HMGB1促进类风湿性关节炎滑膜成纤维细胞表达基质金属蛋白酶-13

目的:探讨高迁移率族蛋白1(HMGB1)能否激活类风湿性关节炎滑膜成纤维细胞(RASF)表达基质金属蛋白酶-13(MMP-13)。方法:HMGB1与RASF共孵育后,采用流式细胞术、实时RT-PCR、ELISA分别检测细胞周期和MMP-13的基因表达量以及蛋白表达水平。结果:1μg/ml HMGB1刺激滑膜成纤维细胞3小时,MMP-13 mRNA的表达明显增加;HMGB1刺激滑膜成纤维细胞48小时后MMP-13蛋白分泌增高,并且滑膜成纤维细胞增殖周期速度加快。结论:HMGB1可以激活滑膜成纤维细胞,使其增殖周期速度加快,MMP-13表达增加,提示HMGB1通过作用于滑膜成纤维细胞表达MMP-13加强关节结构破坏。     

Activation of histamine H<Subscript>1</Subscript>-receptor enhances neurotrophic factor secretion from cultured astrocytes

Objective:Histamine stimulates nerve growth factor (NGF) secretion from cultured astrocytes. Histamine H₁-receptor antagonists completely block its effect. In the present study, we determined the involvement of histamine-receptor subtypes in this process.Materials and methods:Radioligand-binding assay was used to establish the presence of histamine H₁- and H₂-receptors on new-born rat cortical astrocytes in primary culture. Histamine H₁-, H₂- and H₃/H₄-receptor ligands, and highly selective protein kinase C (PKC) inhibitor were used to influence NGF secretion from cultured astrocytes. NGF, released into the culture medium, was measured by NGF-ELISA.Results:Histamine H₁-receptor agonists (histamine, selected histaprodifens) increased the secretion of NGF from cultured astrocytes in a concentration-dependent manner. H₁-receptor antagonists/inverse agonists (mepyramine, triprolidine) and PKC inhibitor completely blocked the effect of histamine. Histamine H₂- and H₃-receptor agonists did not enhance NGF secretion significantly. In addition, H₂- and H₃/H₄-receptor antagonists did not diminish histamine-stimulated NGF release.Conclusions:Our results indicate that histamine H₁-receptor and PKC are involved in the signal transduction pathway, responsible for histamine-stimulated NGF secretion from cultured astrocytes.Received 25 July 2003; returned for revision 4 November 2003; accepted by A. Falus 23 December 2003     

Effect of H1- and H2-receptor antagonists on the hemodynamic changes induced by the intravenous administration of ketamine in sevoflurane-anesthetized cats

Objective: The anesthetic ketamine has been reported to cause both an increase of the plasma histamine concentration, notably in cats, and a cardiovascular depression. The latter has been described in humans and in other species. However the relevance of the histamine fluctuation for the ketamine-induced hemodynamic changes has not been determined.Subjects and treatment: We studied the contribution of histamine to the hemodynamic effects induced by IV ketamine (7 mg/kg) in 12 sevoflurane anesthetized cats, of which half had been pre-treated with combined H₁- and H₂ -receptor antagonists.Methods: The mean arterial pressure (MAP) and the heart rate (HR) from both untreated (group C) and pre-treated (group AH) cats were recorded before and after the ketamine administration. The plasma histamine concentration was also measured.Results: Plasma histamine fluctuations in the control and the antihistamine-treated group followed a similar pattern (no statistical differences); an initial rise that peaked 2 min after ketamine injection (from 0.63 ± 0.11 ng/ml to 2.22 ± 0.69 ng/ml in the C group, and from 0.71 ± 0.10 ng/ml to 1.09 ± 0.28 ng/ml in the AH group) followed by an immediate decrease in plasma concentrations. As for the hemodynamic variables under analysis, in the control group ketamine administration was followed by an early 30.3 ± 8.1% reduction (p < 0.005) in the MAP with no associated changes in the HR. In the antihistamine pre-treated group, ketamine caused a further decrease of the MAP (41.7 ± 2.3%), and a significant (p < 0.01) 11.6 ± 2.9% reduction of the HR.Conclusion: Ketamine in anesthetized cats triggers histamine release and induces cardiovascular depression. The depression is more pronounced under the blockade of histamine activity through histamine receptor antagonists.Received 22 October 2004; returned for revision 5 January 2005; accepted by A. Falus 14 February 2005     

Presynaptic histamine H2 receptors modulate the sympathetic nerve transmission in the isolated rat vas deferens; no role for H3-receptors

The modulatory activity mediated by histamine receptors on the sympathetic nerve transmission was investigated in the rat vas deferens. Agonists and antagonists acting at the different histamine receptor subtypes (H₁, H₂ and H₃) were tested on electrically-driven preparationsin vitro. Low-frequency stimulation (0.1 Hz) evoked muscle contractions almost completelysustained by ATP release, while at high-frequency stimulation (5–10 Hz) norepinephrine was mainly involved. The H₁ receptor agonists, pyridilethylamine and 2-(2 aminoethyl)thiazole, enhanced the electrically evoked twitch responses, but not contractions induced by exogenously-applied norepinephrine and ATP. These effects were prevented by the H₁-blocking drugs, mepyramine and phenyramine, but only at high concentrations (10 mol/l). All these H₁-antagonists strongly enhanced muscle response to electrical stimulation. The H₂ receptor agonists, dimaprit, amthamine and impromidine, reduced the contractions evoked by field stimulation, but not by exogenously applied norepinephrine and ATP, the effect being antagonised by H₂-blocking drugs, ranitidine and famotidine. The H₃ receptor agonist,R()-methylhistamine, reduced the electrically evoked muscle contractions, the effect being not modified by the selective H₃-blocking drug, thioperamide, but prevented by famotidine. These data suggest that rat vas deferens contains presynaptic histamine H₂ receptors, able to mediate inhibitory effects on the sympathetic transmission, while histamine H₃ receptors are apparently not involved. On the contrary, the role of H₁ is still unclear, since both agonists and antagonists may have the same effects.     

Increased desensitization by picomolar phorbol ester of the endothelium-mediated effect of histamine in the perfused rat mesenteric bed

The vasodilatatory, endothelium-mediated, effect of histamine (H), through H₁ receptor, in the isolated and perfused mesenteric bed of the rat, undergoes strong desensitization during perfusion or repetitive injections of noradrenaline (NA) and H. The mesenteric bed completely desensitized to H is responsive to carbachol (C) and this latter compound does not affect the H desensitization. The homologous desensitization to C effect is very small, attaining less than 10% after 30 min of continuous perfusion. In this work the effect of inhibitors or activators of protein-kinase(s)-C (PKC) are studied during continuous perfusion of H or C in preparations preconstricted by NA. Staurosporine antagonizes the onset of the H desensitization, while the rate of desensitization in increased by phorbol-12-myristate-13-acetate (PMA). PMA, at a concentration from 10^–12 to 10^–10M, selectively enhances the homologous desensitization of H, while at 10^–8 M it also produces a desensitization to C. At least two different PKC isoenzymes might be involved in the desensitization of the vasodilatatory effect of H and C in the isolated and perfused rat mesenteric bed.accepted by W. Lorenz     

Pharmacological characterisation of cardiovascular histamine receptors in man in vivo

Summary Data from pharmacological studies carried out in healthy subjects using systemic histamine or impromidine and their antagonists are reviewed. Exogenous histamine by rapid injection appears to stimulate only H₁-receptors. Chlorpheniramine alone antagonised the responses to histamine.The effects of cardiovascular H₂-receptor stimulation are demonstrated best by a sustained and large dose of histamine given by infusion. If it be considered desirable to antagonise all the cardiovascular responses to endogenous histamine, the available pharmacological data in man suggest this would be achieved best by a combination of an H₁- and H₂-receptor antagonist.     

Dysrhythmias caused by histamine release in guinea pig and human hearts

Summary Histamine is released into the systemic circulation during anaphylaxis, by drugs and by surgical procedures. Studies in animal models have conclusively demonstrated that released cardiac histamine is a major mediator of arrhythmias that occur during anaphylaxis and following the administration of histamine-releasing drugs. Several lines of evidence suggest a similar arrhythmogenic role for cardiac histamine in humans: (1) The human heart is rich in histamine; (2) cardiac histamine can be readily released from human heartin vitro by therapeutic concentrations of drugs; (3) histamine has potent arrhythmogenic effects on the human heartin vitro.Arrhythmogenic effects of histamine include enhancement of normal automaticity, induction of abnormal automaticity, induction of triggered tachyarrhythmias, depression of atrioventricular conduction, and increase in the vulnerability of the ventricles to fibrillation. A combination of H₁ and H₂ antihistamines is needed to block the arrhythmogenic effects of histamine. Certain arrhythmogenic effects of histamine (e.g. induction of slow responses and delayed afterdepolarizations) can also be blocked by drugs which inhibit the influx of cations through slow channels. In contrast, the commonly-used drug digitalis potentiates the arrhythmogenic effects of histamine.We propose that histamine release produced by drugs and surgical procedures may be an overlooked factor in fatal cardiac arrhythmias. Experimental studies suggest that selective pharmacological methods can be developed to block the arrhythmogenic effects of histamine.Post-Doctoral Research Fellow supported by 1F32 HL 05536     

Effects of histamine on hippocampal pyramidal cells of the rat in vitro

Summary The actions of bath applied histamine on CA1 pyramidal cells were investigated in hippocampal slices of the rat. Histamine caused a) a slight depolarization but no significant change in resting membrane conductance; b) an abbreviation of long afterhyperpolarizations after single action potentials, bursts of action potentials or TTX resistant spikes; c) a loss of accommodation of firing. In the presence of TEA or barium, histamine prolonged and increased the size and number of the slow TTX resistant spikes. A depolarizing plateau which follows such spikes was also increased by histamine. Evoked synaptic potentials were unaffected by histamine, but the population spike was increased. The frequency of spontaneous chloride dependent potentials, which reflect interneurone firing, was also increased. These effects considerably outlasted histamine application and were mimicked by the H₂-agonist impromidine but not the H₁-agonist thiazolethylamine, and blocked by the H₂-antagonists cimetidine and metiamide but not the H₁-antagonists mepyramine or the beta-antagonist propranolol. It is concluded that histamine, by activating H₂-receptors, antagonizes a calcium mediated potassium conductance in hippocampal pyramidal cells without affecting calcium current. By this mechanism histaminergic afferent fibres could effectively regulate cortical responsiveness by selectively potentiating large excitatory inputs of target neurones.     

Effects of histamine on the circulatory system

Summary Experiments have been made in anaesthetised cats and dogs and in healthy, human volunteers to compare the changes in blood pressure and heart rate during systemic administration of histamine.Histamine, 1×10^–9 to 1×10^–7 mol/kg/min, lowered blood pressure in a similar dose-dependent fashion in all three species. In man and the cat this was accompanied by clear dose-dependent tachycardia whereas in the dog heart rate changes were minimal.Pharmacological analysis of the depressor responses to histamine in all three species and the reduction in total peripheral resistance in the cat and dog showed that the immediate responses to histamine in all three species involved H₁-receptors and that sustained responses involved H₂-receptors. Abolition of responses to histamine throughout infusions required H₁- and H₂-receptor blockade.Histamine antagonists, used in doses which cause abolition of cardiovascular responses to large doses of histamine, do not cause any significant change in the resting cardiovascular system.     

Reticuloendothelial system function and histamine release in shock and trauma: Relationship to microcirculation

Summary The material reviewed, and presented, here lends credence to the concept that the severity or course of the shock syndrome can be evaluated, quantitatively, at a tissue level by assessing RES phagocytic function. In general, the available data indicate that RE cell stimulants can adapt animals (and probably man) to the insults of circulatory shock and trauma; such substances could have important value in pretreating patients scheduled for massive surgery. The fact that a number of biologically active materials with vasotropic, and RE cell depressant, effects appear in the tissues and blood in shock, particularly when the organism becomes refractory to therapy, suggests that the final functional deterioration of the cardiovascular system may be due to the specific action of one or more of these biologically active materials; such a contender is, without doubt, histamine.Histamine has all the attributes of a typical shock-toxin. Evidence is presented that histamine can be a potent splanchnic (shock target-organ) arteriolar (microcirculatory) dilator even in physiologic (circulating) concentrations. Concentrations of histamine found in plasma of shocked animals and human subjects would produce extremely potent splanchnic vasodilator actions at the microcirculatory level. Evidence is also presented to indicate that microvessels can synthesize and release free, pharmacologically-active histamine.Endogenous release of histamine (e.g., with compound 48/80) produces dose-dependent and lethal shock-like anaphylactic actions; such release also produces, dose-dependently, RES phagocytic depression. Repeated administration of the histamine releaser, compound 48/80, results in almost a 400% enhancement of RES phagocytic function and cross-tolerance to lethal doses of whole-body trauma. Such results raise the possibility that the RES plays a pivotal role in the circulatory manifestations of compound 48/80 and anaphylactic-type (histamine release) shock syndromes.Evidence is presented to indicate that H₁-receptor antihistamines can ameliorate circulatory shock (and trauma) and prevent RES phagocytic depression, whereas H₂-receptor antihistamines do the reverse. Direct in situ microscopy revealed that the former types of histamine receptor blockers prevent tissue ischemia, whereas H₂-receptor blockers exacerbate tissue ischemia in circulatory shock. Histamineinduced vasodilatation via H₂-receptors may thus be a beneficial effect in circulatory shock and trauma; one must think seriously about the potential value of antihistamines as adjuvant drugs in the treatment of low-flow states and as preoperative medication.Collectively, the data reviewed herein could be taken as strong support for a pivotal role for the release (and possible synthesis) of free, pharmacologically-active histamine in shock.Supported by Research Grants H1 18002, HL 18015 and DA 02339 from the U.S.P.H.S.     

Anti-oedematous action of some H1-receptor antagonists

The oedema disk technique was used to study the effects of orally administered H₁-receptor antagonists (cetirizine, chloropyramine, clemastine, cyproheptadine, dimethindene, loratadine, mequitazine and terfenadine) on the inflammation induced with capsaicin or croton oil in the mouse ear, and the effect of topically applied dimethindene maleate gel on the inflammation induced with croton oil in the mouse ear.In rats of the Wistar strain, oedema was induced in the hind paw by the subplantar injection of dextran or compound 48/80. Preliminary antihistamine treatment inhibited the development of oedema in the mouse ear, and of oedema in the rat paw, to statistically significant extents, in a dose-dependent manner. In all experiments, the most potent drugs, were loratadine and cyproheptadine.     

Association between matrix metalloproteinase-1 (MMP-1) protein level and the risk of rheumatoid arthritis and osteoarthritis: a meta-analysis

Recent publications have investigated the potential role of the protein level of matrix metalloproteinase-1 (MMP-1) in the susceptibility to rheumatoid arthritis (RA) and osteoarthritis (OA). However, no unanimous conclusion was obtained. Therefore, we carried out a meta-analysis to explore the association between MMP-1 expression and these two clinical disorders. After database searching and screening, we enrolled a total of eighteen articles for the pooled analysis. We observed a significant association between RA cases and controls in the whole population [SMD (standard mean difference)=1.01, P=0.017]. There were similar positive results in the subgroup analysis of “population-based control” (SMD=1.50, P=0.032) and “synovial fluid” (SMD=1.32, P=0.049). In addition, we observed an increased risk in OA cases, compared with controls, in the overall analysis (SMD=0.47, P=0.004) and subsequent subgroup analysis of “knee OA” (SMD=0.86, P<0.001), “Asian/China” (SMD=0.76, P=0.003), “cartilage-Asian/China” (SMD=1.21, P<0.001), and “synovial fluid-Asian/China” (SMD=0.73, P=0.004). In summary, a high protein level of MMP-1 in synovial fluid may be associated with the susceptibility to RA, and the high MMP-1 level in the cartilage tissue or synovial fluid may be related to the pathogenesis of knee OA in the Chinese population. This should be confirmed by larger sample sizes.     

Contractile activity of human greater saphenous vein mediated by P2-receptors

In vitro experiments were carried out to measure the contractile responses to P₂-receptor agonists in the greater saphenous vein isolated from patients with obliterating vascular atherosclerosis and varicose veins of the legs. In patients with varicose veins, the contractile responses of the greater saphenous vein to ATP, ,-methylene-ATP, and UTP were significantly lower than in patients with obliterating atherosclerosis, while the responses to ADP, adenosine, and 2-methylthio-ATP were similar in both groups. These data attest to the presence of P₂-receptor-mediated contraction component in the greater saphenous vein, which are pronouncedly weakened during varicose disease.     

Inhibitory effect of triptolide on interleukin-18 and its receptor in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To determine the effects of triptolide (TP) on the expression of interleukin-18 (IL-18) and its receptor in phorbol 12-myristate 13-acetate (PMA)-stimulated rheumatoid arthritis synovial fibroblasts (RASF). MATERIALS AND METHODS: RASF were obtained from the synovial tissue of patients with RA. RASF were pretreated with TP (0~100 ng/ml) for 2 h before stimulation with PMA (50 ng/ml). The bioactivity of IL-18 in the supernatant was detected based on IFN-gamma secretion from IL-18-responding human myelomonocytic KG-1 cells. IL-18 level was analyzed by ELISA. In situ expression of IL-18Ralpha was determined by immunofluorescence assay. To estimate the protein and mRNA expression of IL-18 and IL-18Ralpha in RASF, western blot and quantitative RT-PCR were performed. Nuclear factor-kappaB (NF-kappaB) activity in the whole-cell extract of treated RASF was also measured using an ELISA-based method. RESULTS: TP effectively inhibited the bioactivity of IL-18 in PMA-stimulated RASF. The expression of IL-18 and IL-18R at protein and gene levels was reduced by TP. NF-kappaB activity in PMA-stimulated RASF was profoundly suppressed by TP. These effects showed a high correlation with TP concentration (0~100 ng/ml). CONCLUSION: TP effectively inhibited the expression of IL-18 and its receptor in PMA-stimulated RASF. These results suggest a mechanism of TP in RA therapy.     

Blockade of brain histamine metabolism alters methamphetamine-induced expression pattern of stereotypy in mice via histamine H1 receptors

The administration of methamphetamine (METH, 10 mg/kg, i.p.) to male ICR mice induced stereotyped behavior consisting of nail and/or wood chip biting (86.0%), continuous sniffing (12.0%), head bobbing (1.1%), and circling (1.0%) during the observation period of 1 h. Pretreatment of the mice with metoprine (2, 10, and 20 mg/kg, i.p.), a selective inhibitor of histamine N-methyltransferase (HMT), which metabolizes histamine in the brain, significantly increased and decreased METH-induced continuous sniffing (20.5, 51.3, and 80.3%) and nail and/or wood chip biting (77.4, 45.3, and 14.2%), respectively, in a dose-dependent manner. The hypothalamic contents of histamine and its metabolite N(tau)-methylhistamine were significantly increased and decreased by metoprine (10 mg/kg, i.p.), respectively. The metoprine action on METH-induced behavior was completely abolished by pyrilamine (10 and 20 mg/kg) and ketotifen (10 mg/kg), selective, centrally acting histamine H(1) receptor antagonists, but not by fexofenadine (20 mg/kg), zolantidine (10 mg/kg) and thioperamide (10 mg/kg), a peripherally acting histamine H(1) receptor antagonist and a selective, brain-penetrating antagonist for histamine H(2) and H(3) receptors, respectively. The metoprine action was mimicked by SKF 91488 (100 microg/animal, i.c.v.), another HMT inhibitor, and the action of SKF 91488 was also blocked by pyrilamine. The frequency of the expression of METH-induced total stereotypic patterns was unchanged after metoprine pretreatment. Mice pretreated with metoprine displayed no anxiety-like behavior in the elevated plus maze test. These results suggest that brain histamine, increased by agents such as metoprine and SKF 91488, binds to histamine H(1) receptors in the brain, resulting in the modulation of dopaminergic transmission associated with stereotyped behavioral patterns induced by METH.     

Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B₁ (AFB₁) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB₁ became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB₁ is potentially useful for studying mechanisms of chemically induced immortalization.     

Analysis of histamine and modeling of ligand-receptor interactions in the histamine H<Subscript>1</Subscript> receptor for Magic Angle Spinning NMR studies

Objective and Design: Investigation of the principles of ligand-receptor interaction in histamine receptors can help to provide a solid foundation for structure-based drug design. Stable isotope labelling of the ligand 'Histamine' has been performed and 1D ¹³C CP MAS and 2D Radio Frequency Dipolar Recoupling (RFDR) spectra for the ligand are presented. Hyperfine signals were well spread and did not suffer from any sizable line broadening. The production of H₁ receptor for Magic Angle Spinning NMR studies is currently in progress. Treatment: An agonist binding domain is proposed using homology modeling, database searches and mutagenesis data for the H₁ receptor. Methods: Homology modeling, Database searches for Expressed sequence Tag (ESTs), Magic Angle Spinning Nuclear Magnetic Resonance analysis of the ligand histamine. Results: The three-dimensional receptor model and mutagenesis studies suggest that the amine of the agonist histamine may form an ion pair with the TM III Asp, whereas the imidazole ring of histamine may associate with TM V Asp and Thr. Conclusions: Homology modeling studies confirms the absence of TM VIII in the H₁ receptor. According to the model the histamine in particular interacts with the transmembrane (TM) regions of the H₁ receptor structure, in particular TM helix III and V. This is in line with recent mutagenesis studies. Database search methods for ESTs have been used for electronic prediction of tissue distribution of H₁ receptor expression. The results indicate that the H₁ expression is highest in heart and skeletal muscle, which may be of importance for drug targeting.Received 3 June 2003; accepted by A. Falus 3 June 2003     

The histamine H4 receptor antagonist JNJ7777120 induces increases in the histamine content of the rat conjunctiva

Objectives:  Although the H₄ receptor localisation in the eye is unresolved, this study aimed to investigate the effects of the H₄ receptor antagonist JNJ7777120 in a model of experimental conjunctivitis. Methods:  JNJ7777120, at 0.005–1 mmol/l, was instilled into the lower conjunctival fornix of normal and compound 48/80 (C48/80)-challenged eyes of male Wistar rats, in the absence or presence of 40 mg/ml disodium cromoglycate (DSCG). Conjunctival histamine content was quantified 20 min post-challenge. Statistical analyses were performed by ANOVA. Results:  JNJ7777120 increased dose-dependently (r = 0.784, p < 0.001) the conjunctival histamine content. In the C48/80-challenged eye no effect of the antagonist was observed. Co-administration of JNJ7777120 with DSCG resulted in a biphasic action of JNJ7777120, implying a competitive action of the two agents. Conclusions:  These data suggest a functionality of the H₄ receptor in the rat eye and address questions on the localization and the role of the receptor in ocular inflammation. Received 15 October 2008; returned for revision 11 November 2008; received from final revision 18 November 2008; accepted by A. Falus 22 November 2008     

Evidence for a selected humoral immune response encoded by VH4 family genes in the synovial membrane of a patient with rheumatoid arthritis (RA)

The analysis of rearranged antibody-encoding genes from B cell foci in rheumatoid synovial tissue has characterized these cells as highly mutated memory B cells with a high proportion of members of the V_H4 family. In order to characterize further the V_H4 response in one patient, B cell-rich areas from different sections of synovial membrane (SM) were identified by CD20 staining, isolated by microdissection and pooled, in order to analyse highly enriched B cells without selection by in vitro culture procedures. From DNA of about 5 × 10³ B cells rearranged V_H genes were amplified by polymerase chain reaction (PCR) and cloned. Sequencing of 11 clones containing rearranged V_H4 gene products revealed that seven were potentially functional, and all were mutated with 84–96% homology to known germ-line (gl) genes and V_H4 gl genes amplified from the patient’s genomic DNA. Analysis of the complementarity determining region (CDR) 3 revealed that two products represented members of one B cell clone which differed by five nucleotide changes. Three of the five mutations encoded amino acid replacements in CDRs indicating antigen-driven expansion of one specific clone. Additional analyses of 25 members of three B cell clones from isolated aggregates showing intraclonal diversity in one of three clones provided further evidence that antigen selection takes place in the SM. Overall, the pattern of mutations and the replacement to silent (R:S) ratios were diverse, with six products indicating antigen selection by their high R:S ratios in CDRs. Although DNA analysis does not allow a characterization of antibody specificities, we can conclude from our analysis of antibody-encoding genes that selection by antigen and expansion of specific clones occur in the SM against the background of polyclonal activation.