The novel HLA-B*15:180 allele appears to be a recombinant B*08/B*15 allele.

Human leukocyte antigen B-*15:180 is a B*08/B*15 recombinant allele similar to B*15:29 with substitutions positions at 97, 292, 538, 539.

Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML.

Killer Ig-like receptor (KIR) is a major cluster of the natural killer cell receptors and may play a role in the outcome of hematopoietic cell transplants. A total of 65 AML cases transplanted with T-replete hematopoietic cells from unrelated donors were retrospectively KIR-genotyped by a multiplex PCR method of our own design. The KIR gene frequency and genotype patterns in these 130 samples were consistent with the data in the literature. Based upon overall inhibitory and activating KIR genes in both donors and patients, we developed an algorithm to calculate a compatibility score for each transplant case as plus, zero or minus. Significantly higher incidence (18/20, 90%) of acute (a) GVHD (grade II-IV) was found in the transplant cases with plus scores than that (25/45, 56%) in the cases with zero or minus scores (P < 0.01). When the scores are sorted in the opposite way, fewer cases (13/26, 50%) of aGVHD were found in the transplants with minus scores than that (30/39, 77%) in the transplants with zero or plus scores (P < 0.05). The difference of aGVHD prevalence between the plus score and minus score groups is highly significant (P < 0.01). KIR genotype compatibility calculated by this algorithm may predict aGVHD incidence and be helpful in choosing donors.

A sensitive screening method of detecting anti-granulocyte antibodies employing radiolabeled staphylococcal protein A.

We describe an improved method of detecting anti-granulocyte antibodies utilizing radiolabeled staphylococcal protein A (SPA). The results of this SPA assay were compared to data obtained with leukoagglutination tests and granulocyte indirect immunofluorescence techniques. We have shown that the SPA assay is highly sensitive and reproducible. In addition, absorption studies confirmed that the assay is specific for granulocytes. The SPA assay is performed in microtiter plates, and requires significantly fewer granulocytes and less test sera than previously described techniques. Also, we have shown that granulocytes prepared for this assay can be separated and stored for up to 48 h. Therefore, the SPA assay described herein is particularly useful for screening of sera and is one of the most sensitive assays available for detecting anti-granulocyte antibodies.

Visual detection of granulocyte surface antigens using the avidin-biotin complex.

A visual test for detection of granulocyte surface markers using the avidin-biotin complex (ABC) has been developed. That this assay is highly specific, reproducible, and sensitive was determined by studying the expression of HLA antigens on granulocytes with monoclonal antibodies. Further, using granulocyte specific alloantisera, the results of the ABC test compared well to data from leukoagglutination assays and indirect immunofluorescence tests. The assay is particularly advantageous in that granulocytes can be stored, only small amounts of cells and sera are needed, and heterogeneous cell populations can easily be studied.

Simplified alpha1-antitrypsin phenotyping by immunofixation of acid-starch gels.

Immunofixation of acid-starch gels represents a simplified method for alpha1-antitrypsin phenotyping. This technique reduces the amount of work and time involved in phenotyping by eliminating the need for crossed-immunoelectrophoresis. Its major purpose is to directly enhance the staining quality of the minor bands, especially the Z bands on the acid-starch gel. It is hoped that this modified method will allow more wide-spread use of antitrypsin phenotyping, especially in the routine clinical chemistry laboratory.

Racial distribution of alpha1-antitrypsin variants among junior high school students.

A survey of alpha1-antitrypsin phenotypes and serum trypsin inhibitory capacities was performed in 1,841 seventh grade junior high school students. Antitrypsin deficiency states were detected only in white subjects (3.04) per cent) and not in 461 subjects of other races or of Mexican origin. A screening level of serum trypsin inhibitory capacity at 75 per cent of normal or less included all of the deficient phenotypes, but phenotyping was necessary to eliminate a small percentage of normal and unimportant variant phenotypes from this group. It is suggested that future programs for detecting antitrypsin deficiency and for counseling students who are genetically prone to develop pulmonary emphysema be aimed primarily at white populations.

Interference with alpha-antitrypsin studues in stored serum by presumed bacterial proteases.

Contamination of werum by certain gram-negative bacteria has been shown to spoil the serum for measurement of trypsin inhibitory capacity (STIC) or for antitrypsin phenotyping. Such sera develop intense fibrinolytic activity when the STIC has dropped to itsminimal level, but antitrypsin concentration as measured by radial immunodiffusion remainsconstant. Cultures of ENTEROBACTER, Klebsiella, Bacillus subtilis, and Pseudomonas species were shown to have this capability, but production of the fibrinolytic enzyme by the bacteria was most proficient in the presence of human serum. The enzyme is believed to be of bacterial origin because of its lack of esterase activity, and because activation of serum plasmin by streptokinase did not affect the STIC. Care mustbe taken to avoid bacterial contamination of blood that is to be submitted for an STICassay and/or antitrypsin phenotyping. Serum should be prepared quickly, frozen soon,and stored and transported in a frozen state.

A new deficient variant of alpha1-antitrypsin (MDUARTE). Inability to detect the heterozygous state by antitrypsin phenotyping.

A new molecular variant of alpha1-antitrypsin was discovered in the family of a woman with severe antitrypsin deficiency and bullous emphysema. The variant resembles the Z variant in most respects in that it results in severe antitrypsin deficiency with the homozygous state and intermediate deficiency with the heterozygous state, and is associated with diastase-resistant, periodic acid-Schiff-positive globules in the liver cells. It differs from the usual Z variant, however, by having normal mobility on acid-starch electrophoresis so that the heterozygous state with the normal M form cannot be distinguished by phenotyping procedures on either acid-starch or alkaline-agarose electrophoresis. The variant has been labeled MDUARTE. A review of phenotype patterns in all patients previously classified as having a homozygous ZZ phenotype reveals extra, fast-moving bands on acid-starch suggestive of an MDUARTEZ heterozygous state in 7.9 per cent of such cases. When intermediate antitrypsin deficiency occurs in the presence of a normal phenotype pattern, one must consider that the patient has inherited either a null gene for antitrypsin synthesis or an MDUARTE variant.

Molecular abnormality of human alpha1-antitrypsin variant (Pi-ZZ) associated with plasma activity deficiency.

A human alpha1-antitrypsin variant protein was purified to homogeneity from homozygous variant subjects (Pi-ZZ) who had a deficiency of plasma trypsin inhibitory capacity. Molecular weight, specific trypsin inhibitory capacity, and immunologic activity of the variant protein were identical to those of normal. Amino acids, N-acetylglucosamine, and hexose contents were closely similar in the normal and variant proteins, but the sialic acid content in the variant protein was significantly lower than normal. The structural difference between the normal and the variant alpha1-antitrypsin was elucidated by fingerprinting of their tryptic peptides. Two amino acid substitutions, i.e., glutamic acid in the normal protein to lysine in the variant protein, and glutamic acid in the normal protein to glutamine in the variant protein, were found.

The detection of cross-reacting material for alpha-antitrypsin in liver extracts by acid-starch electrophoresis.

Purified extracts of alpha-antitrypsin cross-reacting material (CRM) from liver of patients with MM, ZZ, and MZ phenotypes were studied by acid-starch electrophoresis and crossed immunoelectrophoresis. It is believed that CRM-M from liver is derived primarily from postmortem digested serum antitrypsin, whereas CRM-Z is a product of postmortem digested antitrypsin from hepatocytes. All extracts produced small bands of antitrypsin on the cathodal side of the gel: A single M band migrated faster than the single Z band, whereas CRM from an MZ heterozygote produced a combination of 2 mobile bands. The CRM-Z (from a ZZ phenotype) did not migrate toward the anode, in contrast to the presence of a broad plateau of antitrypsin protein with CRM-M (from an MM phenotype) or CRM-MZ. Thus, most of the CRM-Z protein could not be accounted for on routine acid-starch electrophoresis. However, an anodal peak could be seen on starch gels with CRM-Z at less acidic pH, suggesting that the anodal-migrating CRM-Z protein is denatured and loses antigenicity at lower pH.

Identification of a new HLA-B*78 allele in a Hispanic family.

A new B*78 variant, B*7804, was detected in three members of a Hispanic family. The novel B*78 sequence differs from B*78021 by two substitutions: T at nucleotide 527 (all other B*78s have A) and T at nucleotide 583 (all other B*78s have a C). Both nucleotide substitutions encode amino acid changes at codons 152 and 171, respectively.

Human granulocytes lack red cell Kx antigen.

We have investigated the presence or absence of the red cell Kx antigen on human granulocytes by measuring specific uptake of anti-Kx using three techniques: direct measurement by 125I-staphylococcal protein A (125I-SPA) and avidin-biotin-complex (ABC) immunoperoxidase staining and also, an indirect measurement using granulocyte adsorption of anti-Kx. Our results with all three methods indicate that the Kx antigen is not present on normal human granulocytes. Prior to adsorption of the anti-Kx serum with purified, pooled, normal human granulocytes, 11 of 21 (53%) of normal granulocytes were non-reactive by 125I-SPA and 16 of 20 (80%) by ABC. This pattern of reactivity was shown to be due to contamination of our anti-Kx serum with an antibody to a granulocyte-specific antigen unrelated to the Kx antigen. After adsorption, there was no diminution in the reactivity of the adsorbed anti-Kx compared to the unadsorbed antiserum against red cells which express strong Kx antigen, i.e. Ko and DTT-modified normal human red cells, by either serologic or 125I-SPA techniques. Likewise, reactivity with McLeod red cells, which have weak expression of the Kx antigen, was not changed using either the unadsorbed or adsorbed anti-Kx. The adsorbed anti-Kx was nonreactive with all 12 normal donors' granulocytes tested by 125I-SPA and with 10 normal donors' granulocytes tested by ABC. Furthermore, granulocytes from a Ko individual were nonreactive using either unadsorbed or adsorbed anti-Kx. These studies indicate that Kx antigen is not present on normal human granulocytes. Further, additional adsorption studies using granulocytes from a boy with X-linked chronic granulomatous disease (CGD) indicated that these granulocytes also do not possess the Kx antigen. In contrast to previous reports, these data suggest that Kx antigen is most probably a red cell-specific antigen and that the red cell Kx antigen has no direct relationship to the biochemical defect in CGD.

Detection of herpes simplex virus DNA sequences in human blood and bone marrow cells.

Herpes simplex virus type 1 (HSV1) establishes latent infections in neural tissues of humans and experimental animals. Utilizing a sensitive polymerase chain reaction (PCR) assay we detected HSV DNA sequences in blood cells of healthy prospective bone marrow transplant (BMT) donors and patients. In three healthy individuals studied, HSV DNA sequences were found in all blood cell types and also in bone marrow cells as well as in stem cell progenitor colonies isolated from in vitro cultures. Studies of BMT donor-recipient pairs suggested that HSV reactivation may occur in hematopoietic cells after transplantation, as the PCR signal intensity increased over time simultaneous with an increased antibody titer to HSV. In a mouse model for HSV infection, HSV DNA sequences were found in blood and bone marrow cells at the latent stage of infection, after intravenous (IV) inoculation, but not after ocular inoculation. These studies suggest that bone marrow cells may be an additional site of HSV latency capable of reactivation after BMT. These studies have broad implications for understanding pathogenesis of HSV disease and are of particular significance in situations where allogeneic bone marrow cells are given therapeutically.

The red cell antigens A, B, D, U, Ge, Jk3 and Yta are not detected on human granulocytes.

We report the inability to detect the following red blood cell antigens on human granulocytes: A, B, D, U, Gerbich (Ge), JkaJkb (Jk3) and Cartwright (Yta). To study each antigen, granulocytes were purified on density gradients, fixed in glutaraldehyde, and the uptake of specific antisera measured using two direct immunological techniques: 125I-staphylococcal protein A (125I-SPA) binding and avidin-biotin-complex (ABC) immunoperoxidase staining. Glutaraldehyde fixation was shown not to affect the antigenicity when the antisera were tested using red blood cells. Using three anti-A, three anti-B and three anti-A,B antisera, our 125I-SPA results of 47 tests with granulocytes from group A individuals and 39 tests with granulocytes from group B individuals indicate that A or B antigens are not expressed on human granulocytes. Tests using ABC were also negative with 37 and 36 granulocytes from group A or B individuals, respectively. In addition, no positive results using 125I-SPA were obtained with granulocytes from individuals having antigen positive red cells when tested with two anti-D (number of tests performed (n = 22), three anti-Ge (n = 22), three anti-U (n = 20), two anti-Jk3 (n = 17), and three anti-Yta (n = 25); control anti-NA1 or -NB1 antisera were invariably positive. Also, using these antisera, no positive results were obtained by ABC except with one anti-Yta antiserum which was positive with one of seven granulocytes tested. This anti-Yta was also positive with three of 10 granulocytes by 125I-SPA. This activity was shown to be due to a granulocyte-specific antibody; adsorption of the antiserum with human granulocytes removed all activity against granulocytes but did not reduce the activity against red cells. Thus, our results are in agreement with recent reports which demonstrated the absence of the A, B and D antigens on human granulocytes. However, we have been unable to confirm previous reports which indicated the presence of the U, Ge and Jk3 antigens on human granulocytes. Also, we have been unable to detect the Yta antigen on human granulocytes.

A monoclonal antibody to hemoglobin F.

A monoclonal antibody BMU7-17 which recognizes an antigenic site unique to the cyanogen bromide fragment, CB2 (residues 56 through 133) of the gamma-chain of human hemoglobin has been produced using cell hybridization techniques. The antibody does not crossreact with hemoglobin A, the isolated alpha- or beta-chains, or with hemoglobin of various mammals. Affinity chromatography on Staphylococcal protein A-Sepharose 4B and radial immuno-diffusion revealed the antibody to be of the mouse IgG 1 class. BMU7-17 identifies F cells in adult and in cord blood specimens, as demonstrated by an indirect immunofluorescence assay. The dissociation constant of the antibody as determined using tritiated hemoglobin F with a double antibody radioimmunoassay has a Kd = 1.2 X 10(-9) moles by Scatchard plot analysis.

Optimizing reference values for the measurement of alpha 1-antitrypsin in serum: comparison of three methods.

We studied three methods (rate nephelometry, radial immunodiffusion, and trypsin-inhibitory capacity) for their ability to detect those individuals with a deficiency of alpha1-antitrypsin. The phenotype represented in 170 serum samples was determined by isoelectric focusing as the reference method. All three methods correctly identified Pi Z, Pi S, and Pi SZ phenotypes but varied in their ability to detect Pi MZ and Pi MS phenotypes. The rate-nephelometric method was the least sensitive in detecting Pi MZ and Pi MS variants because of the inappropriately low reference interval suggested by the manufacturer. We found that the three screening methods are comparable when the limiting values are properly selected. We suggest that the reference value for the rate-nephelometric method be increased from 0.85 g/L to 1.40 g/L to improve the sensitivity of the test.

Molecular abnormality of PI S variant of human alpha1-antitrypsin.

Alpha1-antitrypsin variant protein was purified to homogeneity from a PI S-S subject with a mild deficiency of plasma trypsin inhibiting capacity. Molecular weight, specific trypsin inhibitory activity, and composition of amino acids and carbohydrates were similar to the proteins purified from Pi M-M individuals with normal alpha1-antitrypsin activity. The structural difference between the normal and the variant alpha1-antitrypsin was elucidated by peptide mapping of their tryptic digests. An amino acid substitution of glutamic acid in the normal protein to valine in the variant protein was found. The result is consistent with the previously reported amino acid substitution in Pi S-Christchurch.

Bovine gammadelta T-cell responses to the intracellular protozoan parasite Theileria parva.

T cells bearing the gammadelta antigen receptor (gammadelta T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that gammadelta T cells are involved in immune responses against Theileria parva. gammadelta T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of gammadelta T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the gammadelta T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the gammadelta T-cell lines and clones. These gammadelta T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of gammadelta T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine gammadelta T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion.