The effect of epithelial remnant and whole organ grafts of thymus on the recovery of thymectomized irradiated mice.

Work has been presented which suggests that thymus epithelial reticular cells are not effective in restoring the microscopic morphology of lymphoid tissues and their immunologic capacities. They function in recruiting precursors of thymus lymphocytes from the host animals to produce an organ which, after it becomes architecturally normal, can reconstitute the defective host. Intact thymus grafts in situ from 10-14 days, but not for shorter periods of time, have been shown to result in a return toward normal of these two parameters. Evidence is offered to show that few dividing cellular components in the lymphoid tissue originate from the thymus remnant grafts, and that a minor cellular component is contributed by the intact grafts. These data support the concept that the structural and functional development of the lymphatic tissue in thymectomized animals is dependent on thymus lymphoid cells and/or their products, and that the epithelial-reticular cells do not have a direct action in peripheral lymphoid reconstitution.

Effects of short-term epithelial reticular cell and whole organ thymus grafts in neonatally thymectomized mice.

Neonatally thymectomized mice were implanted with thymus grafts composed of epithelial reticular cells for periods of 7 and 14 days. Regardless of whether the grafts were placed immediately after thymectomy, or at 3 wk of age, there was little recovery of the lymphocyte depletion and impaired immunologic responsiveness, characteristically found in a neonatally thymectomized host. The findings were similar in animals studied at 2 months or 2 wk after graft removal. Many of the short-term remnant grafts were populated with lymphocytes and had attained the morphologic appearance of thymus by 14 days. A lesser degree of lymphocyte depletion and impaired responsiveness to SRBC occurred if thymectomy was delayed until 7 days of age, if remnant grafts were removed after 2 months, and if intact neonatal thymus was used for the short-term grafts. Complete normality was found in some of the animals in all of these groups. These observations suggest a direct role for mature thymus lymphocytes in reconstituting the neonatally thymectomized host and indicate that epithelial cell function is to direct the maturation of cells that ultimately behave as thymus lymphocytes.

Red sea: detailed survey of hot-brine areas.

A bathymetric and geophysical survey of the Red Sea rift valley between 21 degrees 10' and 21 degrees 30'N has defined three separate pools of hot brines. The brines and their associated heavy metals are believed to be periodically discharged from the eastern side of the largest deep, Atlantis II. Cores taken from the flanks of the deeps show repetitive cycles of sedimentation of hydrous amorphous iron oxides which fill most of Atlantis II Deep.

Gangliosides shed by tumor cells enhance tumor formation in mice.

The role of tumor cell membrane gangliosides in tumor formation was probed using a series of cloned murine AKR lymphoma cell lines. Tumor formation was directly related to high expression and shedding of membrane gangliosides. In vivo, as little as 1 pmol of purified total gangliosides of highly tumorigenic cells, injected intradermally with poorly tumorigenic cells (which lacked and did not shed gangliosides), markedly increased the tumorigenicity of these cells in syngeneic normal mice. Thus, gangliosides shed by tumor cells are a previously unrecognized, extremely potent enhancer of tumor formation in vivo.

Spermatogenesis associated 22 is required for DNA repair and synapsis of homologous chromosomes in mouse germ cells.

Analysis of the N-ethyl-N-nitrosourea (ENU)-induced repro42 mutation previously identified spermatogenesis associated 22 (Spata22) as a gene required for meiotic progression and fertility in both male and female mice, but its specific contribution to the process was unclear. Here, we report on a novel, null allele of Spata22 (Spata22Gt ) and confirm its requirement for germ cell development. Similar to repro42 mutant mice, histological and mating analyses indicate that gametogenesis is profoundly affected in Spata22Gt/Gt males and females, resulting in infertility. Cytological examination confirms that germ cells do not progress beyond zygonema and meiotic arrest is linked to impairment of both synapsis and DNA repair. Analysis of SPATA22 distribution reveals that it localizes to foci associated with meiotic chromosomes during prophase I and that the number of foci peaks at zygonema; there are also more SPATA22 foci in oocytes than in spermatocytes. Furthermore, SPATA22 co-localizes with a number of proteins involved in meiotic recombination, including RAD51, DMC1, and MLH1, and is present until mid-pachynema, suggesting a role in resolution of recombination intermediates. In fact, SPATA22 co-localizes with MLH1 in more than 20% of foci at pachynema. Analysis of Spata22Gt/Gt meiocytes confirms that SPATA22 is required for localization of MEIOB but not RPA (two proteins known to interact with SPATA22), and immunoblotting corroborates that production of MEIOB is indeed decreased in the absence of SPATA22. Together, these data suggest that SPATA22 is required for both meiotic recombination and synapsis during meiosis in mice.

Therapy of transplanted lymphomas.

Allogeneic cells or cell products containing murine leukemia virus were effective in curtailing growth of a transplanted CBA lymphoma, which was originally induced by Gross virus and then maintained by syngeneic cell passage. All allogeneic cells or products effective in therapy of this lymphoma expressed virus as tested by the XC cell in vitro infectivity assay. Some of the materials also expressed virus as assayed by oncogenicity in vivo. A therapeutic effect was not demonstrated with allogeneic cells or cell products laking these virus activities. Syngeneic lymphoma cells were ineffective. Protected animals did not develop lymphoma when rechallenged several months later. Mechanisms for this therapy with virus were proposed.

Development of virus-accelerated thymic lymphoma in AKR mice.

The progression of prelymphoma cells (PLC) in the bone marrow to lymphoma cells (LC) in the thymus in the presence of the lymphomagenic retrovirus SL3-3c was studied in a model system of virus-accelerated thymic lymphoma in AKR/J mice. On the average, a single LC appears in the thymus 30 days after the neonatal ip inoculation of SL3-3c virus; 50 days later, thymic lymphoma is clinically detectable. PLC in the bone marrow and oncogenic virus in the thymus are continuously present during this period before lymphoma develops. Biologically active oncogenic virus in the thymus increases as the animal nears the time of lymphoma development. Intrathymic inoculation, but not ip inoculation, of SL3-3c virus results in accelerated thymic lymphoma in 4- to 6-week-old AKR mice. PLC defined as a population of bone marrow-derived thymocyte progenitor cells susceptible to malignant transformation by oncogenic retrovirus after homing to thymus were further studied and characterized. PLC, like normal bone marrow thymocyte progenitors, were found to be radiosensitive and glucocorticoid resistant. Thymocytes of 21- to 28-day-old AKR mice, neonatally inoculated with SL3-3c virus, were studied for PLC. They could not be detected. It is concluded that lymphoma development is the final outcome of a series of events in which bone marrow-derived thymocyte progenitors are transformed after entering the thymus by virus in the thymic environment.

Virus expression and immunoprophylaxis of a murine lymphoma.

CBA mice tested with a rapidly lethal transplanted lymphoma could survive challenge when pretreated with allogeneic lymphoma cells or other material expressing murine leukemia virus (MuLV). Protection resulted when recipients were given injections of AKR and C3H transplanted lymphomas or selected normal AKR and SJL tissues, filtrates of which give positive assays for MuLV by in vitro and/or in vivo tests. There was no protection when recipients were given injections of C3H lymphomas or C3H normal tissues that failed to have positive assays for virus.

Spontaneous leukemia viruses: lymphomagenic ecotropic viruses of AKR mice.

The spontaneous leukemia (SL) viruses are ecotropic lymphomagenic viruses isolated from AKR spontaneous lymphomas. These viruses are produced stably by continuous cell lines from spontaneous lymphomas and by a cell line derived from the bone marrow stroma of an AKR mouse neonatally inoculated with an SL virus. All cell lines cloned from the parent lymphoma cell lines consistently produce SL viruses. These viruses can be passaged in vivo and maintain their leukemogenic properties. Cloned isolates of SL viruses accelerate lymphoma in AKR mice and induce thymic lymphoma in mice of other strains. Thus their lymphomagenic properties are conclusively shown. In a study with the use of a sensitive host range assay, xenotropic and/or dual-host range viruses are consistently found in spontaneous lymphoma and cell lines derived from them. However, viruses able to replicate in mink lung cells are not expressed in SL virus-induced lymphomas or their derived cell lines.

Autocrine growth of murine lymphoma cells.

By using an assay system in which small numbers of murine T lymphoma cells are stimulated to grow in serum-free medium, we have continued and expanded our previous studies of an autocrine growth factor that we call leukemia-derived growth factor (LDGF). We show that a T lymphoma cell line of immature phenotype, adapted to growth in serum-free medium, produces and responds to LDGF. LDGF activity is distinct from activities of 10 highly purified or recombinant hematopoietic growth factors including IL-1 and IL-2. However, growth-stimulating activity for the murine lymphoma cells is provided by a partially purified human LDGF.

SL12 murine T-lymphoma: a new model for tumor cell heterogeneity.

It has been observed that subclones from the spontaneous murine AKR/J T-lymphoma cell line SL12 with similar in vitro growth characteristics exhibit stable differences in tumorigenicity. The cell line is composed of at least three distinct cloned cell types that are highly, moderately, or poorly tumorigenic in syngeneic host animals. When healthy, young, syngeneic host animals were given iv injections with the same number of viable growth phase cells, each cloned cell type had a different tumor incidence, latent period, and pattern of tumor spread. The unusual stability of the cloned cell lines is shown by a similar incidence, latency, and spread of the tumors when studied after more than 1 year of continuous in vitro culture. The SL12 clones also differ in several phenotypic characteristics commonly used to classify thymocyte maturation, e.g., a) the expression of three of seven surface antigens examined, b) the cellular response to glucocorticoid hormone, and c) the expression of terminal deoxynucleotidyl transferase.

Virion interaction in mouse lymphomas.

Virion expression in filtrates of lymphomas in AKR/J or C3H/HeJ mice was assayed by two techniques: (a) in vitro infectivity (XC assay), or (b) acceleration of oncogenicity in vivo (O assay). The two assays appeared to detect different virus populations or activities. This was shown when the same filtrates were tested in parallel by the XC and O tests and opposite results were obtained on the two assays, e.g., XC+ 0- or XC- O+. It was postulated that two viruses, termed "XC+" and "O" to correspond to the assays used to detect them, were involved in oncogenesis. When lymphomas originally virus induced were passaged by cells in 2- to 3-month-old syngeneic AKR or C3H mice, oncogenic activity of filtrates of these transplanted lymphomas decreased as cell passages increased. These filtrates from C3H lymphomas also had decreased XC activity as cell passages increased. Normal lymphoid tissue from 1- to 5-month-old AKR mice was XC+ O-, while from C3H mice it was XC- O-. Thus, the two strains modified activities lacking in their normal tissues. Filtrates highly oncogenic in XC+ newborn AKR mice were oncogenic in C3H newborn mice only when sufficient XC+ virus was in the inoculum received by the XC- C3H mice. Thus the XC+ virus appeared to play a synergistic role with a postulated O virus in oncogenesis.

A discrepancy in XC and oncogenicity assays for murine leukemia virus in AKR mice.

Studies of murine leukemia virus expression in AKR mice are presented. Material from in vivo and in vitro sources of normal tissues and lymphomas was assayed for in vitro infectivity, using the XC plaque assay, and for oncogenicity, by assessing lymphoma-accelerating capacity after inoculation into newborn animals. Normal tissues from healthy young AKR mice up to 7 months of age were found to have XC but not oncogenic activity. XC activity persisted, and weak oncogenic activity appeared in older mice. Cocultivation of normal young cells with NIH Swiss mouse embryo cells did not result in the appearance of oncogenic activity, although XC virus increased in titer. A cell-free filtrate of a virus-accelerated lymphoma was studied for host range. Virus as measured by polymerase and gs antigen was found to be propagated on NIH Swiss mouse embryo and wild mouse embryo cells, but not on human rhabdomyosarcoma, normal rat kidney, rabbit corneal, and BALB/c embryo cells. Virus as measured by the XC assay grew better on NIH Swiss mouse than on BALB/c embryo cells. Both of these cell lines propagated virus as measured by the oncogenicity assay. Supernatants from an in vitro cell line from a virus-accelerated lymphoma did not produce XC plaques but were oncogenic. Those from two cell lines of spontaneous lymphomas were negative with both assays. Cultivation of supernatants from these cultured lymphoma cells with NIH Swiss mouse embryo cells resulted in material which produced small plaques on the XC assay. These findings are interpreted as showing the presence of two viruses in AKR mice. One is XC positive and present throughout life. The other is oncogenic, appears later in life, and could be a separate virus or a variant of the first one.

Transferrin-like activity produced by murine malignant T-lymphoma cell lines.

Transferrin has been considered to be an essential requirement for hematopoietic cell proliferation in culture. We have isolated two cloned lymphoma cell lines, SL 12.1 and SL 12.4, which grow and adapt in serum-free medium without added transferrin. Antibody to the transferrin receptor blocks the growth of these cells. We have also demonstrated that transferrin-free conditioned medium from the cells will compete with transferrin for binding. Furthermore, conditioned medium from SL 12.1 and SL 12.4 cells induces and supports exponential growth of a transferrin-dependent lymphoma cell line, SL 12. We conclude that these two transferrin-independent cloned lines produce transferrin-like activity which plays a crucial role for cell proliferation.

Presence of prelymphoma cells in the bone marrow of the lymphomagenic virus-treated AKR mouse.

Prelymphoma cells (PLC) are defined as cells which give rise to lymphoma cells but are not in themselves autonomous. They are present in the bone marrow of young AKR mice, a strain with a high natural incidence of thymic lymphoma. PLC are identified by transfer of AKR bone marrow into 400-rad-treated F1 recipients, one of the parents being AKR. Normal 1-month-old AKR bone marrow cells result in thymic lymphomas of AKR type in the hybrid recipients after latent periods of 6 to 16 months. In the present study, PLC resulting in lymphoma of AKR type 3 to 4 months after inoculation to irradiated hybrids are described. They are consistently found in the bone marrow of 21- to 28-day-old AKR mice treated at 3 to 5 days of age with a lymphomagenic virus, SL3-3. Long-term culture of these bone marrow cells allows the survival of totipotent hemopoietic stem cells but not of PLC. Thymic stromal remnants prepared from the 21- to 28-day-old virus-treated mice efficiently replicate oncogenic virus; however, they do not contain PLC. This was determined by grafting the remnants to irradiated and nonirradiated hybrid recipients. No AKR type lymphomas developed in the grafted mice. We conclude that the bone marrow of young oncogenic virus-treated AKR mice contains PLC modified by oncogenic virus so that they can produce thymic lymphomas after a shorter latent period than can PLC found in 1-month-old normal AKR mice. The modified PLC are not derived from totipotent marrow stem cells or sequestered in the thymic stroma of virus-treated mice.

Mechanisms of thymic lymphomagenesis by the retrovirus SL3-3.

These studies report changes occurring in the thymus of AKR and NFS/N mice after infection with the lymphomagenic retrovirus SL3-3. In virus-infected AKR fetal thymus, the programmed cell death caused by treatment with antibody to CD3 was remarkably diminished. A method of establishing thymic stromal cultures from mice of 1 to 3 wk of age is described. Using this method, it was found that SL3-3 virus infection by neonatal inoculation allowed establishment of thymic stromal cultures from organs removed from AKR mice of 30 to 50 days of age and from lymphomas, whereas thymic stromal cultures could not be established from control mice after 30 days of age. Using NFS/N mice which have no endogenous virus, it was shown that infection of thymic stroma precedes infection of thymocytes and that thymocytes are permissive for infection with SL3-3 virus but not for the nononcogenic retrovirus, Akv, yet Akv virus replicates efficiently in thymic stroma. SL3-3 virus integrates randomly in each lymphoma induced by this virus. The lymphomas are clonal or oligoclonal. Pim-1 and c-myc genes commonly rearranged in other virus-induced thymic lymphoma showed rearrangement in only a few lymphomas. A theory is proposed, based on the work presented here and in recent studies, which states that SL3-3 virus infection of thymic stroma allows infection of thymocyte progenitors entering from the bone marrow. These cells are then altered so that their maturation is delayed and their intrathymic survival is prolonged. This permits virus integration and reintegration that results in the genetic changes which transform the cell.

A new murine model system for the in vitro development of thymoma cell heterogeneity.

We have established and characterized a continuous T-cell line derived from the bone marrow of an AKR mouse with disseminated lymphoma. The original tumor cell line is heterogeneous with respect to several markers of thymocyte differentiation. Clones from the line differ in the expression of ThB, Pgp-1, and H-2Kk surface antigens. These clones also differ in their sensitivity to glucocorticoid-induced cell lysis. The quantity, affinity, and nuclear translocation properties of the glucocorticoid receptor are similar in the hormone-sensitive and -resistant clones. Furthermore, dexamethasone-resistant T-cells can be selected in vitro from freshly cloned cells sensitive to hormone-induced lysis at high frequency and without mutagenesis. Of several randomly sampled, spontaneously arising, independently derived dexamethasone resistant clones, all show a coordinate reduction in cell surface Thy-1 and ThB expression with no detectable changes in glucocorticoid receptor properties. Following treatment with the DNA-demethylating agent 5-azacytidine, the original dexamethasone-resistant T-cell line as well as the dexamethasone-resistant derivatives obtained in vitro regain sensitivity to lysis. These results collectively suggest a role of DNA methylation in hormone resistance and are consistent with a model of thymocyte differentiation in which a glucocorticoid-sensitive cell is the progenitor of hormone-resistant T-cells.

Genetic complexity of glucocorticoid-induced lysis of murine T-lymphoma cells.

Several well characterized murine T-lymphoma cell lines were used in somatic cell hybridization experiments to study the genetic regulation of glucocorticoid-induced lysis. Cell fusions were carried out among the SL12-derived cloned lines and between the W7 and SAK8 lines all of which have functional hormone receptors. These cell lines differ in their sensitivity to glucocorticoid-induced lysis. The resultant hybrids were characterized by their growth response to 1 microM dexamethasone, their hormone receptor content, their chromosome number, and the expression of surface antigens. Fusion of the hormone-sensitive W7 parent to a number of glucocorticoid-resistant cell lines resulted in hybrids which were of the sensitive phenotype. In contrast the fusion of another hormone-sensitive clone, SL12.4, with glucocorticoid-resistant SL12 clones or with SAK8 always resulted in hybrids resistant to glucocorticoid lysis. These results reveal a complex genetic regulation of the hormone response or the requirement for multiple gene activity in the mechanism for glucocorticoid-induced cell lysis.

Development of lymphoma in the thymus of AKR mice treated with the lymphomagenic virus SL 3-3.

A chronological study of the individual thymic lobes of young AKR mice after neonatal inoculation of the oncogenic AKR retrovirus SL 3-3 was performed. 100% of mice treated in this manner develop lymphoma between 60 and 100 days of age. A search for early lymphoma cells in individual thymi was carried out by inoculating the thymocytes subcutaneously in syngeneic and intrathymically in syngeneic and semisyngeneic recipients. Tumor progression was observed in animals between 48 and 60 days of age. These animals have: (a) normal weight lobes, in which no lymphoma cells could be detected, (b) thymus-dependent lymphoma cells, in one or both normal weight lobes; (c) thymus-independent lymphoma cells, found in lobes of normal weight as well as in thymi enlarged by lymphoma cells. Thymocyte characteristics of virus-treated animals of 21 to 63 days of age were compared with those of age-matched controls. Beginning at 28 days a concordant, progressive with time, increase of thymocyte surface staining for the viral envelope glycoprotein gp70 was seen in all lobes from virus-treated animals. Evaluation of cell surface markers by two-color fluorescence with antibodies to CD4 and CD8 showed that after 50 days of age, thymic lobes with and without lymphomas had nonspecific, but marked, alterations of the typical thymocyte surface marker pattern. No characteristic CD4, CD8 surface phenotype was found in primary lymphomas. Using probes for the T-cell receptor J beta 2 gene segments and the Akv ecotropic virus gp70 envelope genes, oligoclonality in J beta 2 rearrangements and clonality using the Akv env genes was demonstrated in thymi with the thymus-dependent phenotype. In lymphomas T-cell receptor beta gene probes showed either oligoclonality or clonality. Clonal virus integrations were found in these lymphomas. These experiments suggest the following series of events in virus-accelerated AKR lymphomagenesis. First, lymphoma cells arise which are initially thymus-dependent and can appear in one or simultaneously in both thymic lobes. These progress to become thymus-independent, fully autonomous, tumor cells. Thymocytes close to or at the time of the initial transformation event show a marked disorder of differentiation defined by the alterations in the CD4, CD8 surface phenotype distribution.

In vivo antitumor activity of the bitter melon (Momordica charantia).

The in vivo antitumor activity of a crude extract from the bitter melon (Momordica charantia) was determined. The extract inhibited tumor formation in CBA/H mice which had been given i.p. injections of 1.0 X 10(5) CBA/Dl tumor cells (77% of the untreated mice with tumors versus 33% of the treated mice with tumors after 6 weeks). The extract also inhibited tumor formation in DBA/2 mice which had been given i.p. injections of either 1 X 10(5) P388 tumor cells (0% of untreated mice survived after 30 days versus 40% survival of the treated mice) or 1 X 10(5) L1210 tumor cells (0% survival of untreated mice versus 100% of treated mice after 30 days). The in vivo antitumor effect required both the prior exposure of tumor cells to the extract (2 hr) in vitro and i.p., biweekly injections of the extract into the mice. The optimum dose for tumor inhibition (8 micrograms protein, biweekly, i.p.) was not toxic to mice for at least 45 days of treatment. This same treatment caused a marked enhancement of C3H mouse thymic cell response to concanavalin A in vitro. When compared to the untreated control mice, the bitter melon-injected animals exhibited a 4-fold-higher incorporation of tritiated thymidine into trichloroacetic acid-precipitable material after 48 hr of exposure to 50 micrograms of concanavalin A. Nylon wool-purified spleen cells from these same bitter melon-treated mice exhibited an enhanced mixed lymphocyte reaction when exposed to irradiated P388 stimulator cells (186% of the untreated control mice). These data indicate that in vivo enhancement of immune functions may contribute to the antitumor effects of the bitter melon extract.