Dietary beta-carotene and ultraviolet-induced immunosuppression.

Ultraviolet (UV)-induced immunosuppression is a critical step in UV carcinogenesis, permitting tumour outgrowth. We investigated the effect of dietary beta-carotene on UV suppression of contact hypersensitivity (CHS) to trinitrochlorobenzene (TNCB) in BALB/c mice. Mice were fed for 10-16 weeks chow alone or supplemented with 1% beta-carotene or placebo as beadlets. Serum beta-carotene was detectable by high performance liquid chromatography (HPLC) analysis only in beta-carotene-fed mice (2.06 +/- 0.15 micrograms/ml). Serum retinol was 0.22-0.27 micrograms/ml in all three groups. Mice (n = 41/dietary group) were irradiated with 0, 4.5, 9 or 18 kJ/m2 of UVB and the CHS response was measured. Decreased CHS responses were observed in all UV-irradiated groups compared with unirradiated controls. UV dose-responses for suppression of CHS derived by first-order regression analyses of plots of percentage suppression of CHS as a function of log10UV dose showed significant slopes (P < 0.02) for all three dietary groups and similar residual variances between groups, P > 0.05. The UV dose for 50% suppression of CHS was 6.3 kJ/m2 for control, 6.4 kJ/m2 for placebo, and 5.5 kJ/m2 for beta-carotene-fed mice. No significant differences in slopes or elevations between UV dose-responses were observed, P > 0.05. Skin levels of the initiator of UV-induced immunosuppression, cis urocanic acid, were determined by HPLC in mice given 0 or 9 kJ/m2 of UV (n = 28/dietary group). No significant differences were observed between dietary groups (range 35.2-41.1 ng/mg skin, P > 0.15) We conclude feeding beta-carotene to BALB/c mice does not alter susceptibility to UV immune suppression, in contrast to human studies.

Control of UVB immunosuppression in the mouse by autosomal and sex-linked genes.

Irradiation with UVB (290-320 nm) initiates a systemic immunosuppression detectable as suppression of contact hypersensitivity (CHS). We investigated susceptibility to UV suppression in reciprocal F1-hybrid and backcross mice derived from BALB/c (low susceptibility) and C57BL/6 (high susceptibility) inbred strains. CB6F1 male mice exhibited high susceptibility and B6CF1 male mice exhibited low susceptibility, indicating a major X-linked effect in the genetic control of UV immune suppression. Females of either F1 hybrid showed intermediate suppression, consistent with random X-inactivation. A model of monogenic X-linked control was not sufficient, and evidence for the action of two genetically unlinked autosomal genes was found in parental backcross animals. Both sexes of (BALB/c x CB6F1) mice showed a 1 high:1 low ratio of phenotypes, indicating control by a major autosomal locus, Uvs1, confirmed by propagation of the high phenotype through selective backcrossing for nine generations to BALB/c. Uvs1 was not genetically linked to 12 chromosomal markers including the pigment genes b (brown) and c (albino). Backcross animals (C57BL/6 x CB6F1) showed a significant sex difference, male mice giving a 3 high:1 low ratio of phenotypes, compatible with the action of a second autosomal locus, Uvs2, in this hybrid. The findings are compatible with a model in which high phenotype (Uvs1b/Uvs1b) is dominant when subjected to recessive epistatis by the X-chromosome locus Uvs3, or by the autosomal locus Uvs2. The finding of genetic control by interacting autosomal and X-linked genes is unique. Genetically determined high susceptibility to UV immunosuppression may be an important risk factor for UV-related human diseases.

Susceptibility to immunosuppression by ultraviolet B radiation in the mouse.

Irradiation with ultraviolet B (UVB; 290-320 nm) initiates systemic immunosuppression of contact hypersensitivity (CHS). UV dose-responses for suppression of CHS to trinitrochlorobenzene were established in 18 strains of inbred mice. Three phenotypes with significantly different susceptibilities to UV suppression were identified. The phenotypes were: high (HI) susceptibility, 50% suppression with 0.7-2.3 kJ/m2 UV (C57BL/6, C57BL/10, and C57L and NZB females); low (LO) susceptibility, 50% suppression with 9.6-12.3 kJ/m2 UV (BALB/c, AKR, SJL and NZW), and intermediate (INT) susceptibility, 50% suppression with 4.7-6.9 kJ/m2 UV (DBA/2, C57BR, C3H/HeJ, C3H/HeN, CBA/N and A/J). UV suppression was not correlated with skin pigmentation or with the magnitude of the CHS response in non-irradiated animals. Major histocompatibility complex (MHC) haplotype was not correlated with UV suppression in MHC congenic strains B10.D2/oSnJ, B10.D2/nSnJ, B10.BR/SgSnJ, and A.BY/SnJ. There were no sex differences in UV suppression in BALB/c, C57BL/6, or NZW animals. In the autoimmune NZB strain, however, male mice (LO) were seven times less sensitive to UV suppression than NZB female mice (HI). Both sexes of (NZB x NZW)F1 and (NZW x NZB)F1 mice were HI, supporting dominance of HI over LO. Thus there are genetic factors and interacting sex-limited factors determining susceptibility to UV suppression. These findings may be of relevance to UV-related diseases such as photosensitive lupus and skin cancer.

Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride.

This report describes a microprocedure that may be used for direct measurement of proteoglycans and glycosaminoglycans, after chromatographic elution with chaotropic reagents. The assay is based on the ability of the sulfated glycosaminoglycans to bind to the cationic dye, dimethylmethylene blue, in solution. Inclusion of guanidinium chloride (0.24 M) in the assay resulted in a stable dye-proteoglycan interaction, but eliminated the interference of other anionic macromolecules such as DNA. The assay is rapid, sensitive, and reproducible and therefore useful for processing several samples. Finally, the procedure can be used for quantitative determination of several types of proteoglycans and glycosaminoglycans.

An investigation of genetic variation within a series of congenic strains of mice.

2 congenic strains of mice, B6N.AKN-Ahk and D2N.B6N-Ahb, imported from the USA, were found to be either segregating or fixed for an incorrect allele at a number of biochemical loci. B6N.AKN-Ahk, supposedly congenic with C57BL/6N, had the wrong genotype at 6 out of 12 biochemical loci; D2N.B6N-Ahb, supposedly congenic with DBA/2N, was segregating at 3 out of 9 loci. There was genetic variation in mandible shape within the 2 strains but no abnormal coat colours were found and no hybrid vigour in breeding performance was detected. Analyses in the USA confirmed these results and showed that 2 other congenic strains, C3N.D2N-Ahd and AKN.B6J-Ahb, were also segregating at a number of loci. Some of the alleles found in the C3N.D2N-Ahd mice must be the result of a genetic contamination. The simplest explanation for this breakdown in the backcrossing programme is genetic contamination with other congenic strains or recombinant inbred lines under development in the same laboratory. These findings emphasize the importance of continual genetic monitoring of all genetic stocks at regular intervals and in particular during the development of congenic and recombinant lines.

An intervention to reduce the rate of hospital discharges against medical advice.

The initiation of a patient advocacy program in a private psychiatric hospital in 1980 was effective in significantly reducing the rate of hospital discharges against medical advice relative to the rate in 1979 and in 1978. There was also a concomitant increase in the rate of clinically approved discharges relative to that in 1979 and in 1978. The authors view the patient advocate as an objective intermediary who represents an acknowledgment of patients' rights and affords patients an opportunity to have some autonomy and an impact on the hospital system.

Susceptibility to fluorescent light-induced chromatid breaks associated with DNA repair deficiency and malignant transformation in culture.

The increased susceptibility of mouse cells to fluorescent light-induced chromatid damage following their spontaneous malignant transformation in culture could result from loss or inactivation of catalase that decomposes the photoproduct H2O2 or from impaired capacities to repair DNA damage. No consistent change in catalase activity with respect to neoplastic state could be established. To interpret the cytogenetic damage in terms of DNA strand breaks, we determined the incidence of chromatid breaks induced by light exposure during the G1 and late S-G2 phases of the cell cycle in normal and malignant derivatives of a C3H mouse cell line. Chromatid breaks at metaphase following light exposure during G1 would result from DNA strand breaks, cross-links, or base damage, whereas breaks following exposure during late S-G2 would result from single-or double-strand breaks. Both G1 and late S-G2 were susceptible in malignant cells but only G1 in normal. Since caffeine inhibits DNA repair, we compared its effects on light-induced chromatid damage in the normal and malignant cells to assess their DNA repair capacities. Treatment of normal cells with caffeine (50 microgram/ml) directly following five hr of light exposure in G1 increased the chromatid damage to that in malignant cells exposed with or without caffeine. Similarly, treatment of normal cells with caffeine during late S-G2 exposure increased chromatid damage to a level not significantly different from that in malignant cells exposed without caffeine. Caffeine had little influence on chromatid damage in malignant cells. The increased susceptibility of malignant mouse cells to fluorescent light-induced chromatid breaks thus appears to result from impaired capacities to repair DNA damage.

Polyamine synthesis in bone marrow granulocytes: effect of cell maturity and early changes following an inflammatory stimulus.

Enriched fractions of mature and immature neutrophil granulocytes, isolated from guinea pig bone marrow, were assayed for ornithine decarboxylase activity and polyamine content. The results show that immature granulocytes contain at least ten times more ornithine decarboxylase activity and two times more spermidine than mature granulocytes. The incorporation of 14C-ornithine into putrescine and spermidine of intact immature granulocytes was three to four times and ten times, respectively, that of mature granulocyte preparations. Six hours after an inflammatory stimulus, transient increases of 14-fold and 3-fold in the activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, respectively, were observed in immature bone marrow granulocytes. At this time the incorporation of 14C-ornithine into putrescine and spermidine in bone marrow granulocytes from stimulated animals was 14 times that of cells from controls. A maximum increase in DNA synthesis in these cells during the inflammatory response occurred 6 hr after the maximum increase in the polyamine synthetic activity. Together these data suggest that polyamine synthesis in the granulocyte compartment of the bone marrow is associated chiefly with immature proliferating cells and that increased polyamine synthesis precedes increased granulocyte proliferation in the bone marrow following an inflammatory stimulus.

Location of theSas-1 locus on mouse chromosome 1.

Using three sets of recombinant inbred strains (BXD, BXH, and BXJ), we found the locus controlling an antigenic substance (Sas}-1) in murine serum to be closely linked to the Chromosome-1 marker,Dip-1. This linkage was confirmed by an analysis of backcross linkage. The BXD and backcross data suggest that the gene order isId-1-Dip-1-Sas-1-Mls. Data from the three sets of RI strains and the 32 backcross mice lead to the estimate that the recombination frequency betweenDip-1 andSas-1 is 0.030 ±0.015.

Genetic studies of murine catalase: regulation of multiple molecular forms of kidney catalase.

Starch gel electrophoresis of kidney catalase in inbred strains C3H and C57BL/6, their F1 hybrid, and first and second backcross generations demonstrated that single-component (type A) v. multiple-component (type B) electrophoretic patterns are controlled by a single locus. The type A electrophoretic pattern is dominant. Twenty-five inbred strains of mice were classified according to their kidney catalase electrophoretic pattern. The data indicate that the segregating genetic factor determines a specific substance in the type A kidney which affects the electrophoretic mobility of catalase. A comparison of the F1 hybrid enzyme with a 1:1 mixture of C3H and C57BL/6 enzyme showed that the alteration of electrophoretic mobility is the result of posttranslational modification of the catalase molecule. An association of kidney catalase electrophoretic pattern and the H-2kappa haplotype indicates that the locus controlling the electrophoretic pattern is most likely located on chromosome 17 in close proximity to the H-2 complex.

New mutant and congenic mouse stocks expressing the murine leukemia virus-associated thymocyte surface antigen GIX.

For several reasons the G(IX) antigen (1) has a prominent place in current work on murine leukemia virus (MuLV): In the prototype G(IX+) mouse strain 129, the G(IX) trait is mendelian, and is expressed selectively (though not exclusively) on thymocytes. Thus, expression of this cell surface component is under the control of cellular genes and is subject to the controls governing the differentiation of T lymphocytes (2). Although the 129 mouse produces no demonstrable leukemia virus such as that found in the AKR strain, it was soon realized that G(IX) antigen must in some way be related to MuLV, because productive infection with MuLV is frequently associated with appearance of G(IX) antigen on cells that are genotypically G(IX-), most notably on MuLV-infected rat cells, or cells that belong to other differentiation pathways (1). The basis of this connection between G(IX) and MuLV has recently become clear from the demonstration that G(IX) is one of MuLV envelope. Therefore, our working hypothesis is that the presence of G(IX) is one of the antigens present on gp69/71 (3,4), the major glycoprotein component of the MuLV envelope. Therefore, our working hypothesis is that the presence of G(IX) antigen always denotes the presence of gp69/71 (though not all variants of gp69/71 need necessarily carry G(IX)). Study of the circumstances under which G(IX) is expressed on the cell surface is thus potentially a powerful approach to understanding how the expression of C-type viral genomes is controlled. Such studies are greatly facilitated by the availability of mutant and congenic strains of inbred mice which differ from the nonmutant or partner strains only with respect to one or another manifestation of the viral genome. It is for this reason that we record here (Table I) some details of two G(IX) mutant and two G(IX) congenic stocks derived in our colonies at Memorial Sloan-Kettering Cancer Center (MSKCC). In addition, to these four strains, Table I includes data for the three relevant partner strains, and for strain AKR, for comparison. These eight strains all differ from one another with respect to one or more MuLV-related traits.

Genetic regulation of the expression of catalase activity in murine red blood cells.

The specific activity (k'1) and concentration of red blood cell catalase from four inbred strains of mice (BALB/c, C57BL, C57BL/6, and NBL) were measured to determine the mechanisms responsible for interstrain variations in enzyme activity. The specific activities of RBC catalase in NBL and the C57BL sublines are equal (2.5 x 10(7) M-1 sec-1), while that of BALB/c (4.0 x 10(7) M-1 sec-1) is 67% greater. The relative concentration of catalase is approximately 30% lower in NBL erythrocytes compared to the other three strains. The activity of BALB/c RBC catalase is due to a high k'1 coupled with a high intracellular concentration; RBC catalase activity in the C57BL sublines is the result of a low k'1 and high concentration. A low k'1 and a low concentration are responsible for the low catalase activity levels found in NBL erythrocytes.