Steven L. Spitalnik

cDNA cloning and expression of UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase T1 from Toxoplasma gondii ☆

We report the cloning, expression, and characterization of the first UDP-GalNAc:polypetide N-acetylgalactosaminyltransferase (ppGalNAc-T) from the human disease-causing parasite, Toxoplasma gondii. This enzyme is also the first characterized ppGalNAc-T of protozoan origin. This type of enzyme catalyzes the initial step of mucin-type O-glycosylation, that is, the transfer of GalNAc in O-glycosidic linkage to serine and threonine residues in polypeptides. We used polymerase chain reaction amplification with degenerate primers and hybridization screening of a T. gondii cDNA library to identify this enzyme. The resulting 84-kDa type II membrane protein contains a 49-amino acid N-terminal cytoplasmic domain, a 22-amino acid hydrophobic transmembrane domain, and a 680-amino acid C-terminal lumenal domain. Conceptual translation of the cDNA sequence reveals a relatively long (i.e. 135 amino acids) stem region and the presence of several important sequence motifs. The latter include a glycosyltransferase 1 (GT1) motif containing a DXH sequence, a Gal/GalNAc-T motif, and a region homologous to ricin lectin. Northern blot analysis identified a single 5.5-kb ppGalNAc-T transcript. Comparison of the cDNA and genomic DNA sequences reveals that this transferase is encoded by 10 exons in a 10 kb region. When the recombinant construct was expressed in stably transfected Drosophila melanogaster S2 cells, the purified protein exhibited transferase activity in vitro. The identification of this enzyme in T. gondii demonstrates that this human parasite has its own enzymatic machinery for the O-glycosylation of toxoplasmal proteins.

CORRELATION OF HUMORAL IMMUNITY TO LEWIS BLOOD GROUP ANTIGENS WITH RENAL TRANSPLANT REJECTION

Recent studies suggest that the Lewis blood group antigens are important for human renal transplantation. A possible mechanism by which Lewis blood group incompatibility could influence allograft rejection was investigated by measuring Lewis antibodies in sera from renal transplant recipients and appropriate controls using conventional hemagglutination and a sensitive and specific kinetic enzyme-linked immunosorbent assay (k-ELISA). The two methods yielded identical results with 14 positive control sera known to contain Lewis antibodies and with 43 sera from negative controls. Antibody was not detected in 16 Lewis-positive transplant patients. However, antibody was detected by k-ELISA in 4/11 Lewis-negative transplant candidates and in 8/8 Lewis-negative recipients of Lewis-incompatible grafts. All 8 grafts were rejected. The one Lewis-negative recipient of a Lewis-negative graft did not develop Lewis antibody nor reject his graft. Of the 12 renal patients in whom antibody was detected by k-ELISA, in only 4 was antibody also demonstrable by hemagglutination. These results indicate that hemagglutination may not be sensitive enough for antibody detection and that Lewis antibodies may play a role in renal allograft rejection.

A technetium-99m red cell survival technique for in vivo compatibility testing

A technique for labeling red cells with Technetium‐99m was developed using in vitro reduction by stannous pyrophosphate. The use of Technetium‐99m and Chromium‐51 enabled simultaneous determination of 60‐minute autologous and heterologous red cell survival using small aliquots. In vivo pretransfusion red cell compatibility in a patient with a panagglutinin was identical to that with 51Cr‐labeled cells. Simultaneous testing of two heterologous donor units or testing multiple units in the course of a few days was also feasible. In nine studies (one patient and six healthy controls) no significant elution of the 99mTc was observed over 1 hour.

A new technique in quantitative immunohematology: solid-phase kinetic enzyme-linked immunosorbent assay.

Abstract. A solid‐phase kinetic enzyme‐linked immunosorbent assay has been developed to measure binding of antibodies to purified or synthetic blood group antigens and tested in the Lewis blood group system. Chemically synthesized Lewis a antigen is used as the target for the binding of serum antibody in this sandwich‐type assay. Kinetic, rather than endpoint, determinations are used to calculate the amount of specific antibody. Data are presented showing the assay to be quantitative, sensitive, and specific. It can separately quantitate the amount of IgG or IgM anti‐Lewis a present in patient sera. The assay uses commercially available reagents and is semiautomated. Thus, it will be useful for studies in quantitative immunohematology as other blood group antigens become available in purified form.

Detection of IgG Anti‐Lewis (a) Antibodies in Cord Sera by Kinetic Elisa

Abstract. Lewis blood group antibodies rarely, if ever, cause hemolytic disease of the newborn. This observation has been attributed to the absence both of Lewis antigens on fetal cells and of maternal IgG Lewis antibody. In the present study, sera from 13 mother‐infant pairs were tested for the presence of anti‐Lewis (a) by hemagglutination and by a sensitive and specific kinetic enzyme‐linked immunosorbent assay. By routine hemagglutination methods, anti‐Lea was present in all maternal samples but absent in all cord samples. By kinetic enzyme‐linked immunosorbent assay, IgG anti‐Lea was present in 13 of 13 maternal samples and in 12 of 13 cord samples. These results indicate that IgG anti‐Lea antibodies are common and do cross the placenta. This suggests that they do not cause hemolytic disease of the newborn because of the low levels of Lewis antigens on fetal red cells.

The serology of Sda effects of transfusion and pregnancy.

Abstract: Pre‐ and posttransfusion antibody titers were performed on 6 patients with anti‐Sda transfused with incompatible blood. In 3 of these patients a significant rise in IgG antibody titer was found. The data suggest that in occasional patients the Sda antigen does evoke a secondary immune response. We evaluated 245 pregnant women for the presence of Sda and found that 30% were Sd(a‐). This incidence was significantly higher than that found in normal blood donors (4%), but was lower than that described in previous reports. We found that 22% of pregnant women in their first trimester were Sd(a‐) whereas at term 36% were Sd(a‐). These significantly different incidences of antigen positivity suggest decreased antigen expression with progressing pregnancy, as seen in the Lewis system. No difference was found in the incidence of anti‐Sda between pregnant women, either during their first trimester or at term, and normal donors.

Multidisciplinary Pediatric Pathology Rotations in a Residency Training Program

The pediatric pathology residency rotations described herein represent an innovative multidisciplinary approach to residency education that combines concepts from anatomic pathology and laboratory medicine, and utilizes faculty members from pathology, pediatrics, and obstetrics/gynecology to teach pathology residents the clinicopathological highlights of antenatal, perinatal, and postnatal pathology. Training is provided through a combination of didactic interactions, laboratory experiences, and current clinical cases. As such, it can be a model for other multidisciplinary residency rotations that could span graduate medical education in pathology to permit a more thorough, informative, and stimulating residency experience.

Posttranslational Modifications of the Amyloid Precursor Protein

Many studies have demonstrated the importance of amyloid precurser protein (APP) in the pathogenesis of Alzheimers disease. Nonetheless, the exact mechanism by which APP contributes to the pathogenesis of Alzheimers disease is still not clear. Because APP is a glycoprotein, and because glycosylation can be important in the cell biology of individual glycoproteins (for review, see refs. 1 and 2), it is possible that changes in APP glycosylation during development and aging are important in APP biosynthesis, proteolysis, and degradation. However, few studies have addressed this issue (3 -8). This chapter provides methods for analyzing the glycosylation of APP that is actively synthesized by living cells in tissue culture. These methods can be applied to primary cultures, continuous cell lines, and transfected cell lines expressing recombinant APP.

The fine specificity of Lewis blood group antibodies. Evidence for maturation of the immune response.

Abstract. Eight human Lewis blood group antibodies were characterized for their fine specificity by the use of specific immunoadsorbents and a kinetic enzyme‐linked immunosorbent assay technique. Examination of sera following immunoglobulin fractionation showed IgM anti‐Lea exhibiting broad cross‐reactivity with structures biochemically related to the Lewis antigens. IgG anti‐Lea binding was restricted to Lea and Leb. These findings are consistent with the concept of affinity maturation of the immune response, which has been previously demonstrated only in animal model systems.

Detection of Anti‐Lea in Le(a‐b+) Individuals by Kinetic ELISA

Abstract. Utilizing a kinetic enzyme‐linked antiglobulin binding assay, eight examples of anti‐Lea were detected in individuals whose Lewis phenotype is Le(a‐b+). These antibodies were not detectable by routine hemagglutination techniques, but were indistinguishable from anti‐Lea made by Le(a‐b‐) individuals in terms of their immunologic fine specificity. However, unlike most anti‐Lea antibodies from Le(a‐b‐) individuals, none of the anti‐Lea antibodies had an IgG component. Anti‐Lea antibodies in Le(a‐b+) individuals may represent immune responses on the continuum between autoimmunity and alloimmunity.