Fatty acid binding protein 4 is expressed in distinct endothelial and non-endothelial cell populations in glioblastoma.

AIMS: Glioblastoma (GBM) is the most common and aggressive primary brain tumour in adults. Angiogenesis and vasculogenesis play key roles in progression of GBMs. Fatty acid binding protein 4 (FABP4) is an intracellular chaperone for free fatty acids. FABP4 is detected in microvascular endothelial cells (ECs) in several normal tissues and promotes proliferation of ECs. The goal of this study was to characterize the tissue distribution pattern of FABP4 in GBMs. METHODS: Immunohistochemistry for FABP4 was performed on paraffin-embedded tumour sections and the intensity and distribution of FABP4 immunoreactivity were analysed. Double immunofluorescence was employed for detailed characterization of FABP4-positive cells. RESULTS: FABP4 immunoreactivity was absent in normal brain tissue sections. FABP4-positive cells were detected in 33%, 43%, 64% and 89% of Grade I, Grade II, Grade III and Grade IV glial tumours, respectively. Thus, the percentage of FABP4-positive cells in GBMs was significantly higher than lower-grade gliomas. In general, FABP4-expressing cells were distributed in a non-homogenous pattern, as 'hot spots' in glial tumours. FABP4 expression was detected in a subset of vascular ECs as well as some non-ECs. CONCLUSION: FABP4 is expressed in a significantly higher percentage of GBMs in comparison to both normal brain tissues and lower-grade glial tumours. FABP4 is expressed in some tumour ECs as well as non-ECs in glial tumours. As FABP4 promotes proliferation of ECs, detection of FABP4 in GBM-ECs, but not normal brain ECs suggests that FABP4 may play a role in the robust angiogenesis associated with GBMs.

Characterization of monoclonal antibodies directed against squamous cell carcinoma antigens: report of the TD-10 Workshop.

Thirteen monoclonal antibodies directed against squamous cell carcinoma antigens (SCCA1 and SCCA2) were obtained from five international collaborating laboratories participating in the ISOBM TD-10 Workshop. Native and recombinant forms of SCCA were used in a wide variety of approaches to determine the reactivity and specificity of these antibodies. Based on reactivity, the antibodies could be divided into three groups: the SCCA1-reactive group containing those that reacted only with recombinant SCCA1 (rSCCA1) and native SCCA1 (nSCCA1) antigens, the SCCA2-reactive group containing those that reacted only with recombinant SCCA2 (rSCCA2), and the pan-reactive group containing those antibodies that reacted with rSCCA1, nSCCA1, and rSCCA2. Binding to radioiodinated rSCCA1 showed that all reactive antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Binding to labelled rSCCA2 demonstrated that five antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Antibody reactivity on Western blots was tested with nonreduced and reduced native and recombinant SCCA1 and SCCA2. In general, these findings showed that reduction had little effect on binding to SCCA1, but often a strong effect on the binding to SCCA2. Binding of antibodies to rSCCA1 and rSCCA2 in complexes with cathepsin L and G, respectively, was used to assist in the localization of epitope regions in enzyme-complexed SCCA. Cross-inhibition experiments showed that SCCA1-reactive antibodies represent two different epitope groups, and this is supported by their ability to make SCCA1-specific assays by combining antibodies from the two epitope groups. The SCCA2-reactive group represents two related antibodies and one unique as seen in cross-inhibition, but they do not form successful assay combinations. Classification of the pan-reactive antibodies is more difficult, as some epitope groups differ when results from rSCCA1 are compared with rSCCA2 as the target. However, two antibodies are outstanding, SCC107 and SCC113, as they are high-affinity antibodies which react equally well with free and protease complexes of SCCA1 and SCCA2. The precise location of epitopes was further studied using sequential overlapping peptides and homology modelling. The findings from this workshop strongly indicate that the recombinant antigens (rSCCA1 and rSCCA2) are very similar in epitope structure to the native counterparts in saliva, and squamous epithelium from normal and cancer tissues. Therefore, it is reasonable to conclude that the specificities found are reliable and have application for antibody measurement of all forms of squamous cell carcinoma in serum except SCCA2 in complex with its protease.

Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.

Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.

SERPINB12 is a novel member of the human ov-serpin family that is widely expressed and inhibits trypsin-like serine proteinases.

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 +/- 1.6 x 10(5) and 1.6 +/- 0.2 x 10(4) M(-1) S(-1), respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.

SCCA1 expression in T-lymphocytes peripheral to cancer cells is associated with the elevation of serum SCC antigen in squamous cell carcinoma of the tongue.

Squamous cell carcinoma (SCC) antigen has been used for the management of SCC arising in various cites including head and neck region. However, the true mechanism of the elevation of this protein in the serum of patients with SCC is still unknown. SCC antigen belongs to the superfamily of serine protease inhibitors. Recently, molecular studies show that serum SCC antigen is transcribed by two nearly identical genes (SCCA1 and SCCA2), and is mainly produced by SCCA1. The objective of this study is to clarify the mechanism of the elevation of SCC antigen in oral tongue SCC patients and to identify cells histologically, which are responsible for serum SCC antigen production. In this study, we examined SCCA1 expression in a series of four head and neck SCC (HNSCC) cell lines, and found that all expressed equal to low SCCA1 protein as compared with the normal human oral keratinocyte. Using the double immunohistochemical technique to examine the expression pattern of SCCA1 in 86 cases of oral tongue squamous cell carcinoma, SCCA1 immunostaining was observed in the cytoplasm of cancer cells and T-lymphocytes peripheral to cancer cells. We also compared the clinicopathological features including serum SCC antigen level of the oral tongue SCC cases with the immunohistochemical SCCA1 expression pattern, and found that elevated serum SCC antigen level was significantly correlated with SCCA1 expression not in cancer cells, but in T-lymphocytes peripheral to cancer cells. These results suggest that T-lymphocytes peripheral to cancer cells may be responsible for serum SCC antigen production in HNSCC patients.

Circulating serpin tumor markers SCCA1 and SCCA2 are not actively secreted but reside in the cytosol of squamous carcinoma cells.

An elevation in the circulating level of the squamous-cell carcinoma antigen (SCCA) can be a poor prognostic indicator in certain types of squamous-cell cancers. Total SCCA in the circulation comprises 2 nearly identical, approximately 45 kDa proteins, SCCA1 and SCCA2. Both proteins are members of the high-molecular weight serine proteinase inhibitor (serpin) family with SCCA1 paradoxically inhibiting lysosomal cysteine proteinases and SCCA2 inhibiting chymotrypsin-like serine proteinases. Although SCCA1 and SCCA2 are detected in the cytoplasm of normal squamous epithelial cells, neither serpin is detected normally in the serum. Thus, their presence in the circulation at relatively high concentrations suggests that malignant epithelial cells are re-directing serpin activity to the fluid phase via an active secretory process. Because serpins typically inhibit their targets by binding at 1:1 stoichiometry, a change in the distribution pattern of SCCA1 and SCCA2 (i.e., intracellular to extracellular) could indicate the need of tumor cells to neutralize harmful extracellular proteinases. The purpose of our study was to determine experimentally the fate of SCCA1 and SCCA2 in squamous carcinoma cells. Using subcellular fractionation, SCCA-green fluorescent fusion protein expression and confocal microscopy, SCCA1 and SCCA2 were found exclusively in the cytosol and were not associated with nuclei, mitochondria, lysosomes, microtubules, actin or the Golgi. In contrast to previous reports, metabolic labeling and pulse-chase experiments showed that neither non-stimulated nor TNFalpha/PMA-stimulated squamous carcinoma cells appreciably secreted these ov-serpins into the medium. Collectively, these data suggest that the major site of SCCA1 and SCCA2 inhibitory activity remains within the cytosol and that their presence in the sera of patients with advanced squamous-cell carcinomas may be due to their passive release into the circulation.

Pulmonary hypertension associated with nonketotic hyperglycinaemia.

Nonketotic hyperglycinaemia (NKH) is an autosomal recessive disorder characterized by defective glycine degradation by the mitochondrial glycine cleavage system. The clinical features include lethargy, hypotonia, apnoea, seizures and severe psychomotor retardation, all attributed to the accumulation of glycine in the nervous system. Pulmonary hypertension (PHN) has not been reported in NKH. We describe four patients with NKH who had PHN in addition to the characteristic manifestations of NKH. This newly recognized association might provide additional insight into the underlying pathophysiology of PHN.

Development of specific monoclonal antibodies and a sensitive discriminatory immunoassay for the circulating tumor markers SCCA1 and SCCA2.

The squamous cell carcinoma antigen (SCCA) serves as a serologic marker for advanced squamous cell carcinomas (SCC) of the uterine cervix, lung, esophagus, head and neck and vulva. Elevations in serum levels of SCCA following treatment for SCC correlate with tumor relapse or metastasis. Recent molecular studies show that SCCA is transcribed by two nearly identical genes (SCCA1 and SCCA2) that encode for members of the high molecular weight serine proteinase inhibitor (serpin) family. Despite a high degree of similarity in their amino acid sequences, SCCA1 and SCCA2 have distinct biochemical properties: SCCA1 is an inhibitor of papain like cysteine proteinases, such as cathepsins (cat) L, S and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, catG and mast cell chymase. In this paper, we report the generation and characterization of anti-SCCA1 and anti-SCCA2 specific monoclonal antibodies (MAbs). Using these MAbs, we developed an enzyme-linked immunoassay (ELISA) that discriminated between SCCA1 and SCCA2 without any cross-reaction. This assay measured both the native and complexed forms of SCCA1 and SCCA2. The sensitivity of detection of SCCA1 and SCCA2 assays were 0.17 ngml(-1) and 0.19 ngml(-1), respectively. Mean inter- and intra-assay coefficients of variation were 12.1% and 9.9% for SCCA1 assay and 12% and 8.8% for SCCA2 assay, respectively. Recovery and parallellism studies indicated that SCCA1 and SCCA2 were detected in the plasma and amniotic fluids without any major interference by the biologic fluid components. This assay provides a simple and accurate procedure for the quantitation of total SCCA1 and SCCA2.

Co-expression of the squamous cell carcinoma antigens 1 and 2 in normal adult human tissues and squamous cell carcinomas.

Squamous cell carcinoma antigen (SCCA) serves as a serological marker for advanced squamous cell carcinomas (SCCs) and as an indicator of therapeutic response. Recent molecular studies show that the SCCA is transcribed by two almost identical tandemly arrayed genes, SCCA1 and SCCA2. These genes are members of the high molecular weight serine proteinase inhibitor (serpin) superfamily. Although SCCA1 and SCCA2 are 92% identical at the amino acid level, they have distinct biochemical properties. Paradoxically, SCCA1 is an inhibitor of papain-like cysteine proteinases, such as cathepsins L, S, and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, cathepsin G, and mast cell chymase. Using a new set of discriminatory monoclonal antibodies (MAbs) and polymerase chain reaction (PCR) assay, we showed that SCCA1 and SCCA2 were co-expressed in the suprabasal layers of the stratified squamous epithelium of the tongue, tonsil, esophagus, uterine cervix and vagina, Hassall's corpuscles of the thymus, and some areas of the skin. SCCA1 and SCCA2 also were detected in the pseudo-stratified columnar epithelium of the conducting airways. Examination of squamous cell carcinomas of the lung and head and neck showed that SCCA1 and SCCA2 were co-expressed in moderately and well-differentiated tumors. Moreover, there was no differential expression between these SCCA "isoforms" in normal or malignant tissues. In contrast to previous studies, these data indicated that the expression of SCCA1 and SCCA2 was not restricted to the squamous epithelium and that these serpins may coordinately regulate cysteine and serine proteinase activity in both normal and transformed tissues.

SCCA1 and SCCA2 are proteinase inhibitors that map to the serpin cluster at 18q21.3.

The genes for the squamous cell carcinoma antigen (SCCA) were found flanking a deletion breakpoint from a patient with the 18q-syndrome. The genes are <10 kb apart, tandemly arrayed in a head-to-tail fashion, and approximately 10 kb in size. Both genes also contain 8 exons and identical intron-exon boundaries. The cDNAs encode for proteins that are 92% identical and 95% similar. Amino acid comparisons show that SCCA1 and SCCA2 are members of the high-molecular weight serine proteinase inhibitor (serpin) family. Physical mapping studies show that the genes reside within the 500-kb region of 18q21.3 that contains at least four other serpin genes. The gene order is cen-maspin (PI5), SCCA2, SCCA1, PAI2, bomapin (PI10), PI8-tel. Biochemical analysis of recombinant SCCA1 and SCCA2 proteins shows that SCCA1 is a potent cross-class inhibitor of papain-like cysteine proteinases such as cathepsins L, S and K, whereas SCCA2 is an inhibitor of chymotrypsin-like serine proteinases such as cathepsin G and mast cell chymase. These findings suggest that SCCA1 and SCCA2 are capable of regulating proteolytic events involved in both normal (e.g., tissue remodeling, protein processing) and pathologic processes (e.g., tumor progression).

Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis.

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.

Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase.

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the high molecular weight serpin superfamily of serine proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in primary structure, the reactive site loop of each inhibitor suggests that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine proteinases. The purpose of this study was to determine whether SCCA2 inhibited a different family of proteolytic enzymes. Using recombinant DNA techniques, we prepared a fusion protein of glutathione S-transferase and full-length SCCA2 . The recombinant SCCA2 was most effective against two chymotrypsin-like proteinases from inflammatory cells, but was ineffective against papain-like cysteine proteinases. Serpin-like inhibition was observed for both human neutrophil cathepsin G and human mast cell chymase. The second order rate constants for these associations were on the order of approximately 1 x 10(5) M-1 s-1 and approximately 3 x 10(4) M-1 s-1 for cathepsin G and mast cell chymase, respectively. Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like serine proteinases.

A family with one child with acrocallosal syndrome, one child with anencephaly-polydactyly, and parental consanguinity.

A 2-year-old boy with typical features of the acrocallosal syndrome is presented. His parents are first degree cousins and their first pregnancy resulted in spontaneous abortion whereas the offspring of their second pregnancy was an anencephalic baby with bilateral post-axial polydactyly of the hands. The possibility of including anencephaly in the spectrum of the CNS malformations of the acrocallosal syndrome is suggested.

The effect of bolus methylprednisolone in prevention of brain edema in hypoxic ischemic brain injury: an experimental study in 7-day-old rat pups.

One of the major mechanisms responsible for tissue injury in hypoxic ischemic brain damage is the formation of free radicals. We studied the efficacy of methylprednisolone, which is claimed to cause rapid congealing of membranes, and to protect the cells against the free radicals present in the environment, in preventing the brain edema that occurs in hypoxic ischemic brain injury. Hypoxic ischemic brain injury to the right hemisphere in 7-day-old rat pups is produced by cauterization of the right common carotid artery followed by hypoxia in 8% oxygen and 92% nitrogen for 3 h. The animals were divided into the groups. One group received methylprednisolone at 30 mg/kg body weight and the other an equal volume of saline 40 min before hypoxia. At 42 h of recovery, some of the rats were killed for measurement of water content in the right and left hemispheres whereas others were killed at 40 days for neuropathological examination. The water content in the right hemisphere was significantly lower in the 19 methylprednisolone-treated pups than it was in 20 saline-treated pups, mean +/- S.E.M. was 83.913 +/- 0.313% and 86.681 +/- 0.377%, respectively. The differences between the means were significant (P less than 0.001) according to the Mann-Whitney U-test. The difference of the mean water contents between the two hemispheres was significantly higher in the saline-treated group (2.695 +/- 0.372%) than it was in the methylprednisolone-treated group (0.432 +/- 0.088%), P less than 0.0001 by independent samples t-test. Neuropathological study was performed on 4 rat pups.(ABSTRACT TRUNCATED AT 250 WORDS)