Partial characterization of a T cell-derived factor that suppresses the initiation of the humoral immune response in vitro.

Specific anamnestic stimulation of spleen cells from mice immunized 7 days earlier with horse erythrocytes (HRBC) generated the release of a soluble factor that was capable of suppressing the initiation of the in vitro primary gammaM immune response to sheep red blood cells (SRBC), as well as to the immunogen that elicited its formation. Moreover, the suppressive macromolecule (mol. wt yields to 34,000), derived from antigen-activated, HRBC-primed T lymphocytes (but not B cells), inhibited the secondary gammaM and gammaG anti-SRBC plaque-forming cell responses of SRBC-primed spleen cells. The active material was resistant to treatment with DNase and RNase, but was inactivated by protease (10 microgram/ml, 30 min) or exposure to mild heat (56 degrees, 30 min). The antibody initiation suppressor factor (AISF) was concentrated and partially purified by gel filtration, followed by poly-acrylamide gel electrophoresis.     

Immunosuppressor T Cells in Tumor Bearing Hosts

Thymus or spleen cells of tumor bearing animals (TBA) (methyl-cholanthrene induced sarcoma bearing A/Jax mice) were shown to possess immunosuppressor cells regulating the immune response to the tumor in syngeneic animals. The immunosuppressive activity of these cells of TBA was totally abolished by the in vitro treatment of anti-t serum and complement. Soluble factor(s) with identical suppressive activity was isolated from the thymus cells of TBA and its molecular size was estimated to be lower than that of serum albumin in terms of its elution behaviour on gel filtration through a Sephadex G-200 column. These immunosuppressor T cells or their soluble factor(s) may be largely responsible for the growth of antigenic tumors.     

Immunological and physicochemical properties of a highly purified allergen from Dirofilaria immitis.

Allergen in crude extract of Dirofilaria immitis was purified and separated from IgG-inducing antigens by a combination of DEAE-Sephadex A-50 chromatography, Sephadex G-200 gel filtration and starch gel zone electrophoresis. The purified preparation was proved to be one protein band by sodium dodecyl sulfate polyacrylamide gel (SDS-gel) electrophoresis and one precipitin arc by immunodiffusion. The molecular weight of the purified allergen was estimated to be approximately 20,000 by gel filtration and 15,000 by SDS-gel electrophoresis. The carbohydrate content of the preparation was apparently low, about 2%. The allergen was positively charged, and its determinant group was protein in nature. It was resistant to tryptic, pepsic and chymotryptic digestion, periodate oxidation and DNase and RNase digestion but very sensitive to pronase digestion. Allergen was inclined to aggregate each other in the buffered solution. It was also very resistant to vibration, heat (80 degrees C for 1 h) and acid (pH 2.5) and alkali (pH 11.0) treatments. Rats as well as mice immunized with allergen developed only a reaginic antibody and no hemagglutination antibody.     

Characterization of a murine cytomegalovirus-induced immunosuppressive factor

Summary Murine cytomegalovirus infection in spleen cultures resulted in the production of a soluble factor, VISF (virus-induced suppressive factor), which inhibited concanavalin A mitogenesis in fresh spleen cells. Its production was specific for MCMV, since infection of spleen cultures by Sindbis virus, or bacteriophages PM 2 and T 4, and the phagocytosis of latex beads, all failed to elicit VISF. Maximum appearence of the factor occurred within 24 hours p.i. in spleen cultures, and its source was identified as the population of spleen cells which adhered to a plastic culture dish within two hours at 37° C. Non-adherent cells did not produce the factor. Its production was not inhibited by indomethacin. VISF could be concentrated by ultrafiltration on a YM 2 membrane filter, and it was readily fractionated by chromatography on sephadex G-25. In relation to peptides of known molecular weight it appeared to be smaller than 1,400 daltons. Its ability to suppress concanavalin A mitogenesis was largely removed by digestion with proteinase K. Thus VISF appears to be a relatively small peptide or peptide-containing substance. It was purified further by HPLC.     

Purification and some properties of Aeromonas hydrophila hemolysin

A hemolysin produced by a strain of Aeromonas hydrophila isolated from a patient with diarrhea was purified by acid precipitation and quarternary aminoethyl-Sephadex chromatography. The molecular weight of the hemolysin was estimated at 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 48,000 by Sephadex G-100 gel filtration. In polyacrylamide gel electrophoresis at pH 4.0 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the hemolysin migrated as a single band, whereas electrophoresis at pH 9.4 and thin-layer isoelectric focusing demonstrated multiple bands. The results may indicate charge isomers of the hemolysin. The purified hemolysin had a hemolytic activity of 134 hemolytic units per microgram of protein on rabbit erythrocytes. It caused fluid accumulation in infant mouse intestines and rabbit ligated ileal loops. Purified hemolysin also elicited cytotoxicity to Vero cells and lethal toxicity to mice. All these biological activities were lost on heating for 5 min at 56 degrees C. These findings support the notion that A. hydrophila hemolysin is a cytotoxic enterotoxin.     

Interferon production-stimulating factor

A factor stimulating interferon production (FSIP) has been isolated from cells treated once or twice (at an interval of 6-8 hours) with poly(rI).poly(rC) followed by actinomycin D (2 microgram/ml) or dehydrorifampicin (50 microgram/ml). In a dilution 1:512 the preparation stimulated interferon production 4-fold. In higher concentrations, the preparation induced interferon yields of 1280-2560 IE50/ml, i.e. exceeded the control values 16-fold. The factor does not affect the antiviral activity of interferon and has the tissue specificity. Its stimulating effect was not manifested with inducers of other nature. All the properties of FSIP studied show it to be similar with homologous interferon and repressor of its production but, unlike the latter, it was thermolabile and sensitive to acid pH.     

Isolation of a low-molecular-weight antibacterial system from human amniotic fluid.

A low-molecular-weight antibacterial system has been isolated from human amniotic fluid. The bacterial inhibitor requires the metal cation zinc and a peptide with a molecular weight of 630. The peptide component was purified using ultrafiltration, gel filtration, and ion-exchange chromatography. It can be inactivated by digestion with carboxypeptidase. The amino acid composition of the peptide is: 3 glutamine-glutamic acid, 2 glycine, and 1 lysine. Removal of zinc from the peptide has been shown to remove bacterial inhibitory activity.     

Immunosuppressive Factors in Ascites Fluids From Ovarian Cancer Patients

ABSTRACT: The nonspecific immunosuppressive effect of ascites fluids from ovarian cancer patients was examined and compared with that of noncancerous abdominal effusion and sera from ovarian cancer patients. The malignant ascites fluids produced a noncytotoxic, dose-dependent suppression of DNA synthesis of phy-tohemagglutinin-stimulated human peripheral blood lymphocytes. The suppression was higher than that observed in sera from cancer patients. No suppressive effect was seen in control abdominal effusion. The factors responsible for inhibition of in vitro lymphocyte function were partially purified from ascites fluid by lentil lectin affinity chromatography and gel filtration. Major active factors had a high molecular weight (440–1500 kilodaltons), an affinity to lentil lectin, and were stable against heat and acid treatment.     

Role of Uteroglobin and Transglutaminase in Masking the Antigenicity of Implanting Rabbit Embryos*

ABSTRACT: Using rabbit as a model, the roles of uteroglobin (UG) and transglutaminase (TG) in masking the antigenicity of early developing mammalian embryo have been investigated. Maternal lymphocytes in vitro, when mixed with mitomycin-C inactivated blastomeres, incorporated H³-thymidine, suggesting recognition of embryonic antigens by these cells. However, pretreatment of blastomeres with pregnant uterine fluid (PUF) or with UG alone or in combination with TG (coagulation factor XIIIa), resulted in a significant and dose-dependent suppression of H³-thymidine incorporation into these lymphocytes. A complete suppression was achieved at a concentration of 250 μg of UG/ml, in the absence of TG. However, in the presence of TG, only 1.0 μg of UG/ml was required for total suppression. Neither nonpregnant uterine fluid (NPUF), nor myoglobin, a nonspecific protein similar in molecular weight to UG, had any suppressive effect. Incubation of uteroglobin with anti-UG or TG with its antiserum prior to the pretreatment of blastomeres eliminated the suppressive effect of these proteins. Inhibition of TG by neopentyl chloroethyl nitrosourea (NPCNU) also eliminated the suppressive effects of uteroglobin on H³-thymidine incorporation into maternal lymphocytes. These results suggest that in the pregnant uterus UG in conjunction with TG may play a specific role in masking the antigenicity of developing embryos during implantation.     

Ubiquitin-like polypeptide inhibits the proliferative response of T cells in vivo

The monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic Ag-nonspecific suppressive functions. Recently, we demonstrated that the recombinant form of the ubiquitin-like segment (rUbi-L) of MNSFbeta, a 15.6 kDa-protein consisting of a polypeptide with 36% homology with ubiquitin fused to the ribosomal protein S30, presented an antigen-nonspecific immunoregulatory action in a manner similar to native MNSF. Although this cytokine has been characterized in vitro, little is known about its effects in vivo. Thus, we investigated whether rUbi-L shows a suppressor activity in vivo. The proliferative response of Con A (5 microg/ml)-stimulated splenocytes of mice treated with rUbi-L (500 ng/body) was notably decreased in a dose-dependent manner (max. 57+/-20%). In contrast, administration of high dose ubiquitin (50 microg/body) showed a little, but significant, effect (30+/-7%). Interestingly, concomitant addition of ubiquitin inhibited Ubi-L-induced suppression. Mice injected with rUbi-L without gelatin did not show any suppressive effect. NA4 (1microg/body), a neutralizing monoclonal antibody against rUbi-L, abolished the Ubi-L-mediated suppression. Therefore, ubiquitin-like polypeptide may be implicated in the immune responses in vivo.     

Suppression of murine lymphocyte proliferation induced by a small RNA purified from theTaenia solium metacestode

A substance fromTaenia solium metacestodes that decreases lymphocyte proliferation induced by concanavalin A was isolated. The molecular weight of this substance was estimated to be slightly more than 1,450 Da. Crude metacestode factor was fractionated through a Bio-gel P-6 column. Peak 1 showed suppressive activity. After incubation with RNase the substance lost its activity. Incubation of this material with trypsin or papain increased its suppressive activity. It was stable at boiling temperature for 10 min. The incubation of this substance with murine macrophages had no effect on [³H]-thymidine uptake by cocultured fresh splenic lymphocytes stimulated with concanavalin A. Conversely, cocultures of lymphocytes pretreated with the substance and fresh splenic lymphocytes showed a decreased incorporation of [³H]-thymidine. These results suggest that this substance is a RNA-peptide molecule whose RNA moiety accounts for its suppressive activity. The findings also suggest that in vivo the factor may be a modulator of the immune response.     

Purification and characterization of an enterotoxin from Bacteroides fragilis.

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.     

Suppression of lymphocyte responses by cephalosporins.

Human peripheral blood lymphocytes were cultured in several concentrations of each of several cephalosporins. Responses to phytohemagglutinin were compared with that of duplicate cultures containing penicillin-streptomycin, chloramphenicol, or no antibiotics. Possible effects of cephalosporins on responses of lymphocytes to concanavalin A and pokeweed mitogen were similarly determined. Significant suppression of responses to phytohemagglutinin and concanavalin A were seen in cultures containing 50 microgram each of cephalothin, cephalexin, or cephradine per ml. Lymphocyte responses to pokeweed mitogen were suppressed by 50 microgram of cephalexin, cephradine, or cefoxitin per ml. A higher concentration (100 microgram/ml) of all cephalosporins except cefoxitin and cefazolin suppressed the phytohemagglutinin response to less than 20% that of controls. Chloramphenicol (50 microgram/ml) did not inhibit the response to any mitogen used. These findings suggest that cephalosporins should not be used for prevention of bacterial overgrowth in certain cell cultures. Since many of the cephalosporins were suppressive in therapeutically attainable concentrations, these results may have potential clinical significance.     

Immunological activity of a T hybrid line. I. Production of an H-2-related suppressor factor with specificity for sheep red blood cells.

T lymphocyte hybrid lines have been produced by fusion of the thymoma BW 5147 with spleen cells of C57BL/10 mice primed to sheep red blood cells (SRBC). The supernatant (culture fluid) of a T hybrid designated A 1 was able to suppress the primary (IgM) and secondary (IgM and IgG) antibody responses to SRBC in vitro. The suppressive activity of supernatants could be titrated to 50% end points at final dilutions of up to 1 : 270. The suppression affected only SRBC and haptens coupled to SRBC, except when used at high concentration when some nonspecific suppression was observed. Absorption of the A 1 supernatant with SRBC removed all activity, while several other species of red cells failed to do so. The suppressor factor present in A 1 supernatant was not removed by anti-Ig adsorbents, but was removed by anti-H-2 antibodies reacting specifically with the haplotype of the spleen cells used in the fusion (H-2b). The cellular target of action of the factor was apparently a B cell, based on absorption with different cell populations. No genetic restrictions in the activity of the factor were found. A 1 cells carried H-2 and Thy-1 alleles of both parental cells and formed rosettes with SRBC.     

Immunosuppressive activity of mouse amniotic fluid

Immunosuppressive activity of mouse amniotic fluid was investigated. Swiss mice were treated from birth through young adulthood with intraperitoneal injections of homologous mouse amniotic fluid. A marked suppression of the primary splenic plaque-forming cells antibody response to sheep red blood cells was demonstrated. A pronounced suppressive effect by amniotic fluid was noted on the gamma A and gamma G plaque-forming cells, with variable degrees of immunosuppression observed on the gamma M response. It is postulated that the immunosuppressive effect of amniotic fluid may be related to its alpha-fetoprotein content.     

L-Lysine α-oxidase from Trichoderma viride i4. Purification and characterization

A L-lysine α-oxidase (LOD) has been purified to homogeneity in a two-step procedure with 300-fold enrichment and 60% recovery from the culture extract of Trichoderma viride i4. The enzyme catalyzes the reaction between L-lysine and molecular oxygen forming 2-oxo-6-aminocaproate, ammonia and hydrogen peroxide. Numerous substrates have been tested. The K_m value for L-lysine was found to be 0.026 mM. Its apparent molecular mass is 110000 Da when determined by gel filtration on Sephadex G-200, the estimated molecular weight of the subunits being 55000 Da in the SDS-PAGE. The enzyme is a glycoprotein and shows absorption maxima at 276, 386 and 463 nm. It was found to contain 1 mol of FAD per subunit. The coenzyme is bound non-covalently. Its isoelectric point is at pH 4.3. The enzyme is stable at extreme pH values, at relatively high temperatures and in diluted hydrogen peroxide. The enzyme described here differs from two other known L-lysine oxidases previously characterized with regard to its amino acid composition and its substrate specificity.     

Immunosuppressive factor from Actinobacillus actinomycetemcomitans down regulates cytokine production.

A cytoplasmic soluble fraction of Actinobacillus actinomycetemcomitans Y4 was isolated and characterized as suppressing mitogen-stimulated proliferation of and cytokine production by C3H/HeN mouse splenic T cells. This factor, designated suppressive factor 1 (SF1), was isolated from the supernatant of sonicated whole bacteria and purified by Q-Sepharose Fast Flow column chromatography, DEAE-Sepharose Fast Flow column chromatography, hydroxyapatite high-pressure liquid chromatography (HPLC), and Protein Pack 300 & 125 gel filtration HPLC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified SF1 migrated as a single band corresponding to a molecular mass of 14 kDa. This molecule was protease labile, heat resistant, and noncytotoxic. N'-terminal sequence analysis revealed no homology with any known peptides of periodontopathic bacteria or with any host-derived growth factors. Purified SF1 suppressed the proliferation of mouse splenic T cells which had been stimulated with concanavalin A, as well as suppressing the production of interleukin-2 (IL-2), gamma interferon, IL-4, and IL-5 from CD4+ T cells as 0.1 microgram/ml or more. These data suggest that SF1 produced by the periodontal pathogen A. actinomycetemcomitans functions as a virulence factor by down regulating T-cell proliferation and cytokine production at local defense sites.     

Molecular and functional identification and purification of complement component factor D from urine of patients with chronic renal failure

Urine proteins of normal subject and patients with impaired renal function were analyzed by two-dimensional polyacrylamide gel electrophoresis. As a result, a clear spot was detected specifically in urine from patients with obvious renal dysfunction. The isoelectrical point of this unique spot was pH 7.1-7.2 and the flow-rate (Rf) was 0.50-0.55 as that of albumin was 1.0. Partial amino acid sequence analysis revealed that the NH2-terminal to 22nd amino acid sequence was identical with that of complement factor D. We purified 22 mg of this protein (factor D) from 5000 ml of urine from a patient on hemodialysis by three chromatographic steps using DEAE-Sephadex A-50 and Sephacryl S-200. The purified urine factor D gave a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis at the position of 23 kD, and displayed normal factor D hemolytic activity. The concentrations of factor D estimated by hemolytic assay were 1.9 micrograms/ml of normal serum, less than 0.1 microgram/ml of normal urine, 15 micrograms/ml of patient serum and 50 micrograms/ml of patient urine.     

Regulatory substances produced by lymphocytes

A factor which suppresses DNA synthesis in rat lymph-node cells stimulated with phytohemagglutinin (PHA) and in mouse fibroblasts (L cells) was obtained from the culture supernatant of rat lymph-node cells stimulated with ovalbumin in vivo and rechallenged with the same antigen in vitro. The factor is protein in nature, as evidenced by its sensitivity to trypsin. It is relatively heat stable and its activity is lost after periodate treatment, suggesting that it is glycoprotein. On Sephadex gel filtration, it gives 2 peaks with estimated molecular weights of 80,000 and 160,000–200,000 daltons. Their isoelectric points were estimated as 2.7 and 3.0 by isoelectric focusing analysis. The factor had almost no effect on the synthesis of RNA and protein, but inhibited DNA synthesis completely. This inhibition followed multihit kinetics with a multiplicity estimated as 20–40. The suppressive effect of the factor was reversible when it was removed within 14 h after addition of PHA to cultures of rat lymph-node cells, but became completely irreversible by 30 h. Between 14 and 30 h the effect was partially reversible. These results suggest that the factor acts by inhibiting a process or processes occurring shortly before the initiation of DNA synthesis. They also imply that the factor may play a role as a short-range regulator of immune responses.