Immunological studies of an organic anion-binding protein isolated from rat liver cell plasma membrane.

The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.

Genetic control of natural resistance to Trypanosoma rhodesiense: transfer of resistance with bone marrow or spleen cells.

Inbred strains of mice differ greatly in their innate resistance to Trypanosoma rhodesiense, the etiologic agent of African sleeping sickness. BALB/c mice are very susceptible to this organism, whereas C57BL/6 mice are highly resistant. This difference is regulated by at least three distinct genes. An adoptive transfer study was performed in order to determine the tissue site of expression of these genes. Three inbred mouse strains (C57BL/6J, C3H.SW/SnJ, and BALB.B) that differ in resistance to T. rhodesiense, but are histocompatible at the H-2 locus, were used in the study. The adoptive transfer of normal bone marrow cells from C57BL/6J (resistant) mice into X-irradiated BALB.B (susceptible) mice rendered the recipient mice resistant to a subsequent experimental challenge with T. rhodesiense. Conversely, the transfer of bone marrow cells from BALB.B mice into irradiated C57BL/6J mice rendered the latter mice susceptible. Resistance could also be adoptively transferred from C57BL/6J mice to a second susceptible strain, C3H.SW/SnJ, by using either bone marrow or spleen cells. These findings demonstrate that although multiple genes control innate resistance to T. rhodesiense, all or most of these genes appear to control the development or function of cells whose progenitors reside in the spleen and bone marrow.

Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi.

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.

Trypanosoma rhodesiense: analysis of the genetic control of resistance among mice.

Inbred mouse strains differ in their resistance to infection with the human pathogen Trypanosoma rhodesiense. Of the strains tested, C57BL/6 (B6) mice were the most resistant, and BALB/c (C) mice were among the most susceptible. The genetic basis underlying the different susceptibility of these two strains was analyzed. (CXB6)F1 progeny of either sex were more resistant than the BALB/c parent. Also, the backcross of F1 mice to the susceptible male or female BALB/c parent resulted in 52.0% susceptible (i.e., death on or before day 24) progeny, as compared with only 0.64% susceptible F1 progeny. The data suggested that resistance was the dominant phenotype and that the resistant allele was carried by the B6 parent. The presence of another locus regulating resistance to death was suggested by the facts that only a small percentage of F2 mice were susceptible and that a number of F1 and F2 mice were more resistant than their B6 parent. The locus responsible for these phenomena was presumably hypostatic in nature and carried by BALB/c mice, and its effects were only evident in the presence of other resistance genes. In addition, the observation that many of the susceptible individuals among F2 and backcross mice were more resistant than the BALB/c mice suggested that other minor genes also modulated the response of mice to infection. A set of CXB recombinant inbred mice was tested as well, and the individual strains within this set could also be placed into four groups: susceptible, intermediate, resistant, or hyperresistant. These findings are compatible with the multigenic model suggested by the Mendelian analyses.

Trypanosoma rhodesiense infection in mice: sex dependence of resistance.

There is large variation in the survival of inbred mouse strains infected with Trypanosoma rhodesiense (EATRO 1886). Of those strains that survived for at least 22 days postinfection, female mice were markedly more resistant than male mice. The longer a strain survived, the greater was the difference in survival between male and female mice. Parasite counts were higher in male mice than in females, suggesting that the decreased resistance of males was due to their relative inability to control parasite growth. To determine the possible role of an X-linked resistance gene, resistant (C57BL/6) and susceptible (BALB/c) mice were mated, and their F1 progeny were infected with T. rhodesiense. There was no difference in the resistance between reciprocal F1 male mice (C57BL/6 X BALB/c versus BALB/c X C57BL/6), indicating that an X-linked gene does not account for the difference in resistance between susceptible and resistant mice.

Antibody-dependent phagocytosis of Trypanosoma rhodesiense by murine macrophages.

Murine resident peritoneal adherent cells bound and ingested Trypanosoma rhodesiense in the presence of specific rat or mouse antiserum. Serum which mediated this phenomenon was obtained as early as 3 days after mice were immunized with gamma-irradiated parasites, with peak levels of activity obtained on day 7. A second injection of gamma-irradiated trypanosomes resulted in a secondary elevation in activity. Fresh normal serum, as a source of complement, enhanced phagocytosis in the presence of otherwise suboptimal antiserum concentrations. P388D1 cells, which like peritoneal macrophages possess Fc and complement receptors, also bound trypanosomes in the presence of antiserum. This in vitro model reflects events that occur in vivo in hosts immunized against T. rhodesiense.