Human transfer factor. II. Clinical results with the large pool transfer factor

21 patients with diseases due to immunological causes were treated 6 times at an interval of four weeks by administering 5 E TF or 1 E TF each per 10 kg of body weight. TF consisted of 5 different large pool TF charges of 420 to 822 buffy-coats of fresh stored whole blood. Clinical and immunological investigations as well as biochemical ones in the laboratory were made before and after treatment. Large pool TF is clinically effective in 9 from 18 patients and immunologically in 16 from 18 patients. There is a greater effect in immunodeficiencies than in autoimmune diseases. TF is not able to remove the defect for a long time. Repeated administrations are required. TF therapy may be regarded as a substitution therapy. At first, the intervals have to be chosen according to clinical parameters (recidive of the disease or crisis respectively). There is a good compatibility of TF. Side-effects could not be observed. The frequent immunological conversions after score evaluation indicate, however, that in comparison to the clinical appearance the course of the disease must be seen to be much more complex than it can be expressed by in vitro correlates of immunological responses. Statistically ensured correlations of single tests concerning the clinical course could also not be found. Large pool TF provides favourable conditions for controlled therapy trials in order to elucidate those findings of therapy which hitherto had been a subject of controversy (e.g. autoimmune diseases, tumours).     

Isolation of homologous restriction factor from human urine. Immunochemical properties and biologic activity

A soluble form of homologous restriction factor (HRF-U) was isolated from normal human urine. With respect to m.w. (65,000) and immunoblotting characteristics, it resembled membrane HRF (HRF-M) that had been isolated from human E membranes. The protein exhibited limited cross-reactivity with the channel-forming proteins of C and cytotoxic lymphocytes. It inhibited reactive lysis of E by human C5b-9. Inhibition occurred at the attachment stage of C5b-7 to target cells, rather than at the C8 or C9 stage of membrane attack complex assembly which is inhibited by HRF-M. In this respect, HRF-U acts analogously to S protein of serum, but no immunochemical relationship between these two proteins was detected. HRF-U might be derived from the soluble HRF detected in cytoplasmic granules of killer lymphocytes.     

Dog secretin: sequence and biologic activity

Secretin was purified from the first 50 cm of proximal intestine of a single dog. The purification sequence included extraction in acid-ethanol, differential precipitation with acetone, Sephadex gel filtration and three successive HPLC steps. Dog secretin has the following sequence: HSDGTFTSELSRLRESARLQRLLQGLV. The underlined amino acid represents a substitution of Glu for Asp at position 15 from the NH2-terminus of pig secretin. Dog secretin is equal in potency to pig secretin in stimulating pancreatic ductal flow in a rat bioassay system.     

Inhibitory activity of medium-dialyzed transfer factor on lymphocyte blastogenesis.

Human transfer factor was prepared from normal donors with marked skin reactivity against common microbial antigens by dialysis of lymphocyte extracts versus tissue culture medium (TFmd). Several batches of TFmd were tested for their ability to specifically increase the DNA synthesis of unsensitized lymphocytes in vitro in the presence of the corresponding antigens (PPD, SKSD, Candida, Thistoplasmin). No transfer or immunologic reactivity by TFmd was observed. There was, however, a significant unspecific inhibition by TFmd of lymphocyte cultures stimulated by antigens, PHA or allogeneic cells.     

Murine transfer factor. III. Specific interactions between transfer factor and antigen

The interactions between dialyzable transfer factor and antigens have been studied. Incubation of transfer factor-containing dialysates from ferritin-sensitized mice or ferritin-coated plastic surfaces removed the antigen-sensitizing activity; incubations of the same preparations on cytochrome c-coated surfaces did not. Similar results were obtained when cytochrome c-transfer factor was studied. Incubation on cytochrome c-coated surfaces removed the activity, but incubation on ferritin-coated surfaces did not. Specific transfer factor activities could be recovered by elution with 8 M urea or acetonitrile. The finding of interactions between transfer factor and antigens provides evidence for a molecular basis of the specificity of the immunologic effects of transfer factor. This technique may also enable us to obtain amounts of specific material that are adequate for chemical analysis.     

Human factor VIII procoagulant activity and phospholipid interaction

The interaction between purified human factor VIII and phospholipid vesicles was investigated. The binding of factor VIII to an equimolecular mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) was studied by sucrose gradient ultracentrifugation (10–40% w/v saccharose in 0.01 M Tris-HCl/0.15 M NaCl buffer (pH 7). In the absence of phospholipids all factor VIII activities (VIII : C, VIII R : WF and VIII R : AG) were found in the zone of highest sucrose density including the factor VIII related protein subunit (200 000 molecular weight). In the presence of an equimolecular mixture of PS/PC VIII R : WF activity, VIII R : AG and a factor VIII related protein still migrated to the bottom of the tube, while VIII : C activity remained at the top where phospholipids were found. Thus a dissociation phenomenon between VIII : C and the other factor VIII relateda activities was apparent in the presence of phospholipids. These results also demonstrate the binding of factor VIII : C to certain active phospholipids.     

Testicular GnRH-like factors: characterization of biologic activity

Observations that gonadotropin releasing hormone and its agonists directly inhibit gonadal function by binding to receptors on the Leydig cells had led to search for testicular GnRH-like peptide(s). This communication presents evidence that GnRH-like factors isolated from rat testis by immunoaffinity chromatography and previously characterized by radioimmunoassay and radioreceptor assay possess biologic activity. The partially purified material led to dose dependent inhibition of oLH stimulated testosterone production in a mixed Sertoli-Leydig cell monolayer culture. Pre-incubation of the cells with a potent GnRH antagonist prevented the inhibitory effects of the partially purified material suggesting that inhibition of oLH stimulated testosterone production may be receptor mediated.     

Antigen-specific human T cell factors. II. T cell suppressor factor: biologic properties

The in vitro induction of an ovalbumin-specific human T cell suppressor factor is described (TsF120-OA). The antigen-specific suppressive component can be purified by affinity chromatography from supernatants derived from Marbrook-Diener type cultures of peripheral blood T cells stimulated with a high dose of ovalbumin. TsF120-OA suppresses the antigen-induced PFC formation of human blood B cells in vitro in an antigen-specific way. The target of TsF120-OA activity is shown to be the T helper cell. No genetic restriction in the action of the factor is observed.     

Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets

The plasma and serum of humans and various animal species exert an actin-depolymerizing activity. Human actin-depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE-cellulose and blue-Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin-sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin-A-Sepharose column. Incubation of ethanol-fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90-kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes.     

Modulation of the biologic activities of IgE-binding factor. II. Physicochemical properties and cell sources of glycosylation-enhancing factor

T lymphocytes of rats treated with Bordetella pertussis vaccine (BP) formed a soluble factor that enhanced the glycosylation of IgE-binding factors during their biosynthesis, and provided the latter factors with the biologic activity to potentiate the IgE response. The present experiments demonstrated that pertussigen (leukocytosis-promoting factor) from BP induced normal rat spleen cells to form the glycosylation-enhancing factor. The same factor was obtained by incubation of normal spleen cells with 5 micrograms/ml, but not 2 micrograms/ml, concanavalin A. When normal rat mesenteric lymph node cells were incubated with the glycosylation-enhancing factor together with IgE, IgE-binding factors formed by the cells selectively potentiated the IgE response. The IgE-binding factors formed by the same cells upon incubation with IgE alone neither enhanced nor suppressed the IgE response. The glycosylation-enhancing factor changed the nature of IgE-binding factors formed by the rat-mouse T cell hybridoma, 23A4. IgE-binding factors induced by IgE alone lacked affinity for lentil lectin, whereas those induced by IgE in the presence of the glycosylation-enhancing factor had affinity for the lectin. The cell source of the glycosylation-enhancing factor appeared to be W 3/25+ Fc gamma R+ T cells. The glycosylation-enhancing factor was protein in nature and had a m.w. of about 25,000. The factor had affinity for acid-treated Sepharose and could be recovered from the beads by elution with lactose. The factor was different from interleukin 2 with respect to both its affinity for galactose and its isoelectric point.     

Synthesis and biologic activity of conformationally constrained analogs of L-363,301.

We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L-363,301, c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11] (numbering as in the native hormone somatostatin-14). The d-Trp in position 8 was replaced with (2R,3S)- and (2R,3R)-beta-MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d-Trp upon binding to human somatostatin receptors hsst1-5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3. Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.     

Woodchuck interleukin-6 gene: structure, characterization, and biologic activity

Woodchuck is an important animal model for studying human hepatitis B virus (HBV) infection. Within the cytokine network, interleukin-6 (IL-6) plays an important role in immune responses that may lead to viral clearance. To further understand woodchuck IL-6 biology, we cloned and characterized the IL-6 gene from white blood cells. The complete woodchuck IL-6 gene is about 7 kb and consists of five exons and four introns. The IL-6 gene organization of the woodchuck is similar to those of the human, rat, and mouse. Also several elements are highly conserved in the 300 bp promoter region of the IL-6 gene, including a nuclear factor kappa B (NF-kappaB) binding site. The woodchuck IL-6 gene encodes a polypeptide of 207 amino acids in a precursor form and 189 amino acids in the mature form. The expressed protein was 23 kDa according to SDS-PAGE. To demonstrate biologic activity, we expressed woodchuck IL-6 and showed that the purified recombinant protein induced terminal differentiation, as reflected by upregulation of Fcgamma receptor expression, and substantially inhibited proliferation of M1 cells, a murine myeloid leukemia cell line. The inhibitory effect of woodchuck IL-6 on M1 cells was blocked by an anti-gp130 monoclonal antibody, suggesting that woodchuck IL-6 activity is specifically mediated by signaling through the IL-6 receptor complex. Cloning of the woodchuck IL-6 gene and demonstrating biologic activity of the gene product will facilitate studies of human hepatitis B virus using the woodchuck model.     

Human transfer factor. I. Production by the great pool technic and the biochemical and immunological characteristics of the preparation

A great pool procedure for producing human transfer factor preparations from buffy-coats of stored whole blood which has not been used up till now is described. From an initial tool size of about 1,500 units of whole blood the procedure represented makes it possible to gain higher amounts of uniform and in their immunological efficacy in vitro standardizable preparations for controlled long-term therapy studies. By means of various immunological in vitro tests, stimulating as well as suppressing activities of the charges obtained could be identified. Starting from the differentiated immunological effects of various charges of the transfer factor on T-cell populations in vitro, the possibility of influencing T-cell subpopulations through well-conceived therapeutic measures by a transfer factor is discussed.     

Full-length chicken parathyroid hormone. Biosynthesis in Escherichia coli and analysis of biologic activity.

Chicken parathyroid hormone (cPTH) has been reported to stimulate adrenal steroidogenesis and to have unusual potency on traditional PTH target tissues. To evaluate these properties, chicken PTH-(1-88) has been expressed in Escherichia coli using a plasmid encoding a fusion protein which links together growth hormone, a factor Xa recognition site, and chicken PTH-(1-88). The growth hormone-cPTH fusion protein required the presence of 0.02% sodium dodecyl sulfate to remain in solution and be cleaved by factor Xa. The high performance liquid chromatography-purified recombinant cPTH-(1-88) and chemically synthesized cPTH-(1-34) had similar potency in rat osteosarcoma (ROS 17/2.8) cells, opossum kidney (OK) cells, and dispersed primary chicken kidney cells. The biologic potencies of cPTH-(1-34) and cPTH-(1-88) in radioreceptor binding and cAMP generation in both bone- and kidney-derived cell lines were less than those of human (h)PTH-(1-34). In dispersed chicken kidney cells, cAMP production by cPTH-(1-34) and cPTH-(1-88) was similar to that stimulated by human PTH-(1-34). No stimulation of steroidogenesis could be detected when recombinant chicken PTH-(1-88) was added to dispersed chicken adrenal cells. The biologic activity of recombinant chicken PTH-(1-88) purified from E. coli was comparable with that of chicken PTH-(1-88) expressed by mammalian COS cells. Thus, the full-length chicken PTH did not exhibit enhanced potency, when compared with human PTH in ROS 17/2.8, OK cell lines, and dispersed chicken kidney cells and did not demonstrate the novel steroidogenic action previously reported in adrenal cells. The successful production of chicken PTH-(1-88) will enhance our understanding of the structure-activity relationships for PTH, particularly the sequence-dependent metabolism of the hormone.