The analysis of the complement activation product SC5 b-9 is applicable in neonates in spite of their profound C9 deficiency.

Native complement factors and complement activation products were measured in healthy neonates (n = 72) and in a group of infants with premature prolonged rupture of the membranes (PPROM) without sepsis (n = 10). Vitronectin concentration in normal cord blood was not correlated with gestational age, and the median value was 86.0% of adult values. This was markedly higher than other native complement factors studied (factor B: 35.9%, C4: 45.1%, C3: 56.2%). The concentration of C9 showed a positive correlation with gestational age and was very low, 10.8% of normal adult values in cord blood and 8.3% in the patients. Fifteen percent of the neonates had C9 levels lower than 2% of adult values. The complement activation products Bb and SC5 b-9 were significantly elevated in the patients (159% and 130% of control values, respectively), indicating alternative and terminal pathway activation. In contrast, C4 bc and C3 bc levels were not increased. The maximum amount of SC5 b-9 which could be generated in the neonatal sera by cobra venom factor was highly correlated with C9 concentration (rs = 0.86, p = 0.0001) The profound C9 deficiency found in neonates is correlated with gestational age, limits the capacity to form bacteriolytic C5 b-9 (m) and may predispose for severe invasive bacterial infection. The plasma level of SC5 b-9 under normal conditions was very low, only 0.3% (0.1%-3.0%) of the values obtained after CVF activation of the same samples. Therefore, we suggest that the analysis of SC5 b-9 is applicable also in neonates, in spite of their extremely low C9 levels.

Membrane attack complex formation on yeast as trigger of selective release of terminal complement proteins from human polymorphonuclear leukocytes.

It has recently been shown that measurable amounts of complement proteins, C6 and in particular C7, are released from human polymorphonuclear leukocytes (PMNs). The aim of the present study was to investigate the impact of opsonized Candida albicans on this release. Stimulation with opsonized C. albicans led to a rapid and sustained increase of C6 and C7 in the cell culture supernatant beginning within 5 min of placing in co-culture, whereas co-culture with unopsonized C. albicans or C. albicans mock-opsonized with inactivated human serum did not affect the release. In contrast, even after stimulation employing opsonized C. albicans, no release of the complement component C8 and only trace amounts of C9 were detected. The presence of the membrane attack complex (MAC) on C. albicans after opsonization was demonstrated by indirect immunofluorescence. Opsonization of C. albicans with human serum deficient in or depleted of a terminal complement component resulted in only minor stimulation of C6 and C7 release, although C3 deposition on the surface of C. albicans was not affected as determined by direct immunofluorescence. Detailed analyses with inactivated or deficient sera showed that detection of C6 and C7 was not due to insufficient washing of the opsonized yeast prior to co-culture and suggest that only a small proportion of these proteins was derived from the membrane bound and then cleaved off MAC. Thus, these findings imply that MAC on the fungal surface may represent an additional trigger for the release of C6 and C7 from PMNs, suggesting a new role for the terminal complement complex (TCC) on target membranes as modulator of PMN functions locally at the site of inflammation.

17 beta-Oestradiol and 1 alpha,25-dihydroxycholecalciferol modulate constitutive and bone matrix-induced interleukin-1 beta (IL-1 beta) production by peripheral blood mononuclear cells isolated from postmenopausal women.

Local production and release of interleukin-1 (IL-1) may be of importance for bone remodeling, since this cytokine is known to stimulate bone resorption. We have studied the effect of bone matrix constituents on IL-1 beta production by peripheral blood mononuclear cells (PBMCs) isolated from 20 postmenopausal non-osteoporotic women. Hydroxyapatite (0.5 mg/ml) and heat-denaturated collagen (25 micrograms/ml) stimulated IL-1 beta production 5-fold and 520-fold, respectively, compared to control (p < 0.01). In contrast, transforming growth factor-beta (TGF-beta, 10 ng/ml), a cytokine which is abundant in bone matrix, suppressed median IL-1 beta release to 13% of control value (p < 0.01). The bone matrix-induced changes in IL-1 beta production were modulated by 10 nmol/1 17 beta-oestradiol and 10 nmol/1 1 alpha,25-dihydroxy-cholecalciferol (1,25(OH)2D3). Specifically, 17 beta oestradiol stimulated constitutive IL-1 beta release with 89% (p < 0.01) and nullified the suppressive effect of TGF-beta. Moreover, 1,25(OH)2D3 had a synergistically stimulatory effect with both hydroxyapatite and collagen, although there was no effect of this hormone when added alone. The adherent cells were slightly more elongated after treatment with 1,25(OH)2D3 and collagen, while TGF-beta and 17 beta-oestradiol had no effect on cellular morphology. Addition of hydroxyapatite resulted in long and spindle-shaped cells, and phagocytosis of the particles occurred. The modulatory effects of oestrogen and vitamin D on constitutive and bone-matrix induced IL-1 beta production by PBMCs may be of importance for bone remodelling during postmenopausal bone loss and at a site of fracture.

Infection with human cytomegalovirus (HCMV) stimulates monocyte production of complement factor 3.

Complement biosynthesis in monocytes is stimulated by different microorganisms including Gram negative bacteria and yeasts. We have tested the effect of human cytomegalovirus (HCMV) on complement factor 3 (C3) production by cultured human monocytes. The monocytes were challenged with either a crude or a purified HCMV preparation obtained from the supernatant of HCMV-infected fibroblasts. When the monocytes were infected with 2 pfu/cell of virus and cultured for 2 days, the increase in C3 production compared to control ranged from 3% to 162%, median 62% (p < 0.01). However, crude HCMV was even more potent in stimulating C3 production, as the increase in C3 values ranged from 104% to 507%, median 247% (p = 0.001). This indicates the presence in the crude HCMV preparation of a substance which acts synergistically with HCMV on the C3 production. When monocytes were stimulated by lipopolysaccharide (LPS), a well known inducer of C3, infection with crude or purified HCMV did not further increase C3 production. Both HCMV and substances produced during the propagation of HCMV in fibroblasts are able to stimulate C3 production in monocytes. Complement production by inflammatory cells may be of importance in host resistance against viral infections.

[New diagnostic and therapeutic possibilities in neonatal sepsis]. / Nye muligheter for diagnostikk og behandling ved neonatal sepsis.

Neonatal sepsis remains a major clinical problem in neonatology, with high morbidity and mortality rates. The host defence against infections is immature in the newborn infant, and this makes the child more susceptible to invasive infection. The neutrophil storage pool and various granulocyte functions are impaired. In addition, the levels of immunoglobulins and complement are low. The detection of raised levels of complement activation products and cytokines may be of diagnostic help at an early stage of neonatal infection. Rapid treatment with antibiotics is essential for a favourable outcome. Possible adjuvant treatment may be to reduce the relative immunodeficiency by giving immunoglobulins or colony-stimulating factors which increase the production of leukocytes. Further, the potent inflammatory reaction initiated by the microorganisms may be suppressed by various therapies. In spite of much research in this field, no such adjuvant treatment has so far been shown to improve the outcome of neonatal sepsis.

Human monocytes stimulation by particles of hydroxyapatite, silicon carbide and diamond: in vitro studies of new prosthesis coatings.

Aseptic loosening due to wear and debris formation constitutes the major problem in longevity of joint replacements. Diamond coated onto the prosthesis surface may reduce wear, owing to its excellent tribological properties. A thin diamond coating may be brittle, and we plan eventually to reinforce it with silicon carbide whiskers (SiC). In the present study we compared particles of diamond, SiC and hydroxyapatite (HA) in serum-free cultures of human monocytes. All particles were found to be phagocytozed, and monocyte morphology changed except after the ingestion of diamond. Interleukin-1 beta production was increased on average 30-fold and 38-fold in cultures exposed to HA and SiC, respectively, compared to control and diamond cultures (n = 6). Addition of the phagocytosis inhibitor cytochalasin B inhibited the morphological changes of the monocytes and reduced interleukin-1 beta production. In some experiments particles of polymethylmethacrylate were also included, and the interleukin-1 beta stimulation was in the same range as after HA and SiC stimulation. The results show that diamond particles in serum-free monocyte culture are inert, while SiC and HA have a stimulatory effect comparable to polymethylmethacrylate. With its excellent tribological and biocompatible properties, future studies with diamond coating are warranted.

Serum clusterin and vitronectin in alcoholic cirrhosis.

Clusterin and vitronectin are multifunctional regulatory proteins which both serve as complement lysis inhibitors. Previous data have strongly suggested that serum vitronectin is mainly produced in the liver, whereas the biosynthetic origin for serum clusterin has not been determined. In the present study we aimed to determine the role of the liver in producing these proteins and to evaluate the proteins as possible markers of liver failure. We therefore quantified clusterin and vitronectin in serum from patients suffering from alcoholic liver cirrhosis (n = 83), and in serum-free culture supernatants from the hepatoma cell line HepG2. The median clusterin concentration was 0.20 g/l in cirrhosis and 0.37 g/l in the controls, whereas corresponding vitronectin values were 0.19 and 0.26 g/l, respectively. The concentration of both proteins showed significant correlation (p < 0.0001) with disease severity and with established plasma markers of hepatic synthetic function, such as albumin and prothrombin complex. The clusterin level, but not the vitronectin level, correlated with survival (p = 0.005). The rates of synthesis of clusterin, vitronectin and C3 from HepG2 cells were 0.02, 0.21 and 1.9 micrograms/10(6) cells/24 h, respectively. From the present data we conclude that clusterin (as vitronectin and C3) is mainly produced in the liver and may be a useful marker in the evaluation of severity of liver disease and prognosis of patients with alcoholic cirrhosis.

Respiratory burst of intestinal macrophages in inflammatory bowel disease is mainly caused by CD14+L1+ monocyte derived cells.

Macrophages play a crucial role in intestinal mucosal defence, forming dense subepithelial aggregates, particularly in the colon. One of their important bactericidal mechanisms is production of oxygen radicals but this may damage the intestinal epithelium, perhaps as an early step in inflammatory bowel disease (IBD). The potential for release of oxygen radicals from mucosal macrophages in IBD was measured and whether a difference exists between newly arrived (CD14+L1+) monocyte-like cells and resident macrophages (CD14(-)L1-), without or with additional priming in vitro, was investigated. Lamina propria mononuclear cells from six patients with IBD and five with a normal intestine were isolated with an ethylenediaminetetra acetic acid/collagenase/dispase technique and cultured for three days. The cells were tested with or without interferon gamma (200 U/ml) priming in the presence or absence of lipopolysaccharide (1 microgram/ml) for the last 48 hours in cultures. Samples from inflamed IBD mucosa depleted of CD14+ cells by immunomagnetic beads were compared with their undepleted counterparts and with samples from virtually normal mucosa from the same patients. The production of oxygen radicals was measured as the amount of reduced cytochrome C 2.5 hours after triggering with phorbol 12-myristate 13-acetate. The oxygen radical production in macrophages from moderately or severely inflamed mucosa was reduced by median 69% (range 22%-79%, p < 0.027) after depletion of CD14+ cells, reaching a level similar to that found for virtually normal samples from the same IBD patients. Furthermore, this production did not increase significantly in mucosal macrophages from normal reference mucosa and from virtually normal or inflamed IBD mucosa after priming with interferon gamma with or without addition of lipopolysaccharide. Upregulation of a respiratory burst in subepithelial resident macrophages os not a likely pathogenetic step in IBD. The increased oxygen radical production shown by macrophages from IBD lesions can, however, be ascribed to recently extravasated CD14+L1+ monocyte-like cells. Inhibition of extravasation of these reactive cells may form part of a therapeutic approach in the future.

There is no difference in spontaneous and 17 beta-estradiol-induced interleukin-1 beta release by peripheral blood mononuclear cells from nonosteoporotic women with different rates of early postmenopausal bone loss.

Interleukin-1 (IL-1) is a potent stimulator of bone resorption, and a causal role for IL-1 has been suggested in postmenopausal bone loss. We have examined IL-1 beta release in vitro by peripheral blood mononuclear cells (PBMC) isolated from nonosteoporotic women 9-15 yr after menopause. These women had presented 6 yr previously with significant differences in the rate of early postmenopausal bone loss. Ten women with low rates of bone loss (median 2.0% per year) and 10 women with high rates of bone loss (median 4.9% per year) were included in the study. The women with a high rate of bone loss had a significantly lower bone mass of the lumbar vertebrae compared with that of the other group, but there were no differences in biochemical markers of bone metabolism between the groups (pyridinoline/creatinine ratio in urine and collagen 1 c-terminal telopeptide and bone gla protein in serum). Moreover, there was no difference in spontaneous IL-1 beta release by PBMCs between the two groups and no correlation between IL-1 beta release and present bone turnover, as judged by biochemical markers. Treatment of PBMCs with 10 nmol/L 17 beta-estradiol in vitro significantly stimulated IL-1 beta production in both groups. We conclude that IL-1 beta production by PBMCs in vitro does not correlate with the rate of early postmenopausal bone loss.

Human polymorphonuclear leukocytes store large amounts of terminal complement components C7 and C6, which may be released on stimulation.

Secretion of the C factors C7, C6, and C3 by human polymorphonuclear leukocytes (PMNs) and PBMCs was studied by ELISA and immunoblot. The release of C7 and C6 by PMNs during 24 h of culture was 16-fold and 6-fold higher, respectively, than the C3 release, with median concentrations of 50.2 ng/ml, 18.3 ng/ml, and 3.1 ng/ml, respectively. In PBMC cultures, C release was considerably lower, and there was a different secretory pattern with a 6-fold higher release of C3 compared with C7 and C6. Stimulation with PMA led to a more rapid and complete secretion of the components to the culture media, whereas treatment with unopsonized Candida species did not affect the release. PMN release of C factors was not dependent on protein biosynthesis, and there was no indication of a selective uptake of C7 from serum as demonstrated by incubating PMNs from a subject with allotype C7 N in C7 M serum. Thus, the C components were probably produced by the PMNs or their bone marrow precursors before ex vivo culture. In cell lysates of freshly isolated cells, median C7, C6, and C3 contents of 1 x 10(7) PMNs were 149.7, 60.1, and 10.4 ng/ml, respectively, whereas the corresponding values for 1 x 10(7) PBMCs were 3.2, 2.6, and 14.6 ng/ml, respectively. The C6 and C7 were shown to incorporate into the terminal complement complex, and their molecular integrity was supported by identical m.w. to C6 and C7 present in normal serum. PMNs may represent a major source of C7 and C6 and may be more important than monocytes or macrophages in contributing terminal C components at a site of inflammation. This suggests a new role for the PMN as a C membrane attack modulator.

Various Candida and Torulopsis species differ in their ability to induce the production of C3, factor B and granulocyte-macrophage colony-stimulating factor (GM-CSF) in human monocyte cultures.

The incidence of infections with Candida albicans and also with non-albicans yeast species is increasing rapidly, particularly in immunocompromised patients. Eight Candida and Torulopsis species were compared for their ability to stimulate production of complement components C3 and factor B by monocytes. In addition, the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) was determined, because this cytokine affects monocyte complement production. The highest ranked pathogenic yeasts, i.e., C. albicans, C. tropicalis and C. parapsilosis, were the most effective inducers of C3, factor B and GM-CSF production. C. krusei and T. glabrata showed intermediate activity, whereas C. kefyr, C. guilliermondii and T. candida had only a moderate stimulatory effect on C3 production and did not affect either factor B or GM-CSF release. The stimulated cytokine and complement production in response to the yeasts was highly variable in monocytes from different donors, but there was a consistent inverse relationship between C3 and GM-CSF concentrations in the monocyte supernates. This is in agreement with the previously described suppressive effect of GM-CSF on yeast-induced C3, but not factor B production. The monocyte responses elicited by a specific yeast species may be linked to its pathogenicity, and may also explain the predilection of some yeasts for particular underlying diseases.

Polymyxin B stimulates production of complement components and cytokines in human monocytes.

Polymyxin B (PmB), an agent often used to neutralize the effects of bacterial lipopolysaccharide (LPS), was shown to exert a dose-dependent stimulatory effect on the biosynthesis of C3, factor B, interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in human monocytes. A low dose of PmB (1 to 5 micrograms/ml) efficiently suppressed the LPS-induced (1 or 100 ng/ml) production of IL-6, GM-CSF, and factor B, but not the C3 production induced by 100 ng of LPS per ml. A reduced level of GM-CSF may have contributed to the persisting high C3 concentrations and the apparent lack of LPS inhibition in the latter situation, since GM-CSF is an inhibitor of monocyte C3 biosynthesis.

Transforming growth factor beta modulates C3 and factor B biosynthesis and complement receptor 3 expression in cultured human monocytes.

Complement biosynthesis in monocytes is stimulated by different pathogens and modulated by a variety of cytokines, but little is known about the possible effect of transforming growth factor beta (TGF-beta) on this monocyte function. We therefore studied the effect of TGF-beta 1 and TGF-beta 2 on constitutive, lipopolysaccharide (LPS)- and Candida albicans-induced monocyte biosynthesis of complement components C3 and factor B. Under all three conditions, both forms of TGF-beta (20 ng/ml) induced a two- to fourfold increase in C3 concentration in monocyte supernatants harvested after 2 or 5 days of cell culture, an effect that was abrogated by cycloheximide. In contrast, constitutive and pathogen-induced production of factor B was suppressed by TGF-beta. The effects of TGF-beta on complement production were neutralized by a monoclonal anti-TGF-beta antibody. Moreover, TGF-beta suppressed the pathogen-induced release of granulocyte-macrophage colony-stimulating factor and down-regulated the expression of complement receptor 3 (CD11b/CD18), while the expression of CD11a/CD18, a related beta 2 integrin, was unaffected. These novel effects of TGF-beta emphasize the immunomodulatory significance of this cytokine.

Granulocyte-macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, suppresses basal and lipopolysaccharide-stimulated complement factor production in human monocytes.

Monocyte/macrophage contribution of C biosynthesis is important, particularly during inflammation. Since granulocyte-macrophage CSF (GM-CSF) and macrophage-CSF (M-CSF) exert a variety of stimulatory effects on monocyte/macrophage functions in vitro, we studied their impact on the biosynthesis of the C components C3 and factor B by human monocytes in culture. GM-CSF at doses of 10 ng/ml and higher inhibited the basal C3 synthesis. This effect was most pronounced when the cytokine was added to freshly isolated monocytes. No effect was found on the basal production of factor B. Furthermore, GM-CSF abrogated the LPS-stimulated production of both C3 and factor B. These suppressive effects were neutralized by a polyclonal anti-GM-CSF antibody. Moreover, when anti-GM-CSF was added to unstimulated or LPS-stimulated cells, their C3 production increased. This indicates that both spontaneous and LPS-triggered release of monocyte-produced GM-CSF has an autocrine function in regulating monocyte C3 biosynthesis. GM-CSF also down-modulated the expression of CD14 at an early stage of cell culture. This might be the mechanism through which the LPS-effects are suppressed because CD14 has been shown to be a LPS receptor. Contrary to this, M-CSF at doses of 100 U/ml and higher stimulated the synthesis of C3, whereas the basal production of factor B and the LPS-stimulated production of C3 and factor B were unaffected. Granulocyte-CSF (G-CSF) did not influence monocyte C biosynthesis, and neither anti-M-CSF nor anti-G-CSF influenced the LPS-induced C3 production. The effects of GM-CSF and M-CSF on C biosynthesis may be important in regulating the availability of C components during an inflammatory response, and these observations may also have implications for the clinical use of CSF.

Increased C3 production in human monocytes after stimulation with Candida albicans is suppressed by granulocyte-macrophage colony-stimulating factor.

Activation of the complement system is an important part of host resistance against fungal infections. When human monocytes, cultured for 2 days or more, were treated in vitro with Candida albicans for 24 h, an enhancement of their biosynthesis of the complement components C3 and factor B was found. However, when C. albicans was administered to freshly isolated monocytes, a consistent stimulation of factor B biosynthesis occurred, while the C3 production was increased in about 50% of the donors. C. albicans also induced the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from the cultured cells, apparently in larger amounts in the donors in whom no stimulation of C3 production was found. An antibody to GM-CSF administered with the yeast at the initiation of the monocyte culture caused an increase in the C3 production. Furthermore, when monocytes were treated with recombinant human GM-CSF either at the same time as or 4 days prior to the addition of C. albicans, the increase in C3 production was suppressed or neutralized, while factor B biosynthesis was unaffected. Taken together, these results indicate that monocytes respond to C. albicans with an increased production of complement factors. This may be an important mechanism both for opsonization of the fungus and for initiation of an inflammatory reaction. At an inflammatory site, this complement response may be suppressed by locally produced GM-CSF.

Rupture of the trachea: an unusual complication of delivery.

We describe a neonate whose trachea ruptured during delivery. The baby developed respiratory symptoms shortly after delivery and became dramatically ill after 6 h. This particular birth trauma appears to be very unusual: there are few cases reported in the literature. Rupture of the trachea should be considered as a differential diagnosis in a neonate who develops pneumomediastinum after a complicated delivery.