Immunopathogenesis of hematopoietic tissues in response to Vibrio parahaemolyticus (VPAHPND) infection in Macrobrachium rosenbergii.

In crustacean, hemocytes are known as crucial components of crustaceans' innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VPAHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC50 dose of VPAHPND; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VPAHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VPAHPND-infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6-12 h and declined by 24 h. This evident related to the almost VPAHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VPAHPND-infected prawn was caused by an acute self-destructive immune response by hematopoietic cells.

An internal deletion enhances the transcriptional activity of a recombinant retrovirus in hematopoietic cells in vivo.

Lv-myc is a recombinant retrovirus that spontaneously arose during experiments designed to express the provirus LNAv-myc in the hematopoietic system of bone marrow-reconstituted mice (L. Bonham, K. MacKenzie, S. Wood, P. B. Rowe, and G. Symonds, Oncogene 7:2219-2229, 1992). The recombinant provirus is of interest because it is able to promote long terminal repeat-initiated transcription in hematopoietic cells in vivo, whereas the parental provirus, LNAv-myc, is transcriptionally repressed in the same cells. Here we report that Lv-myc was generated by precise deletion of the neomycin resistance gene (neo) and the human gamma-actin promoter from LNAv-myc. In comparison with LNAv-myc, no sequence alterations in the viral regulatory regions of Lv-myc were detected. Thus, it appears that neo and/or the gamma-actin promoter exerted a cis-acting repressor effect on the long terminal repeat of LNAv-myc in vivo. The origin of Lv-myc was also investigated, and it was shown that Lv-myc was harbored as a productive provirus in a G418-resistant subpopulation of the LNAv-myc producer cell line, psi 2AV. It appears that Lv-myc arose during propagation of the psi 2AV cell line. Repeated sequence detected at the sites of the deletion suggest that Lv-myc was generated by a template misalignment during reverse transcription of LNAv-myc.

Partial cloning of the genome of infectious hypodermal and haematopoietic necrosis virus, an unusual parvovirus pathogenic for penaeid shrimps; diagnosis of the disease using a specific probe.

The infectious hypodermal and haematopoietic necrosis virus (IHHNV), pathogenic for penaeid shrimp, is an icosahedral unenveloped particle, 22 nm in diameter, with an ssDNA linear genome, and proposed to be a member of the Parvoviridae. A large majority of minus-strand DNA is incorporated into the capsids compared to the plus-strand. A small amount of reannealed plus- and minus-strands (dsDNA) obtained after nucleic acid extraction was blunt-ended and cloned into the system pUC18/Escherichia coli strain DH5 alpha. Selected clones were studied and characterized using restriction enzymes. One of them, BQ31, was used to construct different sized probes labelled with digoxigenin-11-dUTP. These probes failed to hybridize with DNA of some insect parvoviruses and with DNA of a parvo-like virus of shrimp. They reacted strongly with dilutions of homogenized IHHNV-infected shrimp tissues and, conversely, did not react with uninfected shrimp tissues. They hybridized in situ, in sections of infected animals, labelling strongly the target cells and particularly the nuclear Cowdry type A inclusion body, which is the most diagnostic characteristics of this disease.

The in vitro infection of the hematopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus.

Viral hemorrhagic septicaemia virus (VHSV) infected the hematopoietic stromal cells (7,8) derived from pronephritic tissue of the rainbow trout, Oncorhynchuss mykiss, W., at their ninth passage in vitro. Viral infection resulted in the development of lytic cytopathic effects on confluent in vitro tridimensional network stromal cell cultures. Replication of VHSV in the stromal cell cultures was demonstrated by the increase in infectivity by epithelioma papulosum cyprini (EPC) cell culture assays and by the increase of the nucleoprotein antigen of VHSV by ELISA. By using anti-VHSV monoclonal antibodies (MAbs), flow cytometry studies demonstrated that only the infected stromal cells contained cytoplasmic viral antigens. The lytic infection of trout hematopoietic stromal cells in vitro could be relevant to the hemorrhagic pathology seen in the kidney of fish infected with VHSV.

The viral and cellular forms of the Abelson (abl) oncogene.

The precision of molecular biology has allowed a better definition of the components of the Abelson system. We know the gene structures and gene products for the cellular and viral forms of this family of related tyrosine kinases. However, many basic issues first identified in the early biological observations of Abelson, Rabstein, and others remain unanswered. The precise pathway for transformation in biochemical terms remains unknown for Ab-MLV and all of its relatives. Relatively little can be said to explain the preferential growth stimulation for certain hematopoietic cell types by the viral and other altered forms of the oncogene, and no clear insights into the function of the normal cellular forms of the abl oncogene are available. Future progress will certainly depend on the intensive efforts by many workers in the broader field of cellular growth control mechanisms.

[Reaction of the hematopoietic system in mice to the administration of virulent and avirulent strains of Shigella flexneri 2A]. / Reaktsiia krovetvornoi sistemy myshei na vvedenie virulentnogo i avirulentnogo shtammov shigell Fleksnera 2A.

The influence of live bacteria and antigenic preparations of virulent and avirulent stains of Shigella flexneri 2a on the endogenous splenic colony formation in murine hematopoietic cells has been studied. The different stimulating activity of live microbial cells of virulent and avirulent strains Shigella flexneri 2a and their antigenic preparations on the endogenous colony formation has been shown. The effect observed depends on the preparation doses and time of their administration before irradiation of the animals. The stimulating influence on the development of hemopoiesis endogenous foci may be conditioned by the action of heat-labile products of the live microbial cells and closely correlates with the virulence of strains studied.

Two families of abl-related transcripts in human haematopoietic cells differing in their homology to v-abl.

The transforming gene of the Abelson murine leukaemia virus, v-abl, contains two open reading frames (orf). The 5' orf encodes a tyrosine-specific protein kinase while the 3' orf has the capacity to code for an 18,000 Mr protein. However, no 3' orf product has yet been identified. Using probes capable of distinguishing between the 5' and 3' orfs of v-abl, we have examined the abl-related transcripts present in human haematopoietic cells and leukaemia-derived cell lines, including the chronic myeloid leukaemia-derived cell line K562. Our results indicate that transcripts of 6 kb, 7 kb and 8 kb (kilobase, 10(3) base-pairs) show strong homology to v-abl 5' protein kinase-encoding orf sequences, but are devoid of any sequences from the v-abl 3' orf. In addition, transcripts of 5 kb, 3 kb, 1.6 kb and 1.4 kb, reacting with both 5' orf and 3' orf probes, were observed. The latter species, with coding sequences from both the tyrosine kinase and the putative 18,000 Mr protein, must be transcribed from the human c-abl gene as this is apparently the only human gene containing sequences homologous to the v-abl 3' orf. The 6 kb, 7 kb and 8 kb transcripts may arise either from the c-abl gene through differential splicing, or from one of the three other regions of the human genome with sequences homologous to the 5' orf of v-abl. Examination of genomic DNA from the K562 cell line revealed that the amplification of abl-related sequences, which is presumed to result in the elevated levels of the 8 kb transcript found in this cell line, does not involve sequences homologous to the v-abl 3' orf. This lends credence to the idea that the 8 kb transcript may derive from an abl-related gene other than c-abl. While the significance of the 3' orf of v-abl remains unknown, the data presented strongly suggest the existence of at least two distinct abl-related proteins in human haematopoietic cells.

Binding of adult T-cell leukemia virus to various hematopoietic cells.

A newly found human retrovirus, adult T-cell leukemia virus (ATLV) was shown by means of membrane immunofluorescence to bind to various hematopoietic cells including T-, B- and non-T, non-B-cell lines. Partially purified viral gp46 from culture fluids of ATL virus producer lines also bound efficiently to an ATLV-negative T-cell line, CCRF-CEM cells. When the viruses were pre-incubated with anti-ATLV-positive human sera, ATLV binding to the cells was clearly inhibited but not by pre-incubation with anti-ATLV-negative sera. These data suggest that: (1) ATLV binds not only to T-cells but also to multiple types of cells of hematopoietic origin; (2) anti-ATLV antibody-positive human sera have the blocking antibody for the binding of ATLV to lymphoid cells.

In vivo infectivity of the fibrotropic C-type viral isolates from C57BL/Ka mice.

Of the three fibrotropic C-type viral isolates from C57BL/Ka mice, only the BL/Ka(B) virus is capable of infecting normal hematopoietic and lymphoid cell populations of C57BL/Ka mice in vivo, and none are tumorigenic. Inoculation of this virus alone into neonates resulted in transient replication in the bone marrow, spleen, and occasionally the thymus. Thymocytes could, however, be permanently infected in such animals if BL/Ka(B) were coinoculated with the xenotropic BL/Ka(X) virus. Neonatal injection of BL/Ka(B) prior to fractionated wholebody irradiation yielded an increase in the percentage of virus-productive radiogenic lymphomas and a decrease in incidence of such tumors. Injection of BL/Ka(B) into normal adult C57BL/Ka mice did not yield overt expression of virus replication in any of the tissues tested; latent infection could, however, be detected in the marrow and in the reticuloepithelium of the thymus. Whole-body X-irradiation of adults with 400 rads partially restored the neonatal susceptibility of bone marrow cells to infection by the isolate. BL/Ka(B) injection after fractionated whole-body irradiation of weanling C57BL/Ka mice increased the percentage of virus-positive lymphomas and revealed that a bone marrow cell subpopulation permissive for infection by the virus increases greatly in abundance soon after irradiation.

Reticulum cell neoplasms induced in C57BL/6 mice by cultured virus grown in stromal hematopoietic cell lines.

Thirty-one adherent cell lines have been established from the spleens, lymph nodes, and bone marrow of C57BL/6 mice carrying radiation leukemia virus (Duplan isolate)-induced reticulum cell neoplasms (RCN). The cell lines had a stable epithelial or fibroblastoid morphology, Supernatant virus from these lines induced splenic and lymph node RCN in 100% of inoculated C57BL/6 mice within 30 days. The disease was generalized and involved many organs. The monolayer cells themselves were not tumor cells and induced RCN through infection of the host with RCN virus. Simultaneous inoculation of in vitro-grown RCN-inducing virus any thymic lymphosarcoma virus induced each disease independently with unaltered incidence, latency period, and organ involvement; no mutual enhancement or inhibition was found, thus two separate mechanisms of action were indicated. Reextraction of the viruses from spleen, lymph nodes, and thymus gland indicated the specific organotropism of each agent. All the adherent cell lines that were derived from hematopoietic tissues produced ample, potent RCN-inducing virus. This high success rate suggests that in the hematopoietic organs the stromal, fibroblastoid cells are a natural habitat for the RCN-inducing virus. The RCN-inducing virus species may well be synthesized in these hematopoietic stromal cells. RCN-inducing virus from culture supernatants contained high-titer infectious ecotropic and xenotropic virus that was titrated. The cultures are being used to clone the RCN-inducing virus and to establish the virologic and molecular properties that endow it with specific RCN-inducing capacity.

Electron microscopic study of virus particles in yolk sac and placenta and in hematopoietic organs of newborn C3Hf mice.

Examination of yolk sac from a C3Hf and a C3H mouse with the electron microscope revealed the presence of C-type virus particles in the blood islands. Particles were observed budding from the plasma membrane of hemocytoblasts, from erythroblasts, and occasionally from reticulocytes. C-type particles were also found in similar cells in hematopoietic foci in the liver, spleen, and bone marrow of embryos, and they continued to be present in newborn C3Hf mice up to 11 days of age. Particles consistently appeared in the thymus, even in older suckling mice. A comparison is made between the presence of C-type particles in organs of embryonic, newborn, and adult C3Hf mice. C-type particles were not observed in the chorioallantoic placentas from mice that were given injections of mouse leukemia virus (Gross) or from normal noninjected mice; however, intracisternal A-type particles were present in cytotrophoblast cells from these placentas.

[Immunologic study of suspension cultures prepared from hematopoietic organ cells of baboons with lymphoma]. / Immunologicheskoe izuchenie suspenzionnykh kul'tur, poluchennykh iz kletok krovetvornykh organov bol'nykh limfomoi pavianov

The cytoplasmic antigen was determined by the indirect immunofluorescence test using serum from a Burkitt lymphoma patient with a titer of antibody to Epstein-Barr virus capsid antigen of 1.320, in the cytoplasm of 2--6% of cells of suspension cultures obtained from baboons with lymphoma. There was a correlation between the number of antigen-containing cells in this cultures and the number of cells in which herpes virus was determined by electron microscope studies. Sera from baboons with lymphoma from which the suspensions cultures had been prepared contained high titers of antibody to herpes virus of baboons (HVB) and to Epstein-Barr virus (EBV). Specific differences between the antigens produced in the cultures by HVB and EBV were minimal, indicating their immunological similarity. Lymphoblastoid cultures prepared from hemopoietic organs of baboons with lymphoma belong to B cell type.

Fine structure of the asexual stages of Plasmodium elongatum.

Plasmodium elongatum, an avian malarial parasite, differs from other such parasites by infecting both the circulating red blood cells and the hematopoietic cells. The exoerythrocytic development of P. elongatum occurs mainly in these red cell precursors. The fine structure of the asexual stages of P. elongatum has been studied in the bone marrow and peripheral blood of canaries and compared with that of the asexual stages of other avian malarial parasites. With minor differences, the merozoites of P. elongatum possess the same organelles as those in the exoerythrocytic merozoites of P. fallax and the erythrocytic stages of P. cathemerium, P. lophurae, P. fallax, and P. gallinaceum. The developmental sequence is also essentially similar to that of other avian malarial parasites, in that upon entry into a new host cell, the dedifferentiation, growth, and redifferentiation phases take place. However, we have found some important differences in the feeding mechanism of P. elongatum. The cytostome is involved in the ingestion of host cell cytoplasm in both exoerythrocytic and erythrocytic stages, in contrast to P. fallax, in which the cytostome is inactive in the exoerythrocytic stages. In P. elongatum, host cell cytoplasm is ingested through the cytostome, and "boluses" are formed and incorporated into a large digestive vacuole. Subsequently, the digestion of the boluses takes place in this digestive vacuole. Thus, in regard to the function of the cytostome, the exoerythrocytic stages of P. elongatum appear to be closely related to the erythrocytic stage which has a feeding mechanism similar to that of the erythrocytic stage of other avian malarial parasites.