Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection.

The release of microparticles from eukaryotic cells is a well-recognized phenomenon. We demonstrate here that the chemokine receptor CCR5, the principal co-receptor for macrophage-tropic human immunodeficiency virus (HIV)-1, can be released through microparticles from the surface of CCR5+ Chinese hamster ovary cells and peripheral blood mononuclear cells. Microparticles containing CCR5 can transfer the receptor to CCR5- cells and render them CCR5+. The CCR5 transfer to CCR5-deficient peripheral blood mononuclear cells homozygous for a 32-base-pair deletion in the CCR5 gene enabled infection of these cells with macrophage-tropic HIV-1. In monocytes, the transfer of CCR5 could be inhibited by cytochalasin D, and transferred CCR5 could be downmodulated by chemokines. A transfer of CCR5 from peripheral blood mononuclear cells to endothelial cells during transendothelial migration could be demonstrated. Thus, the transfer of CCR5 may lead to infection of tissues without endogenous CCR5 expression. Moreover, the intercellular transfer of membrane proteins by microparticles might have broader consequences for intercellular communication beyond the effects seen for HIV-1.

Phenotypic changes induced by wild type and variant c-src genes carrying C-terminal sequence alterations.

We previously reported the isolation of PR2257, a novel avian sarcoma retrovirus which transduced the c-src protooncogene. The v-src gene of PR2257 differs from the c-src gene by a sequence change after amino acid 525, resulting in the replacement of tyrosine 527 by a valine, and an extension of the open reading frame into the non coding region of c-src. We investigated the respective roles of Tyr527 mutation and of the C-terminal extension in activating the oncogenic properties of c-src. Therefore we overexpressed the wild type c-src gene and c-src variants, carrying either a substitution of tyrosine 527 or an extension of the C-terminus or both modifications in combination, in chicken embryo fibroblasts and post mitotic neuroretina (NR) cells, using replication defective retroviruses. We also used in vivo inoculation of plasmid DNA to assess the tumorigenicity of the various c-src genes. We report that, in contrast to previous results, overexpression of c-src is sufficient to induce NR cell division. While mutation of tyrosine 527 alone significantly activates c-src transforming and tumorigenic properties, its combination with the C-terminal extension of PR2257 confers to this gene full oncogenic properties and increased metastatic potential as compared to the v-src of Rous sarcoma virus strains.

Biology of the chicken MHC (B complex).

The major histocompatibility complex (MHC) of chicken is the B complex, originally described as a blood group system. Its three classes of cell membrane antigens have been clearly defined by serological, histogenetic, biochemical, and molecular biological methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L respectively), while the third--B-G antigen--has not so far been detected in mammals. The possible role of this antigen is discussed. The genes of the MHC play important roles in the regulation of immune response, disease resistance, and regression of Rous sarcomas.

Expression of nonspecific esterase (NSE) by thyroid follicular epithelium as a marker for the target organ susceptibility to immune system attack.

Spontaneous autoimmune thyroiditis in obese strain (OS) chickens provides an excellent animal model for the study of Hashimoto's autoimmune thyroiditis in humans. The data presented in this paper indicate that nonspecific esterases (NSE) may play a role in or serve as a marker for the target organ susceptibility. Experiments have shown that follicular epithelial cells and interfollicular macrophages in connective tissue stain positively for NSE as early as the first day after hatching, a time at which infiltrating lymphocytes are not yet observed. We also have observed NSE positivity of follicular cells in the vicinity of mononuclear cell infiltration in all OS chickens, as well as weaker positivity in 6-month-old, avian leukosis virus free, Brown Leghorn outbred chickens, which appears in each case to correlate with infiltration of lymphocytes. In F2 hybrids between OS and healthy CB inbred chickens, the intensity of NSE staining was more variable than in OS chickens. Using specific inhibitors eserine, Na-taurocholat and p-hydroxymercuribenzoic acid, we were able to inhibit in vitro the NSE positivity of thyroid gland follicular epithelium, indicating that this staining was not an artifact. Experiments are currently in progress to clarify the relationship between the presence of NSE in follicular epithelium and the predisposition to spontaneous autoimmune thyroiditis.

Graft versus host reaction induced by thymectomized and bursectomized chicken blood.

Graft versus host reaction in chicken embryos was induced by blood taken from thymectomized or bursectomized or nonoperated donors. The intensity of graft versus host reaction induced by the blood of thymectomized chickens was increased comparing to the reaction induced by blood from control donors. No such result was recorded when the blood donors had the bursa of Fabricius removed.

The influence of bursocytes and thymocytes on graft versus host reaction and IgG level in chickens.

Graft versus host reaction was induced by the blood of chickens previously transferred by the bursa and thymus cells. These bursa and thymus cells were taken from bursectomized or thymectomized chicken. It was occurred that graft versus host reaction was influenced by transfer of bursa and thymus cell. IgG level was affected by the transfer of these cells too.

Genes of chicken MHC regulate the adherence activity of blood monocytes in Rous sarcomas progressing and regressing lines.

The influence of the chicken major histocompatibility (B) complex (MHC) on the adherence potential of monocyte-derived macrophages was examined using the congenic chicken lines CB and CC. These lines represent well-defined genetic models for the study of resistance (CB) or susceptibility (CC) to the progressive growth of Rous sarcomas. Using a monoclonal antibody specific for chicken monocytes/macrophages, CB and CC chickens were shown by flow cytometry analyses to have similar proportions of peripheral blood monocytes. However, when the glass-adherence potential of these cells was compared during incubation in tissue culture medium over 24, 48 and 72 h at 40 degrees C, significant differences were seen between cells from these two inbred lines. After 24 and 48 h, glass-adherence by CB cells was 2-3 fold higher than that of CC cells. After 72 h this difference decreased to 1.5 fold. At 24 and 48 h, the adherent CB macrophages also appeared about 1.5 times larger than those of CC chickens. Genetic analysis using F1 hybrids (CBxCC) showed that this trait is regulated by a dominant gene that segregates with the B12 haplotype in the backcross generation F1xCC. From the results obtained with the recombinant congenic lines CB.R1 and CC.R1, we conclude that the gene regulating adherence potential is localized within the B-F/L region of the chicken MHC. About 50% of adherent cells were able to phagocytose opsonised FITC-labelled Zymosan particles. The level of nitric oxide production in vitro by CB and CC macrophages was equal. The importance of cells of the mononuclear phagocyte system for the response to Rous sarcoma virus (RSV) infection was studied in CB chickens using the anti-macrophage agents silica, carrageenan, and C12MDP, encapsulated in liposomes. In those chickens treated with silica and carrageenan, we observed progressive growth of RSV-induced tumors. The graft-versus-host reactivity of peripheral blood lymphocytes (PBL) of treated chickens was comparable to controls. In vitro nitric oxide production by macrophages from silica-treated chickens was higher than by macrophages from untreated controls.

The chicken--a laboratory animal of the class Aves.

Prague inbred lines of chickens represent a unique system of MHC(B) congenic partners differing in the immune-based resistance/susceptibility to v-src-induced oncogenesis. Mapping in chickens can be facilitated by the availability of inbred lines, since many well described differences in disease susceptibility and MHC(B) haplotypes exist among the defined lines. Long-term intensive research on human, mouse, and rat MHC has established a canonical picture of this multigene complex. The chicken MHC(B) is clearly the best characterized outside the mammals and it was the first MHC clearly different from the paradigmatic structure of the above mentioned mammalian species. Chickens were in many aspects the poor relatives of mice, and they had to wait for introduction of molecular biology methods. But, when it happened, the newly gained data could be easily reconciled with classical genetic studies using available congenic chicken lines. We have established permanent tumor cell lines from ex vivo tumors induced by the LTR, v-src, LTR provirus in inbred chickens. These cells express a high level of the v-src oncogene and are of defined MHC(B) genotype. We witness a dramatic acceleration of the development of chicken (avian) genomics. The chicken is not only a good comparative model for basic science, but it is also an object of the poultry industry, which is threatened by several avian diseases. The reason for genome mapping in chickens is thus more than academic.

Age-dependent complementation of the B (MHC) alleles and a non-B allele controlling resistance to progression of Rous sarcomas, and genetic independence of resistance to induction of tumours in the model of inbred lines of chickens.

The effect of age on the resistance to progression of Rous sarcomas was investigated in chickens of different inbred lines and their hybrids. This genetic resistance depends on complementation of B-linked genes and complementation of genes outside the B complex. The reduction of tumour inducibility is controlled by genes outside the B complex, and this type of resistance is independent of the resistance to tumour progression that is primarily controlled by B-linked genes.

An analysis of the response of recombinant congenic lines of chickens to RSV challenge provides evidence for further complexity of the genetic structure of the chicken MHC (B).

We found that CB (B12/B12) and CB.R1 (B12r1/B12r1) congenic chicken lines differing from each other only in the B-G region of the MHC(B), although both of them could be characterized as regressors of Rous sarcomas induced by PR-C and BH-C, are distinguishable in their response to the challenge with these two viruses by means of some parameters of tumour growth. This points further to the concept of functional and structural complexity of the B-G region. Both CB and CB.R1 lines are highly susceptible to progressive growth of sarcomas induced by BH-D virus. This suggests a crucial role of helper viruses of different antigenic subgroups used for complementation of the Bryan high-titre pseudotype (BH) of RSV in the pathogenesis of tumours in this experimental system. Furthermore, attempts were made to analyse both primary and secondary response to the challenge with different strains of RSV in the genetic model encompassing the Prague congenic lines CB (B12/B12), CC (B4/B4), CB.R1 (B12r1/B12r1), CC.R1 (B4r1/B4r1) and their F1 hybrids, and a number of backcross matings. The data led to the view that interacting genes within both B-F/L and B-G regions of the MHC(B) govern the observed hierarchy of the response to RSV challenge.

The B-G region genes of the chicken MHC are responsible for lethal graft-versus-host disease in newly hatched chickens.

Using the genetic model of Prague recombinant congenic lines of chickens we found that incompatibility in the B-G region of the major histocompatibility complex (MHC) causes very severe graft-versus-host reaction (GVHR)-associated haemolytic anaemia in newly hatched chickens. Unexpectedly, mild or no signs of this GVH disease are elicited when a recipient chick and an adult donor of lymphocytes are incompatible in the whole B haplotype (B-F/L + B-G regions). On the other hand, the B-G region incompatibility alone (as has been described previously) is not sufficient to produce any GVH splenomegaly in embryos at 14 days of incubation. However, GVH splenomegaly in the donor-recipient combinations with the difference in the whole B haplotype (B-F/L + B-G regions) is significantly greater than in those with the B-F/L region difference only. These results confirm that the B-G region genes of chicken MHC are also involved in the histocompatibility reactions. Furthermore, a new hypothetical model for the structure of the chicken MHC is discussed.

Hierarchy of the B (MHC) haplotypes controlling resistance to rous sarcomas in a model of inbred lines of chickens.

The effects of individual B haplotypes on regression of Rous sarcoma virus-induced tumours were studied in a genetically defined model of congenic chicken lines differing at the B complex (MHC). Regression of Rous sarcomas is genetically controlled mainly by B genes the effects of which are qualitative in nature. In the model described which encompassed congenic inbred lines and their F1 hybrids and a number of various combinations of progeny from backcross matings, the genetic interactions among individual B haplotypes and the modifying influence of the genetic background, as manifested by quantitative variability of tumour growth parameters, could also be investigated. Some specific combinations of backcross progeny exhibited significant shifts with regard to basic classification of the degree of resistance of groups with a given B genotype in the congenic lines and their F1 hybrids. Based on these differences, further more detailed studies of the complex genetic mechanism operative in the control of tumour growth may be undertaken. Discriminant analysis, as part of the known series of the biomedical programs BMDP75, was used to evaluate the results.

Failure to confirm the role of immunological tolerance to B (MHC) alloantigens in the growth of RSV-induced tumours in chickens.

Chickens of the inbred "regressor" line CB (B12/B12) were made tolerant to the B13 (MHC) alloantigen of the congenic "progressor" line CC and then challenged with Prague strain of Rous sarcoma virus of subgroup C. No changes in tumour growth between tolerant and control groups were observed. These results suggest that there is no cross-reactivity between B13 alloantigen and RSV-induced tumour antigens.

Chicken cells--oncogene transformation, immortalization and more.

Domestic chicken as a laboratory animal as well as chicken cells in vitro have been highly evaluated in several fields of experimental biology. Retrovirology and experimental oncology traditionally use this model, whose comparative aspects are still inspirative. The first (retro)viral aetiology of a tumour was recognized in the chicken and the first quantitative in vitro measurement of oncogenic transformation was developed using the chicken cells. Chicken cells (like human and primate, but unlike rodent cells) have a long primary life-span, during which they remain genetically stable. While this property is advantageous for several types of experiments, it correlates with a low propensity of the chicken cells to immortalization. Recent establishment of several continuous chicken cell lines, however, has surmounted this drawback. Furthermore, the chicken B cell line DT40 was proved to be extremely useful for gene disruption studies because of a high frequency of gene targeting not found in any vertebrate cells. In the present communication, we have tried to review several traditional achievements accomplished using the chicken model and point to newly opened areas, where chicken cells appear to be an efficient tool, particularly in cell transformation and immortalization.

Genetic control of Rous sarcoma regression in inbred lines of chickens.

The fate of tumours induced by PR-RSV-C was investigated in highly inbred chicken lines congenic at the B complex, the major histocompatibility complex of the chicken. The results demonstrated that Rous sarcoma regression in congenic CB and CC lines was controlled by a gene within the B region of the chromosome. Another congenic pair of the WA and WB lines also differed in the ability to regress RSV-induced tumours and a gene within the B complex also appeared to have a significant role in the regression of Rous sarcomas.

Location of the gene responsible for Rous sarcoma regression in the B-F region of the B complex (MHC) of the chicken.

We studied the fate of tumours induced by PR-RSV-C and B77-C in inbred chicken lines CB, CC and CB.R1. Rous sarcomas regressed in the CB and CB.R1 lines which are identical in the B-F region of the B complex. In contrast, progressive tumour growth was observed in the CC line which differed in the B-F region from the CB and CB.R1 lines and was identical in the B-G region with the CB.R1 line. These results suggest that the gene responsible for Rous sarcoma regression is located in the B-F region of the B complex.

Syngeneic lines of chickens. VII. The lines derived from the recombinants at the B complex (MHC) of Rous sarcoma regressor and progressor inbred lines of chickens.

Based on recombinants between the B haplotypes of chickens of inbred lines CB, CC, and CB.I-B7, three new inbred lines homogeneous in transplantation and erythrocyte antigens were established by backcrossing. These newly established recombinant lines, CB.R1, CC.R1, and CC.R2, together with the original inbred lines, which differ at the B complex (MHC) and in the degree of genetically controlled resistance to progressive growth of RSV-induced tumours, represent a model system for study of the structure of the B complex and of the functions of individual B regions in the control of carcinogenesis.

A spontaneous chicken chimaera with serologically and morphologically different types of erythrocytes.

Among hybrids of the inbred chicken lines, cock 1357 was found to be chimaeric in red blood cells. This cock possessed three serologically distinct types of erythrocytes and two types different in shape and size.

Concomitant immunity leads to retardation or acceleration of the growth of Rous sarcomas in genetically resistant or susceptible inbred lines of chickens.

We report results on the age-dependent effect of the B (MHC) genotype on resistance to the growth of a second RSV tumour in chickens from highly inbred lines genetically resistant or susceptible to progressive growth of primary RSV-induced tumours. Furthermore, the second inoculation of RSV caused either accelerated or retarded growth of the primary tumours induced by the same virus. These two types of response to a second challenge with RSV were controlled by alleles of the chicken major histocompatibility (B) complex. The retarded growth of the primary tumours was found to be thymus-dependent. Furthermore, a clear-cut trans-complementation effect of two B alleles on the response to RSV challenge was demonstrated.

Resistance to Marek's disease is controlled by a gene within the B-F region of the chicken major histocompatibility complex in Rous sarcoma regressor or progressor inbred lines of chickens.

Rous sarcoma "regressor" chicken lines CB and CB.R1 identical in the B-F region of the B complex (MHC) are susceptible to Marek's disease. On the contrary, the Rous sarcoma "progressor" line CC different in the B-F region from the CB and CB.R1 lines and identical in the B-G region with the CB.R1 line is considerably resistant to MD. The inbred chicken line M is susceptible to MD, although it carries the B21 allele which is generally considered to determine MD resistance. Analysis by means of sera specific for B-F and B-G antigens revealed that the B haplotype of the M line is composed of the B-G region coding for the typical B-G21 antigen and of a different B-F region coding for B-Fx antigen of thus far unknown specificity. Thus genetic resistance to MD is controlled by a gene located within the B-F region of the chicken MHC.