Effects of in ovo injection with selenium on immune and antioxidant responses during experimental necrotic enteritis in broiler chickens.

This study was conducted to investigate the effects of in ovo injection of Se on modulating the immune system and antioxidant responses in broiler chickens with experimental necrotic enteritis. Broiler eggs were injected at 18 d of embryo age with either 100 µL of PBS alone or sodium selenite (Na2SeO3) in PBS, providing 0 (SS0), 10 (SS10), or 20 (SS20) µg of Se/egg. At 14 d posthatch, PBS-treated and uninfected chickens were kept as the control group, whereas the remaining chickens were orally infected with 1.0 × 10(4) sporulated oocysts of Eimeria maxima (SS0, SS10, SS20). At 18 d posthatch, E. maxima-infected chickens were orally infected with 1.0 × 10(9) cfu of Clostridium perfringens. Infected control SS0 group showed significantly decreased BW compared with the uninfected control. However, SS20 group showed significantly increased BW compared with the infected control SS0 group, whereas the BW were similar among uninfected control and infected SS10 and SS20 groups. The SS10 group showed significantly lower intestinal lesions compared with the SS0 group, and oocyst production was decreased in both SS10 and SS20 groups. Serum malondialdehyde level and catalase activity were also decreased in both SS10 and SS20 groups, whereas the superoxide dismutase level was significantly lower in the SS10 group compared with the SS0 group. The SS20 group showed significantly higher levels of transcripts for IL-1ß and IL-6 in intestine, and SS10 and SS20 groups had higher levels of transcripts for IL-8 and inducible nitric oxide synthase expression and decreased glutathione peroxidase 7 mRNA levels compared with the SS0 group. The SS10 and SS20 groups also showed increased serum antibody levels to C. perfringens α-toxin and NetB toxin compared with the SS0 group. These collective results suggest that the injection of Se into the amniotic cavity of developing eggs may be beneficial for enhancing immune and antioxidant responses in the hatched chickens exposed to the necrotic enteritis-causing pathogens.

In vitro effects of plant and mushroom extracts on immunological function of chicken lymphocytes and macrophages.

1. The present study was conducted to examine the effects of organic extracts from milk thistle (Silybum marianum), turmeric (Curcuma longa), reishi mushroom (Ganoderma lucidum), and shiitake mushroom (Lentinus edodes) on innate immunity and tumor cell viability. 2. Innate immunity was measured by lymphocyte proliferation and nitric oxide production by macrophages, and the inhibitory effect on tumor cell growth was assessed using a non-radioactive assay. For measuring the cytokine levels in the HD11 macrophages which were treated with extracts of turmeric or shiitake mushroom, the levels of mRNAs for interferon-alpha (IFN- alpha), interleukin-1beta (IL-1beta), IL-6, IL-12, IL-15, IL-18, and tumor necrosis factor superfamily 15 (TNFSF15) were quantified by real time RT-PCR. 3. In vitro culture of chicken spleen lymphocytes with extracts of milk thistle, turmeric, and shiitake and reishi mushrooms induced significantly higher cell proliferation compared with the untreated control cells. Stimulation of macrophages with extracts of milk thistle and shiitake and reishi mushrooms, but not turmeric, resulted in robust nitric oxide production to levels that were similar with those induced by recombinant chicken interferon-gamma. All extracts uniformly inhibited the growth of chicken tumor cells in vitro at the concentration of 6.3 through 100 microg/ml. Finally, the levels of mRNAs encoding IL-1beta, IL-6, IL-12, IL-18, and TNFSF15 were enhanced in macrophages that were treated with extracts of turmeric or shiitake mushroom compared with the untreated control. 4. These results document the immunologically-based enhancement of innate immunity in chickens by extracts of plants and mushrooms with known medicinal properties in vitro. In vivo studies are being planned to delineate the cellular and molecular mechanisms responsible for their mechanism of action.

Association of resistance to avian coccidiosis with single nucleotide polymorphisms in the zyxin gene.

Our previous genetic studies demonstrated that resistance to avian coccidiosis is linked with microsatellite markers LEI0071 and LEI0101 on chromosome 1. In this study, the associations between parameters of resistance to coccidiosis and single nucleotide polymorphisms (SNP) in 3 candidate genes located between LEI0071 and LEI0101 [zyxin, CD4, and tumor necrosis factor receptor super family 1A (TNFRSF1A)] were determined. The SNP were genotyped in 24 F(1) generation and 290 F(2) generation animals. No SNP were identified in the TNFRSF1A gene, whereas 10 were located in the zyxin gene and 4 in the CD4 gene. At various times following experimental infection of the F(2) generation with Eimeria maxima, BW, fecal oocyst shedding, and plasma levels of carotenoid, nitrite plus nitrate (NO(2)(-) + NO(3)(-)), and interferon-gamma (IFN-gamma) were measured as parameters of resistance. Single marker and haplotype-based tests were applied to determine the associations between the 14 SNP and the parameters of coccidiosis resistance. None of the CD4 SNP were correlated with disease resistance. However, by single marker association, several of the zyxin SNP were significantly associated with carotenoid or NO(2)(-) + NO(3)(-) concentrations. These were the SNP at nucleotide 149 associated with carotenoid at d 3 postinfection (PI), nucleotide 187 with carotenoid at d 6 and 9 PI, and nucleotide 159 with carotenoid between d 3 and 9 PI. In addition, the zyxin SNP at nucleotide 191 was significantly associated with increased levels of NO(2)(-) + NO(3)(-) at d 3 PI. By haplotype association, the zyxin SNP also were found to be highly associated with NO(2)(-) + NO(3)(-) at d 3 PI and increased IFN-gamma at d 6 PI. These results suggest that zyxin is a candidate gene potentially associated with increased resistance to experimental avian coccidiosis.

Immune-related gene expression in two B-complex disparate genetically inbred Fayoumi chicken lines following Eimeria maxima infection.

To investigate the influence of genetic differences in the MHC on susceptibility to avian coccidiosis, M5.1 and M15.2 B-haplotype-disparate Fayoumi chickens were orally infected with live Eimeria maxima oocysts, and BW gain, fecal oocyst production, and expression of 14 immune-related genes were determined as parameters of protective immunity. Weight loss was reduced and fecal parasite numbers were lower in birds of the M5.1 line compared with M15.2 line birds. Intestinal intraepithelial lymphocytes from M5.1 chickens expressed greater levels of transcripts encoding interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, IL-15, IL-17A, inducible nitric oxide synthase, and lipopolysaccharide-induced tumor necrosis factor-alpha factor and lower levels of mRNA for IFN-alpha, IL-10, IL-17D, NK-lysin, and tumor necrosis factor superfamily 15 compared with the M15.2 line. In the spleen, E. maxima infection was associated with greater expression levels of IFN-gamma, IL-15, and IL-8 and lower levels of IL-6, IL-17D, and IL-12 in M5.1 vs. M15.2 birds. These results suggest that genetic determinants within the chicken MHC influence resistance to E. maxima infection by controlling the local and systemic expression of immune-related cytokine and chemokine genes.

Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa.

Recently, we cloned and characterized a full-length cDNA of the hamster Muc1 gene, the expression of which appears to be associated with secretory cell differentiation (Park HR, Hyun SW, and Kim KC. Am J Respir Cell Mol Biol 15: 237-244, 1996). The role of Muc1 mucins in the airway, however, is unknown. In this study, we investigated whether cell surface mucins are adhesion sites for Pseudomonas aeruginosa. Chinese hamster ovary (CHO) cells not normally expressing Muc1 mucin were stably transfected with the hamster Muc1 cDNA, and binding to P. aeruginosa was examined. Our results showed that 1) stably transfected CHO cells expressed both Muc1 mRNA and Muc1 mucins based on Northern and Western blot analyses, 2) Muc1 mucins present on the cell surface were degraded by neutrophil elastase, and 3) expression of Muc1 mucins on the cell surface resulted in a significant increase in adhesion of P. aeruginosa that was completely abolished by either proteolytic cleavage with neutrophil elastase or deletion of the extracellular domain by mutation. We conclude that Muc1 mucins expressed on the surface of CHO cells serve as adhesion sites for P. aeruginosa, suggesting a possible role for these glycoproteins in the early stage of airway infection and providing a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and cystic fibrosis in particular.

A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis.

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

Serum antibodies reactive with non-human primate retroviruses identified in acute onset schizophrenia.

Schizophrenia is a pervasive neuropsychiatric disease of uncertain etiology. Previous studies have postulated that retroviruses may contribute to the etiology of some cases of schizophrenia. We examined the possible relationship between retroviral infection and schizophrenia by measuring antibodies to a number of different primate retroviruses in the sera of individuals undergoing their first hospitalization for this disease. Sera from patients with first onset schizophrenia and matched healthy controls were analyzed by immunoblot and enzyme linked immunosorbent assays using purified retrovirus antigens to identify and quantify antibodies reactive with retrovirus proteins. A significantly increased incidence of antibodies reactive to gag encoded proteins of Mason-Pfizer monkey virus (MPMV), baboon endogenous virus (BaEV) and simian retrovirus type 5 (SRV-5) was observed in the sera of schizophrenia patients compared to controls. The reactivity of the cases and controls displayed the greatest differences in terms of antibodies to the proteins of Mason-Pfizer monkey virus. Employing an algorithm of enzyme linked immunosorbent assay reactivity followed by immunoblot confirmation, we found that MPMV antibodies in 28.9% of the individuals with first episode schizophrenia patients as compared to 3.7% of the unaffected controls (P<0.009, Fisher's Exact Test). These studies are consistent with the occurrence of retrovirus replication in some individuals who are undergoing their first episode of schizophrenia.

Chicken macrophages and thrombocytes share a common cell surface antigen defined by a monoclonal antibody.

A monoclonal antibody (mAb), designated K1, reacted with a cell surface antigen shared on chicken macrophages and thrombocytes. By immunofluorescence staining, the mAb K1 was reactive with 31.8% of peripheral blood lymphocytes (PBL) separated on Histopaque. In contrast, only 3.2% of PBL separated by slow-speed centrifugation were K1 positive. This antibody did not react with B- or T-lymphocytes, as demonstrated by the very small percentage of positive cells in thymus, bursa and spleen. Furthermore, no staining was observed with avian T-cell (MDCC-RP1 and SK3) or B-cell (LSCC-RP9) lines. Adherent cells derived from PBL separated on Histopaque and cultured for 48 h in plastic cell culture dishes were 81.5% positive with K1. These cells were also 82.6% positive with an antibody detecting the major histocompatibility complex (MHC) Class II antigen in chickens, indicating that they were monocyte-derived macrophages. Sheep red blood cell phagocytosis by these cells could be demonstrated, further supporting their macrophage lineage. In addition, K1 stained virtually 100% of HD11 cells, a chicken macrophage cell line, as well as 86.7% of peritoneal exudate cells. Eighty five percent of plastic adherent cells from PBL collected after 2 h of adherence reacted with the mAb K1, but only 8% of these cells were MHC Class II positive. These cells were morphologically identified as thrombocytes. Immunoprecipitation analysis demonstrated that the K1-reactive antigen consisted of a heterodimer with constituent polypeptide chains of 135 kDa and 61-68 kDa.

Virion-associated trans-regulatory protein of human T-cell leukemia virus type I.

Western blot analysis of HTLV-I virus particles from HUT-102 cells revealed a 40-kD protein strongly reactive with Tax-specific rabbit antisera. This protein subsequently was isolated from density gradient purified virions by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), purified from comigrating Gag and human cellular proteins by reversed-phase high-performance liquid chromatography (HPLC) and identified as the tax-encoded gene product by amino acid composition analysis. Among extracellular virions from five HTLV-I producing cell lines, only those from HUT-102 and C10MJ cells contained a detectable Tax protein, although all cells expressed Tax mRNA and protein intracellularly. To investigate the diagnostic implications of virion-associated Tax protein, sera from HTLV-I-infected individuals were compared on HUT-102 and MT-2 virus Western blots. The seroprevalence of antibodies to Tax, but not Gag or Env proteins, was substantially higher among adult T-cell leukemia and tropical spastic paraparesis patients using HUT-102 viral proteins. Thus, immunoassays utilizing HUT-102 virus are most sensitive for detection of Tax-reactive antibodies.

Development and evaluation of a human T-cell leukemia virus type I serologic confirmatory assay incorporating a recombinant envelope polypeptide.

A recombinant protein derived from the gp21 region of the human T-cell leukemia virus type I (HTLV-I) env gene was synthesized in Escherichia coli and purified by reversed-phase high-performance liquid chromatography. The purified protein was free of contaminating bacterial proteins and retained reactivity with human HTLV-I- and HTLV-II-positive sera and a gp21 monoclonal antibody. An immunoblot procedure using the recombinant polypeptide in conjunction with native viral proteins was more sensitive than the conventional immunoblot and radioimmunoprecipitation confirmatory assays for detection of antibodies to HTLV-I and HTLV-II env-encoded gene products. The recombinant protein was equally reactive with sera from polymerase chain reaction-confirmed HTLV-I or HTLV-II infections. Furthermore, on the basis of the differential reactivities of gp21-positive sera with the HTLV-I p19 and p24 gag-encoded proteins, an algorithm was proposed to distinguish exposure to HTLV-I from exposure to HTLV-II. These results establish the utility of a modified immunoblot assay incorporating a recombinant envelope polypeptide as an alternative to existing HTLV-I-confirmatory assays.

Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies.

Seven monoclonal antibodies (mAb) were used to characterize antigens present on chicken T lymphocytes and on natural killer cells by flow cytometry, radioimmunoprecipitation and by effects on cell-mediated cytotoxicity and mitogen-induced proliferation. mAb CTLA8 and 5 stained 73% of thymus, 44% of spleen and 51% of peripheral blood lymphocytes (PBL), respectively, and immunoprecipitated 65- and 45-kDa proteins from detergent extracts of 125I surface-labeled thymocytes. Pretreatment of splenic lymphocytes with mAb CTLA5 and 8 in the presence of rabbit complement (C) eliminated the concanavalin A (Con A)-induced T cell proliferative responses. mAb CTLA3, 4 and 9 stained 43% of thymus, 36% of spleen and 18% of PBL, and immunoprecipitated 33-35-kDa proteins. Pretreatment of spleen cells with mAb 4 or 9 plus C reduced, but did not eliminate, the Con A-induced proliferative response and significantly reduced both major histocompatibility complex (MHC)-restricted and non-MHC-restricted cellular cytotoxicity. mAb CTLA1 and 6 stained 58% of thymus, 13% of spleen and 19% of PBL. mAb CTLA1 and 6 immunoprecipitated a 65-kDa protein. mAb CTLA1 and 6 had no effect on the Con A-induced blastogenesis and CTLA6 caused no decrease in virus-specific cytotoxic T lymphocyte and natural killer activity. These results indicate that (a) mAb CTLA5 and 8 identify antigens on mature T lymphocytes that are similar in tissue distribution, molecular mass and function to the mammalian CD5 antigen; (b) mAb CTLA3, 4 and 9 detect the avian homologue of CD8 antigen; and (c) mAb CTLA1 and 6 identify the avian homologue of CD4 antigen.

The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors.

Seven human immunodeficiency virus gag polypeptides were identified in the purified virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing of immune-purified proteins. Two gag polyproteins of 55,000 (p55) and 41,000 (p41) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag open reading frame (ORF) as NH2-p16 (MA)-p24 (CA)-p9 (NC)-p7-COOH. The myristoylated p16 (matrix, MA) protein was processed from the myristoylated p55 gag precursor protein. The immunoreactivity of the p16 (MA) protein with region-specific gag antisera and the conservation of the N-terminal myristyl group of the p55 precursor protein in p16 (MA) confirmed its position as the N-terminal-most protein. The p9 (nucleocapsid, NC) protein was localized to residue 378 of the gag ORF, next to the C terminus of the p24/p25 (core antigen, CA) protein. The p9 protein had a repeating Cys residue containing motif which is found in the nucleic acid-binding Cys residue-containing proteins of retroviruses. The p24 (CA) protein, which was localized to residue 133 of the gag ORF, was apparently derived by C-terminal processing of an intermediate polypeptide, p25. Both the mature p24 (CA) and p16 (MA) proteins were phosphorylated at Ser residue(s). We also identified two forms of gag p41 species, one resulting from the C-terminal processing of p55 and the other originating either from N-terminal processing of p55 or from de novo synthesis.

Quantitative differences in Ia antigen expression in the spleens of 15I5-B congenic and inbred chickens as defined by a new monoclonal antibody.

A monoclonal antibody (MAb), designated P2M11, that detects a monomorphic determinant of chicken class II antigens was produced from the fusion of P3X63 myeloma cells with spleen cells from BALB/c mice immunized with chicken splenic lymphocytes. Flow cytometric analyses of lymphocytes from the SC and FP strains of chickens showed 30 to 50% staining of bursa cells, 15 to 20% staining cells, and less than 5% staining of thymus cells. Addition of MAb P2M11 to splenic of T cell cultures stimulated with allogeneic cells or concanavalin A resulted in a significant inhibition of the T cell proliferation responses. Immunoprecipitation of 35S-methionine-labeled spleen cell extracts using MAb P2M11 identified molecules with apparent molecular weights of approximately 28,000, 30,000, and 32,000 by sodium dodecyl-polyacryl-amide gel electrophoresis. Taken together, these data indicate that the antigens detected by MAb P2M11 are similar in cell distribution and structure to chicken Ia antigens encoded by B-L genes. Using this MAb, a strain difference was demonstrated in the tissue distribution of Ia antigen positive lymphocytes in the spleens but not the thymuses of 15I5-B congenic and inbred strains of chickens. This difference may be due to the genes associated with B-complex genes.

Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus.

We have purified a 10,774-dalton protein from human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame (ORF). Radiochemical amino acid microsequencing identified 12 amino acids from the stretch of 39 N-terminal residues of this protein, beginning with a PQITLW sequence at position 69 of the pol ORF. Radiosequencing of selected tryptic peptides of the protein identified 11 additional residues (Leu-9 and Val-2) in six peptides encompassing the entire molecule of 99 residues. A protein of similar size and identical N-terminal sequence (determined through the first 39 residues) was present among the processed HIV pol gene products in Escherichia coli which expressed the entire HIV pol ORF. The C terminus of both the viral and E. coli-expressed proteins was inferred to be contiguous with the N terminus of the p64-p51 reverse transcriptase on the basis of tryptic mapping and specific immunoreactivity with an antiserum against a dodecapeptide located upstream of the reverse transcriptase. Thus, the initial processing of the pol precursor that generates the native protease is apparently preserved across phylogenetic barriers. Although the purified viral protease lacked measurable proteolytic activity, the bacterial extracts were capable of processing an HIV gag precursor protein synthesized in E. coli.

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice.

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.

Alternative protein products with different carboxyl termini from a single class I gene, H-2Kb.

In addition to mRNA encoding the canonical form of the murine class I antigen H-2Kb (348 amino acid residues), mRNA that would encode a shortened form of H-2Kb (missing 9 amino acids from the C-terminus) has been identified in C57BL/6 spleen cells by RNase-protection studies. The alternative transcripts of H-2Kb arise through the use of different AG acceptor splice sites for exon VIII. The existence of a shortened H-2Kb protein was demonstrated by sequential immunoprecipitation. Lysates of spleen cells that had been labeled with [35S]methionine and [3H]histidine were precleared with rabbit anti-peptide serum reactive with the C-terminus of the canonical H-2Kb. The shortened form of H-2Kb was immunoprecipitated from this lysate with H-2Kb alloantiserum. Both forms of H-2Kb were isolated by NaDodSO4/PAGE. Tryptic peptide mapping confirmed that these molecules differed only at their C-terminus. The shortened form of H-2Kb is also found in a B-cell line (R8) but not in three cloned T-cell lines or in a T-cell lymphoma (EL4), suggesting that regulatory events are involved in producing the two forms of H-2Kb. Putative lariat branch points involved in these alternative splicing events for the 3' coding region of H-2 class I pre-mRNAs are proposed.

Normal and abnormal aspects of proteinuria. Part I: Mechanisms, characteristics and analyses of urinary protein. Part II: Clinical considerations.

Part I highlights the mechanisms of glomerular filtration and tubular reabsorption of plasma proteins, selected characteristics of urinary proteins based upon electrophoretic properties and recent advances in clinical laboratory analysis of proteinuria. Both structural characteristic of the glomerular capillary wall and molecular properties of plasma proteins are important determinants of glomerular filtration. Proteins filtered by the glomerulus subsequently appear in urine only after escaping the efficient mechanisms of tubular reabsorption. Albumin is one such protein and constitutes the major protein in normal urine although trace amounts of alpha, beta, and gamma globulins are also detectable. Several techniques of protein analysis have thus been developed to specifically measure albumin as well as other plasma proteins. Other methods have been adapted to measure total urinary protein content enabling the clinician to readily monitor renal function in health and disease. The second part of this review will consider conditions associated with proteinuria in both asymptomatic individuals and patients with renal disease. Asymptomatic proteinuria encompasses states of excess protein excretion during conditions of orthostasis, exercise, travel to high altitude of fever. Proteinuria during renal disease has received considerable interest as a means to monitor kidney function. It is therefore classified according to the type of damage incurred: (1) glomerular-type where large molecular weight proteins are excreted (2) tubular-type where small molecular weight proteins are excreted and (3) mixed-type characterized by both large and small molecular weight proteinuria.

Structural definition of a family of Ld-like molecules distributed among four of seven haplotypes compared.

Comparative tryptic peptide analyses were performed on 12 different D region molecules representing seven different haplotypes. The Dd, Dq, and Dw16 regions were shown to encode multiple, antigenically distinct molecules (Dd Ld, Dq Lq Rq, and Dw16 Lw16, respectively). In addition, each of these molecules was found to have a unique primary structure, implying that they are the products of separate genes. However the previously described Rd molecule, which was identified by sequential immuno-precipitation and 2-D gel analyses, was indistinguishable from Ld by tryptic peptide mapping, implying that these two molecules may be products of the same gene. The Db, Ddx, Dk, and Dp regions were found to determine a single molecule with the reagents tested. Intra- and/or inter-haplotype comparisons of the peptide maps of each of these D region molecules revealed widely disparate structural relationships. For example, the Db, Dq, Lq, Rq, Dw16, and Lw16 molecules all showed striking homology with the Ld molecule. Members of this family share between 43 to 55% peptide homology with Ld, indicating a high conservation of primary structure (greater than 90%). However, because Dq and Dw16 region-encoded molecules show no exceptional relationship to each other, the portion of the conserved sequence is not the same for each of these Ld-like molecules. By contrast, comparisons of the Dk, Dd, Ddx, and Dp molecules with Ld or with each other revealed tryptic peptide homologies ranging from 22 to 38%, suggesting a sequence homology of 70 to 85%. When compared with the Kb molecule, each of the D region molecules showed between 21 to 36% peptide map homology (70 to 85% sequence homology). These studies indicate, therefore, that there is a family of Ld-like molecules representing several distinct haplotypes. This definition of a highly homologous family of D region molecules suggests that many D-region molecules have evolved from an Ld-like primordial gene and that in different haplotypes different portions of this prototypic structure have been maintained.