ABSTRACT
In vitro amplification of DNA by PCR is a powerful tool to detect small amounts of DNA. It is now widely used for detection of pathogenic agents from extracellular fluids and organs. The use of anion exchange HPLC to quantify the PCR product resulting from the specific amplification of the DNA from the replicative-defective viral DNA responsible for MAIDS is described. This technique allows precise quantification of MAIDS virus DNA in different organs and circumvents the use of radioactivity and gel electrophoresis.
Subject(s)
Chromatography, High Pressure Liquid , DNA, Viral/analysis , Leukemia Virus, Murine/isolation & purification , Lymph Nodes/virology , Murine Acquired Immunodeficiency Syndrome/virology , Polymerase Chain Reaction/methods , Animals , Calibration , DNA Primers , Female , Leukemia Virus, Murine/genetics , Mice , Mice, Inbred C57BLABSTRACT
The murine-acquired immunodeficiency syndrome (MAIDS) is caused by a mixture of murine leukemia viruses (LP-BM5 MuLV). The influence of perinatal contact with retroviruses or their Ags on the response to infection was tested by infecting with LP-BM5 (MuLV) the adult offspring of mice with MAIDS. These offspring were resistant to disease after virus challenge. Most of them were free of defective viral DNA, and even those with molecular evidence of infection had lymphoid cells with a lower infectious capacity to cause MAIDS in naive recipients. No ecotropic, xenotropic, or mink cell focus-forming (MCF) virus expression was found at the age of 5 wk, which is the time of LP-BM5 (MuLV) challenge. However, at 22 wk of age, one-half of the offspring from MAIDS mothers had ecotropic virus-expressing cells in their spleens. At the time of suckling, offspring from infected mothers had enhanced percentages of B cells and CD4 and CD8 T cells in the spleen, possibly followed by a slight persistent splenomegaly. These results suggest that immune reactivity, rather than tolerance to the virus, is responsible for resistance to disease after challenge. The offspring of MAIDS mice could clear the virus after challenge. This clearance was mediated by CD8 T cells, as continuous CD8 T cell depletion initiated at the time of viral challenge abrogated the resistance of these mice to MAIDS.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Leukemia Virus, Murine/immunology , Murine Acquired Immunodeficiency Syndrome/immunology , Animals , Base Sequence , CD8 Antigens/immunology , DNA Primers/chemistry , Defective Viruses , Female , Hyaluronan Receptors/analysis , Immunity, Cellular , Immunophenotyping , Leukemia Virus, Murine/pathogenicity , Male , Maternal-Fetal Exchange , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Pregnancy , Proviruses/chemistry , Spleen/immunology , Virus ReplicationABSTRACT
A non-radioactive PCR method was developed to quantify the development of malaria parasites in the infected host. This was achieved by using Plasmodium genus-specific primers corresponding to the parasite's small subunit ribosomal RNA genes. The quantification of the PCR product was performed by high performance liquid chromatography, and calibration curves were obtained by amplification from defined quantities of purified Plasmodium genomic DNA. Using this method, it was possible to quantify development of P. berghei and P. yoelii blood-stage parasites from blood and brain samples of infected mice, and of hepatic stage parasites, from liver samples of mice infected with different numbers of sporozoites.
Subject(s)
DNA, Protozoan/analysis , Malaria/parasitology , Plasmodium berghei/growth & development , Plasmodium yoelii/growth & development , Polymerase Chain Reaction/methods , Animals , Brain/blood supply , Brain/parasitology , Capillaries/parasitology , DNA, Protozoan/blood , Erythrocytes/parasitology , Female , Liver/parasitology , Mice , Mice, Inbred Strains , Plasmodium berghei/genetics , Plasmodium berghei/isolation & purification , Plasmodium yoelii/genetics , Plasmodium yoelii/isolation & purification , RNA, Protozoan/genetics , RNA, Ribosomal/genetics , Species Specificity , Spleen/parasitologyABSTRACT
Characterization of cells present in the extravascular compartment of murine liver was performed after different immunization procedures against the malaria parasite Plasmodium yoelii. Mice were immunized with live or irradiated sporozoites or with parasitized erythrocytes. Whatever the immunization protocol used, the mice were protected against a sporozoite challenge but each immunization procedure induced a specific profile of cell types. Immunization with irradiated sporozoite induce a significant increase in CD8+ lymphocytes, parasitized erythrocytes stimulates production of monocytes/macrophages and CD8+ lymphocytes while, after live sporozoites immunization, polymorphonuclear cells, macrophages/monocytes, B cells and a range of T cell subsets were increased in number.
Subject(s)
Immunization , Liver/immunology , Lymphocyte Subsets/immunology , Malaria/immunology , Plasmodium yoelii/immunology , Animals , Leukocyte Count , Liver/parasitology , Macrophages/immunology , Malaria/parasitology , Mice , Monocytes/immunology , Neutrophils/immunology , Plasmodium yoelii/growth & developmentABSTRACT
The retrovirus LP-BM5 murine leukemia virus induces murine AIDS in C57BL/6 mice that has many similarities with human AIDS; Plasmodium berghei ANKA causes experimental cerebral malaria in the same strain of mice. The outcome of malaria infection was studied in mice concurrently infected with the two pathogens. The retrovirus significantly reduced the gravity of the neurological manifestations associated with Plasmodium berghei ANKA infection. The protection against experimental cerebral malaria induced by murine AIDS increased with duration of viral infection and, hence, with the severity of the immunodeficiency. Interleukin 10, principally from splenic T cells, was shown to play a crucial role in this protection.
