Passive administration of antibodies during the primary immunization. The influence on the secondary response

Groups of mice were injected with different quantities of sheep red blood cells (SRBC: 10⁸, 10⁹ and 10¹⁰) and simultaneously with different quantities of antibodies against SRBC. The response was tested 15 and 30 days after the primary dose and 4 days after the secondary response. The action of antiserum regulates the quantity of antigen available for the immunization process. With a large dose of antiserum it is possible to inhibit the primary, as well as the secondary response. A smaller dose of antiserum suppresses primary antibody formation but the process of preparing the secondary response is not inhibited. An inhibitory dose of antiserum injected 24 and 48 hours after antigen significantly depresses the primary response but is followed by a pronounced secondary response. When the antigen is bound 24 and 48 hours after the primary stimulus, the secondary response is only of the 19S type. If the antigen is present at least 72 hours after the primary dose of antigen cells forming 7S antibodies appear also in the secondary response. Experimental data support the hypothesis that there is a common precursor cell for the 19S and 7S Ab-forming cell; during limited proliferation the cellular basis for the 19S secondary response and with intensive proliferation (only after 6 or 7 generations) the precursors for 7S antibody forming cells, appear. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Ontogenetic development of antibody formation in response to different doses of gram-negative microorganisms in young rabbits

Rabbits at different stages of development were immunized with different doses of heatinactivated suspension ofEscherichia coli 086 andSalmonella paratyphi B. The dynamics and the site of formation of bactericidal and haemolytic antibodies during the primary reaction was investigated. An increase and an acceleration of antibody formation after increasing the dose of antigen was found in the serum and at the cellular level. The magnitude of the response and the rate of the reaction were directly proportional to the age of the experimental rabbits. The site of antibody formation depends on the character, route of administration, antigen dose and age of rabbits. After intraperitoneal and also after intravenous immunization withEscherichia coli andSalmonella paratyphi B antigens the site of antibody production in 15-day-old rabbits was the lymphatic tissue of the intestine, the appendix, and mesenteric lymph nodes. As the antigen dose was increased and the age of rabbits rose, i.e. in correlation with the increase of the antibody response, antibody formation shifted to the spleen which is the chief site of antibody production following immunization by these bacterial antigens from the first month of life of rabbits. In contrast with this type of antigen, after intraperitoneal or intravenous immunization with sheep erythrocytes of new-born or older rabbits antibody formation was concentrated in the spleen. The development of the immunological competence and the significance of intestinal lymphatic tissue as one of peripheral type is discussed.     

CD8+ T cells Are Preferentially Activated during Primary Low Dose Leishmania major Infection but Are Completely Dispensable during Secondary Anti-Leishmania Immunity

We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity.     

Competitive Al Inhibition of Net Mg Uptake by Intact Lolium multiflorum Roots : II. Plant Age Effects

Rhizotoxicity of Al is more pronounced in younger plants. Effects of Al on nutrient uptake by plants of different age are poorly understood. The depletion technique was used to monitor net Mg²⁺ uptake from nutrient solutions by intact 15- and 35-day-old plants of two ryegrass (Lolium multiflorum Lam.) cultivars. Lowering the pH from 6.0 to 4.2 decreased the maximum net ion influx without affecting K_m. Aluminum at 6.6 micromolar Al³⁺ activity increased K_m indicating competitive inhibition. The effects of pH and 6.6 micromolar Al³⁺ on net Mg²⁺ uptake were much larger in 15- than in 35-day-old plants. Aluminum at 26 micromolar Al³⁺ activity competitively inhibited net Mg²⁺ uptake by 35-day-old plants, while causing time- and external Mg²⁺ activity-dependent net Mg²⁺ efflux from 15-day-old plants. The equilibrium constant (K_i) of a reversible combination of postulated plasmalemma Mg²⁺ transporter and Al³⁺ was calculated to be 2 and 5 micromolar Al³⁺ activity for 15-day-old plants of Wilo and Gulf ryegrass, respectively, and 21 micromolar Al³⁺ activity for 35-day-old plants of both cultivars. The Al³⁺-mediated increase in K_m was larger for 15-day-old plants of the Al-sensitive cultivar `Wilo' than of the more Al-tolerant cultivar `Gulf,' while Al³⁺ affected 35-day-old plants of both cultivars to the same extent.     

Photo-induced electron transport and water state in Rhodospirillum rubrum chromatophores

It is shown that in bacterial chromatophores the pronounced changes in the free water content with a proton spin-spin relaxation time (T₂) of 10^?3—10^?2 s does not influence the efficiency of electron transfer from the photosynthetic reaction centre to the membrane pool of secondary acceptors. An abrupt inhibition of this process occurs only after the loss of the water with faster proton spin-spin relaxation time (T₂ of 10^?4 s). The process is reversible. The water fraction in question is obviously bound to the chromatophore proteins and forms the primary hydration layer.     

Thymus cell traffic induced by antigen stimulation

Spleens of adult mice immunized with either RSA or RGG responded in vitro to DNP-RSA or DNP-RGG, respectively, at a significantly higher rate than spleens of untreated mice. Stimulation in vitro could be achieved by short pulses of the antigen (5–15 min). It was found that thymectomy prior to injection of the carrier protein interfered with the subsequent response in vitro to the hapten-carrier conjugate, and that this was much more pronounced in 8- to 10-day-old mice than in older mice. It is suggested that antigen stimulation in vivo triggers thymus cell migration. Although this is by no means the only mechanism accountable for manifestation of the carrier effect, it may represent a device for amplification of the immune response in vivo.     

Studies on transplantation immunity: III. The secondary response to dissociated allogeneic cells

The secondary response of CBA mice to dissociated (A X CBA)F₁ lymphoid cells has been studied with the aid of the ⁵¹Cr-labeling technique. The effect of a secondary dose is independent of the time interval between primary and secondary doses over a wide range, due presumably to the remarkably stable status induced by a single stimulus. No clear evidence of a true secondary has been found for the liver or spleen responses, which are largely mediated by antibody production. The lymph node response, which is largely cell mediated, does possess a true secondary. The dose response data for the lymph nodes show that the primary response has an initial impetus into tolerance, reversed by sensitisation; and that the secondary response is simpler in form than the primary. Only a restricted range of primary and secondary doses are capable of eliciting a secondary response. Various peculiarities of the secondary suggest that it may arise as an in vivo form of “mixed lymphocyte reaction.”     

Cellular requirements for the expression of IgM. Immunological memory in vitro

In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10⁴ RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro. The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.     

Estrogen-induced synthesis of riboflavin-binding protein in immature chicks kinetics and hormonal specificity

The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay proceudre. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h. reaching peak levels around 48 h and declining thereafter. A two-fold amplication of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulat ions with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered ¹²⁵I-labelled protein. Simultaneous administration of progestrone did bot affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively countered the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the anti-esterogens per se were completely ineffective in substituting for estrogen in the inductive ptrocess.     

Milk fat depression and energy balance in stall-fed dairy goats supplemented with increasing doses of conjugated linoleic acid methyl esters

Feeding dietary supplements containing trans-10, cis-12-conjugated linoleic acid (t10,c12-CLA) has been shown to induce milk fat depression in cows, ewes and goats. However, the magnitude of the response is apparently less pronounced in lactating goats. The objective of this study was to evaluate the effects of increasing doses of CLA methyl esters (CLA-ME) on milk production, composition and fatty-acid profile of dairy goats. Eight Toggenburg goats were separated in two groups (four primiparous and four multiparous) and received the following dietary treatments in a 4×4 Latin Square design: CLA0: 45 g/day of calcium salts of fatty acids (CSFA); CLA15; 30 g/day of CSFA+15 g/day of CLA-ME; CLA30: 15 g/day of CSFA+30 g/day of CLA-ME; and CLA45: 45 g/day of CLA-ME. The CLA-ME supplement (Luta-CLA 60) contained 29.9% of t10,c12-CLA; therefore, the dietary treatments provided 0, 4.48, 8.97 and 13.45 g/day of t10,c12-CLA, respectively. Feed intake, milk production, concentration and secretion of milk protein and lactose, body condition score and body weight were unaffected by the dietary treatments. Milk fat secretion was reduced by 14.9%, 30.8% and 40.5%, whereas milk fat concentration was decreased by 17.2%, 33.1% and 40.7% in response to CLA15, CLA30 and CLA45, respectively. Secretions of both de novo synthesized and preformed fatty acids were progressively reduced as the CLA dose increased, but the magnitude of the inhibition was greater for the former. There was a linear reduction in most milk fat desaturase indexes (14:1/14:0, 16:1/16:0, 17:1/17:0 and 18:1/18:0). Milk fat t10,c12-CLA concentration and secretion increased with the CLA dose, and its apparent transfer efficiency from diet to milk was 1.18%, 1.17% and 1.21% for CLA15, CLA30 and CLA45 treatments, respectively. The estimated energy balance was linearly improved in goats fed CLA.     

The secondary antibody response induced in tissue cultures

The conditions for induction of memory cells (B-MC) and evocation of the secondary antibody (Ab) response in tissue cultures (TC) were estimated.(1) In vivo primed B-MC cells were isolated 6–150 d after priming and stimulated in TC with different doses of sheep red blood cell (SRBC) antigen. The Ab response has a strict time and dose dependence: only small doses (10⁵) evoke a secondary response, high doses (10⁸, 10⁹) a state of immediate tolerance. (2) Antigen added to TC directly with B-MC rescued their Ab production for a long period. Addition of the antigen 1 or 2 d after setting the TC, follows the Ab-response decay, comparable with virgin cells (B-ICC). (3) Primed B-MC stimulated in TC responded preferentially with an IgM secondary response; the same cell suspension adoptively transferred into isologous recipients switched into IgG cells. (4) Virgin, immunocompetent, B-ICC were primarily stimulated in TC with a small dose of antigen (10⁵ SRBC); after 7 d of cultivation the cells were transferred into isologous recipients, SCID mice and into TC. In all cases, the secondary response of IgM was determined, 10 times higher than in the primary controls.     

Isolation of Citreoviridin from Penicillium charlesii Cultures and Molded Pecan Fragments

The extraction and systematic fractionation of Penicillium charlesii Smith cultures and contaminated pecan fragments yielded the yellow mycotoxin citreoviridin. Citreoviridin proved acutely toxic to 1-day-old chickens, with an oral 50% lethal dose of 37.5 mg/kg, and showed plant growth inhibition in wheat coleoptiles even at concentrations as low as 10⁻⁴ M. It was toxic to corn seedlings but did not affect young tobacco seedlings.     

Allogeneic T-cytotoxic reactivity of senescent mice: affinity for target cells and determination of cell number

Experiments were performed to determine the cause(s) of the reduced T-cytotoxic-cell response observed in senescent mice. The cytotoxic cells studied developed in mixed lymphocyte cultures (MLC) of 6 × 10⁶ C57B1/6J (H-2^b) spleen cells from mice of various ages which were stimulated by doses of irradiated Balb/c (H-2^d) cells giving responder to stimulator ratios of 10:10 and 10:1. The cytotoxic response, as determined in a ⁵¹Cr-release assay against P815 (H-2^d) mastocytoma cells in culture, declines with age. This age-related decline is more pronounced with the lower dose of stimulator cells (10:1). The cytotoxic response developing in 10:10 and 10:1 MLC of spleen cells from young mice is comparable in magnitude, whereas the lower dose (10:1) is much less stimulatory in cultures of spleen cells from mice above 12 months of age. In order to better understand this age-dependent decline in cytotoxic response, the affinity of effector cells to their target and the percentage of cytotoxic cells which develop in the cultures of spleen cells from mice of various ages were determined. The affinity of cytotoxic cells developing in 10:10 MLC does not change with age. The affinity of cytotoxic cells developing in 10:1 MLC from young mice is significantly higher than the affinity of those developing in 10:10 MLC. This dose-dependent increase in affinity is not apparent in 20-month-old mice, which show equal affinity of cytotoxic cells in 10:10 and 10:1 MLC. The percentage of cytotoxic cells in the cultures was found to decrease with age. This decrease was more pronounced after 10:1 stimulation. Thus the decline with age in cytotoxic response can be attributed to a decrease in number of functional cytotoxic cells developing in MLC cultures, regardless of stimulator cell dose and a decrease in affinity for target cells at low stimulator cell doses.     

The effect of T-2 toxin on bone microstructure in rabbits

T-2 toxin is the most toxic of the trichothecene mycotoxins. Its effect on bone microstructure is still unknown. This study focuses on acute effects of the T-2 toxin on compact and trabecular bone tissues structure of rabbits after a single intramuscular administration. Experimental E group (n?=?4) consisted of animals which were intramuscularly injected with T-2 toxin at dose 0.08 mg.kg^?1 body weight 72 h before slaughter. Group C (n?=?4) without T-2 toxin application served as a control. An absence of primary vascular longitudinal bone tissue near endosteal surfaces, its deposition on periosteal surfaces and a lower density of secondary osteons in the middle part of the substantia compacta were observed in both females and males injected with T-2 toxin. On the contrary, morphometrical analysis of the compact bone showed no demonstrable alternations in the sizes of primary osteons’ vascular canals, Haversian canals or secondary osteons between rabbits from E and C groups. Also, no significant effects of the T-2 toxin on trabecular bone morphometry and cortical bone thickness were observed between rabbits of either sex. The single intramuscular application of T-2 toxin at the dose used in our study affects only qualitative histological characteristics of the compact bone in rabbits.     

Efficacy of recombinant anthrax vaccine against Bacillus anthracis aerosol spore challenge: Preclinical evaluation in rabbits and Rhesus monkeys

This report describes the immunogenicity and protective efficacy of Escherichia coli-expressed recombinant protective antigen (rPA) in New Zealand White rabbits and Rhesus Macaques against an aerosol challenge with Bacillus anthracis spores (IVRI strain, tox+cap+). A dose-ranging study was performed in which it became evident that the level of anti-PA IgG and toxin-neutralizing antibody titer was directly proportional to the dose of rPA administered. However, the onset time of primary and secondary immune response was not dependent on the dosage. Revaccination of primed animals with the same threshold dose yielded a robust and rapid secondary response. Quantitative differences in peak titers were obtained for both the animal models, in addition to qualitative differences in the immune kinetics. In spite of a weak priming response, the secondary response in rabbits peaked earlier than that in macaques once the booster dose was administered. However, evaluation of the post-challenge quantitative anti-rPA ELISA titer measurements indicated higher titers for non-human primates as compared to the lagomorphs. Importantly, 100% protection was seen for the dosage groups that received ≥25 μg rPA, following a challenge against a target dose of 1000 LD₅₀ of aerosolized spores of Bacillus anthracis.     

Immune response in dog 2 effect of dose of antigen on antibody production.

The primary antibody response of dogs and rabbits to both 'H' and 'O' antigens of Salmonella typhosa following intravenous injection with a formalin killed vaccine from 2.4 x 10(6) to 2.4 x 10(10) organisms/kg body weight was analysed. The animals were restimulated 80 days later with various vaccine concentrations. The lgM anti-'O' and lgG anti-'H' and 'O' antigens in the dogs, were significantly weaker in both primary and secondary response than the comparable rabbit group. Primary lgM anti-'H' response in the dog was found to be greater, equal, or less than that observed in the rabbit. A closer analysis of the primary response indicated that both animal species show the same latent period and doubling time in respect of anti-'H', and the differences observed are probably the result of the number of progenitor cells stimulated by the antigen. On the other hand the suppressed response of the dog to 'O'-antigen is the result of an overall weaker response of this animal to the antigen. The secondary anti-'H' lgM response was found to be greater than, equal to, or less than the primary response in the same animal. The significant inhibition of this response was observed in those animals which received a high primary dose of antigen.     

The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10⁴ and 10⁶ pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.     

Occurrence of an oxalate oxidase in Sorghum leaves

An oxalate oxidase found in the 15 000 g supernatant of 10-day-old sorghum leaves exhibited a pH optimum of 5 and a temperature optimum of 45° and was unaffected by Na⁺. The enzyme activity remained linear up to 10 min and the apparent K_m for oxalate was 2.4 × 10^?5 M. The enzyme activity was strongly inhibited by sodium dithionite and α,α′-dipyridyl. Inhibition by the latter was specifically reversed by Fe²⁺. The activity of the dialysed enzyme was restored by the addition of Fe²⁺ and FAD. Inhibition of the enzyme by iodoacetate, p-chloromercuribenzoate and N-methylmaleimide revealed that SH groups at the active site are essential.     

γ-Aminobutyric acid-stimulated chloride permeability in crayfish muscle

γ-Aminobutyric acid selectively increased Cl^? permeability in isolated strips of crayfish abdominal muscle. Muscle fibers incubated in VAn Harreveld's solution at room temperature took up ³⁶Cl^? to the extent of 700 ml/kg wet weight with a halftime of 2.5 min. During 15-s incubations, the control ³⁶Cl^? uptake space was 131 ± 4 ml/kg (n = 60) and this was significantly increased by γ-aminobutyric acid at 200 μM or higher concentrations to 177 ± 4 ml/kg (n = 48, P < 0.05). This effect was specific for chloride since γ-aminobutyric acid did not increase the uptake by crayfish muscle of radioactive sucrose, inositol, or propionate. γ-Aminobutyric acid stimulation of ³⁶Cl^? uptake is mediated by receptor-ionophore function since the process shows pharmacological properties virtually identical to those observed by electrophysiological techniques. The γ-aminobutyric acid stimulation of Cl^? permeability is dose dependent with 50% of the maximal effect at 40 μM γ-aminobutyric acid and the dose vs. response curve is somewhat sigmoid. The γ-aminobutyric acid agonist muscimol causes the same maximal effect on Cl^? uptake as γ-aminobutyric acid, but acts at 5-fold lower concentrations, i.e. is more potent. However, the partial agonist γ-amino, β-hydroxybutyric acid produced little or no stimulation of ³⁶Cl^? flux. The response to γ-aminobutyric acid was blocked by 2 mM β-guanidinopropionate or γ-guanidinobutyrate, 0.5 mM bicuculline, and 10 μM picrotoxinin. Picrotoxinin inhibition was dose dependent with 50% inhibition occurring at 4 μM. Antagonists did not affect control ³⁶Cl^? uptake. These results confirm electrophysiological observations that the postsynaptic response to the inhibitory neurotransmitter γ-aminobutyric acid involves a rapid increase in membrane permeability to Cl^?     

The number of chlorophyll-less spots and the primary leaf area as criteria of radiation damage in soybean (Glycine max merill)

The number of chlorophyll-less spots occurring on the primary leaves as well as the primary leaf size were investigated in two soybean cultivars, differing genetically in radiosensitivity, after irradiation of seed with ⁶⁰Co γ-rays. A high correlation was found between increasing number of spots, decreasing leaf size and seedling growth inhibition.The number of spots can be used to monitor radiation effects over the small dose range where the growth inhibition is not pronounced. Primary leaf size can be used as a convenient criterion of seedling growth inhibition. Possible causes of leaf spotting are discussed.