Delineation between T- and B-suppressive molecules from human seminal plasma: I. Partial characterization of a 180-kD protein inhibiting the B response to T-independent antigens.

The immunosuppressive activity of fractionated human seminal plasma (SP) was investigated both in vitro (on human lymphocytes) and in vivo with Balb/c mice. SP fractionation by dialysis allowed delineation of the major suppressor factors according to their respective sizes--small (less than 12 kD) or large (greater than 12 kD). In vitro, large molecules were found to suppress the B-cell proliferative response induced by the Nocardia mitogen, while small molecules suppressed the T-cell proliferation induced by phytohemagglutinin. In vivo, immunosuppression was obtained almost exclusively on T-independent responses after preliminary treatments either with unfractionated SP or with large SP molecules. Both type 1 and type 2 T-independent responses were suppressed, as evidenced by plaque-forming cells and antibody assays. In contrast, no immunosuppression was found in vivo after treatment by small SP molecules. Purification of the B-cell suppressor by gel filtration and high-performance liquid chromatography, as well as by preparative isofocusing, indicated that its molecular weight was 180 kD and its isoelectric charge was between pH 5 and 6. This factor is a protein, as evidenced by pronase digestion. A possible role for this molecule in the protection of sperm against the female immune system is discussed.

Dysregulation of telomerase activity and expression in lymphokine-activated killer cells from advanced cancer patients: possible involvement in cancer-associated immunosuppression mechanism.

There exists cancer-associated immunosuppression, and the generation of lymphokine-activated killer (LAK) cells is impaired in patients with advanced cancer. Telomerase has been reported to be upregulated in the activation of lymphocytes to proliferate against immune stimulation as well as in the malignant transformation of immortal cancer cells. We attempted to clarify the involvement of telomerase in the impairment of LAK cell generation in patients with advanced cancer. LAK cells were generated by stimulation with interleukin (IL)-2 and immobilized anti-CD3 antibody (IL-2/CD3 system) from peripheral blood mononuclear cells of healthy volunteers (he-LAK) or patients with advanced cancer (ca-LAK), and proliferative potential of LAK cells was evaluated on the basis of population doubling level (PDL). Telomere length and telomerase activity of LAK cells were measured by the hybridization with oligonucleotide (TTAGGG)4 and by the telomeric repeat amplification protocol (TRAP) assay, respectively. Effects on telomerase activity in LAK cells of serum from cancer patients, transforming growth factor (TGF)-beta, and IL-10 were also examined. The lifespan of ca-LAK (15.2 +/- 5.1 PDLs) was significantly shorter than that of he-LAK (22.6 +/- 8.3 PDLs) (p = 0.0358). There were no significant differences between he- and ca-LAK in telomere length before IL-2/CD3 stimulation and maximal telomerase activity induced. The telomerase activity induced in ca-LAK failed to elongate sufficiently the telomeric ends (-35.2 +/- 46.2 bp) compared with that in he-LAK (16.8 +/- 41.5 bp) (p = 0.0448). The telomerase activity was initially detectable on day 2 in all he-LAK, whereas 8 (61.5%) of 13 ca-LAK expressed telomerase activity on day 3 or later following the stimulation, showing a significant retardation of telomerase expression (p = 0.0116). The addition to the LAK cell generation system of serum from cancer patients, as well as IL-10, but not transforming growth factor (TGF)-beta, suppressed the telomerase activity. This serum-induced suppression of telomerase activity in LAK cells was abrogated with the addition of anti-IL-10 antibody but not with anti-TGF-beta antibody. It is suggested that the dysregulation of telomerase activity and expression exists in LAK cells of cancer patients, resulting in the impairment of LAK cell generation in patients with advanced cancer. Serum IL-10 may be involved in the impairment of LAK cell generation by the suppression of telomerase activity of lymphocytes in vivo. Thus, the dysregulation mechanism of telomerase activity and expression in lymphocytes of cancer patients may be attributable, in part, to cancer-associated immunosuppression.

Use of acidic and basic pH and calcium ion addition in the capillary zone electrophoretic characterization of recombinant human deoxyribonuclease, a complex phosphoglycoprotein.

This paper describes the analysis of recombinant human deoxyribonuclease (rhDNAse), an acidic and complex phosphoglycoprotein, by capillary zone electrophoresis (CZE). Separation performance was found to be dramatically improved by the addition of calcium ions to the CZE running buffer, due to the influence of calcium binding on the charge and the electrophoretic behavior of rhDNAse. The pH dependent calcium binding effects on the electrophoretic separation were demonstrated at both acidic and basic pH, resulting in a two-dimensional (pH 4.8 and 8.0) calcium aided analysis that achieved multipeak resolution of the complex, glycosylation based, charge microheterogeneity of rhDNAse. Two-dimensional investigation of neuraminidase- and alkaline phosphatase-digested protein further demonstrated that the acidic pH resolved acidic charge heterogeneity and that the basic pH discriminated neutral heterogeneity. This work demonstrates the resolving power of CZE for the analysis of a complex microheterogeneous glycoprotein, and emphasizes the importance of employing multiple separation conditions in accordance with known structural characteristics of the protein.

Human DNase I contains mannose 6-phosphate and binds the cation-independent mannose 6-phosphate receptor.

DNase I isolated from human urine (hDNase) or expressed in Chinese hamster ovary (CHO) cells contains mannose-phosphorylated oligosaccharides. hDNase binds to a column of immobilized cation-independent mannose 6-phosphate receptor, with the strongest binding exhibited by the protein bearing diphosphorylated oligosaccharides. The binding is inhibited by 5 mM mannose 6-phosphate, and can be prevented by prior treatment of hDNase with alkaline phosphatase. Phosphorylated high-mannose oligosaccharides were observed at both sites of glycosylation in hDNase by high-performance liquid chromatography-mass spectrometry of a tryptic digest. These results indicate that hDNase, though not an acid hydrolase, may enter the lysosomal trafficking pathway, and may have evolved from a lysosomal enzyme.

Immunoglobulins of the human amniotic fluid.

PROBLEM: Except for the description of a secretory immunoglobulin (S-Ig) of a low size, no recent study has investigated the molecular status of antibodies in the human amniotic fluid. METHOD: After separation with a high performance chromatography, we analyzed the different isotypes of amniotic Igs by immunoblotting and ELISA. RESULTS: IgG is found to be the major isotype and to contain mother-derived tetanus antitoxins. IgA is much less abundant, whereas no IgM can be detected. IgA is monomeric, with a low level of secretory IgA and with various amounts of free secretory component (SC). The presence of a low level of SC-containing immunoglobulin of a low size is confirmed during the last trimester of pregnancy. This molecule contains no alpha chain but includes a Fabgamma fragment noncovalently associated with SC. IgG, IgA, and SC are detected in the fetal urine and, therefore, can reach the amniotic fluid by this route. CONCLUSION: In addition to the predominant maternal IgG, the amniotic fluid contains different molecular forms of fetal immunoglobulins. Their function as an immune barrier against infection and against mother-derived autoantibodies is discussed.

Protein sorting by high-performance liquid chromatography. I. Biomimetic interaction chromatography of recombinant human deoxyribonuclease I on polyionic stationary phases.

Chromatographic separations can be tailored to exploit specific interactions between a stationary phase ligand and a protein structural feature of interest. Variations in this feature then form the basis for sorting a mixture of closely related proteins into defined subpopulations. This report describes the sorting of variants of recombinant human deoxyribonuclease I (rhDNase) that differ in the occurrence of deamidation at a single residue. rhDNase, an enzyme that non-specifically hydrolyzes DNA, is glycosylated and exhibits considerable charge heterogeneity owing to the sialylation and phosphorylation of its N-linked oligosaccharides. This heterogeneity obscures the relatively subtle differences between deamidated and intact rhDNase, preventing separation on this basis in conventional ion-exchange HPLC. Published structural information on bovine DNase reveals that the analogous labile asparagine residue is involved in DNA binding, so stationary phases containing polyanionic ligands mimicking nucleic acids were employed to separate the deamidation variants of rhDNase. Electrostatically immobilized DNA, a "tentacle" cation exchanger (TCX) and immobilized heparin columns all resolved the deamidated and intact forms of rhDNase when operated at pH 4.5. The ligands of the TCX and heparin columns are sufficiently long, flexible and polyanionic to interact with rhDNase in a manner similar to DNA and to sort rhDNase variants on the basis of the charge difference of a single residue involved in that interaction. A non-hydrolyzable double-stranded oligonucleotide analogue of DNA was also synthesized and immobilized to an HPLC support. This column, operated at pH 6, where rhDNase is active, resolved the two isomeric products of deamidation of rhDNase, i.e., variants of the enzyme containing either aspartate or isoaspartate in lieu of asparagine at the deamidation site in rhDNase. This is the first reported separation of intact variants of a glycoprotein differing on the basis of these isomeric products of deamidation through the common cyclic imide mechanism.

Mucosal immunity induced by intramuscular administration of free peptides in-line with PADRE: IgA antibodies to the ELDKWA epitope of HIV gp41.

To improve the mucosal antibody response against a short amino acid (aa) sequence (ELDKWA) of HIV gp41, we have investigated a construction including this peptide in-line with the Pan DR epitope (PADRE). ELDKWA is a conserved peptide playing a key role in the pathogenicity of HIV transmission. PADRE is a non-natural peptide with multipotential immunogenic properties. The results show striking differences between mucosal and systemic immune systems, with a preferential response of the mucosal organs. In contrast with most mucosal immunizations, the intracellular response persists for over two months after the last injection. This strongly suggests that further investigations of conserved key epitopes from various pathogens may lead to safe and chemically defined mucosal vaccines with synthetic peptides. These candidate vaccines with free peptides may be suitable for mass campaigns even in developing countries.

Protein sorting by high performance liquid chromatography. 2. Separation of isophosphorylates of recombinant human DNase I on a polyethylenimine column.

Anion exchange HPLC with a polyethylenimine (PEI) column separates recombinant human deoxyribonuclease I (rhDNase) glycoforms according to the extent and positions of phosphorylation of mannose residues in N-linked oligosaccharides. The separation provides a selectivity unavailable by anion exchange HPLC with other columns or by isoelectric focusing gel electrophoresis and can be used to quantify the phosphate content of preparations of rhDNase. Tryptic mapping of fractions collected from the column and treated with alkaline phosphatase was used to identify the sites of phosphorylation. Unnatural forms of rhDNase, bearing oligosaccharide structures at only one of the two sites of glycosylation, were prepared by cleaving the phosphate-containing high mannose and hybrid structures from the purified isophosphorylates fractionated on the PEI column. The separation of rhDNase isophosphorylates on the PEI column mimics the relative affinities for the mannose 6-phosphate receptor that traffics acid hydrolases to lysosomes and provides a useful example of protein sorting by biomimetic interaction chromatography.

Human amniotic IgA inhibits natural IgG autoantibodies of maternal or unrelated origin.

We show that the natural autoantibody activity of amniotic IgG dramatically increases after purification, and that the IgG-depleted fraction can suppress the activity of IgG natural antibodies from amniotic fluid or from the maternal serum. This suppression is also observed towards serum IgG from unrelated adults but does not impair the tetanus antitoxin activity of serum-derived IgG. Absorption experiments and immunoglobulin separation by gel permeation demonstrate that this suppression is due to monomeric immunoglobulins of the IgA isotype. The inhibition is associated with an anti-F(ab')2 activity of the amniotic IgA, involving hypervariable regions of the IgG as demonstrated by different reactivities towards monoclonal IgG sharing the same family of VH and Vkappa domains. These results indicate that the inhibition of natural autoantibodies not only occurs with fetal and adult serum IgM, as reported by other groups, but also with amniotic IgA, suggesting a general and important phenomenon. In the case of the amniotic fluid, IgA could protect the fetus against maternal IgG autoantibodies without interfering with simultaneously translocated antigen-induced IgG antibodies to pathogens.

Homology of partial primary sequences between alpha-enolase and a suppressive lymphokine from human T cells.

A relationship is described between TS--a human suppressive lymphokine produced by hybridoma T cells--and alpha-enolase from the U937 monocyte human cell line. A strong homology (92%) was observed by comparing the internal amino acid sequences of 6 TS peptides, corresponding to 52 residues, with the complete sequence of alpha-enolase. The molecular masses of these two molecules were found to be of about 47 kDa and both were detected with the same monoclonal antibody to TS. In contrast, no immunosuppressive activity was detected with the purified enolase fraction, whereas TS did not show any enolase activity. A suppressive monokine secreted by U937 cells was found to be unrelated with TS. Theses results suggest that the TS immunosuppressive factor despite its absence of enzyme activity, belongs to the enolase family.

Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans.

Secretory immunoglobulin A (S-IgA) was investigated in human secretions for the presence of natural antibodies (Abs) acting as the first "immune barrier" to infection before induction or boosting of specific responses. These molecules could be the secretory counterpart of the natural Abs in serum that were previously shown by our laboratory to be polyreactive to autoantigens. Significant levels of S-IgA Abs to human actin, myosin, tubulin, and spectrin were detected in 10 saliva and 8 colostrum samples from normal subjects. Computer-assisted analysis of immunoblots of extracts from human muscles showed these Abs to react with a large number of autoantigens. Their polyreactivity was confirmed by cross-inhibition and by immunoblotting studies of affinity-purified natural Abs, assayed against a large variety of surface or secreted antigens from Streptococcus pyogenes. The thiocyanate elution method showed that functional affinities of some natural Abs can be of the same order of magnitude as those of tetanus vaccine antitoxins. Moreover, nonimmune binding of these natural Abs to the gut protein Fv (Fv-fragment binding protein) can enhance their effector functions. This demonstrates that human secretions contain polyreactive auto-Abs which can also react with pathogens. These secretory Abs of "skeleton key" specificities are possibly produced by a primordial B-1-cell-associated immune system and can be involved in a plurispecific mucosal protection against pathogens, irrespective of the conventional immune response.

Characterization of a tryptic digest by high-performance displacement chromatography and mass spectrometry.

High-performance displacement chromatography (HPDC) provides a means of increasing the capacity of a chromatographic column, while maintaining the resolution afforded by high-performance liquid chromatographic (HPLC) instruments. The high capacity and high resolution of HPDC can be exploited in tryptic mapping to facilitate the characterization of a protein preparation. In this manner, minor constituents of the mixture, which may be difficult to isolate by conventional chromatographic methods, can be obtained in sufficient amounts to permit chemical characterization by established techniques. The isolation by HPDC of peptides obtained by digestion of recombinant human growth hormone (rhGH) and the subsequent characterization of the peptides are described. The identification of certain of these peptides revealed information on the specificity of trypsin for the substrate, rhGH, and for autolysis. Fractions from the HPDC tryptic map were collected and analyzed by electrospray ionization mass spectrometry (ESI-MS) either directly or following further separation by gradient elution HPLC. Fragment ions observed in the ESI mass spectra facilitated identification of peptides obtained by HPDC tryptic mapping.

Protection by human serum from the immunosuppression induced by spermine in vitro.

The in vitro suppressive activity of spermine to PHA-induced proliferation of human T lymphocytes is shown to be abolished by normal human serum. This protection acts within the first 4 hr of culture and is due to a protein of 67 kDa, showing an isoelectric charge of pH 4.9. This protein does not bind, or modify, spermine and does not inhibit spermine oxidase activity, an enzyme required for the in vitro suppression. Results with acrolein confirm that this eventual cleavage product is probably not involved in the spermine-induced suppression. Nevertheless, since serum also reduces acrolein-induced suppression, it is probable that both these protective mechanisms are related. Such a protection of T lymphocytes by a serum factor may play an important role by preventing diffusion, outside the genital tract area, of a potential spermine-induced immunosuppression.

Different dysregulations of the natural antibody repertoire in treated and untreated HIV-1 patients.

To investigate a possible dysregulation of the autoantibody network in AIDS patients, the relative activity of representative natural antibodies was measured in serum IgG and IgM. These immunoglobulins were purified from two cohorts of 20 HIV-infected patients undergoing, or not, a triple combination therapy. A cohort of 20 normal patients was used as a control. Marked alterations of the natural antibody repertoire were observed, varying according to the isotype and specificity of the antibody studied. For the classical self-protein antigens, human actin and myosin, the changes observed in the untreated cohort were absent in the treated cohort. In contrast, no changes, or even increased changes of the activity of antibodies to special antigens, DNA and TNP, occurred in the treated cohort. The differences were highly significant, indicating that this repertoire is regulated and not randomly modified by the disease. These results suggest the presence of different factors of dysregulation of the B cell repertoire of natural antibodies associated with the disease as well as with the treatment. These major dysregulations may favor the autoimmune phenomena observed during HIV infection.

High affinity serum-derived Fab fragments as another source of antibodies in the gut lumen of both neonates and adults.

The authors have investigated the presence of serum-derived immunoglobulin G (IgG) fragments in the human intestine at various ages, these fragments possibly representing another source of antibodies in addition to secretory IgA (SIgA). Fab fragments of the gamma isotype were found to be the major molecular form of immunoglobulins in the meconium (median value: 3.7 mg/g of stools), as compared with Fab alpha (75 micrograms/g) and IgM (2.6 micrograms/g). These fragments provided by molecules of the maternal serum displayed a strong antibody activity to the tetanus toxoid and were also found in the stools of 1-week-old babies fed with formula milk. The release of serum antibodies into the digestive lumen occurs largely via hepatobiliary secretions, as suggested by the presence of IgG antitoxins in the bile of children operated on extrahepatic biliary atresia. In adults, the Fab antitoxins were also detected in most stool extracts. Affinity of these molecules was found to be similar to that of their serum counterpart with a Ka of 2.1 x 10(10) M1 (median value). These mucosal antibody fragments, associated with the normal pathway of serum IgG catabolism, could provide additional immune defences against pathogens, and be of importance to supplement an immature or deficient secretory immune system.

Susceptibility of rhDNase I to glycation in the dry-powder state.

The accumulated data on the glycation process in rhDNase I, formulated with lactose and stored in the dry-powder state, indicates that this protein becomes covalently modified with lactose at five of the six lysines (2, 50, 77, 157, 260) and to a lesser extent on the amino terminus. Analysis of the three-dimensional protein structure indicates that the reported requirements for the specificity of site reactivity, site accessibility, and the presence of a proton donor/acceptor group near the reaction site, are maintained in this protein. A chemical reaction in the dry-powder state may become permissible simply due to the close packing and resultant high concentrations of the reactant molecules. The reaction between reducing sugar and protein in the dried state indicates that the arrangement of molecules within the dry-powder particle allows for direct contact between all the reactants, including contacts between protein molecules, which may contribute to the completion of the covalent reaction at a surface-accessible reactive site in which the required surrounding microenvironment for self-catalysis is available on only 35% of the individual molecules. These findings should indicate that the use of reducing sugars in the formulation of an excipient for the stabilization of dried protein is not advisable, as reaction clearly occurs between the reducing sugar and protein because of the potentially reactive nature of the proteins' accessible amino groups.

Suppressor factor secreted by T hybridoma established from peripheral blood lymphocytes of a bone marrow transplantation patient. I. Establishment of human T-cell hybridoma and partial characterization of suppressor factor.

A stable human T-cell hybridoma was established by cell fusion between activated human peripheral blood lymphocytes from an allogeneic bone marrow transplantation patient and the JD1-17 cell line, a subclone of the human T leukemia Jurkat cell line. This hybrid clone 1-8, which bore the surface phenotype of suppressor cells (CD8+HNK1+), spontaneously secreted a factor which, at high dilutions, suppressed the responses of T and B cells induced by mitogens and alloantigens. This suppressor factor was found to be heat-resistant (56 degrees C, 30 min), stable at alkaline but not acid pH, unaffected by 2-mercaptoethanol, and sensitive to trypsin. Preparative isoelectric focusing revealed an isoelectric point of 5.35. The suppressor activity was selectively absorbed by blast T cells. By gel filtration on Sephacryl S-200 and HPLC, the suppressor activity was found in two peaks corresponding to 40-45 kDa (monomer) and 90-95 kDa (dimer).

Delineation between T and B suppressive molecules from human seminal plasma: II. Spermine is the major suppressor of T-lymphocytes in vitro.

The nature of the human semen T-suppressor was investigated in vitro on human lymphocyte proliferations induced by phytohemagglutinin (PHA) or by alloantigens. Purification by ion-exchange chromatography, followed by butanol extraction, showed this factor to be present only in the polyamine-containing fractions. The purified product, obtained by preparative thin-layer electrophoresis, contained almost exclusively spermine and exhibited the same suppressive activity as this polyamine. Human T-lymphocyte suppression occurred in the presence of fetal calf serum, but it did not occur in a serum-free medium. No suppression was observed after preincubation of the fetal calf serum with hydroxylamine, a spermine oxidase inhibitor, whereas a nondialyzable fraction, from normal human serum, decreased the suppression. The semen factor did not act by direct cytotoxicity, as there was no effect of preincubation and suppression could be induced only within the first 6 hr of mitogen activation. These data demonstrate that the in vitro T-suppressive activity of semen can be assigned mainly to spermine and show that in vivo this suppression must require locally the presence of a spermine oxidase or related enzyme.