Cross-reactivity of a normal human cell surface antigen with primate retrovirus glycoproteins

Normal human cells express a human-specific antigen, HuLy-m5 (defined by the E4.3 monoclonal antibody), cross-reactive with determinants of the primate retroviruses, MPMV(Mason Pfizer monkey virus) and GALV (gibbon ape leukemia virus). Purified virus preparations of MPMV and GALV absorbed E4.3 antibody activity while antisera to these retroviruses blocked the binding of E4.3 antibody to human target cells. Sequential immunoprecipitation and two-dimensional gel analysis both indicated that the anti-primate retrovirus sera recognize the same molecular entity (a two-chain glycoprotein of Mr60 and 69Kd) as does the E4.3 antibody. These results suggest that normal human cells express primate retroviral proteins (most probably viral envelope glycoprotein, gp69) at the cell surface.     

Expression of tumor-associated Thomsen-Friedenreich antigen (T Ag) in Helicobacter pylori and modulation of T Ag specific immune response in infected individuals

We tested the hypothesis that the gastric cancer associated bacteria, Helicobacter pylori (H. pylori) express the cancer-related Thomsen-Friedenreich (T) antigen. We also analysed whether infection with H. pylori alters the amount of natural anti-T antibodies in the patients' sera. Cell surface membrane extracts of H. pylori NCTC 11637 strain and clinical isolates of H. pylori (n = 13) were analysed by immunoblotting and cell-ELISA with five different T antigen-specific monoclonal antibodies (MAbs). Two major protein bands of approximately 68 kDa and 58 kDa were immunostained on blots of H. pylori extracts with T specific MAbs but not immunostained with unrelated MAb. The specificity was shown in that immunostaining was blocked with peanut agglutinin (PNA) and rabbit antiserum to T antigen. The binding of T specific MAb to the 58 kDa protein band was also blocked by rabbit antiserum against heat shock proteins of H. pylori. The relative expression of T antigen-related proteins differed among H. pylori strains, with 68 kD associated T antigen expression higher in patients with more severe pathology. The level of IgG antibody to T epitope in patients with gastric cancer (n = 66) and normal blood donors (n = 62) were compared and the level of anti-T Ab in gastric cancer patients was significantly lower than that in normal blood donors. A significant positive correlation between T specific antibody in serum and H. pylori IgG antibody level was found in H. pylori-infected normal blood donors (P < 0.001), but this correlation was not found in H. pylori-infected cancer patients. In summary, the cancer related T epitope is expressed in H. pylori and modulation of T antigen-specific immune response in H. pylori-infected individuals suggests that H. pylori infection may alter natural immune mechanisms against cancer.     

Studies on the Thomsen-Friedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen.

The lectin Amaranthin has been shown to be highly specific for the galactose beta 1,3 N-acetylgalactosamine-alpha and sialic acid alpha 2,3 galactose beta 1,3 N-acetylgalactosamine-alpha sequence which represents the Thomsen-Friedenreich (T) antigen and its cryptic form, respectively. Previously, we demonstrated the usefulness of gold-labeled Amaranthin for the histochemical detection of the T antigen and its cryptic form. Application of the galactose oxidase (GO)-Schiff sequence abolished lectin binding to the T antigen but not its cryptic form, and therefore permitted their differentiation. In the present study we have analyzed by light and electron microscopy the distribution and subcellular localization of Amaranthin binding sites in normal, dysplastic and neoplastic colonic epithelium. Furthermore, a monoclonal antibody raised against synthetic galactose bera 1,3 N-acetylgalactosamine-alpha-bovine serum albumin was applied as a reagent for the T antigen. In normal colonic mucosa, two different Amaranthin staining patterns existed: (a) reactivity restricted to the lower portion of the crypts which was principally observed in the left colon, and (b) reactivity along the entire length of the crypts and in the surface epithelium with goblet cell staining in the upper portion of the crypts which was principally observed in the right colon. This Amaranthin staining was resistant to GO-Schiff treatment. No immunostaining with the monoclonal anti-T antigen was observed. Investigation of transitional mucosa, adenocarcinomas of different degrees of differentiation and mucinous carcinomas as well as adenomas with different degrees of dysplasia all revealed positive Amaranthin staining. The lectin staining was resistant to GO-Schiff treatment, and immunolabeling with the monoclonal antibody against the T antigen was absent. These results indicate that only the cryptic form of the T antigen is expressed in normal, dysplastic and neoplastic human colonic epithelium.     

Asthma and urticaria induced by seminal plasma in a woman with IgE antibody and T-lymphocyte responsiveness to a seminal plasma antigen

A patient with asthma urticaria and angioedema induced by allergy to seminal plasma was examined at intervals for 10 years. Before treatment her anaphylactic susceptibility to seminal plasma was manifested by very strong prick-test responses, IgE antibody to an allergenic fraction of seminal plasma determined by RAST, and by antigen-induced histamine release from her blood leucocytes. The skin test and in vitro lymphocyte tests indicated concomitant delayed hypersensitivity to the same allergen. The patient's lymphocytes treated with seminal plasma allergen fraction showed much increased incorporation of thymidine, and also synthesis of a product (NIF) that inhibited migration of neutrophils from a normal donor. The allergen fraction of seminal plasma had about five components in the range of 20000–40000 daltons molecular weight; the major fraction binding IgE appeared to be a glycoprotein. The patient was successfully desensitized by injections of her husban?s seminal plasma. Desensitization was not associated with persistent amounts of antigen-specific IgG antibodies.     

Presence of antibodies to sperm YLP(12) synthetic peptide in sera and seminal plasma of immunoinfertile men

Using an enzyme-linked immunosorbent assay (ELISA), sera (n = 15) and seminal plasma (n = 30) from antisperm antibody-positive immunoinfertile men (n = 45) and from fertile men (n = 45), were tested for the immunoreactivity with the synthetic YLP(12) sperm peptide. Of the 15 immunoinfertile sera tested, 46% were positive for immunoglobulin (Ig)M, 73% for IgG, and 40% for IgA. Of the 30 samples of immunoinfertile seminal plasma tested, 10% were positive for IgM, 20% for IgG, and 43% for IgA. None of the sera or seminal plasma from fertile men showed a positive reaction. There was no significant correlation between the sperm immobilization technique (SIT) or tray agglutination technique (TAT) titres or percentage binding in immunobead binding technique (IBT) and the antibody reactivity for any class in the ELISA. The YLP(12) peptide conjugated to bovine serum albumin-Sepharose 4B beads pulled out IgG antibodies from the serum of the immunoinfertile, but not the fertile, men. The beads pulled out IgA antibodies from the immunoinfertile, but not the fertile, seminal plasma. The immuno-affinity purified antipeptide antibodies reacted with a specific band of 72 +/- 5 kDa in the human testis and with a specific band of approximately 50 +/- 5 kDa in the human sperm extracts. The YLP(12) peptide may have applications in the specific diagnosis and treatment of male infertility and in contraceptive vaccine development.     

Tn epitopes, immunoreactive with ordinary anti-Tn antibodies, on normal, desialylated human erythrocytes and on Thomsen-Friedenreich antigen isolated therefrom

Hybridoma generation, using specifically, maximally desialylated human blood group O erythrocytes (T RBC) as immunogen, and biochemical studies suggested the presence of immunogenic Tn epitopes. GalNAc alpha-O, on T RBC. We therefore investigated by immunochemical means whether or not Tn-specific epitopes immunoreactive with anti-Tn antibodies present in ordinary human sera occur on T RBC and on Thomsen-Friedenreich (T) antigen prepared from them. We did detect the Tn epitope with such antibodies, in addition to the T epitope, on isolated T antigen. T RBC absorbed specifically, under standard conditions, 25-60% of the heterogeneous anti-Tn antibody populations in ordinary human sera of appropriately adjusted titer score. The anti-Tn eluted from T RBC had scores ranging from 6.5 to 35% of those of the unabsorbed parent sera. The varying fine specificities of eluted anti-Tn were demonstrated by inhibition of Tn RBC agglutination with putative haptens and antigens. Tn-specific haptens and antigens were the most powerful inhibitors. Depending on the serum used to prepare the anti-Tn eluates, the antibodies could be divided into those that were inhibited well exclusively by GalNAc alpha-O derivatives and those that were also inhibited by Gal, notably by Gal alpha-O derivatives and more strongly by GalNAc and Me-alpha-GalNAc. In the two reciprocal hemagglutination inhibition systems used, Tn-specific haptens were considerably more active than the T-hapten Gal beta 1----3GalNAc alpha-O, and desialylated ovine submaxillary mucin (AS-OSM) had higher activity than T antigen. Inhibition of Tn RBC agglutination by haptens was uniformly more efficient than that of T RBC; this is, at least in part, due to the much higher negative charge of Tn as opposed to T RBC. In microprecipitin tests, Helix pomatia lectin was nearly as powerful a precipitin of T antigen as of AS-OSM. The importance of the terminal GalNAc alpha of T antigen for its precipitation with the Helix lectin was demonstrated by the very high and virtually exclusive inhibitory activity of Me-alpha-GalNAc and GalNAc. Our findings may contribute to comprehension of the significance of uncovered Tn in most carcinomas, and the role of anti-Tn as a "natural" anti-carcinoma antibody. They may also help illuminate the rare heterozygous, autosomal, apparently premalignant spot mutation that leads to Tn RBC in vivo.     

Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes

The CAMPATH-1 (CD52) antigen is a 21–28 kDa glycopeptidewhich is highly expressed on lymphocytes and macrophages andis coupled to the membrane by a glycosylphosphatidylinositol(GPI) anchoring structure. The function of this molecule isunknown. However, it is an extremely good target for complement-mediatedattack and antibody-mediated cellular cytotoxicity. The humanizedCAMPATH-1H antibody, which is directed against CD52, is veryefficient at mediating lymphocyte depletion in vivo, and iscurrently being used in clinical trials for lymphoid malignancyand rheumatoid arthritis. It is therefore important to examinethe functional effects of this antibody on different lymphocytesub-populations. Because several other GPI-linked moleculesexpressed on the surface of T lymphocytes are capable of signaltransductlon resulting in cell proliferation, we have investigatedwhether the CAMPATH-1 antigen can also mediate these effects.In the presence of phorbol esters and cross-linking anti-lgantibodies, mAbs specific for CD52 induced proliferation andlymphokine production in highly purified resting CD4⁺ and CD8⁺T lymphocytes. The ret lgG2c YTH 361.10 anti-CD52 antibody,however, was able to activate resting CD4⁺ and CD8⁺ T cellsdirectly without cross-linking or phorbol myristate acetatein the absence of Fc-bearlng cells. Anti-CD52 antibodies alsoaugmented the anti-CD3 mediated proliferatlve response of CD4⁺and CD8⁺ T cells when the two antibodies were co-immobilizedonto the same surface or cross-linked in solution by the samesecond antibody. Both CD4⁺CD45RA and CD4⁺CD45RO T cells werestimulated to proliferate by anti-CD52 antibodies in the presenceof appropriate co-stimulatory factors. Antl-CD52 mAbs did not,however, synerglze with anti-CD2 or CD28 mAb to induce CD4⁺T cell proliferation. The activation of CD4⁺ T cells by antl-CD52antibodies was inhibited by cyclosporin A, suggesting a rolefor the calcineurin-dependent signal transduction pathways.Although CD52 could transduce a signal In T cells, anti-CD52antibodies did not inhibit antigen-specific or polyclonal Tcell responses, suggesting this molecule does not play an essentialco-stimulatory role in normal T cell activation.     

Autoimmune T cell responses to seminal plasma in chronic pelvic pain syndrome (CPPS)

The aetiology of chronic prostatitis is not understood. The aim of this study is to investigate an autoimmune hypothesis by looking for T cell proliferation in response to proteins of the seminal plasma. We studied peripheral blood mononuclear cell proliferation from 20 patients with chronic prostatitis and 20 aged-matched controls in response to serial dilutions of seminal plasma (SP) from themselves (autologous SP) and from a healthy individual without the disease (allo-SP). We found that the patients have a statistically greater lymphocyte proliferation to autologous SP at the 1/50 dilution on day 6 compared to controls (P = 0 x 01). They also have a greater proliferation to allo-SP on both day 5 (P = 0 x 001) and day 6 (P = 0 x 01) at the same dilution. Using a stimulation index (SI) of 9 to either autologous SP or allo-SP on day 6 at the 1/50 dilution as a definition of a proliferative response to SP, then 13/20 patients as compared to 3/20 controls showed a proliferative response to SP (P = 0 x 003, Fishers exact test). These data support an autoimmune hypothesis for chronic prostatitis.     

Serine protease inhibitors (serpins) in human seminal plasma: Concentrations and inhibition of acrosin

Acrosin is a trypsin-like serine protease present in its zymogen form in the acrosome of spermatozoa. The activated enzyme is thought to digest a pathway for the sperm through the zona pellucida of the ovum during the process of fertilisation. We have shown that in a purified system boar acrosin was inhibited by human protein C inhibitor with an apparent second order rate constant (k_app) of 3.7×10⁴M⁻¹s⁻¹ 1. Protein C inhibitor is present in high concentrations in seminal plasma and endogenous protein C inhibitor was found in the immediate vicinity of disrupted acrosomal membranes of washed human spermatozoa. This serpin could therefore function as a scavenger of prematurely activated acrosin in the male reproductive tract.Since little is known about the interaction of acrosin with other serpin type inhibitors, we analysed in this study the interaction of boar acrosin with other purified human serpins. Antithrombin III, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and α₁-antitrypsin inhibited acrosin activity. The following apparent k_apps were calculated: Antithrombin III: 19.5×10⁴ M⁻¹s⁻¹, plasminogen activator inhibitor-1: 21.5×10⁴M⁻¹s⁻¹, plasminogen activator inhibitor-2: 3.3×10⁴M⁻¹s¹, α₁-antitrypsin: 0.09×10⁴M⁻¹s¹. α₂-antiplasmin and heparin cofactor II did not inhibit acrosin. SDS-stable acrosin/serpin complexes were only seen with antithrombin III; all other acrosin inhibitors were cleaved by the enzyme.As determined by enzyme-linked immunosorbent assays, the concentrations of protein C inhibitor, plasminogen activator inhibitor-1, and plasminogen activator inhibitor-2 in individual seminal plasma samples from healthy donors were 5.33±0.47 μM, 88±24 pM and 163±17 pM (means±SD), respectively. The concentrations of antithrombin III in seminal plasma were ⩽50 nM (semiquantitative immunoblotting). Therefore considering both, the k_app calculated for the inhibition of boar acrosin in a purified system and the concentration of each serpin in seminal plasma, protein C inhibitor seems to be the best candidate to function as a physiological acrosin inhibitor in the male reproductive tract.     

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.     

Immunogenetic studies of soluble Fc receptor (CD 16) antigens in human seminal plasma

Abstract: Two forms of CD 16 Fc receptors (FcRMIII) have been defined on human leukocytes. Type 1, present on polymorphonuclear neutrophils (PMN), expresses the NA alloantigen system and type 2, present on natural-killer (NK) cells and macrophages, has no known polymorphic variations. We have described the presence of soluble FcRMIII antigens in human seminal plasma (SP) (see Ref. 9). These SP antigens retain an affinity for IgG-Fc and are biochemically distinct from leukocytic FcRMIII. Their origin within the male reproductive tract and how they relate to FcRMIII types 1 and 2 were not known. By using monoclonal antibodies CLB/Gran 11 and Leu lie that differentially react with FcRMIII type 1 and type 2 from different individuals, we studied how SP FcRMIII antigens relate to leukocytic FcRMIII. CLB/Gran 11 and Leu 11c reactivities with PMN and NK cells from a selected panel of male donors were compared with enzyme-linked immunosorbent assay reactivities of CLB/Gran 11 and Leu lie with SP samples from these donors. The reactivity pattern with SP samples was shown to be different from findings with donors'PMN, but it corresponded with their NK cells. Our results indicate that SP FcRMIII antigens do not manifest the polymorphic variations that are detected by CLB/Gran 11 and Leu 11c on PMN. These findings suggest that SP FcRMIII antigens do not originate from PMN.     

Radioassays of blood group M, N and T (Thomsen-Friedenreich) antigens.

Radioassays employing the double-antibody or Farr techniques were developed for the M, N and T antigens. Blood group glycoproteins were isolated by butanol extraction of red cell stroma and iodinated by the chloramine-T technique. The final purity of glycoprotein was over 75% as judged by radioimmunoassay (RIA). T activation of glycoprotein was obtained with neuraminidase. A specific RIA was obtained for the M antigen and was sensitive to approximately 10 ng of glycoprotein or glycopeptide. In the RIA system rabbit anti-M displayed a higher affinity for M glycoprotein than for M glycopeptide. A RIA that was entirely specific for the N antigen, could not be obtained. A radioassay, obtained for the T antigen with peanut agglutinin in the Farr technique, was sensitive to approximately 100 ng of T antigen and was readily inhibitable by monosaccharides. A RIA, obtained for the T antigen with rabbit anti-T, was entirely specific and sensitive to approximately 1 ng of T activated glycoprotein or glycopeptide but was not inhibitable by monosaccharides.     

Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma.

We report on the existence of two kinds of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in human seminal plasma. Partial purification of the proteinases was achieved by two steps, consisting of chromatography on a gel-filtration column and then on a gelatin affinity column. Proteinase activities in the chromatography extracts were shown to hydrolyse a fluorescent substrate specific to MMPs (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2). The proteinases were detected using gelatin-zymography, but were not detected using casein-zymography, and were also inhibited by EDTA, EGTA and o-phenanthroline. Molecular weights of the proteinases were determined by SDS-PAGE, gelatin-zymography and Western blot to be approximately 92, 84, 72, 67, 52 and 45 kDa. Gelatin-zymography showed three major bands of activity at 72, 67 and 52 kDa and minor bands at 92, 84 and 45 kDa. Apart from the two smallest bands, these proteinases were all recognized by the polyclonal antibodies for MMP-2 or MMP-9. These results indicate that two kinds of pro-form and active-form matrix metalloproteinases, MMP-2 and MMP-9, and their degradation products, are present in human seminal plasma.     

Expression of CD176 (Thomsen-Friedenreich antigen) on lung, breast and liver cancer-initiating cells

The cancer-initiating capacity of most malignant tumours is considered to reside in a small subpopulation of cells. Therapeutical interventions should target these cells rather than the tumour mass. Numerous studies have shown that the carbohydrate antigen structure CD176 (Thomsen-Friedenreich antigen, core-1) is present in many types of cancer and absent in normal adult human tissues. In this study, we assessed whether CD176 is co-expressed with CD44 or CD133 [markers of cancer-initiating cells (CIC)] in human lung, breast and liver carcinoma. A variety of human cancer cell lines and surgical specimens of these malignancies were examined. It was found that in most cases the majority of tumour cells stained strongly for CD44 by immunohistochemistry and flow cytometry, whereas CD133 expression was found on a smaller, but varying proportion of cells. Co-expression of CD176 with CD44 was found at a surprisingly high percentage of cancer cells in vitro and in vivo. Co-expression of CD176 with CD133 was also detected, although at a lower rate. Tamoxifen treatment of MDA-435 breast cancer cells enhanced the CD44(+) /CD176(+) phenotype. Evidence is provided through a new sandwich solid-phase enzyme-linked immunosorbent assay (ELISA) suggesting that CD44 is a carrier molecule for CD176 not only in colorectal cancer as previously reported, but also in lung, breast and liver cancer. The expression of CD176 in CIC suggests that it may represent an effective target for tumour therapies.     

Comparative study of antigen binding T cells separated by Vicia villosa or streptococcal antigen and the effect of HLA class II antigens

The antigen binding capacity and function of T cells which adhere to the lectin Vicia villosa (VV) or to streptococcal antigen (SA) have been studied. VV-adherent T8+ cells (T8+ VV+) bind 125I-SA whereas VV non-adherent T8+ cells (T8+ VV-) bind little SA. Similarly, SA-adherent T8+ cells (T8+ SA+) bind 125I-SA specifically, whereas SA-non-adherent T8+ cells (T8+ SA-) show little binding of 125I-SA. The SA binding T8+ VV+ or T8+ SA+ cells can present the antigen to T4+ helper cells which generate helper factors and these enhance anti-DNP-antibodies, when incubated with mouse spleen cells and DNP-SA. Parallel reconstitution studies with either T8+ VV- or T8+ SA- cells have revealed that both subpopulations of cells can suppress T4+ helper cell activity. Further reconstitution experiments between the T8+ VV+ (or T8+ SA+) antigen presenting cells and T8+ VV- (or T8+ SA-) suppressor cells suggest that the former can also prevent the latter cells from inhibiting T4 helper cells, so as to function as contrasuppressor cells. Cross-over, reconstitution studies between the VV and SA separated cells have confirmed that the T8+ VV+ and T8+ SA+ cells have similar functions, in SA binding, presenting and contrasuppressor activities. The dose-response curve of binding 125I-SA to T8+ cells is dependent on the HLA-DR type of cells and the binding of 125I-tetanus toxoid was similar to that of 125I-SA. Whereas DRw6- T cells bind predominantly 1,000 ng SA or tetanus toxoid, DRw6+ T cells bind 1 ng and to a lesser extent 1,000 ng of either antigen. HLA-DRw6+ subjects can be considered as high responders for their T cells bind optimally at low concentrations of antigens which induce helper and contrasuppressor functions. In contrast, HLA-DRw6-subjects can be considered as low responders, as their T cells bind optimally at high concentrations of antigens and only the latter induce helper and contrasuppressor functions.     

The Australia (hepatitis-associated) antigen in fibrinogen and other fractions of human plasma

Human plasma containing the Australia (hepatitis-associated) antigen was fractionated by the cold ethanol method of Cohn, Strong, Hughes, Mulford, Ashworth, Melin, and Taylor (1946) and small aliquots were examined for the presence of this antigen by immunodiffusion and by electron microscopy. The findings were in general agreement with the postulated risk of transmitting hepatitis by blood derivatives. The Australia (hepatitis-associated) antigen was detected in fibrinogen, thrombin, and antihaemophilic globulin as well as in other fractions. The antigen was not found in gamma globulin (immunoglobulin fraction) nor in albumin.The use of radioiodinated fibrinogen for the diagnosis of deep vein thrombosis is discussed and it is concluded that the use of fibrinogen for diagnostic procedures should be assessed against the possible risk of hepatitis.     

Excretion patterns of glycosaminoglycans and glycoproteins in normal human urine

Glycosaminoglycans and glycoproteins in the urine of 100 healthy, active, human subjects were examined by cellulose acetate electrophoresis and salt gradient, ion-exchange, column chromatography. The cetylpyridinium chloride (CPC) turbidity and uronic acid:creatinine ratio was also studied. Fractions were identified by electrophoretic mobility, staining reactions, susceptibility to enzyme digestion, identification of amino- and neutral sugars, hexosamine, uronic acid, and sulphate assays, and optical rotation.The CPC turbidity is relatively high in childhood, falling to lower levels in adults, but rising again to relatively high levels in old age. The uronic acid: creatinine ratio is high in children, falling to a low level in adult life, and rising only slightly in old age.Three major electrophoretic fractions, corresponding with glycoprotein, heparan sulphates, and chondroitin sulphates, were identified in every urine sample. Hyaluronic acid was identified in some samples. A small amount of keratan sulphate was present in the ;heparan sulphate' fraction.Chondroitin sulphate excretion is high in children. Adults excrete relatively less chondroitin sulphate and more heparan sulphate. In old age, the proportion of glycoprotein increases. The excretion pattern in the first few days of life resembles that of the adult. It is stressed that extreme caution must be exercised in interpreting the urinary glycosaminoglycan pattern of a child.     

Immunochemical characterization of human TL-like (T48) and Ly 1-like (T72) glycoproteins using two-dimensional polyacrylamide gel electrophoresis

Xenoantisera, designated AT48 and AT72, were developed by immunizing rabbits with human thymus cell membrane and guinea-pigs with a T-cell glycoprotein purified from leukaemic T-cell membrane. Whereas AT48, after appropriate absorption, reacted exclusively with the majority of thymocytes (mainly cortical thymocytes) among normal lymphoid populations, AT72 reacted with virtually all of the thymus and T cells but not with B cells. Thymocytes, which were strongly reactive with AT72, existed in the thymic medulla, but cortical cells were also very weakly reactive with AT72. When cultured T-cell lines, all of which were derived from patients with T-cell-type acute lymphatic leukaemias, were tested for their reactivities with AT48 and AT72 by immunofluorescence, we found that AT48 stained certain T-cell lines, whereas AT72 stained all of the T-cell lines tested so far. Immunochemical data showed that AT48 precipitated a 48K molecular weight (mol. wt) glycoprotein from 125I-labelled thymus cell surface glycoproteins, which appeared to be very weakly associated with a 12K mol. wt component. These 48K and 12K mol. wt components precipitated by AT48 showed almost identical isoelectric points (pI) to those of HLA heavy chain and beta 2-microglobulin respectively. AT72, on the other hand, precipitated a 72K mol. wt glycoprotein from thymus and T cells as well as from leukaemic T cells. A less prominent 65K mol. wt glycoprotein was also precipitated by AT72 from thymus and T cells but not from leukaemic T cells. These two components showed almost identical pI ranging approximately from 4 to 7, and this marked charge heterogeneity observed was reduced by neuraminidase treatment, suggesting that it reflects the heterogeneity in sialylation of this molecular species. We concluded from these data that AT48 and AT72 used in this work could detect human homologues of mouse TL and Ly 1 antigens respectively.     

Presence of a 16 kd protein in human seminal plasma counteracts the effects of the anti-fertility agent, gossypol

Gossypol inhibited lactate dehydrogenase (LDH) noncompetitively in human spermatozoa. The inhibitory effect of gossypol on LDH was cancelled by the addition of human serum albumin, human gamma-globulin, bovine serum albumin or human seminal plasma. Seminal plasma was at least 10 times more effective than the other three proteins, when expressed on a per mg protein basis. Attempts were made to purify the active fraction from human seminal plasma. The purification steps included gel filtration, ammonium sulphate precipitation, centrifugal microconcentration and fast-performance liquid chromatography. A single active protein of Mr = 16,000 was purified to a final yield of 0.18%. The 16 kd protein was not observed in male blood plasma. The protein was found to be heat-stable and leucine-rich (16% of the molecule), and has been designated 'gossact'. The inhibitory effect of gossypol on the LDH reaction was completely blocked by the addition of gossact (5 micrograms/ml); human blood plasma (25 micrograms/ml) and human serum albumin (200 micrograms/ml) were far less potent in this assay. In addition, gossact bound 1.4 mol of gossypol/mol of protein with the dissociation constant (Kd) = 3.06 x 10(-5) M. The role of gossact in the protection of LDH from gossypol is discussed.     

Leucocyte common antigen expression on T cells in normal and inflamed human gut.

The expression of the 220,000 MW (p220) glycoprotein component of the leucocyte common antigen (LCA) family by intestinal mucosal lymphocytes was studied using the CD45R monoclonal antibody WR16. In normal intestine, a proportion of CD3+ mucosal T cells were WR16+ and this population resided predominantly in the mid-villus and crypt region of lamina propria. In the inflammatory infiltrates of both coeliac disease and Crohn's disease the CD3+, WR16+ population was markedly reduced. The monoclonal antibody UCHL1 identifies the 180,000 MW member of the LCA family and is expressed on T cells and in macrophages. CD3+ lymphocytes expressing this marker were widespread in normal lamina propria and epithelium. In contrast with WR16, UCHL1+ cells remained at a high level in coeliac disease and Crohn's disease. Our results support the view that loss of the p220 molecule occurs upon T-cell activation in inflammation.