Mechanism of Rh prophylaxis: an experimental study on specificity of immunosuppression.

The mechanism by which Rh immunization is prevented by IgG anti-D was investigated by studying the specificity of immunosuppression. 62 D-negative Kell(K)-negative male volunteers were given two successive stimuli of 1 ml D-positive K-positive red cells. Thirty-one of the volunteers were also given 13-14 mug of IgG anti-K immediately after each stimulus, the others acting as controls. Anti-D developed in 11 of the 31 controls and in one of the 31 volunteers who had received anti-K. This marked suppression of the anti-D response by IgG anti-K was accompanied by the rapid clearance of the injected red cells to the spleen. This shows that the predominant mechanism that must be operating when IgG anti-D prevents Rh immunization is not antigen specific but is one that must involve the whole red cell, probably through destruction within splenic macrophages.     

Efficacy of rhesus antibodies (Anti-Rh0 (D)) in autoimmune thrombocytopenia: correlation with response to high dose IgG and the degree of haemolysis

We have compared the efficacy of high-dose IgG with that of Rhesus antibodies (anti-Rh0 (D)) in 5 patients with autoimmune thrombocytopenic purpura (3 adults and 2 children). Although only transient, high-dose IgG (0.4 g/kg X 5 days) was effective in all patients (peak values 50-200 X 10(9)/1), whereas anti-Rh0 (D) (11-20 micrograms/kg X 5 days) led to comparable results in only 3 patients (165 X 10(9)/1, 72 X 10(9)/1, 33 X 10(9)/1). This response to anti-Rh0 (D) was neither related to the degree of induced haemolysis (increase of LDH and decrease of haptoglobin) nor to the amount of IgG antibodies bound to red blood cells, as quantified by the 125-I-antiglobulin test. A decrease of platelet-associated IgG was recorded in 3 patients: 2 of them showed an improvement of platelet counts and in one of them there was no response. We conclude that the therapeutic response of high-dose IgG and anti-Rh0 (D) is independent of the degree of induced haemolysis and may not be predicted from the effectiveness of either therapy alone.     

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for sup-pressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human trans-formed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.     

Deaths from rhesus haemolytic disease in England and Wales in 1982 and 1983.

Examination of death certificates and the clinical notes of the patients concerned showed that the number of deaths from rhesus (D) haemolytic disease in England and Wales was 44 and 34 during 1982 and 1983, respectively, a substantial decrease from the figure of 106 for 1977. Of the 78 women whose infants died in 1982 and 1983, 49 had not received anti-Rh immunoglobulin after previous pregnancies with Rh positive infants; most of these deaths would presumably have been prevented had postnatal anti-Rh immunoglobulin been given. In 13 women anti-D was detected during, or immediately after, a first pregnancy, and in 15 women rhesus immunisation developed despite administration of anti-Rh immunoglobulin postnatally. One or two apparent failures of treatment may have been due to underdosage, but it must be concluded that about one third of the deaths in 1982 and 1983 could have been prevented only by giving anti-Rh immunoglobulin antenatally as well as postnatally.     

Cell Response to Surface Stimulation

The injection of anti-D into Rh-negative subjects who have Rh-positive red cells in the circulation results in the inhibition of immunization against the D-antigen¹. On the other hand, subjects who have had a primary anti-D response to Rh-positive red cells frequently give a good secondary response to small doses of red cells despite the presence of anti-D in their plasma. The difference in action between the passively-administered and the actively-produced anti-D might lie in the fact that the injected IgG anti-D is derived from a pool of donors and therefore contains a number of IgG antigens which are foreign to the recipient, compared with the autologous nature of the anti-D present after a primary response.     

Is an expression system for producing therapeutic antibodies with immunosuppressive properties found at last? Comment to letter by Dr. Quagliaroli

The prophylaxis of the hemolytic disease of the newborn — a mandatory procedure in obstetrics — requires significant amounts of plasma-derived polyclonal anti-D immunoglobulin. Despite numerous attempts, the proper technology for mass production of effective monoclonal anti-D is still not available. LFB Biotechnologies is currently performing clinical trials with recombinant anti-D antibody that has low fucose content and is expressed in the cells of rat myeloma YB2/0. It was shown that this drug is well tolerated, accelerates fast clearance of D+ red blood cells, and can inhibit anti-D immune response in Rhesus-negative volunteers.     

Direct Antiglobulin Reaction in ABO-Haemolytic Disease of the Newborn

The minimum number of IgG anti-A (or anti-B) molecules detectable on A or B red cells by the antiglobulin reaction was found to be the same—that is, about 150 molecules per red cell—with newborn as with adult cells. Furthermore, the ratio of anti-IgG bound to IgG anti-A (or anti-B) molecules was the same whether the anti-A (or anti-B) molecules were present on newborn or on adult cells and was similar to that found for anti-IgG bound to IgG anti-Rh.In 15 infants (11 group A, 4 group B) with haemolytic disease of the newborn due to ABO-incompatibility the amount of anti-A or anti-B on the red cells ranged from 0·25 to 3·5 μg antibody per ml red cells, corresponding to 90-1,320 antibody molecules per cell; only five infants had more than 0·55 μg antibody per ml of red cells. These amounts are far smaller than those found in most moderate or severe cases of Rh-haemolytic disease.It is concluded that the weak direct antiglobulin reactions observed in ABO-haemolytic disease are due simply to the fact that the number of anti-A (or anti-B) molecules on the infant''s red cells is at the lower limit of sensitivity of the test. Since ABO-haemolytic disease can be quite a severe process it seems probable that IgG anti-A and anti-B molecules are more effective than anti-Rh molecules in bringing about red cell destruction.     

Quantitative study of chimpanzee and gorilla counterparts of the human D antigen

The reactivities of three human anti-D monoclonal antibodies (mAbs) with human, chimpanzee, and gorilla red blood cells (RBCs) were compared by quantitative radioimmunology and indirect immunofluorescence methods. The number of antigenic sites varies widely in gorillas (from 48,000-283,000), while in chimpanzees this number is very close to that observed in human R₁R₂ RBCs. The affinity of the anti-D antibodies was slightly lower with ape RBCs than with D-positive human RBCs. In chimpanzee, the D-like epitopes recognition is enhanced by papain while the gorilla D-like epitopes are destroyed by enzyme treatment.     

An enzyme-linked antiglobulin test for assessing anti-D immunoglobulin preparations

Two enzyme-linked antiglobulin tests (ELAT) for assessing anti-D IgG preparations are described; one is performed in tubes and the other in microtitre plates. An anti-human IgG alkaline phosphatase conjugate and the substrate p-nitrophenyl phosphate are used. Both methods were sensitive and reproducible, with variations coefficients of 7.8 and 8.6% for enzyme immunoassay in tubes and microplates, respectively. The linear relationship between the amount of red cell-bound anti-D and the optical density shows that the method is suitable for quantitative studies. Results obtained by the two methods show a very good correlation (r = 0.99) in 12 of the 14 samples assayed, and both give good agreement with results obtained in automated haemagglutination. Since microtitre plate ELAT has numerous advantages over the tube method, it could provide an alternative method for assessing anti-D activity of specific IgG preparations in control laboratories.     

抗-D抗体及其应用

Rh血型是仅次于ABO血型系统的人类红细胞抗原系统,至今已发现40多种抗原,但与临床密切相关的是D,C、c、E、e等5种抗原,其中最主要的是D抗原。相应的抗-D抗体无论是在临床输血检测,还是在Rh(D)新生儿溶血病、溶血性输血反应等的防治方面均具有非常重要的意义。传统的抗-D抗体的制备需用人的血清,来源受限。各种抗-D人源性单克隆抗体和基因工程抗体已经成为发展方向。     

WinRho: Rh immune globulin prepared by ion exchange for intravenous use.

An Rh immune globulin [Rh IgG] for intravenous use, WinRho, has been prepared by the Winnipeg Rh Institute by a modification of the ion-exchange column method of Hoppe and colleagues. When administered to Rh-negative male and nonpregnant female volunteers WinRho was found to be nonpyrogenic, nontoxic, safe and protective against Rh alloimmunization. In a clinical trial with 240 microgram given at about 28 weeks'' gestation and 120 microgram given after delivery to Rh-negative women at risk of Rh immunization WinRho was effective in preventing Rh immunization. Of the 870 women carrying Rh-positive fetuses who were treated with WinRho during pregnancy and were not tested several months after delivery 14 would have shown evidence of Rh immunization by the time of delivery if WinRho had been ineffective; none showed such evidence. Of the 1122 women carrying Rh-positive fetuses who were retested 4 to 6 months after delivery 83 would have shown evidence of Rh immunization at that time if WinRho had been ineffective; only 1 showed such evidence. The efficiency of yield of anti-D with the modified method of production, the fct that it can be given intravenously (a route that causes the patient less discomfort and immediately results in high anti-D levels) and the lower levels of contaminating IgA and IgM make WinRho the preparation of choice for preventing Rh immunization.     

Rhesus Isoimmunization after Abortion

Out of 177 Rh(D)-negative patients studied, 96 were successfully followed-up after spontaneous or therapeutic abortion. Rh antibodies were detected by the indirect Coombs test in two patients and by an enzyme technique only in a further seven, an overall incidence of 9·4%.The prophylactic use of anti-D immunoglobulin is now recommended for Rh-negative non-immunized patients undergoing abortion, but the dose could be less than 75 μg. The Kleihauer test was of no value in predicting the risk of isoimmunization.     

Idiotypy of anti-Rh antibodies

Anti-id sera to Rh antibodies were produced by injecting rabbits with purified Rh antibodies. These sera were shown to agglutinate O Rh+ RBC coated by the immunizing antibody and--in some cases--by other anti-D antibodies. Id and cross-reactive id were shown to be located in the antigen-binding and in the non-antigen binding regions of Rh antibodies. An unique example of evolution of idiotypic specificities on human antibodies has been reported. Lastly, we have demonstrated by rosette assay, presence on some PBL of receptors for Fab'2 anti-Rh coating O Rh+ red cells. Rosettes could not be obtained with lymphocytes of a donor and Fab'2 anti-Rh of another individual. Rosettes appeared at a period of time in which the amount of antibody was decreasing.     

Molecular characterization of a human anti-Rh(D) antibody with a DH segment encoded by a germ-line sequence.

The lambda-light-chain and lambda-heavy-chain variable-region genes of an anti-Rh(D) (Rh, Rhesus; D, heavy-chain diversity region) human monoclonal antibody secreted by lymphocytes transformed by the Epstein-Barr virus have been cloned and sequenced. Sequence comparison of the anti-Rh(D)mAb lambda-chain variable region with those of the other available human lambda chains revealed that it belonged to the human V lambda I (V lambda, variable region of lambda chain) subgroup. The greatest sequence similarity (80%) was observed with that of another anti-Rh antibody lambda-chain directed against the Rh(c) antigen. For the VH (VH, variable region of heavy chain) sequence, the highest similarity (86%) was observed with the germline VHG3 gene which belongs to the VHI subgroup. The expressed DH sequence of the anti-Rh(D) antibody is also of germline origin and complementarity-determining region 3 is thus produced by VH-DH and DH-JH (J, joining region) joining without recombination of multiple DH gene segments.     

Crossmatch difficulties following the prophylactic use of Rh immune globulin.

With increasing demand for platelet transfusion the need to use platelets from Rh-positive persons for Rh-negative individuals often arises. The administration of Rho(D) immune globulin (human) in this situation has been recommended, but may cause serologic difficulties owing to the recipient''s passive acquisition of antibodies other than anti-D.     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

High dose intravenous immunoglobulin repertoire versus anti-D. Disproportions in quantity and quality.

Here we consider certain therapeutic effects that intravenous administration of pooled high dose immunoglobulin and anti-D IgG share. Despite million-fold difference in doses such an effect occurs at least in idiopathic thrombocytopenic purpura (ITP). We postulate that spontaneous bleeding events may remit even when platelet numbers show refractoriness. We also mention the possible sparing of anti-D antibody-coated red blood cell (RBC) destruction and, finally, an acceleration of fibrotic involution. Fc receptors (FcRs) play a central role; beyond the well-established interactions with the immunoglobulin Fc fragment, FcRs are supposed to display special cognitive properties that enable them to pick out the therapeutic molecules from the recipient's IgG pool. Such subtle selection suggests some disarray in the host. On the other hand it may explain why the often-encouraging outcome of IVIG therapy remains unpredictable.     

Recombinant IgA Is Sufficient To Prevent Influenza Virus Transmission in Guinea Pigs

A serum hemagglutination inhibition (HAI) titer of 40 or greater is thought to be associated with reduced influenza virus pathogenesis in humans and is often used as a correlate of protection in influenza vaccine studies. We have previously demonstrated that intramuscular vaccination of guinea pigs with inactivated influenza virus generates HAI titers greater than 300 but does not protect vaccinated animals from becoming infected with influenza virus by transmission from an infected cage mate. Only guinea pigs intranasally inoculated with a live influenza virus or a live attenuated virus vaccine, prior to challenge, were protected from transmission (A. C. Lowen et al., J. Virol. 83:2803–2818, 2009.). Because the serum HAI titer is mostly determined by IgG content, these results led us to speculate that prevention of viral transmission may require IgA antibodies or cellular immune responses. To evaluate this hypothesis, guinea pigs and ferrets were administered a potent, neutralizing mouse IgG monoclonal antibody, 30D1 (Ms 30D1 IgG), against the A/California/04/2009 (H1N1) virus hemagglutinin and exposed to respiratory droplets from animals infected with this virus. Even though HAI titers were greater than 160 1 day postadministration, Ms 30D1 IgG did not prevent airborne transmission to passively immunized recipient animals. In contrast, intramuscular administration of recombinant 30D1 IgA (Ms 30D1 IgA) prevented transmission to 88% of recipient guinea pigs, and Ms 30D1 IgA was detected in animal nasal washes. Ms 30D1 IgG administered intranasally also prevented transmission, suggesting the importance of mucosal immunity in preventing influenza virus transmission. Collectively, our data indicate that IgG antibodies may prevent pathogenesis associated with influenza virus infection but do not protect from virus infection by airborne transmission, while IgA antibodies are more important for preventing transmission of influenza viruses.     

Maternal anti-D concentrations and outcome in rhesus haemolytic disease of the newborn.

The relation between maternal anti-D concentrations, measured against the British working standard, and outcome of rhesus-sensitised pregnancies was studied. There is a clear relation between increasing anti-D concentrations and the chance of a severely affected baby. Of those pregnancies (78) where serial anti-D concentrations remained below 4 IU/ml, no baby had a cord haemoglobin below 10 g/dl and three had exchange transfusions. In contrast, of those mothers (106) with anti-D concentrations above 4 IU/ml, 23 had babies with a cord haemoglobin below 10 g/dl and 79 babies had exchange transfusions. It is suggested that those pregnancies where anti-D concentrations remain below 4 IU/ml represent a relatively safe group in which amniocentesis may be avoided.     

Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations

Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.