Dose dependency of the responses in draining lymph nodes and skin to repeated applications of oxazolone. A quantitative and histological study in mice.

The skin reactivity and lymph node responses to a large scale of different doses of oxazolone, were followed under continuous stimulation for a period of 12 days. It was found that the lack of a continuous high blast cell activity in the paracortex was a physiological response that could be observed with high dosage as well as with very low dosage. Low doses gave distinct stimulation of the paracortex without any detectable reaction in the cortex and in the medulla. Manifest stimulation of these compartments required considerably higher doses. A marked paracortical response was the only lymph node change required for development of a typical delayed type skin response. When germinal centres and plasma cells had developed, the skin reactivity was also found to change characteristically.     

Responses of the thymus and the paracortex of draining lymph nodes to repeated applications of oxazolone to mouse skin.

The oxazolone-induced response in the paracortex of draining lymph nodes is characterized by an early increase in the proliferative activity that decreases to control levels when stimulation is continued. The possibility that this may be a toxic side effect of the concentrated oxazolone solution used was investigated by simultaneous registration of the changes taking place in the thymus. These were found to be different from toxin-induced changes and compatible with cell loss due to massive emigration of cells. Repopulation of the thymus took place over the last 1 1/2 week of stimulation. It was concluded that the changes in the thymus as well as the decline of the proliferative activity in the paracortex, are most likely physiological responses. The most important factor in maintaining a high production of paracortical lymphocytes under chronic stimulation is the increase in the lymphocyte mass in the paracortex.     

Requirements for the adoptive transfer of antibody responses to a priming antigen in man

Antibody responses to recall vaccines can be adoptively transferred after marrow transplantation in man. Transfer of responses to priming Ag has not been successful, although this would broaden the range of organisms to which recipients could be protected. To investigate the importance of T cells and Ag in such transfer we primed marrow donors with keyhole limpet hemocyanin (KLH) 1 or 3 wk before marrow harvesting. B cells secreting IgM and IgG anti-KLH antibody were present in donor marrow at both 1 and 3 wk after immunization. After T cell depletion, donor marrow was infused into chemo-irradiated recipients, half of whom were immunized pretransplant with KLH. We found no evidence for the transfer of the IgM component of the response. Clonal expansion of the transferred IgG antibody-secreting cells with a corresponding rise in recipient serum IgG antibody levels was seen only when donors were primed 3 wk before marrow harvest and when the recipients were also immunized. IEF and immunoblotting demonstrated that successful transfer coincided with maturation of the IgG primary response from a polyclonal to an oligoclonal pattern and confirmed that donor oligoclonal bands appeared in the recipient serum. We conclude that the immunization protocols required for the transfer of antibody responses to priming Ag reflect the initial dependence of unprimed B cells on T cell help and on prolonged Ag stimulation. Ag-stimulated primary B cells in T cell-depleted marrow respond only to the noncognate growth and differentiation signals available in the chemo-irradiated recipient after an initial period of clonal selection and expansion in the donor which is both T cell and Ag dependent. Even after this initial selection, continued expansion of antibody-secreting clones in recipients retains an absolute dependence on Ag stimulation. Immunization techniques to protect transplant recipients against organisms such as Pseudomonas and CMV may need to be modified accordingly.     

Cellular composition of various structural zones of the iliac and mesenteric lymph node of pregnant rats

The dynamic of cellular reactions demonstrates certain changes in functional activity of all structures of the node during pregnancy. A similar trend of processes in the iliac (regional for the uterus) and mesenteric lymph nodes has been defined. At early stages of pregnancy, lymph nodule are the most active, this is demonstrated as an increasing portion of lymphoblasts, macrophages and dividing cells. During this period, cell composition of the cortical plateau is relatively stable. For the paracortical zone of the mesenteric lymph nodes a rather significant decrease in the portion of middle lymphocytes and reticular cells is characteristic. There is not any significant change in the relative amount of the cells in the same functional zone of the iliac lymph nodes during the same period of pregnancy. The medullar cords demonstrate an increasing number of blast forms and young plasmocytes. However, as the pregnancy develops, the structure of the paracortical zone undergoes an essential change--progressively increases the portion of lymphoblasts and large lymphocytes. The blastic reaction in the mesenteric lymph nodes is proved to depend, to some extent, on that in the iliac lymph nodes of the same animal. Mature plasma cells become the dominating cellular element in the medullary cords. At the end of the pregnancy a relative amount of the reticular cells increases in all structural zones of the node.     

Stimulation of regional lymphatic and blood flow by epicutaneous oxazolone.

The application of the epicutaneous antigen oxazolone results in persistent induration and erythema; however, the relative changes in lymph and blood flow in the inflammatory skin are largely unknown. To define the contribution of lymph and blood flow to the clinical appearance of cutaneous inflammation, we studied the sheep ear after the application of oxazolone. As a model for the study of these changes, the sheep ear had several experimental advantages: 1) a simplified superficial vascular network, 2) defined lymphatic drainage, and 3) an avascular and alymphatic cartilaginous barrier. Lymph flow was continuously monitored by cannulation of the prescapular efferent lymph duct. Blood flow, as reflected by cutaneous erythema, was noninvasively measured by use of a visible-spectrum spectrophotometer. The application of the epicutaneous oxazolone resulted in increased ear thickness for >7 days. The lymph flow from the oxazolone-stimulated ear peaked between 24 and 48 h after oxazolone stimulation. Spectrophotometric evaluation indicated that the cutaneous erythema peaked 72-96 h after application of oxazolone. Corrosion casting and scanning electron microscopy of the microcirculation at 96 h after antigen stimulation demonstrated significant dilatation of the superficial vascular network. These results suggest a biphasic response to oxazolone stimulation: 1) an early increase in vascular permeability associated with increased lymph flow and 2) a subsequent increase in relative blood flow associated with a dilated inflammatory microcirculation.     

Characteristics of the cellular reactions of the T- and B-dependent areas of the regional lymph nodes in dogs to the administration of immunodepressants

Mesenteric, bifurcational, axillary and popliteal lymph nodes have been studied in 22 healthy mature male dogs. Amount of blast cells, small lymphocytes, plasma cells and macrophages has been taken into account in the paracortical zone, in the germinative centers and in the medullary cords. For two weeks to one group of the animals every day imuran in turn with aurantin (10 mg/kg and 25 mg/kg) are injected, or antilymphocytic serum (ALS) intraperitoneally every other day (0.1 ml/kg). The combined injection of imuran and aurantin produces a more pronounced toxic effect to the hemopoietic organs than ALS. ALS is more specific for T-dependent zones of the lymph nodes. In the dose and interval mentioned ALS is an immunostimulating preparation for the immunocompetent cells of the germinative centers of the lymph nodes. The reaction of the lymph nodes depends on their regional belonging.     

Acceleratory Synapses on Pacemaker Neurons in the Heart Ganglion of a Stomatopod, Squilla oratoria

The pacemaker neurons of the heart ganglion are innervated from the CNS through two pairs of acceleratory nerves. The effect of acceleratory nerve stimulation was examined with intracellular electrodes from the pacemaker cells. The major effects on the pacemaker potential were an increase in the rate of rise of the spontaneous depolarization and in the duration of the plateau. The aftereffect of stimulation could last for minutes. No clear excitatory postsynaptic potential (EPSP) was observed, however. On high frequency stimulation, a small depolarizing response (the initial response) was sometimes observed, but the major postsynaptic event was the following slow depolarization, or the enhancement of the pacemaker potential (the late response). With hyperpolarization the initial response did not significantly change its amplitude, but the late response disappeared, showing that the latter has the property of the local response. The membrane conductance did not increase with acceleratory stimulation. The injection of depolarizing current increased the rate of rise of the spontaneous depolarization, but only slightly in comparison with acceleratory stimulation, and did not increase the burst duration. It is concluded that the acceleratory effect is not mediated by the EPSP but is due to a direct action of the transmitter on the pacemaker membrane.     

The synthesis of immunoglobulins by lymphoid cells in the sheep. The contribution made by the lymphoblasts to the total immunoglobulin and antibody production by lymphoid cells of the popliteal node during a secondary response to bacterial lipopolysaccharide.

The production of immunoglobulin (Ig) and antibody by lymphoid cells of sheep popliteal lymph node was studied in vivo and in vitro during a secondary response to lipopolysaccharide extracted from Salmonella muenchen. The maximum production of Ig in vitro occurred on the fourth and fifth days of the response and the proportion of the Ig expressing affinity for the antigen was also maximal at this time. By infusing [³H]leucine into the node via a chronic fistula, it was possible to assess the relative contributions made by the blast cells to the total Ig production by the node at various times in the response. It was found that there were three phases of Ig production. It is proposed that the blast cell may be the primary antibody-producing cell involved in providing protection to the animal against the initial antigenic assault. The role of the plasma cell would be one of maintaining levels of specific antibody in the blood and extravascular spaces for a period of time after the main antigenic challenge has passed. Alternatively, the plasma cell may simply be an end cell.     

Comparison of rice lines derived through anther culture and the pedigree method in relation to blast (Pyricularia grisea Sacc.) resistance

Crosses were made between Fanny (highly susceptible to blast) and 11 cultivars differing in blast resistance. Using the pedigree method (PM) segregating generations were evaluated and selected for blast resistance. Via anther culture (AC), doubled-haploids were obtained from F₁ plants and from F₂ blast-susceptible plants. Pedigree and anther culture-derived lines were planted together and evaluated for blast resistance under rainfed conditions at the Santa Rosa Experiment Station, Villavicencio, Colombia. The principal objective was to compare PM and AC in terms of their efficiency in producing rice lines resistant to blast. Results of a stratified analysis showed an association between method and blast resistance. Results of the logit-model analysis showed that AC produced a significantly (P=0.0001) higher proportion of lines with initial blast resistance (leaf- and neck-blast reaction 4) than did PM across all cross types. Stable blast resistance was assessed based on field performance over 3 years. AC was superior to PM in generating stable resistance for only some cross types. Consequently, with a few exceptions, AC can be used as effectively as PM to develop rice cultivars resistant to blast, with savings in time and labor. Additionally, blast-resistant lines were obtained either by the pedigree method or by anther culture from crosses between blast-susceptible cultivars (Fanny/CICA4 and Fanny/Colombial). This excludes somaclonal variation as a possible mechanism responsible for this resistance and suggests that a recombination of minor genes could have occurred and was fixed through either method. However, the stability of the resistance was greater in pedigree-derived lines. The implications of these findings for rice blast-resistance breeding are discussed.     

Characterization and Ca2+ Requirement of Histamine-Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50 = 3 x 10(-7) M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (greater than or equal to 10(-4) M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura-2-loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L-type voltage-sensitive Ca2+ channel, i.e., Bay K 8644 (1 microM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca(2+)-channel blockers (10 microM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]i rise. When the external Ca2+ level was elevated, both sustained [Ca2+]i rise and continuous secretion were enhanced in a similar Ca(2+)-dependent manner, showing saturation with around 1-3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L-type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of the O and P cell lines in the embryo of the leech. II. Genealogical relationship of descendant pattern elements in alternative developmental pathways

The p blast cells are a group of embryonic precursors found in the ectodermal cell layer of the leech germinal band. Each p blast cell normally undergoes the same invariant sequence of cell divisions and gives rise to a precisely defined set of uniquely identifiable neuronal and epidermal descendants in the mature leech. In the present paper, various of the p blast cell progeny were injected with a fluorescent lineage tracer in order to characterize the cellular composition of their descendant clones, and the results show that there is a stereotyped segregation of descendant cell fates through the first three p blast cell divisions. Previous work has shown that neurons and epidermal specializations which normally descend from the p blast cell will arise from a different precursor--the o blast cell--in response to ablation of the neighboring P cell line and that if the o blast cell is at a certain stage of differentiation when the ablation is performed it will produce only a subset of the normal P descendants. Comparison with the present findings indicates that under those conditions the o blast cell clone is not simply recapitulating a branch of the normal p blast cell lineage, but rather manifests an alternative lineage in which P descendants exhibit an abnormal genealogical relationship. Thus, even though normal leech development comprises a nearly invariant cell lineage, lineage relationships are open to considerable reorganization under experimental conditions.     

Development and function of B cells in monolayer islet cells of 3-week-old rat

Monolayer cultures of pancreatic B cells of 3-week-old rats were kept for 7 days in medium with 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose or for 4 days in medium with 5.5 mM glucose alone, following exposure for 3 days to a medium with 5.5 mM glucose plus 5 microM iodoacetic acid. Addition of the deoxyglucose or iodoacetic acid caused a selective deletion of fibroblasts, yielding large clusters that consisted mostly of islet cells. At the early stage of culture in medium with 16.7 mM glucose (day 4), the response of B cells to 16.7 mM glucose included only a small rise in insulin secreted during the first and second phases, and that to 10 mM of leucine and 2-ketoisocaproate was monophasic. After culturing for 7 days, these three secretagogues markedly stimulated insulin secretion by B cells cultured in both media, with a significant rise in secondary phase secretion. However, quantitative relationships differed. Thus, the response (total insulin secreted during a 30-min stimulation) of B cells in 2-deoxy-2-fluoroglucose to glucose was 155%, to leucine 185% and 2-ketoisocaproate 126% of that of cells exposed to iodoacetic acid. In conclusion, the present results suggest that B cells of 3-week-old rat may be immature, and that medium containing 2-deoxy-2-fluoroglucose is beneficial to continued maturation of the response in vitro.     

Correlation between keratinocyte expression of Ia and the intensity and duration of contact hypersensitivity responses in mice

Langerhans cells are the only cells within the epidermis that normally express immune response-associated antigens (referred to as Ia in mice and HLA-D in humans). However, in the epidermis of patients with allergic contact dermatitis or individuals undergoing a delayed-type hypersensitivity response, the keratinocytes at the reaction site are induced to express HLA-DR. In this study the inducible expression of Ia by the keratinocytes of mice was found to be directly correlated with the intensity and duration of experimentally induced contact hypersensitivity (CH) responses. During a CH response in animals that were sensitized on the belly and challenged on the ear with the contact-sensitizing (CS) agent oxazolone, the keratinocytes in the challenged, but not the unchallenged, ear were induced to express Ia. In comparison with animals that were sensitized and challenged at different sites, an intensified expression of Ia by the keratinocytes was associated with a twofold increase in ear swelling in mice that were sensitized and challenged with oxazolone at the same site. Curiously, the challenge of oxazolone-sensitized ears with dinitrofluorobenzene (an unrelated CS agent), croton oil (a nonspecific inflammatory agent), or acetone/olive oil (a noninflammatory agent) also induced both a marked keratinocyte expression of Ia and an enhanced CH response. These results suggest that residual antigen at the original sensitization site may be mobilized to function as the challenge stimulus to elicit a CH response, in association with keratinocyte expression of Ia, when CS-sensitized skin is perturbed with a nonspecific agent. Further evidence of an association between CH responsiveness and keratinocyte expression of Ia came from the following observations. First, the magnitude and duration of a CH response was markedly increased in pertussis toxin (PT)-treated mice. These enhanced responses were associated with intense Ia expression by the keratinocytes in the epidermis at the reaction site. Because PT is known to have an adjuvant effect on delayed-type hypersensitivity reactions, as well as to alter the normal regulatory mechanisms associated with this type of response, it is possible that Ia+ keratinocytes play a synergistic role in the enhanced CH responses that are observed in PT-treated animals. Second, a direct correlation between keratinocyte expression of Ia and CH responsiveness was observed in athymic nude mice that were challenged with oxazolone after receiving an adoptive transfer of lymphoid cells from oxazolone-primed normal syngeneic donors.(ABSTRACT TRUNCATED AT 400 WORDS)     

A study of lymphocyte subsets in human normal tonsil and lymph node

A series of T and B lymphocyte specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulation in frozen and paraffin tissue sections of human normal tonsil and lymph node by means of immunocytochemical technique. In the paracortical and interfollicular area of tonsil and lymph node, most lymphocytes reacted with Leu 1, Leu 3 a, Leu 4 and OKT4. The numbers of Leu 2 a and OKT8 positive cells were rare in tissue. These cells were not only limited in paracortical area, they also appeared in considerable numbers in medullary cords of lymph nodes. Leu 2 a and OKT 8 positive cells decreased with prominent follicular hyperplasia of tonsils. In addition, substantial leu 3 a and Leu 4 cells were found in the germinal centers. This finding supports the importance of these lymphocyte subsets in regulation of human immune response. In the mantle zone of secondary follicles, the majority of lymphocytes were positive for OKB 2 and BA 1, whereas, the IgM positive cells were predominately observed in the cytoplasma and extracellular substance of B lymphocytes in the germinal centers, but the lymphocytes bearing sIgM were rarely observed. In the mantle zone, the IgM were frequently found on the surface of membrane of small lymphocytes, however, the staining intensity was much than that in the germinal centers.     

Studies on the role of antigen-presenting cells in the systemic suppression of contact hypersensitivity by UVB radiation

Exposure of mice to UVB radiation produces a highly selective, systemic immunosuppression associated with the appearance of suppressor T lymphocytes. Suppression of delayed hypersensitivity to hapten-coupled syngeneic cells has been shown to result from an altered distribution of antigen-presenting cells. The purpose of this study was to determine whether an alteration in the activity of antigen-presenting cells could account for the systemic suppression of contact hypersensitivity (CHS) by UVB radiation. Fluorescein isothiocyanate (FITC) was used for contact sensitization because it uses different antigen-presenting cells than does oxazolone to induce CHS. Our previous studies demonstrated that CHS to oxazolone was suppressed by UVB irradiation. In these studies, we show that exposure of mice to UVB radiation before epicutaneous application of FITC onto unirradiated skin markedly decreased the CHS response to FITC painted on unexposed ears. Cyclophosphamide-sensitive suppressor T cells were detectable in the spleens of mice exhibiting decreased CHS. The antigen-presenting activity of cells in lymph nodes draining the site of epicutaneous sensitization (DLN cells) was assessed by injecting them into the hind footpads of syngeneic recipients and measuring the CHS response to FITC 6 days later. Viable DLN cells from UVB-irradiated, FITC-sensitized mice were equal to those from unirradiated, FITC-sensitized mice in their ability to induce CHS in normal recipients. No sensitization resulted when killed DLN cells were used for immunization, indicating that sensitization was not caused by reprocessing of antigen by host cells. We conclude that impairment of the CHS reaction in UVB-irradiated mice does not appear to be blocked at an initial step of antigen uptake, processing, or presentation, but must be impaired at some other step in the immunologic pathway.     

In vitro immune response by murine bone marrow cells stimulated against soluble immune complexes.

Synchronized nonadherent bone marrow lymphocytes were stimulated with soluble immune complexes, in antigen excess formed by C3H/HeJ antibodies and various noncross-reacting protein antigens, in a suspension culture which allowed longterm cultivation. On binding of these complexes, lymphocytes underwent blast transformation with mitosis and formation of plasma cells which secreted specific antibodies to the antigen; a cyclic sequence of lymphocytes, blasts, and plasma cells was observed until the majority of the cell population appeared to be plasma cells. The relative percentage of mature plasma cells then decreased leaving mostly small lymphoid cells among which evidence suggests the presence of memory cells. Complexes at equivalence stimulated for the first few days, whereas antibody excess caused stimulation only initially followed by inhibition of the response. Antibodies passively added to the cultures inhibited the proliferative reaction; free antigen induced a typical secondary-type response.     

Unitary responses of the caudate nucleus to direct stimulation

Unitary responses of the caudate nucleus to stimulation of various parts of it were investigated by extracellular recording. Latent periods of response discharges varied from 3.5 to 40 msec. Most neurons were excited by stimulation of the most rostral part of the head of the caudate nucleus. Irrespective of the site of stimulation, in most cases responses consisted of initial excitation in the form of one or, less frequently, two discharges followed by a period of depression of spontaneous activity. Recovery of activity took place gradually, without postinhibitory facilitation. No afterdischarges or periodic repetitions of spikes were observed after the initial response. Repetitive stimulation of the caudate nucleus showed that the neurons of this nucleus reproduce frequencies of stimulation badly above 30/sec, and under these circumstances in many cases they continued to discharge on average at a frequency of 5–15/sec. The results are examined from the standpoint of participation of the caudate nucleus in the formation of spindle activity.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 5, pp. 497–506, September–October, 1976.     

Nonspecific inhibitor of DNA synthesis elaborated by T acceptor cells. I. Specific hapten- and I-J-driven liberation of an inhibitor of cell proliferation by Lyt-1-2+ cyclophosphamide-sensitive T acceptor cells armed with a product of Lyt-1+2+-specific suppressor cells

Lyt-1+2+ hapten-specific T suppressor cells (Ts) from mice injected and then painted with picryl or oxazolone derivatives produce hapten-specific T suppressor factors (TsF) in vitro. Stimulation by painting with contact sensitizer (which need not be specific) gives rise to Lyt-1-2+, I-J+, cyclophosphamide-sensitive T acceptor cells (Tacc). When the Tacc population is armed with TsF and then is exposed to specific antigen in the context of I-J-controlled determinants (antigen-presenting, haptenized spleen cells and Ts sharing the same I-J subregion), a nonspecific inhibitor of DNA synthesis (nsINH) appears in the supernatant. This inhibitor suppresses the primary DNA synthetic response to concanavalin A, lipopolysaccharide, and alloantigens in both syngeneic and allogeneic lymphocytes. The nsINH is only effective when added to lymphocyte cultures less than 8 hr after the stimulation with concanavalin A. The nsINH, however, affects neither primary nor secondary cytotoxicity in vitro. These data suggest the mouse immune system is capable of selective regulation of the response to specific antigen by the production of nonspecific soluble suppressor factor(s).     

Modulation by zinc chloride of concanavalin A binding to rat thymocytes and early inhibition of lectin-induced blastogenesis.

Zinc chloride was shown to modulate the number and mobility of concanavalin A receptors of rat thymocytes at 4 °C. Zinc was able to induce the exposition at the cell surface of the same number of receptors that can be exposed by high doses of concanavalin A. It only weakly restricted their mobility. Moreover, zinc chloride was shown to inhibit rat thymocyte stimulation by succinyl-concanavalin A. This inhibition occurred most effectively if zinc was present during the first few hours after mitogen addition to cell cultures, as assessed by [³H]uridine incorporation in RNA and [³H]thymidine incorporation in DNA. The time at which DNA synthesis took place was not modified by zinc. Blast transformation in the presence of zinc was characterized by fewer blast cells which otherwise appeared identical to blast cells in control cultures. The possible action on membrane-cytoskeleton interactions is discussed.     

Contributions of memory B cells to secondary immune response

The secondary immune response is one of the most important features of immune systems. During the secondary immune response, the immune system can eliminate the antigen, which has been encountered by the individual during the primary invasion, more rapidly and efficiently. Both T and B memory cells contribute to the secondary response. In this paper, we only concentrate on the functions of memory B cells. We explore a model describing the memory contributed by the specific long-lived clone which is maintained by continued stimulation with a small amount of antigens sequestered on the surfaces of the follicular dendritic cells (FDC). The behavior of the secondary response provided by the model can be compared with experimental observations. The model shows that memory B cells indeed play an important role in the secondary response. It is found that a single memory cell in a long-lived clone may not be long-lived. In the present note, the influences of relevant parameters on the secondary response are also explored.