Mouse Ascitic Fluid Research Articles

Phosphorylation of the Consensus Sites of Protein Kinase A on α1D L-type Calcium Channel

The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.

Production of Monoclonal Antibody Supernatant and Ascites Fluid

This unit details methods for production of monoclonal antibodies. Two methods are given for production of hybridoma supernatants, including one for large-scale production. A protocol for large-scale production of hybridomas or cell lines is presented for use in isolation of cellular proteins. Finally, a method is given for producing and obtaining mouse ascites fluid containing monoclonal antibody. Recommendations developed by the Committee on Methods of Producing Monoclonal Antibodies (Institute of Laboratory Animal Research, National Research Council) on the use of animals for producing monoclonal antibodies are also discussed. These recommendations are designed to foster the judicious use of animals and to strongly encourage the use of in vitro methods whenever possible.

Her2-targeted pegylated stealth liposomal doxorubicin (PLD) retains its specific targeting ability to Her2-expressing tumor cells after in vivo circulation and extravasation to mouse malignant ascites

13097 Background: Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted PLD retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to the ascitic fluid of mice with malignant ascites. Methods: PLD was grafted with a pegylated lipophilic conjugate of an anti-Her2 scFv antibody fragment (F5) at a ratio of 15 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites. When abdominal swelling developed, Her2-targeted PLD and nontargeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 hr later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the liposome-containing ascitic fluid was tested in vitro against Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of Her2-PLD and PLD, using as parameter the amount of cell-associated doxorubicin. Results: Plasma and ascitic fluid levels of Her2-PLD were only slightly below those of PLD indicating that the Her2 ligand did not cause any significant change in the clearance rate of PLD. Her2-PLD and PLD bound to an equal extent to J6456 cells in vivo. The in vitro binding of Her2-PLD from ascites to Her2- expressing cells was increased 10 to 20-fold above that of PLD from ascites, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. Conclusions: Her2-targeted PLD demonstrates similar circulation time to that of nontargeted PLD. After in vivo passage, the targeted liposome retains most of its original binding capacity to Her2 expressing cells indicating that the ligand is stably maintained in association with the doxorubicin liposomal carrier. Targeting of PLD using this Her2 antibody fragment should provide an important means of selective drug delivery to tumors expressing the Her2 receptor. [Table: see text]

A Novel Purification Method for Monoclonal Antibodies

Immobilized Protein A and Protein G are commonly used for immunoglobulin purification. Problems with these affinity methods include selective binding between different species and their subclasses ranging from strong binding to no binding at all, the number of steps involved in the purification protocol, and the harsh elution buffers containing primary amines at low pH. Here we report the use of a resin that overcomes the weaknesses of monoclonal antibody purification. The resin and protocol have been optimized to allow the extraction of non-relevant proteins from tissue culture supernatant and ascites fluid often present in high abundance such as albumin and transferrin, thereby enriching the final product with immunoglobulins without further diluting the sample. This flow-through purification approach can be completed in less than 15 minutes for volumes less than 30 ml and 2.1 hours for volumes up to 1L. These purification times are considerably shorter than the labor-intensive hours of affinity purification. The method uses a mild buffer at physiological pH, unlike immobilized Protein A and Protein G that require a harsh elution buffer that is known to denature antibodies due to low pH. Additionally, with our resin the purified antibody is in a buffer free of primary amines and is ready for use or further processing without subsequent dialysis. Monoclonal antibodies produced in tissue culture supernatant as well as mouse and rat ascites fluid have been used for purification studies with Melon Gel. The results in all cases of purity and recovery were better than or equal to those obtained with immobilized Protein A and Protein G. Pierce Biotechnology Inc.

Development of Competitive Direct Enzyme-linked Immunosorbent Assay for the Detection of Gentamicin Residues in the Plasma of Live Animals

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities (CR50) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/B0 ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/B0 ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life (t1/2) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 10 : 1498-1504)

The Turnover Kinetics of Major Histocompatibility Complex Peptides of Human Cancer Cells

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. Thus, the repertoire of these peptides (the MHC peptidome) should correlate better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. To test the validity of this statement and to determine whether the majority of MHC peptides are derived from short lived proteins, from defective ribosome products, or from regular long lived cellular proteins we analyzed in parallel the turnover kinetics of both MHC peptides and cellular proteins in the same cancer cells. The analysis was performed by pulse-chase experiments based on stable isotope labeling in tissue culture followed by capillary chromatography and tandem mass spectrometry. Indeed only a limited correlation was observed between the proteome and the MHC peptidome observed in the same cells. Moreover a detailed analysis of the turnover kinetics of the MHC peptides helped to assign their origin to normal, to short lived or long lived proteins, or to the defective ribosome products. Furthermore the analysis of the MHC peptides turnover kinetics helped to direct attention to abnormalities in the degradation schemes of their source proteins. These observations can be extended to search for cancer-related abnormalities in protein degradation, including those that lead to loss of tumor suppressors and cell cycle regulatory proteins.

Plasma Membrane Residence of Hyaluronan Synthase Is Coupled to Its Enzymatic Activity

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.

Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN)-mediated Enhancement of Dengue Virus Infection Is Independent of DC-SIGN Internalization Signals

Dengue virus (DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pH-dependent fusion into DCs.

Purification of IgG and insulin on supports grafted by sialic acid developing “thiophilic-like” interactions

We developed the synthesis of new supports for the purification of insulin and IgG by affinity chromatography. The preparation of such an affinity support is performed in two steps. First, silica beads are coated with dextran polymers carrying a calculated amount of positively charged diethylaminoethyl groups in order to mask negative charges at its surface. Second, ligand is immobilized using a coupling agent. This support combines the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. The existence of N-acetylneuraminic acid (sialic acid) in insulin receptor and in the antigenic determinant of IgG suggests that such an acid may develop specific interactions usable in affinity chromatography. Therefore, N-acetylneuraminic acid was used as an active ligand. The immobilization of sialic acid can be carried out by using the conventional coupling agent: the carbonyldiimidazole. The performances of these supports grafted by sialic acid were studied by high-performance liquid affinity chromatography (HPAC). The optimization of the chromatographic conditions (support characteristics and mobile phase) enabled us to observe a behavior of the type “thiophilic” of the support, which does not contain sulfone group. This new affinity support allowed a one-step separation of the IgG from mouse ascitic fluids and also allowed the insulin purification from a pancreatic extract with a good purification yields.

NapF Is a Cytoplasmic Iron-Sulfur Protein Required for Fe-S Cluster Assembly in the Periplasmic Nitrate Reductase

The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His(6)-tagged NapF protein was overproduced in Escherichia coli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF(-) strain of R. sphaeroides. The His(6)-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2'-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF(-) strain phenotype, and the NapF-dependent reactivation of the 2,2'-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.

Carboxy-terminally truncated Dengue 4 virus envelope glycoprotein expressed in Pichia pastoris induced neutralizing antibodies and resistance to Dengue 4 virus challenge in mice.

We have expressed a recombinant Dengue 4 virus envelope glycoprotein (E4rec), truncated at its C-terminus by 53 amino acids, in Pichia pastoris. The presence of E4rec was confirmed by Western-blot using anti-DEN 4 hyper immune mouse ascitic fluid. E4rec migrated during SDS-PAGE as a 64 kDa protein. Treatment with endoglycosidases showed that the E protein was modified by the addition of short mannose chains and the absence of hyperglycosylation. When administered to BALB-C mice, E4rec elicited a DEN 4 neutralizing antibody response haemagglutination inhibition antibodies and specific memory T cell response. Mice immunized were also significantly protected against lethal DEN 4 virus challenge (86.6%, p < 0.001).

Purification of the specific immunoglobulin G 1 by immobilized metal ion affinity chromatography using nickel complexes of chelating porous and nonporous polymeric sorbents based on poly(methacrylic esters) : Effect of polymer structure

Ni 2+ complexes of the chelating nonporous and porous bead sorbents based on methacrylic esters crosslinked with ethylene dimethacrylate were used in isolation of the horseradish peroxidase-specific immunoglobulin IgG 1 from the crude mouse ascitic fluid by immobilized metal ion affinity chromatography (IMAC). Iminodiacetic and aspartic acids were attached to porous poly(glycidyl methacrylate) beads differing in size, morphology and chemical composition. Ethylenediaminetriacetic acid and quinolin-8-ol chelating groups were attached mainly to the surface hydroxyl groups in nonporous poly(diethylene glycol methacrylate) beads through spacers. The latter sorbents exhibited better kinetic characteristics than the former but a very low IgG 1 sorption capacity. In a single-step IMAC procedure, the best efficiency in the specific IgG 1 purification was obtained with porous sorbents (recovery 92%, purity 73%). Differences in IMAC separations are discussed from the point of view of morphology of polymer beads as well as of the type and concentration of chelating ligands.

Integrated self-calibrationvia electrokinetic solvent proportioning for microfluidic immunoassays

On-board generation of a set of calibration standards was demonstrated within a microfluidic device designed to perform immunoassay. Electrokinetic flow was used to proportionally mix the antibody (Ab) to bovine serum albumin (BSA) and a diluting buffer, to provide varying Ab concentrations for downstream mixing with fluorescently labeled BSA (BSA*). Mixing ratios were determined from electrical impedance modeling of the fluidic network using P-SPICE software, and peak heights for the labeled species were analyzed relative to the concentration calculated from the model. For dilution and separation of fluorescently labeled amino acids, a linear calibration curve was obtained for mixing ratios of 0.118 to 7.46. A linear calibration curve was obtained for the immunoassay calibration using dilution ratios between 0.197 and 5.077. Deviations were observed at larger extremes, possibly due to leakage effects at intersections. Peak height reproducibility was +/- 3% for the immunoassay, using diluted monoclonal Ab in mouse ascites fluid as the analyte. Recovery for on-chip calibration was 92 +/- 6% versus calibrants prepared off-chip, indicating a small bias.

The Presence of Nipah Virus in Respiratory Secretions and Urine of Patients during an Outbreak of Nipah Virus Encephalitis in Malaysia

Objectives: To study the excretion of Nipah virus in the upper respiratory secretions and urine of infected patients in relation to other clinical features.Methods: Isolation of Nipah virus from the respiratory secretions and urine was made in Vero cells and identified by indirect immunofluorescence assay using anti-Hendra specific hyperimmune mouse ascitic fluid and FITC-conjugated goat anti-mouse IgG.Results: During the peak outbreak of Nipah virus encephalitis in Malaysia, Nipah virus was isolated from the upper respiratory secretions and urine in eight of 20 patients who were virologically and/or serologically confirmed to be infected with the virus. From these eight patients, Nipah virus was isolated from six throat swab specimens, three urine specimens and only one nasal swab specimen. The positive virus isolation rate was related to the collection of these specimens during the early phase of the illness (P= 0.068). The presence of serum anti-Nipah specific IgM appeared to reduce the chance of isolating the virus (P= 0.049). There was no significant difference in the isolation rate with respect to the age, gender, ethnic group and clinical features associated with grave prognosis and mortality outcome of the patients.Conclusion: This study shows that it is possible to be infected from secretions of infected patients, but epidemiological survey on close contacts so far did not suggest that human-to-human transmission is common.

Inhibition of pig granulosa cell adhesion and growth in vitro by immunoneutralization of epithelial cadherin

Epithelial cadherin (E-cadherin) is a member of the cadherin family of calcium-dependent cell adhesion molecules and is present in the ovary. Although expression of E-cadherin is high in healthy pig granulosa cells and low in granulosa cells of atretic follicles, the importance of E-cadherin-mediated adhesion in granulosa cell function is unclear. The aim of the present study was to determine the impact of immunoneutralization of E-cadherin on granulosa cell adhesion, DNA synthesis and cell proliferation in vitro. Before attachment, pig granulosa cells were exposed to a monoclonal E-cadherin antibody (DECMA-1) which blocks E-cadherin function. Controls included substitution of the antibody with either mouse ascites fluid or another E-cadherin antibody directed against the cytoplasmic domain and which was therefore inaccessible in intact cells. Both granulosa cell proliferation and insulin-like growth factor I-induced DNA synthesis were inhibited significantly in the presence of DECMA-1 compared with controls (P < 0.05). Control granulosa cells in culture formed large clusters with many cells packed tightly together. However, after 48 h exposure to the function-perturbing E-cadherin antibody, there was a significant decrease in the size of the granulosa cell clusters (P < 0.05) and the degree of cell-cell contact was reduced compared with control cultures. No effects on DNA synthesis, cell proliferation or cell adhesion were observed when DECMA-1 was substituted with either mouse ascites fluid or the antibody specific for the cytoplasmic domain of E-cadherin. In conclusion, these data provide evidence to support the hypothesis that E-cadherin is important for maintaining granulosa cell contact, DNA synthesis and cell proliferation in vitro. These results indicate that E-cadherin plays a fundamental role in maintaining both the structure and function of ovarian follicles.

High-performance sodium dodecyl sulfate-capillary gel electrophoresis of antibodies and antibody fragments

High-performance sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) has been used to separate antibodies and their fragments according to size. Under non-reduced conditions, murine monoclonal antibodies generally show a predominant peak with five to six apparent fragment peaks. The magnitude of the apparent fragmentation is temperature-dependent and is more pronounced with rabbit, sheep and bovine immunoglobulin G's than murine monoclonal antibodies. In addition to temperature, pH and buffer also affect the fragmentation. Without heat treatment during the preparation of the SDS–antibody complexes, the observed fragments become nearly absent; however, some murine monoclonal antibodies exhibit several peaks that group near the expected migration time of an immunoglobulin G, presumably due to their anomalous interaction with sodium dodecyl sulfate. The method can also be used to monitor the progress of peptic digestions to generate murine F(ab′) 2 antibody, to perform “gel-shift” assays, and to separate mouse ascitic fluid. This high-performance electrophoretic technique is suitable for quality control as well as the characterization of the antibodies under experimental conditions.

Sedimentation field-flow fractionation application to Toxoplasma gondii separation and purification

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii, an intracellular protozoa of micronic size range (4–10 μm). Its classical purification processes are complex and often associated with low recovery. All investigation procedures concerning this parasite require its isolation and purification from at least the mouse ascitic fluid. For this purpose, a recently developed laboratory technology was used, i.e. sedimentation field-flow fractionation. This chromatographic-like separation technology was demonstrated to be particularly selective for isolation and separation of micron-sized biological particles. Sedimentation field-flow fractionation operated on the steric-hyperlayer mode was used to isolate the parasite from the remanent ascitic contaminants of different origins and from red blood cells. With this technology, 86% recovery with 97% viability was obtained in less than 30 min.

Molecular cloning and characterization of the Ehrlichia chaffeensis variable-length PCR target: an antigen-expressing gene that exhibits interstrain variation.

A clone expressing an immunoreactive protein with an apparent molecular mass of 44 kDa was selected from an Ehrlichia chaffeensis Arkansas genomic library by probing with anti-E. chaffeensis hyperimmune mouse ascitic fluid. Nucleotide sequencing revealed an open reading frame (ORF) capable of encoding a 198-amino-acid polypeptide. The ORF contained four imperfect, direct, tandem 90-bp repeats. The nucleotide and deduced amino acid sequences did not show close homologies to entries in the molecular databases. PCR with primers whose sequences matched the sequences flanking the ORF was performed with DNA samples extracted from cell cultures infected with nine different isolates of E. chaffeensis, blood samples from seven patients with monocytic ehrlichiosis, and Amblyomma americanum ticks collected in four different states. The resulting amplicons varied in length, containing three to six repeat units. This gene, designated the variable-length PCR target, is useful for PCR detection of E. chaffeensis and differentiation of isolates.

Development of ELISA Method for Aflatoxin B1 Using Monoclonal Antibody Precipitated from Mouse Ascitic Fluid by Ammonium Sulfate

Development of ELISA Method for Aflatoxin B1 Using Monoclonal Antibody Precipitated from Mouse Ascitic Fluid by Ammonium Sulfate

Preclinical models for Chlamydia pneumoniae and cardiovascular disease: hypercholesterolemic mice

Seroepidemiologic studies have shown an association of Chlamydia pneumoniae antibody with atherosclerosis. Compelling additional evidence has accumulated, in that the organism has been found within atherosclerotic lesions throughout the arterial tree by multiple methods. C. pneumoniae has also been isolated from coronary and carotid atheromatous plaques. Although these studies support a potential role for C. pneumoniae in atherogenesis, confirmation of a causal relationship requires the use of animal models and intervention studies. We have focused on the evaluation of mouse models to address the hypothesis that, following upper respiratory tract infection, lung macrophages are infected, disseminate to the aorta, and alter the onset or progression of atherogenesis. ApoE-deficient knock-out and C57BL/6J mice were used. The apoE-deficient mouse develops atherosclerotic lesions spontaneously on a regular diet in a time- and age-dependent manner. This knock-out strain was developed on the background of the C57BL/6J mouse, which only develops atherosclerosis on a high-fat/high-cholesterol diet. To investigate whether infected macrophages constitute a vehicle for dissemination of C. pneumoniae in vivo, mice were inoculated intranasally or intraperitoneally. The organism was detected in harvested alveolar and peritoneal macrophages at all time points following intranasal and intraperitoneal inoculations, respectively, and in peripheral blood mononuclear cells following inoculation by both routes. In another experiment, alveolar and peritoneal macrophages from intranasally and intraperitoneally inoculated mice were adoptively transferred by intraperitoneal injection to uninfected mice. Subsequently, C. pneumoniae was detected in lung, spleen, abdominal lymph nodes and/or thymus of recipient mice. In control experiments, UV-inactivated C. pneumoniae DNA was not detected in alveolar or peritoneal macrophages beyond 5 min after inoculation in vivo. These cumulative results demonstrate that C. pneumoniae infects macrophages in vivo and that macrophages can serve as a vehicle for dissemination to other sites. To answer the question of whether the organism disseminates to and persists in the aorta, 8-week-old mice were infected intranasally. Following single or multiple inoculations in apoE-deficient mice, C. pneumoniae was detected in the lung and aorta for 20 weeks postinfection. In contrast, in C57BL/6J mice, the organism did not persist in the aorta following a single intranasal inoculation, but could be detected up to 7 weeks postinfection in multiply inoculated mice. Significantly, in apoE-deficient mice with developed atherosclerotic lesions, the organism was found in foam cells within the lesions by immunocytochemical staining. These studies show that persistent C. pneumoniae infection occurs in atherosclerotic lesions in the aorta in the apoE-deficient knock-out mouse model. Infection of the aorta also occurred in C57BL/6J mice but was more transient. Both models should be useful in studying the pathogenic role of C. pneumoniae in atherogenesis and for determining if therapy prevents dissemination of infection. To this end, we have evaluated the susceptibility of C. pneumoniae to roxithromycin, a new macrolide, in vitro in cell culture and in vivo in the apoE-deficient mouse model. In vitro results compare favorably with other new macrolides, quinolines, and tetracyline. Preliminary studies in the apoE model of persistent infection suggest that the organism is detected less frequently in the lung and aorta by PCR following treatment. These promising preliminary studies warrant further investigation.