Demonstration of Isoantigenic Receptors on Human Epidermal Cells and Established Cell Lines

This paper describes mixed agglutination and serum absorption experiments for the demonstration of A, B, H, M, N, D, C, E, c and e isoantigens inhuman epidermal cells and cultured cells. It was found that only A, B andH antigens are present on human epidermal cells and that this does not appearto be related to the secretor status of the donor. The same antigens were alsoexamined on six different established human cell strains (HeLa, EE, ERK-1,Maben, Chang conjunctiva, Chang liver), and one established mouse cellstrain (L). It was found that the H antigen persists in established human cellcultures. The M antigen which is not seen in normal epithelial cells can bedemonstrated on HeLa cells by mixed agglutination reaction and by absorption experiments. Guinea pig antisera against established cell lines of humanorigin were found to contain a small fraction of agglutinins with H specificity,but no anti-M, anti-N or anti-Rh agglutinins.

Submitted on December 30, 1963 Accepted on April 17, 1964     

Cell Antigens and Cell Specialization. IV. On the H Blood Group Antigen of Human Platelets and Nucleated Cells of the Human Bone Marrow

This article describes the specific agglutination of normoblasts, recitulocytes, megakaryocytes and platelets of human bone marrow with anti-H sera.It also describes the erythrocyte-platelet mixed agglutination reactions demonstrating H antigen receptors on all human platelets regardless of the ABOgroup of the donor with the exception of the Bombay blood.

Submitted on November 15, 1963 Accepted on January 24, 1964     

Studies on the Dynamics of the Reaction Between Rh Antibody and Antigen

The anti-D antibody of two human sera was found to be transferred inpart from sensitized group A Rh (D)-positive cells to normal group B Rh (D)-positive cells when the cell suspensions were mixed and incubated in a salinemedium.

The amount of anti-D antibody transferred from sensitized to normal cellsin a given period of time was found to be directly related to the concentrationof antibody to which the group A donor cells were originally exposed. It isconcluded that the amount of antibody transferred to recipient cells understandard conditions is related to the concentration of antibody on the surfaceof the donor cells.

Submitted on January 18, 1960 Accepted on April 21, 1960     

In Vivo Localization of Heterologous Anti-Erythrocyte Antibodies in the Rat Bone Marrow and Their Effect on the Proliferative Capacity of Immature Erythroid Cells

The in vivo localization of heterologous anti-erythrocyte antibodies in therat bone marrow was determined by the I¹³¹-labeled antibody technic. I¹³¹-labeled anti-erythrocyte antibodies localized specifically in the bone marrowindicating the presence of localizing antibody. Both the localizing antibodyand the incomplete antibody were thermostable, whereas hemolysins andhemagglutinins were thermolabile. Following an intravenous injection of antierythrocyte antibodies in rats, hemolysins and hemagglutinins were clearedrapidly from the plasma. The incomplete antibodies became attached to circulating red cells within 6 hours and red cell sensitization persisted for 1 week.The localizing antibody localized in the bone marrow within 30 minutes, leaving no activity in plasma.

The anti-erythrocyte antibodies markedly reduced the uptake of tritiatedthymidine by erythroblasts in vitro, demonstrating their inhibitory effect onthe proliferative capacity of erythroblasts.

Submitted on February 3, 1964 Accepted on May 12, 1964     

The "Infectious Mononucleosis Cell" A Cytochemical Study

The abnormal cells in the peripheral blood of patients with infectious mononucleosis were studied by available histochemical technics and with immunofluorescent and autoradiographic technics. The findings indicated a closerelationship of these cells to lymphocytes, although the strong acid phosphatase reaction observed in the infectious mononucleosis (IM) cells wasnot usually seen in normal lymphocytes, but rather in monocytes and reticulum cells. There was no evidence of antibody production by the IM cells ofthe peripheral blood.

Studies of the lymph nodes showed two abnormal cell types: one, similarto the IM cells in the peripheral blood but more immature, and the second,a primitive cell with various indications of immunologic competence.

Submitted on February 28, 1963 Accepted on July 8, 1963     

Mixed agglutination of leukocytes and erythrocytes in relation to studies on leukocyte antigens

Serologic experiments are described in which was studied the agglutination ofleukocytes from donors representing various ABO, Rh, and Lewis erythrocytegroups an certain canine blood groups, in the presence of corresponding antisera.

Appropriate mixing of antisera with leukocytes and erythrocytes from donorsof different groups was seen to produce clumping of leukocytes which did notconform to the reactions of the erythrocytes from the leukocyte donor. Whenviewed under phase microscopy, certain of these leukocyte clumps, which appeared homogeneous with ordinary light microscopy were found to be clumps ofleukocytes mixed with ghosts of erythrocytes reacting with the antibody present.

Factors contributing to this apparently immunologically non-specific clumping were the presence of complement-fixing, potentially hemolytic antibody andthermolabile components of serum. A possible relationship with erythrophagocytosis is suggested.

These observations indicate that certain results of this and other "leukoagglutination" technics, which have been interpreted as demonstrating thepresence of A and B antigens on human leukocytes, deserve re-evaluation andemphasize the importance of developing methods of preparing homogeneousleukocyte suspensions.

     

Identification of Blood Group Antigens and Minor Cell Populations by the Fluorescent Antibody Method

It is possible to produce fluorescence of erythrocytes as the result of specificantigen-antibody reactions of various blood group antigens; thus far, thefactors A and B, a variety of Rh antigens and Kidd, have been successfullydemonstrated with this method. It is important to realize that in the presenceof adequate negative controls, low intensity fluorescence like that obtained withRh antigens is nevertheless specific in systems involving erythrocytes.

The method discriminates between A₁ and A₂ cells. More antibody must beattached to the red cell for fluorescence than for agglutination. The relativepaucity of antigenic sites of Rh substance as compared to A and B antigens isreflected by the difference in intensity of fluorescence.

The fluorescent antibody technic has been used successfully for the demonstration, and, to some extent, quantitation of minor cell populations in in vitromixtures and in vivo. Injected Rh-positive erythrocytes were demonstrated inthe blood of an Rh-negative volunteer during a period of 20 days. Fetal Rh-positive erythrocytes were demonstrated in several Rh-negative women, bothwith and without antibody, in the last trimester of gestation.

Submitted on December 10, 1959 Accepted on February 1, 1960     

Globulin Coating of Neoplastic Lymphocytes in Chronic Lymphocytic Leukemia

1. Employing the technic of specific mixed cell agglutination (SMCA), ithas been demonstrated that leukemic lymphocytes, as well as red cells, maybe coated with globulins which are reactive with antihuman (Coombs) serum.

2. Moreover, in vitro, these proteins may dissociate from neoplastic lymphocytes and reassociate on normal erythrocytes rendering them Coombs-positive.

3. The dissociation-reassociation phenomenon appears to be time- andtemperature-dependent and may be augmented by complement or a relatedheat-labile serum or cell factor.

4. It is suggested, by analogy to instances in which the normal lymphocytemediates certain immunologic reactions, that the neoplastic lymphocyte mayparticipate actively in the acquired hemolytic anemia of chronic lymphocyticleukemia.

Submitted on December 8, 1962 Accepted on March 1, 1963     

The Effect of Amelioration of Anemia on the Synthesis of Fetal Hemoglobin in Sickle Cell Anemia

Two adult patients with sickle cell anemia of blood group A and A₂Brespectively, received sufficient transfusions of group O blood to maintainnearly normal hemoglobin concentrations for 4 months or longer.

Serial samples of the erythrocytes of each recipient were obtained by agglutination with anti-A (and anti-B) serum. The proportion of Hb F in theagglutinated erythrocytes was determined. Early in the transfusion period, amarked rise in the proportion of Hb F was noted. This rise was attributed toprolonged survival of erythrocytes which contained larger proportions of Hb F.In the later part of the transfusion period, the proportion of fetal hemoglobindeclined to pre-transfusion levels or below. However, significant amounts offetal hemoglobin in the erythrocytes of each patient were demonstratedthroughout the period of study, and Fe⁵⁹ incorporation into Hb F in vivo wasdemonstrated in one patient after 4 months of transfusion therapy. Under theconditions of these studies, synthesis of Hb F continued despite prolongedcorrection of the anemia. A decline in the proportion of Hb F in the erythrocytes of one patient after 5 months of transfusions suggested that Hb F synthesis may ultimately be depressed by transfusions. It was suggested that theproportion of fetal hemoglobin observed in the erythrocytes might in certaindiseases reflect the degree of anemia present many months before.

Submitted on February 26, 1964 Accepted on April 5, 1964     

Quantitative Serologic and Isotopic Studies on the Rho Variant--Du

1. Type D^u cells take up less than 10 per cent of the I-131 anti-D boundto D positive cells, so that the quantity of cell bound I-131 did not differ significantly from that bound nonspecifically to D negative cells.

2. Papain modification of D^u cells results in an almost threefold increasein the uptake of I-131 anti-D, whereas, D negative cells show no consistentincrease after such treatment. Enzyme modification, therefore, serves to clearlydelineate the two cell types.

3. The range of uptake of I-131 anti-D to enzyme treated D^u cells is fromapproximately 7 to 17 per cent of that by R₁R₂ cells. Most of these results wereobtained from studies on Mendelian D^u types. Studies on several bloods frommembers of a family having Rh genotypes typical of the "gene interaction" D^uconfiguration (C in transposition to D) revealed no significant reduction inI-131 anti-D uptake when compared with D positive cells.

4. Decreased antibody binding to D^u cells was confirmed by the serologicmethods used. Absorption of an anti-D serum and antiglobulin inhibitionwith sensitized D^u cells indicated that the quantity of D antigen was lessthan that observed with the isotopic technic. Elution studies did not show asmarked a reduction of D antigen on the D^u cell as the other serologic technicsindicated. Possible reasons for these differences have been discussed.

5. Although, from both the serologic and isotopic studies, the data indicate that the type D^u red cell is quantitatively deficient in D antigen, theyare inadequate to rule out other explanations for their reduced D antigenactivity. Irrespective of the mechanism(s) responsible, the effective bindingof anti-D by D^u cells is reduced several fold; this would appear to be theprimary cause of the weak second stage of red cell antigen-antibody reactionwhich characterizes type D^u blood.

Submitted on July 28, 1964 Accepted on October 8, 1964     

A New Property of Iso-Agglutinins of the ABO Blood Group System

A new agglutination phenomenon in the ABO group system is described.This phenomenon consists of the development of agglutinates by interactionbetween a single specifically sensitized cell and other unsensitized, nonantigenic cells. Aggregates of cells formed in this way are tentatively describedas "anomalous mixed agglutinates."

Some of the properties of anomalous mixed agglutinates have been investigated, and the impact of these properties on the current theories ofagglutination is briefly discussed.

Submitted on November 21, 1958 Accepted on January 7, 1959     

Specificity of lytic factors for erythrocytes,leukocytes and platelets in a case of pancytopenia

A case of chronic idiopathic pancytopenia in a young girl is presented, in whichthe pancytopenia was shown to be due to increased destructions of all 3 blood celltypes. Antileukocyte and antiplatelet antibodies were demonstrated by transfusion methods as well as by in vitro agglutination, while differential agglutination provided evidence of a plasma factor causing increased red cell destruction.

Cross absorption experiments demonstrated the presence in the patient’sserum of at least 2 separate and distinct antibodies, specific for leukocytes andplatelets respectively.

Observations on the phagocytic behavior of leukocytes and on the electrophoretic mobility of leukocytes and platelets exposed to the patient’s serum arereported.

Submitted on October 21, 1955 Accepted on March 24, 1956     

The Action of Two Sulfhydryl Compounds on Normal Human Red Cells: Relationship to Red Cells of Paroxysmal Nocturnal Hemoglobinuria

Treatment of normal human red cells with AET and cysteine, under suitableexperimental conditions, modifies them in such a way that their behavior inin vitro hemolysis tests becomes similar to that of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

It is felt that alteration of the red cells is due to the -SH groups possessedby both substances. A possible mechanism of action is hypothesized.

Submitted on April 7, 1964 Accepted on June 17, 1964     

Tissue Culture of Cells Already in DNA Synthesis from Patients with Infectious Mononucleosis

1. Peripheral blood cultures from patients with infectious mononucleosiscontained approximately 50 times more cells incorporating tritiated thymidineat time 0 than normal cultures.

2. Cultures from these patients showed active cell division within thefirst 24 hours. No cell division was seen during this time in cultures of normal cells.

3. Cells dividing within the first 24 hours were identified as cells whichhad incorporated thymidine at time 0 by observation of metaphases containingtritiated thymidine.

4. Cell division ceased after 18-24 hours in the absence of phytohemagglutinin.

5. The data suggest that a population composed of immature lymphocyticcells in the circulation of patients with infectious mononucleosis is in theprocess of cell division. Proliferative capacity of peripheral blood cells cannot be studied adequately by ordinary morphologic technic.

Submitted on March 16, 1964 Accepted on November 11, 1964     

Investigation on monoclonal antibodies against two serovars of the icterohaemorrhagiae serogroup of leptospira

By means of the cell fusion technique, two hybridoma cell lines, V-1 and H₂-1 have been obtained. V-1 cells secrete monocloncal antibody against serovars icterohaemorrhagiae and dakota. The H₂-1 cell line secretes serovar-specific monoclonal antibody against serovar H₂.These monoclonal antibodies have been successfully used in serovar-typing of leptospires isolated in China. The results of identification of leptospires by using monoclonal antibodies showed total coincidence with that by the traditional cross agglutinin absorption test and factor antiserum method.It was confirmed by using monoclonal antibody that the serological agglutination totally paralleled with animal protection. On the basis of the study, a concept was proposed that the agglutination in vitro and the protection in vivo are different manifestations in different reaction systems from the same antibody (antibodies) stimulated by a component(s) of the surface antigen of leptospires.     

Studies on the survival of incompatible cells in patients with hypogammaglobulinemia

1. Four adult patients are described whose sera showed a complete lackof gamma globulin by paper electrophoresis.

2. All four patients might have been considered "universal recipients" because of the absence of isohemagglutinins on routine laboratory tests.

3. Weak isohemagglutinins were detectable in the sera from three of thefour subjects when the sensitivity of the in vitro test was increased.

4. Following intravenous administration of 4 ml. of ABO-incompatible cells,red cell survival was shortened in all of the patients, with a wide range ofhalf-survival times from <10 minutes to as long as 9 days.

5. Although in vitro tests for antibody activity revealed no qualitative differences among the sera from the three patients with detectable isohemagglutinins, two different mechanisms of red cell removal were observed, onewhich entailed nearly equal activity by both liver and spleen, the other beingprimarily a function of the spleen.

6. The survival of incompatible cells under conditions of antibody "excess"and antibody "insufficiency" was compared in one of the patients. The findings emphasize the sensitivity of in vivo survival studies employing smallvolumes of incompatible cells to detect minute quantities of circulating antibody.

7. A fall in anti-A titer following the administration of group B cells toone hypogammaglobulinemic subject is interpreted as a possible in vivoexample of the absorption of "cross-reacting" antibody in the ABO system.

8. In the light of the in vivo and in vitro findings, none of the 4 hypogammaglobulinemic patients in the present series could be categorized as "universalrecipients."

Submitted on March 10, 1958 Accepted on May 20, 1958     

Erythropoietin in Vitro. II. Effect on "Stem Cells"

The in vitro effect of sheep erythropoietin on bone marrow cells wasstudied in order to elucidate its point of action. It was concluded thaterythropoietin in vitro acts primarily or exclusively on stem cells and doesnot have a measurable effect on differentiated nucleated red blood cells oron reticulocytes. Attempts to isolate and study stem cells by freezing orby long or short term culture were not successful.

Submitted on December 5, 1963 Accepted on February 28, 1964     

The Detroit strains of human epithelial-like cells from nonleukemic peripheral blood

1. The development of 6 strains of Ep-L cells from human blood was reported. In each instance the Ep-L cells appeared in cultures which had someconnection with the administration of estrogenic materials, either to the donorof the blood or to the primary explant of blood in vitro. The possible role ofestrogens was discussed, but no final conclusions were reached.

2. The development of stable human Ep-L cells did not require the participation of cells from leukemic blood.

3. Morphologic, karyotypic, and mitotic data on the Detroit blood cellstrains were presented.

4. The morphogenesis of the Ep-L cells was described. The most likelysource of the Ep-L cells in blood cultures appeared to be lymphocytes, butthe role of monocytes could not be ruled out.

5. The significance of our findings with respect to transplantation studieswas indicated.

Submitted on February 28, 1958 Accepted on April 12, 1958     

Erythroid Homograft Following Leukocyte Transfusion in a Patient with Acute Leukemia: II. Serologic and Immunochemical Studies

The establishment of a hematopoietic graft of stem cells from a donor withchronic myelogenous leukemia in a patient with acute leukemia took placein the face of ABO red cell group incompatibility. The donor was group Aand the recipient who was group O gradually increased his red cell mass tobecome 80 per cent group A.

There was both active and passive immunity to A present at the time ofinduction of the graft. The graft flourished despite persistent anti-A agglutininsand an immune response in the B agglutinin and hemolysin system.

Failure of the graft coincided with a fall in antibody levels and was followed by a second immune response which included marked elevation of 7Sgamma globulin levels. Red cell incompatibility was not a barrier to thisgraft and failure of the graft was probably due to other immune mechanisms.

Submitted on September 14, 1964 Accepted on February 2, 1965     

Life Span of Reticulocytes in Paroxysmal Nocturnal Hemoglobinuria

Two patients with PNH were transfused with young and old Cr⁵¹-labeledred cells. The young red cells (reticulocytes) were destroyed more rapidlythan the older red cells. In vitro studies also revealed that the reticulocytewas more susceptible to acid hemolysis in acidified serum.

In contrast the Cr⁵¹-labeled reticulocytes in 3 patients with perniciousanemia responding to cyanocobalamin showed a normal life span.

In the patients with PNH and pernicious anemia, no sequestration of Cr⁵¹-labeled reticulocytes was noted in the liver or spleen by surface scanning.

Submitted on June 2, 1964 Accepted on September 18, 1964