Search

Neutrophils are the most abundant nucleated cells in blood circulation and play important roles in the innate and adaptive immune responses. Neutrophil-specific antigens, only expressed on neutrophils, are glycoproteins originally identified in studies on neonatal neutropenia due to fetal-maternal incompatibility and autoimmune neutropenia of infancy. The most investigated neutrophil–specific antigens are the NA and NB antigens that their incompatibilities also cause transfusion-induced febrile reactions and acute lung injury, a potentially fatal reaction, and in bone marrow transplantation, causing graft rejection. NA antigens are members of the immunoglobulin superfamily and are low-affinity Fc-receptors FcγRIIIb (CD16b). Fc receptors connect the F(ab), the antigen-binding fragment of the antibody molecules, to neutrophils and lead them to recognize and phagocytize the targeted antigens. The NB (CD177) antigen belongs to the urokinase-type Plasminogen Activator Receptor Superfamily (uPAR, CD59, Ly6), but its specific functions have not been fully determined. It is known, however, that NB antigen binds proteinase-3 (PR3 to the neutrophil membrane), a serine protease. In clinical studies, it was also demonstrated that NB expression is highly elevated in Polycythemia Vera and is unexpectedly expressed in some cancer tissues. Neutrophil-specific antigens are examples of antigens that have important biological and clinical activities beyond antigenicity.

The immune system has been described to play a role in the development of Alzheimer’s disease (AD), but the distribution of immunoglobulins and their subclasses in brain tissue has not been explored. In this study, examination of pathologically diagnosed frontal cortex gray matter revealed significantly higher levels of IgM and IgG in late-stage AD (Braak and Braak stages V and VI) compared to age-matched controls. While levels of IgG2 and IgG4 constant region fragments were higher in late-stage AD, concentration of native–state IgG4 with free Fc regions was increased in AD III and VI. RNA analysis did not support parenchymal B-cell production of IgG4 in AD III and V, indicating possible peripheral or meningeal B-cell involvement. Changes in the profile of IgM, IgG and IgG subclasses in AD frontal cortex may provide insight into understanding disease pathogenesis and progression.

Introduction Mutations in brain tissues that cumulate with age may contribute to Alzheimer's disease (AD). Abnormal glycoprotein and Tn antigen expression have been demonstrated in AD. We identified C1GALT1C1/COSMC mutations in AD and age‐matched normals without AD. The COSMC coding mutations resulted in a significant reduction in T‐synthase activity in advanced AD cases. Methods Identification of COSMC mutations, Real‐Time Quantitative Reverse Transcription PCR (Q‐RT‐PCR), western blotting, and T‐synthase activity assays. Results COSMC mutations were detected in the promotor, coding region and 3′UTR in AD and normals. COSMC coding mutations demonstrated a correlation with AD progression. T‐synthase levels were significantly elevated in advanced AD compared to AD III (P = 0.03) and normals (P = 0.002). T‐synthase activity in advanced AD {Braak and Braak (B&B) stages V and VI} with COSMC coding mutations was 3‐fold lower than advanced AD without mutations, and 1.3‐fold lower than normal (P = 0.001) and AD B&B stage III (P = 0.01) with coding mutations. Discussion COSMC coding mutations significantly diminished T‐synthase activity in advanced AD, potentially causing defective galactosylation.

A new method for extraction and concentration of organic dyes that uses a reagent composed of a nonionic detergent mixed with an alcohol is described. We have observed that water-soluble organic dyes are also soluble in nonionic detergents and can be extracted by adding salt, which separates the dye-detergent component from the aqueous phase. We have also found that mixing nonionic detergents with alcohols markedly reduces their viscosity and produces stable, free-flowing, and effective reagents for color extraction. On the basis of these observations, we used a mixture of Triton X-100 and 1-butanol and observed that water-soluble natural and synthetic chromophores, as well as dyes generated in biochemical reactions, can be extracted, concentrated, and analyzed spectrophotometrically. Trypan blue and phenol red are used as examples of synthetic dyes, and red wine is used as an example of phenolic plant pigments. Applications for quantification of nitric oxides and sialic acids are described in more detail and show that as little as 0.15 nmol of nitric oxide and 0.20 nmol of sialic acid can be detected. A major advantage of this method is its ability to concentrate chromophores from dye-containing solutions that otherwise cannot be measured because of their low concentrations.

Doctor Parviz Lalezari, currently a clinical professor of Medicine and Pathology at Albert Einstein College of Medicine in New York, describes highlights of his research career since 1958. He became the director of the blood bank at Montefiore Hospital in New York City in 1961, director of the Division of Immunohematology until 1996, and then until 2001, was President and chief executive officer of the Bergen Community Regional Blood Center in New Jersey. Doctor Lalezari was born in Iran in 1931, and after graduation from Medical School, he came to the United States in 1956. His initial research was on leukocyte antibodies. After modifying the available antibody detection techniques, he discovered that like hemolytic disease of the newborn and neonatal immune thrombocytopenia, fetal-maternal neutrophil incompatibility can cause neonatal neutropenia. He identified the targets of these antibodies and showed that they were expressed only on peripheral blood neutrophils. Doctor Lalezari also discovered that a common form of neutropenia in early childhood was caused by development of autoantibodies, which surprisingly were directed against the same neutrophil-specific antigens involved in fetal-maternal incompatibility. In 1959, a heparin-neutralizing drug (Polybrene) was introduced to be used after open-heart surgery. Lalezari discovered that Polybrene, a quaternary ammonium polymer, reacted with sialic acid molecules on the red blood cell (RBC) surface, causing the RBCs to aggregate. Later, realizing that the repelling forces generated by the RBC surface membrane charges were responsible for failure of the small IgG antibody molecules to agglutinate the RBCs, he used Polybrene to neutralize the RBC surface negative charge to allow the IgG antibody molecules to induce hemagglutination. This became The Polybrene test, which is to be used in RBC antibody detection.

The reducing agent dithiothreitol was used to deoxygenate human haemoglobin, and the time course observed via visible light spectroscopy. A decrease in the time required for deoxygenation was found for exposures to both a pulsed radiofrequency signal currently in clinical use for treatment of pain and edema, and a 150 mT static magnetic field.

The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40tax in their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40 tax . Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1 tax sequences and antibodies to p40 tax , while they lacked sequences and antibodies related to gag, pol ,o renv. Thus, it seemed important to determine whether the “tax-only” state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegative tax-only-positive MF patients or from healthy tax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits became tax sequence positive. These observations suggest that HTLV-1 tax can be transmitted by tax-only-positive mononuclear cells. The human T-cell lymphotropic virus (HTLV) type 1 (HTLV-1) is causally associated with adult T-cell leukemia and lymphoma (27) as well as with the nonneoplastic conditions tropical spastic paraparesis (5) and HTLV-associated myelopathy (22). The vast majority of patients with these diseases as well as healthy carriers of HTLV-1 have antibodies to the structural proteins of this virus. Because infection has been shown to be transmitted by transfusion (18, 21), all blood collected for this purpose has been screened for antibodies to this virus since 1988 (2). However, subsequent studies with patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) revealed that the majority carry the proviral tax sequence of HTLV-1 in their peripheral blood mononuclear cells (PBMCs) and skin-infiltrating lymphocytes, while they have no antibodies to the structural proteins of the virus (14, 24). This raised the question of whether the “tax-only”-positive state may also pertain to some healthy, serologically negative blood donors. Indeed, on the basis of serologic tests for 250 randomly selected donors who presented at the New York University Medical Center blood bank, 8.6% proved to harbor proviral HTLV-1 tax in their PBMCs and also had antibodies to p40 tax , the protein encoded by this sequence (36, 39). Therefore, a study to determine whether the tax-only state could be transmitted by transfusion of tax-positive cells seemed to be indicated. It should be recalled here that HTLV-1 tax functions in the transcriptional transactivation of the HTLV-1 long terminal repeat and the transactivation of numerous cellular genes, particularly those involved in inflammation and cell proliferation, such as interleukin-1 (IL-1), IL-2, the a subunit of the IL-2 receptor, IL-6, granulocyte-macrophage colony

Structural abnormalities in the cytoplasmic region of the G-CSF receptor (G-CSF-R) or defects in signal transduction pathways triggered by the G-CSF-R or both have been implicated in the development of neutropenia and increased prediposition to leukemia in patients with severe congenital neutropenia (SCN). To assess the structural integrity of the G-CSF-R in SCN patients, the transmembrane and cytoplasmic regions of the G-CSF-R from 5 SCN patients were cloned and sequenced. DNA mutations (point, deletion, frame-shift, and silent) were observed in 3 patients. In 2 of these, the DNA mutations resulted in altered G-CSF-R protein sequences, including additions of novel C-terminal sequences. Three of the 5 mutant receptor clones lacked 115-121 amino acids in the cytoplasmic region, and 2 showed complete loss of the transmembrane and cytoplasmic regions. Neutrophils from 1 patient expressing these mutant receptors showed normal binding of radiolabeled G-CSF. G-CSF-R in 2 other patients with SCN showed no mutations. Our results indicate that structural abnormalities in the G-CSF-R may be present in some SCN patients. They may not affect the binding of G-CSF to the receptor but may contribute to the pathogenesis of SCN through impaired signal transduction pathways of the mutant G-CSF-R.

The growing body of clinical and experimental data regarding electromagnetic field (EMF) bioeffects and their therapeutic applications has contributed to a better understanding of the underlying mechanisms of action. This study reports that two EMF modalities currently in clinical use, a pulse-modulated radiofrequency (PRF) signal, and a static magnetic field (SMF), applied independently, increased the rate of deoxygenation of human hemoglobin (Hb) in a cell-free assay. Deoxygenation of Hb was initiated using the reducing agent dithiothreitol (DTT) in an assay that allowed the time for deoxygenation to be controlled (from several min to several hours) by adjusting the relative concentrations of DTT and Hb. The time course of Hb deoxygenation was observed using visible light spectroscopy. Exposure for 10–30 min to either PRF or SMF increased the rate of deoxygenation occurring several min to several hours after the end of EMF exposure. The sensitivity and biochemical simplicity of the assay developed here suggest a new research tool that may help to further the understanding of basic biophysical EMF transduction mechanisms. If the results of this study were to be shown to occur at the cellular and tissue level, EMF-enhanced oxygen availability would be one of the mechanisms by which clinically relevant EMF-mediated enhancement of growth and repair processes could occur.

T, Tn, and sialyated Tn (sTn) are pancarcinoma antigens, and increased expression of these carbohydrate epitopes has been correlated with a poor prognosis in several epithelial malignancies. Ten murine monoclonal antibodies have been generated to these antigens, and compared by ELISA and immunohistochemistry to established mAbs reactive with these antigens. Nine mAbs (3 IgM and 6 IgG) reactive with synthetic T-human serum albumin (T-HSA) were produced after immunizing BALB/c mice with a synthetic T-keyhole limpet hemocyanin glycoconjugate (T-KLH). An additional IgM mAb (145.22) was produced in mice immunized with erythrocytes isolated from a patient with Tn syndrome. Three IgM and six IgG1 mAbs reactive with T-HSA did not react with natural T antigen present on desialyated glycophorin. All three IgM and several IgG1 mAbs, however, did react with LS-174T, a mucinous colon carcinoma cell line, 647V, a human bladder carcinoma cell line, and TA3Ha, a murine mammary carcinoma cell line as well as fresh frozen colon carcinomas. MAb 145.22 reacted with both natural and synthetic sources of sTn and Tn, as well as with LS-174T cells and mucin deposits in 10/11 colon carcinomas on fresh-frozen sections. MAb B72.3 reacted strongly with ovine submaxillary mucin (OSM) and sTn-HSA, while mAb CC49, a second-generation mAb to TAG-72 carcinoma mucin, reacted strongly with OSM, less strongly with desialyated OSM, and only weakly with sTn-HSA, suggesting that the epitope specificity for mAb CC49 is distinct from that of B72.3.

Recent evidence suggests a link between brain injury and the autonomic release of pro-inflammatory cytokines by resident macrophages in the spleen. This phenomenon, termed “brainspleen inflammatory coupling,” has garnered attention from scientific and medical communities interested in developing novel treatments for traumatic brain injury (TBI). Cholinergic stimulation of the α7-subunit nicotinic acetylcholine receptor (α7NAchR) on splenic macrophages has been shown to inhibit their release of pro-inflammatory cytokines. This inhibition, mediated by the parasympathetic nervous system, has been shown to improve outcomes in animal models of sepsis, stroke, and TBI. As evidence of a beneficial role of splenic inhibition grows, new treatment strategies might be applied to many medical conditions involving neuroinflammation, a process that contributes to further neurological deterioration.

Publisher Summary Neutropenia is defined by the fall of absolute neutrophil and band forms below 1.5*109/L. Immunologically induced neutropenias are probably as common as the more familiar autoimmune disorders of red cells and platelets, with clinical manifestations varying from being asymptomatic to severe forms complicated by overwhelming sepsis. Autoimmune neutropenia is divided into primary and secondary forms, depending on whether or not neutropenia is part of a complex autoimmune disease. Furthermore, NA and NB neutrophil-specific antigens are often the target of autoantibodies in autoimmune neutropenias. These antigens are anchored to the outer leaflet of the lipid bilayer of neutrophils via a glycosyl phosphatidyl inositol structure. The chapter also discusses the mechanism of cell destruction. Many drugs cause dose-dependent neutropenia due to myelotoxicity and interference with protein synthesis and cell replication. Some drug-induced neutropenias are not dose related and are considered to be immunologic in origin. Finally, CSFs have become the treatment of choice for neutropenias regardless of their etiology.

Tn polyagglutinability syndrome is an acquired condition where erythrocytes express Tn neo-antigen and become susceptible to hemagglutination by the naturally occurring anti-Tn present in normal sera. Early studies had indicated that O-linked N-acetyl galactosamine was the sole serologic Tn determinant, but more recently O-linked NeuNAc alpha 2, 6GalNAc also has been implicated as a Tn antigen (sialosyl-Tn). However, none of these studies were performed on purified glycoproteins. In this report we examine oligosaccharides of glycophorins A and B purified from Tn erythrocytes of two affected individuals to establish how N- and O-linked saccharides differ from normal. Analysis of carbohydrate composition and treatment with N- glycanase showed that the Asn-linked unit of glycophorin A was not affected. O-linked oligosaccharides were obtained by beta-elimination in the presence of tritiated sodium borohydride. The reduced radiolabeled products were fractionated by Bio-Gel P-2 chromatography, and their structures were investigated by comparison with standards, by monosaccharide quantification, and by neuraminidases of known specificities. The results show that Tn glycophorins from both donors contain intact and truncated forms of trisaccharide and tetrasaccharide NeuNAc alpha 2,3Gal beta 1,3GalNAc and NeuNAc alpha 2,3Gal beta 1,3- (NeuNAc alpha 2,6)GalNAc usually present in glycophorins A and B. The truncated forms include the protein O-linked monosaccharide, GalNAc and disaccharide, NeuNAc alpha 2,6GalNAc (major isomer). The presence of intact glycans in the total population of Tn erythrocytes was confirmed by their susceptibility to T activation after treatment with neuraminidase. The proportion of the four species was not identical in glycophorins of these two donors but, in both, the truncated units predominated and the amount of the disaccharide was approximately one half of that of the monosaccharide. The data are consistent with alterations in UDPGal:GalNAc beta 1,3galactosyl transferase that may have multiple molecular origins and with induction of a specific GalNAc protein alpha 2,6 sialosyl transferase in Tn hematopoietic precursor cells. The molecular basis for these alterations awaits further study.

A rapid manual Polybrene test for detection of red blood cell antibodies have been devised which uses standard laboratory equipment. Red blood cells are incubated with the test sera in a low ionic medium at room temperature for one minute. Polybrene, a quaternary ammonium polymer, is then introduced to cause nonspecific red blood cell aggregation. The test tubes are centrifuged, the cell free supernatant fluid decanted, and the Polybrene effect on the cells is neutralized by adding a dilute sodium citrate-glucose solution. The hemagglutination results are evaluated macroscopically and microscopically. The entire procedure is completed in less than three minutes. In the Rh system, the test is 10--160-fold more sensitive than the antiglobulin reaction. In other systems tested, except for the Kell, a high sensitivity is achieved. The sensitivity for the Kell system is markedly increased, however, by performing a supplementary antiglobulin reaction on the sensitized, Polybrene-treated, red blood cells. The antiglobulin reagent used for this purpose should lack anti-C4 and anti-C3 activities. Sensitivity for cold reactive antibodies is augmented by cooling the cells for 30 seconds before citrate-glucose reagent is added.

The electrochemical and rheologic behaviors of red blood cells (RBCs) from a patient with acquired polyagglutinability were studied. The N-acetylneuraminic acid content of the RBCs averaged less than one-half normal. The majority of the patient’s RBCs (over 90%) showed a reduction in electrophoretic mobility and a decrease in binding of positively charged colloid with a widening of the charge-free areas, but a small population exhibited normal surface charge density. Dextran 40, which does not induce significant aggregation of normal RBCs, caused the patient’s RBCs to form rouleaux. Dextran 80 induced a stronger aggregation of the patient’s RBCs than that of normal RBCs. The increased tendency of the patient’s RBCs to be aggregated by macromolecules was associated with an elevation of blood viscosity. This study on a clinical patient has provided evidence for the conclusions previously derived from experimental studies that the stability of RBC aggregation depends upon a balance between macromolecular bridging force and electrostatic repulsive force and that alterations in RBC surface charge may affect cell aggregation and blood viscosity.

Thrombocytes from HLA-A1 and HLA-A2 negative donors were incubated with plasma obtained from respective HLA-positive donors. The plasma-treated thrombocytes were shown to acquire HLA specificity of the plasma as demonstrated by their ability to absorb lymphocytotoxic activity of monospecific anti-HLA sera. The data are consistent with the hypothesis that the expression of HLA antigens in cells of hematopoietic origin diminishes in the course of differentiation and maturation and that the expression of HLA antigens on thrombocytes is primarily due to absorption of these antigens from plasma.

New examples of the Rhd (D, category III) red cell phenotype are described in three siblings. One of these individuals who had previously been transfused, received three units of Rho(D) positive blood several years later and developed a high titer anti-RhD antibody. This anamnestic response was associated with the development of an autoantibody which persisted for a period of at least 8 months. This observation represents another example of autoimmunization which has rarely been observed to accompany alloimmunization.

In exploring the immunologic causes of chronic neutropenia, we identified a persistent neutrophil-specific antibody with NA2 specificity in the blood of a nontransfused two-year-old girl with severe neutropenia. No such antibody was detected in the maternal blood. The antibody was first studied and identified when the patient was 11 months old, but she had had clinical manifestations since the age of one month. After a trial of steroid therapy, a marked but temporary reduction in the antibody titer occurred, accompanied by the rise of the neutrophils to normal level. Neutropenia reoccurred, however, when the antibody titer began to rise, despite continuation of steroid therapy. This transient response allowed the patient's neutrophils to become available and identified as NA2-positive. Although the cause of this disorder remains obscure, the data presented indicate that the anti-NA2 autoantibody is responsible for the neutropenia observed. (N Engl J Med 293:744–747, 1975)

Cold insolubility of a serum IgA cryoimmunoglobulin was found to be inhibited by the addition of 1.5 mM sodium decanedicarboxylate in vitro. The patient with the cryoglobulin had advanced multiple myeloma complicated by severe hyperviscosity that caused lethargy and episodic loss of consciousness. Decanedicarboxylic acid administered orally resulted in transient relief of symptoms and the loss of cryoprecipitability of the paraprotein. Further in vitro studies revealed that sodium salts of long-chain monocarboxylic acids with a minimum of eight carbons, and dicarboxylic acids with a minimum of 12 carbons inhibited cryoprecipitation. Salts of short-chain carboxylic acids, by contrast, enhanced cryoprecipitation. Sodium phenolate and sodium salts of benzoic acid, 2,4-DNP, phenylpropionic acid, and salicylic acid were also inhibitory. These latter compounds, which have a ring structure, did not cause precipitation at any concentration. It was demonstrated that the presence of a free carboxylic group was required for these activities; conversion of carboxylic acid to amide resulted in the loss of both the inhibitory and cryoprecipitation-enhancing effects. Normal plasma, or plasma from five other patients who had IgG, IgM, or mixed-type cryoglobulinemia, were not affected by any of these compounds. It is suggested that in selected cases of hyperviscosity syndrome associated with cryoglobulinemia, some of these compounds, especially monocarboxylic acids with appropriate chain lengths, or those with a ring structure, may have therapeutic applications.

The selective scavenging capacities of 19 important oxygen radical scavengers were determined by adding them individually to each of the four oxy radical standards, (superoxide, hydroxy, alkoxy and hydroperoxy, and singlet O2), calculating the percent chemiluminescence inhibited, and extrapolating O2 equivalents neutralized from baseline. The sensitivity (0.01 nm/ml) and selectivity of this method not only allows identification of individual oxygen free radical species but also quantitates the efficiency of free radical scavengers.

Summary. A new method is described for direct determination of specificity of red cell-bound antibodies without the need for antibody elution. This method employs Polybrene in an Auto Analyzer continuous flow system. Washed antibody-coated cells are mixed, in different proportions, with uncoated indicator cells of known blood type. If the indicator cells possess appropriate receptor sites for the cell-bound antibody, ‘coagglutination’ occurs between the antibody-coated and indicator cells. In contrast, in the absence of specific receptor sites, indicator cells not only fail to coagglutinate but physically interfere with the expected agglutination of antibodycarrying cells. Quantitative data are provided which appear to be related to the amount of cell-bound antibody. The technique has been used to identify antibodies with Rh and penicillin specificities and antibodies associated with acquired haemolytic anaemia.

A family is reported with multiple cases of neonatal neutropenia. The first child died of severe infection, the second was asymptomatic, the third and the fourth children had a benign course of agranulocytosis which lasted about 10 weeks and was followed by spontaneous recovery. A potent leukoagglutinin was found in the maternal serum which agglutinated leukocytes obtained from the father and all three available children but failed to agglutinate the mother’s own cells. Identical leukoagglutinin was found in the last baby’s serum; it disappeared after the baby recovered. The disorder is tentatively named "isoimmune neonatal neutropenia" and is considered to represent a condition analogous to erythroblastosis fetalis.

The third child of a 34-year-old Caucasian woman presented at two weeks of age with pyoderma of five days' duration and severe neutropenia. The neutropenia persisted for at least 28 days. A neutrophil-specific antibody was demonstrated in both the infant's and mother's blood. Muramidase determinations on the infant's serum revealed an initial level of 3.8 μg per milliliter at four weeks of age with a gradual fall to 2.2 μg per milliliter at eight weeks; this decrease possibly reflected neutrophil degradation. This infant represents a case of immune-induced neutropenia following maternal isoimmunization.

In vitro lysis of fibrin, as indicated by increased fibrinogen-fibrin-related antigen (FR-antigen) in serum is usually seen when whole blood, or plasma, or highly purified fibrinogen prepared by several different procedures is clotted and kept at temperatures above 0 degrees C. This increase is both time and temperature dependent, occurs despite the addition of various plasmin and cathepsin inhibitors, and is probably caused by thrombin evolved during clotting and/or added in vitro. In these experiments, the FR-antigen was measured by a sensitive, reproducible hemagglutination inhibition immunoassay adapted to the AutoAnalyzer. Serum from whole blood contained more than serum from plasma, and fibrin rather than fibrinogen proved to be essential for the in vitro lysis. The phenomenon was also caused by Arvin or Reptilase, suggesting that splitting of one or more arginine or lysine bonds in fibrin may be at least partially responsible. To obtain minimal levels of FR-antigen (< 0.5 mug/ml), plasma is clotted for 4 hr at 0 degrees C with 1.0-5.0 U/ml thrombin, CaCl(2) (0.0125 mole/liter), and epsilon aminocaproic acid (0.05 mole/liter). Slightly higher levels, probably adequate for clinical diagnosis, are obtained by 10-30 min clotting at room temperature. Since endogenous and/or exogenous thrombin is essential for the collection of serum FR-antigen, all the FR-antigen found in normal serum probably results from an irreducible amount of in vitro lysis rather than from continuous intravascular clotting and fibrinolysis.

The thermal and immunochemical properties of seven examples of anti-s and one example of anti-U have been studied. It is shown by the antiglobulin, as well as the automated Polybrene technique, that these antibodies are cold-reacting and share this property with antibodies of the rest of the MNS system. It is furthermore demonstrated that the antibodies studied are IgG immunoglobulins. These data are in conformity with the hypothesis that the thermal properties of various antigen-antibody reactions are primarily determined by the antigen and not by the antibody.

designated Ke (10), was also obtained from a multiparous female, none of whose children ever presented any evidence of neutropenia. This antibody had previously been shown to detect an antigen common to leukocytes, platelets, and several solid tissues. Of these donors only Mrs. Ke had received a tranfusion-1 U of blood 6 years before the present study, 30 years after her last pregnancy.

Intravenous gammaglobulin (IVIgG) was used to treat autoimmune neutropenia of infancy in two males with repeated infections. The neutrophil count increased significantly in both patients with the initial IVIgG therapy; 1 patient went into remission. The neutrophil count in the other remained above baseline for 3 wk, and a subsequent booster infusion also caused the neutrophil count to increase. The patients have remained clinically well since their treatment began. Serial studies of antineutrophil antibody and serum lysozyme, performed to elucidate the mechanism of action, suggested decreased neutrophil destruction, perhaps by Fc receptor blockade, as well as decreased synthesis of antineutrophil antibody. Neutrophil function was not impaired after the neutrophil count increased. Many patients with immune neutropenia have a benign course, but those who have significant infections could be treated, acutely or prophylactically, with intravenous gammaglobulin.

Two episodes of immune hemolysis occurring following the administration of tetracycline-HCI are reported. The patient was found to be symptom-free and to have normal hematologic studies during drug-free intervals. The antigenic specificity of the drug-red blood cell complex was proven by conventional AGT results, as well as the more sensitive Polybrene technique. An in vitro technique for conjugation of tetracycline with human red blood cells is described which provides cells suitable for use for the detection of this unusual antibody.

A series of 2-[4-[[[(substituted-phenyl)amino]carbonyl]amino]phenoxy]-2- methylpropionic acids and other substituted phenoxyacetic acids were synthesized and tested for their ability to reduce the affinity of hemoglobin for oxygen. 2-[4-[[[(3,4,5-trichlorophenyl)amino]carbonyl]amino]phenoxy]-2- methylpropionic acid was found to be the most potent compound known. Structure-activity relationships of the compounds synthesized are discussed.

A method which uses microquantities of reagents is described for detection of agglutinating leukocyte antibodies. The leukocyte preparations are devoid of platelets, and have little red cell contamination. Both serum and plasma can be used as the source of antibody. Leukocyte antibodies, particularly those specific for neutrophils, are detected by this method.

Serum proteins affect the performance of the manual hexadimethrine bromide (Polybrene) test. Red cells suspended in the low-ionic medium adhere to the surface of test tubes when centrifuged. This adherence is prevented by serum components apoprotein B, fibronectin, von Willebrand factor, or serum diluted less than 75-fold, but not by serum albumin or immunoglobulin fractions. A high concentration of serum reduces the sensitivity of the test and destabilizes antibody-dependent aggregation. Disaggregation time (DT), defined as the time required for antibody-dependent aggregates to disperse, was used as an indicator of agglutination stability. DT was often shortened when antibodies were in native serums, but could be prolonged if serums were diluted with 0.85 percent saline. Other factors that influenced DT included antibody concentration, temperature, specificity, and sources of antibodies within specificities. When antibodies were present in a mixture, they produced various DTs related to individual specificities. Determination of these differences allowed the successful identification of multiple antibodies. The procedure, called differential disaggregation time, was also used for typing red cells coated by autoantibodies. Additional modifications improved the performance of the antiglobulin phase.

Erythrocyte-bound immunoglobulins have been characterized by a PVP- potentiated antiglobulin test in 11 patients who had developed antibodies after treatment with alpha-methyldopa. Serologic profiles were recognized that could distinguish between the hemolyzing and nonhemolyzing patients: IgM antibodies together with the first component of complement (C1q) were demonstrated on erythrocytes of all eight hemolyzing patients. By contrast, these immunoproteins were absent from the cells of nonhemolyzing patients and became undetectable when the hemolyzing patients recovered. IgG and its subclasses were variably present on erythrocytes of all patients regardless of hemolytic activity. Eluates prepared from erythrocytes of the hemolyzing patients were shown to contain both IgG and IgM, and fixes C1q, C3, and C4. Eluates from the nonhemolyzing patients contained only IgG. The IgM antibodies differed from the commonly occurring cold agglutinins in that they were warm-reactive and were mainly concentrated on the patients' cells rather than being free in the serum. Because of their nonagglutinating property, it is suggested that they are monomeric IgM. It is concluded that the high affinity, warm- reactive IgM and not the IgG antibodies are primarily responsible for clinically manifest anemia in patients receiving alphamethyldopa and that the hemolytic activity is probably mediated by the classic pathway of complement activation.

Neutrophil antibodies were demonstrated in 119 of 121 infants and young children with chronic neutropenia, estabilishing the diagnosis of autoimmune neutropenia of infancy. The median age at diagnosis was 8 months (range 3 to 30 months), and the female/male ratio was 6:4. Autoimmune neutropenia of infancy was manifested by recurrent fever and infection. All patients had selective neutropenia (absolute neutrophil count 0 to 500), and many had monocytosis. Fifteen of 16 patients tested falled to respond to epinephrine and hydrocortisone stimulation. Bone marrow had myeloid hyperplasia and reduced mature neutrophils. Recovery occurred in all 81 patients who passed the age of 5 years, except for one patient who is recovering at 6 1/2 years. The median age at recovery was 30 months; 95% recovered before 4 years. The estimated median duration of disease was 20 months. Neutrophil antibodies were detected early in the neutropenic phase by a combination of immunofluorescence and agglutination tests. Ten percent of these antibodies had specificity for NA1 or NA2. Ten of the 12 serum samples with a strong reaction in the flow cytometer reacted only with neutrophils. Two also reacted with an unidentified subpopulation (30%) of lymphocytes. Lymphocyte subsets were normal in 10 patients investigated, and abnormal levels of circulating immune complexes were detected in sera from 11 of 25 (44%) patients tested. Temporary remission was induced in all of eight patients who received intravenous IgG therapy. Autoimmune neutropenia of infancy is probably the most common cause of chronic neutropenia in infancy and early childhood, can be diagnosed by immunologic techniques, and requires only conservative management; spontaneous cure appears to be the rule.

In 1926, Charles Doan observed that sera from some patients agglutinated leukocytes from other individuals. This was the beginning of modern leukocyte immunology. The first report on autoimmune leucopenia was that of Francke, who attributed the disorder to the “leukolysine” he found in a patient’s serum. The efforts of an international cooperative research, initiated in 1964, led to the discovery of the HLA, the antigens that are expressed on lymphocytes and many tissues and are markers for histocompatibility. The finding of neutrophil-specific antigens suggested that other nucleated cells may also express “organ-specific” antigens. Autoimmune neutropenia was then established as a definable clinical entity when neutrophil-specific antigens were demonstrated to be a target in primary autoimmune neutropenias. More recently, the structure, genetics, and functions of neutrophil-specific antigens were determined and the finding that NA is a receptor for IgG was helpful in better understanding its clinical implications. Finally, the availability of colony-stimulating factors for treatment has had a major impact on the management of all neutropenias.

Cold-induced insolubility of serum cryoglobulins can be inhibited by various carbohydrates and aminocarboxylic acids. Among the inhibitors, Epsilon amino caproic acid (EACA) was found to be effective at therapeutic levels in a patient who had mixed-type cryoglobulinemia. In vivo administration of EACA completely prevented cryoprecipitation; the inhibition being accompanied by a parallel decrease in the viscosity of the precooled serum. The data along with kinetics of serum EACA levels suggest specific binding of the drug to the cryoglobulins in this patient. This binding may be the basis for the observed inhibitory activity. The in vivo use of specific inhibitors of cryoglobulin precipitation may prove to have therapeutic application in the management of cryoglobulinemic patients.

A modification of the Polybrene technique for red blood cell antibody characterization has been employed to differentiate the panhemagglutinins arising during methyldopa administration from those accompanying other disease states. Dissociation characteristics of methyldopa-associated antigen-antibody complexes were determined by temperature gradient dissociation technique. Data obtained by this technique for cell-bound antibody were found to distinguish this antibody from those accompanying systemic lupus erythematosus (SLE) and Pronestyl therapy. Graphic data derived from temperature gradient dissociation curves at varying antibody concentrations were obtained for the methyldopa-induced serum antibodies. Results obtained with samples from all six patients were found to be relatively uniform in relation to each other, and different from similarly derived results for red cell antibodies accompanying idiopathic autoimmune hemolytic anemia and Hodgkin’s disease. By means of these procedures, as well as standard blood banking techniques, distinguishing features are described that permit in vitro segregation of these distinct groups of red cell autoantibodies.