Induction of lymphocyte blast transformation by purified fibronectin in vitro

Purified rat plasma fibronectin (Fn) induces a dose-dependent, nonspecific proliferation of lymphoid cells isolated from spleen and lymph nodes, but has no effect on thymocytes. Proliferation of cells occurred after 4 to 5 days of incubation and was generally 5- to 10-fold greater than control cells cultured without Fn or in the presence of the same concentrations of rat serum albumin. The responding cell appears to be the T cell and requires the presence of adherent accessory cells (macrophages). Although purified T and B cells failed to demonstrate a significant increase in blastogenesis in the presence of Fn, reconstitution of T, but not B, cells with irradiated peritoneal exudate macrophages restored the stimulatory effect of Fn. Furthermore, comparison of the effect of various concentrations of macrophages on the restored T cell response shows that proliferative activity in the presence of Fn is dependent on a critical number of macrophages. Adding higher numbers of macrophages beyond the optimal concentration results in a sharp decline in lymphocyte responsiveness to Fn, although the proliferation of control cells continues to increase at these macrophage concentrations. Macrophages pulsed with Fn for 1 hr failed to evoke an increase in T cell responsiveness after 3 or 5 days of incubation. In addition, incubation of Fn-pulsed T cells with macrophages that had been precultured with or without Fn also failed to result in T cell activation. The effect of Fn on T lymphocyte transformation appears to be mediated indirectly through its interaction with the macrophage, because 125I-Fn, which shows significant binding to peritoneal exudate macrophages, fails to interact with purified T cells. Radiolabeled Fn also demonstrated little or no binding activity for unseparated lymph node cells.     

Immunologic activity of human lymph cells in the lymphocyte blast transformation reaction

The immunologic activity of lymph cells obtained by curative draining of the chest lymph drainage was studied in 14 patients with subhepatogenous jaundice of different origin. The lymphocyte blast transformation reaction showed that lymphocytes of the lymph can form blasts under the effect of phytohemagglutinin, antilymphocyte gamma-globulin and some bacterial antigens. The reaction of the highest intensity was recorded in lymphocyte cultures of the lymph collected for this purpose on the 2--3d day from the commencement of lymph drainage. Lymphorrhea produces a decrease in the capacity of stimulated lymph cells for blastformation. During lymph drainage no blastformation takes place in stimulated blood cell cultures of the patients. Therefore, the fundamental part of antigen sensitive lymphocytes is stored in the lymphoid tissue.     

Thymus-derived inhibitor of lymphocyte proliferation. II. Tissue specificity in vitro and in vivo

A thymus crude factor (TCF) isolated from bovine thymus tissue has been tested for its effects on the proliferation of various murine cells. Specific inhibition in vitro has been found for DNA synthesis in murine T and B lymphocytes which appears not to be based on cytotoxicity. Moreover, TCF, when administered to mice, also interferes with the DNA synthesis in lymphoid tissue in vivo. Our data are suggestive for the presence in TCF of an endogenous 'chalone-like' inhibitor of lymphoid cell proliferation in vitro and in vivo.     

Mefloquine, an antimalaria drug with antiprion activity in vitro, lacks activity in vivo

In view of the effectiveness of antimalaria drugs inhibiting abnormal protease-resistant prion protein (PrP-res) formation in scrapie agent-infected cells, we tested other antimalarial compounds for similar activity. Mefloquine (MF), a quinoline antimalaria drug, was the most active compound tested against RML and 22L mouse scrapie agent-infected cells, with 50% inhibitory concentrations of approximately 0.5 and approximately 1.2 microM, respectively. However, MF administered to mice did not delay the onset of intraperitoneally inoculated scrapie agent, the result previously observed with quinacrine. While most anti-scrapie agent compounds inhibit PrP-res formation in vitro, many PrP-res inhibitors have no activity in vivo. This underscores the importance of testing promising candidates in vivo.     

N-benzyloxycarbonyl-L-proline: an in vitro and in vivo inhibitor of prolidase

Prolidase deficiency (PD) is a recessive disorder of the connective tissue caused by mutations in the prolidase, a specific peptidase, cleaving the dipeptides with a C-terminal prolyl and hydroxyprolyl residue. PD is a complex syndrome characterized mainly by intractable skin lesions, recurrent respiratory infections and mental retardation. The relation between prolidase biological functions and the disease is still largely unknown. We studied the effect of a prolidase inhibitor, N-benzyloxycarbonyl-l-proline (Cbz-Pro), in vitro on prolidase from human fibroblasts and in vivo on murine erythrocytes prolidase. A 90% inhibition was detected incubating cellular extracts at 1:1 ratio of Gly-Pro substrate: Cbz-Pro inhibitor. Pulse experiments performed incubating human fibroblasts with 6 mM Cbz-Pro revealed that the inhibitor uptake was completed in about 1 min. The Cbz-Pro uptake was saturable and pH dependent. Long-term incubation of fibroblasts with Cbz-Pro caused mitochondria depolarization and increased cellular death as reported for long-term culture of fibroblasts from PD patients. An inhibitory effect of Cbz-Pro has also been shown in vivo. Our results demonstrated that Cbz-Pro is a potent inhibitor of prolidase in cultured fibroblasts and it can be used in vivo to better characterize the prolidase enzyme and further investigate PD physiopathology.     

Effect of cerebrocrast on the lymphocyte blast transformation activity in normal and streptozotocin‐induced diabetic rats

Both IDDM and NIDDM are characterized by deviations in peripheral T and B lymphocyte count, T_helper:T_suppressor ratio, as well as by impaired T_suppressor function. These abnormalities may promote insulin antibody and other antibody production, contributing to overt diabetes mellitus development in early stage of the disease. In the present study we explored the effects of cerebrocrast (1,4‐dihydropyridine derivative) administration on Con A‐ and IL‐2‐stimulated tissue lymphocyte blast transformation activity and on the thymus and lymph node mass in normal and streptozotocin (STZ)‐induced diabetic rats. It was established that cerebrocrast, administered four times at the doses of 0·05 and 0·5 mg kg⁻¹, has long‐term (up to 14 days) effects on the immune system and protects against the toxic effect of STZ in STZ‐induced diabetic rats, preventing thymus and lymph node mass loss. We conclude that cerebrocrast administration leads to the increase in number and activity of T_helper and T_suppressor lymphocytes. Glycolysis and DNA synthesis in these cells is augmented under the influence of cerebrocrast administration. We propose that the increase in lymphocyte suppressive activity caused by cerebrocrast administration may prevent the development of IDDM and NIDDM in patients with pre‐diabetes, but in patients with early and overt diabetes mellitus the drug administration may prevent the overexpression of insulin antibodies and other antibodies. The effect of cerebrocrast on the de novo production of insulin and IL‐2 receptors may be beneficial for IDDM and NIDDM patients. Copyright © 1999 John Wiley & Sons, Ltd.     

Specific in vitro lymphocyte transformation with Venezuelan equine encephalitis virus.

Specific lymphocyte transformation to viral antigen was detected in individuals vaccinated with live, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain TC-83. Suspensions of purified, inactivated virus were used an antigen in 250-μ1 lymphocyte cultures, and under optimal conditions the assay demonstrated 10-fold greater incorporation of ¹⁴C-thymidine by lymphocytes from immune than from nonimmune people. The rise in lymphocyte transformation response occurred 1 week after vaccination. The magnitude and range of the lymphocyte transformation response to the VEE viral antigen were similar to the responses seen using antigens derived from five other microbial sources: Francisella tularensis, Coccidioides immitis, Mycobacterium tuberculosis, Streptococcus, and parainfluenza virus. Autologous plasma containing antibody exerts an inhibitory effect on cultures from immune individuals. The onset, magnitude of response, and specificity of this in vitro assay are correlated with the clinical and pathological events of VEE virus infection.     

Suppressive effect of the blast cells of AKR strain mice on antibody formation in vivo and lymphocyte proliferation in vitro

Blast cells obtained from the "erythropoietic spleen" of FG-stimulated young mice and cells accumulating in the spleens of preleukemic AKR mice have a marked suppressive effect on spontaneous and mitogen-induced proliferation of young mouse splenocytes in vitro and suppress the development of humoral immune response in immunized recipients during syngeneic transfer in vivo. Some disturbances in erythron system in preleukemic AKR mice manifested in the accumulation of immature erythroid precursors which are suppressors of immunocompetent lymphocytes are suggested to be a pathogenetic link in the development of leukemia.     

The in vitro detection of antigen sensitivity in contact dermatitis by lymphocyte transformation.

Contact sensitivity induced in rabbits by the topical application of 1-chloro-2,4-dinitrobenzene (DNCB) was evaluated by the in vitro lymphocyte transformation response. The antigen employed to stimulate cultures was a preparation of DNCB conjugated homologous lymphocytes. This antigen preparation specifically stimulated blood lymphocytes from DNCB sensitive animals to transform in vitro. The kinetics of the primary immune response were determined by antigen mediated lymphocyte transformation in rabbits percutaneously sensitized with DNCB. The peak response was reached 12 days following initial exposure to DNCB. Sensitivity decreased but remained at detectable levels through 57 days of the primary response. Skin testing the animals late in the primary response stimulated an increase in the number of circulating sensitive lymphocytes. Serum proteins, conjugated with DNCB, were not suitable antigens for the in vitro detection of contact sensitivity.     

Proliferative activity of lymphocyte cultures due to the mutagenic action of thiophosphamide in vivo and in vitro

Thiophosphamide is capable of inhibiting the cell cycle after the treatment of rabbit blood lymphocytes in vivo and in vitro. To assess the proliferative activity of the cells during cultivation, use was made of the index of the mean division number which was experienced by the cells after mutagenic exposure prior to fixation. As the mutagen dose was raised in vivo and in vitro, the mean division number decreased linearly, namely by approximately the same value with an equivalent dose variation. This indicates that thiophosphamide produces the same cytotoxic action both in vivo and in vitro.