Regulation of antibody response in vitro. X. Biphasic effect of cyclic AMP on the secondary anti-hapten antibody response to anti-immunoglobulin and enhancing soluble factor.

The effect of elevation of an intracellular cyclic AMP level on in vitro anti-hapten antibody response was studied, by using mesenteric lymph node cells of rabbits which were primed with dinitrophenylated Ascaris antigen (DNP-Asc) or DNP-ragweed antigen (DNP-Rag). The anti-hapten antibody response was induced by stimulation of the primed B cells by either DNP-heterologous carrier conjugate or anti-immunoglobulin (anti-Ig) for 24 hr (first stage), followed by 6-day culture of the activated cells in the presence of nonspecific enhancing factor (second stage). The stimulation with anti-Ig induced IgG anti-hapten antibody response and enhanced the formation of total IgG. Addition of dibutyryl cyclic AMP or aminophylline with anti-Ig or DNP-heterologous carrier during the first stage enhanced IgG anti-hapten antibody response. The optimal concentration of these reagents for the enhancement was 5 x 10(-4) M to 10(-3) M. The presence of 5 x 10(-6) M prostaglandin E1 during the first stage also enhanced the antibody response. Similarly, the presence of dibutyryl cyclic AMP or aminophylline during the stimulation of DNP-Rag-primed cells with homologous antigen (first stage) enhanced the antibody response. If the same concentration of dibutyryl cyclic AMP or aminophylline was added together with enhancing soluble factor during the second stage after the stimulation of the primed cells with anti-Ig, both the antibody response and the formation of IgG were suppressed. The antibody response of DNP-Rag-primed cells stimulated with homologous antigen was also suppressed if dibutyryl cyclic AMP or aminophylline was added during the subsequent culture (second stage). Evidence was obtained that suppression of antibody response by cyclic AMP during the second stage is probably due to inhibition of the proliferation of B cells. Neither of these drugs suppressed the formation of enhancing soluble factor from the carrier-specific cells stimulated with the homologous carrier. The results obtained in the present experiments suggested that stimulation of hapten-primed B cells with anti-gamma chain in the presence of an optimal concentration of dibutyryl cyclic AMP resulted in the formation of a significant amount of IgG anti-DNP antibody without participation of T cells.     

Immunization of rabbits with a midgut extract of the human body louse Pediculus humanus humanus: the effect of induced resistance on the louse population

Abstract. Resistance to human body lice, Pediculus humanus humanus L, induced by feeding on rabbits immunized with an extract of louse gut was studied. The mortality of lice fed on immunized rabbits was 73%, significantly higher than that of lice fed on control rabbits (52%) ( P < 0.01). The proportion of dead nymphs and female lice with ruptured guts was significantly higher in lice fed on immunized rabbits (P<0.01). The size of the bloodmeal was 35% greater in female lice fed on control rabbits than on immunized rabbits. Lice fed on immunized rabbits laid 40% less eggs than those fed on the controls, they also demonstrated a significant decrease in the number of eggs per female over time ( P < 0.01). 86% of the eggs laid by lice fed on immunized animals hatched, compared with 92% hatching of eggs laid by the lice fed on control animals (P< 0.01). With the exception of the first bloodmeal the percentage of hatched eggs which were laid between any two bloodmeals was significantly smaller (p<0.01) in the lice fed on immunized rabbits than in the control group. The first nymphal stage of lice fed on immunized rabbits took an average of 5.2 days to moult to the second stage, compared with 4 days for those fed on control rabbits.     

Phagocytic response to fully controlled plural stimulation of antigens on macrophage using on-chip microcultivation system

To understand the control mechanism of innate immune response in macrophages, a series of phagocytic responses to plural stimulation of antigens on identical cells was observed. Two zymosan particles, which were used as antigens, were put on different surfaces of a macrophage using optical tweezers in an on-chip single-cell cultivation system, which maintains isolated conditions of each macrophage during their cultivation. When the two zymosan particles were attached to the macrophage simultaneously, the macrophage responded and phagocytosed both of the antigens simultaneously. In contrast, when the second antigen was attached to the surface after the first phagocytosis had started, the macrophage did not respond to the second stimulation during the first phagocytosis; the second phagocytosis started only after the first process had finished. These results indicate that (i) phagocytosis in a macrophage is not an independent process when there are plural stimulations; (ii) the response of the macrophage to the second stimulation is related to the time" delay from the first stimulation. Stimulations that occur at short time intervals resulted in simultaneous phagocytosis, while a second stimulation that is delayed long enough might be neglected until the completion of the first phagocytic process.     

Effect of tolerance and of antibody mediated immune suppression on the avidity of the cellular and humoral immune response

Partial tolerance induction in adult rabbits although resulting in a marked depression of circulating antibody concentration, had no effect on either the avidity of the antibody synthesized at 2 weeks after immunization or the magnitude of the response of lymph node cells to stimulation by antigen in culture. A modest depression in the avidity of the cells responding to antigen in culture by an increase in thymidine incorporation was observed. Partial antibody mediated immune suppression was found to result in an increase in avidity of the residual circulating antibody and had no effect on the magnitude of the proliferative response induced by antigen in culture. Thus suppression appears to affect predominantly B-lymphocytes.     

Frequency potentiation in the neonatal rabbit hippocampus

The formation of properties of frequency potentiation in the entorhinal afferent pathway of the hippocampus was studied in unanesthetized rabbits aged from 1 to 15 days. In areas CA₁ and CA₃ of the dorsal hippocampus in newborn rabbits repetitive (1–20 Hz) electrical stimulation of the perforant path led to an increase in amplitude of the slow wave of the field potential by 20–100% compared with the control and to an increase in the probability of response discharges from the neurons from 0–0.5 in the control to 0.8–1.0 during tetanization. In rabbits aged 2–3 days potentiation was more marked at a frequency of 4–6 Hz, whereas depression of the responses developed rapidly to a higher frequency of stimulation. The frequency optimum of 4–15 Hz was established on the 5th day. Potentiation of the first component of the field potential was observed starting from the 8th–10th day of life. The experimental results show that the property of frequency potentiation in the cortical afferent connections of the hippocampus is found in rabbits actually at birth, and it acquires the adult form at the beginning of the second week of life.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 533–539, November–December, 1979.     

Changes in immunity to Ixodes ricinus by rabbits infested at different levels

Abstract. Two groups of rabbits were infested twice with different numbers of Ixodes ricinus adults: one group (high infestation) with twenty-five females and twenty-five males and the other group (low infestation) with five pairs. A third infestation was performed in both groups with fifteen adult pairs. Tick biology was monitored for resistance effects. At the second infestation, the feeding and the egg production were more perturbed in ticks fed on high infestation rabbits. The embryogenesis was only affected in ticks from high infestation rabbits. At the third infestation, resistance was increased only in low infestation rabbits, which became more resistant than high infestation rabbits. The blastogenic response of peripheral blood lymphocytes and antibody production against ticks were assessed. A salivary gland extract and an integumental antigen from Lricinus adult females were able to initiate lymphocyte proliferation. The response was significantly higher in high infestation rabbits, especially at the end of the second infestation, and higher in low infestation rabbits during the third infestation. Non-specific proliferation with concanavalin A was temporarily decreased in both rabbits groups during the first and the second infestations. Specific antibody response to salivary and integument antigens were always the highest in high infestation rabbits. The involvement of tick-induced immunosuppression is discussed.     

Influence of antigen stimulation on the oxidative stress parameters in outbred and inbred rabbits

The influence of antigen stimulation on the oxidative stress parameters in two groups of rabbits-inbred and outbred were explored by evaluation of the level of lipid peroxidation products (MDA) in the plasma membrane, and the activity of erythrocyte antioxidant defense enzymes superoxide dismutase (SOD) and catalase (CAT). There was not a significant difference between levels of MDA in inbred and outbred rabbits before immunization. However, SOD activity in inbred rabbits was significantly increased in comparison with that of outbred (p = 0.006). Significantly higher plasma levels of lipid peroxidation products were detected in both inbred and outbred rabbits during immune response in comparison to the corresponding groups before immunization (p = 0.008 and p = 0.002). SOD and CAT activities in erythrocytes of rabbits during immune response were also significantly increased compared to that before immunization. In addition, during immune response SOD and CAT activities were found to be positively correlated to each other in both inbred and outbred rabbits (r = 0.727 and r = 0.916). In conclusion, our results suggest the presence of an increased oxidative stress during the antigen stimulation accompanied by an adaptive increase of SOD and CAT activities. 30 days after immunization, the plasma levels of MDA and the activities of SOD and CAT in erythrocytes decreased and reached values close to the controls.     

Secretion of very low density lipoproteins by cultured rabbit hepatocytes with hypercholesteremia, induced by purified cholesterol and containing autooxidation products

To evaluate the impact of oxidized derivatives of cholesterol on the development of hypercholesterolemia in rabbits, the secretion of very low density lipoprotein (VLDL) apoproteins and lipids was studied in cultures of hepatocytes obtained from: i) control rabbits, ii) rabbits fed on purified cholesterol (PCH), and, iii) rabbits fed on old commercial cholesterol (OCH) containing 5% of oxidized cholesterol derivatives. The rabbits fed on OCH for 6 weeks revealed a 5-fold increase in the serum cholesterol level compared with that in PCH-fed rabbits. The secretion of VLDL apoproteins and lipids by hepatocytes of two cholesterol-fed groups was similar, but was 2-3 times as high as that of cells from control rabbits. The cholesterol ester content in hepatocytes and the secretion of VLDL cholesterol esters by hepatocytes from OCH-fed rabbits was dramatically increased in comparison with hepatocytes from control and PCH-fed rabbits. These effects appear to be caused by the activation of cholesterol esterification by oxidized cholesterol derivatives. The rapid development of hypercholesterolemia in OCH-fed rabbits is at least partly associated with the stimulation of hepatic VLDL production.     

Modulation of the thalamocortical responses of the sensorimotor cortex during stimulation of the lateral hypothalamus in rabbits in ontogeny

It has been demonstrated that rhythmic stimulation of the relay thalamic nucleus in rabbits at the second half of the first month of postnatal life does not significantly affect the amplitude of the second and third positive components (PC-2 and PC-3) of the positive phase of the thalamocortical responses. Combination of rhythmic stimulation of this nucleus and that of lateral hypothalamic structures is accompanied by a significant increase of the amplitude of both positive components of the thalamocortical responses, the degree of potentiation of PC-3 being higher than that of PC-2. The described potentiation is most evident at the third week of postnatal development of rabbits.     

Characterization of antigen purified from type 3 strains of Pasteurella multocida and its use for an enzyme-linked immunosorbent assay

Surface antigens were purified from a type 3, 4 rabbit isolate of Pasteurella multocida designated as R11146. Two protein peaks were obtained by gel filtration with Sephadex G-200 from crude saline extract. Major antigenic activity was detected in the first peak. The first peak was absorbed onto DEAE-cellulose and eluted by a linear gradient of NaCl. Four fractions eluted from the column contained a single antigen which was identical to an antigen purified from another type 3 strain, P-1059. Also, they uniformly contained two protein species of molecular weights of 44,000 and 25,500. Six Pasteurella-free rabbits were infected intranasally with R11146 isolate and antibody response was determined by an enzyme-linked immunosorbent assay (ELISA) with the use of an antigen purified from P-1059 strain. Serum samples from the infected rabbits showed ELISA titers at the plateau stage by 21 or 28 days post-inoculation. Highest titers ranged from 1:15,000 to 1:16,000, while all the preinoculation sera had titers lower than 1:10. The high titers generally persisted for longer than 98 days after the infection. These results indicate that ELISA using a purified type 3 antigen is useful to detect P. multocida infection in rabbits by a type 3-related strain.     

感染流行性出血热病毒家兔病毒血症的研究

本文采用流行性出血热病毒114株实验感染家兔,用免疫荧光法及病毒培养技术研究了家兔病毒血症动态,发现感染后第6天,病毒抗原首先在淋巴细胞及单核细胞中出现;次日,亦可见于粒细胞中,第10—12天的抗原反应较强,第15天则明显减弱至消失。而在红细胞及血小板中始终未见明显的抗原反应。从感染后第3—13天的血浆中分离出病毒;感染后第6—15天,外周血单核细胞病毒分离阳性。结果表明,流行性出血热病毒在接种局部增殖后,侵入血液,并在白细胞中复制增殖,可能使病毒随血循环播散至全身其它组织脏器,造成因血传播引起的靶器官感染。     

In vitro proliferation of rabbit bone marrow-derived and thymus-derived lymphocytes in response to vaccinia virus

Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in vivo. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.     

The morphofunctional characteristics of the neurons in the sensorimotor cortex of old rabbits during the trace assimilation of rhythm]

Extracellular neuronal activity was recorded from 460 neurons from alert young (5-7 months), middle-aged (54-65 months) and old (66-85 months) rabbits. Trace rhythmic activity of sensorimotor cortical neurons was examined after long-lasting (10-20 min) rhythmic (0.5-2 Hz) electrocutaneous stimulation of the contralateral forelimb. Spectral analysis of spike activity showed age-related differences in capability of producing a rhythm of previous stimulation in spontaneous neuronal activity. In young animals propriate rhythmic fluctuations of firing rate appeared after the first or second sessions of stimulations (on the first experimental day), in middle-aged ones--after 2-4 sessions (on the second or third days); cortical neurons in old rabbits did not exhibit trace rhythmic activity. Significant morphological changes in glial and neuronal cells were observed in sensorimotor cortex of old rabbits. It is proposed that morphological deteriorations may be the reason of the impairement of trace processes during aging.     

Role of C3 in the regulation of a splenic PFC response in rabbits

The effects of in vivo C3 depletion on the immune response were examined in rabbits by assaying for splenic PFC after immunizing normal or cobra venom factor-treated animals with aggregated human gamma-globulin. The response to this T-dependent antigen has previously been shown to be regulated such that several cycles of PFC appear following a single intravenous injection of antigen. C3 depletion had no effect on the first peak of PFC (appearing 5 days after injection), but resulted in depression of the second peak of PFC (day 13). In rabbits depleted of C3, antigen localization in splenic germinal centers was markedly decreased. Delaying C3 depletion until after antigen localization had occurred resulted in no depression of the second peak of PFC. These results suggest that one mechanism by which C3 affects immune responses in vivo is via its role in influencing the persistence of antigen. In the absence of C3, no significant localization of antigen occurs, resulting in interference with the cyclical production of antibody.     

Raised excitability produced in cortical neurons by reinforced stimulation of the lateral hypothalamus

Response was recorded in the pyramidal tract (PT) under three experimental situations modelling the shaping of conditioned reflex (CR) during experiments on unrestrained but unanesthetized rabbits. The first paradigm consisted of direct stimulation of two points on the sensorimotor cortex, the second of the same stimuli combine with electrical stimulation (used as additional reinforcement) of the lateral hypothalamus (LH), and the third of LH stimulation in response to a rise occurring in PT response to above control level (modelling instrumental CR). An overall increase in the monosynaptic wave indicative of altered efficacy of synaptic connections was most commonly observed under all these conditions. Increase in the component directly reflecting pyramidal neuronal excitation appeared significantly more pronounced in the second and third than in the first experimental paradigm. The data obtained would point to reinforced efficacy of excitatory synaptic connections as the principal mechanism of CR, while the changed quality of electrical excitability at the membrane of cortical neurons reflects mechanisms underlying the contribution of reinforcement triggered by LH activation in cortical reordering of the motivational/emotional component of reinforcement.Higher Nervous Activity and Neurophysiology Research Institute, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 805–811, November–December, 1989.     

Effect of cold stimulation during slow-wave sleep on sleep cycle

To clarify the effect of cold stimulation during slow-wave sleep (SWS) on the sleep cycle, we conducted a sleep experiment. Five healthy males slept on a bedding system we developed to make the inside of bedding cooler. When the subject was sleeping deeply in the second and fourth SWS, the system cooled their bedding. When the subject's sleep condition shifted toward arousal, the cold air was stopped. As a result, all subjects’ sleep stage shifted to light sleep and reached arousal. After stopping stimulation, they immediately returned to the SWS at the first stimulation. But at the second stimulation, the sleep state did not return to the SWS.     

Comparison of the physiological and biochemical indices of the phagocytosis of ram erythrocytes by macro- and microphages]

Morita and Perkins' method was applied to the study of the stage of ingestion and destruction of an antigen (sheep erythrocytes) in the macrophages of peritoneal exudate of rabbits and rats and in the microphages of rabbit pleural exudate. Ingestion and intracellular destruction of the antigen were accompanied by intensified respiration and glycolysis of phagocytes. Respiration of the three types of phagocytes at two stages of phagocytosis and also the digestive capacity of microphages proved to be sensitive to cyanide and colchicine. The latter failed to influence the ingestion of the antigen by the three types of phagocytes and its digestion by macrophages. The differences in the metabolism routes of macro- and microphages in intracellular destruction of the antigen was postulated. An intensification of the phagocytic activity after the immunization was characteristic of rabbit and rat macrophages.     

The inflammatory reaction in the airways in an animal model of the late asthmatic response

The late asthmatic response is defined as airway obstruction that occurs hours after antigen exposure in some atopic asthmatics. The importance of this reaction is that the airway obstruction may be severe, prolonged, and difficult to control unless corticosteroids are employed. In addition, this response may lead to an increase in airway reactivity. To investigate the immunopathogenesis of this disorder, an animal model in rabbits was developed. In this model, antigen-specific IgE was associated with the late asthmatic response and antigen-specific IgG was associated with blunting of the reaction. Antigen challenge of immune rabbits led to edema within the large airways shortly after antigen exposure, with infiltration of inflammatory cells (neutrophils and eosinophils) into the large and small airways during the late response. The infiltrates became more mononuclear with time and resolved over 10 days. As in humans, the late response was associated with an increase in airway reactivity and correlated temporally with infiltration of the airways with neutrophils and eosinophils. The contribution of granulocytic cells to the airway responses to antigen was studied by granulocyte depletion, which prevented both the late response and the heightened airway reactivity. In addition, transfusion of a neutrophil-rich population of white cells into granulocytopenic immune rabbits restored both responses. Thus, in this animal model, the antigen-induced late asthmatic response and subsequent increase in airway reactivity were dependent on the presence of granulocytes at the time of exposure to antigen.