Prospects of AIDS therapy by thymic humoral factor, a thymic hormone

Research performed in our laboratory has established that thymic humoral factor (THF), a peptide hormone isolated from calf thymus, leads to clonal expansion, differentiation and maturation of T-cell lymphocytes. THF augments the response to T-cell lectins, mixed lymphocyte reactions, graft-versus-host reactivity, antibody response to SRBC, cytotoxic responses and production of interleukin-2. Clinical data based on the use of THF of natural sources in about 200 patients indicate that it regulates differentiation of T-cell precursors, leading to normalization of the ratio between helper and suppressor/cytotoxic subsets. THF reconstitutes the defective cell-mediated immunity in patients affected by various types of neoplasms and suffering from secondary immune deficiencies, as a result of chemo and/or radiotherapy. THF-gamma 2 was purified and synthesized and found to be an octapeptide which retains all the biological activity of the thymus extracts. It is presently available for extensive clinical use.     

Influence of thymic humoral factor(s) on haemopoiesis in mice

Postirradiation administration of Leukotrophin to whole-body irradiated mice was associated with increased LD50/30 and DRF. As indicated by 59Fe uptake and ESC number, haemopoiesis was significantly stimulated in spleen and bone marrow after Leukotrophin application to irradiated mice. DNA content and the uptake of 3H-thymidine in DNA was significantly enhanced in the thymus and bone marrow of irradiated and Leukotrophin-treated mice. The micronucleus test confirmed that Leukotrophin is a therapeutic agents, while administered before irradiation it does not influence the initial radio-lesions.     

Degradation of thymic humoral factor gamma2 by human plasma: involvement of angiotensin converting enzyme

The degradation of thymic humoral factor-gamma2 (THF-gamma2), an immunoregulatory octapeptide important for T-lymphocyte regulation, by enzymes present in human plasma, was investigated. THF-gamma2 was metabolized through two steps that involved the detaching of N-terminal amino acid leucine followed by hydrolysis of the Lys(6)-Phe(7) bond. The THF-gamma2 cleavages were sensitive to aminopeptidase and metalloproteinase inhibitors. The degradation was completely blocked by amastatin and specific inhibitors of angiotensin converting enzyme (ACE). The cleavages occurred independently, with two different kinetics, faster for the N-terminal hydrolysis than for that of the Lys(6)-Phe(7) bond. Purified human plasma ACE was used to characterize the hydrolysis of Lys(6)-Phe(7) bond. The K(m) and K(cat) values for THF-gamma2 hydrolysis were 0.273 mM and 107 s(-1), respectively. The optimum of chloride concentration was 300 mM, while that of pH was 7.6. The presence of ACE in circulating mononuclear cells raises the possibility that it may play a role in modulating the THF-gamma2 activity.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

Antigen-dependent induction of a non-specific humoral factor blocking rosette-forming cells in experiments in vitro and in vivo

Isologous serum of CBA mice immunized with rabbit immunoglobulins (ARIS) contained a factor capable of inactivating rosette-forming splenocytes (RFC) in vitro from the same strain of mice immunized with SRBC. When mouse SRBC immunization was carried out against the background of ARIS injection the court of RFC to SRBC at the peak of immune response was 30% of that of mice injected with SRBC and normal isologous serum. A decrease of RFC count was the result of disappearance of the theta-negative RFC. Passive ARIS immunization failed to influence the antigen-induced proliferation of the antibody-forming cells and the synthesis of antibodies against SRBC.     

Synthesis and immunological effect of thymic humoral factor-gamma 2 analogues

Nine analogues of thymic humoral factor (THF)-gamma 2 were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin(PHA)-stimulated T-lymphocytes of uremic patients with infectious diseases were examined. The results were as follows: [Arg6]-THF-gamma 2 exhibited higher restoring activity than that of our synthetic THF-gamma 2. [Sar4]-, [Val1]-, [Arg3]-, [Gly5]-, and [Asn3]-THF-gamma 2 were also active, but less potent than that of our synthetic THF-gamma 2. Three other peptides, [beta Ala4]-, [Arg2]-, and [Gln2]-THF-gamma 2, did not show any restoring activity on the impaired blastogenic response of uremic patients with infectious disease.     

Enhancement of natural killer cell activity in mice by treatment with a thymic factor

Summary The administration of a thymic factor, thymostimulin (TP-1), to mice resulted in considerable augmentation of natural killer (NK) cell activity as measured in a short-term assay against ⁵¹Cr-labeled YAC-1 target cells. Conditions suitable for detection of the thymostimulin-induced boosting of NK included multiple daily exposures to TP-1 (50 g/kg), and peak levels of reactivity were observed at 2–4 days after discontinuation of treatment. A strict age-dependency of the effect was also observed, with optimal augmentation of NK-cell activity when TP-1 was administered to mice at 4–6 weeks of age. The effect was not limited to TP-1 treatment but was also observed on administration of another thymic factor (thymosin ₁). The activated cells responsible for the increased natural cell-mediated cytotoxicity appeared to be typical murine NK cells, judging by both functional and antigenic criteria.     

Synthesis and immunological effect of thymic humoral factor-gamma-2 analogues

Nine analogues of thymic humoral factor (THF)-gamma-2 were prepared by the solid-phase method, and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin(PHA)-stimulated T-lymphocytes of uremic patients with infectious diseases were examined. The results were as follows: [Arg6]-THF-gamma-2 exhibited higher restoring activity than that of our synthetic THF-gamma-2. [Sar4]-, [Val1]-, [Arg3]-, [Gly5]-, and [Asn3]-THF-gamma-2 were also active but less potent than that of our synthetic THF-gamma-2. Three other peptides, [beta-Ala4]-, [Arg2]-, and [Gln2]-THF-gamma-2, did not show any restoring activity on the impaired blastogenic response of uremic patients with infectious disease.     

Potentiation of the primary humoral immune response in vitro by C5a anaphylatoxin

The effect of the anaphylatoxin C5a on the primary humoral immune response to SRBC was studied in culture of spleen cells from C3H mice. The addition of human C5a to antigen-stimulated cultures resulted in a significant, dose-dependent augmentation of the primary PFC response to antigen. The specificity of this effect was affirmed by the ability of C5ades Arg, but not of the structurally analogous C3a anaphylatoxin, to act in a parallel fashion. Enhancement could be observed over a range of doses of antigen. Brief preincubation of macrophages, but not of lymphoid cells, with C5a was sufficient to cause subsequent enhancement of the primary humoral immune responses. The duration of preincubation required for this effect closely paralleled that for binding of C5a to peritoneal cells. Immunopotentiation by C5a appears to involve the function of C5a receptor-bearing, Ia- accessory cells as well as Ia+ antigen-presenting cells. Immunopotentiation could be observed when the addition of C5a was delayed for up to 24 hr after initiation of culture. Additionally, augmentation of tritiated thymidine uptake in mixed lymphocyte reactions was enhanced by the addition of C5a in a fashion parallel to that for the primary response to SRBC. These observations support a role for C5a as a modulator of cellular immunity in addition to its role in acute inflammation. Possible cellular mechanisms and implications for immunomodulation of immune responses are discussed.     

Inhibition of phospholipid methylation by a cytosolic factor.

Rat liver cytosol contains a heat-stable factor which inhibits phospholipid methylation by rat liver microsomes. The effect of this factor on lipid methylation was dose- and pH-dependent. This factor has an Mr of approx. 3200 as estimated by gel filtration. It could not be extracted by chloroform/methanol (2:1, v/v), and its action was inhibited by incubation with subtilisin.     

Inhibition of cholinesterases by fluoride in vitro

1. Series of colorimetric dynamic assays allowed the study of the inhibition of cholinesterases by F(-) ions in vitro, by using, as sources of enzyme, whole human blood, human serum, homogenized rat brain and two preparations of red blood cells (human and bovine) whose enzymic purity was ascertained. 2. The first evidence of inhibition of human serum pseudocholinesterase by fluoride was noticed at 15-25mum-fluoride. Ten times as much fluoride was needed to start inhibition of acetylcholinesterase of the red blood cells. 3. The action of fluoride on the enzymic reaction was immediate. The reversibility of the inhibition was shown by dialysis and dilution. 4. Kinetic measurements showed that the inhibition under study was not dependent on the substrate concentration and was of the uncompetitive type, similar to that observed in the presence of a heavy metal (cadmium). 5. The activity of serum cholinesterase did not change in the absence of Mg(2+) and Ca(2+) ions. Fluoride was shown to inhibit the enzyme in the absence of these ions as well as of phosphate. 6. Fluoride could inhibit cholinesterases in the presence of three different substrates and had no action on the non-enzymic hydrolysis. 7. It is thought that the halide is bound reversibly to the enzyme molecule, with the probable exclusion of the active site, but no firm conclusion could be reached on this point.     

Thymic epithelium in vitro. III. Cytokine production by a thymic epithelial subset

We have previously shown that two populations of thymic epithelium can be separated in culture on the basis of their differential growth rates and their adherence to the culture substratum, and maintained as long-term, morphologically distinct cell cultures, TECs and TECL. We have also described the effects of supernatants from the small epithelial cell (TESs) on the proliferative responses of thymocytes cocultured with mitogen and TESs over a 72-hr period. We now describe the effects in thymic epithelial supernatants (TESL) of soluble factors produced by TECL (the large epithelial cell) on thymocytes costimulated with mitogen and compare their effects to those derived from TECs. Both TESL and TESs suppress optimally stimulated thymocytes and enhance the proliferative responses of suboptimally stimulated thymocytes over a 72-hr period. The suppressive activities produced by TECL and TECs appear distinct, based upon markedly different molecular weights, but have similar sensitivities to heat treatment. The enhancing activities are of similar molecular weight, but have different sensitivities to heat treatment. In addition, TECL synthesize four- to fivefold less PGE2 than TECs. These results provide additional distinctions between the two cell types, and taken in conjunction with data on the anatomic distribution of similar cells, suggest that although they have similar functional effects in vitro, they may prove to have separable roles in vivo.