To determine if human vascular endothelium influences the growth of smooth muscle cells (SMC), we have investigated the effect of media conditioned by cultured human vascular endothelial cells on smooth muscle cell proliferation. Cell growth was measured by [3H]thymidine incorporation in endothelial and smooth muscle cells. The conditioned medium of confluent endothelial cells contains factors that inhibit the growth of actively dividing endothelial and smooth muscle cells. Media conditioned by proliferating endothelial cells or confluent smooth muscle cells were inactive. The generation of the inhibitory activity was time-dependent, not correlated with cell lysis and was demonstrated in both serum-free and serum-containing medium. Protein synthesis was involved in the generation of the inhibitory activity. Upon gel filtration, the inhibitory activity was associated with a substance having a molecular weight (MW) of >105. Treatment with trypsin or alpha chymotrypsin did not abolish the activity. Furthermore, the activity remained stable after incubation for 30 min at 56 °C, whereas only 50% of the activity remained present after heating for 2 min at 100 °C. It is speculated that this inhibitory activity plays an important role in the regulation of smooth muscle cell proliferation.

The expression of HLA-A and -B antigens on peripheral blood lymphocytes and blood platelets was measured using monoclonal antibodies in a semi-quantitative ELISA technique. Reactively of monoclonal anti-HLA-A2 and anti-HLA-B7, with lymphocytes as well as platelets, was in agreement with the presence of these antigens as detected by conventional HLA typing of lymphocytes. When the actual amount of HLA antigens was measured, a gene-dose effect was seen: cells from HLA-B7-homozygous individuals bound more monoclonal anti-HLA-B7 antibodies compared to their HLA-B7-heterozygous siblings. At the same time, cells of different donors showed only very small differences in binding of monoclonal antibody against an HLA-"backbone" determinant. Relative to total HLA-A, -B and -C expression, the amounts of HLA-A2 on lymphocytes and platelets were similar. On the other hand, the expression of HLA-B7 on platelets was diminished compared to that on peripheral blood lymphocytes.

Peripheral blood lymphocytes from antibody-producing Rh- donors were fused with mouse myeloma cells, and the hybrids were screened for anti-Rh antibody production. Although the resulting hybrids were not stable in long-term culture, they provided a useful test system for an investigation of ways to stimulate human lymphocytes in vitro before fusion in order to maximize the recovery of hybrids producing antibodies with a desired specificity. Although 6-day culture with PWM or with antigen (Rh+ erythrocytes) increased slightly the yield of desired hybrids, exposure of the PBL to antigen in the presence of human endothelial cells was dramatically more effective. This effect was not produced with endothelial cell culture supernatants (HECS) that had been shown previously to enhance hybridoma growth. Culture of PBL before fusion with both PWM and antigen also improved the proportion of active hybridomas, and incubation with antigen plus endothelial cells plus PWM resulted in a very high number of hybridomas, with a very high proportion producing antibody to the immunizing antigen. There are several alternatives to explain the mechanisms involved, but in any case, the technique should be particularly useful when the possibility of fusion of human PBL with a human myeloma line becomes more widely available.

The activity of a thymus-dependent factor (SF) was determined in the sera of 145 healthy controls and 111 patients with myasthenia gravis. Evidence for an age-related decline in the activity was obtained, both in normal donors and in the patients. SF activity appeared to be absent from most of the thymectomized patients: after thymectomy the SF activity decreased rapidly, reaching very low levels within a few days. No clear correlation was found between the activity of SF in the sera of the patients and their clinical stage of disease.

The influence of adult thymectomy on several parameters of immunocompetence in patients with myasthenia gravis (MG) was investigated. Since incomplete thymectomy may lead to the presence of thymic remnants, we determined the activity of a thymus-dependent factor in the sera of the MG patients. As measured by several parameters, MG patients showed a normal immunocompetence compared with healthy controls, except in the response to DNCB sensitization in vivo. When tested at least 5 years after thymectomy, MG patients were found to have decreased response to mitogens, and a decreased cytotoxic T cell response in cell-mediated lympholysis. The response to challenge with DNCB in vivo was decreased both in thymectomized and nonthymectomized MG patients. No difference was found in a) the percentage of circulating T, B, non-B/non-T cells; b) the response to allogeneic cells (MLR); c) the antibody-dependent lymphocytotoxicity; d) the production of immunoglobulins in vitro by pokeweed mitogen-stimulated cells; and e) the anamnestic response to antigens in vitro. We conclude that adult thymectomy results in a decrease in the function of some subpopulations of lymphocytes.

Mouse thymocytes were incubated in vitro in the presence of a thymus-dependent serum factor. This factor increases the level of cellular cyclic AMP but does not affect the number of proliferating cells. After incubation with the factor, C57BL/6J thymocytes were injected into new born (C57BL/6J × C3H)F 1 hybrids. The capability of thymocytes to elicit a graft-versus-host reaction in vivo was evaluated by the spleen weight assay. Thymocytes treated with the thymic factor were found to induce a more effective graft-versus-host reaction as compared with untreated thymocytes and with thymocytes treated with inactivated thymic factor. These results are consistent with the existence of a thymocyte maturation pathway which does not involve cell proliferation and is under the influence of thymic factors.

Human endothelial culture supernatant (HECS) had strong growth-promotion activity for hybridoma cells: the yield of hybridomas after fusion was increased at least 2-fold over that in the presence of feeder cells, such as mouse macrophages and spleen cells. Moreover, HECS could substitute for feeder cells when hybridomas were cultured at the single-cell level, and strongly enhanced the proliferation of hybrid cells. furthermore, the presence of human endothelial cells prolonged the stability of human-mouse hybridomas, producing human immunoglobulin, hybrids known to survive poorly during culture in vitro.

Rudimentary uterine horn pregnancy is a rare and serious type of ectopic pregnancy and is hard to diagnose due to a lack of typical clinical symptoms at the early stage. A 35-year-old woman who was17 weeks pregnant and had a complicated history of infertility came to our hospital complaining of abdominal pain without vaginal bleeding. Computed tomography scan after 12 hours showed that her pregnancy was in the small cavity of a rudimentary uterine horn, which had ruptured. Noncommunicating rudimentary uterine horn pregnancy is an extremely rare form of ectopic gestation, and its diagnosis and management remain challenging. Nevertheless, physician awareness of various forms of unicornuate uteri and rudimentary uterine horn can save lives.

By means of direct measurement of intracellular cyclic AMP in thymocytes in vitro we demonstrated the existence in human serum of a thymus-dependent factor (SF). This activity of SF appeared to be due to a very low M.W. (less than 500) peptide distinct from other circulating thymic peptides. SF was found to act selectively on immunologically immature, hydrocortisone (HC)-sensitive peanut lectin-agglutinating thymocytes. In a period of 240 min SF sequentially induces in thymocyte synthesis of cyclic AMP, protein synthesis and synthesis of phosphorylated non-histone chromatin proteins of high molecular weight. Because SF does not induce a change in DNA synthesis, it seems likely that the events induced by SF are associated with a process requiring DNA translation, but not DNA replication. The biochemical events induced by SF are accompanied by the acquisition of immunological maturation by thymocytes. This has been shown by the acquisition of HC resistance, by the expression of FC receptors for IgM, by the decrease of terminal transferase activity and by the induction of the capacity to elicit a graft-versus-host reaction. We conclude that, most likely, SF acts on immature thymocytes inducing a part of them to acquire some properties of mature T cells.

The activity of thymus-dependent serum factor (SF) was measured in 11 patients with subacute sclerosing panencephalitis (SSPE). A significantly decreased activity was found in 6 out of 11 patients, whereas the level of cyclic AMP in lymphocytes of the patients, a parameter for the maturation of peripheral blood lymphocytes, showed low values in 9 out of 11 patients. These findings were compared with the clinical stage of the disease and with some lymphocyte functions in vitro. No correlation was found between the SF levels and the amount of cyclic AMP in the lymphocytes or between these two parameters and the lymphocyte functions in vitro. It is concluded that a thymic humoral dysfunction is unlikely to play a role in the pathogenesis of SSPE; it appears that such a dysfunction is rather a secondary event in this disease.

Human serum contains a thymus-dependent factor that raises cyclic AMP levels in thymocytes. We found that this factor stimulates protein synthesis in thymocytes cultured in vitro. This activity of serum factor is thymus-dependent, because it is absent in sera from thymectomized donors; furthermore, this effect is predominantly found on precursors of mature T cells. Incubation of thymocytes with other agents that increase cyclic AMP, induces an increase in protein synthesis similar to that observed with serum factor. Most likely, the increase in protein synthesis is one of the events following stimulation of adenylate cyclase in thymocytes that leads to cell differentiation.

Supernatants from thymic epithelial cultures (TES), a putative analogue of thymic humoral function, are shown to induce strong cytolytic T lymphocyte (CTL) responses when added to primary mixed lymphocyte cultures (MLC) with thymocytes as responder cells. No enhancing effects of TES are observed with spleen cells or hydrocortisone-resistant thymocytes. These effects of TES on functional T cell parameters are accompanied by a reduction in part of the thymocytes in Thy 1 antigen density and sensitivity to hydrocortisone (HC). Inasmuch as immunocompetent T cells are found only among the HC-resistant thymocytes expressing a low Thy-1 antigen density, these findings suggest a TES-induced maturation process of thymocytes. In addition, we attempted to answer the question of whether the enhancing effects of TES on T cell-proliferative capacity and T cell killer function of thymocytes are due to an effect on previously unresponsive immature T cells or merely to extra stimulation of already responsive cells. Using the peanut agglutinin (PNA)-agglutination technique as a reversible probe, we could now demonstrate that TES affects the T cell-proliferative and CTL responses of the PNA-agglutinating thymocytes only. These findings support the concept that TES induces maturation of immature thymocytes, inasmuch as the PNA-agglutinating cells represent the immunoincompetent cells which are rich in Thy 1 and poor in H-2 antigens and HC sensitive. The PNA-nonagglutinating thymocytes exhibited high reactivity in all the functional assays investigated (like HC-resistant thymocytes), and this could not be further enhanced by TES. Together with the observation that supernatants from nonthymic epithelial cultures or from thymic lymphocyte cultures exhibit no effects on any of the abovementioned parameters, these findings suggest that TES represents a relevant in vitro model for studying thymic humoral function.

Previously, it has been shown that a human thymus-dependent serum factor (SF), isolated from peripheral blood and acting on precursors of mature T lymphocytes, induces an increase in the synthesis of cyclic AMP and proteins in thymocytes. We have now investigated the action of SF on the incorporation of 3H-leucine and 32P-orthophosphate into nuclear proteins of thymocytes after 15 to 240 min of culture. SF induced a rapid increase in the synthesis and phosphorylation of nuclear proteins, especially in the phosphorylated nonhistone chromatin proteins (P-NHCP). Electrophoretic patterns in polyacrylamide gels of the P-NHCP fractions, extracted from the chromatin of the stimulated cells, showed that proteins with m.w. higher than 50 x 10(3) were synthesized to a larger extent as compared with unstimulated cells. These data suggest that SF acts specifically on the synthesis of P-NHCP and may in this way control DNA-template activity.

Peripheral blood lymphoid cells of 29 patients with acute lymphocytic leukaemia (ALL) at onset were studied for characterization of B and T membrane markers and phytohaemagglutinin responsiveness. 24 cases (83%) were classified as "null" cell ALL and 5 (17%) as T-cell ALL. No relationship could be found between cytological presentation and immunological classification. Moreover, no correlation has been demonstrated between clinical-immunological parameters and prognosis, indicating that in our series of patients, assessment of cell size and surface markers were not a reliable predictor of prognosis.

Thymosin has no effect in vitro on cyclic AMP levels in thymocytes. However, when thymosin was injected into patients lacking "serum factor" (SF) activity, it induced the appearance of SF or SF-like activity and the disappearance of target cells for SF among the peripheral blood lymphocytes of the treated patients. On the other hand, when thymosin was injected into one patient with normal SF activity, it induced a marked decrease of SF activity and the appearance of target cells for SF among the peripheral blood lymphocytes of this particular patient.

Direct measurements of SF was performed by a cAMP assay using mouse thymocytes (1) in 15 patients with several forms of infantile immunodeficiency diseases. In addition we investigated, among patients' lymphocytes, the presence of “target cells” for SF, i.e. lymphocytes which could be stimulated by SF to increase intracellular cAMP, which is a property of precursors of mature T cells. Results were the following:The detection of SF activity and of “target cells” for SF provides an additional for diagnosis of primary immunodeficiencies and a way to evaluate thymic function also in view of treatment with tronsplantation(s) and/or thymic hormone(s).1) Astaldi, A. et al., Nature 260, 713-715 (1976).

THE action of factors which have been extracted from the thymus or found circulating in peripheral blood and which are active in in vitro lymphocyte differentiation assays1 has been extensively studied. Astaldi et al.2 have described a factor (SF) of molecular weight (MW) less than 50,000 present in the serum of normal donors; a thymic site of production was suggested because it was not found in serum from athymic donors. It also significantly elevated cyclic AMP levels in mouse thymocytes. Because of the potential significance of this finding to the study of thymic factor action, we have attempted to corroborate these findings but, as reported here, we have observed that a serum fraction from both normal and athymic individuals can raise cyclic AMP levels in mouse thymocytes. Neither serum fraction, however, produced the degree of stimulation of cyclic AMP levels described by Astaldi et al.2. Increases in cyclic AMP were much lower than those observed using the β-adrenergic agonist isoproterenol. We are therefore unable to confirm the existence of a circulating thymic factor in humans which raises cyclic AMP levels in mouse thymocytes.