Estimating cell depth from somatic mutations

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.     

Tolerance induction by veto CTLs in the TCR transgenic 2C mouse model. II. Deletion of effector cells by Fas-Fas ligand apoptosis

The direct assay of veto CTLs in the 2C mouse model enables monitoring, by FACS, the fate of the TCR transgenic effector CD8(+) T cells, the transgene of which can be stained with clonotypic Ab 1B2. After the addition of veto cells, CD8(+)1B2(+) effector cells increasingly express annexin V, and maximal apoptosis is attained 72 h after initiation of MLR. This veto activity can be partially blocked by anti-CD8 Abs directed against the allele expressed by the veto CTLs, but not by the effector cells. When effector CD8(+) T cells were from 2C mice, which lack Fas expression ((2CX lpr)F(2)), deletion of effector cells was not exhibited by veto cells. The protein levels of the apoptosis inhibitors FLIP and Bcl2 in purified CD8(+)1B2(+) effector cells at different time points after MLR showed an initial up-regulation of these inhibitors, with marked reduction of FLIP, but not of Bcl2, by 48 h after initiation of culture. Taken together, these results are in accordance with a Fas-FasL-based mechanism in which prolonged binding between the effector cell and the veto cell might be required to allow FLIP to be down-regulated. Such prolonged interaction might be afforded through the interaction of CD8 molecules on the veto cell with the alpha3 domain of H2 class 1 on the effector cell.     

Changes in sialyltransferase activity during murine T cell differentiation

The main aim of our study was to investigate whether the marked decrease in the expression of peanut agglutinin (PNA) receptors during T-cell maturation in the mouse is accompanied by increased activity of sialyltransferase. By differential agglutination with PNA, mature thymocytes (PNA-) were separated from immature ones (PNA+) and the separated fractions were tested for their sialyltransferase activity with asialofetuin as acceptor. In parallel, sialyltransferase activities of hydrocortisone-resistant thymocytes and untreated thymocytes were also compared. Optimization of the enzyme assay revealed that previous results in the literature were obtained under suboptimal conditions. Using manganese chloride instead of magnesium chloride, we have now found that hydrocortisone-resistant thymocytes contain 3.3-fold more sialyltransferase activity compared to untreated thymocytes. PNA- thymocytes contain 8.1-fold more enzyme activity compared to the PNA+ cells. Studies with fluorescein conjugated PNA of the agglutinated and unagglutinated thymocyte fractions suggest that the trace amount of sialyltransferase activity found in the PNA+ fraction may result from 5 to 10% cross-contamination with PNA- cells. These results suggest that the cellular levels of sialyltransferase specific for asialofetuin may play an important role in T-cell differentiation.     

Booster irradiation to the spleen following total body irradiation. A new immunosuppressive approach for allogeneic bone marrow transplantation

Graft rejection presents a major obstacle for transplantation of T cell-depleted bone marrow in HLA-mismatched patients. In a primate model, after conditioning exactly as for leukemia patients, it was shown that over 99% of the residual host clonable T cells are concentrated in the spleen on day 5 after completion of cytoreduction. We have now corroborated these findings in a mouse model. After 9-Gy total body irradiation (TBI), the total number of Thy-1.2+ cells in the spleen reaches a peak between days 3 and 4 after TBI. The T cell population is composed of both L3T4 (helper) and Lyt-2 (suppressor) T cells, the former being the major subpopulation. Specific booster irradiation to the spleen (5 Gy twice) on days 2 and 4 after TBI greatly enhances production of donor-type chimera after transplantation of T cell-depleted allogeneic bone marrow. Similar enhancement can be achieved by splenectomy on day 3 or 4 after TBI but not if splenectomy is performed 1 day before TBI or 1 day after TBI, strengthening the hypothesis that, after lethal TBI in mice, the remaining host T cells migrate from the periphery to the spleen. These results suggest that a delayed booster irradiation to the spleen may be beneficial as an additional immunosuppressive agent in the conditioning of leukemia patients, in order to reduce the incidence of bone marrow allograft rejection.     

Factor VIII and factor IX in a twin population. Evidence for a major effect of ABO locus on factor VIII level.

In order to establish the relative importance of genetic factors on the variation in plasma concentration of coagulation factors VIII and IX, these parameters were determined in 74 monozygotic and 84 like-sexed dizygotic twin pairs. The twins belonged to two age groups: 33-39 years and 57-62 years. Factor VIII was determined as factor VIII coagulant antigen (VIIICAg) and as factor VIII-related antigen (VIIIRAg). Factor IX was determined as factor IX antigen (IXAg). A higher value for each coagulation factor was found in the older-age group compared to the younger group, whereas no difference was found between the sexes. A significant correlation was found between values for VIIIRAg and VIIICAg (r = .56). For VIIICAg, it could be demonstrated that the age effect was secondary to the age effect on VIIIRAg. The concentration of VIIICAg and VIIIRAg varied among ABO blood types, being lowest in type O individuals, higher in A2 individuals, and highest in A1 and B individuals. The effect of the ABO locus on VIIICAg was secondary to an effect on VIIIRAg. Analysis of variance revealed a significant genetic influence on the variance of VIIICAg and VIIIRAg with a heritability estimate of .57 for VIIICAg and .66 for VIIIRAg. This is in agreement with a previous hypothesis of an effect of several autosomal genes on factor VIII concentration. Thirty percent of the genetic variance of VIIIRAg was due to the effect of ABO blood type. The ABO locus is therefore a major locus for the determination of factor VIII concentration. No significant genetic effect on the variation in plasma concentration of IXAg could be detected.     

The phenotypic range of hemophilia A carriers.

We have described the study of a small kindred with X-linked hemophilia A. It was ascertained through a clinically affected female, the daughter of a man with moderately severe hemophilia. The pedigree and the proband's phenotype suggest that she may be a heterozygote in whom most of the normal alleles at the VIII-1 locus are not active. She has two sisters, also obligatory carriers. The three sisters exhibit the three phenotypes possible for heterozygous females: clinically affected, clinically normal but phenotypically abnormal as determined by laboratory tests, and clinically and phenotypically normal.     

Apparent relatedness of the main component of ovine 1.714 satellite DNA to bovine 1.715 satellite DNA

The nucleotide sequence of the principal component of ovine 1.714 g/cm3 satellite DNA was determined from a monomeric fragment inserted at the BamHI site of pBR322 and cloned in Escherichia coli strain RR1. The 816-bp tandemly repeated sequence contains a number of small repeated sequences dispersed within it, one group of which forms a pentameric tandem repeat of a 13-bp segment (positions 548-612). A 20-bp region (60-79) shows an 85% homology with the reverse-complement of the sequence from 455 through 474. There are two regions of 67 bp (75-141) and 59 bp (755-813) which show greater than 70% homology with regions of bovine 1.715 g/cm3 satellite DNA (1402 bp; positions 1218-1284 and 1079-1137, respectively) while a 31-bp region (ovine 62-92, bovine 133-163) shows 80% homology. Quasi-correlation coefficients (Qr) were determined using the triplet numbers of the sheep satellite versus all sequences in the National Biomedical Research Foundation and EMBL nucleotide sequence data bases. Qr equals 0.85 for ovine 1.714 g/cm3 satellite versus bovine 1.715 g/cm3 satellite. The next highest Qr for a bovine satellite segment was 0.58. Thus, the ovine 1.714 g/cm3 and bovine 1.715 g/cm3 satellite appear demonstrably related. Taking into account that sheep and cattle diverged 18-20 million years ago, this suggests that the material may be functional and that its function is related to its sequence.     

Inhibition or acceleration of tumor growth by subpopulations of thymus cells separable by a peanut lectin.

The effect of different lymphocytes subpopulations on tumor growth in mice was investigated using an in vivo adoptive neutralization test (Winn test). Thymocytes from non-tumor-bearing mice accelerated the growth of the tumors tested [Lewis lung carcinoma (3LL), thymoma 40-127-299 and fibrosarcoma P-14] when injected into syngeneic or F₁ mice in a mixture with the tumor cells. The thymocytes were separated with the aid of peanut agglutinin into immunologically mature and immature subpopulations (Reisner Y., Linker-Israeli and Sharon N., Cell. Immunol. 25, 129, 1976). The immature thymocytes accelerated tumor growth to an extent similar to that of the unfractionated cells, whereas the mature subpopulation exhibited pronounced inhibitory activity. Our findings demonstrate that the murine thymus contains two thymocyte subpopulations with opposite activities on tumor growth and that the mature thymocytes have an inhibitory effect on tumor growth similar to that of spleen cells.