Presence Of Fresh Human Serum Research Articles

Synthesis and aggregation properties of a novel enzymatically resistant nucleoamino acid

In this work, we describe the synthesis, evaluation of some biological properties, such as DNA- and RNA-binding ability and in sero stability, as well as the supramolecular assembly of a novel nucleoamino acid based on L-spinacine. More particularly, a thymine-containing L-spinacine derivative was synthesized in liquid phase by a simple peptide-coupling procedure. Subsequently, nucleic acid and Cu(2+)-binding ability, as well as self-assembly properties of the novel nucleoamino acid, were investigated by spectroscopy (CD and UV) and laser light scattering which furnished interesting information on the assembly of supramolecular networks based on the peptidyl nucleoside analog. Finally, nucleoamino acid enzymatic stability was studied and a half life of about 7days was found in the presence of fresh human serum.

Cytochrome P450 2B1 Mediates Complement-dependent Sublytic Injury in a Model of Membranous Nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

The detection of anti-granulocyte antibodies by chemiluminescence.

Ten sera containing polyspecific anti-HLA antibodies, a rabbit antihuman leucocyte antiserum and a monoclonal anti-granulocyte antibody were used in the development of a semi-quantitative technique for the detection of anti-granulocyte antibodies. The assay measures the metabolic response of human monocytes to antibody coated (opsonized) granulocytes. Immune complexes were opsonic only in the presence of fresh human serum. Monocyte response was strongly inhibited by normal human serum and purified free immunoglobulin. The assay is simple, rapid and sensitive, and all antibodies investigated were readily detected.

Human IgM Monoclonal Antibodies Reactive with HIV‐1‐Infected Cells Generated Using a Trans‐Chromosome Mouse

The trans-chromosome (TC) mouse that we used harbors human chromosomes 2, 14 and/or 22, and has undergone knock-out of its endogeneous genes coding for mu-and kappa-chains of immunoglobulin. One of these TC mice was immunized with HIV-1-infected U937 cells, and spleen cells from the immunized animal were fused with the mouse myeloma cell line to generate hybridoma cells. We selected hybridomas that produce human IgM antibodies (Abs) reactive with HIV-1-infected MOLT4 cells but not with uninfected MOLT4 cells. Two hybridoma cell lines were established termed 9F11 and 2G9. Although 0.4 mug/ml of 9F11 was able to induce complement-mediated cytolysis of the infected cells in the presence of fresh human serum, 2G9 could not. There was no difference between the two monoclonal Abs in the base sequences of cDNAs coding for the constant regions of mu-and kappa-chains. Therefore, we speculate that the ability to activate complement on homologous cell membranes might reflect the structural presentation of antigenic molecules, which could facilitate the binding of an IgM Ab to multiple binding sites resulting in escape from restriction by species-specific inhibitors of complement such as DAF (CD55) and CD59. On the other hand, 2G9 induced apoptosis of HIV-1-infected cells, including latently infected OM10.1 cells, although the Ag for 2G9 remains to be identified. Since both of the Abs had reduced reactivity toward HIV-1-infected MOLT4 cells following cultivation in the presence of tunicamycin, the responsible antigens would involve a sugar moiety.

A novel IgM class autoantibody to a hepatocyte-related 190 kDa molecule in patients with type 1 autoimmune hepatitis

It has been reported that autoantibodies to hepatocytes are frequently found in patients with autoimmune hepatitis (AIH). To elucidate the nature of these hepatocyte-specific autoantibodies, we attempted to generate a hepatocyte-specific monoclonal antibody (MoAb) from Epstein-Barr virus-transformed peripheral blood mononuclear cells obtained from a patient with AIH. We established a single clone, 2E3, that continued to produce an immunoglobulin M (IgM) antibody (lambda-type). This MoAb had the following properties: it reacted mainly with hepatocyte-derived cell lines, rather than with other cell lines, and it reacted with liver tissue but not with other tissues. By immunoblot analysis, we found that this MoAb recognized a 190 kDa molecule on hepatocytes. The MoAb was able to kill hepatocyte-derived cell lines in the presence of fresh human serum. This cytotoxic effect was completely abrogated by heat inactivation of human serum prior to its addition to cell lines. In addition, an IgM autoantibody that recognized a 190 kDa molecule was also found in patients with AIH but not in those with chronic hepatitis C; its titer correlated significantly with serum alanine aminotransferase (ALT) levels in patients with AIH. In conclusion, we generated a human MoAb that recognizes a 190 kDa molecule on hepatocytes. Because of its ability to mediate complement-dependent cytotoxicity and the presence of similar IgM autoantibody in patients with AIH, we hypothesize this autoantibody may play a role in the immunopathogenesis of AIH.

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.

Microencapsulation of neonatal porcine islets: protection from human antibody/complement-mediated cytolysis in vitro and long-term reversal of diabetes in nude mice.

Recently, we have developed a simple and reliable method to efficiently isolate large numbers of neonatal porcine islets (NPI). We and others have shown that NPI are susceptible to cytolysis by the activation of human complement in vitro. Microencapsulation of islets may be one strategy to protect NPI from this form of rejection. We examined whether microencapsulation can prevent lysis of NPI induced by human antibody and complement in vitro and also assessed their ability to reverse hyperglycemia in diabetic nude mice. NPI were microencapsulated with purified alginate, cultured for 2 days, then tested for sensitivity to fresh human serum using an established in vitro cytotoxicity assay or transplanted into alloxan-induced diabetic nude mice. Incubation of nonencapsulated NPI for 24 hr in the presence of fresh human serum resulted in a 53% loss of cellular insulin content, a 51% reduction in recoverable DNA content, and a marked reduction of insulin secretory responsiveness when compared with controls cultured in heat-inactivated human serum. In contrast, exposure of encapsulated islets to fresh human serum had no cytotoxic effect on the islets. Transplantation of 2000 encapsulated NPI i.p. into diabetic nude mice (n=16) corrected hyperglycemia in all mice within 8 weeks. Similar results were obtained when 2000 nonencapsulated NPI were implanted under the kidney capsule (n=10); however recipients of nonencapsulated NPI placed i.p. failed to obtain euglycemia and survived for only 3 weeks posttransplantation. Microencapsulation protects NPI from the cytotoxic effects of human antibody and complement and allows for long-term reversal of diabetes in nude mice.

Human IgM monoclonal antibody to ganglioside GM2 and complement suppress virus propagation in ex vivo cultures of lymphocytes from HIV-1 infected patients.

HIV-1 infection induces aberrant ganglioside GM2 expression on infected cell lines, and human IgM anti-GM2 monoclonal antibody (L55 Ab) together with normal fresh human serum (FHS) as a source of complement causes complement mediated cytolysis of HIV-1 infected cells as well as HIV-1 particles. We report here that high expression of GM2 was also detected on HIV-1 infected lymphocytes from HIV-1 seropositive patients. L55 Ab effectively suppressed the generation of HIV in the presence of FHS in primarily cultured lymphocytes from HIV-1 infected patients in ex vivo experiments, and the suppression was enhanced additively by AZT. These data suggest that L55 Ab may increase the therapeutic effect of chemotherapy.

The selective cytotoxicity of cobra venom factor immunoconjugate on cultured human nasopharyngeal carcinoma cell line.

The selective cytotoxicity of a CVF immunoconjugate on human nasopharyngeal carcinoma cell line was reported. Cobra venom factor (CVF), a C3b-like glycoprotein, was linked to BAC5, a murine monoclonal antibody directed against a human nasopharyngeal carcinoma-associated membrane antigen, by a disulfide bond. The high affinity to cultured human nasopharyngeal cells (CNE2) and the complement activating potency retained in CVF immunoconjugate. Although the equimolar concentration of BAC5 or CVF alone was harmless to CNE2 cells, the CVF immunoconjugate in the presence of fresh human serum exhibited selective cytotoxicity on CNE2 cells in a concentration- (IC50 3.07 x 10(-7) mol/L) and time-dependent manner. No cytotoxicity occurred on either CNE1 (another human nasopharyngeal carcinoma cell line) or MGC-803 (human gastric carcinoma cell line) cells. Furthermore, direct lytic factor (DLF, cardiotoxin) separated from cobra venom, augmented CVF immunoconjugate-induced cytotoxicity significantly. These results indicate that the CVF immunoconjugate has complement-mediated selective cytotoxicity on CNE2 cells, which can be potentiated by DLF.

Complement-Mediated Anti-HIV-1 Effect Induced by Human IgM Monoclonal Antibody Against Ganglioside GM2

HIV-infected cells aberrantly express a high level of antigenic glycosidic structures such as GM2 and Gg4. Some normal sera containing natural IgM Abs to GM2 and/or Gg4 cause C-mediated cytolysis of HIV-infected cells. In the present study we demonstrated that a human IgM anti-GM2 mAb (L55 Ab) can induce cytolysis of HIV-infected cells. Increased GM2 expression by HIV-1 infection of a human T cell line (MOLT4), a human monocyte cell line (U937), and human lymphoblastoid cells was confirmed by immunofluorescence staining with L55 Ab. These infected cells were readily lysed by L55 Ab in the presence of fresh human serum as a C source that alone did not cause cytolysis. L55 Ab also had the ability to destroy HIV-1 particles via C-mediated lysis. By adding L55 Ab together with human C to mixed culture of HIV-infected cells and naive cells, HIV-1 replication was significantly suppressed, and this effect was synergistic when L55 Ab was combined with a reverse transcriptase inhibitor and a proteinase inhibitor. Therefore, a human IgM anti-GM2 mAb may be effective in treating HIV-infected patients, especially when used together with chemotherapeutic agents.

Serum proteins facilitate neutrophil induction of endothelial leukocyte adhesion molecule 1.

Although the individual actions of neutrophils and serum proteins such as complement in acute inflammation are well characterized, less is known about their effects in combination. We investigated the combined effects of neutrophil contact and active serum proteins on the expression of endothelial leukocyte adhesion molecule 1 (ELAM-1). Confluent monolayers of human umbilical vein endothelial cells were incubated with neutrophils in the presence and absence of fresh human serum. Flow cytometry was used to assess expression of endothelial intercellular adhesion molecule 1 (ICAM-1) and ELAM-1. In addition, neutrophils were retained in a semipermeable insert, which allowed their secretions to contact the endothelium but restricted neutrophil-endothelial contact. ELAM-1 expression was significantly increased on the cells coincubated with neutrophils and fresh human serum (25.8%; p < 0.001). There was no significant change in ELAM-1 expression on endothelial cells incubated with fresh human serum alone (3.9%; p > 0.01) or in those incubated with neutrophils and heat-inactivated serum (9.3%; p > 0.01). In the absence of neutrophil contact, ELAM-1 expression was increased only in the presence of fresh human serum (9.6%; p < 0.05). These findings suggest that serum proteins may potentiate the volume or potency of neutrophil-derived diffusable mediators of ELAM-1 expression. These effects are eliminated with the heat inactivation of serum proteins, implicating a heat sensitive mediator such as the complement cascade.

Serum proteins facilitate neutrophil induction of endothelial leukocyte adhesion molecule 1

Background: Although the individual actions of neutrophils and serum proteins such as complement in acute inflammation are well characterized, less is known about their effects in combination. We investigated the combined effects of neutrophil contact and active serum proteins on the expression of endothelial leukocyte adhesion molecule 1 (ELAM-1). Methods: Confluent monolayers of human umbilical vein endothelial cells were incubated with neutrophils in the presence and absence of fresh human serum. Flow cytometry was used to assess expression of endothelial intercellular adhesion molecule 1 (ICAM-1) and ELAM-1. In addition, neutrophils were retained in a semipermeable insert, which allowed their secretions to contact the endothelium but restricted neutrophil-endothelial contact. Results: ELAM-1 expression was significantly increased on the cells coincubated with neutrophils and fresh human serum (25.8%; p < 0.01). There was no significant change in ELAM-1 expression on endothelial cells incubated with fresh human serum alone (3.9%; 0.01) or in those incubated with neutrophils and heat-inactivated serum (9.3%; p > 0.01). In the absence of neutrophil contact, ELAM-1 expression was increased only in the presence of fresh human serum (9.6%; p < 0.05). Conclusions: These findings suggest that serum proteins may potentiate the volume or potency of neutrophil-derived diffusable mediators of ELAM-1 expression. These effects are eliminated with the heat inactivation of serum proteins, implicating a heat sensitive mediator such as the complement cascade. (Surgery 1998;123:199-204.)

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.

Collagen synthesis by human fibroblasts. Regulation by transforming growth factor-β in the presence of other inflammatory mediators

We have examined the combined effects of transforming growth factor-beta (TGF-beta), serum and gamma-interferon (gamma-IFN) on collagen synthesis by fibroblasts and compared the response of fibroblast subpopulations to TGF-beta. Human diploid fibroblasts were treated with TGF-beta alone and with serum of gamma-IFN. Cells were labelled with radioactive amino acids, and collagen production was measured as collagenase-digestible radioactivity. Collagen mRNA was determined by a solution-hybridization assay using procollagen-alpha 1[I] cDNA clone HF 677. The results showed that either serum or TGF-beta increased incorporation, collagen production and mRNA by fibroblasts approx. 2-fold; however, collagen synthesis relative to total protein synthesis and collagen mRNA relative to total polyadenylated [poly(A)+] RNA were not affected. Only serum activated cell growth. Collagen production increased approx. 4-fold in cells exposed to both TGF-beta and serum, and this increase was equal to that expected for an additive effect by both components. Treatment with gamma-IFN decreased collagen production and collagen mRNA to 44 and 40% respectively, whereas total incorporation and poly(A)+ RNA were affected only marginally. Cells exposed simultaneously to both gamma-IFN and TGF-beta produced less collagen and contained less mRNA than did those treated with TGF-beta alone. The gamma-IFN decreased collagen synthesis in control and TGF-beta-treated cultures to a similar extent, and TGF-beta increased collagen synthesis 2-fold in cells pre-treated with gamma-IFN. Fibroblast strains obtained in medium containing plasma-derived serum synthesized approximately half as much collagen as did cells derived from the same explant in the presence of fresh human serum, and TGF-beta stimulated collagen production and mRNA in both cell strains. We conclude that TGF-beta, serum and gamma-IFN regulate collagen synthesis by independent mechanisms, and that the combined action of these components plays a significant role in regulating collagen synthesis during wound healing and tissue repair.

Monoclonal opsonic mouse antibodies specific for streptococcal IgG Fc-receptor.

Spleen cells from mice immunised with group-A type-M15 streptococci and boosted with purified IgG Fc-receptor (FcR) from this type were fused with Sp 2/0 mouse myeloma cells. The resulting hybridomas were screened by ELISA for antibody production. Two IgM-secreting cell lines were selected. The monoclonal antibodies and ascites fluids inhibited the binding of 125I-labelled human IgG and IgG Fc-fragments to group-A type-M15 streptococci. The monoclonal antibodies also displaced purified FcR towards the anode in electrophoresis. They opsonised group-A type M-15 streptococci for phagocytosis by human granulocytes in the presence of fresh human serum. It was concluded that FcR is important for group-A streptococcal virulence.

Complement-dependent cellular cytotoxicity: lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum.

Lymphocyte-mediated lysis of cells of the Raji, Daudi, Jijoye, and Bjab lines was elevated when fresh human serum was added to the assay. A higher proportion of effector-target conjugates was observed in the presence of human serum. In similar experiments lysis of 1301, Rael, and P3HR-1 cells was unaltered. All cell lines activated the alternative pathway of complement but they varied in the expression of receptors for complement component 3 (C3) and in the ability to fix the C3 cleavage products on their membrane. The enhancement of lysis in the presence of human serum occurred only with those cells that bound C3. This characteristic was correlated to the expression of C3 receptors. Analysis of the nature of the deposited C3 was performed with Raji cells. Raji cells exposed to human serum bound C3b as indicated by the immunoadherence test. The C3b was further processed to C3bi, because the immunoadherence declined with time and conjugate formation increased with Daudi cells, which carry the C3 receptors CR2 and CR3. This suggests that in the lytic assay lymphocytes with C3bi receptors are recruited in the presence of human serum. We assume that the bridge of C3 molecules between targets and effectors increases the avidity of their interaction.

Effects of lipopolysaccharide on human endothelial cells in culture

Lipopolysaccharide (LPS) in concentrations up to 10 ug/ml did not induce detectable direct cytotoxicity in human umbilical vein, pulmonary artery, or pulmonary vein endothelial cells. By contrast, significant cytotoxicity was observed in bovine aortic endothelial cells exposed to LPS 0.01 ug/ml. Transmission electron microscopy of human umbilical vein cells exposed to LPS 10 ug/ml for 4 days revealed no significant ultrastructural abnormalities compared to control cells. Whereas human umbilical vein endothelial cell cytotoxicity was observed when neutrophils were stimulated with phorbol myristate acetate, LPS-stimulated neutrophils did not induce significant cytotoxicity even in the presence of fresh human serum as a complement source. Moreover, human umbilical vein endothelial cell factor VIII-antigen and fibronectin release, angiotensin-converting enzyme activity, and PGI 2 release were unaffected by a 24-hour exposure to LPS. Cytotoxicity, however, was produced when human umbilical vein endothelial cells were coincubated with LPS and cycloheximide. The proliferation of human umbilical vein endothelial cells was also inhibited after prolonged, continuous exposure to LPS 10 ug/ml. We conclude that LPS with or without complement or neutrophils does not induce significant human endothelial cell lysis or detachment. Moreover, brief exposure to LPS has minimal, direct effect on several functions of human endothelial cells in vitro.

Interaction of inflammatory cells and oral microorganisms. IX. the bactericidal effects of human polymorphonuclear leukocytes on isolated plaque microorganisms.

Polymorphonuclear leukocytes (PMNs) come in contact with plaque microorganisms in the gingival sulcus. However, little is known about the ability of PMNs to kill or inhibit the growth of plaque microorganisms. In this study, the bactericidal effects of human PMNs and serum against S. mutans 6715, S. sanguis M5, and A. viscosus T14 virulent and avirulent were examined. Plaque microorganisms grown to stationary phase were incubated with human PMNs and/or serum for 90 minutes at 37°C. The killing and growth inhibition of bacteria were determined by measurement of percent reduction in colony forming units and percent inhibition of 3H‐uridine uptake by the microorganisms. A. viscosus T14 virulent was the only microorganism significantly killed by human PMNs in the absence of serum. In the presence of fresh but not heat‐inactivated (56°C, 30 minutes) human serum, human PMNs had a significant bactericidal effect against all of the microorganisms tested. Exposure of S. mutans 6715, but not S. sanguis M5, to sucrose inhibited the bactericidal effect of human PMNs in the presence of fresh human serum but not in the presence of rabbit anti‐S. mutans immunoglobulin. Cytochalasin B markedly inhibited phagocytosis of S. mutans 6715 by human PMNs in the presence of serum. However, cytochalasin B‐treated PMNs still were significantly bactericidal for S. mutans 6715 in the presence of serum. The data suggests that human PMNs may kill or inhibit the growth of certain plaque microorganisms in the gingival crevice. The microbial killing may be influenced by bacterial extracellular polysaccharides and the presence of serum antibodies and complement, both resulting in phagocytosis and extracellular lysosomal release from PMNs.

The interaction in vitro between human polymorphonuclear leukocytes and Neisseria gonorrhoeae cultivated in the chick embryo.

Cultivation of Neisseria gonorrhoeae in the allantoic cavity of 10-day chick embryos ensured the following necessary properties for subsequent quantitive in vitro phagocytosis studies of viable gonococci: log phase of growth, resistance to the cidal effect of fresh human serum, maintenance of colonial type, and absence of clumping. Employing a modification of the Maaloe technique, phagocytosis of log-phase type 1 and 2 gonococci by human PMN leukocytes did not occur in the presence or absence of serum. These findings indicate that log-phase type 1 and 2 gonococci possess antiphagocytic surface factors Stationary-phase organisms of the same colonial type were ingested and rapidly killed by human PMN leukocytes under similar experimental conditions, thus emphasizing the necessity to employ log-phase gonococci in the study of phagocytosis and antiphagocytic surface factors. Log-phase type 4 gonococci were ingested and rapidly killed by human PMN leukocytes in the presence of fresh human serum but not heat-inactivated serum or in the absence of serum. Morphologic studies demonstrated that log-phase viable gonococci attach to the surface membrane of human PMN leukocytes. Interiorization of avirulent but not virulent organisms was observed in the presence of fresh human serum. Gonococci-human PMN leukocyte interactions thus provide a model for the investigation of the nonimmunologic and immunologic parameters associated with the attachment and ingestion stages of phagocytosis.

Chemotaxis of eosinophils and neutrophils by aggregated immunoglobulins

Human myeloma proteins of all recognized immunoglobulin classes, including IgE, were purified and chemically aggregated. The chemotactic properties of these aggregated immunoglobulins for enriched preparations of human eosinophils and neutrophils were studied in vitro with the use of a modification of the Boyden chamber. Significant chemotactic effects were exerted on eosinophils and neutrophils by IgG and IgM in the presence of fresh human serum. IgE did not attract eosinophils either alone or in the presence of fresh serum. Inhibition of chemotaxis was observed when fresh human serum was heated at 56 °C. for 30 minutes and when the same concentration of chemotactic agents was placed in both compartments of the chambers. The results indicate that aggregated IgG and IgM, in the presence of fresh human serum, generate chemotactic factor(s) for eosinophils and neutrophils and that heatlabile serum factors are essential for chemotaxis of these cells. These results are discussed in terms of the eosinophil and neutrophil chemotactic factors derived from the complement system, ECF-C and NCF-C, and the eosinophil chemotactic factor of anaphylaxis, ECF-A, associated with immediate-type hypersensitivity reactions.