Calretinin is not a marker for subdivisions within the suprachiasmatic nucleus.

In this study, we report the immunocytochemical localization of the calcium-binding protein calretinin (CAL) in the suprachiasmatic nuclei (SCN) of male and female rodents including rats, mice, golden hamsters, and Arvicanthis niloticus. The results revealed that CAL is present in different subdivisions of the SCN in the different species studied and CAL can, therefore, not be considered a marker for particular subdivisions within the SCN. No differences were found between males and females.

Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time.

The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.

Genetic control of prion incubation period in mice.

The prion gene complex (Prn) is located on mouse chromosome 2 between the beta-2-microglobulin (B2m) and agouti (A) genes. Within this complex are the prion protein gene (Prn-p), which encodes the only identified macromolecule (PrP) that purifies with infectious scrapie agent, and a scrapie incubation time gene (Prn-i). Using a variety of restriction endonucleases, six allelic forms of the Prn-p gene have been distinguished by their patterns of restriction fragment length polymorphisms. We had previously shown that the exceptionally long scrapie incubation period of I/LnJ mice inoculated with the Chandler isolate (over 200 days) was due to the effects of a scrapie incubation time gene tightly linked to Prn-p. So far, this long scrapie incubation time allele has been found only in those inbred mouse strains (I/LnJ, P/J and IM) that have the b allele of Prn-p. It is not known whether the incubation time gene and prion protein gene are two distinct loci or are one and the same. Putative recombinants between the incubation time phenotype and Prn-p genotype have been observed, but this could be due to effects of other genes segregating in the population. Regardless of whether or not the incubation time and PrP genes are identical, if any differences were found in the amino acid sequences of PrP encoded by the different Prn-p alleles there would be important implications for interpretation of results on 'strains' of scrapie agent. It would not be necessary to invoke nucleic acid as the informational macromolecule of the scrapie agent because differences in prion 'strains' recovered from mice with different Prn-p genotypes need not be the result of host selection but could be due to differences in host-encoded PrP.

Linkage of prion protein and scrapie incubation time genes.

A single gene (Prn-i) that affects scrapie incubation period in mice has been identified. I/LnJ mice have a very long incubation period after inoculation of scrapie prions (200-385 days) and NZW/LacJ mice have a short one (113 +/- 2.8 days). (NZW X I/Ln)F1 hybrid mice had incubation times of 223 +/- 2.8 days indicating longer incubation times were dominant. Incubation periods in the backcross progeny of (NZW/LacJ X I/LnJ)F1 X NZW/LacJ segregated into two groups (64 mice, 130 +/- 1.1 d; 66 mice, 195 +/- 1.9 d) indicating single gene control. NZW/LacJ and 20 other inbred strains have the Prn-pa allele which is identified as a 3.8 kb Xbal fragment using a hamster PrP (prion protein) cDNA probe. I/LnJ and three other Prn-pb mouse strains have a 5.5 kb Xbal restriction fragment. Analysis of DNA from 66 backcross mice indicated Prn-i is tightly linked to Prn-p, the structural gene for PrP.

Early events in natural resistance to bone marrow transplantation. Use of radiolabeled bone marrow cells.

Natural resistance against the proliferation of splenic colony-forming units (CFU-S) is seen in certain combinations of bone marrow donors and irradiated hosts. In order to examine the early events following bone marrow transplantation and to determine whether genetically determined CFU-S repression is due to elimination of the transplanted cells from the spleen or to inhibition of their proliferation, we labeled proliferating cells in freshly isolated bone marrow or long-term bone marrow cultures (LTBMC) with radioactive I-iododeoxyuridine prior to injection. A heterogeneous mixture of cells was labeled in both freshly isolated marrow and LTBMC; CFU-S precursors were among the cells labeled as indicated by reduction of CFU-S numbers in the radiolabeled cell population. Radiolabeled cells from C57BL/6J, DBA/2J, and B6D2F1 mice were injected into syngeneic, semisyngeneic, and allogeneic irradiated mice that were killed at various times after injection to determine the amount of radioactivity remaining and the organ distribution of the labeled cells; additional mice were killed 7-8 days later to count CFU-S. Retention of label in the spleen was predictive of subsequent CFU-S numbers, suggesting that killing or elimination of the transplanted cells from the spleen is the cause of CFU-S depression in resistant animals. Further genetic analysis of the survival of the radiolabeled cells in the spleen indicated that mismatching of H-2 homozygous donor cells with the host at H-2D was a prerequisite for resistance and that H-2 heterozygous cells were not resisted in spite of H-2 mismatching. Although natural killer (NK) cell-deficient beige mice were able to resist H-2-nonidentical bone marrow cells, pretreatment of the host with anti-asialo GM-1 antibodies completely abrogated natural resistance as assessed by splenic survival or radiolabeled cells. The findings that splenic survival or radiolabeled bone marrow cells reflects the immunogenetic specificity of CFU-S repression and that abrogation of CFU-S repression increases the splenic survival of the labeled cells strengthen the postulate that repression of CFU-S proliferation involves events occurring within the first 24 hr after injection.

Adaptive immune defects and delayed rejection of allogeneic tumor cells in beige mice.

The effect of the beige (bg) mutation on adaptive allogeneic tumor rejection was examined by monitoring tumor cell survival in vivo using [131I]iododeoxyuridine-prelabeled cells. Accelerated elimination of allogeneic tumor cells normally begins 8 days after ip injection and is due to active immune responses. Two independent mutations to beige on two different inbred backgrounds (C57BL/6J bgJ and DBA/2JCo bg8J) were tested, and bg/bg mice showed a 1-day delay in immune elimination of allogeneic cells. This delayed rejection was not due to a defect in clearing label from dead cells, nor to an inability to effect antibody-induced killing in vivo. Both humoral and cell-mediated responses against the allogeneic tumor cells were significantly lower in bg/bg than in +/bg mice.

A genetic analysis of natural resistance to nonsyngeneic cells: the role of H-2.

The genetic control of natural resistance in vivo to four natural killer (NK) cell-resistant H-2 homozygous lymphoid tumor cell lines was investigated by following the survival and organ distribution of cells prelabeled with radioactive iododeoxyuridine. Backcross mice derived from DBA/2J and CBA/J parents were injected with H-2d tumor cells and tumor cell elimination was lowest in H-2d homozygotes. Natural killer cell activity was also reduced in mice with the H-2d haplotype, but no direct correlation between NK cell levels against YAC-1 or SL2-5 lymphoma cells and natural resistance in vivo was demonstrable. Analysis of 23 BXD recombinant inbred strains indicated that natural resistance to H-2d tumors was restricted to H-2b strains. There was no direct association of NK cell activity with H-2 type in the BXD strains and NK cell levels did not correlate with tumor survival in vivo. By comparing natural resistance to H-2d and H-2b tumors in DBA/2, C57BL/6, B6D2F1, and B10.D2 mice we found that H-2 nonidentity between the tumor and the host, rather than the host H-2 haplotype, determined whether natural resistance occurred. Again, NK cell activity against YAC-1 cells was not predictive of tumor survival in these strains. These results provide genetic evidence that NK cells alone cannot account for natural resistance to H-2 nonidentical cells of hemopoietic origin.

Stimulation of immunoglobulin biosynthesis in human B cells by wheat germ agglutinin. I. Evidence that WGA can produce both a positive and negative signal for activation of human lymphocytes.

Wheat germ agglutinin (WGA), previously regarded strictly as a nonmitogenic or anti-mitogenic lectin, can under appropriate conditions markedly stimulate in vitro synthesis and secretion of immunoglobulin (Ig) by human B lymphocytes. Stimulation of Ig production by WGA is 1) confined to a narrow lectin dose range (2 to 10 micrograms/ml; 2) abrogated by the simple sugar N-acetyl-D-glucosamine but not by a variety of other monosaccharides; 3) effective only after early additions of WGA within the initial 72 hr of 12-day cultures; 4) detected in the presence of B and T cells but not B cells alone; and 5) polyisotypic in nature, as indicated by augmented synthetic rates of Ig in each of 3 major classes (IgG, IgA, and IgM). With few exceptions, WGA produces equivalent or greater rates of Ig production as obtained in cultures activated with pokeweed mitogen (PWM), a well-recognized T-dependent polyclonal activator of human B cells. Furthermore, periperal blood lymphocytes from select individuals that respond weakly to PWM are markedly stimulated with WGA. In contrast to these stimulatory effects of WGA on Ig production by lymphocytes exposed to low lectin concentrations, addition of WGA in amounts greater than 15 micrograms/ml to PWM-stimulated human lymphocyte cultures produces marked suppression of the expected level of Ig synthesis. These data indicate that varying doses of WGA can produce contrasting stimulatory and inhibitory effects on human B cell metabolism.

Analysis of the effects of lipopolysaccharide on macrophages: differential phagocytic responses of C3H/HeN and C3H/HeJ macrophages in vitro.

The phenomenon of lipopolysaccharide (LPS)-induced in vitro macrophage cytotoxicity has been reported by a number of investigators but has often been difficult to reproduce and to quantitate. In this report, we have examined the effect of LPS on the ability of macrophages to ingest 51Cr-labeled, opsonized sheep erythrocytes as a method for examining the direct toxic effects of LPS on macrophages in vitro. By using this assy, we can clearly discriminate between LPS responder C3H/HeN macrophages and LPS nonresponder C3H/HeJ macrophages and demonstrate that LPS induces a profound inhibition of Fc-mediated phagocytosis in LPS responsive macrophages. Furthermore, low concentrations of LPS stimulate phagocytosis in macrophages derived for C3H/HeJ mice. The lipid A moiety of the LPS is responsible for the observed enhancement or inhibition of Fc-mediated phagocytosis. This assay was more sensitive than LPS-induced cytotoxicity, since inhibition of phagocytosis was detectable in cultures of LPS-sensitive macrophages even when cytotoxicity, assessed by trypan blue exclusion, was not. Thus, this assay represents an extremely sensitive method for analyzing the direct effects of LPS on macrophages.