The simultaneous production of two classes of cytoplasmic immunoglobulins by single cells in Xenopus laevis

The classes of cytoplasmic immunoglobulins of individual lymphoid cells from the spleen and peripheral blood of normal and immunized Xenopus laevis were investigated. Immunofluorescence microscopy and simultaneous double staining of the cytoplasm, using a mixture of class-specific TRITC-anti-19S Ig and FITC-anti-7S Ig conjugated antisera, showed that 70% of the immunoglobulin producing cells are “double producers” and contain the two classes of 19S and 7S immunoglobulins. The remaining 30% are composed of “single producers.” The proportion of splenic cells containing either 19S Ig or 7S Ig, respectively, was unequal, and reversed between two groups of animals, one examined at the primary immune response (29% 19S Ig and 4% 7S Ig) and the second group examined at the secondary immune response (8% 19S Ig and 30% 7S Ig). A spontaneous lymphoid tumor with an increased serum level of 19S Ig was examined in the same way. The tumor cells appear to produce 19S Ig exclusively.     

Structural and genetic studies on chicken 7S immunoglobulin allotypes. II. Distribution of allotypes on the 7S immunoglobulin of homozygous and heterozygous chickens.

We have previously reported that chicken 7S immunoglobulin (Ig) heavy (H) chain allotypes (CS-1 locus) segregate as phenogroups in F2 progeny. Specificity CS-1.1 formed a phenogroup with CS-1.4 in inbred chicken line UCD 2, and a second phenogroup with CS-1.3 in line UCD 3. To determine whether these phenogroups were formed by combinations of specificities on the same, or on separate subclasses of 7S Ig, their distribution on the 7S Ig molecules of birds homozygous for 7S Ig allotypes was analyzed by radioimmunoassay. Anti-CS-1.1 and anti-CS-1.3 alloantisera each bound more than 94% of line UCD 3 1252-7S Ig. Similar results were obtained with alloantisera to CS-1.1 and CS-1.4 WITH 125 I-7S Ig from line UCD 2. These results indicate that both phenogroups were formed by combinations of specificities present on the same H chain. Thus, each phenogroup described, probably is the product of a single structural gene which is responsible for more than 94% of the 7S Ig H chain constant regions. In F hybrids with the genotype CS-1.3, 1.3/CS-1.2, two populations of serum 7S Ig molecules were detected by direct and sequential binding analysis with specific alloantisera. One population of 7S Ig contained specificities CS-1.1 AND CS-1.3, but not CS-1.2; while the second population was exclusively the product of one parental allele. Consistent with a genetic regulatory mechanism involving allelic exclusion, no MS Ig containing allotypes produced by both alleles was detected.     

Structural and genetic studies on chicken 7S immunoglobulin allotypes. IV. The presence of an unexpected chicken immunoglobulin heavy chain allotype: subclass or pseudoallele?

Low concentrations of allotypic specificity CS-1.1 were detected in the sera of two inbred chicken lines [University of California, Davis (UCD) 7 and Regional Poultry Research Laboratory 15I4] previously reported to lack this specificity. The CS-1.1 alloantigen in 15I4 chickens has the same specificity as the major allotype in a line of chickens (UCD 2) in which it was initially defined. In 15I4 chickens, CS-1.1 allotype is present on a population of molecules distinct from those which carry the major allotype; thus a second 7S Ig H chain locus, CS-2, is proposed. The concentration of CS-1.1-bearing molecules determined by two different methods was 7 microgram/ml and 230 microgram/ml in 15I4, whereas UCD 2 chickens had 4 mg/ml of CS-1.1 molecules. The levels of CS-1.1 inhibitory activity in 15I4 birds remained relatively constant over a 30-day period. The presence of two 7S Ig populations in 15I4 chickens may be interpreted as evidence either for 7S Ig subclasses with shared allotypes or for a pseudoallelic organization of genes controlling expression of 7S Ig H chains. The results were consistent with the presence of redundant C region genes, differing in allotypes, whose expression is under the control of an as yet undefined regulatory mechanism.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey, pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey,pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

Interaction and release of soulble immune complexes from mouse B luymphocytes

Soluble immune complexes (¹²⁵I BSA-anti-BSA-C) bind to B lymphocytes and accumulate at one pole of the cells (“caps”). The complexes remain on the membrane after incubation of the cells at 37 °C in tissue culture medium for several hours. The ¹²⁵I BSA can be quantitatively removed from the cell surface by incubation with excess BSA but not with excess antibody to BSA or preformed BSA-anti-BSA-C complexes. The release of ¹²⁵I BSA is probably due to the removal of the complexes from the cell membrane and not to an exchange between unlabeled BSA in the medium and the labeled BSA present in the membrane-bound complexes. Release of ¹²⁵I BSA by excess BSA is temperature dependent. The membrane-bound complexes can also be removed by incubating the cells with papain fragments of rabbit antibody to mouse Ig (anti-γ₁, γ₂, and k Ig chains). However, after exposure to divalent [F(ab′)² or 7S Ig] rabbit antibodies to mouse Ig, the complexes remain associated with the cells. In addition, after such treatment the complexes cannot be removed by excess BSA or by Fab anti-Ig.     

DNA polymerases ν and θ are required for efficient immunoglobulin V gene diversification in chicken

The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis–dependent point mutations (Ig hypermutation) and homologous recombination (HR)–dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polν and Polθ led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN^−/−/POLQ^−/− cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Polη has also been previously implicated in Ig gene conversion. We show that a POLH^−/−/POLN^−/−/POLQ^−/− triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polν and Polθ in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.     

Surface immunoglobulin of mouse thymus cells and its in vitro biosynthesis

Surface immunoglobulin (Ig) was demonstrated on thymocytes from BALB/c and CS7BL mice by lactoperoxidase radioiodination of the cells. Active synthesis of Ig in these cells was demonstrated in short-term tissue culture using ¹⁴C-labeled amino acids. The demonstration of intracellular and surface Ig required procedures that minimize proteolytic degradation.Monomeric α chains and light chains were found in the cytoplasm and on the surface of BALB/c thymocytes, whereas monomeric μ chains and light chains in the cytoplasm and on the surface of CS7BL thymocytes. Cytotoxic tests with an alloantiserum revealed that in the thymus of BALB/c mice the IgA monomeric subunits are synthesized by the θ⁺ cells.     

Reverse regulation of soluble receptor for advanced glycation end products and proinflammatory factor resistin and S100A12 in Kawasaki disease

Introduction

Kawasaki disease (KD), an acute febrile disease, characterized by systemic vasculitis, predominantly affects infants and children under 5 years of age. Coronary artery lesions (CALs) are its most critical complication, and the etiology remains unknown yet. In order to explore the value of resistin, S100A12 and soluble receptor for advanced glycation end products (sRAGE) in the pathophysiology of KD, we studied the serum levels of resistin, S100A12 and sRAGE in different stages of KD.

Methods

Serum levels of resistin, S100A12 and sRAGE were measured by enzyme-linked immunosorbent assay (ELISA) method in 15 healthy children and 40 KD patients at acute, afebrile and subacute stage.

Results

The resistin and S100A12 levels, including the ratio of resistin to sRAGE and S100A12 to sRAGE increased significantly in the acute stage, and decreased progressively in the afebrile and subacute stage. However, the sRAGE levels decreased significantly in the acute stage, and increased progressively in the afebrile and subacute stage. In the acute, afebrile and subacute stage, the resistin levels were higher in intravenous immunoglobulin (IVIG) non-responders (0.64 ± 0.30, 0.48 ± 0.35, 0.28 ± 0.19, × 10² ng/ml) than in IVIG responders (0.35 ± 0.24, 0.21 ± 0.19, 0.12 ± 0.05, × 10² ng/ml). In the acute and subacute stage, the S100A12 levels were higher in IVIG non-responders (7.92 ± 2.61, 4.98 ± 4.75, × 10² ng/ml) than in IVIG responders (5.05 ± 3.22, 2.35 ± 2.26, × 10² ng/ml). In the afebrile and subacute stage, the sRAGE levels were lower in IVIG non-responders (3.51 ± 2.64, 3.65 ± 3.27, × 10² pg/ml) than in IVIG responders (6.00 ± 2.78, 7.19 ± 2.88, × 10² pg/ml). The resistin levels were positively correlated with S100A12 levels. The sRAGE levels were negatively related with S100A12 and resistin levels.

Conclusions

Resistin, S100A12 and sRAGE are involved in the pathophysiology of KD.     

Intermediary Metabolite Concentrations in Xylulose- and Glucose-Fermenting Saccharomyces cerevisiae Cells

Glucose and xylulose fermentation and product formation by Saccharomyces cerevisiae were compared in batch culture under anaerobic conditions. In both cases the main product was ethanol, with glycerol, xylitol, and arabitol produced as by-products. During glucose and xylulose fermentation, 0.74 and 0.37 g of cell mass liter⁻¹, respectively, were formed. In glucose-fermenting cells, the carbon balance could be closed, whereas in xylulose-fermenting cells, about 25% of the consumed sugar carbon could not be accounted for. The rate of sugar consumption was 3.94 mmol g of initial biomass⁻¹ h⁻¹ for glucose and 0.39 mmol g of initial biomass⁻¹ h⁻¹ for xylulose. Concentrations of the intermediary metabolites fructose-1,6-diphosphate (FDP), pyruvate (PYR), sedoheptulose 7-phosphate (S7P), erytrose 4-phosphate, citrate (CIT), fumarate, and malate were compared for both types of cells. Levels of FDP, PYR, and CIT were lower, and levels of S7P were higher in xylulose-fermenting cells. After normalization to the carbon consumption rate, the levels of FDP were approximately the same, whereas there was a significant accumulation of S7P, PYR, CIT, and malate, especially of S7P, in xylulose-fermenting cells compared with in glucose-fermenting cells. In the presence of 15 μM iodoacetate, an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), FDP levels increased and S7P levels decreased in xylulose-assimilating cells compared with in the absence of the inhibitor, whereas fermentation was slightly slowed down. The specific activity of transaldolase (EC 2.2.1.2), the pentose phosphate pathway enzyme reacting with S7P and glyceraldehyde-3-phosphate, was essentially the same for both glucose- and xylulose-fermenting cells. It was, however, several orders of magnitude lower than that reported for a Torula yeast and Candida utilis. The presence of iodoacetate did not influence the activity of transaldolase in xylulose-fermenting cells. The results are discussed in terms of a competition between the pentose phosphate pathway and glycolysis for the common metabolite, glyceraldehyde-3-phosphate, which would explain the low rates of xylulose assimilation and ethanol production from xylulose by S. cerevisiae.     

Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l⁻¹ at day 10.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

Characterization of human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) expressed in transgenic rice cell suspension cultures

The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig^P) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig^M), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig^P and hCTLA4Ig^M had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig^P and hCTLA4Ig^M had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-γ, but did not suppress the expression of macrophage-derived cytokines, including TNF-α and IL-1β, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig^P is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig^M.     

Caloric restriction reduces IgA levels and modifies cytokine mRNA expression in mouse small intestine

The aim of this study was to determine the effect of caloric restriction (CR) in mouse small intestine on the production and secretion of immunoglobulin (Ig) A, the population of lymphocytes in the lamina propria, and the expression of cytokines that mediate and regulate innate and adaptive immunity. One group of young Balb/c mice was fed ad libitum, while the CR group was fed ad libitum and fasted on alternate days. When mice were six months old, IgA levels in the proximal small intestine were quantified by enzyme-linked immunosorbent assay, while the number of IgA containing cells, CD4⁺ T cells and CD8⁺ T cells in the duodenal mucosa was determined by immunohistochemistry. Furthermore, the expression of several intestinal cytokines, the genes for α-chain IgA, and the polymeric Ig receptor (pIgR) were analyzed by real-time polymerase chain reaction. CR decreased the levels of IgA in the intestine, apparently a consequence of a reduced number of IgA⁺ cells in the lamina propria that decrease the production and secretion of this Ig, and a reduced secretion of S-IgA into the bile, which in turn discharges into the proximal intestine. Contrarily, CR increased the expression of genes for α-chain IgA, and the pIgR, indicating that transport of IgA was not a key factor in the decrease of this Ig. Additionally, CR modified the expression of genes for tumor necrosis factor-α, interferon-γ, tumor growth factor-β, interleukin (IL)-2 and IL-10, all of which regulate the synthesis of IgA and pIgR, the inflammatory response, and the immune response in the intestine.     

Production of Italian Dry Salami: Effect of Starter Culture and Chemical Acidulation on Staphylococcal Growth in Salami Under Commercial Manufacturing Conditions

The effect of starter culture and chemical acidulation on the growth and enterotoxigenesis of Staphylococcus aureus strain S-6 in Italian dry salami under commercial manufacturing conditions was studied. The experimental design included two levels of S. aureus (10⁴ and 10⁵/g), three levels of starter culture (0, 10⁵, and 10⁶/g), three levels of initial pH (pH₀) (6.1, 5.5, and 4.8), two manufacturing plants, and three replications. S. aureus growth in the salami was affected significantly (P < 0.005) by pH₀, initial levels of S. aureus (staph₀) and lactic acid bacteria (LAB₀), day of fermentation, and by the interactions of pH₀ × day, pH₀ × LAB₀, LAB₀ × staph₀, pH₀ × staph₀, and pH₀ × location of fermentation. In general, the lower the pH₀ and the higher the LAB₀, the greater the inhibition of S. aureus. The LAB levels during the fermentation were affected significantly (P < 0.005) by pH₀, LAB₀, day of fermentation, location, LAB₀ × pH₀, and LAB₀ × day. Derived regression equations related level of S. aureus and LAB at any day of fermentation to a number of microbiological and chemical variables. Close similarity of observed and predicted levels of S. aureus and LAB growth demonstrated the usefulness of the experimental approach in evaluating the safety of a process. No detectable enterotoxin or thermonuclease was found at any stage of processing even when S. aureus reached levels of 10⁷/g of salami.     

Regulated Secretion of Acid Sphingomyelinase: IMPLICATIONS FOR SELECTIVITY OF CERAMIDE FORMATION*

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase^WT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C₁₆-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase^WT exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase^S508A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C₁₆-ceramide. Taken together, these data support a crucial role for Ser⁵⁰⁸ in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.     

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems

Summary Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first 2 weeks while an increasing number of monocytes and macrophages appeared in the media of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO₂ concentrations, i.e. 2%, 5%, 10%; similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera. Supported by U.S.P.H.S. Grant CA-5834 from the National Cancer Institute. Department of Medicine A. Department of Cell Physiology Department of Immunology and Immunochemistry.     

The secreted immunoglobulin domain proteins ZIG-5 and ZIG-8 cooperate with L1CAM/SAX-7 to maintain nervous system integrity

During nervous system development, neuronal cell bodies and their axodendritic projections are precisely positioned through transiently expressed patterning cues. We show here that two neuronally expressed, secreted immunoglobulin (Ig) domain-containing proteins, ZIG-5 and ZIG-8, have no detectable role during embryonic nervous system development of the nematode Caenorhabditis elegans but are jointly required for neuronal soma and ventral cord axons to maintain their correct position throughout postembryonic life of the animal. The maintenance defects observed upon removal of zig-5 and zig-8 are similar to those observed upon complete loss of the SAX-7 protein, the C. elegans ortholog of the L1CAM family of adhesion proteins, which have been implicated in several neurological diseases. SAX-7 exists in two isoforms: a canonical, long isoform (SAX-7L) and a more adhesive shorter isoform lacking the first two Ig domains (SAX-7S). Unexpectedly, the normally essential function of ZIG-5 and ZIG-8 in maintaining neuronal soma and axon position is completely suppressed by genetic removal of the long SAX-7L isoform. Overexpression of the short isoform SAX-7S also abrogates the need for ZIG-5 and ZIG-8. Conversely, overexpression of the long isoform disrupts adhesion, irrespective of the presence of the ZIG proteins. These findings suggest an unexpected interdependency of distinct Ig domain proteins, with one isoform of SAX-7, SAX-7L, inhibiting the function of the most adhesive isoform, SAX-7S, and this inhibition being relieved by ZIG-5 and ZIG-8. Apart from extending our understanding of dedicated neuronal maintenance mechanisms, these findings provide novel insights into adhesive and anti-adhesive functions of IgCAM proteins.     

Reactivities of Shark 19S and 7S IgM Antibodies to <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

LOWER vertebrates such as sharks can synthesize humoral antibodies in response to antigenic stimulation with a wide variety of antigens¹. Physicochemical studies have shown that sharks can synthesize both 19S and 7S immunoglobulins and that these two proteins belong to the same immunoglobulin class, which seems to be structurally homologous to IgM as defined for higher animals. Thus the shark immunoglobulins have been designated 19S IgM and 7S IgM^2–4. Because the predominant immunoglobulin (IgG) of most mammals is absent from sharks, the shark monomeric (7S) IgM might be functionally analogous to IgG. One example of the functional differences between IgM and IgG antibodies is the greater reactivity of the former in agglutination and bactericidal reactions^5,6. We have isolated and characterized functionally the relatively high levels of agglutinating antibodies which the nurse shark, Gingly-mostoma cirratum, synthesizes in response to Salmonella typhimurium “O” antigens.