Suppressor T-cell activity in responder x nonresponder (C57BL/10 x DBA/1)F(1) spleen cells responsive to l-glutamic acid(60)-L-alanine(30)-L-tyrosine (10)

The ability of spleen cells from (responder X nonresponder)F(1) mice immunized with various GAT-M, GAT-MBSA, and soluble GAT to develop IgG GAT-specific PFC responses in vitro after stimulation with responder and nonresponder parental and F(1) GAT-M, was investigated. F(1) spleen cells from mice immunized with F(1) GAT-M or GAT-MBSA developed secondary responses to responder and nonresponder parental and F(1) GAT- M, but not to unrelated third party GAT-M. Spleen cells from F(1) mice immunized with either parental GAT-M developed secondary responses to F(1) GAT-M and only the parental GAT-M used for immunization in vivo. Soluble GAT-primed F(1) spleen cells responded to F(1) and responder parental, but not nonresponder parental, GAT-M. Simultaneous immunization in vivo with the various GAT-M or GAT-MBSA plus soluble GAT modulated the response pattern of these F(1) spleen cells such that they developed secondary responses only to F(1) and parental responder GAT-M regardless of the response pattern observed after immunization with the various GAT-M or GAT-MBSA alone. These observations demonstrate the critical importance of the physical state of the GAT used for immunization in determining the subsequent response pattern of immune F(1) spleen cells to the parental and F(1) GAT-M. Further, suppressor T cells, capable of inhibiting primary responses to GAT by virgin F(1) spleen cells stimulated by nonresponder parental GAT-M, were demonstrated in spleens of F(1) mice immunized with soluble GAT, but not those primed with F(1) GAT-M. Because responder parental mice develop both helper and suppressor T cells after immunization with GAT-M, and soluble GAT preferentially stimulates suppressor T cells whereas GAT-M stimulate helper T cells in nonresponder parental mice, these observations suggest that distinct subsets of T cells exist in F(1) mice which behave phenotypically as responder and nonresponder parental T cells after immunization with soluble GAT and GAT- M.     

The role of H-2-linked genes in helper T-cell function. I. In vitro expression in B cells of immune response genes controlling helper T-cell activity

The ability of murine helper T cells primed to the antigen, sheep erythrocytes (SRBC) to cross-react with burro erythrocytes (BRBC) in the in vitro anti-trinitrophenol (TNP) response to TNP-RBC was shown to be under genetic control. Although non-H-2 genes were shown to influence the absolute level of helper activity assayed after SRBC priming, the extent of cross-reaction of SRBC-primed helpers with BRBC was shown to be controlled by an H-2-1inked Ir gene(s). H-2 haplotypes were identified which determined high, intermediate, or low response to the cross- reacting determinants and the gene(s) controlling the cross-reaction tentatively mapped to the K through I-E end of the H-2 complex. Helpers primed in F(1) mice of high x intermediate or high x low responder parents were tested for cross-reaction using B cells and macrophages (M) of parental haplotypes. In each case the extent of cross-reaction was predicted by the H-2 haplotype of the B cells and M, establishing the expression of the Ir gene(s) in B cells and/or M a t least, but not ruling out its expression in T cells as well. The low cross-reaction seen when T cells from F(1) mice of high × low responder parents were tested on low responder B cells and M was not increased by the presence of high responder M, indicating the Ir gene(s) is expressed in the B cell a t least although it may be expressed in M as well. These and our previously reported experiments are consistent with the hypothesis that helper T cells recognize antigen bound to the surface of B cells and M in association with the product(s) of Ir gene(s) expressed on the B cell and M.     

Inactivation and Inhibition of Replication of the Enveloped Bacteriophage φ6 by Fatty Acids

The enveloped bacteriophage 6 has been shown to be an interesting model system for the study of chemical agents that might have specific antiviral effects against lipid-containing mammalian viruses. In this report, we describe two types of antiviral activity exhibited by several fatty acids against bacteriophage 6. Oleic acid (18:1) and palmitoleic acid (16:1) were potent inactivators of the virus. Treatment with either fatty acid at 50 μg/ml at 25 or 0°C for 30 min reduced the virus titer to about 0.1% of the initial titer. Oleic acid at a concentration as low as 3 μg/ml (~10⁻² mM) reduced the virus titer to <1% of the initial titer within 30 min. Ultracentrifugation analyses of ¹⁴C-amino acid- and ³²P-labeled virus treated with oleic acid indicated that the virion is largely disassembled by the treatment. Myristic acid (14:0) and palmitic acid (16:0) did not inactivate 6 at 50 μg/ml, but nevertheless did prevent 6 plaque production. Single-step virus growth experiments in which fatty acid was added at various times before or after infection indicated that it was an early stage of the 6 replication cycle that was inhibited by the presence of myristic acid and that the inhibition occurred only if the myristic acid concentration in the extracellular growth medium was 10 μg/ml. 6 could attach to its host cell in the presence of myristic acid at 50 μg/ml. We conclude that the fatty acids that prevent 6 replication probably do so by interfering with the entry of the viral genome into the host cell.     

The Macrophage Scavenger Receptor Type A Is Expressed by Activated Macrophages and Protects the Host Against Lethal Endotoxic Shock

During gram-negative bacterial infections, lipopolysaccharide (LPS) stimulates primed macrophages (M) to release inflammatory mediators such as tumor necrosis factor (TNF)-α, which can cause hypotension, organ failure, and often death. Several different receptors on M have been shown to bind LPS, including the type A scavenger receptor (SR-A). This receptor is able to bind a broad range of polyanionic ligands such as modified lipoproteins and lipoteichoic acid of gram-positive bacteria, which suggests that SR-A plays a role in host defense. In this study, we used mice lacking the SR-A (SRKO) to investigate the role of SR-A in acquired immunity using a viable bacillus Calmette Guérin (BCG) infection model. We show that activated M express SR-A and that this molecule is functional in assays of adhesion and endocytic uptake. After BCG infection, SRKO mice are able to recruit M to sites of granuloma formation where they become activated and restrict BCG replication. However, infected mice lacking the SR-A are more susceptible to endotoxic shock and produce more TNF-α and interleukin-6 in response to LPS. In addition, we show that an antibody which blocks TNF-α activity reduces LPS-induced mortality in these mice. Thus SR-A, expressed by activated M, plays a protective role in host defense by scavenging LPS as well as by reducing the release by activated M of proinflammatory cytokines. Modulation of SR-A may provide a novel therapeutic approach to control endotoxic shock.     

The immune response of allophenic mice to the synthetic polymer L-glutamic acid, L-lysine, L-phenylalanine. II. Lack of gene complementation in two nonresponder strains

The genetic control of the immune response of inbred strains of mice to certain antigens has been demonstrated to be governed by a set of Ir genes linked to the major histocompatibility complex (H-2) of mice (1,2). Until recently, the control was thought to be governed by single, dominant genes, located within the I region of the H-2 complex. Merryman et al. (3) originally demonstrated that the immune response to the synthetic terpolymer L-glutamic acid, L-lysine, L-phenylaline (GL) is under dominant, H-2-linked Ir gene control (4-7). This was shown both by crossing two nonresponder parental strains to produce responder offspring in the F(1) generation, and by the analysis of appropriate recombinant stains of mice. The two complementing genes have been mapped in the IA and IC regions of the H-2 complex, and have been termed β and α, respectively (5,6). Thus, any strain of mouse may contain neither, one, or both genes. Only mice containing both genes are capable of responding to GL. It has been shown using F(1) hybrid and recombinant strains of mice, that the α- and β-genes can complement each other in either the cis (on the same chromosome) or in the trans (on different chromosomes) position (8). In this paper we report the results of studies aimed at answering the question of whether or not the α- and β- genes can complement each other when they are present in different lymphoid cells. To this end we have constructed allophenic mice composed of two nonresponder strains (A and C57BL/6), which show gene complementation in the F(1) generation. Allophenic mice are chimeras containing two cell types coexisting in a “normal” environment. The mice were tested for the specific cellular composition of the two parental cell types and were found to possess a complete range in the relative proportion of the two cell types. This report demonstrates that regardless of the mixture of cell types present in the allophenic mice, none of them were responders to GL. Thus no complementation of the α- and β-genes is seen when the two genes are present in different cells.     

Down-regulation of mannosyl receptor-mediated endocytosis and antigen F4/80 in bacillus calmette-guerin-activated mouse macrophages. Role of T lymphocytes and lymphokines

Bacillus Calmette-Guerin (BCG) infection alters the surface and endocytic properties of mouse peritoneal macrophages (PM) compared with thioglycollate- elicited (TPM) or resident PM (RPM). Expression of Ia antigen (Ag) is enhanced up to fourfold, but plasma membrane receptors that mediate binding and uptake of mannosyl/fucosyl-terminated glycoconjugates (MFR), Fc receptors, and the macrophage (m)-specific Ag F4/80 are reduced by 50-80 percent. Levels of Mac-1 remain relatively stable. These changes are accompanied by enhanced secretion of O(2)(-), after further stimulation with phorbyl myristate acetate, and of plasminogen activator. Both these products are released by TPM, but not RPM. The characteristic surface phenotype of BCG-PM can also be induced by injection of C. parvum, another m- activating agent, but not by thioglycollate broth, lipopolysaccharide, or proteose peptone. Purified protein derivative (PPD) and N-acetylmuramyl-L- alanyl-D-isoglutamine. 2H(2)0 are soluble agents with partial activity. Alteration of m markers by BCG infection depends on T lymphocyte function, although studies with nude mice indicate that other pathways may also serve to modify the surface of the m. M from uninfected animals displayed all markers of activation after adoptive transfer of specifically-sensitised lymphocytes with PPD, intraperitoneally, or after co- cultivation. Treatment of primed lymphocytes with anti-Thy-1 antibody and complement ablated this effect. Lymphokines obtaned by Ag or mitogen stimulation induced similar changes in TPM and RPM. Mannose-specific endocytosis decayed rapidly, time 1/2 approximately equal to 16 h and stabilized at approximately 25 percent of control values. Single-cell analysis showed that residual MFR activity was uniform in the target population. Loss of Ag F4/80 after activation by lymphocyte and PPD was less marked than after infection (35 percent vs 80 percent), unlike MFR activity, which declined to a similar extent. Induction of m Ia by lymphokine reached a peak after 2-3 d and was lost within 2 d of its removal. Recovery of MFR and F4/80 was incomplete under these conditions. These studies establish that activated m known to display enhanced antimicrobial/anticellular activity express markedly different surface properties distinct from elicited or resident cells. The role of antigen- stimulated T cell products in regulating m function is confirmed, and down-regulation of mannosyl-receptor-mediated endocytosis provides a sensitive, quantitative, and cell-specific new marker to study their properties and mechanism of action. Extensive, but selective remodeling of m plasma membrane structure could play an important role in controlling recognition and effector mechanisms of the activated m.     

Studies of the cell surface of mouse dendritic cells and other leukocytes

The surface of dendritic cells (DC) has been analyzed by means of monoclonal antibodies (Ab) and lactoperoxidase (LPO)-mediated radioiodination. Antigens and other exteriorily disposed polypeptides of purified spleen DC were compared with those of tissue macrophages (M), monocytes, and other bone marrow-derived elements. Quantitative binding studies and autoradiography with (125)I-Ab established that DC expressed high levels of I-A and H-2D, 2 × 10(5) and 1 × 10(5) Ab binding sites per cell, respectively. DC from conventional, germ-free, and specific pathogen-free mice were all rich in Ia. Expression of Ia on B cells was 5-10 percent of that on DC and increased fivefold during lipopolysaccharide mitogenesis. More than 70-90 percent of purified M and monocytes from specific pathogen-free mice were Ia negative, but increased levels of Ia were noted on cells from mice reared under conventional conditions. Thus large amounts of Ia on DC is a constitutive trait, whereas the expression of Ia by other cell types may be governed by the environmental and immunological status of the host. The 2.4G2 Fc receptor Ag was not detected on DC. Peritoneal and spleen M had 10(5) 2.4G2 binding sites/cell, whereas monocytes and lymphocytes were less reactive (1 × 10(4)-3 × 10(4) binding sites/cell). Four other M-related antigens were evaluated. Each had a distinctive tissue distribution and none bound exclusively to M and monocytes. Neither 1.21J (Mac-1) nor F4/80 reacted with DC. Immunoprecipitation studies of externally ((125)I) and biosynthetically ([(35)S]methionine)dabeled cells confirmed the binding data. Sensitive binding assays with (125)I-Ab confirmed previous observations that DC lack Ig and Thy-1. Lyt-1 was also not found on DC, but 5-12 percent of the cells in purified DC preparations expressed both Lyt-2 and Ia. All DC expressed the leukocyte common antigens at levels similar to other leukocytes. The spectrum of surface polypeptides labeled by LPO-mediated iodination was different on M, DC, and lymphocytes. Polypeptides migrating at molecular weights of 155,000, 85,000, and 62,000 appeared to be restricted to DC. These observations establish that the cell surface of DC differs considerably from other leukocytes, including the blood monocyte, and suggest that the DC is part of a unique Ia-rich leukocyte differentiation pathway.     

Role of arginase in killing of schistosomula of schistosoma mansoni

Nonspecific resistance to the multicellular organism Schistosoma mansoni can be induced in mice by several infectious agents. We utilized the observed genetic restriction of such acquired resistance to study the mediators of killing of the larval stage of S. mansoni in vitro. Adherent peritoneal cell monolayers from Corynebacterium parvum-treated C57BL/6J but not from C. parvum-treated BALB/cJ mice killed an increased proportion of schistosomula in 24 h. Activated macrophages (M) from both strains exhibited enhanced H(2)0(2) production after incubation with the parasites or phorbol myristate acetate. Thus H(2)0(2) production was not associated with schistosomula killing. Moreover, schistosomula killing was unaffected by catalase or superoxide dismutase. In contrast, activated C57BL/6J (but not BALB/cJ) M released fourfold more arginase into supernates than control M. Schistosomula killing by these M correlated with arginase content of the supernates, was exaggerated in arginine-poor medium, and could be blocked by the addition of arginine. Exogenous bovine arginase added to Fischer's medium without macrophages produced comparable parasite mortality. Our data suggest that arginase is a critical mediator of in vitro killing of this multicellular organism by activated macrophages.     

Synthesis and Secretion of Cystic Fibrosis Ciliary Dyskinesia Substances by Purified Subpopulations of Leukocytes

Cultured peripheral blood leukocytes (PBL) from individuals homozygous or heterozygous for the defective gene causing the inherited disease cystic fibrosis (CF) secrete three different ciliary dyskinesia substances (CDS), which can be detected by their activity in vitro in a rabbit mucociliary bioassay. Their PBL also release substances that promote mucus expulsion and destruction of the ciliated epithelium. In the present study the relative numbers of lymphocytes (T, B, and null), monocytes-macrophages (M), and polymorphonuclear neutrophils were found to be normal in subjects with the CF gene, as were the responses of their PBL to phytohemagglutinin and pokeweed mitogen. Using purified subpopulations of leukocytes, we obtained evidence that both monocytes and T lymphocytes can secrete CDS in vitro with no requirement for cooperation with other lymphocyte subsets, whereas B and “null” lymphocytes probably require either differentiation or cellular cooperation for optimal secretion of CDS. Mucus expulsion and tissue destruction were produced by substances released primarily from polymorphonuclear neutrophils and secondarily from M. Using cycloheximide and actinomycin D, we obtained evidence that CDS accumulation requires active protein synthesis and is not dependent on newly synthesized RNA, at least in short-term cultures. Gel filtration chromatography of active culture supernates showed that T lymphocytes synthesized only a CF-specific CDS, whereas M synthesized all three CDS found in PBL cultures. Evidence is presented that one CDS is related structurally to C3a, since it can be removed with rabbit antisera specific for human C3a.     

Inactivation of Lipid-Containing Viruses by Long-Chain Alcohols

This report describes the inactivation of lipid-containing viruses by several long-chain alcohols. A striking peak in antiviral activity was found for saturated alcohols having chain lengths from 10 to 14 carbons. Viruses having different membrane structure showed different susceptibilities to alcohols having different chain lengths and structural features. Decanol, dodecanol, and tetradecanol readily inactivated herpes simplex virus and the enveloped bacterial virus 6. The lipid-containing virus PM2 was susceptible to decanol and dodecanol but comparatively unsusceptible to tetradecanol. The branched-chain alcohol phytol, a naturally occurring component of chlorophyll, was active against 6 and herpes simplex virus but not against PM2. Polyoma virus and the bacteriophage 23-1-a, which do not contain lipids, were not susceptible to inactivation by any of the alcohols tested. Experiments were also carried out to determine the effects of these compounds on cells. At 0.5 mM, decanol lysed human embryonic lung cells, erythrocytes, and the bacterial hosts for 6 and PM2. Dodecanol, tetradecanol, and phytol at this concentration were less damaging to cells. At 0.05 mM, none of the alcohols caused observable cytopathic effects on human embryonic lung cells, although several of the alcohols at this concentration were active against herpes simplex virus. Our findings suggest that dodecanol, tetradecanol, and phytol may warrant further studies as potential antiviral agents, particularly for topical application to virus-infected areas of the skin.     

ANTIBODY FORMATION : IV. FORMATION OF RAPIDLY AND SLOWLY SEDIMENTING ANTIBODIES AND IMMUNOLOGICAL MEMORY TO BACTERIOPHAGE φX 174

Injection of a sufficient dose of bacteriophage X 174 into guinea pigs results in the formation of rapidly sedimenting antibody molecules (19S), and later, slowly sedimenting molecules (7S). Above a threshold dose of antigen, the relative rate of 19S formation is maximal and dose-independent; below this dose, slower relative rates are obtained. The time for doubling the serum 19S level is as short as 6 to 8 hours, suggesting that the absolute rate of antibody formation per cell is increasing in addition to proliferation of antibody-producing cells. Synthesis of 19S after injection of 10¹⁰ X virtually ceases at 10 days after which 19S antibody activity disappears from the circulation with a half-life of approximately 24 hours. A second injection of X on day 5 or 9 prolongs 19S synthesis, indicating that antigen not only can regulate the relative rate, but also is essential for continued synthesis of 19S. 19S synthesis is also prolonged in guinea pigs by injection of X with endotoxin or by 400 r whole body x-irradiation 24 hours after injection of phage into rabbits. The primary 7S response is not detected until approximately 1 week after immunization and relative rates are antigen-dependent. Primary 7S synthesis can continue for many months and leads to preparation for a secondary antibody response (immunological memory) during which only 7S is detected. In contrast, in animals that form precipitating 19S without detectable 7S, a second injection of phage 1 month later results in a second 19S response which closely resembles the first. These findings have led to the suggestion that formation of 19S does not lead to persisting immunological memory.     

THE CARRIAGE OF IMMUNOLOGICAL MEMORY BY SMALL LYMPHOCYTES IN THE RAT

Lymphocytes were obtained from the thoracic duct of rats 1½ to 15 months after primary immunization with a single dose of bacteriophage X 174. An intravenous injection of these lymphocytes conferred on heavily X-irradiated rats the ability to form antibody in a secondary-type manner after a first injection of X. Negligible responses were obtained after cell transfer if the recipients were not challenged with antigen. Thoracic duct cells from some immunized donors were incubated in vitro for 24 hr before transfer in order to destroy selectively the large, dividing lymphocytes. The responsiveness conferred on X-irradiated recipients by such "incubated" inocula was then compared with that given by equal numbers of "fresh" thoracic duct cells. In all such comparisons the recipients of the "incubated" cells gave higher and more rapid antibody responses. It was concluded that the cells in thoracic duct lymph which carried immunological memory were small lymphocytes.     

Selective Breeding for a Behavioral Trait Changes Digit Ratio

The ratio of the length of the second digit (index finger) divided by the fourth digit (ring finger) tends to be lower in men than in women. This 2D4D digit ratio is often used as a proxy for prenatal androgen exposure in studies of human health and behavior. For example, 2D4D ratio is lower (i.e. more “masculinized”) in both men and women of greater physical fitness and/or sporting ability. Lab mice have also shown variation in 2D4D as a function of uterine environment, and mouse digit ratios seem also to correlate with behavioral traits, including daily activity levels. Selective breeding for increased rates of voluntary exercise (wheel running) in four lines of mice has caused correlated increases in aerobic exercise capacity, circulating corticosterone level, and predatory aggression. Here, we show that this selection regime has also increased 2D4D. This apparent “feminization” in mice is opposite to the relationship seen between 2D4D and physical fitness in human beings. The present results are difficult to reconcile with the notion that 2D4D is an effective proxy for prenatal androgen exposure; instead, it may more accurately reflect effects of glucocorticoids, or other factors that regulate any of many genes.     

CHARACTERIZATION OF A NEW INTRA-H-2 RECOMBINANT : SEPARATION OF THEIr-REANDIr-GLT GENES

This report characterizes the intra-H-2 crossover in the D2.GD mouse strain. Recombination occurred within the I region between the immune response (Ir) gene controlling immune responsiveness to ragweed extract (RE). Ir-RE, and the Ir gene governing responsiveness to the random linear terpolymers of glutamic acid and lysine with either tyrosine or pheylalanine (Ir-GLT and Ir-GL). The Ir-RE gene was tentatively located in the Ir-1A subregion and the Ir-GLT and Ir-GL gene(s) tentatively placed in the Ir-1B subregion. The importance of this recombinant strain to further studies on the fine structure of the I region is discussed.     

CD80 (B7-1) Binds Both CD28 and CTLA-4 with a Low Affinity and Very Fast Kinetics

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (K_d values ~12 and ~200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell–APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37°C, soluble recombinant CD80 bound to CTLA-4 and CD28 with K_d values of 0.42 and 4 μM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k_off); sCD80 dissociated from CD28 and CTLA-4 with k_off values of 1.6 and 0.43 s⁻¹, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate dynamic T cell–APC contacts and to facilitate scanning of APC for antigen.     

Lipid Reorganization Induced by Shiga Toxin Clustering on Planar Membranes

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb₃ in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb₃ clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb₃ lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb₃ (65305) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb₃ (4035205) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb₃, STxB-Gb₃ complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.     

LYMPHOID CELLS MEDIATING TUMOR-SPECIFIC CYTOTOXICITY TO CARCINOMA OF THE URINARY BLADDER : SEPARATION OF THE EFFECTOR POPULATION USING A SURFACE MARKER

Peripheral lymphocytes from patients with urinary bladder carcinoma and controls have been separated on the basis of rosette formation with sheep erythrocytes. The fractions were tested for tumor-specific cytotoxicity. The E rosette-forming cells of purity 90% respond well in PHA-induced cytotoxicity but are totally inactive in the tumor assay. The non-E rosette-forming cells (purity 91%) give enhanced activity in the tumor-specific cytotoxicity as well as in antibody-mediated target cell lysis in a model system. These data support the notion that the effector cells in cell-mediated immunity to carcinoma of the urinary bladder are members of the nonthymus-derived population of peripheral lymphocytes.     

ANTIBODY FORMATION : III. THE PRIMARY AND SECONDARY ANTIBODY RESPONSE TO BACTERIOPHAGE øX 174 IN GUINEA PIGS

Injection of a small bacteriophage X 174 into guinea pigs results in an accelerated elimination of phage detectable as early as 24 hours after injection. The immune nature of the accelerated elimination is indicated by its specificity, by the appearance of excess specific serum antibody after phage elimination, and by the prevention of accelerated elimination by 400 r whole body x-irradiation of guinea pigs prior to injection of phage. The early antibody response is considered to be a primary one since an analogous response occurs in newborn guinea pigs, antibody is not detectable in the sera of non-immunized animals, and the second challenge with X stimulates a serum antibody response 100-fold greater than that after primary immunization. The early detection of immune elimination appears to be due, in part, to the small amounts of phage employed, since larger doses of phage delay the time of onset of detectable immune elimination. The early rise of serum antibody in the primary and secondary response appears exponential with a similar rate constant of antibody formation. The rate constant is also independent of dose. These findings have led to the suggestion that during this exponential phase, the relative rate of antibody formation at a cellular level may be constant for a given antigen.     

IMMUNOCOMPETENT CELLS OF THE CHICKEN : I. SPECIFIC SURFACE ANTIGENIC MARKERS ON BURSA AND THYMUS CELLS

Specific antisera to chicken thymus and to bursa of Fabricius were obtained in rabbits. After appropriate absorption and dilution all four anti-thymus sera, in the presence of guinea pig C', killed >90% of thymus and 12% of bursa cells. They were cytotoxic for approximately 50% of spleen cells and did not affect antibody-forming cells. The surface antigen detected by these antisera was named chicken T-lymphocyte antigen (CTLA). Two of four anti-bursa sera, under similar conditions, killed >90% of bursa cells and 10% of thymus cells. These antisera were cytotoxc for a large percentage of antibody-forming cells and killed approximately 30% of spleen cells The other two anti-bursa sera were somewhat less potent but showed similar specificity. The surface antigen detected by these antisera was named chicken bursa-derived lymphocyte antigen (CBuLA). Rabbit antisera to chicken immunoglobulin were cytotoxic for bursa but not for thymus cells and killed a similar percentage of spleen cells as did anti-bursa sera. They were also cytotoxic for antibody-forming cells.     

An O-phosphotransferase catalyzes phosphorylation of hygromycin A in the antibiotic-producing organism Streptomyces hygroscopicus

The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (K_m = 30 ± 4 μM) at the C-2 position of the fucofuranose ring in the presence of ATP (K_m = 200 ± 20 μM) or GTP (K_m = 350 ± 60 μM) with a k_cat of 2.2 ± 0.1 min⁻¹. The phosphorylated HA is inactive against HA-sensitive ΔtolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k_cat and K_m values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5-dihydrohygromycin A and 5-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.