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Rapid assignment of the swine major histocompatibility complex (SLA) class I and II genotypes in Clawn miniature swine using PCR-SSP and PCR-RFLP methods.

BACKGROUND: Inbred miniature swine with defined novel SLA haplotypes will be useful in allo- and xeno-transplantation studies, which can be carried out representing variable combinations of SLA haplotypes. METHODS: In Clawn miniature swine, two haplotypes (c1 and c2) and one crossover haplotype (c3) have been assigned by nucleotide sequence determination of RT-PCR products of the three SLA classical class I genes and two SLA class II genes. To select SLA class I and II homozygotes in Clawn miniature swine individuals, we developed a rapid and simple SLA-class I- and II-DNA typing method by a combination of polymerase chain reaction-sequence specific primer (PCR-SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. RESULTS: Seven allele specific primer pairs were designed for amplification of the second exons of three SLA class I genes, SLA-1, SLA-2, and SLA-3, and one SLA class II gene, DRB1. Furthermore, based on PCR-RFLP patterns in the SLA-DQB1 gene, two allelic variants were recognized in the second exon in the Clawn miniature swine. Three haplotypes, c1, c2 and c3, were simply identified by the combination of PCR-SSP and PCR-RFLP methods in 22 samples from five families. A single allele at each of the class I and II genes was also observed in seven samples as SLA class I and II homozygotes with either the c1 or c2 haplotype. CONCLUSIONS: The combination of PCR-SSP and PCR-RFLP methods facilitate the rapid identification of the three haplotypes and SLA class I and II homozygotes in individual Clawn miniature swine.

Alleles↗

First detection of African swine fever in a swine farm in Taiwan.

INTRODUCTION: African swine fever (ASF) is a highly contagious, high-consequence transboundary animal disease that poses a critical threat to global swine production and agricultural economics. Since its emergence in China in 2018, ASF has spread to over 20 Asia-Pacific countries, causing significant economic disruption. While Taiwan previously detected ASF virus several times in dead pigs drifting offshore, or in illegal pork-related products brought by international tourists and inspected at airport and seaport border controls, no local domestic swine farm had tested positive before this October 2025 ASF event. However, maintaining this disease-free status requires constant vigilance against evolving regional biosecurity threats. METHODS: The first ASF detection in a domestic swine herd in Taichung City, Taiwan, reported on October 22, 2025, was triggered by abnormal alerts of the monitoring system in the rendering plant; the index farm captured a cumulative herd mortality rate of 35.2% that exceeded the predefined threshold (3% mortality daily in the nursery to finish pigs). Five finisher pigs were submitted for diagnostic evaluation, which subsequently confirmed ASFV infection via real-time PCR, pathological examination, immunohistochemistry, virus isolation, and whole-genome sequencing. RESULTS: Affected pigs showed clinical signs including wheezing, sudden death, nasal bleeding, uncoagulated blood in the nostrils, and mild hemorrhage on the skin surface of the neck, abdomen, and buttocks. Histopathological examination revealed severe multisystemic hemorrhagic lesions. Based on assay results of the P72, P54, P30, and CD2v genes, and whole-genome sequence, phylogenetic analysis confirmed that the isolate (ASFV/TWN/2025) is a genotype I/II recombinant strain, most similar to prevailing strains isolated in China and Vietnam, sharing 99.95%-99.97% and 99.92%-99.97% nucleotide similarity of whole genome sequence, respectively. DISCUSSION: Upon confirmation of the ASF case, authorities immediately implemented a nationwide swine movement standstill for 15 days to mitigate transmission risk. To date, no secondary cases have been detected. This article details the early monitoring and rapid diagnosis process of the first case of ASF infection in a farm in Taiwan, and highlights the information from this case to provide lessons for disease diagnosis and prevention in ASF-free areas.

African swine fever↗

Isolation and characterization of Yersinia enterocolitica from swine feces recovered during the National Animal Health Monitoring System Swine 2000 study.

A national study was conducted for the isolation of pathogenic Yersinia enterocolitica in pig feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from September 2000 to March 2001 from 77 production sites in 15 of the top 17 swine-producing states were tested for the presence of pathogenic Y. enterocolitica. After enrichment of swine fecal samples in irgasan-ticarcillin-potassium chlorate broth, the enriched cultures were plated on cefsulodin-irgasan-novobiocin agar for isolation of presumptive Y. enterocolitica. The isolates were confirmed as pathogenic Y. enterocolitica by the fluorogenic 5' nuclease PCR assay targeting the chromosomal attachment invasion ail gene. Of 2793 fecal samples tested, 106 (3.80%) ail-positive strains of Y. enterocolitica were isolated. These 106 ail-positive isolates originated from 7 of the 15 participating states. The predominant serotype O:3 (n = 79 of 106) was distributed in five states (n = 5 of 7). Serotype O:5 (n = 27 of 106) was also found in five states (n = 5 of 7). All isolates contained the virulence plasmid and expressed virulence-associated phenotypic characteristics. These results indicate that swine in the United Stares harbor Y. enterocolitica that can potentially cause human illness.

Animals↗

Respiratory health of workers exposed to swine confinement buildings only or to both swine confinement buildings and dairy barns.

Swine building workers (N = 488) and nonfarming neighborhood referents (N = 216) were enrolled in this study. There was a slight but significant increase in the prevalence of chronic bronchitis (17.49 versus 11.57%) and more evidence of airflow obstruction (forced expiratory volume in 1 s/forced vital capacity 0.75 versus 0.78) among the swine workers when they were compared with the referents. The subjects who spent more than 3 h/d in the swine buildings had a higher prevalence of chronic bronchitis (21.94 versus 13.25%) and airflow obstruction (forced expiratory volume in 1 s/forced vital capacity 0.75 versus 0.76) than those with shorter daily contact. Swine building only workers had no precipitins to antigens found in their environment and no clinical evidence of extrinsic allergic alveolitis. The number of years on the farm, dual exposure with dairy cattle, positive skin prick tests, type of piggery, and type of feeding did not add to the respiratory health impact of swine buildings.

Agricultural Workers' Diseases↗

Monoclonal antibodies reactive with swine lymphocytes. I. Antibodies to membrane structures that define the cytolytic T lymphocyte subset in the swine.

A panel of monoclonal antibodies (mAb) with specificity for swine leukocytes was prepared by somatic cell hybridization with the use of spleen cells from mice immunized with swine thymocytes. The reactivity of two mAb (295/33 and 122/28), which both immunoprecipitated from the surface of swine leukocytes an antigen termed S-L2 with an apparent m.w. of 33 to 35 kilodaltons under reducing and 65 to 70 kilodaltons under nonreducing conditions, was investigated in detail. These mAb were reactive in indirect immunofluorescence with 50 to 60% of thymocytes, 35% of peripheral blood lymphocytes, and 55% of E rosette-positive cells; they were nonreactive with bone marrow cells, Ig+ B cells, nonrosetting lymphocytes, granulocytes, and monocytes. In functional studies, the elimination of S-L2+ cells partially reduced the proliferative response to concanavalin A and pokeweed mitogen but not to Staphylococcus aureus and lipopolysaccharide. The S-L2- subset proliferated well to alloantigens. Both cytolytic T effector cells and precursor cells carried the antigen S-L2 and could be depleted from heterogeneous cell populations by both antibodies in the presence of complement. These data suggest that the mAb 295/33 and 122/28 recognize a specific polypeptide present on the surface of swine cytolytic T cells. These antibodies will be useful in studies on the swine immune system.

Animals↗

Carboxyhemoglobin values in swine relative to carbon monoxide exposure: guidelines for monitoring animal and human health hazards in swine-confinement buildings.

Miniature pigs were exposed, in an environmental chamber, to 55 mg of CO/m3 of air, 110 mg/m3, 220 mg/m3, or 330 mg/m3. Blood samples were taken from the swine every hour over an exposure period of 6 or 8 hours. After the pigs were removed from the chamber, blood samples were taken every 30 minutes for an additional 3 hours. The blood samples were measured by spectrophotometry for the amount of carboxyhemoglobin (COHb). On exposure to CO, the COHb values in the pigs increased in a linear fashion during the first 2 hours, then began to level off, reaching a peak concentration at 6 to 8 hours. The percentage of COHb in the blood after 6 hours' exposure to the different amounts of CO were as follows: 55 mg/m3 - 5%, 110 mg/m3 - 10.5%, 220 mg/m3 - 20%, and 330 mg/m3 - 27.2%. There was a linear relationship between the amount of CO exposure and the peak blood value of COHb. The present data provide guidelines for the use of COHb measurement in swine as a means to monitor the environment in swine-confinement buildings for potentially dangerous amounts of CO for swine and persons and to aid in the diagnosis of CO-induced perinatal disease in swine.

Animals↗

A conserved African swine fever virus IkappaB homolog, 5EL, is nonessential for growth in vitro and virulence in domestic swine.

An African swine fever virus (ASFV) gene with similarity to the cellular inhibitor of NFkappaB (IkappaB) was described in the pathogenic African isolate Malawi Lil-20/1 (ORF 5EL) and a cell-culture-adapted European virus, BA71V (ORF A238L). Recently, this gene was shown to be a functional IkappaB homolog capable of downregulating NFkappaB-regulated gene expression. This observation suggests the gene may be of significance to aspects of ASFV pathogenesis and virulence in domestic swine by interfering with a normal antiviral host response. Here we show, using nucleotide sequence analysis, that 5EL is highly conserved among various African and European pathogenic field isolates and that in all cases its similarity to IkappaB genes is limited to the presence of four low complexity ankyrin repeats in the ASFV gene. The 5EL gene of Malawi Lil-20/1 encodes a 28-kDa protein which was expressed early in virus-infected macrophage cell cultures with maximum levels observed at 3 to 5 hr postinfection. To study gene function, a Malawi Lil-20/1 5EL gene deletion mutant (Delta5EL) was constructed. Growth characteristics of Delta5EL in porcine macrophage cell cultures were indistinguishable from those of the parental virus. And, Delta5EL exhibited an unaltered parental Malawi Lil-20/1 disease and virulence phenotype in domestic swine. Thus, although highly conserved among ASFV isolates, 5EL is nonessential for growth in porcine macrophages in vitro and for viral virulence in domestic swine. A possible role for this gene in transmission of ASFV in nature, a setting which involves the cycling of ASFV between two highly adapted hosts, Ornithodoros ticks and warthogs or bush pigs, in sub-Saharan Africa is discussed.

African Swine Fever↗

Inhibitory effect of African swine fever virus on lectin-dependent swine lymphocyte proliferation.

The incubation of swine peripheral blood mononuclear cells (PBMC) with African swine fever (ASF) virus preparations strongly inhibited the proliferative response of lymphocytes to PHA and other lectins. The inhibition, which persisted after inactivation of the virus by UV radiation, was dependent upon the dose and the time that virus preparations were present in cultures. When virus preparations were fractionated by ultracentrifugation, the inhibitory activity resulted to be soluble, whereas no activity was found in the sedimented viral fraction. However, the preincubation during 4 days of this sedimented fraction with swine PBMC, before the addition of the mitogen, restored the inhibitory activity. The results obtained suggest that the inhibition is mediated by one or more soluble factors released by swine PBMC after coincubation with ASF virus in a time dependent process. These factors show a molecular weight between 40 and 80 kDa by gel filtration chromatography. The inhibitory activity described in the present paper is an indication of inhibition of lymphocyte function produced by ASF virus which can help to understand how this virus escapes from the host immune system.

African Swine Fever↗

Deletion of a CD2-like gene, 8-DR, from African swine fever virus affects viral infection in domestic swine.

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (delta8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, delta8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo, 8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant delta8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with delta8-DR. A delay in spread to and/or replication of delta8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for delta8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with delta8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.

African Swine Fever↗

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.

Animals↗

Analysis of the serologic relationship among San Miguel sea lion virus and vesicular exanthema of swine virus isolates. Application of the western blot assay for detection of antibodies in swine sera to these virus types.

Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to calciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested.

Animals↗

In vitro and in vivo association of African swine fever virus with swine erythrocytes.

The association of African swine fever virus (ASFV) with swine erythrocytes in vivo, in high titers, was verified by inoculating 30 pigs with 17 ASFV isolates and assaying their plasma and washed erythrocyte fractions for residual virus. Viral antigens were specifically localized on the surface of in vitro and in vivo swine erythrocytes, using the fluorescent antibody technique and 3 monoclonal antibodies specific for ASFV. The same monoclonal antibodies immunoprecipitated virus-specific polypeptides of molecular weights 13 kd and 73 kd from ASFV-infected Vero cells. Erythrocytes from viremic swine infected with Lisbon-60, Dominican Republic, Badajoz-M98, or Cameroon isolates of ASFV were studied by transmission electron microscopy. Virus was found in membrane depressions at the surface of erythrocytes. These surface depressions resembled stages of smooth surfaced pits. Erythrocytes from viremic pigs were fragile osmotically.

African Swine Fever↗

Passively transferred African swine fever virus antibodies protect swine against lethal infection.

The role of anti-viral antibodies in homologous protective immunity to a virulent African swine fever virus (ASFV) strain E75 was examined by passive transfer experiments in swine. Eighty-five percent of animals (n = 14) that received anti-ASFV immunoglobulin (Ig) survived challenge infection, while 100% mortality was observed in control group animals (n = 28) that received anti-pseudorabies virus Ig, normal swine Ig, or phosphate-buffered saline. With the exception of a significantly delayed and transient fever response, anti-ASFV Ig group animals remained clinically normal following challenge, whereas control group animals presented with clinical ASF on Day 4 postchallenge. Additionally a significant 3 day delay in onset of viremia and a 10,000-fold reduction in both mean and maximum virus titers were observed for animals given anti-ASFV Ig. These results indicate that anti-ASFV Ig alone will protect swine from lethal infection with virulent ASFV. Further, they support the view that the antibody-mediated protective effect is an early event that effectively delays disease onset.

African Swine Fever↗

Changes in swine macrophage phenotype after infection with African swine fever virus: cytokine production and responsiveness to interferon-gamma and lipopolysaccharide.

Cytokines produced by cells of the immune system, including macrophages, can influence inflammatory responses to viral infection. This has been exploited by viruses, which have developed strategies to direct the immune response towards ineffective responses. African swine fever virus (ASFV) is a double-stranded DNA virus that infects macrophages of domestic swine. In this study, primary cells of monocyte macrophage lineage were obtained from the lungs, peritoneum or blood of domestic swine and, after infection with ASFV, supernatants were tested for cytokines using biological assays. The cytokine transforming growth factor-beta (TGF-beta) was detected after infection of macrophage preparations, but tumour necrosis factor (TNF) and interleukin-1 (IL-1) were not detected. ASFV-infected and uninfected macrophage populations were also tested to assess their ability to respond to cytokines by enhancing production of superoxide in the respiratory burst mechanism. Responses to interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were suppressed in macrophage populations infected with virus, even at low multiplicities of infection. Addition of TGF-beta to uninfected macrophages resulted in a similar suppression of response, but antibody to TGF-beta did not prevent suppression induced by virus. These results are discussed in relation to the pathology of African swine fever.

African Swine Fever↗

In vivo T cell depletion in miniature swine using the swine CD3 immunotoxin, pCD3-CRM9.

BACKGROUND: Partially inbred miniature swine developed in this laboratory provide a unique preclinical large animal model for studying transplant tolerance. The importance of in vivo T cell depletion for establishing stable mixed hematopoietic cell chimerism using a clinically relevant sublethal regimen has been well documented in murine studies (1). Until now, the lack of an effective in vivo T cell-depleting reagent in swine has limited the progress of studies involving hematopoietic cell transplants. METHODS: The swine CD3 immunotoxin, pCD3-CRM9, was prepared by conjugating our porcine-specific CD3 monoclonal antibody 898H2-6-15 to the diphtheria toxin derivative, CRM9. The resultant immunotoxin was administered i.v. to several miniature swine at doses ranging from 0.15-0.2 mg/kg either in a single dose or two doses 2 days apart. T-cell depletion was monitored in the peripheral blood, mesenteric lymph node, and thymus by flow cytometric analysis and histological examination. RESULTS: T cells were depleted to less than 1% of their pretreatment levels based on absolute numbers in the peripheral blood. Fluorescence activated cell sorter analysis and histological examination of serial lymph node biopsies confirmed depletion of the CD3+ T cells rather than down modulation or masking of the surface CD3 expression. Depletion of the CD3 bright medullary thymocytes could also be detected by flow cytometry and histological examination after immunotoxin treatment. CONCLUSIONS: Administration of the immunotoxin i.v. drastically depletes mature T cells from the peripheral blood, lymph node, and thymus compartments of the pig. This first description of an effective in vivo T-cell depleting reagent for the pig provides a valuable tool for studies of transplant tolerance in this large animal model. It also makes possible preclinical studies of T cell depletion with anti-CD3 immunotoxin in this large animal model.

Animals↗

A conserved African swine fever virus right variable region gene, l11L, is non-essential for growth in vitro and virulence in domestic swine.

The right variable region of the African swine fever virus (ASFV) genome is known to contain genes with functions involving virus virulence and host range in swine. A novel open reading frame, ORF l11L, which was absent in the non-pathogenic, cell culture-adapted European isolate BA71V, was identified in the pathogenic African isolate Malawi Lil-20/1. The location of l11L in the right variable region, together with its absence in BA71V, suggested that l11L may have a function in virus virulence and/or host range. Here, we show that the l11L gene is highly conserved among pathogenic African, European and Caribbean ASFV field isolates and that it exists either in a short form, encoding a protein of 77-78 amino acids (9.1 kDa) or in a longer form of 93-94 amino acids (11.1 kDa). The presence of two predicted membrane-spanning segments suggests that l11L is an integral membrane protein. RT-PCR analysis demonstrated that l11L mRNA is expressed late in the virus replication cycle. A recombinant l11L gene deletion mutant, deltal11L, was constructed from the ASFV isolate Malawi Lil-20/1 to examine gene function. Deletion of l11L did not affect virus replication in swine macrophage cell cultures nor virulence in domestic pigs, indicating that l11L is non-essential for growth in vitro and for virus virulence in domestic swine.

African Swine Fever Virus↗

Antibodies to bovine serum albumin in swine sera: implications for false-positive reactions in the serodiagnosis of African swine fever.

Antibodies to bovine serum albumin were detected in swine sera by use of an immunoblotting technique. Such sera had false-positive reactions, as determined by results of African swine fever virus serodiagnostic techniques when bovine serum albumin was a contaminant in the soluble cytoplasmic antigen obtained from infected cells cultured in the presence of bovine serum. The soluble cytoplasmic antigen obtained from cell cultures infected with African swine fever virus in the presence of porcine serum did not react with the false-positive sera and, therefore, was used for African swine fever virus serodiagnostic methods, with 0% false-positive results.

African Swine Fever↗

Swine aortic smooth muscle in tissue culture. Some effects of purified swine lipoproteins on cell growth and morphology.

Smooth muscle cells (SMC) were grown from inner media explants of swine aorta and used as a model for studying the role of lipoproteins in atherogenesis. These cultured cells retain the characteristics of SMC through multiple passages. Cell growth curves, in time, were obtained by using standard counting techniques, SMC grew slowly (0.019 cycle/day) in modified Dulbecco-Vogt medium supplemented with 1.5% swine serum. Purified lipoproteins were prepared from three normolipidemic and two hyperlipidemic (cholesterol-fed) swine. When the medium of 84 growth experiments was supplemented with these lipoproteins, SMC growth rate increased linearly with lipoprotein cholesterol concentration up to 10 mg/dl. At 10 mg/dl of lipoprotein cholesterol, very low density lipoproteins (VLDL) increased growth rate 7.2-fold (P less than 0.01); low density lipoproteins (LDL) 5.7-fold (P less than 0.01); high density lipoproteins (HDL2) 3,4-fold (P less than 0.02); and HDLc, and lipoprotein appearing in the hyperlipidemic swine, 3.0-fold (P less than 0.01). Addition of 10% lipoprotein-free serum stimulated growth rate 6.0-fold (P less than 0.01). There was no difference between normo- and hyperlipidemic lipoproteins with respect to cell growth rate. Factors present in the ultracentrifugal bottom, and factors appearing during the platelet release reaction, were shown to contribute to the SMC growth response. Morphological alterations characteristic of intimal foam cells occurred in SMC grown in VLDL at triglyceride levels in excess of 15 mg per 100 ml. Thus there are distinct parallels between SMC response in this model in vitro and atherogenesis in vivo.

Animals↗