Heat-labile Serum Factors Research Articles

FIV vaccine with receptor epitopes results in neutralizing antibodies but does not confer resistance to challenge

Feline immunodeficiency virus (FIV) is the feline analogue to human immunodeficiency virus (HIV) and utilizes parallel modes of receptor-mediated entry. The FIV surface glycoprotein (SU) is an important target for induction of neutralizing antibodies, and autoantibodies to the FIV binding receptor (CD134) block infection ex vivo; thus highlighting the potential for immunotherapies which utilize anti-receptor antibodies to block viral infection. To determine whether vaccination with CD134-SU complexes could induce protection against FIV infection, cats (n = 5 per group) were immunized with soluble CD134, recombinant FIV-SU protein, and/or CD134+SU complexes. Two trials were performed with different antigen combinations and vaccination schedules. In vivo generation of anti-CD134 and anti-SU IgG antibodies was measured, and in vitro neutralization assays were conducted. Immunization induced production of anti-CD134 and anti-SU antibodies that significantly inhibited FIV infection in vitro. However, no vaccine combination protected cats from FIV infection, and neat serum from vaccinated cats enhanced FIV growth in vitro. CD134+SU vaccinated cats exhibited increased CD4:CD8 ratio immediately prior to challenge, and antibodies were much more efficiently generated against vaccine by-products versus target antigens. Results suggest vaccination against viral and cryptic receptor epitopes yields neutralizing antibodies that synergistically inhibit FIV infection in vitro. Factors contributing to vaccine failure may include: (1) Heat-labile serum factors that enhance viral replication, (2) changes in circulating target cell populations induced by vaccination, and (3) weak immunogenicity of neutralizing epitopes compared to off-target vaccine components. Results reinforce the need to monitor vaccine preparation components and avoid non-specific immune stimulation during vaccination.

Contribution of the complement Membrane Attack Complex to the bactericidal activity of human serum

Direct killing of Gram-negative bacteria by serum is usually attributed to the Membrane Attack Complex (MAC) that is assembled upon activation of the complement system. In serum bactericidal assays, the activity of the MAC is usually blocked by a relatively unspecific method in which certain heat-labile complement components are inactivated at 56°C. The goal of this study was to re-evaluate MAC-driven lysis towards various Gram-negative bacteria. Instead of using heat-treatment, we included the highly specific C5 cleavage inhibitor OmCI to specifically block the formation of the MAC. Using a C5 conversion analysis tool, we monitored the efficacy of the inhibitor during the incubations. Our findings indicate that ‘serum-sensitive’ bacteria are not necessarily killed by the MAC. Other heat-labile serum factors can contribute to serum bactericidal activity. These unidentified factors are most potent at serum concentrations of 10% and higher. Furthermore, we also find that some bacteria can be killed by the MAC at a slower rate. Our data demonstrate the requirement for the use of specific inhibitors in serum bactericidal assays and revealed that the classification of serum-sensitive and resistant strains needs re-evaluation. Moreover, it is important to determine bacterial viability at multiple time intervals to differentiate serum susceptibility between bacterial species. In conclusion, these data provide new insights into bacterial killing by the humoral immune system and may guide future vaccine development studies for the treatment of pathogenic serum-resistant bacteria.

The uptake by blood-borne phagocytes of monosodium urate is dependent on heat-labile serum factor(s) and divalent cations

Accumulation in tissues of post-apoptotic cells is a feature frequently observed in patients with systemic lupus erythematosus and in murine models of systemic autoimmune diseases. One of the endogenous danger molecules released by secondarily necrotic cells is monosodium urate (MSU), which is already established to be the causative agent of gout. Here, we show that MSU is taken up by eosinophils, neutrophils and monocytes in a process involving (a) heat-labile serum factor(s) and divalent cations. The uptake induces the release of the pro-inflammatory cytokines IL-1β/IL-18/TNFα and IL-6/IL-8 by monocytes and PMN, respectively.

Phagocytosis of Apoptotic Cells by Neutrophil Granulocytes: Diminished Proinflammatory Neutrophil Functions in the Presence of Apoptotic Cells

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-gamma results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-alpha and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-alpha, which was independent of NF-kappaB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

Mechanisms of Pancreatic β-Cell Growth and Regeneration: Studies on Rat Insulinoma Cells

Information about the mechanism of beta-cell growth and regeneration may be obtained by studies of insulinoma cells. In the present study the growth and function of the rat insulinoma cell lines RINm5F and 5AH were evaluated by addition of serum, hormones, and growth factors. It was found that transferrin is the only obligatory factor whereas growth hormone, epidermal growth factor, fibroblast growth factor, and TRH had modulating effects. A heat-labile heparin binding serum factor which stimulated thymidine incorporation but not cell proliferation was demonstrated in human serum. Measurements of insulin mRNA content showed that the insulinoma cells only contained about 2% of that of normal rat beta-cells. These results are discussed in relation to the role of growth factors, oncogenes, and differentiation in the growth and regeneration of beta-cells.

Role of heat-labile serum factor or host complement in the inhibition ofPlasmodium falciparumsporogonic stages inAnopheles stephensiby gametocyte carriers' serological factors

This study investigated the significance of serum complement on transmission-reducing activity (TRA) of field sera from 24 infected Plasmodium falciparum gametocyte carriers (from Cameroon) against cultured NF54 P. falciparum. Laboratory-reared Anopheles stephensi were given infectious blood meals prepared either with sera from naïve Dutch donor (AB type) or pair-matched field serum samples, both with and without active complement. TRA of serum factors and host complement on mosquito infection rate and oocyst intensity were divided into the various components involved in the early stages of sporogony. The majority (>80%) of sera tested showed positive antibody titres to Pfs230, the relevant complement-dependent target of transmission-reducing mechanisms. Regardless of the presence of active complement, bloodmeals with field sera exhibited significantly lower infection rates and oocyst intensity than the control group. Serological reactivity in Capture-ELISA against Pfs230 was significantly correlated with the reduction of parasite infectivity. Contrary to our expectation, the presence of active complement in the mosquito bloodmeal did not increase parasite losses and therefore the magnitude of transmission reduction by individual immune sera. Our findings on P. falciparum are consistent with previous studies on animal hosts of Plasmodium, indicating that early P. falciparum sporogonic stages may be insensitive to the antibody-dependent pathways of complement in human serum.

Phagocytosis of apoptotic cells by macrophages from NOD mice is reduced.

Macrophages limit inflammatory responses by clearing apoptotic cells. Deficiencies in apoptotic cell phagocytosis have been linked to autoimmunity. In this study, we determined the efficiency with which macrophages from diabetes-prone NOD and diabetes-resistant NOR, Idd5, Balb/c, and C57BL/6 mice phagocytose apoptotic thymocytes and NIT-1 insulinoma cells. Peritoneal and bone marrow-derived macrophages from NOD mice engulfed fewer apoptotic thymocytes than macrophages from Balb/c mice (P < 0.05). Peritoneal macrophages from NOR and Idd5 NOD congenic mice were more proficient at engulfment than their NOD counterparts. Annexin V blockade diminished apoptotic thymocyte clearance and heat-labile serum factors augmented clearance. Binding of apoptotic thymocytes to NOD macrophages was also reduced, suggesting that the deficiency in phagocytosis may be partly attributable to a recognition defect. Peritoneal macrophages from female Balb/c and NOD mice were equally efficient in the engulfment of microspheres, suggesting that the phagocytic deficiency observed in NOD mice was specific for apoptotic cells. In summary, we have demonstrated a deficiency in phagocytic function of macrophages from NOD mice. Normal and diabetes-prone neonatal rodents have a wave of beta-cell apoptosis coincident with the onset of target organ inflammation. A constitutive defect in the clearance of apoptotic beta-cells may be contributory to the initiation of autoimmunity.

Expression and characterization of Ov-47, a dominant antigen of Onchocerca volvulus

The expression and characterization of a recombinant antigen termed Ov-47 are described. Ov-47 was identified and isolated from a lambda gt-11 cDNA expression library derived from adult female Onchocerca volvulus mRNA using rabbit antiserum raised against the surface proteins of O. volvulus female worms. The antiserum was earlier found to mediate, in vitro, cytoadherence and cytotoxicity reactions to microfilariae in the presence of heat-labile serum factors. The deduced amino acid sequence of the gene was assigned the EMBL GenBank Accession No. Y15993. The open reading frame (1077 bp) of the gene was then subcloned into pQE-60 and expressed in Escherichia coli JM109 cells. The gene encodes a protein with an apparent molecular weight of 47,000 Da as revealed by SDS–PAGE. Up to 100 μg/ml pure Ov-47 recombinant protein could be isolated from E. coli cultures by Ni–agarose affinity chromatography. The 47-kDa protein was recognized by sera from both infected and endemic normal subjects. The parent protein was found to have a molecular weight of 60 kDa. IgG 3 subclass responses to Ov-47 were significantly higher in endemic normals than in infected subjects ( P<0.05). In contrast, IgG 4 responses were higher in infected subjects than in endemic normals ( P<0.05). IgG 2 response exhibited marked age dependency with lower responses in younger patients, which rose to higher levels in elderly patients. IgG 1, IgG 3, and IgG 4 responses did not show any age dependency. This study clearly shows that Ov-47 is a dominant antigen of O. volvulus adult worms with an important role in the host–parasite-interplay. Index Descriptors and Abbreviations: Onchocerca volvulus; nematode; Ov-47 antigen; endemic normals; IgG subclass responses.

Intracellular survival of Leishmania major in neutrophil granulocytes after uptake in the absence of heat-labile serum factors.

The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasite Leishmania is poorly understood. In the present study, the interaction of human PMN with Leishmania major promastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake of Leishmania. However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalize Leishmania even under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake of Leishmania. Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority of Leishmania parasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response to L. major infection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand, Leishmania can survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival of Leishmania within the first hours or days after infection.

Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions.

Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors.

Adherence to and damage of endothelial cells by Cryptococcus neoformans in vitro: role of the capsule.

Escape from the intravascular compartment is likely a critical step in the development of hematogenously disseminated cryptococcal infections, such as meningitis. The capsule of Cryptococcus neoformans is considered to be a virulence factor because of its antiphagocytic properties. To further investigate the role of the capsule in escape from the intravascular compartment, we used isogenic strain pairs, an acapsular mutant, and an encapsulated clinical isolate to determine the effects of the capsule of C. neoformans on adherence to, phagocytosis by, and damage of endothelial cells in vitro. Acapsular C. neoformans adhered significantly more to endothelial cells and caused greater endothelial cell injury than did encapsulated organisms. Coating of an acapsular strain with cryptococcal glucuronoxylomannan decreased both adherence to and damage of endothelial cells by 61.7% +/- 9.1% and 76.6% +/- 10.2%, respectively. Transmission electron microscopy demonstrated internalization of acapsular, but not encapsulated, organisms by endothelial cells. Internalization of an acapsular strain occurred through endothelial cell phagocytosis and was inhibited by cytochalasin D. Phagocytosis required a heat-labile serum factor, probably complement. These results suggest that acapsular or poorly encapsulated C. neoformans may be the form(s) that escapes from the vasculature during initiation of hematogenously disseminated disease.

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

Opsonic effect of globulins on phagocytosis of liposomes by bone marrow cells.

In recent years there has been a great surge in the development of particulate drug carriers for chemotherapy and diagnostic medicine. However, to realise the full potential of various drug carriers such as liposomes, it is necessary to understand how these particles are cleared from the circulation when injected intravenously, and what determines their recognition by the reticuloendothelial cells in the liver, spleen and bone marrow. In the circulation the drug carriers become coated with plasma components and this process is thought to determine their recognition by the reticuloendothelial cells. Liposomes coated in vifro with opsonins such as immunoglobulin, components of the complement system, fibronectin. C-reactive protein etc., when injected intravenously are cleared rapidly from the circulation (1). Recently we have demonstrated the presence of opsonins in serum which exhibit tissue specificity and described their properties which differentiate between 'liver-specific' and 'spleen-specific' opsonins responsible for the enhancement o f phagocytosis of liposomes by Kupffer cells and splenic macrophages respectively (2,3). However, so far there has been no clear indication to suggest the identity of the tissue-specific opsonin involved in phagocytosis of the injected liposomes. Bone marrow is one of the major organs of the reticuloendothelial system and plays a small but significant role in the clearance of foreign particulates including liposomes. In vifro serum is found to enhance the uptake of cholesterol-containing liposomes in the bone marrow cells. This opsonic activity of serum is destroyed when the serum is heated at 55°C for 30 min. This suggests that heat-labile complement may be involved in the phagocytosis of liposomes. However, when dialysed or EGTA-chelated serum is used, a complete loss of its opsonic activity occurs. Addition of the serum dialysate or divalent cations to the dialysed serum does not reinstall its lost opsonic activity (4). Thus these results shed doubt on the possibility of involvement of the complement and hence we searched for another heat-labile serum factor which may be responsible for enhancing phagocytosis of liposomes in these cells. Heat-labile pand y-globulin are known to enhance phagocytosis of certain strains of bacteria (1) and p-globulin is also known to enhance uptake of anionic liposomes into the perfused liver (5). Hence we examined the effect of various serum globulins on the uptake of neutral high cholesterol liposomes in the freshly prepared bone marrow Cells. The results in Table 1 show that y-globulin and immunoglobulin in the absence or presence of serum have little effect on the uptake of liposomes in the bone marrow cells, whereas p-globulin in the absence of serum stimulates the uptake of liposomes by 5-fold as compared to the control and it retains its opsonin activity in the presence of serum. Thus these results suggest that p-gloublin may be the heat-labile serum factor that enhances phagocytosis of liposomes in the bone marrow. Table 1

Preferential binding ofChlamydia trachomatisto subsets of human lymphocytes and induction of interleukin‐6 and interferon‐gamma

The interactions between Chlamydia trachomatis and human blood mononuclear leukocytes were studied using flow cytometry, immunofluorescence, electron microscopy and cytokine assays. Under serum-free conditions, elementary bodies (EB) of C. trachomatis were found to bind to human T lymphocytes as well as to B cells and monocytes/macrophages (M phi). For all cell types the binding was saturable, rapid, temperature-independent and independent of the chlamydia-specific serological status of the donor. Similar proportions of T and B cells bound EB at similar levels. In the T cell population, proportionally less CD8+ cells bound EB. Whereas M phi phagocytosed and destroyed the bound micro-organisms for lymphocytes, the Chlamydia remained at the surface, adherent to morphologically featureless membrane areas and showed no evidence of uptake even after long periods at 37 degrees C. Host molecules modulated these basic binding patterns: a heat-stable serum factor inhibited EB binding to T cells and a heat-labile serum factor enhanced binding to B cells. Stimulation with C. trachomatis EB rapidly elicited cytokine production by lymphocytes including interleukin-6 from B cells and interferon-gamma (IFN-gamma) from T and/or nonT/nonB cells. The responses were irrespective of the serological status of the donor. The findings suggest that C. trachomatis-leucocyte interactions may differ from the interactions of other bacteria and human leucocytes. The possible relationship between leucocyte-binding, cytokine induction, and the pathognomonic development of lymphoid follicles during mucosal C. trachomatis infections is discussed.

Antimicrobial activity of rabbit leukocyte defensins against Treponema pallidum subsp. pallidum

Defensins, which are peptides with broad antimicrobial activity, are major constituents of rabbit neutrophils and certain macrophages. We tested six rabbit defensins, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, for activity against Treponema pallidum subsp. pallidum. Mixtures of T. pallidum and defensin in 10% normal rabbit serum (NRS) or heat-inactivated NRS (HI-NRS) were incubated anaerobically for various time periods ranging between 0 and 16 h and then examined by dark-field microscopy for treponemal motility or inoculated intradermally into rabbits to assess treponemal virulence. Immobilization of T. pallidum by NP-1 (400 micrograms/ml) occurred after 4 and 8 h of coincubation in mixtures containing NRS and HI-NRS, respectively. Similarly, neutralization of T. pallidum by NP-1 occurred more rapidly and was complete when incubations were performed in NRS as compared with that in HI-NRS. Endpoint titration confirmed the augmentation of NP-1 antitreponemal activity by heat-labile serum factors; NP-1 showed neutralizing activity at 4 micrograms/ml (about 1 microM) in NRS and at 40 micrograms/ml in HI-NRS. When NP-1 was tested in serum that was deficient in C6, the T. pallidum neutralizing activity of NP-1 was reduced to levels slightly greater than that observed in HI-NRS. NP-1 that had been reduced and alkylated was inactive against T. pallidum. When NP-2, NP-3a, NP-3b, NP-4, and NP-5 were tested at 400 micrograms/ml, all exerted potent treponemicidal activity, manifested by abrogation or delayed development of cutaneous lesions relative to that of controls. These data suggest that defensins may equip certain macrophages and neutrophils to participate in host defense against T. pallidum, that the direct activity of defensins against T. pallidum is enhanced by heat-labile serum factors (presumably complement), and that conformational factors influence the biological activity of the defensin molecule.

In Vivo and In Vitro Antibacterial Activity of Conglutinin, a Mammalian Plasma Lectin

Conglutinin is a mammalian C-type lectin which agglutinates iC3b-coated erythrocytes. Ingram [13] found that euglobulin from bovine serum may confer partial protection against experimental infections in mice. We now present evidence that the protective activity in euglobulin against infections of BALB/c mice with Salmonella typhimurium is mediated by conglutinin. Conglutinin also demonstrated antibacterial activity against E. coli and S. typhimurium in vitro. The expression of this activity required the presence of heat-labile serum factors and peritoneal exudate or spleen cells, but not antibodies to the bacteria. Antibacterial activity was also demonstrated when the bacteria were pretreated with serum at 37 degrees C before incubation with conglutinin and cells. The activity of conglutinin was not observed when factor I-deficient or EDTA-treated serum was used instead of normal serum. The active peritoneal exudate or spleen cells showed adherence to plastic.

A Role for Complement in Antimycobacterial Defenses

Although evidence of complement (C') activation has been found in body fluids of patients with mycobacterial (AFB) disease, a definite role for C’ in antimycobacterial defenses has not been well defined. We have previously observed that heat-labile serum factors augment AFB phagocytosis by human blood monocytes in vitro (Am Rev Respir Dis 1987;135:A195). We now report further studies to see whether C’ is important in this effect. Peripheral blood from normal volunteers was separated on lymphocyte separation medium. The mononuclear layer was allowed to adhere to 12-mm glass coverslips for 1 h, nonadherent cells removed by washing, and the resulting monocyte-enriched cells (PBM) cultured in either RPMI-1640 medium with 5% autologous human serum or KC2000, a serumless medium. After 24 h, PBM were challenged with AFB at a ratio of 1:1, AFB:PBM. At 2 h, noningested AFB were removed, the coverslips fixed, stained, and examined for percentage of PBM having one or more associated AFB. C’ depletion of serum by heat inactivation or addition of cobra venom factor markedly reduced Mycobacterium avium complex (MAC) ingestion. Preincubation of MAC with serum-EDTA (blocking both classic and alternative pathways) caused a marked reduction of MAC ingestions, but treatment with serum-EGTA-Mg+ + (blocking the classic C’ pathway) did not alter the effect of serum. MAC mixed with normal human serum (NHS) activated ∼50% of C’ as assayed by C’ fixation tests. Fluorescent antibody studies indicated that C3 was deposited on MAC. Similar experiments showed that serum played a similar but less striking role in the ingestion of M tuberculosis by human monocytes. Thus, C’ is a major opsonin in the phagocytosis of MAC in vitro and perhaps in vivo.

Serum-mediated oligodendrocyte cytotoxicity in multiple sclerosis patients and controls.

We examined serum-mediated cytotoxicity on cultured rat oligodendrocytes, using serum from patients with acute or chronic progressive multiple sclerosis and normal controls. We found heat-labile serum factors in serum from MS and also in controls. The cytotoxic effects of MS and normal sera were not restored by adding a source of complement. Despite apparent lack of disease specificity, such factors might damage oligodendrocytes if they gained access to these cells through a damaged blood-brain barrier in MS or other disorders.

The serum bactericidal and opsonizing defect in sickle cell anaemia: restoration of activity by control serum

Thirty-eight homozygous sickler sera were compared with a large pool of serum from healthy African non-sicklers with regard to bactericidal and phagocytic indices. One third of the sera showed reduced bactericidal activity against Salmonella enteritidis which was restored by the addition of 4% control serum; control serum provided both heat-labile (HL) and absorbable (ABS) serum factors. 76% of test era showed greatly defective opsonization as indicated by ingestion by normal human neutrophils. Activity was not readily restored by the addition of control serum which provided only HL factors. Intracellular survival was increased when bacteria were ingested from sickler serum; activity was readily restored by control serum which provided both HL and ABS factors. In the presence of both serum and neutrophils 84% of test sera permitted increased bacterial survival; the defect was not readily reversed by the addition of control serum which provided both HL and ABS factors.

Chemiluminescence response of the human polymorphonuclear neutrophil to lipopolysaccharides

Polymorphonuclear neutrophils (PMN) respond to a variety of stimuli with a sequence of reactions that lead to the production of "active oxygen" species, including H2O2, free radicals, such as superoxide (O2-.) and hydroxyl (HO.), and singlet molecular oxygen (1O2). Some of these can oxidize (5-amino-2,3-dihydrophthalazine 1,4-dione) (luminol) to the ground state aminophthalate ion; this reaction sequence is accompanied by the generation of a photon and forms the basis for the chemiluminescence (CL) response. In this work we used a dedicated photon counting instrument to record CL from PMN incubated with bacterial lipopolysaccharide (LPS). We have studied the CL response to the LPS from Escherichia coli strains 026:B6 and 055:B5, as well as Salmonella minnesota RE 595 and have determined that CL requires heat-labile serum factors, these most likely being intact components of the complement system.