SummaryBackgroundInfliximab (IFX) is a chimeric murine/human anti‐TNF antibody (Ab) used for the treatment of Crohn's disease (CD) and ulcerative colitis (UC). Loss of response is common and associated with development of anti‐IFX Abs during ongoing therapy. However, human anti‐murine immunoglobulin Abs are common and may cross‐react with the murine part of IFX.AimTo investigate if Abs binding to IFX's Fab region (IFX‐Fab) are present in IBD patients before exposure to IFX, and whether they predict efficacy and safety of IFX therapy.MethodsObservational, retrospective cohort study of patients with CD (n = 29) and UC (n = 22).ResultsPre‐treatment levels of IFX‐Fab reactive IgG Abs were significantly lower in CD patients in remission after 1 year of maintenance IFX (median 91 mU/L, n = 8) than in the rest of the patients (639 mU/L, n = 21; P < 0.01), and lower than in patients with secondary loss of response in particular (692 mU/L, n = 7; P < 0.01). A cut‐off concentration of <439 mU IFX‐Fab reactive IgG Ab per litre comprised all patients who later obtained long‐term sustained remission on IFX (sensitivity 100%, specificity 67%). Similar trends were observed in UC. The pre‐treatment levels of IFX‐Fab reactive IgG Abs were markedly higher in patients developing infusion reactions to IFX (1037 mU/L, n = 7) than in the remaining patients (349 mU/L, n = 44; P = 0.036).ConclusionsIFX‐Fab reactive IgG antibodies present in serum from IBD patients before infliximab therapy associate with lack of long‐term efficacy and safety. Assessments of such antibodies may help clinicians to choose between treatment with infliximab and more humanised agents.

Stem cell transplantation is a promising alternative to whole liver transplantation. However, identification and characterization of true liver stem cells has been problematic, because there are no known specific markers. Previously, we reported long-term liver replacement of normal adult rat liver after transplanting unfractionated embryonic day (ED)14 fetal liver stem/progenitor cells (FLSPC). However, hematopoietic stem cells, a major component in unfractionated fetal liver cell preparations, engraft in other organs and may cause undesirable side effects or complications. Therefore, for most clinical applications, highly purified cell fractions will be needed.

Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34(+) cells in 10 of 12 MDS samples compared with CD34(+) cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34(+) hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.

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 polymorphisins (-221 Y/X, -550 H/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. (c) 2005 Elsevier Ltd. All rights reserved.

Fibrosis is frequent in endometriotic lesions; procollagen aminoterminal propeptide type I (PINP) is a degradation product of collagen synthesis. This study aims to measure the levels of PINP in peritoneal fluid (PF) and plasma (PL) of women with and without endometriosis, in order to evaluate whether they correlate with the severity of the disease. Cross-sectional study. PF and PL samples were obtained from 93 women with endometriosis and 68 controls (infertility, n=16; uterine myomas, n=25; ovarian cyst, n=21; pelvic pain, n=5; tubal sterilization, n=1). Only patients of reproductive age were included in the study; none of the subjects had used hormonal therapies in the 6 months before surgery. The extent of endometriosis was scored using the revised American Fertility Society (rAFS) classification. The phase of the cycle was determined according to the menstrual history and progesterone levels. The serum concentration of PINP was measured by a sandwich enzyme-linked immunosorbent assay utilizing purified alpha 1-chain specific rabbit antibodies. PINP concentration was not normally distributed as determined by the Shapiro-Wilks test; therefore, the Mann-Whitney U test was used to compare its concentration between the two groups. The relationship between PL and PF PINP concentration was evaluated by the non-parametric Spearman correlation coefficient. No significant difference was observed in the mean age of patients in the two groups. PINP concentration was significantly higher in PF in women with severe endometriosis (rAFS stage III-IV, mean ± SEM, 1067.2±198.4 ng/mL) than in mild endometriosis (rAFS stage I-II, 266.5±59.7 ng/mL) (p<0.001) and in controls subjects (p=0.015). PINP concentration was significantly higher in the PF than in PL both in women with endometriosis (p<0.001) and in controls (p<0.001). No significant correlation was observed between PL and PF PINP concentration both in women with endometriosis (p=0.780) and in controls (p=0.970). No significant change in PINP PF and PL levels was observed during the menstrual cycle. By contrast, no significant difference (p=0.328) was observed in the PINP PF concentration between all women with endometriosis (780.4±137.0 ng/mL) and controls (526.5±135.1 ng/mL). No significant difference (p=0.967) was observed in PINP PL concentration between women with endometriosis (47.5±2.5 ng/mL) and controls (50.0±2.9 ng/mL). Similarly, no significant difference was observed in PL PINP concentration between women with mild (42.0±4.7 ng/mL) and severe endometriosis (49.5±3.0 ng/mL). PF PINP levels are significantly higher than PL levels both in women with and without endometriosis. Women with severe endometriosis have higher PINP PF levels than subjects with mild endometriosis and controls, these observations being in accord with previous studies showing that PINP levels are associated with increased fibroblast activity.

No studies to date have reported activation of satellite cells in vivo in human muscle after a single bout of high intensity exercise. In this investigation, eight individuals performed a single bout of high intensity exercise with one leg, the contralateral leg being the control. A significant increase in mononuclear cells staining for the neural cell adhesion molecule (N-CAM) and fetal antigen 1 (FA1) were observed within the exercised human vastus lateralis muscle on days 4 and 8 post exercise. In addition, a significant increase in the concentration of the FA1 protein was determined in intramuscular dialysate samples taken from the vastus lateralis muscle of the exercising leg (day 0: 1.89 +/- 0.82 ng ml(-1); day 2: 1.68 +/- 0.37 ng ml(-1); day 4: 3.26 +/- 1.29 ng ml(-1), P < 0.05 versus basal; day 8: 4.68 +/- 2.06 ng ml(-1), P < 0.05 versus basal and control). No change was noted in the control leg. Despite this increase in N-CAM- and FA1-positive mononuclear cells, an increased expression of myogenin and the neonatal isoform of the myosin heavy chain (MHCn) was not observed. Interestingly, myofibre lesions resulting from extensive damage to the proteins within the myofibre, particularly desmin or dystrophin, were not observed, and hence did not appear to induce the expression of either N-CAM or FA1. We therefore propose that satellite cells can be induced to re-enter the cell growth cycle after a single bout of unaccustomed high intensity exercise. However, a single bout of exercise is not sufficient for the satellite cell to undergo terminal differentiation.

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p < 0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti-bovine serum albumin antibodies in the donor sera (r = 0.639; p < 0.0001; n = 103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two-site immunoassays. The apparent presence of human anti-mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.

Serum concentrations of placental protein 14 (PP14), steroids and gonadotrophins were related to the outcome of IVF/intracytoplasmic sperm injection in 195 normogonadotrophic women subjected to the long protocol gonadotrophin-releasing hormone agonist (GnRHa; buserelin) pituitary down-regulation protocol and gonadotrophin stimulation (HMG or rFSH). Pituitary down-regulation was initiated on cycle day 21 and the patients were randomized to either intranasal or s.c. administration of buserelin. After 14 days of down-regulation, the patients were randomized on stimulation day 1 (S1) to ovarian stimulation with 225 IU per day of either human menopausal gonadotrophin (HMG) or recombinant FSH (rFSH) for a fixed period of 7 days. The daily gonadotrophin dose was adjusted on the following day according to ovarian response. Patient's blood was sampled for PP14 and hormone analysis on cycle days 21, S1, S8 and on the day of oocyte retrieval. Mean concentrations of PP14 on day 21 of the cycle were significantly lower in conception than in non-conception cycles, whereas progesterone and oestradiol were similar in conception and non-conception cycles. PP14 concentrations on the first day of stimulation and at oocyte retrieval were significantly higher in conception than in non-conception cycles, whereas concentrations after 8 days of stimulation were similar. Neither mode of GnRHa administration nor type of gonadotrophin significantly influenced PP14 concentrations throughout ovarian stimulation. Circulating PP14 is thus an important physiological signal of the fertility status of the individual in the cycle antecedent to and during ovarian stimulation. Measuring mid-luteal serum PP14 may offer a clinical test helping to decide if infertility treatment should be initiated in the subsequent cycle.

Hepatic regeneration from toxic or surgical injury to the adult mammalian liver, endorses different cellular responses within the hepatic lineage. The molecular mechanisms determining commitment of a cell population at a specific lineage level to participate in liver repair as well as the fate of its progeny in the hostile environment created by the injury are not well defined. Based on the role of the Notch/Delta/Jagged system in cell fate specification and recent reports linking Notch signaling with normal bile duct formation in mouse and human liver, we examined the expression of Notch1, Notch2, Notch3, Delta1, Delta3, Jagged1, and Jagged2, and delta-like protein/preadipocre factor 1/fetal antigen 1 (dlk) in four well-defined experimental rat models of liver injury and regeneration. Although Delta3 and Jagged2 were undetectable by reverse transcriptase-polymerase chain reaction and Northern blot, we observed the most significant up-regulation of all other transcripts in the 2-acetylaminofluorene-70% hepatectomy (AAF/PHx) model, in which liver mass is restored by proliferation and differentiation of transit-amplifying ductular (oval) cells. The most profound change was observed for dlk. Accordingly, immunohistochemical analyses in the AAF/PHx model showed a specific expression of dlk in atypical ductular structures composed of oval cells. Delta-like protein was not observed in proliferating hepatocytes or bile duct cells after partial hepatectomy or ligation of the common bile duct whereas clusters of dlk immunoreactive oval cells were found in both the retrorsine and the AAF/PHx models. Finally, we used dlk to isolate alpha-fetoprotein-positive cells from fetal and adult regenerating rat liver by a novel antibody panning technique.

Increases in procollagen processing within skeletal muscle have previously been reported in small animal models only. While indirect measurements in humans have suggested an increase procollagen processing, no intra-skeletal muscle measurements have confirmed these findings. In this study, eight young healthy male subjects performed a single bout of unaccustomed high intensity eccentric exercise on one leg, with the contralateral leg being the control. A significant increase in the muscle interstitial concentration of the N-terminal propeptide of procollagen type I (PINP) was observed (day 0: 1.96±0.44 ng ml−1, day 2: 1.94±0.32 ng ml−1, day 4: 3.90±1.03 ng ml−1, day 8: 7.23±2.34 ng ml−1*, *P<0.05 vs. basal and control) with no change being noted in the control leg. By day 2 post-exercise, an increase in the histological immunoreactivity of PINP and the N-terminal propeptide of procollagen type III (PIIINP) was also shown in the exercising leg only. Further, from day 2 post-exercise, immunoreactivity for tenascin C and reactive macrophages (CD68+ cells) was seen within the perimysial and endomysial connective tissue of the exercising leg only, indicating a high mechanical load and inflammation. This study shows that following a single bout of high intensity eccentric exercise there is an increase in procollagen processing within skeletal muscle in humans.

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.

Circulating connective tissue components including the aminoterminal propeptides of type III collagen (PIIINP), type I collagen (PINP) and hyaluronan were determined in patients hospitalised for pneumonia of suspected bacterial origin. Ninety patients were included, 64 of these were followed prospectively for up to 21 days after initiation of therapy. Serum PIIINP was determined by RIA, s-PINP by ELISA, and s-hyaluronan by a radiometric assay. S-PIIINP rose significantly above the zero value within 24 h in both pneumococcal pneumonia (T0: 5.3 microg/l, 95% CI: 2.7-8.1 microg/l vs. T1: 6.7 microg/l, 95% CI: 3.8-9.1, P<0.01) and in pneumonia of unknown aetiology (T0: 4.0 microg/l, 95% CI: 3.6-4.8 vs. T1: 4.5 microg/l, 95% CI: 3.8-5.1, P<0.05) followed by a gradual decline. At T1, S-PIIINP was higher in pneumococcal pneumonia compared with pneumonia of unknown aetiology (P<0.05). By contrast, s-PINP tended to decline within 24 h in both pneumococcal pneumonia (T1: 30 microg/l, 95% CI: 23-40, ns) and in pneumonia of unknown aetiology (T1: 32 microg/l, 95% CI: 22-42, ns) followed by a steady increase. The PINP antigen size distribution remained constant throughout the follow-up period. S-hyaluronan in pneumococcal pneumonia paralleled s-PIIINP reaching a peak value on day 1 (121 microg/l, 95% CI: 65-191, P=0.38). There was a positive correlation between s-PIIINP and C-reactive protein (CRP). The study demonstrates, that community-acquired pneumonia elicits a differentiated mesenchymal response, which is turned down in response to successful antibiotic therapy.

SP-D is a lectin involved in the first line of defense against microorganisms. It is primarily found in the lung but also at extrapulmonary sites and in the circulation. An immunoassay for the quantification of SP-D in serum was established and the SP-D concentration was measured in consecutive blood samples from 61 patients hospitalized for community-acquired pneumonia of suspected bacterial origin. On the day of admission to the hospital the serum SP-D concentration was significantly lower than that in healthy subjects. On day 5, the SP-D concentration had increased on average three times the concentration on admission and then slowly declined toward normal levels. CRP was measured simultaneously but no correlation was observed between concentrations of SP-D and CRP. The results show a wide range of serum SP-D concentration in healthy volunteers and indicate that significant changes occur during pulmonary infection.

Preadipocyte factor-1 (Pref-1)/delta-like protein/fetal antigen-1 (FA1) is a member of the epidermal growth factor-like family. It is widely expressed in embryonic tissues, whereas in adults it is confined to the adrenal gland, the anterior pituitary, the endocrine pancreas, the testis and the ovaries. We have previously cloned Pref-1 from neonatal rat islets stimulated by GH. The aim of the present study was to elucidate the biosynthesis and release of Pref-1/FA1 in beta-cells and to determine if Pref-1/FA1 is mediating the mitogenic effect of GH in insulin-producing cells. First we studied the biosynthesis and processing of Pref-1 to the soluble form, FA1, in pancreatic islets and insulinoma cells transfected with Pref-1 cDNA. We measured the release of FA1 by ELISA and the possible effect of FA1 in GH-stimulated beta-cell proliferation by incorporation of bromodeoxyuridine (BrdU) in insulin-positive islet cells. We found that Pref-1 was synthesized in normal islets and in RINm5F insulinoma cells and released into the medium in two forms, of which one corresponded to FA1. Both the expression of the mRNA for Pref-1 and the release of the soluble form(s) were stimulated by GH and prolactin (PRL). Whereas 2 h exposure to high glucose or 3-isobutyl-1-methylxanthine stimulated insulin release, only a small change was seen in FA1 release, suggesting that the FA1 is released by a different pathway than insulin. However, long-term exposure (48 h) to high glucose increased FA1 secretion, indicating that FA1 is regulated by glucose. Neither FA1 nor conditioned medium from GH-stimulated islets depleted for GH was able to increase beta-cell replication and overexpression of Pref-1 resulted in attenuated proliferation of the RINm5F cells. By immunocytochemistry of GH-stimulated islet cells no correlation between high Pref-1 expression and BrdU incorporation was observed and there was an inverse relationship between the levels of insulin and Pref-1. These results indicate that Pref-1/FA1 is not mediating the mitogenic effect of GH and PRL. Therefore the function of Pref-1 in the beta-cell remains unknown.