Single Priming Research Articles

Implicit memory and test awareness.

In three experiments we examined whether normal subjects can perform an implicit test without becoming aware that the test items were previously encountered in the study phase of the experiment. Experiment 1 assessed single word priming with the stem completion task, and subjects who reported awareness/unawareness that the test items were previously encoded in the study task showed equivalent priming. Experiments 2a-c and 3 assessed associative priming with the stem completion task, and in this case, only subjects who were aware that the test items were previously encountered showed associative priming effects. These findings suggest that single word priming and associative priming reflect different memory processes because the former and not the latter effect can be observed in unaware subjects.

Theta pattern stimulation and the induction of LTP: the sequence in which synapses are stimulated determines the degree to which they potentiate

Induction of long-term potentiation (LTP) by asynchronous stimulation of converging afferents was studied in hippocampal slices. Three stimulation electrodes were positioned to activate separate groups of Schaffer-commissural inputs to a population of CA1 pyramidal cells. Patterned stimulation consisted of a single coincident priming pulse to all 3 electrodes followed by a burst of 4 pulses (100 Hz) to the first input (S1) at a delay of 180 ms, to the second (S2) at a delay of 200 ms, and to the third (S3) at a delay of 220 ms. This pattern was repeated 10 times at 5-s intervals. The magnitude of LTP induced (measured 20 min after stimulation) was greatest for the first stimulated input, intermediate for the second, and least for the third. Intracellular recordings indicated that the greatest postsynaptic depolarization occurred during the period of S2 stimulation; thus the magnitude of LTP induced was not simply dependent on the degree of depolarization during afferent activation. Rather, sustained depolarization after synaptic activation could contribute to LTP induction by prolonging the activity of N-methyl- d-aspartate receptor-gated channels. Earlier-arriving bursts may also trigger an inhibitory process that reduces the effectiveness of later bursts for inducing LTP.

Haloperidol blocks reacquisition of operant running during extinction following a single priming trial with food reward

In the present experiment, we examined the anhedonic effects of the neuroleptic haloperidol in an alleyway reacquisition procedure consisting of four consecutive phases: acquisition, extinction, drug injection, and testing. After acquisition training to traverse a straight alleyway for 10 45-mg food pellets (single trial/day), food-deprived rats (/i=8/group) were tested without food reward until running times met an extinction criterion. Then, a single priming trial with food reward was conducted with four groups receiving 0.033, 0.10, or 0.30 mg/kg of haloperidol or vehicle (V+F) injections. A fifth group (V+E) received vehicle injections but no food reward. The test-day trial was given 24 h after the injection-day trial. The last three phases of the experiment were repeated. The results indicated that the food prime resulted in faster test-day running times for the V+F group; however, haloperidol attenuated this reacquisition effect. Since haloperidol is a relatively specific dopamine blocker, the results support the suggestion that brain dopamine systems are involved in reward.

Characteristics of hippocampal primed burst potentiation in vitro and in the awake rat

A pattern of electrical stimulation based on 2 prominent physiological features of the hippocampus, complex spike discharge and theta rhythm, was used to induce lasting increases in responses recorded in area CA1 of hippocampal slices maintained in vitro and from the hippocampus of behaving rats. This effect, termed primed burst (PB) potentiation, was elicited by as few as 3 stimuli delivered to the commissural/associational afferents to CA1. The patterns of stimulus presentation consisted of a single priming pulse followed either 140 or 170 msec later by a high-frequency burst of 2-10 pulses; control stimulation composed of unprimed high-frequency trains of up to 10 pulses had no enduring effect. Of all intervals tested, only 140 and 170 msec delays between the priming and burst stimuli were effective. PB potentiation could be induced both homo- and heterosynaptically. In the latter case, the priming pulse and burst stimuli were delivered to different dendritic fields; under these conditions, the PB effect was confined to the "burst" pathway. PB potentiation is not dependent on somal spiking; dendritic activation appears to be both necessary and sufficient for lasting changes to occur. Two findings indicate that PB potentiation and LTP have common mechanisms: (1) The effects of PB stimulation and LTP were not additive, in that saturation of the enhancement by PB stimulation eliminated any further increases in response with LTP stimulation; and (2) both PB potentiation and LTP were prevented if the N-methyl-D-aspartate antagonists 2-amino-5-phosphonovaleric acid or phencyclidine were added to the in vitro perfusion medium. Recordings from the hippocampus of awake rats demonstrated that PB potentiation of the CA1 population spike and slope of the EPSP are reliably induced under physiological conditions. This extensive characterization of PB stimulation provides novel information regarding the physiological and pharmacological basis of a possible role of endogenous rhythms in the processing and storage of information.

Re-irradiation of mouse skin: Similarity of dose reductions for healing and macrocolony endpoints

The amount of residual injury in mouse tail skin, assessed by the decrease in re-irradiation dose for equal effect, was similar whether assessed using healing or colony endpoints (17–21% after single priming doses). There were tendencies towards an increased sensitivity of the colony-forming cells by a factor of about 2. and less residual injury after multifractionated priming doses. These observations are compatible with a lower α β ratio characterising the response to dose fractionation for residual injury than for the acute healing response.

20] Improved oligonucleotide-directed mutagenesis using M13 vectors

This chapter discusses the improved oligonucleotide-directed mutagenesis using M 13 vectors. An oligonucleotide is synthesized, which is complementary to part of the DNA template but contains an internal mismatch to direct the required change (point mutation, multiple mutation, insertion, or deletion). The simplest approach to mutagenesis in MI3 is “single priming,” where the mutagenic primer is annealed with the single-stranded M13 template and extended with DNA polymerase I. Single-stranded DNA is then prepared from individual plaques and sequenced by the dideoxy method to verify the mutation. In constructing point mutations, often only the required mutation is obtained. However in constructing deletions, usually only a fraction of the hybridization positive clones carry the required mutation. The specificity of priming of the mutagenic oligonucleotide may be checked by using the oligonucleotide in a dideoxy sequencing reaction or in a primer-directed extension reaction followed by restriction endonuclease digestion. The E.coli (EcoK/ EcoB) selection system provides a convenient way of constructing a series of mutations at high yield.

Induction of hippocampal long-term potentiation using physiologically patterned stimulation

Lasting increases in the population spike recorded in area CA 1 of hippocampal slices may be evoked by the patterned presentation of as few as 5 stimulus pulses delivered to the commissural/associational afferents. The most effective pattern of stimulus presentation was a single priming pulse followed 170 ms later by 4 pulses at 100 Hz; control trains of 5 pulses at 100 Hz had no significant enduring effect. This pattern-dependent phenomenon, termed primed burst potentiation, is of lesser magnitude than is the long-term potentation induced by 100 Hz/l s stimulation, but appears to show a similar time course and duration. In addition, the two phenomena are not additive, suggesting that they may share a common mechanism.

A simple nonisotopic method for restriction mapping in single-stranded DNA cloning vectors based on taking timepoints during primed Klenow synthesis.

A fast, simple, and nonisotopic method for restriction mapping inserts in single-stranded cloning vectors (such as M13 or single-stranded plasmids) is presented. The procedure uses a commercially available oligonucleotide sequencing primer to initiate Klenow-mediated, unidirectional DNA synthesis along the single-stranded insert DNA. Aliquots taken at very short timepoints from this reaction are quick-frozen, heat-inactivated, and restriction-digested with the restriction enzyme or enzymes of interest. When the samples are run on an agarose gel and stained with ethidium bromide, the restriction bands appear in the order of their proximity to the priming site. The method's advantages are that it is fast, unidirectional and thus relatively unambiguous, requires neither isotope nor elaborate DNA handling or extraction procedures, and resolves the ambiguities due to "near doublets" that often plaque double-digest mapping and partial-digest mapping. Tetranucleotide restriction maps extending up to 5 kb can be determined from a single priming experiment; more infrequent hexanucleotide restriction sites can be mapped over longer distances. Also, a single aliquot taken at an early timepoint can be restriction-digested to establish the orientation of cloned inserts.

Efficient oligodeoxyribonucleotide-directed deletion mutagenesis using pEMBL vectors: Removal of early region introns from polyoma virus mutants

We have used oligodeoxyribonucleotide-directed deletion mutagenesis to remove early region introns from polyoma virus mutants. To this end we compared single priming, double priming, and gapped duplex approaches using either priming at 37°C or at the critical temperature. The gapped duplex approach, coupled with priming at the critical temperature, resulted in up to 70% yield of the desired product. In conjunction with the use of the pEMBL vector system this method was simplified to yield specific deletions from cloned large DNA fragments with high efficiency. The resulting mutant plasmids could be used directly for biological assays without retransformation or recloning. RNA and protein analyses showed that removal of the large T- or middle T-antigen introns from polyoma early region mutants dl23 and dl8 was specific and resulted in DNA competent for the synthesis of only one T antigen.

Reversibility of inotropic effects of ouabain on canine atrial and ventricular tissues

The experiments reported here were designed to quantitate reversibility of ouabain-induced inotropy in atrial and ventricular trabeculae of the dog heart. At 30 degrees C, 1 Hz, 2 mM Ca2+ and 5.9 mM K+, the positive inotropic effects, measured after a 1 h exposure to a single priming concentration of 0.5 microM ouabain, decreased after the removal of the drug with a half time (t1/2) of washout of 9.52 +/- 2.72 h in atrial and 7.64 +/- 1.58 h in ventricular trabeculae. When toxicity occurred it was of three types: transient toxicity, toxicity resembling a 'fast washout,' and severe toxicity characterized by profound and persistent negative inotropy and contracture. Use of the classical glycoside bioassay (Hatcher's digitalis titration) during and after washout, provided a semiquantitative estimation of ouabain remaining in the trabeculae; after a 4 h washout of the drug, the trabeculae were challenged by the addition of one-half of the initial concentration of ouabain (0.25 microM). Trabeculae, which had responded to ouabain with only positive inotropic effects and no toxicity, still contained substantial concentrations of ouabain. Trabeculae which reacted clearly with toxicity to the priming dose of ouabain, showed a significant loss of contractile force and development of contracture. The fastest ouabain washout we observed was 3.1 h.

Does a single priming injection of clenbuterol alter behavioral response to β-adrenoceptor agonists and antagonists in mice through a time-dependent process?

The present study investigated the extent to which a single priming injection of clenbuterol, a β-adrenoceptor agonist, could alter the behavioral response (locomotor activity recorded for 30 min with photocell counters) of mice to the subsequent administration of β-adrenoceptor agonists or antagonists. In mice pretreated with clenbuterol (0.25 mg/kg), the dose-response curve of clenbuterol (0.06 to 2 mg/kg) for reducing locomotor activity was shifted to the right with minimal alteration of the maximal response. The magnitude of the shift was dependent on both the priming dose of clenbuterol and the time interval between the priming and the test injections (maximum between 15 to 24 h, disappearance for intervals exceeding 72 h). The coadministration of propranolol (0.5; 2; 8 mg/kg) dose dependently antagonized the effects of the priming injection of clenbuterol. A previous injection of clenbuterol was also found to reduce the ability of another β-adrenoceptor stimulant, salbutamol (8 mg/kg), but not apomorphine, to reduce locomotor activity. Finally, previous β-adrenoceptor stimulation resulted in an enhanced ability of two β-adrenoceptor antagonists, propranolol (0.125 to 8 mg/kg) and penbutolol (0.5 to 32 mg/kg), to reduce locomotor activity in mice. All these data add to the evidence concerning the remarkable plasticity of central β-adrenoceptors and indicate that initial stimulation of a class of β-adrenoceptors may trigger a relatively important, time-dependent alteration in the behavioral reactivity to β-adrenoceptor agonists and antagonists.

Oxytocin antiserum delays onset of ovarian steroid-induced maternal behavior

We have previously reported that intracerebroventricular (ICV) administration of oxytocin (OXY) produces a significant increase in maternal behavior in ovariectomized (OVXed) rats given a single priming dose of estrogen. Arginine vasopressin (AVP) has a weaker and more delayed but significant facilitating effect on the onset of maternal behavior. Other investigators have demonstrated that prolonged treatment of OVXed nulliparous rats with estrogen and progesterone followed by withdrawal of progesterone shortens the latency of onset of maternal behavior. We hypothesized that ovarian steroids increase the onset of maternal behavior by a central mechanism involving OXY and possibly AVP. To test this nulliparous Sprague Dawley rats were given SC one Silastic capsule containing 4.4 mg of 17 β-estradiol eight days after OVX and three capsules each containing 40 mg of progesterone ten days after OVX. Progesterone capsules were removed on the 20th day after OVX, 24 hrs before the introduction of three rat pups (1–5 days old). One hr prior to introduction of pups animals received ICV 10 μl of anti-oxytocin antiserum (AOA), anti-arginine vasopressin antiserum (AVA), anti-neurotensin antiserum (ANA), normal rabbit serum (NRS), AOA + 250 ng OXY or no ICV infusion. Animals receiving AOA displayed significantly less maternal behavior compared to animals receiving NRS, ANA or no ICV infusion over the first two, six and 25 hrs of pup contact. OXY significantly reversed the inhibitory effect of AOA in the first two hrs of pup contact. AVA significantly decreased the onset of maternal behavior compared to ANA or no ICV infusion over the first six hrs of pup contact.

Reproductive Senescence in Female C57BL/6J Mice: Ovarian Impairments and Neuroendocrine Impairments that Are Partially Reversible and Delayable by Ovariectomy*

Ovarian and neuroendocrine impairments were examined before and after the age-correlated loss of estrous cycles in C57BL/6J female mice. The role of ovarian secretions in inducing neuroendocrine impairments before and after the loss of estrous cycles was also examined by determining neuroendocrine impairments after prolonged ovariectomy. Young (6-month-old) cycling, middle-aged (12-, 14-, and 16-month-old) cycling or acyclic, and old (18-month-old) acyclic mice were used. Ovarian impairments were assessed by grafting old ovaries into young hosts. Neuroendocrine impairments were assessed by grafting young ovaries into middle-aged and old hosts, ovariectomized either at grafting or 2 months before, and by inducing a LH surge in mice that had been ovariectomized for 4 days, 1 month, or 2 months. One group of 16-month-old and one group of 18-month-old mice were also ovariectomized at 6 months of age; these groups were used to examine the steroid-induced LH surge. The LH surge was induced by inserting sc a single priming estradiol (E2) implant, followed by two more (surge-inducing) implants 6 days later; 33 h after the second implantation, mice were decapitated (1800 h). The number of estrous cycles supported by middle-aged and old ovaries grafted into young hosts was reduced by more than 90% compared with that in young ovaries. The number of estrous cycles supported by middle-aged and old hosts given young ovarian grafts was reduced by 60% and 90%, respectively, compared with that in young hosts. Middle-aged and old hosts also had progressively longer estrous cycles than young hosts. Levels of the E2-induced LH surge in middle-aged and old mice were also reduced by 60% and 90%, respectively, compared with levels in young mice. In old acyclic mice, ovariectomy for 2 months partially reversed impairments in the LH surge and partially restored the ability to support cycles with young grafts; these functions, normally 90% impaired in old mice, were only 60% impaired after prolonged ovariectomy. Moreover, ovariectomy 2 months before grafting in old hosts resulted in shorter estrous cycles and 60% fewer pituitary adenomas. In middle-aged cycling mice, ovariectomy for 2 months did not affect impairments in the LH surge or in the ability to support estrous cycles with young grafts; these functions remained 60% impaired in middle-aged mice ovariectomized for 2 months. If mice were ovariectomized when young, the age-correlated impairments of the E2-induced LH surge at 16 months were largely prevented.(ABSTRACT TRUNCATED AT 400 WORDS)

The induction of thermotolerance in the ear of the mouse by fractionated hyperthermia

The development of thermotolerance in ears of mice was investigated after fractionated hyperthermia. Ears were heated at 43.5°C by immersion in a water bath and the response was measured in terms of the heating time required to cause thermal necrosis in 50% of the ears (NT 50). Three types of treatment were given: (1) single treatments, for which the NT 50 was 42 minutes; (2) priming treatments, which caused little visible effect but induced thermotolerance. These treatments were given as 1–10 daily fractions, the total heating time ranging from 20–630 minutes; (3) test treatments which were given at various times after priming and were varied to estimate the NT 50. Thermotolerance was defined as an increase in the test NT 50 for preheated ears relative to the single treatment NT 50. It has been suggested that thermotolerance induced by a single priming treatment may be increased by giving additional heat treatments which would not be tolerated by normal cells. In the mouse ear, the maximum thermotolerance induced by a single priming treatment of 20 min at 43.5°C was seen after 24 hr when the test NT 50 was about 2.5 times the single NT 50. The effect of giving up to nine additional daily treatments of 70 min, each of which would cause necrosis in ears that had not received prior hyperthermia, was measured. The maximum thermotolerance observed was equal to that after a single 20 minute priming treatment but thermotolerance decreased as the number of 70 min treatments was increased from four to nine. The effects of repeating a treatment (20 min or 5 min) which was tolerated by normal ears and induced maximal or less than maximal resistance were compared. The interval between each fraction (24 hr or 12 hr respectively) was equal to the time at which maximal thermotolerance was observed after one treatment. For each regimen, the degree of resistance seen after 2 to 10 exposures was similar to that after the appropriate single treatment. This resistance was maintained throughout the course of priming treatment and decayed after the last fraction. Thus for this regimen, thermotolerance depended on the duration of each treatment rather than on the number of treatments given.

Regulation of anti-hapten antibody responses in vivo: memory, carrier-specific Lyt-2+ T cells can terminate antibody production without altering the peak of response.

By using the classical hapten-carrier antigenic system (dinitrophenyl-keyhole limpet hemocyanin) (DNP-KLH), and positive immunoselective techniques for purification of lymphocyte subsets, we studied the kinetics and role of KLH-primed T cells in the termination of a secondary in vivo plaque-forming cell (PFC) response against DNP in the mouse. Separation of T cells by Lyt-2 phenotype and recombination of T cell subsets demonstrated that a single injection of KLH in complete Freund's adjuvant (CFA) concomitantly generated both helper and suppressor T cells in the spleens of primed mice. When these KLH-primed, purified T cells were transferred into irradiated recipients along with DNP-primed, purified B cells and DNP-KLH, helper activity was demonstrable first, with the early production of a high anti-DNP PFC response. Suppression of the PFC response was observed only after the response had fully developed; the PFC response was then "terminated" within a few days. Helper activity was obtained with the carrier-primed Lyt-2- T cell population, whereas suppression required the addition of Lyt-2+ T cells. Both activities were generated over a wide dose range, were specific for the priming antigen KLH, and could be detected as early as 8 days or as late as 9 mo after a single priming injection of KLH. Lyt-2+ T cells from unprimed donors or from animals primed with a different carrier did not have the capacity to terminate the response when added with the primed Lyt-2- helper T cells. Thus, we have now demonstrated the existence of carrier-specific, long-lived, "memory" suppressor T cells, which are initially generated in parallel with specific helper T cells. Whereas these memory suppressor T cells do not inhibit the induction of a secondary antibody response, they can effectively terminate it once it has developed, and may therefore account for the normal termination of antibody responses.

Hapten, carrier recognition and response by immunocytes of the primitive vertebrate, Notophthalmus viridescens

These experiments were designed to test whether newt ( Notophthalmus viridescens) immunocytes are able to recognize the hapten, 2,4,6-trinitrophenyl (TNP), separately from a heterologous erythrocyte (RBC) carrier. While a single priming injection with an erythrocyte species which is unrelated to the carrier of the TNP fails to stimulate amplification of the anti-TNP response, sequential (2×) priming provides about 50% as much amplification as a single priming injection of RBC homologous with the carrier. The anti-carrier response is not increased by sequential priming with the unrelated RBC. Moreover, sequential priming with RBC homologous with the carrier species initiates a suppression of the anti-hapten response without affecting anti-carrier activity levels. Thus the recognition and regulation of responses to a hapten and its carrier may be unlinked in this species.

Unilateral localization of hapten-specific B memory cells in lymph node draining a footpad injection of antigen.

In previous studies, rabbits were found to exhibit higher immunological memory responses in lymph nodes draining a footpad injection of antigen than in lymph nodes from the contralateral side1. This difference was demonstrated by the magnitude and quality of responses in vitro and in vivo at various intervals after a single priming injection1–3. In unpublished observations in our laboratory a difference between lymph nodes from the left (L) and right (R) sides could still be observed 2 years after priming. Several authors3,4 have also observed that draining lymph nodes (LNs) may give “spontaneous” antibody responses even without a second challenge with antigen in vitro. This was suggested to be due to persisting antigen, particularly since added antibody could block this response5. Recent studies in mice have re-emphasized a role for persisting antigen in mediating such a prolonged ability of lymph node cells to give responses in vitro or upon transfer without antigen challenge6.

The role of antigen form and function in the primary and secondary intestinal immune responses to cholera toxin and toxoid in rats.

This report describes studies of the mucosal antitoxic response in rats after enteric administration of several forms of cholera toxin or toxoid, proteins which differ primarily in their ability to bind to cell membranes and activate cellular adenyl cyclase. These two characteristics appeared to markedly enhance the local primary response to these antigens. A single dose of toxoid lacking these features was ineffective in local priming even though it was absorbed and induced a systemic immune response. Single dose mucosal priming occurred only with preparations which bind to cell membranes and was enhanced by those which also activate cellular adenyl cyclase. In contrast, single-dose mucosal boosting was best accomplished by materials with these properties but was also seen with a toxoid lacking both of these functions. The property of membrane binding appears to be most advantageous in mucosal priming, perhaps by increasing effective trapping of absorbed antigen in unprimed mucosal lymphoid tissue, whereas the ability to activate adenyl cyclase appears to enhance primary and secondary type responses about equally. Combinations of crude toxoid and toxin were also more effective in mucosal priming than purified materials, a finding which is unexplained. A single dose of this combination induced mucosal priming which was fully developed in 2 wk, undiminished after 4 too, and only modestly diminished after 8 mo, thus demonstrating relatively prolonged memory in the IgA mucosal immune system. Effective two-dose local immunizing regimens were developed, and it was shown that there was no correlation between the mucosal and systemic secondary antitoxin responses provoked by these regimens.

DNA sequencing by partial ribosubstitution

A new rapid method for DNA sequence analysis has been devised. In this method, base-specific cleavage is achieved at partially substituted ribonucleotides which are introduced by DNA polymerase extension in the presence of Mn2 +. Access to a target sequence and label incorporation are achieved by extending a restriction fragment primer with DNA polymerase I. After a short initial incorporation with [α- 32P]deoxynucleotide triphosphates to label the 5′ region of the target sequence, the triphosphates are removed and the reaction mixture is divided four ways for a second primed extension. The second extension is a cold chase in the presence of Mn 2+, all four deoxynucleotides and one of the four ribonucleotides under conditions that result in about 2% ribonucleotides substitution at each position. After cleavage at the restriction site and alkali cleavage at the positions of partial ribosubstitution, each reaction mixture is analysed by electrophoresis on a high-resolution denaturing acrylamide gel. As in the other rapid DNA sequencing methods the extent of DNA sequence that can be determined from a single experiment is limited only by the resolution of the analysing gels. At present some 100 nucleotides of sequence can be determined from a single priming reaction.

Two stages of B-cell memory development with different T-cell requirements.

We present evidence here for two stages in B-memory cell development, the first of which is T independent and the second T dependent. For these studies, we use a new type of T-deficient mouse (allotype suppressed) which specifically lacks T-helper activity (Th) for a subset of memory B cells responsible for approximately 10% of the overall IgG antibody response. We have shown elsewhere that these mice (SJL X BALB/c hybrids suppressed for Ig-1b) lack Th capable of helping Ig-1b memory cells, although they have normal Th activity for all other IgG memory B cells. This selective Th deficiency allows study of the effects of T depletion on memory development and avidity maturation of one population of B cells under conditions where the bulk of the immune response in the animal is proceeding normally, thus obviating environmental problems due to secondary effects of T depletion. With this sytem, we show that after a single priming dose of 2,4-dinitrophenyl-keyhole limpet hemocyanin, the memory B-cell pool in suppressed and nonsuppressed donors is indistinguishable with respect to magnitude and avidity of the response for all IgG antibodies produced, including Ig-1b antibody, despite the fact that expression of Ig-1b memory cells is prevented in intact Ig-1b-suppressed mice by the absence of Th capbale of cooperating with these memory cells. We have shown elsewhere that virtually all of the Ig-1b memory is carried by Ig-1b bearing cells. In contrast with the lack of suppressor T-cell effect on initial Ig-1b memory cell development, our data show that continued Ig-1b memory development is selectively impaired in suppressed mice. When primed mice are boosted repeatedly with the priming antigen, the average avidity of most of the IgG memory cells increases over 100-fold while there is no avidity increase in the Ig-1b component. To explain these data, we suggest that the development of high avidity memory occurs in two stages. The first stage, which occurs as a result of primary antigenic exposure, is the creation of a pool of IgG-bearing memory cells with a relatively low average avidity for the antigen. The appearance of these first stage memory cells does not require help from (post-thymic) Th, although Th are required for the expression of these memory cells (antibody production). The second stage of B-memory development requires both further antigenic stimulation and B-memory cell interaction with competent Th. This is a continuing process in which the number of memory cells in the pool remains relatively constant but the average avidity of these cells increases with continued antigenic exposure.