The effect of heparin on pregnancy associated plasma protein-A concentration in healthy, non-pregnant individuals.

OBJECTIVES: The objective of this study was to determine the differences in pregnancy associated plasma protein-A (PAPP-A) concentrations in heparin naive and heparin treated healthy men and non-pregnant women, to find a possible difference in different age groups, and to determine the response in PAPP-A concentration to repeated injections of unfractionated heparin. DESIGN AND METHODS: Twenty-five healthy, non-pregnant volunteers divided into five groups (determined by gender and age) received 5000 IU unfractionated heparin intravenously. Five young men received an additional 5000 IU after 90 and 180 min. Blood samples to determine PAPP-A concentration and APTT were drawn at different time points. RESULTS: Injection of heparin elicited increase in and rapid normalization of PAPP-A concentrations in all subjects. The group of 20-30-year-old never-pregnant women had lower responses than the individuals of the four other groups. The difference was not significant (p > 0.05). Repeated injections of heparin caused additional peaks in PAPP-A concentration of about the same sizes as the first peak. We observed an increase in time to normalization of PAPP-A concentration (from 75-90 min to 90-150 min) and APTT levels with repeated injections. CONCLUSIONS: We observed a rapid normalization of PAPP-A. Our result has a great similarity to the half-life of unfractionated heparin. This result combined with the finding of equally sized peaks in PAPP-A concentration, and that all of this was found in healthy, non-pregnant individuals, suggests that heparin might compete for a binding-site on PAPP-A or with PAPP-A itself for a common receptor in healthy arterial vessels.

Pregnancy-associated plasma protein-A and the vulnerable plaque.

For more than a decade, pregnancy-associated plasma protein-A (PAPP-A) has been examined for its relation to acute coronary syndrome (ACS) and the vulnerable plaque. This review summarizes the current knowledge of plasma PAPP-A in relation to nonpregnant individuals focusing on patients with ACS, discusses its use as a possible biomarker for diagnosis and prognosis in ACS, briefly describes the challenges in different assay technologies and describes the effect of heparin administration on PAPP-A concentrations in plasma.

Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells.

Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.

Pregnancy associated plasma protein-A as a marker for myocardial infarction and death in patients with stable coronary artery disease: a prognostic study within the CLARICOR Trial.

OBJECTIVE: Pregnancy associated plasma protein-A (PAPP-A) is a potential new marker for vulnerable plaques in the coronary arteries only examined in stable coronary disease (CAD) in patients undergoing coronary angiography. Here we address the prognostic value of serum PAPP-A in unselected stable CAD patients. METHOD: Blood samples were drawn at study entry. Serum PAPP-A values ≥4mIU/L were considered elevated. Mortality and non-fatal myocardial infarction was prospectively registered. The primary outcome was the composite outcome of myocardial infarction and all-cause mortality, secondary outcomes were all-cause mortality and myocardial infarction. RESULTS: Patients (n=4243) were followed for a median of 2.8 years. In a Cox analysis, elevated PAPP-A was significantly related to the composite outcome of myocardial infarction and death (HR 1.99, 95% CI 1.62-2.45, p<0.0005), all-cause mortality (HR 2.42, 1.92-3.06, p<0.0005), and myocardial infarction (HR 1.40, 1.01-1.94, p=0.046). After Holm's correction, the latter significance disappeared. After adjustment for risk factors and medication at entry, elevated PAPP-A remained significantly related to the composite outcome (HR 1.51, 1.22-1.86, p<0.0005) and all-cause mortality (HR 1.68, 1.32-2.13, p<0.0005). CONCLUSION: In patients with stable CAD elevated serum PAPP-A seems promising as aid in identifying patients at high risk for death.

Pregnancy associated plasma protein-A (PAPP-A) is not a marker of the vulnerable atherosclerotic plaque.

OBJECTIVE: To investigate if pregnancy associated plasma protein-A (PAPP-A) was present in the vulnerable plaque, and if not, to find alternative hypothesis for the release of PAPP-A. DESIGN AND METHODS: Vulnerable plaques and control tissues were examined by immunohistochemistry. Volunteers and patients with non-atherosclerotic disease were examined for release of PAPP-A during ischemia and medical treatment. Non-atherosclerotic tissue samples were examined after incubation with heparins. RESULTS: We were not able to detect PAPP-A in vulnerable plaques. Patients and volunteers experiencing ischemic events without atherosclerotic lesions only had elevated PAPP-A when treated with heparin. When tissue from normal artery wall was incubated with heparin, PAPP-A was eluted. This was not the case for non-arterial tissue samples. CONCLUSION: Elevation of PAPP-A in patients with acute coronary syndromes seems to be caused by heparin induced release of PAPP-A from the arterial wall and not due to excretion from vulnerable plaques.

Generation of a C3c specific monoclonal antibody and assessment of C3c as a putative inflammatory marker derived from complement factor C3.

There is a general need for markers of systemic inflammation in acute or chronic diseases, where complement activation is involved. Available methods to monitor complement activation are elaborate and of low sensitivity; they include haemolytic assays (CH50), quantification of fluid phase terminal complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific mAb was tested in different ELISA combinations with various types of in vitro activated sera and with plasma or serum samples from factor I deficient patients. The specificity of the mAb was evaluated in immunoprecipitation techniques and by analysis of eluted fragments of C3 after immunoaffinity chromatography. The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust ELISA that allows for a quick and reliable evaluation of complement activation and consumption as a marker for inflammatory processes. We established the C3c plasma range in 100 healthy Danish blood donors with a mean of 3.47 µg/ml and a range of 2.12-4.92 µg/ml. We believe that such an antibody might be of potential value in the assessment of in vivo complement activity during the inflammatory processes.

Pregnancy-associated plasma protein-A, a marker for outcome in patients suspected for acute coronary syndrome.

OBJECTIVES: To examine if pregnancy-associated plasma protein-A (PAPP-A) in patients with chest pain, could identify patients at risk for death or myocardial infarction. DESIGN AND METHODS: Patients admitted with chest pain and both normal ECG and normal biomarkers were evaluated by serial measurement of PAPP-A. Main outcome measures were mortality and non-fatal myocardial infarction. RESULTS: Median age of patients included (415) was 67years and 43% were women. The risk of death or non-fatal myocardial infarction after 3 months was 15% in the highest quartile of circulating PAPP-A compared with 3% in the lowest quartile (relative risk 3.7, p<0.01). Corresponding numbers after 1 year were 24% and 10% (relative risk 2.4, p=0.01). CONCLUSION: In patients admitted with chest pain and both normal ECG and normal biomarkers PAPP-A seems to be valuable for predicting patients at high risk of death or non-fatal myocardial infarction.

MicroRNA-15a fine-tunes the level of Delta-like 1 homolog (DLK1) in proliferating 3T3-L1 preadipocytes.

Delta like 1 homolog (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal; however, little is known about the underlying mechanisms. We here report that miR-15a modulates DLK1 levels in preadipocytes thus providing a mechanism for DLK1 regulation that further links it to cell cycle arrest and cancer since miR-15a is deregulated in these processes. In preadipocytes, miR-15a increases with cell density, and peaks at the same stage where membrane DLK1(M) and soluble DLK1(S) are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1(M) protein while it increases the amount of DLK1(S) supporting a direct repression of DLK1 and a parallel effect on the protease that cleaves off the DLK1 from the membrane. In agreement with previous studies, we found that miR-15a represses cell numbers, but additionally, we report that miR-15a also increases cell size. Conversely, anti-miR-15a treatment decreases cell size while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1(S). Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling networks.

Smoking attenuates wound inflammation and proliferation while smoking cessation restores inflammation but not proliferation.

Full-thickness 5 mm punch biopsy wounds were made lateral to the sacrum in 48 smokers and 30 never smokers. After 1 week, the wounds were excised and fixed. The smokers were then randomized to continuous smoking or abstinence with a transdermal nicotine patch or a placebo patch. The sequence of wounding and excision was repeated after 4, 8, and 12 weeks. All excised tissue was stained with hematoxylin-eosin and immunohistochemically for macrophages (CD68), procollagen 1 N-terminal propeptide (PINP) in fibroblasts, and endothelial cells (CD31). The cellularity was assessed and scored by two independent histopathologists, and for the analysis, proportional odds models and random effect models for repeated measurements were applied. Macrophages and PINP-stained fibroblasts were reduced in the smokers' wounds (0.28 [0.14-0.58] [OR, 95%CI]; p=0.01 and 0.37[0.19-0.70]; p<0.01, respectively, when compared with never smokers' wounds). Inflammation scores were marginally affected. Following smoking cessation, inflammatory cell infiltration and macrophages in the wounds increased. PINP-stained fibroblasts were unaffected. Neovascularization was not affected by smoking or abstinence. Wound inflammation and fibroblast proliferation were attenuated in smokers, suggesting delayed healing. Abstinence from smoking restores inflammation, but does not affect proliferation. These findings suggest a pathophysiologic mechanism for postoperative wound infection and dehiscence in smokers and why smoking cessation appears to reduce wound infection but not dehiscence.

Usefulness of pregnancy-associated plasma protein A in patients with acute coronary syndrome.

To investigate whether pregnancy-associated plasma protein-A (PAPP-A) is a prognostic marker in patients admitted with high-risk acute coronary syndrome. In patients admitted with high-risk non-ST-segment elevation acute coronary syndrome (NSTE-ACS) and ST-segment elevation myocardial infarction (STEMI), risk stratification is primarily determined by the markers of myocardial necrosis and known demographic risk profiles. However, it has recently been proposed that the presence and extent of vulnerable plaques might influence the prognosis significantly. A marker for the vulnerable plaque could identify patients at high risk who would potentially benefit from intensive treatment and surveillance. Two populations of consecutive patients admitted with high-risk NSTE-ACS (n = 123) and STEMI (n = 314) were evaluated with serial measurements of PAPP-A. The incidence of mortality and nonfatal myocardial infarction was prospectively registered for 2.66 to 3.47 years. In the patients with high-risk NSTE-ACS, PAPP-A was related to the risk of nonfatal myocardial infarction (p = 0.02) and death (p = 0.03). This result was consistent on multivariate analysis of the combination of mortality or nonfatal myocardial infarction (odds ratio 2.65, 95% confidence interval 1.40 to 5.03) but not for mortality alone (p = NS). In patients with STEMI, PAPP-A was related to the risk of death (p = 0.01) but not the composite outcome of myocardial infarction and death. This was also true after adjustment for other univariate predictors of death (odds ratio 2.19, 95% confidence interval 1.16 to 4.16). In conclusion, PAPP-A seems to be valuable in predicting the outcomes of patients admitted with high-risk NSTE-ACS or STEMI.

Characterization of DLK1+ cells emerging during skeletal muscle remodeling in response to myositis, myopathies, and acute injury.

Delta like 1 (DLK1) has been proposed to act as a regulator of cell fate determination and is linked to the development of various tissues including skeletal muscle. Herein we further investigated DLK1 expression during skeletal muscle remodeling. Although practically absent in normal adult muscle, DLK1 was upregulated in all human myopathies analyzed, including Duchenne- and Becker muscular dystrophies. Substantial numbers of DLK1(+) satellite cells were observed in normal neonatal and Duchenne muscle, and furthermore, myogenic DLK1(+) cells were identified during muscle regeneration in animal models in which the peak expression of Dlk1 mRNA and protein coincided with that of myoblast differentiation and fusion. In addition to perivascular DLK1(+) cells, interstitial DLK1(+) cells were numerous in regenerating muscle, and in agreement with colocalization studies of DLK1 and CD90/DDR2, qPCR of fluorescence-activated cell sorting DLK1(+) and DLK1(-) cells revealed that the majority of DLK1(+) cells isolated at day 7 of regeneration had a fibroblast-like phenotype. The existence of different DLK1(+) populations was confirmed in cultures of primary derived myogenic cells, in which large flat nonmyogenic DLK1(+) cells and small spindle-shaped cells coexpressing DLK1 and muscle-specific markers were observed. Myogenic differentiation was achieved when sorted DLK1(+) cells were cocultured together with primary myoblasts revealing a myogenic potential that was 10% of the DLK1(-) population. Transplantation of DLK1(+) cells into lacerated muscle did, however, not give rise to DLK1(+) cell-derived myofibers. We suggest that the DLK1(+) subpopulations identified herein each may contribute at different levels/time points to the processes involved in muscle development and remodeling.

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.

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.

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.

Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver.

BACKGROUND & AIMS: Previously, we showed high-level, long-term liver replacement after transplantation of unfractionated embryonic day (ED) 14 fetal liver stem/progenitor cells (FLSPC). However, for clinical applications, it will be essential to transplant highly enriched cells, while maintaining high repopulation potential. METHODS: Dlk-1, a member of the delta-like family of cell surface transmembrane proteins, is highly expressed in human and rodent fetal liver. Dlk-1(+) cells, isolated from ED14 fetal liver using immunomagnetic beads, were examined for their hepatic gene expression profile and characteristic properties in vitro and their proliferative and differentiation potential in vivo after transplantation into normal adult rat liver. RESULTS: Rat ED14 FLSPC were purified to 95% homogeneity and exhibited cell culture and gene expression characteristics expected for hepatic stem/progenitor cells. Rat ED14 FLSPC are alpha-fetoprotein(+)/cytokeratin-19(+) or alpha-fetoprotein(+)/cytokeratin-19(-) and contain all of the normal liver repopulation capacity found in fetal liver. Hematopoietic stem cells, a major component in crude fetal liver cell preparations that engraft in other organs, such as bone marrow, spleen, and lung, are totally removed by Dlk-1 selection, and Dlk-1 purified FLSPC repopulate only the liver. CONCLUSIONS: This is the first study reporting purification of hepatic stem/progenitor cells from fetal liver that are fully capable of repopulating the normal adult liver. This represents a major advance toward developing protocols that will be essential for clinical application of liver cell transplantation therapy.

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.

Changes of biochemical markers of bone turnover and YKL-40 following hormonal treatment for metastatic prostate cancer are related to survival.

PURPOSE: Elevated serum levels of biochemical markers of bone turnover and YKL-40 in patients with metastatic prostate cancer (PC) at the time of diagnosis are associated to poor prognosis. In this study, we evaluated the value of these biomarkers in monitoring the patients during hormonal treatment. EXPERIMENTAL DESIGN: Serum procollagen type I N-terminal propeptide (PINP), bone-specific alkaline phosphatase (BAP), CTX-I, and YKL-40 were determined by ELISA in a longitudinal study of 106 patients with metastatic PC during treatment with total androgen ablation or parenteral estrogen. Serum samples were collected with 3 months interval. Median observation time was 4.9 years (range, 3.6-6.2). A total of 78 patients died (64 within 7 months following the last blood sampling). RESULTS: After 6 months treatment, serum PINP, BAP, and YKL-40 decreased (P < 0.0001), but not serum CTX-I compared with baseline values. Univariate Cox analysis showed that serum PINP at 6 months [log transformed and treated as a continuous variable; hazard ratio (HR), 2.2; P < 0.0001], serum BAP (HR, 1.8; P < 0.0001), and serum CTX-I (HR, 2.4; P < 0.0001), but not serum YKL-40 (HR, 1.4; P = 0.16) were associated with survival. Multivariate Cox analysis including the biomarkers 6 months after the start of treatment showed that Soloway score (HR, 3.9; P = 0.013), WHO tumor grade (HR, 3.9; P = 0.004), and serum PINP (HR, 2.2; P < 0.0001) were independent prognostic variables of survival. Scoring the biomarkers during treatment as time-dependent covariates in univariate Cox regression analysis showed that increases in serum PINP (HR, 2.0; P < 0.0001), BAP (HR, 2.1; P < 0.0001), and YKL-40 (HR, 2.1; P < 0.0001) were predictors of early death. CONCLUSIONS: Serial monitoring of serum PINP, BAP, CTX-I, and YKL-40 in metastatic PC patients during hormonal treatment provided information of prognosis.

Optimisation of sandwich ELISA based on monoclonal antibodies for the specific measurement of pregnancy-associated plasma protein (PAPP-A) in acute coronary syndrome.

OBJECTIVES: PAPP-A has become the principal biochemical serum marker in first trimester screening for Down syndrome, the original data being based on results of a radioimmunoassay (RIA). Recent observations using sandwich ELISA technology have proposed PAPP-A as a potential marker in patients with acute coronary syndrome (ACS). The aims of the present study were to demonstrate (i) the importance of antibody specificity, (ii) the potential pitfalls in changing assay technology, (iii) the importance of strict definition of technology, and (iv) the application of a well-defined assay technology on sera from patients with ACS. DESIGN AND METHODS: Candidate monoclonal antibodies (Mab) were identified by immunohistochemistry, Western blot and the absence of positive signals (ELISA) with normal, non-pregnant serum as antigen source. The ELISA technology was standardized against the original PAPP-A RIA and the WHO reference preparation (WHO 78/610). Results different from those obtained by the original RIA led to ELISA modifications with respect to dilution buffer and enzymatic digestion of the Mab. RESULTS: The first generation ELISA revealed serum measurements from a pool of non-pregnant (n=103) individuals which, compared to the RIA, seemed to be false positive. The false positive reaction was abolished by addition of bovine serum (BS) to the dilution buffer. Subsequent analysis of individual sera (n=103) indicated that 7/103 were still false positive. This reaction was eliminated by introduction of F(ab')(2)-fragment of the indicator antibody. This modified ELISA revealed that serum PAPP-A levels in ACS were statistically significantly higher than in controls (p<0.001). Moreover, serum PAPP-A in ACS patients with ST-segment elevation (STEMI) were higher (p<0.001) compared to patients without ST-segment elevation (NSTEMI). Immunohistochemical analysis failed to identify PAPP-A in the atherosclerotic plaques.

Association between combined properdin and mannose-binding lectin deficiency and infection with Neisseria meningitidis.

BACKGROUND: Individuals genetically deficient of properdin are more susceptible to meningococcal disease. Likewise low concentration or decreased biological activity of mannose-binding lectin (MBL) is associated with higher incidence of bacterial infections during childhood. In this study we report our findings in a Danish family with a remarkably high incidence of meningococcal meningitis-in total four cases, one of them fatal. METHODS: Properdin and MBL were quantified by ELISA and the properdin gene was screened for sequence variations using denaturing high-performance liquid chromatography (DHPLC) and subsequent sequencing of abnormal patterns. The MBL gene was genotyped for the three known variant alleles (B, C and D) as well as three promoter polymorphisms (-221Y/X, -550H/L and +4P/Q). RESULTS: Two out of six males with undetectable properdin activity had meningitis. They had also low MBL serum levels or carried an MBL variant allele, whereas high MBL concentrations were measured in three out of four properdin deficient males--without meningitis. A splice site mutation in exon 10 (c.1487-2A>G) was found in the properdin gene and co segregated with biochemically measured properdin deficiency. CONCLUSION: Our results indicate that a combined deficiency of both properdin and MBL increases the risk of infection with Neisseria meningitidis and stress the importance of epistatic genetic interactions in disease susceptibility.

Prognostic value of PINP, bone alkaline phosphatase, CTX-I, and YKL-40 in patients with metastatic prostate carcinoma.

BACKGROUND: To examine the prognostic value of markers of bone metabolism (serum PINP, BAP, and CTX-I) and serum YKL-40 in metastatic prostate carcinoma (PC). METHODS: The biomarkers were determined by ELISAs in 153 metastatic PC patients before treatment with parenteral estrogen or total androgen ablation. The median follow-up was 4.9 years. One hundred fifteen patients died. RESULTS: The biomarkers were increased in the patients compared to controls (P < 0.001), and related to performance status and Soloway score (except YKL-40), but not to T-category and WHO tumor grade. PINP was elevated in 87%, BAP (55%), CTX-I (33%), and YKL-40 (43%). Univariate analysis showed an association to survival: PINP (HR = 1.6, P < 0.0001), BAP (HR = 1.4, P < 0.0001), CTX-I (HR = 1.7, P < 0.0001), and YKL-40 (HR = 1.4, P = 0.004). In multivariate Cox analysis performance status, WHO grade, Soloway score, PINP, and YKL-40 were independently predictive factors. CONCLUSIONS: High serum PINP, BAP, CTX-I, and YKL-40 are associated with poor outcome of metastatic PC patients.