Activity In Culture Fluid Research Articles

Screening of proteases produced by Aspergillus micromycetes active against proteins of the hemostasis system.

For these intents, proteases cardiovascular disease is the primary cause of death; hence, accurate diagnosis and treatment are urgently required are regarded as prospective agents . High substrate specificity is needed for an effective enzyme, which makes Aspergillus micromycetes, known for producing proteases with precise action, biotechnologically promising. This study mainly aimed to look at the possibilities of Aspergillus species, which had never been mentioned in terms of general proteolytics. Every species was cultivated in two-stage submerged conditions with two different nitrogen sources; whereupon, proteolytic activity in culture fluid was determined. Chromogenic peptide substrates and fibrin plates were used to evaluate the thrombin, plasmin, factor Xa, urokinase, protein C-like, activating activities towards hemostasis proteins, as well as fibrinolytic and plasminogen-activating activities of these species. It was found that A. aureolatus and A. tennesseensis are active proteolytics exhibiting plasmin-like activities (116.17 and 87.09 U×10-3, respectively), factor Xa-like activity (76.27 and 77.92 U×10-3, respectively) and urokinase activity (85.99 and 59.91 U×10-3, respectively). The thrombin-like activity was found for A. tabacinus (50.37 U×10-3), and protein C-like activity was noticeable for A. creber, A. jensenii, A. protuberus, and A. ruber (62.90, 65.51, 73.37, and 111.85 U×10-3, respectively). Additionally, more than half of species had the ability to directly activate plasminogen or operate as fibrinolytics. New proteolytic strains were discovered, offering hope for the therapy of cardiovascular disorders. The high specificity and activity of fungal enzymes make them useful in a variety of fields, including medicine and diagnostics.

Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34

Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture temperature were agar, yeast extract, and 25 °C, respectively, for agarase production by one-factor-at-a-time design. The nutritional components of the medium and culture conditions were analyzed by Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effects on agarase production ( p < 0.05). The optimum levels of these key variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium composition and culture condition as well as the dominant occupation of agarase with high activity in culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.

Bactericidal activity of culture fluid components of Lactobacillus fermentum strain 90 TS-4 (21) clone 3, and their capacity to modulate adhesion of Candida Albicans yeast-like fungi to vaginal epithelial cells

Antagonistic activities of L. fermentum strain 90 TS-4 (21), L. casei ATCC 27216, and L. acidophilus ATCC 4356 and bactericidal activity of lactobacillus culture fluid towards E. coli strain K12, S. aureus, and S. epidermidis test cultures were studied. The bactericidal effect of L. fermentum strain 90 TS-4 (21) clone 3 culture fluid preparation (pH 6.0) on the test cultures was dose-dependent. Adhesion of C. albicans yeast-like fungi to vaginal epitheliocytes was more pronounced for strains isolated from women with asymptomatic infection than for strains isolated from women with manifest forms. L. fermentum strain 90 TS-4 (21) clone 3 culture fluid preparation modulated adhesion of yeast-like fungi only if the fungal strain was initially highly adherent.

Laboratory investigations for the morphologic, pharmacokinetic, and anti-retroviral properties of indinavir nanoparticles in human monocyte-derived macrophages

The effectiveness of anti-retroviral therapies (ART) depends on its ultimate ability to clear reservoirs of continuous human immunodeficiency virus (HIV) infection. We reasoned that a principal vehicle for viral dissemination, the mononuclear phagocytes could also serve as an ART transporter and as such improve therapeutic indices. A nanoparticle–indinavir (NP–IDV) formulation was made and taken up into and released from vacuoles of human monocyte-derived macrophages (MDM). Following a single NP–IDV dose, drug levels within and outside MDM remained constant for 6 days without cytotoxicity. Administration of NP–IDV when compared to equal drug levels of free soluble IDV significantly blocked induction of multinucleated giant cells, production of reverse transcriptase activity in culture fluids and cell-associated HIV-1p24 antigens after HIV-1 infection. These data provide “proof of concept” for the use of macrophage-based NP delivery systems for human HIV-1 infections.

Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effects on recombinant human antithrombin III integrity.

Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human recombinant antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha (2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha (2-->3)Gal- and Gal beta (1-->4)-GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal recombinant protein concentration is highly recommended to avoid product desialylation.

HIV-1 infection alters monocyte interactions with human microvascular endothelial cells.

HIV infection of monocytes resulted in twofold elevation of adhesion molecule LFA-1 (both alpha L/CD11a and beta 2/CD18 subunits) and LFA-3 (CD58), with no apparent increase in LFA-2 (CD2) or various beta 1-integrins. Homotypic aggregation of monocytes was evident 2 h after exposure to virus and was inhibited by mAbs to both the alpha L- and beta 2-subunits of LFA-1. HIV-infected monocytes also showed a marked increase in adherence to human capillary endothelial cell monolayers derived from brain, lung, and skin. This adherence was inhibited by mAb to either LFA-1 subunit and by mAb to the counter-receptor intercellular adhesion molecule-1. Cocultivation of HIV-infected monocytes with endothelial cells increased permeability of endothelial cell monolayers to 125I albumin in transwell assay systems. The increased endothelial permeability induced by HIV-infected monocytes was associated with a substantial disruption of the endothelial cell monolayer. Morphologic disruption was not a direct toxic effect on endothelial cells, but appeared to be secondary to changes in endothelial cell-cell or cell-matrix interactions. Northern blot analysis showed increased expression of gelatinase B (92-kDa gelatinase), tissue inhibitor of metalloproteinase TIMP-1, and TIMP-2 in the HIV-infected monocytes. Consistent with these Northern analyses, secretion of gelatinase activity in culture fluids of HIV-infected monocytes was also increased and was dependent on the stage of virus replication. Incubation of HIV-infected monocytes with the proteinase inhibitors TIMP-1 and TIMP-2 inhibited the increased permeability of endothelial cell monolayers to 125I albumin. These results suggest possible mechanisms for extravasation of HIV-infected monocytes through vascular endothelium into tissue in early stages of HIV disease.

Therapeutic intervention with chicken egg white ovomacroglobulin and a new quinolone on experimental Pseudomonas keratitis.

Chicken egg white ovomacroglobulin (ovoM) is a potent protease inhibitor with broad-spectrum activity against various proteases. The combined effects of ovoM and the new quinolone, ofloxacin (OFLX) on experimental Pseudomonas aeruginosa keratitis were investigated. The in vitro inhibitory effects of ovoM on protease activity in culture fluid of clinically isolated P. aeruginosa and on activity of human neutrophil elastase and cathepsin G were assayed using azo-casein as substrate. Albino rabbits received intrastromal injection of the isolated Pseudomonas strain (1 x 10(5) colony-forming units). At 16 h after inoculation, three treatment groups--0.1% ovoM alone, 0.3% OFLX alone, and a combination of both--and a non-treatment control group were tested. Protease activity in the culture solution and human neutrophil elastase was inhibited by ovoM, whereas cathepsin G was not inhibited effectively. In vivo additive therapeutic effects of ovoM and OFLX were observed at 96 h (P < 0.05 compared with OFLX alone). The results indicate that inhibition of proteolytic activity with ovoM is useful in preventing stromal degradation in P. aeruginosa keratitis.

Effect of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine on establishment of human immunodeficiency virus type 1 infection in cultured CD8+ lymphocytes

Several groups have shown that peripheral CD8+ lymphocytes can be infected with human immunodeficiency virus type 1 (HIV-1), resulting in noncytopathic infection and persistent production of viral particles. We studied the ability of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI) to inhibit the establishment of HIV-1 infection in CD8+ cells that were derived from cultures of peripheral blood lymphocytes exposed to both virus and drug. In situ infection of CD8+ cells was demonstrated by double flow cytometry analysis by using both anti-glycoprotein 120 (anti-gp120) and anti-CD8 monoclonal antibodies. At higher concentrations of drug (e.g., 0.4 microM AZT), the production of viral particles was inhibited for over 2 months, as assessed by p24 antigen levels in the culture medium. We also performed a time course experiment to determine whether HIV-1 infection of CD8+ cells would be affected by treatment of peripheral blood lymphocytes with AZT or ddI for different intervals following exposure to virus. Quantitative PCR revealed that 0.4 microM AZT, added as late as 24 h after infection, interfered with the formation of proviral DNA in CD8+ cells. Both HIV-1 load and the production of progeny virions by CD8+ cells, as monitored by reverse transcriptase activity in culture fluids, were inhibited by both AZT and ddI in a dose-dependent manner.

Construction of a model secretion system for oral streptococci

A DNA fragment corresponding to the secretory domain from the Streptococcus mutans GS-5 gtfB gene, which encodes the putative 38-amino-acid signal peptide of the glucosyltransferase I (GTF-I) enzyme product, has been constructed. This fragment was fused with the alpha-amylase structural gene from alkalophilic Bacillus sp. strain 707. This hybrid gene as well as the intact amylase gene were introduced into an Escherichia coli-streptococcus shuttle vector consisting of three components: the E. coli replicon p15Aori from pACYC177, an erythromycin resistance gene from pAM beta-1, and the streptococcal replicon from pVA838. Transformation of the oral noncariogenic bacterium Streptococcus gordonii with the chimeric plasmid harboring the hybrid amylase gene resulted in strong extracellular amylase production. By contrast, transformants containing the intact amylase gene exhibited only trace amounts of amylase activity in culture fluids. Since the two signal peptide structures of the GTF-I enzyme and the Bacillus amylase are distinct from each other, these differences might result from the inability of S. gordonii to correctly process the Bacillus signal peptide. Furthermore, culture fluids from transformants of S. mutans as well as Streptococcus milleri harboring the hybrid amylase gene showed only weak amylase activity. Deletion of the gtfB, gtfC, or ftf gene from S. mutans GS-5 did not increase amylase secretion following transformation with the hybrid amylase gene. These results suggest that in contrast to S. gordonii, the inability of S. mutans and S. milleri to secrete hybrid amylase molecules could result from incorrect interaction of the secretory components of these organisms with amylase precursor molecules.

Glial cell-specific mechanisms of TGF-β1 induction by IL-1 in cerebral cortex

Transforming growth factor beta-1 (TGF-β1) immunoreactive product (IRP) has recently been detected in autopsied brains of individuals who died with central nervous system diseases and/or fever but not in normal individuals or in normal rodent brain. However, the mechanism(s) of induction of TGF-β1 in brain and the identity of cells expressing TGF-β1 need to be understood before a role, if any, for this potent pleiotropic cytokine in neuropathogenesis can be discerned. Towards this end we determined that IL-1 stimulated the production of TGF-β1 IRP in cells and TGF-β1 activity in culture fluids of all glial cells, astrocytes, microglial cells, and oligodendrocytes, derived from neonatal rat cortex and grown in cell type-enriched cultures. TGF-β1 production in vitro varied with the cell type and isoform of IL-1. Oligodendrocytes produced the most and astrocytes the least amount of TGF-β1. IL-1α stimulated TGF-β1 production in all glial cell types, whereas IL-1β did not. In vivo, TGF-β1 IRP was detected in human tissues from cerebral frontal cortex and subcortical white matter only when interleukin-1 (IL-1) was elevated in the same tissues. Moreover, the amount of detectable TGF-β1 was positively correlated with the amount of detectable IL-1 (rho = 0.605; P = 0.003), as determined by morphometry. Double-labelling of cells for their phenotypic markers and expression of TGF-β1 indicated that all glial cells, but not neurons, expressed TGF-β1. IL-1α and IL-1β IRPs were also detected in all three glial cell types, most frequently in astrocytes and least frequently in microglial cells. The cells containing both cytokine IRPs were also detected. These results indicate that TGF-β1 may be induced by IL-1 in all glial cells of the frontal cortex, by both autocrine and paracrine mechanisms.

Cytotoxic activity and production of toxic nitrogen oxides by macrophages treated with IFN-gamma and monoclonal antibodies against the 73-kDa lipopolysaccharide receptor.

Abstract The hamster IgM mAb 5D3 is specific for an 73-kDa LPS receptor on murine leukocytes. This mAb inhibits binding of radiolabeled LPS to splenocytes and acts as an agonist for induction of LPS-mediated changes in macrophage function. Resident peritoneal macrophages treated with IFN-gamma and mAb 5D3 developed potent cytotoxic activity against tumor cells. Cells treated with IFN-gamma or mAb 5D3 alone were inactive. Macrophage cytotoxic activity induced by IFN-gamma and mAb 5D3 was inhibited by NGMMLA and coincident with high levels of NO2-released into culture fluids. These data show that mAb 5D3 serves as an effective trigger signal for induction of cytotoxic activity with IFN-gamma-primed macrophages. Indeed, mAb 5D3 exactly mimicked the effects of LPS in these same systems. Unlike LPS, effects of mAb 5D3 on induction of macrophage cytotoxic activity and production of nitrogen oxides was abrogated after boiling, and not affected by addition of polymyxin B. The effects of LPS and mAb 5D3 as a trigger signal for IFN-gamma-primed macrophages were associated with production of TNF activity in culture fluids and inhibited by mAb against rTNF-alpha. Expression of class II MHC on macrophages induced by IFN-gamma treatment was suppressed by both LPS and mAb 5D3. These suppressive effects of LPS and mAb 5D3 were not affected by NGMMLA or mAb against rTNF-alpha. Finally, macrophages treated with LPS or mAb 5D3 before exposure to IFN-gamma and LPS or mAb 5D3 did not develop cytotoxic activity or high levels of NO2- in the culture fluids. These same cells developed both effector activities after addition of rTNF-alpha. These results in toto identify the 73-kDa protein as a receptor that mediates LPS-induced changes in macrophage effector function. The mAb 5D3 serves as a specific and defined reagent agonist for analysis of LPS receptor-linked change.

Interactions between HIV-infected monocytes and the extracellular matrix: HIV-infected monocytes secrete neutral metalloproteases that degrade basement membrane protein matrices.

The frequency of human immunodeficiency virus (HIV)-infected monocytes that spread on a model basement membrane was about twofold greater than that of an equal number of uninfected control cells through the initial 12 to 18 h of culture. By 24 h, virtually all HIV-infected and uninfected control cells spread on the basement membrane gel. The frequency of spread cells in the uninfected control population was less than 10% of total cells by 12 days. In contrast, 30 to 40% of HIV-infected monocytes remained spread through this time interval and formed a dense interdigitated network of cell processes on and into the gel matrix. Invasion of the basement membrane matrix by HIV-infected monocytes suggested increased secretion of proteases able to digest the gel. Indeed, levels of neutral protease activity in culture fluids from HIV-infected monocytes were significantly higher than those from equal numbers of uninfected control cells. High levels of protease activity in culture fluids of HIV-infected monocytes required productive virus infection and were not observed with cells exposed to T cell-tropic HIV isolates. The predominant protease activity in these cultures was a 92-kd neutral metallogelatinase. HIV-induced changes in monocyte metalloprotease activity may be important for extravasation of infected cells to tissue or for the development of AIDS-associated neuropathology, carcinogenesis, and opportunistic infection.

Dual regulation of silent and productive infection in monocytes by distinct human immunodeficiency virus type 1 determinants.

The regulation of human immunodeficiency virus type 1 infection and replication in primary monocytes was investigated by mutagenesis of recombinant proviral clones containing an env determinant required for the infectivity of monocytes. Virus replication was assayed by determination of reverse transcriptase activity in culture fluids and by recovery of virus from monocytes following cocultivation with uninfected peripheral blood mononuclear cells. Three virus replication phenotypes were observed in monocytes: productive infection, silent infection, and no infection. Incorporation of the monocytetropic env determinant in a full-length clone incapable of infection or replication in primary monocytes (no infection) conferred the capacity for highly efficient virus replication in monocytes (productive infection). Clones with the env determinant but lacking either functional vpr or vpu genes generated lower replication levels in monocytes. Mutation of both vpr and vpu, however, resulted in nearly complete attenuation of virus replication in monocytes, despite subsequent virus recovery from infected monocytes by cocultivation with uninfected peripheral blood mononuclear cells (silent infection). These findings indicate a central role for the "accessory" genes vpu and vpr in productive human immunodeficiency virus type 1 replication in monocytes and indicate that vpu and vpr may be capable of functional complementation.

Non-specific natural cytotoxic factor released from bovine peripheral blood lymphocytes.

Natural cytotoxicity against bovine leukemia cells (PC-3 cells) was found in bovine peripheral blood lymphocytes (PBL), and in non-adherent cells but not in adherent cells to nylon-wool column. Natural cytotoxic cells (NCC), which have natural cytotoxic activity, are found in T cell-rich fraction. When NCC were cocultured with PC-3 cells, natural cytotoxic factor (NCF) was released rapidly from NCC, and dose-response curve for NCF was almost linear induction. Cytotoxicity against PC-3 cells by NCC or NCF was increased with an increment of incubation period. Cytotoxicity against K562 cells, CL-1 cells, M1 cells or EL-4 cells by NCF was almost the same level as that against PC-3 cells, but that against those cell lines by NCC was not found. NCF activity in culture fluid from NCC cocultured with K562 cells or CL-1 cells was lower than that from NCC cocultured with PC-3 cells.

Polygalacturonate lyase production byThermomonospora curvata on protein-extracted lucerne fiber

Protein-extracted lucerne fiber was used as carbon and energy source for production of extracellular polygalacturonate lyase byThermomonospora curvata. The optimal fiber concentration was 1.5% (w/v); peal lyase activity in culture fluid occurred after 3 days growth at 53°C. During that time, lyase biosynthesis was controlled through induction; production was accelerated by adding small amounts of pectin or by grinding the fiber to 40-mesh particle size to release more inducer. After 3 days growth, lyase activity decreased; inactivation of the enzyme was delayed by the presence of 1 mM Ca or by inhibition of serine proteases with 0.05 mM phenylmethylsulfonyl fluoride. The molecular weight of the lyase produced during growth on the fiber was 35 kDa compared to 56 kDa for the enzyme produced on pure pectin. TheKm of the 35-kDa form was 0.54% pectin compared to 0.06% for the 56-kDa form. The smaller form was rapidly inactivated at 60°C, the optimal temperature for activity of the larger form.

Regulation of HIV replication in infected monocytes by IFN-alpha. Mechanisms for viral restriction.

In a survey of 15 different virus isolates, no IFN-alpha or IFN-beta activity was detected in culture fluids of HIV-infected T cells or monocytes. Exogenous rIFN-alpha added to T lymphoblast or monocyte cultures induced restriction in replication of the amphotropic HIV that infect both cell types. With IFN-treated HIV-infected T cells, levels of reverse transcriptase (RT) activity in culture fluids were half those in control cultures, but the frequency of infected cells or the levels of p24 Ag released in culture fluids were unchanged. In contrast to the modest effect of IFN on HIV-infected T cells, IFN-induced antiviral activity in monocytes was quite dramatic. Monocytes treated with IFN at the time of virus challenge showed no evidence of HIV infection: no p24 Ag or RT activity, no viral mRNA, and no proviral DNA. In this system, IFN interrupts one or more early event(s) in the virus replication cycle before formation of proviral DNA. Monocyte cultures infected with HIV 7 days before IFN treatment showed a gradual decrease in levels of p24 Ag and RT activity to baseline by 3 wk. HIV-induced cytopathic changes were markedly reduced, and the frequency of productively infected cells was less than or equal to 1% of total cells. Virus particles released 24 h after IFN treatment were 100- to 1000-fold less infectious than equal numbers of control virions. But, monocytes treated with IFN 7 days after HIV infection were not free of the retroviral pathogen: levels of proviral DNA in the IFN-treated and control HIV-infected cells were indistinguishable. The presence of large quantities of proviral DNA in cells with little or no evidence for active transcription documents a situation approaching true microbiological latency.

Restriction of HIV Replication in Infected T Cells and Monocytes by Interferon-α

Human recombinant interferon-alpha (IFN alpha) restricted viral replication in human immunodeficiency virus- (HIV) infected T cells and monocytes. With T cells, reverse transcriptase (RT) activity in culture fluids was reduced threefold from that of control infected cells by IFN treatment, but HIV p24 antigen levels were unchanged. In contrast, levels of p24 antigen and RT activity in lysates of IFN-treated infected cells were threefold greater than those of controls. These differences suggest that the mechanism for IFN-induced antiviral effects in HIV-infected T cells resides in the terminal events (assembly and release) of the virus replication cycle. Monocytes treated with IFN at the time of virus challenge showed no p24 antigen or RT activity, no HIV-specific mRNA, and no proviral DNA in cells for up to 3 weeks after infection. IFN treatment of chronically infected monocytes also decreased virus replication, as assessed by p24 antigen, mRNA and RT detection assays. However, levels of proviral DNA in the IFN-treated and control HIV-infected cells were indistinguishable. The presence of large quantities of proviral DNA in cells with little or no evidence for active transcription documents a situation approaching true microbiological latency.

High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells

Limiting-dilution techniques were employed to derive single-cell clones from U-937 cells that had been chronically infected with human immunodeficiency virus type 1. All clones thus obtained were positive for the presence of viral antigens; however, not all of the clones produced infectious progeny virus, as detected by the presence of reverse transcriptase (RT) activity in culture fluids. Six of these clones were monitored over time to determine whether their phenotype of human immunodeficiency virus type 1 expression was stable. Three clones maintained production of RT activity at a high level and showed a very high percentage of cells positive for viral p24 antigen, as determined by indirect immunofluorescence. The other three clones showed variations in either their levels of RT activity or the number of cells positive for p24, after which they stabilized. Infectious virus could be recovered from only three clones, as assessed by coculture experiments with different cell types. Two other clones were shown to produce noninfectious viruses. Molecular analyses at the DNA, RNA, and protein levels showed extensive variations between the viral isolates recovered from each clone.

Secretion of Neutrophil Attractant/Activation Protein by Lipopolysaccharide-stimulated Lung Macrophages Determined by Both Enzyme-linked Immunosorbent Assay and N-Terminal Sequence Analysis

Alveolar macrophages contribute to acute pulmonary inflammation by secretion of neutrophil chemoattractants. We determined if one of these attractants is neutrophil attractant/activating protein (NAP-1), which is secreted by blood monocytes stimulated by lipopolysaccharide (LPS). Alveolar macrophages were stimulated in tissue culture with 10 micrograms/ml LPS. Culture fluids collected at 24 h were assayed for both neutrophil chemotactic activity and the concentration of NAP-1 as determined by a sandwich ELISA. The concentration of NAP-1 in culture fluid to LPS-stimulated macrophages was 860 +/- 40 ng/ml (SEM for six normal subjects). NAP-1 in fluid of unstimulated macrophages was 40 +/- 15 ng/ml. We confirmed the presence of NAP-1 in culture fluid of LPS-stimulated lung macrophages by immunoaffinity and HPLC-CM column purification. The HPLC-CM elution profile of macrophage NAP-1 was identical to that of monocyte NAP-1, and the N-terminal sequence of the protein in one of the isolated peaks corresponded to that of monocyte-derived NAP-1 beta. Two lines of evidence show that NAP-1 does not account for all neutrophil chemotactic activity in culture fluid of 24-h, LPS-stimulated macrophages. At a dilution of culture fluid that elcited the same chemotactic response as a known concentration of pure NAP-1, the concentration of culture fluid NAP-1 was only one-tenth that of pure NAP-1. (ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic Characterization of Human Interferon Derived from Amniotic Membranes Induced by Virus

The presence of protein(s) with interferon (IFN)-like activity in culture fluid from human amniotic membranes induced by viruses has been described by different groups. However, the antigenic structure of this protein is controversial. Here we report the presence of IFN activity in supernatants of human amniotic membranes induced by Sendai virus. The major component responsible for this antiviral activity seems to be the classical IFN-beta. However, we were able to demonstrate the presence of a protein fraction with antiviral activity that does not bind to an affinity column for IFN-beta. The antiviral activity of this unbound fraction cannot be neutralized by antibodies to IFN-alpha, -beta, gamma, or by a mixture of them. We called this unbound fraction IFN-AM. We also report the development of a monoclonal antibody that does not neutralize the antiviral activity of IFN-alpha or IFN-beta but reduces the antiviral activity of a partially purified preparation of Sendai virus-induced amniotic membrane supernatant. These observations suggest that the IFN-AM (the unbound fraction that lacks reactivity with antibodies against known IFNs) contains a unique antigenic determinant that is not present, or, if so, is not located at the functional domain of IFN-alpha, -beta, or -gamma.