Anette Kliem

Localization of Lung Surfactant Protein D on Mucosal Surfaces in Human Tissues

Lung surfactant protein-D (SP-D), a collectin mainly produced by alveolar type II cells, initiates the effector mechanisms of innate immunity on binding to microbial carbohydrates. A panel of mRNAs from human tissues was screened for SP-D mRNA by RT-PCR. The lung was the main site of synthesis, but transcripts were readily amplified from trachea, brain, testis, salivary gland, heart, prostate gland, kidney, and pancreas. Minor sites of synthesis were uterus, small intestine, placenta, mammary gland, and stomach. The sequence of SP-D derived from parotid gland mRNA was identical with that of pulmonary SP-D. mAbs were raised against SP-D, and one was used to locate SP-D in cells and tissues by immunohistochemistry. SP-D immunoreactivity was found in alveolar type II cells, Clara cells, on and within alveolar macrophages, in epithelial cells of large and small ducts of the parotid gland, sweat glands, and lachrymal glands, in epithelial cells of the gall bladder and intrahepatic bile ducts, and in exocrine pancreatic ducts. SP-D was also present in epithelial cells of the skin, esophagus, small intestine, and urinary tract, as well as in the collecting ducts of the kidney. SP-D is generally present on mucosal surfaces and not restricted to a subset of cells in the lung. The localization and functions of SP-D indicate that this collectin is the counterpart in the innate immune system of IgA in the adaptive immune system.

Collectin 11 (CL-11, CL-K1) Is a MASP-1/3–Associated Plasma Collectin with Microbial-Binding Activity

Collectins play important roles in the innate immune defense against microorganisms. Recently, a new collectin, collectin 11 (CL-11 or CL-K1), was identified via database searches. In present work, we characterize the structural and functional properties of CL-11. Under nonreducing conditions, in gel permeation chromatography recombinant CL-11 forms disulfide-linked oligomers of 100 and 200 kDa. A mAb-based ELISA estimates the concentration of CL-11 in plasma to be 2.1 μg/ml, and the presence of CL-11 in plasma was further verified by Western blotting and mass spectrometry. Mannan-binding lectin-associated serine protease 1 (MASP-1) copurified with CL-11 and the interaction in plasma with MASP-1 and/or MASP-3 was further demonstrated using ELISA. We identified the adrenal glands, the kidneys, and the liver as primary sites of expression. CL-11 lectin activity was demonstrated by ELISA and showed that CL-11 has preference for l-fucose and d-mannose. We finally show that CL-11 binds to intact bacteria, fungi, and viruses and that CL-11 decreases influenza A virus infectivity and forms complexes with DNA. On the basis of the significant concentration of CL-11 in circulation and CL-11’s interaction with various microorganisms and MASP-1 and/or MASP-3, it is conceivable that CL-11 plays a role in activation of the complement system and in the defense against invading microorganisms.

Neurons in the monoaminergic nuclei of the rat and human central nervous system express FA1/dlk.

The gene DLK1 encodes a member of the epidermal growth factor (EGF) superfamily, delta-like (dlk). When exposed in vivo to the action of an unknown protease, this type 1 membrane protein generates a soluble peptide referred to as Fetal antigen 1 (FA1). By acting in juxtacrine as well as paracrine/autocrine manners, both forms have been shown to be active in the differentiation/proliferation process of various cell types. In adults, FA1/dlk has been demonstrated mainly within (neuro) endocrine tissues. In this study we investigated the presence of FA1/dlk in other parts of the developing and adult rat and human CNS. Using immunocytochemistry and in situ hybridization we found that in both species FA1/dlk was expressed in neurons of the Edinger-Westphals nucleus as well as in substantia nigra, ventral tegmental area (VTA), locus coeruleus and in certain parts of the raphe nuclei.

Pregnancy Associated Plasma Protein A, a Novel, Quick, and Sensitive Marker in ST-Elevation Myocardial Infarction

Traditional biomarkers in acute coronary syndromes reflect myocardial necrosis but not the underlying arteriosclerotic disease. Pregnancy-associated plasma protein A (PAPP-A) is a new biomarker in acute coronary syndromes that detects vulnerable plaques in arteriosclerotic disease and identifies acute coronary syndromes earlier than traditionally used biomarkers. Information regarding circulating PAPP-A levels in patients with ST elevation myocardial infarctions (STEMIs) is limited and contradictory. The aim of the present study was to describe the presence and time-related pattern of circulating PAPP-A levels in patients with STEMIs. Consecutive patients (n = 354) referred for primary percutaneous intervention because of STEMI were included in the study. Blood samples for the analysis of PAPP-A, creatine kinase-MB (CKMB), and troponin T were drawn at admission and every 6 to 8 hours until biomarkers of myocardial necrosis were consistently decreasing. PAPP-A was measured using a newly developed sandwich enzyme-linked immunosorbent assay technique based on 2 monoclonal antibodies. In total, 1,091 PAPP-A, 1,049 troponin T, and 1,016 CKMB samples were analyzed. Mean PAPP-A values at admission were significantly higher in patients with STEMIs than in those with non-ST elevation myocardial infarctions or unstable angina pectoris (27.6 vs 12.2 mIU/L, p <0.01). In samples drawn <2 hours after admission, the sensitivity of PAPP-A was superior (93%) to that of CKMB (60%) and troponin T (61%). In conclusion, PAPP-A levels are elevated in >90% of patients presenting with STEMIs if measured <6 hours after the onset of symptoms or <2 hours of primary percutaneous coronary intervention. In the early stages of STEMI, PAPP-A seems to be a more sensitive marker of myocardial infarction than CKMB and troponin T.

Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout (Onchorhynchus mykiss).

The complement system of fish is generally as complex as in mammals, and in addition Teleost fish often possess several genes encoding different subtypes of a given complement component, such as C3-1, C3-3 and C3-4. Initiators of both the classical (C1) and alternative pathway (factor B) have been characterized in the rainbow trout but so far no molecules of the lectin pathway have been identified. Based on the generally accepted idea of complement evolution, which predicts that the alternative pathway predates the two other pathways, and that the lectin pathway developed before the classical, we set out to characterize members of the lectin pathway in fish. We identified and characterized three homologues of mannan-binding lectin (MBL) with a bona fide collectin structure. By means of RT-PCR and immunohistochemistry using monoclonal antibodies we found that they were synthesized in the spleen, the anterior intestine and the liver. In the liver, we saw co-expression with mannan-binding lectin associated serine protease (MASP). The MBL homologues 2 and 3 (MBL-H2,3) were also found in the vascular system of the rainbow trout. By means of gel size exclusion chromatography of serum we found that MBL-H2,3 oligomerized heterogeneously from monomers to tetramers of a trimeric collagenous subunit. Sequence comparison and phylogenetic studies showed that the homologues were more related with MBL than any other collectins, and that two previously characterized trout proteins, designated MBL1 and MBL2, should be reconsidered as MBL candidates.

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis.

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intestinalis. The CioMBLs display similarities with vertebrate MBLs and comprise a collagen-like region, alpha-helical coiled-coils and a carbohydrate recognition domain (CRD) with conserved residues involved in calcium and carbohydrate binding. Structural analysis revealed an oligomerization through interchain disulphide bridges between N-terminal cysteine residues and cysteines located between the neck region and the CRD. RT-PCR showed a tissue specific expression of CioMBL in the gut and by immunohistochemistry analysis we also demonstrated that CioMBL co-localize with an MBL-associated serine protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway.

Pregnancy associated plasma protein A, a potential marker for vulnerable plaque in patients with non-ST-segment elevation acute coronary syndrome

OBJECTIVES To describe the presence and time-related pattern of circulating pregnancy associated plasma protein A (PAPP-A) levels in patients with non ST-segment elevation acute coronary syndrome (NSTE-ACS). DESIGN AND METHODS Consecutively admitted patients (N=573) with clinical signs of NSTE-ACS were included. Blood samples for analysis of PAPP-A were drawn at admission and every 6-8 h until levels of biomarkers of myocardial necrosis showed a consistent decrease. RESULTS High-risk NSTE-ACS was diagnosed in 123 patients (23%). Significantly more patients with high-risk NSTE-ACS (63%) had detectable PAPP-A than did those with low-risk NSTE-ACS (49%) (P<0.001). PAPP-A concentrations were significantly associated with typical angina at admission, significant ST-depressions on the ECG, multivessel disease and presence of high-risk NSTE-ACS. CONCLUSION PAPP-A seems to be a marker ischaemia both in patients with low- and high-risk NSTE-ACS, possibly due to the release of PAPP-A from the vulnerable plaque.

Pregnancy-associated plasma protein A in non-cardiac conditions

OBJECTIVE PAPP-A is a promising new marker in coronary heart disease. It is important to investigate its specificity in order to establish its clinical utility as a marker of coronary heart disease. DESIGN AND METHODS PAPP-A was measured within 24 h following hospital admission in 1448 consecutive patients admitted with diagnoses other than acute coronary syndromes. RESULTS PAPP-A was detectable (> or = 4.0 mIU/L) in 278 (19.2%) patients, among whom the mean level was 6.3 mIU/L (95% C.I., 6.1-6.5 mIU/L). The 95 and 99 percentiles for PAPP-A were 7.3 and 9.4 mIU/L, respectively. There was no difference in the mean PAPP-A of different diagnoses (p=0.33). None of the specific diagnoses known to influence established coronary markers appeared to influence the level of circulating PAPP-A. CONCLUSION PAPP-A is low in patients without known coronary heart disease. PAPP-A levels seem to be a potentially highly specific marker for heart disease.

Release patterns of pregnancy-associated plasma protein A in patients with acute coronary syndromes assessed by an optimized monoclonal antibody assay.

Objective. Pregnancy‐associated plasma protein A (PAPP‐A) is expressed in eroded and ruptured atheromatous plaques, and circulating levels are elevated in acute coronary syndromes (ACS). Our objective was to investigate release patterns of PAPP‐A in ACS and whether they differ among different types of ACS. Methods. In 40 patients, PAPP‐A concentrations were measured in serially collected samples assessed by a novel ELISA technique. The patients were grouped according to type of ACS. Results. Release patterns for ST elevation myocardial infarction (STEMI) patients who underwent primary percutaneous coronary intervention (pPCI) showed a single substantial PAPP‐A increase shortly after pPCI, followed by an abrupt return to normal levels without secondary peaks. STEMI, high‐risk and low‐risk non‐ST elevation myocardial infarction/unstable angina pectoris (NSTEMI/UAP) patients without pPCI showed highly variable patterns with primary peaks followed by secondary PAPP‐A increases. All patients with elevated PAPP‐A levels reached the upper reference level within 24 h. There was a significant difference in median peak levels between STEMI (23.2 mIU/L) and low‐risk ACS patients (6.35 mIU/L) (p = 0.004) and between high‐risk (median = 15.3 mIU/L) and low‐risk ACS patients (p = 0.01). Among high‐risk ACS patients, NSTEMI patients had significantly higher peak levels than UAP patients (p = 0.003). Conclusion. PAPP‐A serum levels increase above normal values within 24 h after onset of symptoms in ACS. There are significant differences in PAPP‐A peak levels and release patterns across the spectrum of ACS patients.

Screening for Epitope Specificity Directly on Culture Supernatants in the Early Phase of Monoclonal Antibody Production by an ELISA with Biotin‐Labeled Antigen

Abstract This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin‐labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti‐mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin‐labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.