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Biomedical subjects

J C Jensenius

Publications and source records attributed to J C Jensenius.

At least 19 recordsLinked to original sources

Flow cytometric investigation of immune-response-related surface molecules on human colorectal cancers.

Our purpose was to clarify whether human colorectal cancer cells are equipped to present tumour-associated-antigens to the immune system, and whether this ability correlates with lymphoid infiltration, the Dukes' stage and Jass classification. Enzymatically dissociated tumour cells from 70 different colorectal cancers were monitored by multiparameter flow cytometry. Gating on EP4+ cells, the expression of the surface molecules HLA class I, HLA class II, CD80 (B7-1), CD54 (ICAM-I) and CD58 (LFA-3) was evaluated. In 60 of 70 tumours, all tumour cells expressed HLA class I, in 10 tumours 15-96% of the tumour cells expressed HLA class I. In 1 tumour, all tumour cells expressed HLA class II, in 67 tumours some expressed HLA class II, in 2 tumours none expressed HLA class II. Expression of CD58 was heterogeneous, and there was no or only sparse expression of CD80 and CD54. Expression of the HLA class I molecules, but not the class II, was correlated with lymphoid infiltration and the Jass classification. Expression of these surface molecules was not correlated with the Dukes' stage. The tumour cells were generally equipped to present antigens to the effector arm of the immune system since HLA class I is expressed, but the tumour cells were not optimal in stimulating an immune response, since HLA class II and CD58 were only marginally expressed and CD80 and CD54 were absent.

Adult

Mannan-binding lectin in human serum, cerebrospinal fluid and brain tissue and its role in Alzheimer's disease.

Mannan-Binding lectin (MBL) is a serum lectin which can activate the classical complement pathway. Complement proteins of the classical pathway have been found in the brains of patients with Alzheimer's disease (AD) in association with AD brain pathology. To investigate the role for MBL in AD we have looked for its presence in the brain by immunohistochemistry and determined the levels of MBL in paired samples of cerebrospinal fluid and serum from AD patients and controls. MBL was detected in association with blood vessels in the brain tissue of both AD patients and control subjects. There was no apparent difference in the distribution of MBL in the brain tissue between the two groups. The mean concentration of MBL in the CSF was 44% lower in AD patients than in controls (AD 154 +/- 35 pg/ml, n = 19; non-AD 276 +/- 50 pg/ml, n = 15, p < 0.05). The levels of MBL in serum were not significantly different in the two groups. Thus, this study shows that MBL is associated with blood vessels in the brains of both AD and control subjects. Moreover, CSF levels of MBL appear to be lower in AD patients than in control subjects which may indicate a higher degree of MBL consumption connected with complement activation in the AD patients.

Aged

Cloning and sequencing of a cDNA encoding chicken mannan-binding lectin (MBL) and comparison with mammalian analogues.

The serum lectin, mannan-binding lectin (MBL) (also denoted mannan-binding protein or mannose-binding protein, MBP) has been identified in mammals (humans, monkey, cow, rabbit, mouse and rat). Upon binding to carbohydrates on the surface of microorganisms, MBL mediates activation of the complement system, leading to killing of the microorganism. MBL thus exerts a role in the innate immune defence. We have described the isolation and partial characterization of an analogous protein in chicken serum. Oligonucleotides based on the N-terminal sequence of this protein were used in a reverse transcription-polymerase chain reaction (RT-PCR) with chicken liver RNA as template. The PCR product was sequenced and found to encode part of the NH2 terminus of chicken MBL. A perfect match probe was synthesized and used to screen a chicken liver cDNA library. The isolated clones carried a cDNA insert of 1692 bp with an open reading frame of 714 bp encoding a mature protein of 238 amino acids including a signal peptide of five amino acids. The deduced amino acid sequence agrees with those determined by conventional amino acid sequence analysis of the peptides except for four residues. We have compared the deduced primary structure of chicken MBL with the mammalian analogues. The phylogenetic analysis indicates that the gene duplication leading to two different MBL forms in mammals occurred after the split from birds and reptiles. This concurs with the finding of only one form of MBL in chickens.

Amino Acid Sequence

The plasma levels of coglutinin are heritable in cattle and low levels predispose to infection.

Conglutinin, like mannan-binding lectin (MBL) and CL-43, is a serum collection involved in the innate immune defence system. In man, low serum MBL concentrations, resulting from mutations in the collagen region, are associated with a common opsonic defect. Plasma levels of conglutinin in cattle were assayed by rocket immunoelectrophoresis to examine whether they were genetically determined. Samples were collected from calves (309 bull-calves and 260 heifers with complex pedigree relationships). The number of respiratory infections from the 42nd to 336th day of life was recorded. The number of infections was found to be genetically determined (heritability: h2 = 0.31 +/- 0.07). A wide concentration range of conglutinin was found in plasma (< 1.25-35 micrograms/ml for females, geometric mean 8.1 micrograms/ml, and < 1.25-47 micrograms/ml for males, geometric mean 15.5 micrograms/ml), and the concentrations was found to be genetically determined (heritability, h2 = 0.52 +/- 0.07). The analysis revealed a negative association between disease frequency and the conglutinin levels (-0.56 +/- 0.18 for female; -0.50 +/- 0.18 for male). Levels of conglutinin below the detection limit of the assay (1.25 micrograms/ml) were found in 2% of the animals. If these animals are assumed to be homozygous for a single recessive allele causing low concentrations a gene frequency of 0.15 could be calculated. These findings suggests that selection for resistance against infectious disease is possible in cattle and that the level of plasma conglutinin may be a helpful trait in such a breeding scheme.

Animals

A comparison of flow cytometry and immunohistochemistry in human colorectal cancers.

In human colorectal cancer it has been reported that some tumours lack the HLA-ABC antigens. This has been interpreted as reflecting tumour escape from the immune system. Earlier data have been obtained by immunohistochemistry. In this study, we compared the expression of HLA-ABC, HLA-DR, CD80 (B7-1) and CD54 (ICAM-1) in 20 tumours using both a conventional immunohistochemistry two-layer technique and multiparameter flow cytometry, gating on an epithelial cell marker. Colorectal cancer tissue used in flow cytometry was dissociated with collagenase, deoxyribonuclease and hyaluronidase. The intensity of expression of HLA-ABC, HLA-DR and CD80 was unaffected by the enzymes, but CD54 was decreased by 30%. The reproducibility of flow cytometry was good. Microscopy of sections revealed that about 5% of each tumour sample consisted of normal epithelium, but even after correction for this, flow cytometry was superior to immunohistochemistry in 33 out of 80 cases, and showed that tumours described as HLA-ABC negative by immunohistochemistry were in fact weakly positive for HLA-ABC. We conclude that flow cytometry and immunohistochemistry are complementary, and that flow cytometry is superior to immunohistochemistry for detecting antigens/epitopes present in low amounts.

Aged

Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment.

Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured on fibronectin, laminin, or collagen IV. In the case of fibronectin, COU-1 staining was particularly enhanced at intercellular junctions. When carcinoma cells were cultured with COU-1 at 37 degrees C for 6 hr, the antibody was found in large perinuclear vesicles and the punctate surface staining was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia, and between colon cancer metastasis in the liver and surrounding normal hepatocytes. Within biopsies of malignant tissue, COU-1 exhibited membrane-associated staining of proliferating cells, while resting cells had a filamentous pattern. Thus, modified cytokeratin at the surface of carcinoma cells may represent a new target for immunoconjugates and may explain the promising results of the phase I/II clinical study.

Amino Acid Sequence

Pilot scale purification of human monoclonal IgM (COU-1) for clinical trials.

No standard procedure is available for the purification of human monoclonal antibodies for human i.v. administration. Here we describe the procedure developed for pilot scale purification of the human IgM monoclonal antibody COU-1 directed against a cancer-associated antigen. The hybridoma cells were grown in protein-free medium and purification from the clarified culture supernatant was carried out in 4 simple chromatographic steps: (1) hydroxylapatite chromatography; (2) hydrophobic interaction chromatography on phenyl-Sepharose: (3) cation-exchange chromatography on sulphonyl-Sepharose; and (4) anion-exchange chromatography on tetraethylamino-Sepharose. The product was substantially pure with regard to protein after step 3, but contained DNA which was removed in step 4. The average recovery of the IgM was 54% with a range of 40-65%. Importantly, the ability of the antibody to bind to its antigen in ELISA was fully maintained during the purification. Subsequently, the purified antibody was isotope labelled and successfully used for in vivo detection of colon, rectal and pancreas carcinomas in patients. The purification procedure described appears to compare favourably with previously published methods, but a critical comparison is not possible due to the lack of necessary information in the available literature.

Antibodies, Monoclonal

A second serine protease associated with mannan-binding lectin that activates complement.

The complement system comprises a complex array of enzymes and non-enzymatic proteins that is essential for the operation of the innate as well as the adaptive immune defence. The complement system can be activated in three ways: by the classical pathway which is initiated by antibody-antigen complexes, by the alternative pathway initiated by certain structures on microbial surfaces, and by an antibody-independent pathway that is initiated by the binding of mannan-binding lectin (MBL; first described as mannan-binding protein) to carbohydrates. MBL is structurally related to the complement C1 subcomponent, C1q, and seems to activate the complement system through an associated serine protease known as MASP (ref. 4) or p100 (ref. 5), which is similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms, including bacteria, yeasts, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis. The level of MBL in plasma is genetically determined, and deficiency is associated with frequent infections in childhood, and possibly also in adults (for review, see ref. 6). We have now identified a new MBL-associated serine protease (MASP-2) which shows a striking homology with the previously reported MASP (MASP-1) and the two C1q-associated serine proteases C1r and C1s. Thus complement activation through MBL, like the classical pathway, involves two serine proteases and may antedate the development of the specific immune system of vertebrates.

Amino Acid Sequence

A rainbow trout lectin with multimeric structure.

A novel lectin has been identified in rainbow trout serum and plasma. The lectin binds to Sepharose (an agarose polymer) in a calcium-dependent manner. Glucose, N-acetyl-glucosamine, mannose, N-acetyl-mannosamine, L-fucose, maltose and alpha-methyl-mannoside are good inhibitors of this binding, whereas glucosamine and D-fucose inhibits to a lesser degree and mannosamine and galactose do not inhibit the binding to Sepharose. When analysed by SDS-PAGE under non-reducing conditions, the lectin appears as a characteristic ladder of bands with approximately 16 kDa between consecutive bands. Upon reduction, the lectin appears as a 16-kDa band. On size-exclusion chromatography of trout serum and plasma, the protein emerges over a broad range corresponding to sizes from about 2000 kDa to less than 200 kDa. The NH2-terminal sequence (AAENRNQXPPG) shows no significant homology with known proteins. Because of the characteristic appearance in non-reducing SDS-PAGE and the lectin activity, we propose to name the protein "ladderlectin."

Animals

Mechanisms of anti-influenza activity of surfactant proteins A and D: comparison with serum collectins.

The present study provides the first direct comparison of anti-influenza A virus (IAV) activities of the collectins surfactant protein (SP) A and SP-D, mannose-binding lectin (MBL), and conglutinin. SP-D, MBL, and conglutinin inhibited IAV hemagglutination activity with a greater potency than and by a distinct mechanism from SP-A. Although isolated trimeric SP-D carbohydrate recognition domains inhibited hemagglutination activity, preparations of SP-D also containing the collagen domain and NH2 terminus caused greater inhibition. In contrast to SP-A (or nonmultimerized SP-D), absence of the N-linked attachment did not effect interactions of multimerized SP-D with IAV. SP-D, SP-A, and conglutinin caused viral precipitation through formation of massive viral aggregates, whereas MBL formed aggregates of smaller size that did not precipitate. All of the collectins enhanced IAV binding to neutrophils; however, in the case of MBL, this effect was modest compared with the binding enhancement induced by SP-D or conglutinin. These studies clarify the structural requirements for viral inhibition by SP-D and reveal significant differences in the mechanisms of anti-IAV activity among the collectins.

Animals

Neutrophil deactivation by influenza A viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies.

Bacterial superinfections are a major cause of morbidity and mortality during influenza A virus (IAV) epidemics. Depression of phagocyte functions resulting from attachment of the IAV hemagglutinin (HA) to cell surface sialo-glycoproteins is a likely contributory cause of these infections. We have proposed that the group of collagenous lectins (termed collectins) present in blood and pulmonary surfactant play a role in initial host defense against IAV. We used here several recombinant human surfactant protein D (RhSP-D) preparations to determine the mechanism through which opsonization of IAV with collectins protects neutrophils against the deactivating effects of IAV on cellular respiratory burst responses in vitro. RhSP-D was markedly more potent than antibodies that inhibited viral hemagglutination activity (anti-HA antibodies) at protecting neutrophils in this assay. Unlike the anti-HA antibodies, RhSP-D was protective at concentrations that minimally inhibited viral hemagglutination activity. Two related features of SP-D--the degree of multimerization and the ability to cause aggregation of IAV particles--were critical determinants of the ability of SP-D to protect neutrophils against deactivation. Similarly SP-D-induced viral aggregate formation resulted in enhanced IAV binding to neutrophils and potentiated the ability of the virus itself to trigger neutrophil respiratory burst responses. In contrast to the case of IAV-antibody complexes, SP-D-IAV complexes attached to and activated neutrophils through a neuraminidase-sensitive mechanism (ie, similar to unopsonized IAV). These results indicate that collectin-mediated viral aggregation per se may be an important host defense mechanism not only by virtue of reducing the number of infectious viral particles, but also by promoting phagocyte responsiveness.

Animals

Glycopeptide mimics of mammalian Man9GlcNAc2. Ligand binding to mannan-binding proteins (MBPs).

A novel and simple approach for rational design of oligosaccharide mimics has been developed. Mammalian high-mannose triantennary structure Man9GlcNac2 has been subjected to molecular modelling using the NMR data available on structural fragments of the oligosaccharide. The analysis indicated four different low energy conformations, and the spatial arrangement of terminal disaccharides of the oligosaccharide antennae were stimulated with glycopeptides carrying disaccharides by applying weak constraints between the saccharide parts in MD-simulations on a large array of tri- to octaglycopeptides. The five glycopeptides exhibiting the best fit with the four minimum energy confirmations of the oligosaccharide were synthesized by solid phase glycopeptide assembly using glycosylated fluoren-9-ylmethyloxycarbonyl-amino acid-O-pentafluorophenyl esters as building blocks. The glycan was acyl protected alpha-D-Man-(1-->2)-alpha-D-Man and Ser, Thr and Hyp were the glycosylated amino acids. The deprotected and purified glycopeptides were subjected to NMR analysis for characterization, and in order to investigate the cis-trans isomerism of the carbimide bonds to Hyp. The glycopeptides were tested for their ability to inhibit binding of mannan-binding protein to mannan from Saccharomyces cerevisiae. They were found to be weak inhibitors showing no indication of multivalent interaction with the mannan-binding protein.

Carbohydrate Conformation

Human autoantibodies against Clq: lack of cross reactivity with the collectins mannan-binding protein, lung surfactant protein A and bovine conglutinin.

The collectins, a group of humoral C-type lectins, have globular and collagen-like regions and share structural features with the complement protein C1q. The question was asked if autoantibodies to the collagen-like region of C1q (anti-C1qCLR) might cross-react with collectins, such as mannan-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin (BK). Anti-C1qCLR antibodies of the systemic lupus erythematosus (SLE) type and anti-C1qCLR antibodies of the hypocomplementemic urticarial vasculitis syndrome (HUVS) type were investigated. Cross-absorption and elution experiments combined with antibody detection by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis gave no evidence of cross-reactive anti-C1qCLR antibodies. However, one serum with HUVS type anti-C1qCLR antibodies contained anti-MBP antibodies that were cross-reactive with SP-A. Judging from results of ELISA inhibition experiments and immunoblot analysis, four SLE sera contained antibodies to native BK, while two sera with HUVS type anti-C1qCLR antibodies contained antibodies to epitopes of denatured BK. This might imply that autoimmunity to collagen-like structures is not restricted to C1qCLR in HUVS and HUVS/SLE overlap syndromes.

Animals

Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins.

Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.

Animals

Difference and ratio plots: simple tools for improved presentation and interpretation of scientific data. Unexpected possibilities for the use of the conglutinin binding assay in inflammatory rheumatic diseases.

11111onglutinin-binding assay (KgBa) has gained widespread use for the detection of circulating immune complexes. A recent paper questioned the interpretation of the results obtained by this method and the validity of the assay (Holmskov et al. (1992) J. Immunol. Methods 148, 225). We now present hitherto unnoted differences between controls and patients with either rheumatoid arthritis or systemic lupus erythematosus. For this we use simple, but unconventional, graphic representations of the data, based on difference plots and ratio plots. Differences between patients with Burkitt's lymphoma and systemic lupus erythematosus from another previously published study (Macanovic, M. and Lachmann, P.J. (1979) Clin. Exp. Immunol. 38, 274) are also represented using ratio plots. Our observations indicate that analysis by regression analysis may often be misleading.

Arthritis, Rheumatoid

Collectin in a non-mammalian species: isolation and characterization of mannan-binding protein (MBP) from chicken serum.

A chicken serum lectin was isolated by affinity chromatography on TSK-75 beads derivatized with the monosaccharide N-acetyl-D-mannosamine (ManNAc). Serum was applied to the column in a Ca(2+)-containing buffer and proteins were eluted with EDTA. After recalcification, the eluate was passed through a new ManNAc-derivatized column. Bound proteins were eluted with 50 mM ManNAc. Anti-carbohydrate antibodies present in the eluate were removed by passage through a rabbit anti-chicken immunoglobulin derivatized column, and the lectin was further purified by ion-exchange chromatography and gel-permeation chromatography. The purified chicken lectin shows an overall structure similar to mammalian mannan-binding protein (MBP). SDS-PAGE revealed two polypeptides of M(r) 33 and 34 kDa (reduced) with identical sequence for the first 30 NH2-terminal residues. The NH2-terminal sequence shows 43% identity with the human MBP. Like mammalian MBP, the polypeptides of the chicken lectin are degraded by treatment with collagenase. Residues 26-30 (G-L-P(OH)-G-D) are likely to represent the beginning of the collagenous region. Mobilities on SDS-PAGE of the COOH-terminal collagenase-resistant fragment under reduced and non-reduced conditions indicate the presence of intrachain disulphide bonds, as are also found in mammalian MBP. Gel chromatography showed an intact mol. wt of 750 kDa. Binding of the chicken MBP to mannan was inhibited by monosaccharides in the following order of potency: ManNAc > L-fucose > mannose > N-acetylglucosamine. Other monosaccharides inhibited poorly or not at all. Chicken MBP, bound to mannan, activated the classical complement pathway in human serum. Electron micrographs show structures and dimensions resembling human MBP. Overall, the results show that the purified lectin is the chicken homologue to mammalian MBP and indicate the presence of a MBP-like clearance system outside mammals.

Amino Acid Sequence

The level of the serum opsonin, mannan-binding protein in HIV-1 antibody-positive patients.

The concentrations of mannan-binding protein (MBP) in consecutive samples from 10 HIV+ persons were estimated using an ELISA based on polyclonal rabbit anti-MBP. The changes in MBP with time were similar in HIV+ and HIV- persons, and did not appear to be of clinical significance. MBP was determined in a further 70 persons found HIV-1+ during a period of 2.5 years (1984-1986). Out of the total of 80 patients, 32 have by now died from AIDS. According to the serum level of MBP the HIV-infected persons were grouped into high (> 650 ng MBP/ml), intermediate (101-650 ng/ml), and low MBP (< 101 ng/ml). At the termination of the study the frequency of deaths/total in each of the groups were: high MBP, 14/39 (36%); intermediate MBP, 12/26 (46%); and low MBP, 6/14 (43%). There was no association between the MBP level of the individual and the progressive loss of CD4+ T cells, and the level of MBP was not predictive for the length of time between the detection of HIV antibodies and development of AIDS, nor for the duration of AIDS before death occurred. The number of HIV+ persons without detectable MBP (10%) was significantly higher than previously reported for healthy persons (2.4%, P = 0.027). The course of HIV infection does not seem to be influenced by the level of MBP, nor does the antimicrobial activity of MBP appear to affect the progression of AIDS. Further studies are required to substantiate the significance of absence of MBP in the susceptibility to HIV.

Adult