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.

Fermi LARGE AREA TELESCOPE OBSERVATIONS OF BLAZAR 3C 279 OCCULTATIONS BY THE SUN.

Observations of occultations of bright γ-ray sources by the Sun may reveal predicted pair halos around blazars and/or new physics, such as, e.g., hypothetical light dark matter particles-axions. We use Fermi Gamma-Ray Space Telescope (Fermi) data to analyze four occultations of blazar 3C 279 by the Sun on October 8 each year from 2008 to 2011. A combined analysis of the observations of these occultations allows a point-like source at the position of 3C 279 to be detected with significance of ≈3σ, but does not reveal any significant excess over the flux expected from the quiescent Sun. The likelihood ratio test rules out complete transparency of the Sun to the blazar γ-ray emission at a 3σ confidence level.

RelA is required for IL-1beta stimulation of Matrix Metalloproteinase-1 expression in chondrocytes.

OBJECTIVE: Interleukin-1beta (IL-1beta) stimulates collagenase-1 (Matrix Metalloproteinase-1 (MMP-1)) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor-kappa B (NF-kappaB) pathway is potently activated in IL-1beta-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-kappaB family members during IL-1beta-induced MMP-1 gene expression have not been defined. RESULTS: To address the relationship between the NF-kappaB pathway and MMP-1 gene activation in chondrocytes, primary cultured human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1beta over a 24-h time course and MMP-1, NF-kappaB1, NF-kappaB2 and RelA gene expression was assayed. IL-1beta-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-kappaB gene expression, and inhibition of NF-kappaB nuclear translocation with dominant-negative IkappaBalpha reduced IL-1beta-dependent MMP-1 gene expression. IL-1beta activated the NF-kappaB pathway in chondrocytes, both through phosphorylation and transient degradation of IkappaBalpha, as well as through sustained phosphorylation of RelA. Small inhibitory RNAs (siRNA) specific for RelA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-kappaB1 and NF-kappaB2 augmented IL-1beta-induced MMP-1 expression. CONCLUSIONS: Our data demonstrate that IL-1beta activation of the NF-kappaB pathway is required for IL-1beta induction of MMP-1 in chondrocytes and that RelA can work independently of NF-kappaB1 or NF-kappaB2 to activate this gene expression program.

Evidence for TeV gamma-ray emission from a region of the galactic plane.

Gamma-ray emission from a narrow band at the galactic equator has previously been detected up to 30 GeV. We report evidence for a TeV gamma-ray signal from a region of the galactic plane by Milagro, a large-field-of-view water Cherenkov detector for extensive air showers. An excess with a significance of 4.5 standard deviations has been observed from the region of galactic longitude l E (40 degrees, 100 degrees) and latitude /b/ < 5 degrees. Under the assumption of a simple power law spectrum, with no cutoff in the EGRET-Milagro energy range, the measured integral flux is phi gamma(>3.5 TeV) = (6.4 +/- 1.4 +/- 2.1) x 10(-11) cm(-2) s(-1) sr(-1). This flux is consistent with an extrapolation of the EGRET spectrum between 1 and 30 GeV in this galactic region.

The efficacy of cyanoacrylate-derived surgical adhesive for use in the repair of lacerations during competitive athletics.

Octylcyanoacrylate (Dermabond) is approved by the Food and Drug Administration for laceration closure. International studies have shown its utility in wound closure and have shown it to be as good or better than suture closure for speed, patient preference, and cosmesis, with no difference in the rate of dehiscence or infection. We sought to determine whether it retains its tensile strength, durability, and skin apposition when an athlete is allowed to reenter competition, where it is subject to recurrent stress, moisture, and trauma. The study was performed at two professional hockey sites. Wounds were anesthetized, irrigated, and debrided. The skin was closed with Dermabond. The athlete was returned immediately to competition. Wounds were examined at the end of competition and again at 7 days. A total of 32 lacerations on 28 players were studied. The mean size of laceration was 2.3 cm (range 0.8 cm to 4.5 cm). The majority (95%) of wounds were on the face. Of the 32 lacerations, 31 (97.6%) had good results at the conclusion of the game. Of these 31, all had good results at 7 days following repair. Dermabond retained its strength, durability, and skin apposition when the athlete was allowed to reenter competition following wound repair.

Thymic dendritic cells are primary targets for the oncogenic virus SL3-3.

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70(+) cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70(+) cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70(+) cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70(+) dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.

Differential tropism of HIV-1 isolates for distinct thymocyte subsets in vitro.

OBJECTIVE: Understanding the interaction between HIV and developing thymocytes is crucial in determining how HIV infection perturbs the immune system. We determined which thymocyte subsets can harbor and express HIV. DESIGN: HIV expression in mature and immature thymocytes obtained from surgical specimens from non-infected children was determined after in vitro infection with the syncytium-inducing, cytopathic NL4-3 and the non-syncytium-inducing, relatively noncytopathic JR-CSF isolates. METHODS: Intracellular staining for the HIV p24gag antigen was combined with cell surface phenotyping to determine thymocyte subsets expressing HIV. Infection was quantitated by polymerase chain reaction on sorted subsets. RESULTS: NL4-3 replicated faster and to higher titers and caused a more severe decrease of all CD4-bearing thymocytes than did JR-CSF. In addition, both immature CD1+ and mature CD1-thymocytes expressed NL4-3, whereas only mature CD1-cells expressed JR-CSF. The tropism of NL4-3 for these immature cells suggests a mechanism for a more profound impact on T-cell maturation than that seen with JR-CSF. We also found that thymocytes lacking cell surface CD4 (CD4-CD8- and CD4-CD8+ subsets) expressed virus with either isolate late in infection, when viral levels were high. The CD4-CD8- cells expressing HIV were mature CD3bright T-cell receptor (TCR) alpha/beta bright cells. CONCLUSIONS: These results show that NL4-3 can be expressed by thymocytes at immature and mature stages of differentiation and cause severe loss of CD4+ cells. Thus, tropism of a virus for immature cells can affect the capability of the thymus to produce new T lymphocytes leading to a greater impact on development and functions of the immune system. It is proposed that this in vitro model can be used to study pathogenic mechanisms in the thymus.

Organization of the V gene segments in mouse T-cell antigen receptor alpha/delta locus.

The mouse T-cell receptor (TCR) alpha/delta locus was mapped using 17 V alpha and 4 V beta subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The alpha/delta locus spans about 1 Mb. The distance between the 3'-most V gene segment (V delta 1) and the delta constant gene (C delta) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual V alpha gene segment subfamilies are dispersed throughout the locus. In contrast, the V delta gene segments V delta 1 to 5 are clustered at the 3' end of the V gene segment cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the alpha/delta locus and on organization features that might influence the expression of specific V gene segments in gamma delta cells.

Ryanodine and theophylline-induced depletion of energy stores in amphibian muscle.

The effects of high and low levels of ryanodine on theophylline-induced energy depletion were studied in isolated frog sartorius muscle. Whereas low concentrations of ryanodine (1-10 microM) did not change high energy phosphate contents (PE) after 60 min, high levels (100 microM) reduced resting energy contents by 60% after 60 min. Subcontracture levels of theophylline (2 mM), in the presence of high ryanodine, produced an 80% PE depletion, suggesting possible additive or synergistic effects of these two agents. In contrast to theophylline-induced depletion, neither the ryanodine-induced depletion nor the theophylline-plus-ryanodine-induced depletion of PE seemed sensitive to inhibition by 1 mM procaine. This suggests that there may be differences in the mechanisms whereby methylxanthines and ryanodine deplete energy stores and evoke contractures in amphibian skeletal muscle.

Effects of cytokines on HIV-1 production by thymocytes.

The thymus is essential for normal T cell development and is particularly active during fetal and postnatal life. Here we describe in vitro studies of HIV-infected thymocytes cultured with cytokines normally produced in the thymus. Virus expression was determined by measuring p24 antigen levels in the culture supernatants. Addition of IL-2+IL-4 and IL-4+IL-7 to the HIV-infected cultures of both fetal and postnatal thymocytes resulted in various levels of synergistic expression of p24 antigen. When differences in phenotype between HIV-infected and non-infected (sham-treated) cultures from the same specimen were evaluated, there was a decrease in the percentages and absolute numbers of CD4-bearing cells in HIV-infected thymocytes cultured with IL-2+IL-4. Studies were done to determine if synergy in HIV expression was mediated by activation, proliferation or induction or suppression of other cytokines. We found a higher percentage of activated CD4+CD8+/high cells in thymocytes cultured with IL-2+IL-4 and IL-4+IL-7 than in thymocytes cultured with IL-2+IL-7. Proliferation was higher in thymocytes cultured with cytokine combinations but did not correlate with those conditions showing synergy. IL-4 reduced IFN-gamma production by thymocytes cultured with IL-2 in both HIV-infected and non-infected thymocytes. In addition, exogenous IFN-gamma decreased p24 expression by HIV-infected thymocytes when cultured with IL-4 alone, with IL-2+IL-4 or IL-4+IL-7. These results suggest that suppression of IFN-gamma by IL-4 may combine with cell activation and proliferation to produce synergy of virus expression observed with IL-2+IL-4 and IL-4+IL-7.

Contributions to transcriptional activity and to viral leukemogenicity made by sequences within and downstream of the MCF13 murine leukemia virus enhancer.

We have identified nucleotide sequences that regulate transcription in both a cell-type-specific and general manner in the long terminal repeat of the MCF13 murine leukemia virus. Besides the enhancer element, we have observed that the region between the enhancer and promoter (DEN) has a profound effect on transcription in different cell types. This effect, however, was dependent on the copy number of enhancer repeats and was detectable in the presence of a single repeat. When two enhancer repeats were present, the effect of DEN on transcription was abrogated except in T cells. DEN also makes a significant contribution to the leukemogenic property of the MCF13 retrovirus. Its deletion from the MCF13 virus dramatically reduced the incidence of thymic lymphoma and increased the latency of disease in comparison with the wild-type virus. This effect was most marked when one rather than two enhancer repeats was present in the mutant viruses. We also observed that the removal of one repeat alone remarkably reduced leukemogenicity by the MCF13 virus. A newly identified protein-binding site (MLPal) located within DEN affects transcription only in T cells, and its deletion attenuates the ability of an MCF13 virus with a single enhancer repeat to induce thymic lymphoma. This observation suggests that the MLPal protein-binding site contributes to the effect of the DEN region on T-cell-specific transcription and viral leukemogenicity. This study identifies the importance of nonenhancer sequences in the long terminal repeat for the oncogenesis of the MCF13 retrovirus.

Observations on lymphomagenesis and lymphoma in AKR mice. A description of prelymphoma changes in the thymus and phenotypic diversity of lymphomas induced by SL3-3 virus.

These studies were designed to look for a correlation of intrathymic survival of virus-infected thymocytes with lymphomagenesis. Cells from the normal-appearing prelymphoma thymus of SL3-3 virus-treated AKR mice were studied. Also, phenotypic properties of the malignant cells from the virus-induced lymphomas are described. In this model system, 100% of mice inoculated with virus at three days of age develop thymic lymphoma between 60 and 90 days of age. The experiments show that cells with malignant potential do not appear in the thymus until 36 days after virus inoculation. These cells are initially thymus-dependent (TD) in that they produce lymphoma of donor-type in recipients after intrathymic inoculation with long latency. They do not produce lymphoma after subcutaneous inoculation in syngeneic hosts. At 39 days after virus inoculation, the first thymus independent (TI) lymphoma cells appear. These cells, like the cells isolated from thymi with overt tumors, produce lymphoma of donor-type after a short latency when inoculated by the intrathymic or subcutaneous route. Thymocytes from normal-appearing thymi of mice at 42 days after virus inoculation, which could be expected to include TD, TI or no lymphoma cells, were evaluated for their ability to survive in a recipient thymus for three weeks after intrathymic inoculation. They were compared to thymocytes from age-matched control mice. Thymi receiving the virus-infected thymocytes showed 15% to 80% donor cells at three weeks. The highest numbers of donor cells were from thymi which were shown to contain TI lymphoma cells. However, cells from thymi with TD and no lymphoma cells could also be detected in significant numbers at three weeks after intrathymic inoculation. Less than 2% of donor-type thymocytes could be found after inoculation of thymocytes from normal control AKR mice. These data provide evidence that virus infection of thymocytes, even before the appearance of cells with lymphomagenic potential, endows them with a capacity for prolonged intrathymic survival. This appears to be a necessary step for tumor progression in this model. A remarkable phenotypic diversity of the virus-induced lymphomas was shown. The effect of various growth environments, intrathymic, subcutaneous, and in vitro on lymphoma cell phenotypic expression revealed individual differences in each tumor and in each environment.

In vitro studies of HIV-1 expression in thymocytes from infants and children.

OBJECTIVE: To determine whether thymocytes from infants and young children can be infected with and their maturation capability altered by HIV-1, and to examine the effects of interleukin (IL)-2 and IL-4 on this process. DESIGN: Serum-free culture medium was used so that cytokine effects could be studied under defined conditions. The primary virus isolates HIV-1JR-CSF and HIV-1JR-FL were used because their effects should most closely resemble those of viruses which might be found in an infected child. METHODS: Thymocytes from infants and young children were infected with virus and cultured in serum-free medium with the cytokines IL-2 and IL-4 alone or in combination. HIV-1 expression was measured after 12 days by p24 levels in culture supernatants. Thymocyte maturation was determined by changes in surface phenotype, as measured by flow cytometry. RESULTS: HIV-1 expression by thymocytes, which increased with time of culture, occurred when thymocytes were cultured with each cytokine. p24 levels were slightly increased when cultured with IL-2, compared with IL-4. Virus expression was remarkably increased when the cytokines were combined in culture. This expression was synergistic rather than additive. The synergy, evident in mature, but not immature thymocytes, was demonstrated with both pharmacologic and physiologic concentrations of cytokines. T-cells from peripheral blood cultured under the same conditions demonstrated lower virus expression in the presence of cytokines and synergy was not shown. Cytokine-induced thymocyte maturation and thymocyte survival in vitro was unimpaired by infection with HIV-1. CONCLUSIONS: These findings indicate that the cytokines IL-2 and IL-4, which are normally present in the thymic environment, can synergize to promote HIV-1 expression by thymocytes infected in vitro. This occurs without impairing the maturation induced by these cytokines. Thus, the mature thymocyte may provide a continuous supply of virus-expressing T-cells to the peripheral blood and lymphoid tissues of the infected child.

Toxicology training in US and Canadian medical schools.

The objective was to determine the extent of toxicology training in US and Canadian Medical Schools. The authors took a phone survey of the medical schools in the United States and Canada. Questions asked included whether school had a required toxicology course, in what context toxicology was taught, whether basic poison management was taught, and whether a doctoral toxicologist was on staff. Quantitation of hours of toxicology instruction and toxicology-related questions was also sought. Of the 142 medical schools in the United States and Canada, 123 schools were contacted (85.4%); 107 of these schools were US schools while 16 were Canadian medical schools. One hundred two schools (82.8%) stated that toxicology was taught in pharmacology or pathology courses, while only six schools (4.9%) had separate formal toxicology courses. An average of 5.04 hours (+/- 4.6 hours) of toxicology was taught in US courses, while the Canadian average was 6.04 hours (+/- 5.2 hours). Basic poison management was taught in 75 of the schools (61%), while a toxicologist (holding either an MD or PhD degree) was on staff in 56 of the 110 schools responding to this question (51%). While no relationship existed between having a toxicologist on staff and whether poison management was taught in US schools, a significant relationship was noted in Canadian schools (P less than .05). The authors conclude that toxicology as a separate discipline (and poison management in particular) is not routinely taught in medical school.

A model system for T-lymphocyte differentiation: regulation of CD4 and CD8 gene expression in SL12.4 T-lymphoma cell clones.

The T-cell surface proteins CD4 (L3T4) and CD8 (Lyt2) are first expressed on thymocytes as they undergo maturation in the thymus. Two immature T-lymphoma cell clones SL12.4 and RS4.2 which constitutively express low or undetectable levels of CD4 and CD8 were used to investigate the activation of CD4 and CD8 gene expression. The protein synthesis inhibitors cycloheximide (CHX) and pactamycin rapidly and reversibly increased CD4 and CD8 mRNA in the cloned cell lines, suggesting that a labile inhibitor protein(s) may regulate the expression of these transcripts. Cell surface CD4 and CD8 proteins were transiently detectable following a pulse of CHX. Thymic epithelial cell lines also induced CD4 and CD8 mRNA and cell surface protein, as well as TCR-alpha mRNA when co-cultivated with SL12.4 T lymphoma cells. The increase in CD4 and CD8 was modest, but stable for at least 22 cell generations after the thymic epithelial inducer cells were removed. Epithelial cells of non-thymic origin did not cause induction of these T-cell differentiation markers in SL12.4 T-lymphoma cells. Since the induction elicited by thymic epithelial cells and protein synthesis inhibitors differed dramatically in kinetics and reversibility, it is likely that these inducers act, at least in part, via different mechanisms. This lymphoma model system may be useful for analysis of molecular events which occur in immature thymocytes undergoing differentiation.

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.

Progression to development of lymphoma in the thymus of AKR mice treated neonatally with SL 3-3 virus.

All AKR mice develop thymic lymphoma between 60 and 90 days of age after neonatal treatment with the oncogenic retrovirus SL 3-3. At 40-50 days of age, in the normal-sized thymus of virus-treated mice, cells appear that produce lymphoma when inoculated intrathymically but not when inoculated s.c. These cells are designated as thymus-dependent (TD) lymphoma cells. TD cells progress to cells that form tumors after both intrathymic and s.c. inoculation; these are designated as thymus-independent (TI) lymphoma cells. In this report, we show that the TD and TI cells can be distinguished as two distinct cell populations. Experiments show that the TD cells reside within the immature CD4- CD8- thymocyte population of the virus-treated mice. In addition, we also show that CD4- CD8- thymocytes from SL 3-3 virus-treated mice do not mature in fetal thymic stromal rudiments. Using three-color flow cytometry to trace maturation of CD4- CD8- thymocytes after intrathymic inoculation into irradiated syngeneic hosts, disregulated thymocyte maturation of this population from virus-treated mice is demonstrated. Thus, altered maturation of and the appearance of TD lymphoma cells in, the most immature population of thymocytes appears to be a first step in a multistep process of thymic lymphomagenesis caused by SL 3-3 virus.

Human thymocytes do not respond to interleukin-2 after removal of mature "bright" CD5 positive cells.

Interleukin-2 receptors (IL-2R) are expressed on minor populations of immature and mature human thymocytes. These studies were designed to determine if immature T cells could respond to the mitogen phytohemagglutinin (PHA-P) plus IL-2 in vitro by increasing the expression of IL-2R and by proliferation. Using monoclonal antibodies to CD5 and magnetic immunobeads we were able to remove all mature, "bright" CD5+ cells from nylon wool-purified thymocytes and to obtain less mature cells which consisted almost completely of cells with the CD4+CD8+ phenotype. These immature cells were mostly "dim" CD5+ and less than 5% CD5- and a small percentage expressed the IL-2R. After culture in serum-free medium with PHA-P, these cells showed only a slight increase in the percentage of IL-2R+ cells and the addition of IL-2 did not increase the percentage of IL-2R+ cells and no proliferation was observed. Unseparated, nylon wool-purified thymocytes contained 14% bright CD5+ cells. These bright CD5+ cells had a mature phenotype of CD4+CD8- (52%) and CD4-CD8+ (27%) cells. A small percentage of these cells were IL-2R+. These bright CD5+IL-2R+ cells were predominantly mature CD4+CD8- cells as measured by three-color flow cytometry. After culture with PHA-P and IL-2, the percentage of IL-2R+ cells increased and they were now found not only on CD4+CD8- but also on CD4-CD8+ and on CD4+CD8+ cells. IL-2 plus PHA-P increased proliferation of these cells as compared to those cultured in medium with PHA-P without IL-2. Thus, we show that human immature thymocytes in contrast to mature thymocytes are not responsive to IL-2 as measured by a lack of IL-2R expression and proliferation. These data indicate that mature thymocytes can express a functional high affinity receptor for IL-2 and suggest that immature thymocytes may not possess a (functional) p75 chain of the IL-2R.

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.

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.