Erythrocyte Membrane Phospahtidylcholine and Rh (D) Antigen Cryolatiency

The extent of binding of anti-D antibody to intact Rh (D) positive human erythrocytes at -2.5° was approximately one-third that at .3-°. An Arrhenius plot of the temperature dependence of antibody binding showed a clear and reproducible discontinuity at approximately 6-8°. Phospholipasc A² digestion of the intact erythrocytes resulted in a diminuticr. of exclusively phosphatidylcholine (PC) from the membrane, and an approximately parallel loss of Rh antigen activity at 37° to about 50% of the original. An Arrhenius slot showed no change in the temperature dependence below 6-8° but significant diminution above that point suggesting that the outer membrane PC is involved in Rh(D) antigen activity manifested above that temperature.     

Expression of Rh0(D) antigen in choriocarcinoma of the uterus in an Rh0(D)-negative patient: report of a case

Choriocarcinoma of the uterus developed in an Rh0(D)-negative woman six weeks after delivery of a normal Rh0(D)-positive child. Although the patient's medical history did not show any previous sensitization, a strong anti-Rh0(D) antibody was detected during evaluation preceding surgical removal of the tumor. The tumor was considered the most probable cause of the Rh0(D) sensitization, since large Rh0(D)-positive areas of tumor were demonstrated by the immunoperoxidase method. This finding indicates that the Rh0(D) antigen is not restricted to erythrocytes, as commonly assumed, but can also be expressed on other tissue, such as tumor tissue.     

H-Y Antigen Expression Patterns in Human X- and Y-Chromosome-Bearing Spermatozoa

PROBLEM: Restricted expression of H-Y antigen on Y-chromosome-bearing sperm has been reported in some species, although such preferential expression for H-Y antigen in human sperm has yet to be described. In this study, an immunomagnetic approach was used to characterize antigen expression patterns as a function of sex-chromosome content. METHOD OF STUDY: Human sperm was treated with monoclonal immunoglobulin (Ig) M antibodies directed against H-Y antigen. This preparation then was incubated with sheep antimouse IgM antibody affixed to paramagnetic beads, which then were exposed to a magnetic field and sorted. X- and Y-chromosome frequencies in the two subgroups of sperm were assayed by multiprobe fluorescent in situ hybridization (FISH). RESULTS: Sperm were immunomagnetically separated into two populations: a reactive group (presumably, H-Y Ag+); and a nonreactive group (presumably, H-Y Ag-). Triple-color FISH analysis of 1,600 spermatozoa (800 in each group) showed the antigen's expression to be somewhat more prevalent among Y-chromosome-bearing sperm (54.1%), but a large proportion of Y-chromosome-bearing sperm (49.0%) did not express this antigen. The difference was not significant (P = 0.43). CONCLUSIONS: The expression of H-Y antigen has a slightly higher frequency in human sperm containing the Y-chromosome, but its expression among X-chromosome-bearing sperm also is considerable. Current immunologic techniques relying on this antigen are unlikely to effect the sex selection of human sperm.     

Acrosin-Dependent Solubilization of Antigenic Peptides From Human Spermatozoa*

ABSTRACT: Human spermatozoal polypeptides have been solubilized by incubation of spermatozoa in phosphate-buffered saline. Inhibition of peptide release by the acrosin inhibitor N-α-tosyl-L-lysin-chloromethyl suggests the direct involvement of the acrosomal enzyme in the liberation of the peptides. The peptides were coupled to activated Sepharose 4B gel and the peptide-absorbed antibody of antispermatozoal antisera was detected with the aid of ¹²⁵I-protein-A tracer. The occurrence of three types of peptides became evident: one that binds to both sperm-agglutinating and sperm-immobilizing antibodies, one that binds to sperm-immobilizing antibodies, and another that binds to sperm agglutinating antibodies only. The peptides might be of interest from an immunological point of view, because the relatively mild procedure of their solubilization could also prevail under physiological conditions, resulting in release of immunogenic peptides from spermatozoa in the genital tracts.     

HLA-DR Antigen on Human Trophoblast*

ABSTRACT: To assess the presence of human leukocyte antigens (HLA) on first trimester human trophoblast cells, frozen sections of villous trophoblast and monolayer cultures of isolated cells from placental villi were prepared and exposed to a mouse monoclonal antibody directed against HLA-DR and then incubated with fluorescein-conjugated goat anti-mouse antibodies. Fluorescence microscopy demonstrated that HLA-DR antigens were present on only the small polygonal epithelioid cells of the monolayer culture. The crescentic staining pattern was consistent with widespread distribution of antigen on the cell membrane. There was no staining over giant multinucleated structures or on fibroblasts of such cell cultures. No HLA-DR was detected when this indirect immunofluorescent technique was applied to tissue sections of villous trophoblast. Existence of high concentrations of hCG in culture supernatants and coincident localization of both hCG and HLA-DR using antibodies conjugated with rhodamine or fluorescein on the polygonal epithelioid cells indicate the trophoblastic origin and expression of HLA-DR antigen under in vitro monolayer culture conditions.     

Effect of Plasma Exchange and Immunosuppression on Rh0(D), Viral,and Bacterial Antibody Titers in Rhlmmunized Women*

ABSTRACT: Pharmacologic immunosuppression and plasmapheresis have both been advocated as adjunctive methods of treatment for Rh immunization, but a combined regimen of the two has not been attempted. We treated four nonpregnant, severely Rh-sensitized female volunteers with intensive plasma exchange and either promethazine 150 mg or prednisone 60 mg per day in an attempt to remove circulating anti-Rh_o(D) and prevent further synthesis of the antibody. Each patient received three or four exchanges. The Rh_o(D) antibody titer decreased by at least one dilution immediately following 11 of 14 plasma exchanges and was ultimately lowered at least two dilutions in all patients. In one case the titer was reduced from 1:512 to 1:16, and a low titer was maintained for the duration of treatment. However, this regimen could increase the risk of infection for a mother and/or infant, as evidenced by the concomitant lowering of viral and bacterial antibody titers in these women.     

HLA phenotypes and severe Rh(D) immunization

Abstract: Blood samples from 24 Rh(D) immunized women were analyzed for antibody titers and quantification of anti-D. The HLA-DR and -DQ polymorphisms were identified as RFLP. In 11 women with titers 16–256 the HLA- DQB1 allele *0201 was found in 18%, i e as in a reference population. In 13 women with titers ≥ 512 the HLA - DQB1 allele *0201 was found in 85% indicating a correlation between severe Rh(D) immunization with high titers/quantification values and the DQB1 allele *0201. In this group the fetus was severely affected by the immunization and treatment during pregnancy was frequently needed. HLA phenotyping of women known to have anti-D antibodies early in pregnancy seems to be an effective way to assess the probability of severe hemolytic disease of the newborn.     

The mode of attenuation of erythrocyte membrane Rh0(D) antigen activity by 5,5'-dithiobis-(2-nitrobenzoic acid) and protection against loss of activity by bound anti-Rh0(D) antibody

The Rh0(D) antigen activity of human erythrocyte membranes is lost on treatment with the aromatic disulfide, 5,5'-dithiobis-(2-nitrobenzoic acid), reacting by specific disulfide interchange with membrane thiols. The inactivation rate of the antigen was first order with respect to inhibitor concentration and a double reciprocal plot of the inactivation rate constants against the sulfhydryl reagent concentration was linear, giving an apparent dissociation constant of 1.25 mM and indicating that a binding step preceded covalent interaction. In a study of the binding characteristics of anti-Rh0(D) to the 2-nitro-5-thiobenzoate derivative of the membrane Rh0(D) antigen, the maximum number of binding sites fell with increasing sulfhydryl reagent concentration but the apparent association constant also diminished at the same rate. The inactivation of the Rh0(D) antigen was greatly retarded by the presence of bound anti-Rh0(D), a protective effect not seen with normal serum. Bound anti-Rh0(D) antibody protects against both the loss of sites and the change in affinity brought about by DTNB. It is concluded that a single critical thiol whose reactivity is altered by bound anti-Rh0(D) antibody is involved in the membrane Rh0(D) antigenic determinant site. These observations may permit extensive probing of the microenvironment of this essential cysteine residue.     

Molecular size of the Rh0(D) antigen of the human erythrocyte In situ by radiation inactivation

Radiation inactivation was used to determine the molecular size of the Rh₀(D) antigen of isolated membranes and of the intact human erythrocyte. Isolated membranes were frozen and irradiated at ?50°C. After thawing, the bound ¹⁴C-anti-D was measured and the log residual Rh(D) antigen activity was plotted against the radiation dose. A mol. wt of 60,000 was calculated. Intact human erythrocytes frozen in the presence of cryoprotective reagents were also studied. Rh(D) antigen inactivation occurred as a single exponential function of radiation dose which also yielded a mol. wt of approx. 56,000 upon analysis by classical target theory.     

On the mechanism of tolerance to the Rh D antigen mediated by passive anti-D (Rh D prophylaxis)

Anti-D prophylaxis is the most successful clinical application of antibody-mediated immune suppression. Passive IgG anti-D is given to Rh D-negative women to prevent immunisation to foetal Rh D-positive red blood cells (RBC) and subsequent haemolytic disease of the newborn. Despite its widespread use and efficacy, the mechanism of action of this therapy is unproven. The known facts about the antigen, antibody response, dose of anti-D, RBC clearance and effects of the passive anti-D on subsequent primary and secondary immune responses are discussed in relation to recent information on ways by which immune responses may be suppressed. Most Rh D antigen sites on RBC are not bound by passive anti-D, and thus epitope masking (which may occur in experimental murine models using xenogeneic RBC) is not the reason why anti-D responses are prevented by administration of prophylactic anti-D. It is hypothesised that although clearance and destruction of the antigenic RBC may be a contributing factor in preventing immunisation, down-regulation of antigen-specific B cells through co-ligation of B cell receptors and inhibitory IgG Fc receptors must also occur.     

Exposure of the Rh0(D) antigen on the surface and cytoplasmic domains of the red cell membrane

Inside-out (IO) and right-side-out (RO) vesicles derived form human red blood cells were tested for their ability to bind 125I-labelled IgG anti-RHO(D). The binding of anti-RHO(D) to RO vesicles from RHO(D)-positive cells was quantitatively similar to that exhibited by intact cells when compared on a membrane surface area basis. There was no significant binding of labelled antibody to IO vesicles from RhO(D)-positive cells or to either RO or IO vesicles derived from RhO(D)-negative cells. The RhO(D) antigen was immunologically accessible on only the plasma side of the membrane in RhO(D)-positive red cells, as has been shown for blood group antigens defined by carbohydrate determinants. No immunologically reactive RhO(D) antigen was present on either RO or IO vesicles derived from RHO(D)-negative red cells.     

An Immunoglobulin M (IgM) Antibody to Carcinoembryonic Antigen (CEA)-like Antigen in Human Pancreatic Cancer

An immunoglobulin M (IgM) antibody to a carcinoembryonic antigen (CEA)-like antigen was isolated from the ascites fluid of a patient with pancreatic cancer by ammonium sulfate precipitation (25–55% saturation) and subsequent purification by gel filtrations on Sephadex G-200 and Sepharose 6B followed by protein A-Sepharose CL-4B chromatography. The IgM antibody was found to be in complex with a CEA-like antigen as revealed by the multiple precipitin lines obtained on immunoelectrophoresis and double diffusion with a mixture of antihuman IgM and anti-CEA. Dissociation of the complex with 0.2M glycine HCl buffer pH 2.5 and further chromatography on Sepharose CL-6B. followed by Sepharose 6B separated the IgM antibody from the CEA-like antigen. The IgM antibody formed an immunoprecipitate upon double diffusion with a homologous CEA cross-reactive antigen isolated from the liver metastasis of another patient with pancreatic cancer. The antigen (molecular weight 70,000 ± 15,000 daltons) which reacted with the IgM antibody was purified by Con A Sepharose affinity chromatography followed by gel filtration on BioGel A 1.5M. The IgM antibody reacted with tumor antigens from pancreatic cancer only. These results suggest that the human IgM antibody response to antigens may be specific to the tumor type involved.     

Rh弱D(Del)检测及其临床意义

目的探讨Rh弱D(Del)的检测及其临床意义。方法采用氯仿/三氯乙烯放散法检测50份Rh(D)血清学阴性样本及PCR-SSP法分析其基因结构。结果50例Rh(D)血清学阴性样本中弱D(Del)11例,其D基因外显子均完整。结论弱D(Del)型血清学筛选容易漏检为Rh(D)阴性;弱D(Del)型如为献血员,应视为Rh(D)阳性血液用于临床输血;如为患者,应视为Rh(D)阴性,必须输注Rh(D)阴性血液。D基因外显子与D抗原表达的关系有待进一步研究。     

Nonspecific Crossreacting Antigen (NCA) in Human Mammary Carcinoma Extracts

Human mammary carcinoma (HMC) homogenates from 28 patients and mammary hyperplasia (MH) homogenates from 20 patients undergoing reduction mammoplasty were used as antigens in preparing antibodies in rabbits. Crossed immunoelectrophoresis of HMC extracts against the corresponding antibodies revealed one major precipitate not present in MH. This antigen was present in separately prepared extracts of breast tissue from 3 out of 10 patients with HMC. Antibody titre was unchanged following absorption with MH, normal human liver, kidney, skin and serum. Immunochemical crossreaction with pregnancy zone proteins, alpha-1-fetoprotein, ferritin, lactoferrin, plasma, extracts of human placenta and fetal and adult liver did not occur. The antigen could not be demonstrated by rocket immunoelectrophoresis of sera from patients with HMC. Small amounts of the antigen were found in extracts from human spleen and renal carcinoma and larger amounts in extracts from human peripheral granulocytes, but not in preparations of mononuclear leucocytes (98% pure). Tandem crossed immunoelectrophoresis of extracts from HMC and carcino embryonic antigen against antibodies prepared against HMC revealed a reaction of partial identity between the two antigens, and antisera prepared against carcino embryonic antigen contained precipitating antibodies against the HMC antigen. The HMC antigen was eventually identified as nonspecific crossreacting antigen (NCA) described by other authors.     

Clinical Significance of Leukemia Associated Antigen (LAA) in Human Serum

Antiserum to serum leukemia associated antigen (LAA) was produced by immunizing rabbits or sheep with leukemic blast cells or fetal cells. This, as well as its presence in amniotic fluid and fetal tissue identifies LAA as another oncofetal component. LAA was present in the serum of 1% of healthy blood donors. In a longitudinal study of leukemia patients, LAA was found, at one stage or the other, in 50–75% of patients with acute leukemia or chronic myelogenous leukemia, but only in 27% of persons with chronic lymphocytic leukemia. A difference between those who obtained and those who did not obtain a remission was observed, since significantly more patients in the latter group had LAA at the initial test. Among these who obtained a remission, appearance of LAA seemed to signal a turn for the worse. These findings with qualitative tests were confirmed by the quantitative 'rocket' technique. The exact nature of LAA is the subject of present studies.     

Immunohistochemical Localization of Concanavalin A and Wheat Germ Lectin Receptors in the Normal Human Spermatozoa*

ABSTRACT: A semiquantitative immunohistochemical technique for the detection of N-acetyl α-D-neuraminic acid, N-acetyl β-D-glucosamine and its β-(1 ? 4)-linked internal chains, α-D-glucopyranosyl and α-D-mannopyranosyl and its α-(1 ? 2)-linked internal chains and sterically related, nonreducing, end-chain residues of oligosaccharide chains of glycoproteins or glycolipids on the surface of membranes was developed using Con A and wheat germ lectins. When this method was applied to the localization of carbohydrate receptors on the membrane of the normal human spermatozoa, it was found that the Con A and wheat germ lectin receptors were mainly located in the equatorial and post nuclear cap with few receptors located in the acrosome and neck. None of them were found in the intermediate segment plus tail. Con A receptors were α-D-mannopyranosyl end-chain residues and wheat germ lectin receptors were N-acetyl β-D-glucosamine (1 ? 4)-linked internal chains. These groups occur together in the oligomannosidic type of N-glycosidic-linked oligosaccharide chains of glycoproteins and so the use of both lectins on desialycated membranes or on those which contain nonclustered N-acetyl neuraminic acid residues may be of help to localize this type of glycoprotein oligosaccharide chains. Con A receptors were not removed after proteases digestion, suggesting the possibility that they are part of intrinsic spermatozoal antigens.     

Further Characterization of a Human Sperm Coating Antigen (gp12)

ABSTRACT: In our previous paper, we identified a novel sperm-coating antigen with molecular weight of approximately 12,000 daltons that is highly specific to sperm, seminal plasma, and milk from the plasma membrane fraction of human spermatozoa by using rabbit antiseminal plasma antiserum. In the present study, this 12,000-daltons component, termed gp12, has been investigated for its tissue distribution and antigenic stability. The largest amounts of the antigen are found in seminal plasma, although individual variation is rather high. In seminal plasma, the gp12 molecule presents in a large form with a molecular weight of approximately 50,000 daltons. Its antigenicity is stable when treated with acid, alkali, heat, and various protein denaturants.