Cell-mediated immunity in experimental filariasis: lymphocyte reactivity to filarial stage-specific antigens and to B- and T-cell mitogens during acute and chronic infection.

To study immunological responses in chronic filarial infections, a model utilizing inbred Lewis rats infected with Brugia pahangi was developed. Microfilaria were found in the bloodstream of over 90% of the rats by 16 weeks of infection. Using in vitro lymphocyte blastogenesis, cell-mediated immune responses of blood, splenic, and mesenteric node lymphocytes were followed during 1.5 years of infection. Lymphocyte responses to antigen prepared from infective stage filarial larvae were detectable in the early weeks of infection, whereas responses to microfilarial antigen only developed late as microfilaremia waned. Lymphocyte responses to antigen from adult filaria vacillated during the infection. With the mitogens, phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide, periods of B and T-cell hyporesponsiveness were demonstrable. Between 16 and 36 weeks of infection node lymphocytes from many rats were unresponsive to all mitogens and antigens. The model of B. pahangi in inbred rats offers advantages for immunological studies of filarial infections.     

Butyrate-induced glycolipid biosynthesis in HeLa cells: properties of the induced sialyltransferase.

CMP-sialic acid:lactosylceramide sialyltransferase is induced in HeLa cells by butyrate which also causes the cells to undergo morphological changes including the extension of neurite-like processes. The activity of this enzyme is more than 20-fold higher in butyrate-treated cells than in cells grown without this short chain fatty acid. In vitro synthesis of hematoside from endogenous acceptors is also elevated in cells grown in the presence of butyrate. The levels of induced enzyme activity are influenced by the pH of the culture medium, being higher in more acidic cultures, but are not affected markedly by varying the cell density over a wide range. Detergent is required for in vitro sialyltransferase activity, and this activity is stimulated almost fivefold by cardiolipid. The optimum pH for in vitro activity is 6.0 and the apparent K_m value for lactosylceramide is 3.5 × 10^?5m. Although there are several sialyltransferase activities in HeLa cells, the induced enzyme is specific for lactosylceramide.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

Cyclic AMP dependent regulation of mitosis in human lymphoid cells

Intracellular levels of cyclic AMP (cAMP), cAMP-dependent phosphodiesterase activity, and adenylate cyclase activity are examined in an established line of human lymphoid cells synchronized by either excess thymidine or by colcemid treatment. cAMP levels and adenylate cyclase activities during the two G periods are high when compared with the values in M. cAMP-dependent phosphodiesterase activity, which is low during early G 2, is shown to increase during G 2 and reach a maximum activity during M. Agents such as dibutyryl cAMP, 1-methyl-3-isobutyl xanthine, noradrenaline, and isopropyl noradrenaline, which increase the levels of intracellular cAMP were examined to determine their effects on mitosis and on DNA synthesis. In thymidine-synchronized cells the onset of mitosis is prevented by increasing or maintaining high levels of cAMP during G 2. The specificity of inhibition of DNA synthesis or mitosis by dibutyryl cAMP is a function of the time, during the cell cycle, when the analogue is added. The elevation of cAMP by methyl xanthine results in a more general inhibition of nucleic acid synthesis and mitosis. Although both catecholamine hormones inhibit mitosis, isopropylnoradrenaline also inhibits DNA synthesis while noradrenaline treatment does not result in such inhibition.     

Trypanosoma cruzi: the T-cell dependence of the primary immune response and the effects of depletion of T cells and Ig-bearing cells on immunological memory

The effects of T-cell depletion on primary infection with Trypanosoma cruzi and on immunological memory to this parasite were studied in a syngeneic mouse system. Exacerbation of T. cruzi infections occurred in thymectomized, irradiated, bone marrow-reconstituted (TX) C57BL/6J mice compared to sham thymectomized, irradiated, bone marrow-reconstituted (STX) mice. Reconstitution of TX mice with thymocytes restored the resistance to a level equivalent to that of STX mice. Immunological memory against T. cruzi present in spleen cells in mice recovered from T. cruzi infections could be ablated by treatment with rabbit anti-brain-associated theta serum but not with rabbit anti-mouse immunoglobulin serum prior to adoptive transfer of immune spleen cells into TX mice. These experiments suggest that modulation of the primary immune response and memory against T. cruzi depends largely on the thymus-derived lymphocyte. The possible implications of this T-cell regulation on previously reported effector mechanisms againt this parasite are discussed.     

A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation

Microsomal fractions from arterial walls of pigs and rabbits and fundus of rat stomach generate from prostaglandin endoperoxides (PGG₂ or H₂) an unstable substance, prostaglandin X (PGX) which is a potent inhibitor of platelet aggregation induced by several different substances.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Preparation, characterization, and functions of rabbit lymph node populations. III. Antigen-induced uptake of thymidine and dibutyryl cyclic AMP: inhibition of thymidine uptake by cholera toxin and dibutyryl cyclic AMP.

Keyhole limpet hemocyanin (KLH)-primed lymph node cell (LNC) populations were incubated with various amounts of KLH and the cellular incorporation of tritiated thymidine ([³H]TdR) or tritiated N⁶, O^2′ dibutyryl cyclic AMP ([³H]DbcAMP) was determined. T LNC responded more vigorously than did complement receptor lymphocytes (CRL), i.e., B cells, at all KLH concentrations, during all time intervals examined, and in the presence or absence of normal rabbit serum (NRS). The depletion of adherent cells from KLH-primed LNC resulted in no significant decrease in KLH-induced incorporation of either [³H]TdR or [³H]DbcAMP in any of the LNC populations. Thus it appeared that variation among LNC populations in the incidence of macrophages did not account for the marked variation in their responses. Cultures containing equal numbers of T and CRL were induced to incorporate more [³H]TdR or [³H]DbcAMP than either population cultured separately or the sum of their individual responses. It was concluded that KLH-induced incorporation of these substances into primed, isolated LNC, was primarily manifested in the T-cell population. The synergism seen in cultures containing mixtures of T and CRL suggested that B cells are induced to incorporate [³H]TdR or [³H]DbcAMP in the presence of antigen and T-cell product(s). KLH-induced incorporation of [³H]TdR into KLH-primed LNC was inhibited by cholera enterotoxin (CT) and DbcAMP as previously reported. However, CT or DbcAMP inhibited this incorporation into T LNC to a greater extent than into CRL or unfractionated LNC.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. III. Inhibition by cytochalasins, colchicine, and vinblastine.

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages.Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function.Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations (<10^?6M) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction.These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.     

A new approach to understanding kinetic cooperativity when the hill coefficients are less than 2

Dihydrofolate reductase from chicken liver has a single sulfhydryl group which reacts stoichiometrically and specifically with a wide variety of organic mercury compounds to yield an enzyme derivative which exhibits up to 10-fold the activity of the unmodified form when measured at pH 6.5, the optimum for the modified enzyme. The sulfhydryl group is apparently not at the active site since a 25-fold excess of either major cosubstrate, dihydrofolate or TPNH, affects neither the rate nor extent of the modification reaction. The reaction is essentially instantaneous and yields an enzyme with altered kinetic properties for all the substrate pairs examined (TPNH/dihydrofolate, TPNH/ folate, and DPNH/dihydrofolate) when tested near their pH optima. V values increased 3- to 10-fold when TPNH was cofactor; K_m values increased 10- to 15-fold for the TPNH/dihydrofolate pair. The mercurial-activated enzyme, unlike the native form, exhibits a markedly increased sensitivity to heat, proteolysis, and the ionic environment, losing approximately 50% of its activity under conditions where there is no loss of activity in the native form. However, substrates can afford protection, the order of effectiveness being identical with the relative affinities of the substrates for the native enzyme (Subramanian, S., and Kaufman, B. T. (1978) Proc. Nat. Acad. Sci. USA75, 3201). Thus, dihydrofolate, with the largest binding constant is the most efficient, protecting completely against trypsin digestion when present at a 1:1 ratio with enzyme. Heating the mercury enzyme in the absence of substrates gives rise to a stable but altered conformation characterized by a time course which shows marked hysteresis. The striking similarity of the properties of the mercurial-activated dihydrofolate reductase to the reductase activated by 4 m urea, a reagent known to affect the tertiary structure of proteins, suggests that covalent binding of organic mercurials to the sulfhydryl group results in a similar conformational change characterized by a marked facilitation of the dihydrofolate reductase reaction.     

Structural changes in light- and dark-adapted compound eyes of the australian earwig Labidura riparia truncata (dermaptera)

The apposition acone eye of Labidura is relatively small—550–600 facets—with a thick corneal lens and shallow retina. The retinula cell columns are each formed of six peripheral cells plus two central cells, a partially fused rhabdom, and dense pigment in two or three cell types. Upon adaptation from light to dark, the most striking photomechanical response is a proximal broadening of the cone cells, which results in a 38-fold increase in cross-sectional area of the aperture. While longitudinal rhabdom movement is small, microvillar diameters swell in response to light and contract in the dark. Irregularities of facet pattern and shape, and in ommatidial alignment were found, particularly towards eye margins. Three types of interommatidial sense organs on the eye surface are described, one of which has not been previously reported. An argument is presented to explain how the field of view and sensitivity are both apparently decreased in the acone eye by exposure to light.     

2-Hydroxyestradiol-17α and 4-hydroxyestradiol-17α, catechol estrogfn analogs with reduced estrogen receptor affinity

2-Hydroxyestradiol-17α and 4-hydroxyestradiol-17α, the catechol derivatives of estradiol-17α, have reduced affinity for hypothalamic, pituitary, and uterine estrogen receptors, but retain a potency for interaction with catechol-O-methyltransferase equal to that of the natural, 17β-hydroxy catechols. This dissociation of receptor binding and catecholamine interactions nay allow the use of the 17α catechols as a probe for the mechanism of action of the catechol estrogens.     

beta-Adrenergic receptor induction in HeLa cells: synergistic effect of 5-azacytidine and butyrate

³H-Labeled leukotriene C₃ was efficiently taken up by the isolated, perfused rat liver and excreted into the bile. The isolated, perfused kidney eliminated leukotriene C₃ from the perfusate slower and excreted only a fraction of the radioactivity into the urine. Isolated hepatic, intestinal and renal cells also took up leukotriene C₃, the renal cells being the most effective in accumulating the label. Anthglutin, an inhibitor of γ-glutamyl transferase, decreased the uptake by kidney cells but had no effect on the uptake by the other cell types. In liver cells, the uptake rate was sensitive to temperature and to cellular ATP content. Chromatographic analyses indicated that renal cells metabolized leukotriene C₃ more rapidly than hepatic and intestinal cells. Leukotriene D₃ and E₃ were formed during the incubations with kidney cells, whereas intestinal cells produced mainly more polar metabolites.     

Tumoricidal responses in vitro of peritoneal macrophages from conventionally housed and germ-free nude mice

Peritoneal macrophages from untreated nude mice were nonspecifically cytotoxic to tumor cells in vitro and were more responsive to chemotactic stimuli than macrophages from normal mice or from phenotypically normal littermates of nude mice. Tumoricidal and chemotactic responses of activated macrophages from nude mice were quantitatively comparable to responses of macrophages from BCG-infected normal mice. Peritoneal macrophages from germ-free nude mice, however, were not tumoricidal in vitro. These observations suggest that environmental stimuli, rather than thymic deficiency per se, induced activated macrophages in nude mice.     

Proteins foretelling head or abdomen development in the embryo of Smittia spec. (Chironomidae,Diptera)

The development of the segment pattern in Smittia embryos can be manipulated experimentally. Centrifugation during intravitelline cleavage leads to a mirror image duplication of most of the head in the absence of abdominal segments (“double cephalons”). Conversely, mirror image duplications of abdominal segments in the absence of head and thorax (“double abdomens”) can be generated by UV-irradiation of the anterior pole before blastoderm formation. By subsequent exposure to blue light, UV-irradiated embryos can be reprogrammed for normal development (photoreversal). We have characterized an “anterior indicator” protein (designated AI₁; M_r ? 35,000; IEP ? 4.9). Its synthesis was restricted to anterior fragments of embryos during a late blastoderm stage (Bl_VI). This protein was synthesized, however, in both anterior and posterior fragments of prospective double cephalons. Conversely, this protein was synthesized neither in anterior nor in posterior fragments of UV-induced double abdomens. Upon photoreversal, the protein was synthesized again in anterior fragments. Thus, synthesis of this protein in a given fragment always indicated development of head and thorax there. Likewise, we have characterized a “posterior indicator protein” (designated PI₁, M_r ? 50,000, IEP ? 5.5). Its synthesis during early blastoderm stages (Bl_I and Bl_II) was restricted to posterior fragments but not to pole cells in normal embryos. In UV-induced double abdomens, PI_I was synthesized in both anterior and posterior fragments at stage Bl_II. Photoreversal again led to restriction of PI_I synthesis to posterior fragments. Thus, the synthesis of PI_I in a given fragment at stage Bl_II always foreshadowed the formation of an abdomen several hours before this can be discerned morphologically. The synthesis of two other proteins (designated a₁ and p₁) was also restricted, during certain blastoderm stages, to anterior or posterior fragments, respectively. However, UV-irradiation or centrifugation had little or no effect on the synthesis of these proteins. Conversely, programming embryos for double abdomen development by UV-irradiation caused a set of reproducible, and mostly photoreversible, changes in the pattern of proteins synthesized in anterior embryonic fragments. However, the synthesis of most of the affected proteins was not region-specific in normal embryos.     

Changes in plasma catecholamines and behavior of rats during the anticipation of footshock

A chronic catheter was inserted into the ventral caudal artery of male Sprague-Dawley rats to allow for sampling of blood and measurement of blood pressure and heart rate in conscious animals without handling. The day after surgery, one group of rats was transferred individually from the home cage to a shock chamber and after 5 min received 60 footshocks (2.5 mA, 0.4 sec in duration, at 5-sec intervals). This procedure was repeated two additional times during the same day. Control animals were handled in an identical manner but were not shocked. Previous experience with footshock had no effect on basal plasma levels of norepinephrine (NE) and epinephrine (EPI) or on resting blood pressure and heart rate as measured 2 days after surgery. When transferred to the shock chamber, previously shocked rats had greater increases in plasma NE and EPI and heart rate. In addition, previously shocked rats were less active and defecated more frequently than did control rats. However, there were no differences in the responses of previously shocked and control rats to 5 min of intermittent footshock. Results of this study demonstrate an activation of the sympatho-adrenal medullary system and attendant changes in the cardiovascular system and behavior of rats during the anticipation of footshocks. This suggests that the functioning of sympathetic nervous system and the adrenal medulla provides a sensitive measure of arousal and fear in rats.     

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate. II. Specificity of hapten-reactive helper T cells.

Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (PAB-MGG) in mice. Spleen and lymph node cells taken from these primed mice could demonstrate their helper activity for anti-DNP antibody production when transferred intravenously into 600R X-irradiated recipient mice along with DNP-primed B cells and the double hapten conjugated carrier, DNP-MGG-PAB. Isologous carrier (MGG)-primed cells could not demonstrate this helper activity. Accordingly, helper cells reactive for a haptenic group are considered to develop by the immunization of hapten-isologous carrier conjugate. Hapten-reactive helper activity was also induced by the immunization of other hapten-isologous carrier conjugates, e.g., MAB-MGG, PABS-MGG or PAB-MSA. These hapten-reactive helper cells were T lymphocytes, as the helper activity of PAB-MGG-primed cells was completely abolished by in vivo ATS-treatment. Helper activity of PAB-MGG-primed cells for DNP-primed B cells was also demonstrated through the double hapten conjugated heterologous carrier DNP-HGG-PAB to be the same as with DNP-MGG-PAB, but weakly through DNP-KLH-PAB. As HGG but not KLH resembles MGG in composition, almost all hapten-reactive helper T cells can be considered to recognize not only haptenic groups but also physicochemical properties of the hapten-conjugated carrier site. However, these helper T cells could discriminate structural differences among related haptenic groups, because PAB-MGG-primed cells clearly responded to DNP-MGG-PAB to demonstrate their helper activity for DNP-primed B cells, but responded only weakly to DNP-MGG-PABS or DNP-MGG-MAB. When the specificity restrictions of T and B cells to the same haptenic group were compared by responsiveness measured after the antigenic stimulation (B cell function by anti-hapten antibody production and T cell function by helper activity), differences were noted, as PAB-MGG-primed T cells could respond not only to DNP-MGG-PAB but also fairly well to DNP-MGG-MAB to demonstrate their helper activity, but PAB-MGG-primed B cells responded to only PAB-MGG. Thus, hapten specificity appears to be much more restricted for B cells than T cells. The difference of this responsivity between B cells and helper T cells was thought to derive from the specificity difference of B cell and helper T cell receptors rather than from any sensitivity differences of the experimental procedure. The differences in the specificity restrictions of receptors of B and helper T cells were discussed in the light of hapten-specificity.     

Radioimmunoassay for pig angiotensin I converting enzyme: a comparison of immunologic with enzymatic activity.

Angiotensin I converting enzyme in body fluids and extracts of various pig tissues was measured by radioimmunoassay and by enzymic assay. Based on the ratios of enzymic to immunologic activity, the extracts could be separated into two groups. One group, with ratios around 4 U/mg, included urine and extracts from the adrenal, choroid plexus, epididymis, gall bladder, heart, liver, retina, spleen, and testis. The other group, with ratios around 12 U/mg, contained serum and extracts from lung and kidney. Explanations are offered for why one group had a lower enzymic to immunologic ratio than the other.     

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.