Competition among Streptococcus pneumoniae for intranasal colonization in a mouse model.

Widespread use of conjugate vaccines against Streptococcus pneumoniae, by reducing carriage of S. pneumoniae serotypes included in the vaccine, may result in an increase in nasopharyngeal carriage of - and disease from - nonvaccine serotypes of the same species. Mathematical models predict that the extent of such replacement will depend positively on the degree to which carriage of vaccine-type S. pneumoniae inhibits acquisition of nonvaccine-type pneumococci, and may depend negatively on the inhibition of vaccine-type pneumococci by nonvaccine-type pneumococci. We used a mouse model of intranasal carriage of pneumococci to test whether such inhibition occurs between different pneumococcal strains. Mice carrying a streptomycin-resistant derivative of S. pneumoniae BG9163 (serotype 6B) as a resident strain showed reduced levels of colonization when challenged intranasally by optochin-resistant derivatives of the same strain and of a serotype 23F pneumococcus, BG8826. Inhibition could be overcome by increasing the dose of the challenge strain. Carriage of optochin-resistant BG9163 did not inhibit acquisition of the streptomycin-resistant variant. Colonization by a challenge strain did not significantly affect the level of colonization with the resident strain. These results provide evidence that is consistent with several hitherto untested assumptions of mathematical models of serotype replacement and suggest that a biological mechanism exists that could account for serotype replacement that is observed in clinical trials. The findings provide a basis for further studies of in vivo interactions between strains of S. pneumoniae.

Selection of an immunogenic and protective epitope of the PsaA protein of Streptococcus pneumoniae using a phage display library.

Streptococcus pneumoniae is an important pathogen that causes disease in young and elderly individuals. The currently available polysaccharide vaccines have limited efficacy in those age groups most susceptible to pneumococcal infections. This study focuses on mapping the epitopes of a surface protein of S. pneumoniae by biopanning a 15 mer phage display library using 5 different monoclonal antibodies (MAbs) against the Pneumoccal surface adhesin A (PsaA). PsaA is a component of the bacterial cell wall that is highly species specific and is involved in bacterial adherence and virulence. Biopanning of the phage display library reveals three distinct epitopes on the PsaA protein. The sequence homology of these epitopes ranges from two to six amino acids when compared to the native PsaA protein type 2. Two of these epitopes have been evaluated for their immunogeneicity in mice. The peptide selected by the MAbs 8G12, 6F6, and 1B7 is referred to as the consensus peptide and is immunogenic in mice. Optimal anti-PsaA response is observed in mice immunized with 50microg of the consensus peptide complexed to proteosomes in 1:1 ratio. The anti-PsaA response is significantly lower than the response to the PsaA native protein. The peptide selected by monoclonal antibody 4E9 in its lipidated form is significantly protective in mice challenged with S. pneumoniae serotype 2 when compared to mice immunized with the native protein. These results show that the selected epitopes of PsaA protein are immunogenic and protective in mice. These epitopes need to be evaluated further as alternatives to currently available vaccines.

Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli.

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA ( approximately 10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA ( approximately 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [(3)H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.

Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae.

Pneumococcal surface adhesin A (PsaA), with a molecular mass of approximately 37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length psaA sequence and was cloned into a baculovirus expression system. We expressed either cell-associated or cell-free nonfusion PsaA polypeptides using two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High-Five). Recombinant PsaA (rPsaA) polypeptides were partially purified by partitioning in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chromatography. Membrane-bound 'hydrophobic rPsaA' (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of approximately 38 kD by SDS-PAGE and partitioned in a Triton X-114 phase, it reacted with both rabbit polyclonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis. High-Five-cell-expressed 'soluble rPsaA' (srPsaA) with a molecular mass of approximately 37 kD by SDS-PAGE, was isolated from the serum-free culture medium and did not partition in the Triton X-114 phase; it reacted with anti-PsaA rabbit polyclonal and mouse monoclonal antibodies by ELISA and Western blot analysis. Both rPsaA polypeptide forms were immunogenic in Swiss-Webster adult female mice. In an infant mouse model of bacteremia, survival rates for mice given mouse anti-rPsaA immune serum (from mice immunized with High-Five-expressed srPsaA; 20 microl, 1:50,000 titer) 24 h before bacteremic challenge were greater than for the control group (48 h postchallenge, 20 vs. 90% survival rates) when challenged with S. pneumoniae serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.

The anticancer drug edelfosine is a potent inhibitor of neovascularization in vivo.

Edelfosine is an alkyl-lysophospholipid that acts as an anticancer agent in vivo. To test the hypothesis that part of its antineoplastic activity may be due to its ability to inhibit the neovascularization on which the progressive growth of all tumors depends, we evaluated edelfosine in vitro and in vivo for antiangiogenic activity. Edelfosine acted directly on cultured capillary endothelial cells, inhibiting their migration toward the angiogenic factor, basic fibroblastic growth factor (bFGF), at doses of 8-200 nM. When given systemically to rats (20 mg/kg i.p. twice daily), edelfosine was well tolerated and antiangiogenic. The majority of treated animals became unable to mount a corneal neovascular response to a pellet releasing bFGF, whereas vigorous vessel ingrowth was seen in untreated controls.

Culture and characterization of sinusoidal endothelial cells isolated from human liver.

Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.

Comparison of a pneumococcal common protein (PsaA) antibody ELISA and a PsaA immune complex ELISA for detection of pneumococcal serum antibody.

We examined and compared results from three assays, an enzyme-linked immunosorbent assay (ELISA) and two immune complex ELISAs for analysis of the serum antibody response to a native pneumococcal 37-kD common cell-wall protein by using acute- and convalescent-phase sera from 56 patients with community-acquired pneumonia. The sensitivities of the ELISA, the undissociated and dissociated immune complex assays were 85% (23 of 27), 78% (21 of 27) and 67% (18 of 27), respectively. To determine specificity, paired sera from patients with pneumonia of other bacterial etiologies were tested. The specificities were 83, 83 and 72% for the ELISA, undissociated immune complex, and dissociated immune complex, respectively. Based on this study, the sensitivities of the three assays were not statistically different. These tests could be used retrospectively to confirm invasive pneumococcal disease.

Immunoreactivity of five monoclonal antibodies against the 37-kilodalton common cell wall protein (PsaA) of Streptococcus pneumoniae.

Five monoclonal antibodies (MAbs) were produced against the Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) 37-kDa common cell wall protein. These antibodies were used in a dot immunoblot and Western blot study of clinical isolates of S. pneumoniae to detect the presence of the protein. By both assays, the MAbs reacted with clinical isolates representing the 23 type-specific serotypes present in the licensed pneumococcal polysaccharide vaccine. Western blot analysis confirmed the presence of a protein migrating in the gel with a molecular mass of 37 kDa. An extension of the study by using dot immunoblot analysis that included an analysis of the 90 serotypes of S. pneumoniae showed that all five MAbs reacted with 89 of the 90 serotypes tested. MAb 1B6, the exception, did not react with S. pneumoniae serotype 16F. Dot immunoblot analysis of the MAbs with Enterococcus faecalis and viridans streptococci showed varied reactivity patterns, depending on the species. The MAbs against the 37-kDa antigen did not react with Escherichia coli, respiratory pathogens, or nonpathogens representing 22 genera and 29 species of bacteria. All five MAbs also reacted with five multidrug-resistant strains of S. pneumoniae. In summary, these MAbs may be useful for detection of pneumococcal antigen and may lead to the development of diagnostic assays for pneumococcal disease.

BMEC-1: a human bone marrow microvascular endothelial cell line with primary cell characteristics.

Bone marrow microvascular endothelial cells (BMEC) are a functional component of the bone marrow stroma and have been shown to release hematopoietic regulatory factors as well as to selectively adhere and support the proliferation and differentiation of CD34+ hematopoietic progenitors. An early passage of these cells was immortalized by transfection with a vector (pSVT) encoding the large T antigen of SV40. The transformed cell line (CDC/CU.BMEC-1) expresses the SV40 transcript, retains the primary cell expression of Ulex europeaus and vWF/ FVIII, and incorporates acetylated low-density lipoprotein. In addition, BMEC-1 mirrors the phenotype of the primary cells with only a few exceptions. Both cell populations express the cellular adhesion molecules ICAM-1 and PECAM and also VCAM-1 and ELAM-1 after upregulation by tumor necrosis factor-alpha. The fibronectin receptor, hyaluronate receptor, collagen receptor, integrins VLA-alpha 3, VLA-alpha 4, and beta 4, endoglin, collagen IV, CD58, and CD61 are also expressed. The only differences are that BMEC-1 expresses higher levels of ICAM-1, CD58, CD34, CD36, and c-kit than the primary cells. The supernatants of primary cell and BMEC-1 contain stem cell factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-11, and G-CSF. The functional significance of these hematopoietic cytokines was demonstrated in transwell cultures. Both cell populations supported the expansion of progeny from CD34+ cell-enriched cord blood mononuclear cells suspended in the upper chamber. These characteristics, plus the fact that BMEC-1 can be maintained independently of exogenous growth factors and exhibit contact inhibition, indicate that this cell line can be used to further define the role of BMEC in hematopoiesis.

Plasmodium falciparum: involvement of additional receptors in the cytoadherence of infected erythrocytes to microvascular endothelial cells.

The involvement of additional ligands in the cytoadhesion of PRBC to endothelial cells was studied by the use of human microvascular endothelial cells (HMEC-1), brain microvascular endothelial cells (HBEC-51), umbilical vein endothelial (HUVEC), and C32 melanoma cells as well as soluble CD36, ICAM-1, and thrombospondin in the adhesion assays. Immunostaining showed that ICAM-1 and thrombospondin were expressed by all cell lines, whereas CD36 and VCAM-1 were expressed constitutively only by C32 melanoma cells and HBEC-51, respectively; none of these cells had basal expression of E-selectin. Bindings of the parental HB3 parasite strain to HMEC-1 and HUVEC were higher than that to HBEC-51 and C32 melanoma cells. Selections by panning the parental HB3 through HMEC-1 (HB3EC-6 line) or C32 melanoma cells (HB3C32-6 to HMEC-1 was higher than that to C32 melanoma cells. Antibody or peptide blockade against CD36, ICAM-1, and thrombospondin or preincubation of target cells with TNF-alpha and IFN-gamma did not significantly alter the binding intensity of HB3EC-6 to HMEC-1 and HB3C32-6 to C32 melanoma cells. Preincubation of HMEC-1 with IL-4, however, reduced its binding with HB3EC-6. In vitro selection did not enhance the binding of PRBC to plate-bound CD36 or thrombospondin; binding to ICAM-1 was negligible. The binding of both selected lines was inhibited by dextran sulfate and sulfatides, but not by chondroitin sulfate A. These results suggested that in addition to CD36 and thrombospondin, sulfated glycoconjugates were probably concurrently utilized by these PRBC as receptors. Experiments with freshly isolated Kenyan parasites indicated that they also exhibited a similar mechanism of binding to endothelial cells.

The use of a microporous polyvinylidene fluoride (PVDF) membrane filter to separate contaminating viral particles from biologically important proteins.

Viral agents (influenza A virus, 80-120 nm; phage T1, 50 nm head, 150 nm head, 150 nm tail; phage PR772, 53 nm; poliovirus, 28-30 nm; and phage PP7, 25 nm) were used to determine the ability of a newly developed, modified polyvinylidene fluoride (PVDF) membrane filter to remove viruses from several fluids. These included ultrapure water, Dulbecco's modified Eagle minimum essential medium (DMEM) and DMEM with 10% fetal bovine serum (DMEM-10). Small volume (10 ml) filtration experiments were done with 47-mm disks while larger volumes (1 litre) were done with virus suspended in DMEM-10, using cartridge filters with a surface area of 1.63 m2. With 47-mm disks, influenza A virus and phage T1 were removed to below detectable limits in all fluids tested (titre reduction [Tr] > 2.0 x 10(6) and > 5.8 x 10(8), respectively). The retention of phage PP7 and poliovirus was consistent but fluid dependent. The greatest concentration of phage PP7 and poliovirus was removed from ultrapure water (phage PP7, Tr = 2.1 x 10(7); poliovirus, TR > 3.2 x 10(4), while the removal efficiency from DMEM-10 was substantially lower (phage PP7, Tr = 2.3; poliovirus, Tr = 2.1 x 10(2)). Results of cartridge challenges in DMEM-10 were comparable to the corresponding small disk challenges. These results demonstrate that this PVDF membrane filter was very effective (Tr > 10(6)) in removing viral particles (> 50 nm); smaller viruses (< 50 nm) were also consistently removed, but the level of removal depended on the virus and type of fluid tested. In separate experiments, the recovery of purified albumin (69,000 Da) and IgG (150,000 Da) in the filtrate was also determined at approx. 0.015 mg/ml and approx. 10 mg/ml. Recovery of albumin and IgG was > 90%. Efficient virus retention coupled with high recovery of protein < 150,000 Da suggest potential applications of this membrane filter, when protection against adventitious viral contaminants is desired.

Endotoxin removal using 6,000 molecular weight cut-off polyacrylonitrile (PAN) and polysulfone (PS) hollow fiber ultrafilters.

The removal of pyrogenic and microbial contaminants from high purity water and parental solutions during production is a concern to pharmaceutical manufacturers. In a previous study, 6000 molecular weight cut-off (MWCO) polyacrylonitrile (PAN) and polysulfone (PS) ultrafilters were shown to remove poliovirus (Titer reduction [Tr], > 6 logs) and phages T1 and PP7 (Tr, 7 logs) from ultrapure water, 0.85% saline with 1% trypicase soy broth, and Dulbecco's Eagle minimal essential medium with 10% fetal bovine serum (15). In this study, we evaluated the ability of the 6,000 MWCO PAN and PS ultrafilters to remove purified endotoxin (Escherichia coli lipopolysaccharide) which was added to commercial Water for Irrigation. An endotoxin concentration reduction of > 6 logs was achieved with both PAN and PS ultrafilters (detection sensitivity, 0.0031 EU/ml) when a minimum of 3.92 x 10(3) EU/ml of endotoxin was added to Water for Irrigation. These results indicate that the 6,000 MWCO PAN and PS ultrafilters are very effective in removing endotoxin from fluids such as Water for Irrigation.

Hemostatic properties of the SV-40 transfected human microvascular endothelial cell line (HMEC-1). A representative in vitro model for microvascular endothelium.

HMEC-1 is a SV-40T transfected human microvascular endothelial cell line that constitutively expresses RNA transcripts for plasminogen activator inhibitor 1 (PAI-1), tissue-type plasminogen activator (t-PA), protein S (PS), von Willebrand factor (vWF), and thrombomodulin. Tissue factor (TF) can be induced in response to stimulation with tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1alpha) and phorbol 12-myristate 13-acetate (PMA). Proteins corresponding to PAI-1, t-PA, protein S and vWF genes were constitutively released in the culture supernatant. This cell line is a model that will be useful to investigate coagulation/fibrinolytic properties of microvascular endothelium.

Seeding of intracoronary stents with immortalized human microvascular endothelial cells.

Intracoronary stents are effective in decreasing the complications associated with acute closure during coronary angioplasty. A major complication associated with the use of coronary stents is acute thrombotic occlusion. It has been postulated that the stent loses its thrombogenic potential after it becomes covered with a layer of endothelial cells. Human dermal microvascular endothelial cells were transfected with a plasmid containing the simian virus 40 large T-antigen gene. Stents were placed in culture media with cells for 2 weeks. Seeding efficiency of the stent with the endothelial cells was assessed by scanning electron microscopy. Balloon-expandable coronary stents placed in cell culture with immortalized human microvascular endothelial cells showed near-complete coverage after 2 weeks. After balloon inflation, persistence of cells on the stent was noted only on the lateral aspect of the balloon-expanded stents. If these stents were placed in culture, complete recovery of the monolayer was noted after 3 days. Stents were then covered with endothelial cells and frozen for 4 days. After thawing, the cells adhered to the devices and divided to form a monolayer in tissue culture. Seeded balloon-expandable stents were frozen for 4 months, thawed, and then implanted in a pig coronary artery. Human endothelial cells were identified on the stent 4 hours after deployment. These studies demonstrate the feasibility of using a human microvascular endothelial cell line to seed an uncoated metal stent. The cells remain adherent to the stent, are functional after freezing, and remain on the stent at least 3 hours after intracoronary implantation.

The removal of phages T1 and PP7, and poliovirus from fluids with hollow-fiber ultrafilters with molecular weight cut-offs of 50,000, 13,000, and 6000.

We tested the ability of hollow-fiber ultrafilters with molecular weight cut-offs (MWCOs) of 50,000, 13,000, and 6000 to remove and detect viral agents (phage T1, 50-150 nm; phage PP7, poliovirus, 28-30 nm) from ultrapure water, 0.85% saline with 1% trypticase soy broth, and Dulbecco's modified Eagle minimum essential medium with 10% fetal bovine serum (DMEM-10). Virus diluted in saline and DMEM-10 were tested to evaluate filter performance under conditions that minimize the adsorption of viral particles to the filter matrix. During filtration, the retentate was returned to the input reservoir, and the permeate was removed to a separate vessel. Thus, the virus concentration in the feed increased over the course of filtration. Filter performance was evaluated by comparing the concentration of infectious virus in the initial virus suspension with the virus concentration in the permeate and retentate. Very efficient removal of phages T1 and PP7 was observed with the filters with MWCOs of 13,000 and 6000 (titer reduction > 7 logs) for all three fluids tested. No poliovirus was detected in the permeate of the ultrafilters with MWCOs of 13,000 or 6000 (titer reduction > 6 logs). These results indicate that the ultrafilters with MWCOs of 13,000 and 6000 were very effective in removing small viral particles (25-30 nm) by size exclusion. The recovery efficiency of the virus in the retentate varied by fluid type. However, filtration with virus diluted in DMEM-10 resulted in consistent recovery of the viruses tested. The results suggest that these ultrafilters may have the dual potential of removing viral contaminants from fluids and concentrating virus in the retentate.

Seeding of vascular grafts with an immortalized human dermal microvascular endothelial cell line.

Small-caliber vascular grafts (< 6 mm) for arterial bypass frequently fail owing either to acute thrombosis or long-term fibrosis. One strategy to enhance patency is the coverage ("seeding") of luminal polymeric graft surfaces with endothelial cells (EC), which may in themselves be thromboresistant and antiproliferative, or which could be transfected with genes whose products are thrombolytic or growth-inhibitory. Advances in understanding of EC-biomaterial interaction have led to improvements in cell coverage and retention, but the sources of EC for such procedures have been limited to large vessels (autologous veins) and microvascular endothelium isolated from autologous adipose tissue. Before the practice of graft seeding can gain widespread clinical acceptance, the practical constraints of EC harvest, EC culture, and quick access to the seeded prosthesis for the surgical procedure must be overcome. Ideally, an EC line with a high proliferative capacity could be preestablished on the grafts, which could then be cryopreserved and made available as needed. The authors have seeded Dacron graft material with an immortalized human dermal microvascular EC line, HMEC-1. These cells were initially transfected with simian virus 40A large T antigen and have been passaged more than 100 times without signs of senescence. They also express von Willebrand factor, take yp acetylated low density lipoproteins, and rapidly form tubes when cultured on matrigel. Confluent coverage of Dacron graft segments, either untreated or coated with gelatin, was achieved in two weeks. The cells formed a monolayer over topographically elevated regions or appeared to be > one layer thick in other areas. Cells were also shown to remain viable after freezing.2+These results suggest a potential practical method for

Selective inhibition of adhesion molecule expression by edelfosine (ET-18-OCH3) on human umbilical vein or microvascular endothelium.

An abundance of data is accumulating that suggest that if one can block endothelial cell-leukocyte binding or inhibit cell adhesion molecules (CAM), inflammatory events can be greatly diminished. In this report, we demonstrate that an alkyl-lysophospho-lipid compound (ET-18-OCH3) can decrease adhesion molecule expression on cultured human micro- and macrovascular endothelial cell lines. ET-18 selectively decreased CAM expression; CD31 was decreased, however. Vascular CAM-1 tumor necrosis factor-alpha-induced expression was not altered. Intercellular adhesion molecule 1 expression was decreased, but endoglin expression was not affected. Thus, we have demonstrated nontoxic downmodulation of vascular CAM expression in vitro. Whether this compound will have anti-inflammatory properties needs to be clarified in animal models.

Removal of human immunodeficiency virus by an 0.04-micron membrane filter.

We tested the ability of a 0.04-micron nylon membrane filter to remove human immunodeficiency virus (HIV) from tissue culture media containing 10% fetal calf serum. Endpoint titrations of infectious virus (ID50) were performed on lymphocyte cultures. The presence of virus in the cultures was determined using an enzyme-linked immunoabsorbant assay (ELISA) of the HIV-1 P24 core antigen. In repeated experiments, filtration of the virus suspension resulted in the removal of HIV below detectable limits. The titer reduction was estimated to be greater than 8.5 x 10(2). These results suggest that this filter is effective in removing HIV from fluids containing serum or serum products.

Detection by polymerase chain reaction of all common Mycoplasma in a cell culture facility.

The identification of cell cultures contaminated with organisms from the class Mollicutes has led us to examine the effectiveness of polymerase chain reaction (PCR) for detecting these organisms in genomic DNA. We developed a previously identified nested PCR primer set and compared its ability to detect Mycoplasma with that of a commercially available PCR kit for detecting Mycoplasma. We found that although the commercial system detected and identified a few of the most common Mycoplasma species, the primer set (GPO-1, GPO-2, MGSO) detected the presence of all the common Mycoplasma species and many of the rare mycoplasma species previously encountered in tissue culture.