Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat.

Type beta transforming growth factor (TGF-beta), a factor produced by many cell types, is a potent inhibitor of hepatocyte DNA synthesis in vitro. To determine whether TGF-beta can influence hepatocyte proliferation in vivo, its effects were examined on the regenerative response of liver to partial hepatectomy (PH) in the rat. Porcine platelet-derived TGF-beta 1 (0.5 micrograms), administered intravenously at the time of PH and 11 hr later, reduced the fraction of hepatocytes engaged in DNA synthesis 22 hr after PH by 67% and inhibited the rate of hepatic [3H]thymidine incorporation by 50%. TGF-beta 2 produced a similar effect. A single dose of 0.5 micrograms of TGF-beta 1 given 11 hr after PH reduced liver [3H]thymidine incorporation by 32%; 4.5 micrograms of TGF-beta 1 or TGF-beta 2 inhibited DNA synthesis by 88% and the labeling index by 86%. Although sensitive to TGF-beta administered 11 hr after PH, late in the G1 phase of the cell cycle, a single dose of 0.5 micrograms given at the time of PH did not significantly influence DNA synthesis 22 hr after PH. The inhibitory effects of TGF-beta were transient; rats treated with two 0.5-microgram doses of TGF-beta at 0 and 11 hr had completely restored their original liver DNA mass 8 days after PH. Administration of 0.5 microgram of either TGF-beta 1 or TGF-beta 2 every 12 hr for 5 days failed to suppress the recovery of hepatic DNA mass. However, the nuclear labeling index of the TGF-beta-treated animals was significantly higher than that of the controls. There was no evidence of cytotoxicity from TGF-beta, as determined by liver histology and plasma concentrations of glucose, insulin-like growth factor I, and two hepatic enzymes. Thus, TGF-beta 1 and TGF-beta 2 reversibly inhibit the proliferative response of liver to PH and may be important in the modulation of normal liver growth and repair.     

Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity.

In the tumor-bearing host, T cells invariably fail to induce a clinically significant antitumor immune response. Although model systems support the existence of tumor peptide antigens, the molecular interactions critical for antigen presentation by the tumor cell remain unresolved. Here, we demonstrate that human follicular lymphoma cells are highly inefficient at presenting alloantigen despite their strong expression of major histocompatibility complex and low-to-intermediate expression of some adhesion and B7 costimulatory molecules. Activation of follicular lymphoma cells via CD40 induces or up-regulates both adhesion and B7 costimulatory molecules essential to repair this defect. More importantly, once primed, alloreactive T cells efficiently recognize unstimulated follicular lymphoma cells. Thus, correction of defective tumor immunity requires not only expression of major histocompatibility complex but also sufficient expression of multiple adhesion and costimulatory molecules.     

Antibody response to a polysaccharide antigen present in the schistosome gut. II. Modulation of antibody response.

Specific IgM and IgG antibody to a polysaccharide present in the epithelial cells of the gut of adult schistosomes was measured in four groups of infected patients: I) patients with documented acute schistosomiasis; II) Americans exposed to schistosomiasis within the preceding 0--4 years; III) chronically and heavily infected patients, mostly from Puerto Rico, without hepatomegaly or hepatosplenomegaly; and IV) heavily infected Brazilian children with hepatic or hepatosplenic schistosomiasis. Specific IgM and IgG titers were both highest in the acute Group I patients and lowest in the chronically infected Groups III and IV. Total IgG and IgM levels were compared to specific antibody titers. Immunoglobulin levels tended to follow specific antibody titers except in the chronically infected Groups III and IV in which total IgG rose to high levels. The decrease in specific antigen titers over the course of time occurred despite continued antigenic stimulation and suggests a modulation of the humoral response. The mechanism remains obscure.     

Interleukin-3 and granulocyte-monocyte colony-stimulating factor receptors on human acute myelocytic leukemia cells and relationship to the proliferative response

Interleukin-3 (IL-3) and granulocyte-monocyte-colony-stimulating factor (GM-CSF) stimulate proliferation of human acute myeloid leukemia (AML) in vitro, although patterns of response among clinical cases are diverse. Whether regulatory abnormalities related to growth factor responses in human AML may establish the outgrowth of the neoplasm is unclear. We determined receptor numbers and affinity for IL-3 and GM- CSF on human AML cells using human recombinant IL-3 (rIL-3) and GM-CSF (rGM-CSF). In 13 of 15 cases of primary AML high-affinity (kd 26 to 414 pmol/L) receptors for IL-3 were demonstrable on the cells. The average numbers of IL-3 receptors ranged from 21 to 145 receptors per cell. Normal WBCs showed IL-3 receptors on their surface at similar densities. IL-3 receptor positivity often correlated with GM-CSF receptor positivity of AML; GM-CSF receptors were demonstrated on the cells of 11 of 15 cases, although average numbers of GM-CSF receptors were ten times greater. The in vitro response of the cells to exogenous IL-3 or GM-CSF was examined by measuring thymidine uptake. Because IL-3 and GM-CSF were potent inducers of DNA synthesis in vitro, apparently relatively few receptors are required to permit activation of growth. These experiments did not provide evidence for overexpression or increased receptor sensitivity as an explanation for AML growth. In a minority of cases, however, the cells were unable to respond to IL-3 (four of 15 cases) or GM-CSF (four of 15 cases) despite normal receptor availability on the cell surface.     

Role of redox signaling in the autonomous proliferative response of endothelial cells to hypoxia

Endothelial cells exhibit an autonomous proliferative response to hypoxia, independent of paracrine effectors. In cultured endothelial cells of porcine aorta, we analyzed the signaling of this response, with a focus on the roles of redox signaling and the MEK/ERK pathway. Transient hypoxia (1 hour) stimulated proliferation by 61+/-4% (n=16; P<0.05 versus control), quantified after 24 hours normoxic postincubation. Hypoxia induced an activation of ERK2 and of NAD(P)H oxidase and a burst of reactive oxygen species (ROS), determined by DCF fluorescence. To inhibit the MEK/ERK pathway, we used PD 98059 (PD, 20 micromol/L); to downregulate NAD(P)H oxidase, we applied p22phox antisense oligonucleotides; and to inhibit mitochondrial ROS generation, we used the ubiquinone derivate mitoQ (MQ, 10 micromol/L). All three inhibitions suppressed the proliferative response: PD inhibited NAD(P)H oxidase activation; p22phox antisense transfection did not inhibit ERK2 activation, but suppressed ROS production; and MQ inhibited ERK2 activation and ROS production. The autonomous proliferative response depends on the MEK/ERK pathway and redox signaling steps upstream and downstream of ERK. Located upstream is ROS generation by mitochondria, downstream is NAD(P)H oxidase.     

Reduction of syngeneic tumor growth by an anti-I-J-alloantiserum.

Highly significant suppression of the growth of S1509a and Sa-I syngeneic sarcomas was observed in A/J mice following daily intravenous injections of 2 microliter of anti-I-Jk alloantiserum. This effect persisted as long as the anti-I-Jk serum was administered (day 15), In contrast, a control anti-I-Js serum had no discernible effect on the growth of the S1509a tumor. The inhibitory activity of the anti-I-Jk serum on the growth of the tumor was absorbed specifically by B10-BR spleen cells bearing I-Jk determinants. In other experiments, we established that A/J mice treated with anti-I-Jk serum, according to the protocol described above, are no longer a source of tumor-specific suppressor cells for adoptive transfer into immune tumor-bearing recipient mice. We conclude that anti-I-Jk serum inhibits tumor growth in A/J mice by abolishing tumor-specific suppressor activity.     

Differentiation of population of peripheral blood lymphocytes into cells bearing sheep erythrocyte receptors in vitro by human thymic extract.

A small population of human marrow cells has been shown to be differentiated in vitro by thymic extract into cells bearing T-lymphocyte (thymus-derived lymphocyte) characteristics. By a similar method, the differentiation of human peripheral blood lymphocytes has been studied. A discontinuous gradient of bovine serum albumin was used to isolate lymphocytes into four layers and cells from layers I and III demonstrated the greatest potential for differentiation by human thymic extract. Appearance of T-lymphocyte characteristics was recognized by the spontaneous E-rosette technique with sheep erythrocytes. Ability of human marrow cells to be differentiated under the influence of human thymic extract was abolished by specific inhibitors of nucleic acid synthesis, however, had no inhibitory effect on the maturation of peripheral blood lymphocytes during a 2 hr incubation with human thymic extract but puromycin, an inhibitor of protein synthesis, abolished this differentiative step in cells of layer I. It is suggested from these studies that many of the cells in peripheral blood that are differentiable by thymic extract are at a stage of maturation more advanced than those in human marrow that are also differentiable by thymic extract.     

Effect of preincubation on the proliferative response to antigen by cells from leprosy patients and healthy controls

One of the postulated mechanisms contributing to the selective T-cell hyporesponsiveness in patients with leprosy is receptor blockade, the characteristic feature of which is reversibility following preincubation of cells in vitro. To test this hypothesis, peripheral blood mononuclear cells (PBMC) obtained from 17 leprosy patients and 10 healthy Mantoux-positive and -negative controls were cultured freshly or after a period of preincubation in serum-containing medium, and the proliferative responses to Mycobacterium leprae, BCG, and streptokinase-streptodornase (SKSD) were measured. On the basis of the response to M. leprae, the leprosy patients could be divided into low, intermediate and high responders. Preincubation for 2-16 hr resulted in enhanced proliferation by cells from moderate responders, but not from low or high responders. Although the effect was serum dependent, it was neither antigen specific nor was it confined to cells from leprosy patients. Thus, an increase in response to both the crossreactive and unrelated antigens BCG and SKSD occurred, and the same trend was observed when cells from healthy controls were preincubated in serum-containing medium. Furthermore, cells from different individuals displayed varying responses to different antigens following preincubation, suggesting that the effect of this step was neither confined to isolated individuals nor to particular antigens. The addition of pronase to the preincubation step did not further enhance the response to antigen over and above that obtained with preincubation alone. It was therefore concluded that the enhanced proliferative response to antigen following preincubation was an in vitro phenomenon dependent upon the culture conditions employed, was not specific to leprosy, and was not related to receptor blockade.     

Insulin receptors in mouse brain: Reversibility of age-related impairments by a thymic extract

Recently, we have shown that insulin receptors (InsRs) in the brain undergo impairments with aging. Interestingly, age-related alterations of brain InsRs, are not irreparable as thymus grafts are able to recover them. With the present study we verified the possibility that an aqueous extract from calf thymus (TME) can mimic the restoring action of age-related impairments induced by thymus graft. InsR characteristics were assayed in a group of 25 months old BALB/c-nu mice treated with TME: 2μg/g body weight every third day, for total five subcutaneous injections. The last dose was injected the day before animals were killed. Other two groups of young (4 months) and old (25 months) mice received saline solution with the same schedule. A two-sites model analysis of receptor data confirms the age-dependent decrease of InsR number and k_d previously observed in the high affinity population. Furthermore, a statistically significant recovery of number impairment is shown in TME-treated animals. On the contrary, the characteristics of the low affinity receptor subset show no statistically significant differences among the three animal models studied. TME induced recovery of the age-related changes found in brain InsRs, together with previously observed regulatory action of the same thymic extract on the adrenergic system, suggest that thymic gland does not necessarily have to mutually interact with other controlling systems for maintaining or recoving homeostasis of the complex neuroendocrine network during development and aging.     

Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.     

Special proliferative sites are not needed for seeding and proliferation of transfused bone marrow cells in normal syngeneic mice.

The widely held view that transfused bone marrow cells will not proliferate in normal mice, not exposed to irradiation or other forms of bone marrow ablation, was reinvestigated. Forty million bone marrow cells from male donors were given to female recipients on each of 5 consecutive days, 5 to 10 times the number customarily used in the past. When the recipients were examined 2-13 weeks after the last transfusion, donor cells were found to average 16-25% of total marrow cells. Similar percentages of donor cells were found when variants of the enzyme phosphoglycerate kinase determined electrophoretically were used for identification of donor and recipient cells. Evidence is presented that the proportion of donor cells is compatible with a linear dependence on the number of cells transfused over the range tested--i.e., 20-200 million bone marrow cells injected intravenously. Special proliferative sites thus do not appear to be required.     

Effect of graft-versus-host reaction on the immune response to alloantigens and growth of a syngeneic tumor.

The graft-versus-host reaction (GVHR) was evoked in (C57BL/6J times C3H/6J times DBA/2J) F1 hybrids by grafting parental lymphoid cells. Groups of bothe F1 hybrids were immunized with thymocytes carrying Thy-1.1 (theta AKR) antigen and still other groups of (C57BL/6J times C3H/HeJ) F1 hybrids were immunized with thymocytes carrying the H-2-d antigen. Plaque-forming cells (PFC) producing antibodies to the Thy-1.1 antigen, lytic for AKR thymocytes, or antibodies to the H-2-d antigen, lytic for lymphoblasts carrying the H-2-d antigens, were enumerated in spleens of experimental animals. A pronounced suppression of the immune response to the Thy-1.1 antigen was found in animals suffering from GVHR. The suppression could be demonstrated in some animals as late as 180 days after the onset of GVHR. Significant, although less pronounced suppression was found when response to the H-2-d antigens was assayed. After grafting 10-3 or 10-4 DBA/2J derived lymphoma cells (L5178Y) into (C57BL/6J times DBA/2J) F1 hybrids, clinically detectable tumor growth was found earlier and more frequently in hybrids suffering from GVHR than in control animals. The results obtained lend support to the concept that immunosuppression accompanying GVHR may influence the formation and/or growth of the malignant tumors. In addition, a synergistic effect in the suppression of the responsiveness of F1 recipients was seen in some instances when a mixture of parental bone marrow and thymus cells was grafted. The results are consistent with data indicating that more than one cell type is involved in the initiation of GVHR.