A High Frequency of Cold Agglutinins of Anti-IA Specificity in a New Guinea Highland Population

Summary. A high incidence (37 out of 188) of anti-IA cold agglutinins was found in the sera of indigenous New Guineans living at Minj (alt. 1,600-1,800 m) in the Western Highlands District of New Guinea. Anti-IA did not occur in three other populations tested, although these possessed a high incidence of anti-I antibodies. The earlier findings concerning the incidence of specificity of cold haemagglutinins in New Guinea are reviewed and it is suggested that they may be induced by different agents, probably of a parasitic nature, in different localities. It is predicted that cold agglutinins should be found in other populations which are subject to high rates of infection similar to those in New Guinea.     

Haemolysis in Cold Agglutinin Disease: The Role of C''and Cell Age in Red Cell Destruction

The mechanism of red-cell destruction was studied in a patient with cold-agglutinin disease. It was confirmed that the patient's red cells were abnormally resistant to lysis by the anti-I cold agglutinin in the serum. This increased resistance is probably due to the accumulation of C'on the red-cell membrane. It was found that red cells were only protected if C'was brought on to the red cell by anti-I antibodies and not if this was the result of treatment with normal serum at low ionic strength. The resistance was also found only to apply to lysis by anti-I antibodies and not to a haemolytic rabbit anti-human-red-cell serum. These results can be explained by assuming that inactive C'complexes around the I-antigen were blocking receptor sites for new haemolytic C'factors.
It was shown in normal individuals that red-cell age does not effect the susceptibility of the cells to lysis by anti-I serum. In the patient with cold-agglutinin disease, however, the young cells were preferentially lysed, probably because these cells had not yet accumulated C'to the same extent as the more mature cells and were, as a result, less resistant to haemolysis.     

Demonstration of Low-Titer Anti-Pr Cold Agglutinins

Abstract. Hemagglutination tests with protease- and neuraminidase(RDE)-treated red cells are essential to demonstrate anti-Pr cold agglutinins, since RDE and proteases inactivate the Pr antigens in contrast to the I/i antigens. RDE treatment of red cells, however, reveals the T antigen which reacts with anti-T present in anti-Pr sera. Furthermore, anti-I, also present in anti-Pr sera, shows increased reactions with RDE- and protease-treated red cells. Therefore, T-anti-T and I-anti-I reactions mask the loss of anti-Pr activity against RDE- and protease-treated red cells when low-titer anti-Pr sera are studied. Absorption of anti-Pr sera with RDE-treated red cells removes anti-T and anti-I, leaving anti-Pr cold agglutinins which only react with untreated red cells, not with RDE- or protease-treated red cells. Anti-I contaminating anti-Pr in warm eluates from untreated red cells or stroma can be also removed by absorption with enzyme-treated red cells. By eliminating T-anti-T interference from sera, it was possible to show that all Pr determinants known are determined by N-acetylneuraminic acid.     

The simultaneous presence of anti-I and anti-i in sera

The specificity of antibodies that detect the antigens I, i, I^T, IH (10), IA, IB, iH (iO), and IP₁, [23, 10, 21, 11, 12, 3, 16, 17, 2, 5, 18, 22, 4, 20, 6, 7] can be established with little difficulty. There remain, however, many cold agglutinins that do not have specificity directed against any one of these antigens; this paper describes studies on 22 such sera, 3 of which react equally with I and i cells (regardless of ABO group) and 19 of which have anti-I specificity but react with significant strength with i cells as compared to a ‘standard’ anti-I. Absorption and elution experiments with selected erythrocytes show that all 22 sera contain separable anti-I and anti-i.     

Variability of Sheep Red Cells in their Reaction to Agglutinins in Normal Human Sera

S ummary The interaction (agglutination) of 25 normal sera against a panel of 10 different sheep red cells was studied. There were highly significant differences in the agglutinability of different sheep cells to any one normal serum, and the cells could be divided into 'high reactors'and 'low reactors'. Absorption studies confirmed these observations. The significance of these findings is discussed in the light of previous studies on anti-sheep cell haemagglutinins in disease and the selection of sheep cells for all types of haemagglutinin immunoassays.     

The Unitary Nature of "Complete" and "Incomplete" Pathologic Cold Hemagglutinins

Several human pathologic sera containing high titered cold agglutinins werestudied to determine whether the serologic activity ascribed to an "incompletecold antibody" could be separated from the "complete" cold agglutinin activity.Separation was not achieved by physicochemical methods, including zoneelectrophoresis, density gradient ultracentrifugation, and anion exchangechromatography. Both activities were susceptible to destruction by mercaptans.Neither activity could be differentially absorbed from the sera. Using "Bombay" and I-negative ("i") red cells, a difference in specificity of the two activities for the H or I antigen of human erythrocytes could not be demonstrated.The simplest interpretation of these findings is that there is only one antibodyinvolved, the cold agglutinin, and that the serologic manifestation usually attributed to an additional "incomplete cold antibody", i.e. the production of apositive antiglobulin reaction of the "non--globulin" type, results from aninteraction of complement components with the cold agglutinin-erythrocytecomplex.

Three of these cold agglutinating sera were unreactive with I-negativeerythrocytes, in keeping with the reported anti-I specificity of these antibodies. A fourth serum retained moderate, though greatly reduced, activityagainst these cells, and the interpretation of this finding is discussed.

The anti-H specificity of the incomplete cold antibodies in normal humansera was confirmed by their failure to sensitize "Bombay" erythrocytes. Thiswas in sharp contrast to the excellent reactivity of the pathologic sera withthese cells, demonstrating that pathologic cold agglutinins are unrelated tothe incomplete cold antibodies present in most normal sera.

Submitted on October 23, 1961 Accepted on December 2, 1961     

Suppression of chemotactic activity of human polymorphonuclears by monoclonal IgMs with and without biological activity

The influence of monoclonal IgMs on migration of human polymorphonuclears was studied at various temperatures by the use of 19 sera with monoclonal IgMs from patients with macroglobulinemia of Waldenstrom (MW) without obvious biological activity, 29 sera with monoclonal IgM cold agglutinins (18 with anti-I and 11 with anti-i IgMs) and 3 sera with monoclonal IgM rheumatoid factor (RF). Under-agarose migration method and modified Boyden chamber method with double filters and 51Cr-PMNs were used. In under-agarose method, chemotactic differentials for controls, MW, anti-I, and anti-i groups were, respectively, 57 +/- 8 mm, 39 +/- 9 mm, 44 +/- 14 mm, and 32 +/- 16 mm at 37 degrees C and 47 +/- 18 mm, 22 +/- 11 mm, 17 +/- 9 mm, and 15 +/- 12 mm at 24 degrees C. All three sera with IgM RF inhibited chemotaxis. The differences between all groups and controls were significant at p less than 0.01. Random migration was inhibited at 24 degrees C (p less than 0.01) but not at 37 degrees C. Inhibitory concentrations of IgM in the sera tested were equal or less than 0.5 mg/ml. Thirteen sera were tested by the modified Boyden chamber method. At 37 degrees C 8 of 13 sera and at 24 degrees C 11 of 13 sera inhibited significantly chemotaxis at a concentration of IgM of 1 mg/ml. The lowest inhibitory concentration of IgM was 25 micrograms/ml. Eleven chromatographically pure IgMs were tested in the under-agarose assay. At concentrations of 0.4-3.7 mg/ml, eight IgMs inhibited chemotactic differential at 37 degrees C and nine inhibited it at 24 degrees C. At concentrations of 0.6-2.0 mg/ml, all seven pure IgMs tested by the Boyden chamber method significantly inhibited chemotaxis at 24 degrees C and 37 degrees C. Some IgMs inhibited chemotaxis at concentrations as low as 25 micrograms/ml. Ten IgM CA were eluted from the red blood cells. Eluates inhibited strongly chemotaxis at 24 degrees C and 37 degrees C. Heat inactivation did not alter inhibitory activity of IgM, however pepsin digestion or reduction and alkylation of purified IgMs did abolish their inhibitory activity. Inhibition of chemotaxis was not related to the light chain type, the titre, or the thermoamplitude of cold agglutination. However, monoclonal IgMs with anti-i cold agglutinin activity were stronger inhibitors than anti-I. Since 75% of IgMs tested inhibited chemotaxis at 37 degrees C, it is possible that monoclonal IgMs, especially those with anti-i cold agglutinin activity, inhibit PMN migration in vivo.     

Coexisting Anti-I and Anti-Fl/Gd Cold Agglutinins in Infections by Mycoplasma pneumoniae

192 sera containing cold agglutinins of apparent anti-I specificity were reinvestigated for concomitant cold agglutinins (CA) against sialic acid-dependent antigens. 35 cases of additional anti-F1 and 3 cases of additional anti-Gd were detected. 53% of cases with coexisting anti-I and anti-F1/Gd CA had a clinical diagnosis of pneumonia, in 39% IgM antibodies against Mycoplasma pneumoniae could be demonstrated. Since F1 and Gd antigens are identical with the structures identified as receptors for M. pneumoniae, the findings support the hypothesis that postinfectious CA are directed against the receptor of the infectious agent.     

Association between HLA and Red Cell Antigens

Abstract. Cytotoxic HLA antisera of various specificities were examined on the AutoAnalyzer against red blood cells (RBC) from a great number of HLA-typed donors. HLA-associated haemagglutinins of different specificities were demonstrated. In cytotoxic anti-HLA-A9 and anti-HLA-A10, as well as in anti-HLA-B12, anti-HLA-Bw15 and anti-HLA-B17 sera, haemagglutinins of apparently corresponding specificities were found. This indicates that several HLA antigens may be present on RBC, but they are demonstrable only on RBC from a limited number of donors who possess the corresponding antigens on their white cells. Some antigens, like HLA-A28 and HLA-B7, are relatively strong RBC antigens, whereas others, like HLA-B12 or HLA-Bw15 seem to be much weaker. In cytotoxic anti-HLA-A1 and anti-HLA-B13 sera, no corresponding haemagglutinins were demonstrated. However, haemagglutinins of anti-HLA-B7 specificity were shown to be present in two cytotxic anti HLA-A1 and three anti-HLA-B13 sera. No cytotoxic anti-HLA-B7 was found in these sera. Cross-reacting antibodies also seem to be frequent among HLA associated haemagglutinins.     

Endemic Autoimmunity: Cold Auto-agglutinins in Melanesia

Sera from two Melanesian populations, one living at sea level and the other at 7000 feet, were examined for the incidence, strength and specificity of cold agglutinins. The cold auto-agglutination incidence ranged from 48 to 90 per cent for the sea-level group to 94 per cent for the highlanders. Titres of the sera ranged from 10 to 640. The specificity of the antibodies was in the main anti-I, although four probable examples of anti-AI, four examples of anti-I^T and two 'non-specific'cold agglutinins were found. Blood group A subjects in both populations had a higher incidence and titre of cold agglutinins than O subjects.
The light chain types of 160 of 200 cold agglutinins tested were both k and λ, 24 were K only and 16 were λ only. Fingerprints of the H chains of six New Guinea cold agglutinins showed some peptide spots missing compared with normal IgM, but more spots were present than in a lymphoma cold agglutinin, suggesting that the cold agglutinins represented a heterogeneous population of antibodies.
No obvious relationship was found between environmental factors and the occurrence of the cold agglutinins.     

Rapid Purification of Anti-I and Anti-i Cold Antibodies by Affinity Chromatography

A method is described for the rapid purification of serologically active high titer anti-I and anti-i cold antibodies from the sera of patients with chronic cold agglutinin disease (CCAD). The purification procedure is based on thermal affinity chromatography, using desialated orosomucoid (alpha 1-acid glycoprotein)-Sepharose 4B conjugated beads. The nature of the interaction between the cold agglutinins (CA) and the desialated orosomucoid is unknown. Inhibition studies, however, revealed that the cold hemagglutinating activities of all the anti-i sera were inhibited by desialated orosomucoid while only 1 out of 4 of the anti-I sera was similarly affected. Anti-I or anti-i antibodies were separated from whole sera in 7 out of 7 samples with a recovery in most cases of 100% of the cold hemagglutinating activity. The resultant products were purified monoclonal IgM fractions which could react with anti-kappa and anti-mu but not with anti-lambda sera. The homogeneity, purity and specificity of all preparations were confirmed by immunodiffusion analysis against purified I and i blood group antigens isolated from human erythrocyte membranes, zonal and right-angle electrophoresis and hemagglutination or hemagglutination inhibition studies.     

Association between HL-A and Red Cell Antigens

Abstract. Two sera with cytotoxic anti-HL-A2 antibodies and haemagglutinins reacting preferably with red cells from HL-A28-positive² donors, have been absorbed with several white and red cells. Both cytotoxic and haemagglutinating antibodies are shown to cross-react with the two antigens. Absorption experiments have also been carried out in two sera with cytotoxic anti-HL-A8 activity and haemagglutinins. The results indicate that both the haemagglutinating and the cytotoxic antibodies are directed against the HL-A8 antigen. The titration and absorption results show that the haemagglutinins are rather abundant in the sera, while the antigenic sites on the red cells are apparently very few.     

Association between HL-A and Red Cell Antigens. An AutoAnalyzer Study

Abstract. Sera containing cytotoxic HL-A antibodies were examined in order to detect red cell antibodies, using an AutoAnalyzer. In sera with cytotoxic anti-HL-A2 antibodies, we were able to demonstrate haemagglutinins that reacted mainly with red cells from HL-A28 positive donors. In sera with cytotoxic anti-HL-A8 antibodies, we found antibodies that reacted with red cells from some, although not all, HL-A8 positive donors. The haemagglutinins gave indentical and highly reproducible reaction patterns, but there were marked variations in antigenic strength.     

Iso- and Heteroagglutinins in Human Fetal and Neonatal Sera

Maternal AB and sheep cell haemagglutinins were found in the human fetus after the 13th to 14th week of gestation. In fetuses aged 18 weeks or more they were observed as frequently as in full-term newborns, 56.6% of which had isoagglutinins and 57.6% heteroagglutinins. The occurrence of maternal heteroagglutinins in neonatal sera was not dependent on the ABO group of the mother as it is with isoagglutinins. Heteroagglutinins of the IgG class were found equally in mothers of different ABO groups. Isoagglutinins against erythrocytes of the mother's group were detected in 8 out of 44 neonatal sera, but in none of 10 sera from fetuses aged 14 to 19 weeks. IgM concentrations in neonatal sera with and without isoagglutinins of fetal origin revealed that IgM synthesized by the healthy fetus consists mainly of antibodies other than isoagglutinins.     

Horse Anti-Type XIV Pneumococcus Sera Behave as Cold Agglutinins Recognizing Developmentally Regulated Antigens A part from the Ii Antigens on Human Erythrocytes

Haemagglutination studies with horse anti-type XIV pneumococcus sera have shown that they resemble cold agglutinin sera with anti-I specificity reacting more strongly with adult human erythrocytes than cord erythrocytes. However, their substantial reaction with i_adult erythrocytes suggests that they react with other developmentally regulated antigens in addition to the I and i antigens.     

Diagnosis and Pathogenesis of Nairobi Sheep Disease Orthonairovirus Infections in Sheep and Cattle

Nairobi sheep disease orthonairovirus (NSDV) is a zoonotic tick-borne arbovirus, which causes severe gastroenteritis in small ruminants. To date, the virus is prevalent in East Africa and Asia. However, due to climate change, including the spread of transmitting tick vectors and increased animal movements, it is likely that the distribution range of NSDV is enlarging. In this project, sheep and cattle (hitherto classified as resistant to NSDV) were experimentally infected with NSDV for a comparative study of the species-specific pathogenesis. For this purpose, several new diagnostic assays (RT-qPCR, ELISA, iIFA, mVNT, PRNT) were developed, which will also be useful for future epidemiological investigations. All challenged sheep (three different doses groups) developed characteristic clinical signs, transient viremia and virus shedding—almost independent on the applied virus dose. Half of the sheep had to be euthanized due to severe clinical signs, including hemorrhagic diarrhea. In contrast, the course of infection in cattle was only subclinical. However, all ruminants showed seroconversion—implying that, indeed, both species are susceptible for NSDV. Hence, not only sheep but also cattle sera can be included in serological monitoring programs for the surveillance of NSDV occurrence and spread in the future.     

Chemical analyses of variable regions of heavy and light chains of cold agglutinins

Abstract. The variable regions of cold agglutinins with anti-I and anti-Pr specificities were investigated by N-terminal amino acid sequencing and by analyses of pyrrolidone-carboxylic acid-blocked peptides after digestion of polypeptide chains with Nargase. Results showed that the heavy chains of four IgM anti-I cold agglutinins are exclusively VHI subgroups and their light chains are exclusively VKII subgroup. In contrast, the light chains of two cold agglutinins with anti-Pr specificity are not VKII, while their heavy chains are not restricted to a single subgroup. The amino acid sequences at the first hypervariable region of light chains (positions 25–35) are similar in two of the four anti-I cold agglutinins. These sequences are different from that of the light chain of another cold agglutinin with anti-Pr specificity. These results support the concept that only antibodies with the same specificity can share similar primary structure at their antigen combining sites.     

Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.     

Differentiation-dependent expression of sialyl stage-specific embryonic antigen-1 and I-antigens on human lymphoid cells and its implications for carbohydrate-mediated adhesion to vascular endothelium

Expression of two developmentally regulated carbohydrate antigens, the sialyl stage-specific embryonic antigen-1 (SSEA-1) and I-antigens, in human lymphocytes and lymphocytic leukemia cells was investigated using specific monoclonal antibodies. Sialyl SSEA-1 was expressed only on natural killer (NK) cells, and was essentially absent on resting mature T and B cells among normal peripheral lymphocytes. On the other hand, the I-antigen was strongly expressed on virtually all mature B cells, moderately expressed on most mature T cells, but not expressed on NK cells in normal donors. Expression of the two antigens on normal T and B cells was reversible; in vitro stimulation of normal lymphocytes with concanavalin A (Con A) resulted in the loss of I-antigen and appearance of sialyl SSEA-1 on CD3+ T blasts, whereas stimulation with pokeweed mitogen led to loss of I-antigen expression and appearance of sialyl SSEA-1 antigen on CD19+ B blasts. Among lymphocytic leukemia cells, sialyl SSEA-1 was detected primarily on leukemia cells having immature properties such as most common acute lymphocytic leukemia (cALL) blasts, while the I-antigen was frequently expressed on malignant cells having relatively mature properties, such as those found in adult T- cell leukemia or chronic lymphocytic leukemia, and only occasionally on cALL blasts. Among normal peripheral lymphocytes, the sialyl SSEA-1+I- antigen- NK cells selectively underwent E-selectin (ELAM-1, endothelial- leukocyte adhesion molecule-1)-dependent adhesion to endothelial cells, while the I-antigen+sialyl SSEA-1- mature T and B cells did not, in line with the recent finding that sialyl SSEA-1 serves as a specific ligand for E-selectin. Con A blasts, which are sialyl SSEA-1+I-antigen- , also exhibited significant E-selectin-dependent adhesion to endothelial cells. These results indicate that expression of the sialyl SSEA-1 and I-antigens varies alternately depending on the differentiation/activation status of the lymphocytes, and that this at least partly regulates the behavior of lymphocytes at the vessel wall.     

Fertility of the cysts of Echinococcus granulosus in domestic herbivores from Benghazi, Libya, and the reactivity of antigens produced from them.

Unilocular cysts produced by Echinococcus granulosus were recovered from 110 domestic herbivores (60 sheep, 25 cattle, 20 goats and five camels) slaughtered in Benghazi. The proportion of the cysts from the sheep found to be fertile (75%) was higher than that of the cysts from the goats (55%), camels (40%), or cattle (0%). When tested in indirect haemagglutination assays (IHA) with eight sera from human cases of cystic echinococcosis, the fluid from the cattle cysts never gave a positive reaction. Antigens in the fluids collected from sheep or goat cysts did react with the sera, with antigens from each of the two sources giving similar titres with each serum. However, crude somatic antigens (prepared from protoscolices and brood capsules collected from sheep cysts) appeared to be more sensitive for the immunodiagnosis of human cystic echinococcosis than the cyst-fluid antigens.