Biomedical subjects

S J Ewald

Publications and source records attributed to S J Ewald.

At least 19 recordsLinked to original sources

PubMed2005

High-resolution typing for chicken BF2 (MHC class I) alleles by automated sequencing.

Sequence-based typing (SBT) was developed for major histocompatibility complex (MHC) class I and class II alleles in humans. We report here the development and application of a SBT method for alleles of the chicken BF2 locus (the more polymorphic of the two MHC class I loci in chickens). Exon 2 of the BF2 gene was selectively amplified from genomic DNA using a BF2 locus-specific PCR primer. Exon 2 sequences were sufficient to identify the 21 distinct BF2 alleles described in standard B haplotypes of Leghorns and in commercial broiler-breeder lines. Sixty-six samples from MHC typed, pedigreed chickens were tested, including 50 different heterozygous combinations. BF2 sequences from all B homozygotes were successfully amplified, and all combinations of BF2 alleles in heterozygotes were co-amplified equally. The two different BF2 alleles in heterozygotes could be identified unambiguously by distinct sequence motif patterns. In tests of samples of unknown B genotype in commercial broiler-breeder flocks, we identified expected BF2 alleles as well as an allele not previously encountered in one of the lines.

Alleles↗

PubMed2005

The avian major histocompatibility complex influences bacterial skeletal disease in broiler breeder chickens.

This study evaluated bacterial skeletal disease in conjunction with the major histocompatibility complex (MHC) in a genetically pure line of broiler breeder chickens. Chickens from six broiler breeder flocks were examined for skeletal lesions, bacterial pathogens, and MHC genotype. During a 10-week period, eighty-eight, 9- to 21-week-old lame chickens and 34 normal, age-matched controls were selected. Tenosynovitis, arthritis, and femoral or tibiotarsal (or both) osteomyelitis occurred in 86 of 88 (97.7%) lame chickens. Ninety-five bacterial isolates were obtained from 83 of 88 (94.3%) lame birds and 4 of 34 (11.8%) controls. Staphylococcus spp. was isolated from 72.6% of the skeletal lesions, predominantly Staphylococcus aureus (38.9%). MHC B complex genotypes were determined by hemagglutination for 88 lame birds, 34 controls, and 200 randomly selected birds from each of the six flocks (1,200 total). Combined chi-square analysis revealed that the homozygous MHC genotypes B(A4/A4) (chi(2) = 14.54, P = 0.0063) and B(A12/A12) (chi(2) = 42.77, P = 0.0001) were overrepresented in the sample of symptomatic birds compared with random samples from the same flocks. The homozygous A4 and A12 MHC genotypes influenced flock chi-square values more than the corresponding heterozygotes. An MHC B complex influence on bacterial skeletal disease was apparent in this line of broiler breeders.

Animals↗

PubMed2005

Oral infection with chicken anemia virus in 4-wk broiler breeders: lack of effect of major histocompatibility B complex genotype.

The pathologic consequences of chicken anemia virus (CAV) oral inoculation in 4-wk-old broiler breeders of different major histocompatibility B complex (MHC) genotypes were evaluated. MHC B complex was determined by hemagglutination and sequence-based typing. Clinical signs, serology, gross lesions, histopathologic analysis, and CAV genome quantification were used to evaluate disease progression. Clinical disease was not apparent in the inoculated broilers throughout the experimental period. At 14 days postinoculation, antibodies against CAV were detected in 26.4% (29/110) of the inoculated birds. The distribution of percent positive was 34.6% (9/26) and 32.3% (10/31) of the chickens with B A9/A9 and B A9/A4 MHC genotypes, respectively, and seroconversion in six other genotypes was 19% (10/53). These differences among MHC genotypes for specific seroconversion rate were not statistically significant. CAV genomes were detected in the thymus of 87.7% (93/110) of the inoculated birds with no statistically significant differences between MHC genotypes. Mild thymic lymphocytolysis, lymphedema, and medullary hemorrhage were observed in the inoculated chickens. Histomorphometric analysis showed that cortical lymphocyte-to-parenchyma ratios did not differ between inoculated and uninoculated groups or among MHC genotypes. Similar findings have been reported previously in white-leghorn chickens of similar age, suggesting that broilers show a similar resistance to the effects of CAV infection at this age. The absence of significant clinical and pathological changes in the orally inoculated broilers at this age contrasts with CAV-associated thymus damage seen frequently in condemned commercial broilers at harvest.

Administration, Oral↗

PubMed2004

Distinctive polymorphism of chicken B-FI (major histocompatibility complex class I) molecules.

The major histocompatibility complex (MHC) in chickens influences disease resistance, but the mechanism is not understood. In Leghorn lines, the MHC contains 2 closely-linked class I loci, B-FI and B-FIV. Previously, we determined nucleotide sequences of well-expressed class I (B-F) genes from unique MHC haplotypes of broiler chicken lines. More recently, we identified 7 new B-F alpha1alpha2-coding sequences from less well-expressed loci by amplification of genomic DNA from unique broiler haplotypes. Phylogenetic analysis of chicken MHC class I alpha1alpha2-coding sequences resolved 2 clusters (Groups A and B), which appear to correspond to B-FIV and B-FI loci, respectively. Compared with B-FIV locus, B-FI alleles were less polymorphic overall, but nevertheless demonstrated evidence of diversifying selection. The most striking feature of B-FI alleles is a conserved, locus-specific motif in the alpha helix of the alpha1 domain, a region that is highly variable in B-FIV alleles. This distinctive pattern of allelic polymorphism resembles that of the HLA-C class I locus in the human MHC (HLA). The conservation of the alpha helix of the alpha1 domain relates to HLA-C interaction with members of the killer immunoglobulin-like receptors on natural killer (NK) cells that are specific for recognition of HLA-C molecules and function to regulate activation of NK cells. Whereas HLA-C molecules may be dominant ligands for NK cell regulation, HLA-A and -B molecules are more important in presenting antigen to cytotoxic T lymphocytes. We hypothesize that chicken B-FI molecules may be specialized to serve similar functions as HLA-C molecules.

Amino Acid Sequence↗

PubMed2004

Diversity and locus specificity of chicken MHC B class I sequences.

The major histocompatibility complex B (MHC B) region in a standard haplotype of Leghorn chickens contains two closely linked class I loci, B-FI and B-FIV. Few sequences of B-FI alleles are available, and therefore alleles of the two loci have not been compared with regard to sequence diversity or locus specificity. Here, we report eight new B-F alpha 1/alpha 2-coding sequences from broiler chicken MHC B haplotypes, and a unique recombinant between the two B-F loci. The new sequences were combined with existing B-F sequences from Leghorn and broiler haplotypes for analysis. On the basis of phylogenetic analysis and conserved sequence motifs, B-F sequences separated into two groups (Groups A and B), corresponding to B-FIV and B-FI locus, respectively. Every broiler haplotype had one B-F sequence in Group A and the second B-F sequence, if it existed, clustered in Group B. Group B (presumptive B-FI locus) sequences identified in broiler haplotypes resembled the human MHC class I HLA-C locus in their distinctive pattern of allelic polymorphism. Compared with B-FIV, B-FI alleles were less polymorphic and possessed a conserved locus-specific motif in the alpha1 helix, but nevertheless demonstrated evidence of diversifying selection. One B-FI alpha 1/alpha 2-coding nucleotide sequence was completely conserved in four different broiler haplotypes, but each allele differed in the exon encoding the alpha 3 domain.

Amino Acid Sequence↗

PubMed2002

Major histocompatibility complex effect on cellulitis among different chicken lines.

The chicken major histocompatibility complex (MHC) has been implicated in conferring resistance/susceptibility to several bacterial, parasitic, and viral diseases. Investigators have shown that the chicken MHC plays a major role in determining the outcome of a Marek's disease infection, in that standard B(13) is susceptible to the virus while B(21) confers resistance to the virus. Previous work with a broiler line has shown that B(21) is susceptible to an Escherichia coli-induced cellulitis infection and that B(13) conferred resistance to the infection. For this experiment, a broiler and a Leghorn chicken line shown to contain standard B(13) and B(21) were examined in a challenge model for cellulitis. The birds were challenged with a cellulitis-causing E. coli isolate. Homozygous B(21) had the highest incidence of cellulitis development compared with either homozygous B(13) or the heterozygous B(13)/B(21) for both the broiler and Leghorn lines. Additionally, cellulitis lesion severity was measured in both lines and shown to be independent of MHC type.

Animals↗

PubMed2002

Single-strand conformation polymorphism (SSCP) assays for major histocompatibility complex B genotyping in chickens.

We have developed a DNA-based method for defining MHC B system genotypes in chickens. Genotyping by this method requires neither prior determination of allele-specific differences in nucleotide sequence nor the preparation of haplotype-specific alloantisera. Allelic differences at chicken B-F (class I) and B-L (class II) loci are detected in PCR single-strand conformation polymorphism (SSCP) assays. PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the two class I and two class I loci of the B-complex and used to generate DNA fragments that are heat- and formamide-denatured and then analyzed on nondenaturing polyacrylamide gels. PCR primer pairs were tested for the capacity to produce SSCP patterns allowing the seven B haplotypes in the MHC B congenic lines, and seven B haplotypes known to be segregating in two commercial broiler breeder lines to be distinguished. Primer pairs were further evaluated for their capacity to reveal the segregation of B haplotypes in a fully pedigreed family and in a closed population. Concordance was found between SSCP patterns and previously assigned MHC types. B-F and B-L SSCP patterns segregated in linkage as expected for these closely linked loci. We conclude that this method is valuable for defining MHC B haplotypes and for detecting potential recombinant haplotypes especially when used in combination with B-G (class IV) typing by restriction fragment pattern.

Animals↗

PubMed2001

Three new MHC haplotypes in broiler breeder chickens.

Six distinct serotypes of the chicken B blood group system (which encodes the major histocompatibility complex) were identified in a commercial broiler breeder line (Line C). The B serotypes were compared by B-G restriction fragment length polymorphism (RFLP) analysis, allele-specific PCR typing test for B-LBII family genes and nucleotide sequence analysis of expressed B-F and B-LBII family genes. The results indicated the existence of seven distinct B haplotypes. Nucleotide sequence analysis demonstrated that three of the Line C haplotypes encode new B-F and B-LB alleles.

Alleles↗

PubMed1999

The MHC of a broiler chicken line: serology, B-G genotypes, and B-F/B-LB sequences.

Although the major histocompatibility complex of chickens (encoded in the B complex) has been studied for a number of years, almost all work has focused on the White Leghorn breed. Broiler (meat-type) chickens were derived from other breeds, including Cornish and Plymouth Rock. It was our hypothesis that new B haplotypes, not previously identified in White Leghorns, might be present in lines of broiler chickens. Furthermore, alloantisera used to identify B serotypes in Leghorn lines reportedly do not work well outside the line in which they were raised, with the result that broiler B haplotypes have not been incorporated into the universal nomenclature system. Our approach was to use a panel of B alloantisera produced to identify B serotypes within a commercial broiler breeder line (designated line A). B homozygotes identified serologically were compared by B-G genotyping using restriction fragment length polymorphism analysis. Furthermore, reverse transcription-polymerase chain reaction was used to amplify variable domains of expressed B-LB and B-F genes of homozygotes of most of the B serotypes in Line A, followed by cloning and nucleotide sequence determination. Comparison of B-LB and B-F sequences with standard Leghorn haplotypes demonstrated the existence of new alleles of B-L and B-F in a broiler breeder line, as well as the presence of alleles previously identified in Leghorns. In some cases, Leghorn-type alleles were in linkage with different B-G alleles in the broiler line than the common haplotypic associations found in Leghorn lines.

Alleles↗

PubMed1999

A PCR method for typing B-L beta II family (class II MHC) alleles in broiler chickens.

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.

Alleles↗

PubMed1998

Evaluation of GM1 ganglioside-mediated apoptosis in feline thymocytes.

Cats with inherited GM1 gangliosidosis (GM1 mutant cats) have premature thymic involution characterized by decreased total thymocytes primarily affecting the CD4+ CD8+ subpopulation. While GM1 mutant cats have increased cell surface GM1 gangliosides, as determined by cholera toxin B binding, on both thymocytes and peripheral lymph node cells only thymocytes show increased apoptosis. To determine if GM1 gangliosides can increase the occurrence of apoptosis in feline thymocytes directly, we added exogenous GM1 ganglioside (GM1) to feline thymocyte primary cultures and compared the results to apoptotic changes seen in untreated cells or in cells treated with dexamethasone (Dex), a known inducer of thymocyte apoptosis in other species. Incorporation of exogenous GM1 into thymocyte cytoplasmic membranes was confirmed by flow cytometric analyses of cholera toxin B labelling. Apoptosis in feline thymocytes was analyzed by electron microscopy, spectrophotometric evaluation of DNA fragmentation, flow cytometric enumeration of apoptotic nuclei, and gel electrophoretic analysis of degraded DNA. Alterations in percentages of thymocyte immunophenotype following GM1 incorporation were determined by flow cytometric analyses of labelled cell surface markers for feline CD4 and CD8. Because in vitro addition of GM1 gangliosides has been reported in other species to decrease surface expression of CD4 on both thymocytes and peripheral lymphocytes, we evaluated GM1-associated down-regulation of CD4 on the surface of feline thymocytes and peripheral lymph node cells by flow cytometry. Additionally, we compared the apoptotic response of the more mature peripheral lymph node cells to the less mature thymocytes. Our results indicate that incorporation of exogenous GM1 into feline thymocyte cell membranes produces a dose-dependent increase of apoptotic cell death. Although, CD4 expression on both feline thymocyte and lymph node cell membranes was abruptly decreased after introducing exogenous GM1, enhanced apoptotic death was observed only in thymocytes, not in lymph node cells at the same GM1 concentration. Enhancement of thymocyte apoptosis appears to be age-related since cells derived from cats <3 months of age were more vulnerable than those from cats >3 months of age.

Aging↗

PubMed1998

Thymic alterations in feline GM1 gangliosidosis.

GM1 gangliosidosis is an inherited metabolic disease characterized by progressive neurological deterioration with premature death seen in children and numerous animals, including cats. We have observed that thymuses from affected cats greater than seven months of age (GM1 mutant cats) show marked thymic reduction compared to age-matched normal cats. The studies reported here were done to describe alterations in the thymus prior to (less then 90 days of age) and during the development of mild (90 to 210 days of age) to severe (greater than 210 days of age) progressive neurologic disease and to explore the pathogenesis of the thymic abnormality. Although histologic examination of the thymus from GM1 affected cats less than 210 days of age showed no significant differences from age-matched control cats, thymuses from GM1 mutant cats greater than 210 days of age were significantly reduced in size (approximately 3-fold). Histologic sections of lymph nodes, adrenal glands, and spleens from GM1 gangliosidosis-affected cats showed no significant differences. Flow cytometric analyses showed a marked decrease in the percentage of immature CD4+CD8+ thymocytes (p < 0.001) and significantly increased CD4-CD8+ cells (p < 0.01) in GM1 mutant cats greater than 210 days of age when compared to normal age matched cats. Co-labelling with CD4, CD8, and CD5 indicated an increase in the percentage of GM1 mutant cat thymocytes at this age which were CD5high, suggesting the presence of more mature cells. Cytometric analyses of subpopulations of peripheral lymphocytes indicated an increase in CD4-CD8+ cells (p < 0.05) with concurrent decreases in CD4+CD8- and CD4-CD8- cells (which were not significant). Similar analyses of thymocyte and lymphocyte subpopulations from cats < 210 days of age showed no significant differences between GM1 mutant and normal cells. GM1 mutant cats at all ages had increased surface binding of Cholera toxin B on thymocytes, indicating increased surface GM1 ganglioside expression. Increases were highly significant in GM1 mutant cats greater than 210 days of age. In situ labelling for apoptosis was increased in GM1 mutant cats between 90 to 200 days of age when thymic masses were within normal limits. In GM1 mutant cats over 200 days of age, decreased labelling was observed when thymic mass was reduced and the CD4+CD8+ subpopulation, known to be very susceptible to apoptosis, was significantly decreased. These data describe premature thymic involution in feline GM1 gangliosidosis and suggest that increased surface GM1 gangliosides alters thymocyte development in these cats.

Adrenal Glands↗

PubMed1998

Gangliosides enhance apoptosis of thymocytes.

Monosialogangliosides, normal components of cell membranes, regulate cell development and differentiation in several organs. Our previous observation of dramatic premature thymic involution in cats with feline GM1 gangliosidosis, whose thymocytes have abnormally high cell surface gangliosides, suggested that excess GM1 ganglioside (GM1) could modulate thymocyte apoptosis in this disease (Cox et al., "Thymic Alterations in Feline GM1 Gangliosidosis," submitted). In these studies, we added exogenous GM1 to murine primary thymocyte cultures and demonstrated enhanced apoptosis in treated cells by DNA fragmentation, apoptotic body, and electrophoretic analyses. GM1-enhanced apoptosis was blocked by common apoptotic pathway inhibitors including aurintricarboxylic acid (inhibitor of endonuclease activity), actinomycin D (inhibitor of RNA transcription), and cycloheximide (inhibitor of protein synthesis). GM1 treatment primarily affected the immature CD4+ CD8+ subset, as shown by flow cytometric evaluation of fetal thymic organ culture and primary thymocyte cultures. Apoptosis also could be induced by GM2, GM3, and GT1b, whereas asialo-GM1 failed to do so, suggesting that the sialic acid moiety may play an important role in the induction of thymocyte apoptosis.

Animals↗

PubMed1997

Molecular characterization of major histocompatibility complex (B) haplotypes in broiler chickens.

In Leghorn (laying) chickens, susceptibility to a number of infectious diseases is strongly associated with the major histocompatibility (B) complex. Nucleotide sequence data have been published for six class I (B-F) alleles and for class II (B-L beta) alleles or isotypes from 17 Leghorn haplotypes. It is not known if classical B-L or B-F alleles in broilers are identical, at the sequence level, to any Leghorn alleles. This report describes molecular and immunogenetic characterization of two haplotypes from commercial broiler breeder chickens that were originally identified by serology as a single haplotype, but were differentiated serologically in the present work. The two haplotypes, designated BA4 and BA4variant, shared identical B-G restriction fragment length polymorphism patterns, but differed in one B-L beta fragment that cosegregated with the serological B haplotype. Furthermore, the nucleotide sequences of the highly variable exons of an expressed B-L beta II family gene and B-F gene from the two haplotypes were markedly different from each other. Both the B-L beta II family and B-F gene sequences from the BA4 haplotype were identical to the sequences obtained from the reference B21 haplotype in Leghorns; however, in the BA4 haplotype the B-L beta 21 and B-F21 alleles were in linkage with B-G alleles that were not G21. The nucleotide sequences from BA4variant were unique among the reported chicken B-L beta II family and B-F alleles.

Animals↗

PubMed1997

Chronic ethanol exposure alters leukocyte subsets in repopulating spleens, but does not alter negative selection in thymuses of sublethally irradiated mice.

Results from previous in vitro experiments in this laboratory suggested that ethanol may affect selection processes in the thymus. To determine whether ethanol allows escape of potentially autoreactive T-cell clones from negative selection, we fed ethanol to sublethally irradiated, young, adult C57BR mice during the time of thymic and splenic repopulation as a new model of human third trimester fetal alcohol exposure. The mice received a whole-body, sublethal dose (6 Gy) of gamma irradiation at 5 to 6 weeks of age. Feeding of a liquid diet providing 25% of calories as ethanol (EDC) or an isocaloric control liquid diet was begun 3 days after irradiation and was continued for 5 weeks. Each EDC mouse had 2 weight- and age-matched controls, 1 pair-fed (PF), and 1 fed ad libitum (AD LIB). Average blood alcohol concentrations (90 to 440 mg/100 ml) were higher than those reported previously for neonatal mice exposed to ethanol through lactation. At 5 weeks after irradiation, the EDC mice had lower total thymocyte numbers (p < 0.05) and a higher proportion of CD4-CD8-thymocytes than either the PF or AD LIB mice (p < 0.05), which is consistent with findings using in utero models of ethanol exposure. Ethanol exposure also altered the proportion of leukocyte subsets in repopulating spleens. B cells were the most sensitive to the detrimental effects of ethanol and, as a percentage of total nucleated cells in the spleen, B cells were decreased in the EDC group, compared with both the PF and AD LIB groups (p < 0.05). C57BR mice normally delete by negative selection thymocytes bearing v beta 17 T-cell receptors. There was no discernible effect of ethanol exposure during thymic and splenic repopulation on the expression of V beta 17a on thymocytes and splenic T lymphocytes, indicating that ethanol does not affect negative selection.

Alcoholism↗

PubMed1996

B-haplotype control of CD4/CD8 subsets and TCR V beta usage in chicken T lymphocytes.

The major histocompatibility (B) complex of the chicken contains genes similar to Class I (B-F) and Class II (B-L beta) genes in mammals, as well as a highly-polymorphic gene family (B-G) whose exact function is not known. Specific B-haplotypes are strongly associated with resistance to a number of infectious diseases, and with immune responses to soluble and cellular antigens. In mammals, Class I and Class II molecules control development of the T cell repertoire, including selection of CD4+ and CD8+ T cells. One study of chickens reported that low CD4:CD8 ratio was associated with the B4 haplotype, which shares expressed B-F/B-L genes with the B13 haplotype. In studies reported here, chickens of two haplotypes carried in the Auburn R line, B302 and B305 (which is B13-related), were evaluated for percentages of T cells expressing the CD4, CD8, CD3, TCR1, TCR2 and TCR3 antigens in peripheral blood lymphocytes (PBL), thymus, and spleen. These two haplotypes were chosen for comparison because they differ in resistance to Marek's disease (MD) and are closely-related in B-F and B-L genes by restriction fragment length polymorphism analyses. Homozygous birds of each B haplotype were produced from crosses of (B302 x B305)F1 sires and dams. PBL, thymocytes, and splenocytes from B302 homozygotes had higher CD4:CD8 ratios than B305 homozygotes. However, CD4:CD8 ratio differences could not be attributed to haplotype-controlled differences in V beta usage within CD4/CD8 subsets, as has been described for certain V beta families in mice and humans. These results indicate that thymic selection events involving CD4 and CD8 subsets and TCR V beta usage are controlled by a gene or genes closely-linked to the B-complex, which may or may not be Class I or Class II genes.

Animals↗

PubMed1995

Regulation of signal transduction and DNA fragmentation in thymocytes by ethanol.

We demonstrated previously that ethanol enhances apoptosis of murine thymocytes. In this report, we determined intracellular cAMP and cytosolic free calcium ([Ca2+]i) levels in mouse thymocytes following acute exposure to ethanol and investigated the involvement of cAMP, [Ca2+]i, protein kinase A (PKA), and protein kinase C (PKC) in thymocyte apoptotic death induced by ethanol. It was found that ethanol did not alter basal cAMP levels, but produced a dose-dependent, prolonged small [Ca2+]i increase within thymocytes. This dose dependence of [Ca2+]i increase was paralleled by the magnitude of DNA fragmentation induced by ethanol at various concentrations. Additionally, the ethanol-enhanced DNA fragmentation was blocked by H7, a PKC inhibitor, but not by potent PKA inhibitors having little or no effect on PKC. These data suggest that both [Ca2+]i increase and PKC activation triggered by ethanol may belong to the signal pathway(s) leading to thymocyte programmed death.

Animals↗

PubMed1993

Effect of ethanol on development of fetal mouse thymocytes in organ culture.

Exposure of mouse fetuses to ethanol in utero retards thymus development. The direct effect of ethanol on growth and differentiation of thymocytes was studied using organ cultures of 14-day fetal mouse thymuses. Fetal thymus organ cultures containing 0.2 or 0.4% ethanol produced fewer total thymocytes, proportionately fewer CD4+CD8+ (immature) thymocytes, and proportionately more CD4+CD8- (mature) cells than untreated control cultures after 5 days of culture. Total cell numbers and proportions of CD4+CD8+ thymocytes declined in a dose-dependent manner with increasing ethanol concentrations from 0.2 to 0.8%. In time course studies, thymuses cultured with 0.4% ethanol had an increased percentage of CD4+CD8- cells at all days examined between Days 4 and 6. In the same experiments, thymuses exposed to ethanol underwent accelerated loss of the interleukin-2 receptor (a marker of immature prothymocytes) and had higher percentages of cells positive for the gamma delta-T-cell receptor. Exposure to ethanol for 16 to 20 hr increased the percentage of noncycling thymocytes. Furthermore, ethanol increased apoptosis in fetal thymocytes. Acetaldehyde, the immediate product of ethanol catabolism, had no effect on thymocyte subpopulation ratios or cell numbers at a physiologic concentration (50 microM). Results indicate that in a controlled in vitro model of thymus development, ethanol reduced cell numbers and altered proportions of thymocyte subsets defined by differentiation antigens.

Acetaldehyde↗