The Phenotype Rhod: Study of Another Family

A case is described in which an Rh_o-positive Negro woman suffered a transfusion reaction. Her serum was found to contain anti-rh", anti-Fy^a, anti-Jk^b and an antibody resembling anti-Rh_o. A family investigation showed that the propositus and two of her five children belong to the pheno- type Rh_o^d. This evidence gives further support to the theory that the agglutinogen Rh_o^d is determined by a corresponding allelic gene R ^od.     

Reaction of I131 Anti-Rho(D) with Enzyme Treated Red Cells

The effect of proteolytic enzymes (papain, trypsin, ficin and bromelin) on Rh_o(D) positive and negative red cells was studied with I¹³¹ anti-Rh_o(D). Enzymatic pretreatment of red cells results in almost a two-fold increase in the amount of I¹³¹ antiRh_o(D) bound to these cells. The "enzyme exposed Rh_o(D)" antigen by some criteria (stability to hemolysis, absence of an increase in negative cells and studies on the supernatant solutions) appears to have the same specificity as the Rh_o(D) antigen on the untreated cell. The ability of proteolytic enzymes to produce this effect on Rh_o(D) positive red cells does not correlate directly with the ability of these enzymes to digest casein, although the effect appears to be associated with the proteolytic activity of these enzymes. The amount of increase in uptake of I¹³¹ anti-Rh_o(D) induced by enzymes is directly proportional to the quantity of antibody bound by the untreated cell, irrespective of the Rh phenotype or zygosity of the red cell.     

Immunization to Rho(D) Observed in Pregnancy of a Woman with Type rhG(G)

A Negro woman was tested during her third pregnancy and found to have type rh^G(G). When the patient was re-tested during the sixth month of her fourth pregnancy, she was found to have a weak antibody of questionable identity. During the last two months of the pregnancy, the titer of the antibody rose sharply and it was established to be specific for Rh_o(D). The infant, delivered by cesarean section shortly before term, had evidence of mild hemolytic disease; it recovered without transfusion therapy. Since this indicates that isoimmunization to Rh_o can occur in a person of type rh^G, such persons should receive only Rh_o-negative blood for transfusion. They should be regarded as Rh-negative for prenatal purposes, but it might be considered preferable not to use them as blood donors for Rh-negative recipients.     

Further Observations on the Blood Factors RhA, RhB, RhC and RhD

The findings in 18 cases of individuals with Rh_o-positive blood who had antibodies in their serum resembling anti-Rh_o in specificity are briefly summarized. One interesting finding is the relatively high frequency of such individuals among Negroes, especially with the blood type Rh^d_o and Rh^abd_o. The significance of these findings is discussed.     

Familial Autohemolytic Anemia and Runting Syndrome with Rho-specific Autoantibody

A case of autohemolytic anemia with hypergammaglobulinemia and specific anti-Rh_o autoantibody is reported. The patient had the clinical syndrome of simulated runt disease as it occurs in experimental animals. Thymic atrophy and generalized lymphocytic depletion were found at autopsy. Four siblings of the proposita died in infancy of similar affections. The sera of the two living siblings and the parents disclose abnormal serum globulin patterns. It is suggested that dysglobulinemia favors the survival of lymphoid cells acquired trans-placentally from the mother and that the autohemolytic anemia and other pathologic manifestations in this case are the result of a graft-versus-host reaction. The anti-Rh_o specificity of the autoantibody is ascribed to isosensitization of a clone of grafted lymphoid cells which have sustained a loss of Rh genes in the process of somatic mitosis.     

Detection of Fetal Maternal Hemorrhage: An Evaluation of Serological Tests Related to Rho(D) Immune Globulin (Human)

Microscopic examination of 219 indirect anti-human globulin tests using a 1: 1,000 dilution of RhoGAM®* and 259 indirect antihuman globulin tests using appropriate slide test anti-Rh_o(D) sera on specimens simulating fetal maternal hemorrhages (FMHs) of from 15 to 200 ml showed that agglutination was more frequently observed using the slide test anti-Rh_o(D) sera. Tests done on coded specimens sent to several hospital laboratories indicated the need for technologist education in the interpretation of results of these tests on postpartum women. Since the amount of Rh immune globulin needed to treat women with FMHs depends on the volume of fetal bleed, suspected FMHs should be confirmed and quantitated using Kleihauer-Betke smears.     

The Rho Variant-Du

A simplified test for the D^u factor employing a reagent containing 25 per cent serum albumin, the "stick" technic to introduce cells and forceful cen-trifugation has been given an extensive trial in a routine blood bank operation. A routine preliminary test with anti-D gave 5,051 negative reactions among 26,753 bloods tested. The D^u tube test was then performed on the former with an anti-CDE reagent in parallel with the indirect antiglobulin test done with anti-D. All bloods positive by one or both tests were tested further for D antigens by an anti-D elution technic; they were also tested for Rh-Hr factors.
Among the 126 bloods positive by elution for the D^ufactor, 122 and 126 were positive respectively by the antiglobulin and the D^u tube tests. The two tests were in agreement on all eight of the type ccD^uee bloods found in the study. The D^utube test, as done with anti-CDE, gave no false negative reactions. It is, thus, concluded that bloods which are negative with this test will be type ccddee and can be labeled "Rh negative tested for D^u. Serological factors which might account for sensitivity of the D^u tube test's being the equal of the antiglobulin test are considered, as are the probabilities of the existence of D variants so weak that they could not be detected by either test.     

A New Rh Phenotype, Rhorh, G-Negative

A new Rh phenotype, Rh_orh, G-negative is described in a Negro woman who developed anti-G (rhG) antibodies during pregnancy. The baby was Rh_o, G-positive and had mild hemolytic disease with a positive direct antiglobulin test. The mother's titer was 1:1024 at delivery, dropping to 1:8 after 14 months. The baby's direct antiglobulin test became negative after two months. The mother most likely became sensitized alter a blood transfusion five years prior.     

Effects of Albumin on the Radio-immune Assay for Determining the Anti-Rho(D) Content of Rho(D) Immune Globulin

Bovine serum albumin is used as a diluent component in the radio-immune assay for the determination of the Anti-Rh_o(D) content of Rh_o(D) Immune Globulin preparations. The radio-immune assay appears to be more sensitive than other serological assays in the response to differences among albumin preparations; lack of duplication of data and results due to nonspecific binding of immunoglobulin may be pronounced. The effects of bovine and human serum albumin from two commercial sources each were studied. Differences were demonstrated by ultracentrifugal analysis, electrophoresis and serological methods. In addition to monomers and polymers of albumin which are common to both preparations one contained other components. Data presented clearly demonstrate that the choice of albumin preparation is especially critical if the single dilution assay is employed as opposed to the multiple dilution assay.     

The Rho Variant — Du

In a survey of 18,365 bloods for the incidence of the D^u factor, in a population comprised approximately of 72 per cent Caucasoids, 15 per cent Negroids, 10 per cent Mexicans and 3 per cent Orientals, 78 D^u bloods were found of which seven were type ccD^uee. Of the latter, five came from Negroids and two from Caucasoids; the frequency among the two populations approximates respectively one in 6,000 and one in 500. In the general population of this region it is one in 2,500.
The degree of reactivity of the type ccD^uee found in this study was that of the "low grade" variety. However, from the remaining 71 "low grade" D^u bloods, substantially weaker examples were found among type CcD^uee and ccD^uEe. The indirect antiglobulin and ficinized cell method readily detected type ccD^uee; however, among the weaker variants, the antiglobulin method gave the more consistent results.
It is concluded, therefore, that type ccD^uee does occur in this area with a frequency that is considered significant and that its detection rests on the antiglobulin or other equally sensitive methods.     

A Second Example of the Antibody Anti-Jsb of the Sutter Blood Group System

An antibody defined as anti-Js^b of the Sutter blood group system was discovered in the serum of a much transfused Chicago Negro woman of group B, Rh_o (cD^ue), Js (a+b–). Antibodies to rh' (C) and rh" (E) were also present. Tests with 460 Chicago Negro donors, all rh' (C)-negative and rh" (E)-negative, revealed three cases of phenotype Js (a+b–), an incidence of 0.65 per cent.     

Use of Escherichia coli O86: B7 in the Adsorption of Anti-A and Anti-B from Blood Typing Sera

The Gram-negative bacterium, Escherichia coli O₈₆:B7, has somatic A and B antigens that can be utilized to adsorb anti-A and anti-B from blood typing sera. Adsorption of anti-B is highly effective, one adsorption with heat-killed bacteria for one hour being sufficient to remove anti-B from most type A or O sera. Adsorption of anti-A is less efficient, especially with type O sera.
Adsorption with these organisms caused slight loss in activity of an anti-P₁ serum, but no reduction in antibody titer or avidity was detected in adsorptions using a wide range of other human antisera. The bacterial method of processing is a simple and rapid means whereby sera such as anti-Lu^b, anti-k and anti-Vel may be rendered free of anti-B.     

Quantitative Studies of a Two-stage Hemagglutination System Employing the Antiglobulin Reaction

The quantitative analysis of hemagglutination employing an electronic particle counter has been extended to the development of a two-stage system for the study of agglutination of red cells sensitized by incomplete antibodies treated with antiglobulin serum. The agglutination of red cells in an anti-Rh_o (D) system has been studied to demonstrate the relationship between such parameters as per cent agglutination, per cent two-cell aggregates, and mean cell number per aggregate. Demonstrations of applications of this technic to such problems as the measurement of comparative potency and characterization of anti-Rh_o sera, dosage effects, disaggregation patterns with antisera of different specificities, use of chain-specific antiglobulin sera, and antiglobulin consumption studies are given.     

Quantitative Studies of the Rho(D) Antigenic Determinants on Gorilla Erythrocytes

Human and simian red blood cells were tested for ¹²⁵I anti-D binding at equilibrium and for their ability to adsorb ¹²⁵I anti-D. On the basis of the equilibrium quantity of red blood cell-bound anti-D at different ionic strengths, the rate of binding of anti-D, and adsorption studies, evidence has been obtained that D-antigenic specificities occur on the erythrocytes of orangutans ( Pongo pygmaeus ), chimpanzees ( Pan troglocytes ) and gorillas ( Gorilla gorilla ). Gorillas, like chimpanzees, appear to be polymorphic with respect to the presence of D-components on their erythrocytes. Individual animals have been found whose red blood cells take up very little anti-D and are similar to human D-negative cells. The quantity of anti-D bound at equilibrium, as a fraction of that bound to human D-positive red blood cells was: for gorillas 0.6 to 4.0; for chimpanzees 0.08 to 0.51; for orangutans 0.06 to 0.09, and for human D^u red blood cells 0.04. At low ionic strength there was almost a twofold increase in anti-D binding. At low ionic strength, the adsorbing capacities of these erythrocytes, as compared to a human D-positive cell, were: orangutans 10 to 20 per cent; chimpanzees about 50 per cent; human D^u 86 per cent; and gorillas 97 per cent. Evidence is presented to indicate that the discrepancy between adsorbing capacity and the binding of anti-D at equilibrium, for D^u and simian erythrocytes, is due to absence of some of the D-antigenic components found on human D-positive red blood cells.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Prostaglandin E2 (PGE2) induces headache in healthy subjects

The role of prostanoids in nociception is well established. The headache-eliciting effects of prostaglandin E₂ (PGE₂) and its possible mechanisms have previously not been systematically studied in man. We hypothesized that infusion of PGE₂ might induce headache and vasodilation of cranial vessels. PGE₂ (0.40 µg kg⁻¹ min⁻¹) or saline was infused for 25 min into 11 healthy subjects in a cross-over, double-blind study. Headache intensity was scored on a verbal rating scale from 0 to 10. In addition, we recorded mean flow in the middle cerebral artery (V_MCA) by transcranial Doppler and diameter of the superficial temporal artery (STA) by high-resolution ultrasonography. All 11 subjects reported headache on the PGE₂ day and no subjects reported headache on the placebo day ( P  = 0.001). During the immediate phase (0–30 min) ( P  = 0.005) and the postinfusion phase (30–90 min) ( P  = 0.005), the area under the curve for headache score was significantly larger on the PGE₂ day compared with the placebo day. PGE₂ caused dilatation of the STA (23.5%; 95% CI 14.0, 37.8) and the MCA (8.3%; 95% CI 4.0, 12.6). We suggest that PGE₂ induces headache by activation and sensitization of cranial perivascular sensory afferents.     

IgG anti-Jka/Jkb antibodies are unlikely to fix complement

Antibodies in the Kidd blood group system show a great deal of serological variability, are notoriously elusive and hence evoke difficulties in detection. However, they have been regularly reported as causing severe immediate or delayed haemolytic transfusion reactions and this clinical potential has been largely attributed to their complement binding ability.
In initial investigations on 43 anti-Jk^a/Jk^b sera with a range of titres of IgG antibody only a few seemed to fix complement, though following repeated tests on 20 of these sera a further five were shown to bind complement, making a total of 12 (27.9%) showing evidence of complement binding. Twenty-three sera were unavailable for re-testing. Subsequent tests revealed that only those sera which showed direct agglutination or were positive with an anti-IgM reagent in an indirect antiglobulin test (IAT) fixed complement. Evaluations on the IgG fractions of six selected potent anti-Jk^a sera failed to reveal any complement-fixing ability although all the original sera bound complement avidly and contained variable amounts of IgM antibody, some at very low subagglutinating levels.
These findings challenge past perceptions and give cause for reflection on the changing methodologies and strategies which could unduly compromise the detection of these potentially clinically damaging antibodies.     

Endothelin-1 does not modulate O2· release and [Ca2+]i variations in resting or differentiated HL-60 cells

Summary— Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O₂*) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01 – 100 nM) was not able to modulate O₂* generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC₅₀ = 4.24 ± 1.63 nM in the absence and 3.16 ± 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01 – 100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca²⁺]_i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O₂*) or biochemical ([Ca²⁺]_i) response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O₂* generation or [Ca²⁺]_i variations.     

Effects of cyclooxygenase inhibitors, prostaglandins F2α and I2, on isolated coeliac and basilar arteries of alloxan-diabetic dogs

Abstract. The effects of prostaglandin synthetase inhibitors PGF_2α and PGI₂ on the tone of isolated basilar and coeliac arteries were studied in healthy and alloxan-diabetic dogs. PGF_2α (1 μmol l^-1) produced a significantly higher tone in diabetic basilar arteries (1·15 ± 0·16 mN) than in normal cerebral vessels (0·7 + 0·10 mN). By contrast, the contractile responses of normal and diabetic coeliac arteries to PGF_2α did not differ. The cyclooxygenase inhibitors indomethacin (3 μmol l^-1) and suprofen (0·58 μmol l^-1) potentiated the PGF_2α-evoked contractions in all of the vessels studied. The percent potentiation was greater (50–60%) in the basilar arteries from alloxan-treated dogs than in normal basilar vessels (22–30%). There was not such a difference between diabetic and normal coeliac arteries. Prostacyclin produced a concentration-related relaxation in the presence of indomethacin or indomethacin + PGF_2α. The relaxant potencies of PGI₂ were similar in the vessels from metabolically healthy and diabetic dogs. The IC₅₀ values for PGI₂ were 11·6 ± 1·3 and 11·8 ± 1·8 nmol l^-1 in normal and diabetic basilar arteries, respectively; they were 25·4 ± 3·2 and 26·2 ± 3·9 nmol l^-1 in control and alloxan-treated coeliac vessels. These results indicate that normal and diabetic vessels may have differential reactivity to cyclooxygenase inhibitors, this difference being dependent on the vascular region.