspase 3. Using the assay we detected 6.6 ng active caspase 3 per 10(6) apoptotic staurosporine-treated Jurkat cells. Specificity of the assay for caspase 3 was demonstrated by lack of signal with purified caspases 2, 7, 8, and 10 that were modified by a biotinylated inhibitor. Specificity was also demonstrated by lack of signal with apoptotic MCF-7 cells which do not express caspase 3. The ability to discriminate between active and latent caspase 3 was shown by Western blotting with HRP-streptavidin and anti-caspase 3. Although latent caspase 3 was captured it was not covalently modified with the biotinylated inhibitor. The basic principle of using a covalent inhibitor to identify active enzymes and an antibody to differentiate between enzymes with similar activities has potential for quantifying active members of many classes of enzymes.