Search

Arsenicals are painful, inflammatory and blistering causing agents developed as chemical weapons in World War I/II. However, their large stockpiles still exist posing threat to public health. Phenylarsine oxide (PAO), a strong oxidant and a prototype arsenical is tested for its suitability to defining molecular mechanisms underlying arsenicals-mediated tissue injury. Topically applied PAO induces cutaneous erythema, edema and micro-blisters. These gross inflammatory responses were accompanied by the enhanced production of pro-inflammatory cytokines, ROS and unfolded protein response (UPR) signaling activation. To demonstrate the involvement of UPR in the pathobiology of these lesions, we employed chemical chaperone, 4-phenylbutyric acid (4-PBA) which attenuates UPR. 4-PBA significantly reduced PAO-induced inflammation and blistering. Similar to its effects in murine epidermis, a dose- and time-dependent upregulation of ROS, cytokines, UPR proteins (GRP78, p-PERK, p-eIF2α, ATF4 and CHOP) and apoptosis were observed in PAO-treated human skin keratinocytes NHEK and HaCaT. In addition, 4-PBA significantly restored these molecular alterations in these cells. Employing RNA interference (RNAi)-based approaches, CHOP was found to be a key regulator of these responses. These effects are similar to those manifested by lewisite suggesting that PAO could be used as a prototype of arsenicals to define the molecular pathogenesis of chemical injury.

The vast potential applications of biomolecules that bind inorganic surfaces led mostly to the isolation of short peptides that target selectively specific materials. The demonstrated differential affinity toward certain surfaces created the impression that the recognition capacity of short peptides may match that of rigid biomolecules. In the following, we challenge this view by comparing the capacity of antibody molecules to discriminate between the (100) and (111A) facets of a gallium arsenide semiconductor crystal with the capacity of short peptides to do the same. Applying selection from several peptide and single chain phage display libraries, we find a number of antibody molecules that bind preferentially a given crystal facet but fail to isolate, in dozens of attempts, a single peptide capable of such recognition. The experiments underscore the importance of rigidity to the recognition of inorganic flat targets and therefore set limitations on potential applications of short peptides in biomimetics., (Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.)

Haematopoietic stem cells (HSCs) in mouse bone marrow are located in specialized niches as single cells. During homeostasis, signals from this environment keep some HSCs dormant, which preserves long-term self-renewal potential, while other HSCs actively self renew to maintain haematopoiesis. In response to haematopoietic stress, dormant HSCs become activated and rapidly replenish the haematopoietic system. Interestingly, three factors - granulocyte colony-stimulating factor, interferon-alpha and arsenic trioxide - have been shown to efficiently activate dormant stem cells and thereby could break their resistance to anti-proliferative chemotherapeutics. Thus, we propose that two-step strategies could target resistant leukaemic stem cells by priming tumours with activators of dormancy followed by chemotherapy or targeted therapies.

Recent findings support a central role for TLRs in both foreign Ag-driven immune responses and systemic autoimmune diseases mediated by B lymphocytes. In vitro studies have shown that the Ag receptors (BCRs) on B cells specific for nuclear autoantigens can facilitate the delivery of these autoantigens to the endocytic compartment, resulting in activation of the nucleic acid-specific TLRs present in this subcellular locale. If this pathway is operative in vivo it might promote the development, survival, or activation of such autoreactive B cells. To test this idea, we evaluated the influence of a deficiency in the CpG DNA-specific TLR, TLR9, or all MyD88-dependent TLRs on the primary development and foreign Ag-driven immune response of B cells in a line of V(H) knockin mice that contains a high frequency of "dual reactive" B cells specific for DNA-based autoantigens such as chromatin, as well as the hapten arsonate. We found that although development and activation of these B cells in vitro are clearly influenced by DNA-based autoantigens, TLR9 or MyD88 deficiencies had no apparent effect on the primary development and participation in the anti-arsonate response of these B cells in vivo. We discuss these results in the context of previous models for the role of TLR9 and other TLRs in the regulation of antinuclear Ag B cell development and activity.

In this study, germline Abs were used to select clones from a random dodecapeptide phage-display library. This revealed a much greater heterogeneity of binders than could be obtained with mutated daughter Abs that presumably had been selected in vivo by nominal Ag during active immune responses. We demonstrate that the pluripotency of germline Abs can subsequently be optimized by binding interactions that correlate with thermodynamic changes indicative of structural adaptations at the interface. This singular feature confers on each Ab a distinct window of Ag specificities, where the entropic space explored constitutes a thermodynamic signature of that particular Ab. Combining site plasticity may facilitate overlaps in such windows, with independent Abs converging onto common determinants with near identical binding affinities. In addition to providing for an amplified recognition potential, this networking of individual spectra of Ag specificities simultaneously facilitates the rapid recognition of Ag. Importantly, it also ensures that the primary response is composed of Abs with a high degree of "evolvability."

Exposure of RPMI 8226, Karpas 707 and U266 human myeloma-like lines to low doses of As(2)O(3) was followed by a marked increase in lymphokine activated killers (LAK)-mediated killing and up- modulation of CD38 and CD54, two molecules involved in cell-cell interactions. Moreover, simultaneous exposure of effectors and targets to As(2)O(3) yielded the most effective condition for lysis. The expression of CD31 (CD38 ligand) and CD11a (CD54 ligand) was also up-regulated by LAK, suggesting that increased adhesion was responsible for the improved killing. Similar results were obtained using freshly isolated myeloma cells. These findings indicate that As(2)O(3) may be useful to boost the immune system against myelomas.

Mice transgenic for the human immunoglobulin heavy chain minilocus pHCl were developed several years ago to help better understand the mechanisms of VDJ recombination and antibody response. Interestingly, these minilocus transgenic mice develop a polyclonal, extremely diverse mu human immunoglobulin heavy chain repertoire, but when immunized, they exclusively use murine immunoglobulin heavy chains. Here, the data shows that when the minilocus is transferred by cross-breeding onto the muMT background, the resulting mice (HCl-muMT/muMT mice) develop polyclonal, extremely diverse mu and gamma1 human immunoglobulin heavy chain repertoires. Our data indicates that if no antigen specific antibodies are detected in pHCl transgenic mice, it is essentially due to competition with endogenous immunoglobulin heavy chain gene segments. Moreover, the data shows that despite the presence of only one functional V(H) gene segment and despite mu and gamma1 repertoires similar to the early pre-immune human repertoire, HCl-muMT/muMT mice, can develop immune responses against proteins and haptens. Finally, the data shows that in aged HC1-muMT/muMT mice, the generation of new B-cells may be impaired and old mice may mainly rely on B-cell generated earlier in life to mount immune responses.

Whether memory B cells possess altered differentiative potentials and respond in a qualitatively distinct fashion to extrinsic signals as compared with their naive precursors is a current subject of debate. We have investigated this issue by examining the participation of a predominant anti-arsonate clonotype in the primary and secondary responses in the spleens of A/J mice. While this clonotype gives rise to few Ab-forming cells (AFC) in the primary response, shortly after secondary immunization its memory cell progeny produce a massive splenic IgG AFC response, largely in the red pulp. Extensive clonal expansion and migration take place during the secondary AFC response but Ab V region somatic hypermutation is not reinduced. The primary and secondary germinal center (GC) responses of this clonotype are both characterized by ongoing V gene hypermutation and phenotypic selection, little or no inter-GC migration, and derivation of multiple, spatially distinct GCs from a single progenitor. However, the kinetics of these responses differ, with V genes containing a high frequency of total as well as affinity-enhancing mutations appearing rapidly in secondary GCs, suggesting either recruitment of memory cells into this response, or accelerated rates of hypermutation and selection. In contrast, the frequency of mutation observed per V gene does not increase monotonically during the primary GC response of this clonotype, suggesting ongoing emigration of B cells that have sustained affinity- and specificity-enhancing mutations.

The humoral immune response to arsonate (Ars) in normal A/J mice is dominated in the late primary and particularly in the secondary response by a recurrent and dominant idiotype (CRIA) which is encoded by a single canonical combination of the variable gene segments: VHidcr11-DFL16.1-JH2 and Vkappa10-Jkappa1. Accumulation of somatic mutations within cells expressing this canonical combination or some less frequent Ig rearrangements results in the generation of high-affinity antibodies. By contrast, in partially shielded and irradiated A/J mice (autologous reconstitution) immunized with Ars-keyhole limpet hemocyanin (KLH), both the dominance of the CRIA idiotype and the affinity maturation are lost, whereas the anti-Ars antibody titer is not affected. To understand these alterations, we have analyzed a collection of 27 different anti-Ars hybridomas from nine partially shielded and irradiated A/J mice that had been immunized twice with Ars-KLH. Sequence analysis of the productively rearranged heavy chain variable region genes from those hybridomas revealed that (i) the canonical V(D)J combination was rare, (ii) the pattern of V(D)J gene usage rather corresponded to a primary repertoire with multiple gene combinations and (iii) the frequency of somatic mutations was low when compared to a normal secondary response to Ars. In addition, immunohistological analysis has shown a delay of 2 weeks in the appearance of full blown splenic germinal centers in autoreconstituting mice, as compared to controls. Such a model could be useful to understand the immunological defects found in patients transplanted with bone marrow.

We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf). To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30-36 in Ab 36-65 were randomly mutated and expressed as Fab in a bacteriophage display vector. Ab 36-65 is germline encoded, lacking somatic mutations. Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35. All Fab bound Sulf, but not Ars. Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1. At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36-71, while Sulf binding occurs with at least seven different H:35 residues. All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline V(H) sequence. Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.

In this work we have assessed the effect of cell surface anti-immunoglobulin (Ig) of anti-idiotypic B cells on their idiotypic counterparts in vivo and in vitro, as a surrogate for soluble anti-surface Ig, using the well-characterized anti-arsonate system. The response of A/J mice against the hapten arsonate coupled to keyhole limpet hemocyanin (ARS-KLH) is dominated by a closely related family of antibodies sharing the same determinant, named the CRIA idiotype. We show herein that a massive induction of anti-CRIA B cells, subsequent to immunization with the mAb 3665 (CRIA+, arsonate binding) coupled to KLH, mediated a strong and long-lasting inhibition of this dominant oligoclonal response to arsonate. The titer of anti-arsonate antibodies remained, however, unchanged. Adoptive transfers to x-irradiated syngeneic mice showed that anti-CRIA-producing B cells have a direct effect on induction of inhibition. This was supported by the in vitro data where irradiated anti-CRIA B cells could induce inhibition of both antibody production and mitogenesis of their counterparts, CRIA B cells. This inhibitory effect could be decreased when the surface anti-surface Ig were hidden by the 3665 Fab fragments but not by anti-MHC class II antibodies. These interactions between CRIA and anti-CRIA B cells were solely Igh restricted and the inhibition was likely initiated by hyperaggregation of surface Ig. The presence of ARS-KLH-primed T cells in vitro could prevent the growth inhibition but not the suppression of antibody production. A similar profile was noticed in vitro for soluble polyclonal rabbit anti-CRIA Ab. All together, our data suggest that a negative signaling in B cells may be initiated by surface Ig of their idiotypic partners subsequent to a strong cross-linking of their surface Ig receptors.

Male albino rats were given a single oral dose of gallium arsenide (GaAs) (100, 200 or 500 mg/kg). Erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity was inhibited in all the three GaAs-exposed groups accompanied by elevated urinary excretion of ALA. A significant increase in serum aspartate aminotransferase (AST) activity, and gamma-glutamyltranspeptidase (gamma-GT) was observed. A significant increase in hepatic malondialdehyde (MDA) and a decrease in hepatic glutathione contents were also noted. Renal alkaline phosphatase activity, urinary ALA and protein excretion increased significantly on GaAs exposure. These changes were accompanied by significant alterations in almost all the immunological variables, with an increase in gallium and arsenic concentration in blood and soft tissues. While most of the above biochemical alterations were prominent at day 7 following single exposure to 200 and 500 mg/kg GaAs, most of the immunological indices altered with all the three doses and remained high even at day 21. The results suggest only a moderate effect of GaAs on renal and hepatic tissues. By contrast, immunological and haematological systems are the most vulnerable to the toxic effects of GaAs.

Monoclonal antibodies are potentially useful therapeutic agents and can now be produced in hosts such as bacteria. However, it has been found that bacterial expression of some antibody-combining site fragments is greatly diminished. We compared two homologous anti-arsonate antibodies, 36-65 and 36-71, to address the question of why the former but not the latter expresses well as Fab in E. coli. These antibodies are both derived from the same variable region germline genes but differ in affinity due to somatic mutations present in 36-71. To investigate the poor expression of 36-71 Fab, we examined several factors, such as cellular toxicity, induction with isopropylthio-beta-D-galactoside, and growth of transformed bacteria at lower temperatures (30 degrees C), as well as the possibility of E. coli strain-related expression of Fab. However, none of these factors made a significant difference to Fab expression. We next localized a significant portion of the defect in Fab expression to the heavy chain by swapping the heavy and light chains from the two antibodies to construct hybrid Fabs. We used site-directed mutagenesis to engineer amino acids into the variable regions of antibody 36-71, to reproduce those found in 36-65 which is expressed well in E. coli. The defect in expression is due to residues located in the complementarity-determining regions, as mutations of heavy chain framework residues to those present in 36-65 do not enhance expression of 36-71 Fab in E. coli.

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p-azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment.

The TCR is predicted to resemble the Fab fragment of an Ig molecule and by analogy to possess six Ag binding loops that contact MHC proteins bound with antigenic peptides. We have identified residues in the predicted Ag binding loops (beta 1, beta 2, and beta 3) on a TCR beta-chain that are important in the recognition of the MHC/antigenic peptide complex. Using site-directed mutagenesis, we altered the residues forming the predicted Ag binding site on the beta-chain expressed by the T lymphocyte clone D5, which specifically recognizes p-azobenzenearsonate-conjugated peptides presented by the class II MHC molecule I-Ad. Amino acid substitution of individual residues in each loop affected Ag recognition, demonstrating that all three putative Ag binding loops of the D5 TCR beta-chain are important in interaction with I-Ad/arsonate-conjugated Ag. Taken together with our previous work on the D5 TCR alpha-chain (Nalefski et al., J. Exp. Med. 175:1553), these results suggest that all six Ag binding loops of the D5 TCR alpha- and beta-chains interact simultaneously with the MHC/peptide complex. Consequently, the area of interaction between the TCR and the MHC/antigenic peptide complex is predicted to be extensive.

A model of the combining site of a mouse antibody specific for p-azophenylarsonate was tested by oligonucleotide-directed mutagenesis of the proposed hapten-contacting residues, Arg96 in the L chain, and Asn35, Trp47, Tyr50, Ser95, and Tyr100b in the H chain. The affinity and relative affinity for p-azophenylarsonate-N-acetyl-L-tyrosine of mutant antibodies expressed in transfectomas were determined by fluorescence quenching and by inhibition ELISA, respectively. The results show that alteration of the proposed contacting residues has drastic effects on hapten binding, and that the hydroxyl groups of Tyr50 and Tyr100b appear to orient the phenyl rings for optimal aromatic-aromatic interactions with the phenyl ring of the hapten. They further indicate a tight packing of the contacting residues around the hapten, which cannot accommodate changes in the positions of the functional groups of Asn35 and Ser95.

The structure of the antigen-binding fragment (Fab) of an anti-phenylarsonate monoclonal antibody (36-71) bearing a major crossreacting idiotype of A/J mice has been solved and refined to an R factor of 19.3% at a resolution of 2.9 A. An initial electron density map was obtained with phase information from a total of six isomorphous heavy-atom derivatives (from two different compounds) and a molecular replacement solution using the HED10 Fab crystal structure as a model. The structure of the McPC603 Fab was used to provide an initial set of atomic coordinates. The electron density maps are clear and easily interpretable for the entire sequence except for sections from two of the heavy-chain complementarity-determining regions totaling 21 residues. These residues have been left out of the refinement and are not represented in our current model. The antigen-combining site was located by means of a difference Fourier synthesis with one of the heavy-atom derivatives, which contained arsanilic acid. It lies in a small pocket formed by residues from the hypervariable regions of both the heavy and the light chains. Interactions with the hapten from framework residues are also possible.

It has been previously shown that it is possible to modify the expressed repertoire of a given individual using idiotypic manipulation. For example, A/J mice respond to arsonate challenge by synthesizing a dominant idiotype, CRIA, whereas BALB/c mice do not. However, after treatment with rabbit polyclonal anti-CRIA antibodies (Ab2 or anti-idiotypic antibodies) and arsonate, BALB/c mice are able to synthesize a CRIA-like idiotype. To determine whether this modification of repertoire is dependent on the immunoglobulin loci (Ig-h, kappa), we have analyzed the anti-arsonate response after anti-idiotypic treatment of three strains of mice (C58, C.C58, AKR), chosen because they are among a small group of strains which express Kappa V regions not seen in other strains. There are also L chains lacking in these strains which are expressed in other mice. The C58 and C.C58 strains share the same Ig-h locus (Ig-ha) with BALB/c mice but C.C58 are congenic mice, that express the kappa loci on a BALB/c genetic background. AKR mice express the Ig-hd haplotype. AKR, C58 and C.C58 do not produce CRIA positive antibodies in response to arsonate; a defect which has been previously mapped to the kappa locus. These three strains of mice (C58, C.C58 and AKR) were treated with rabbit anti-CRIA and boosted with Ars-KLH. The results show that after such treatment, the C.C58 mice were able to express CRIA-like antibodies which are serologically identical to those of BALB/c.

Rat monoclonal antibodies specific for mouse kappa light chains and mouse gamma heavy chains have been generated. These rat monoclonal antibodies have been biosynthetically labelled with 35S methionine. The free label was dialyzed from the medium and, without further purification, the medium containing the radioactive monoclonal antibody was used in a radioimmunoassay to screen the sera of the immunized animals and hybridomas for specific mouse antibodies of the IgG class.

The experiments presented in this paper demonstrate that the induction of tolerance on the one hand and the induction of delayed sensitivity on the other hand can be accomplished by administration of similar doses of azobenzene-arsonate conjugated to N-chloracetyl tyrosine (ABA-T) to guinea pigs with the determining factor being the absence or presence, respectively, of activating bacterial products in the adjuvant mixture used. Thus, complete, persistent ABA-T-specific T-cell tolerance can be induced in adult guinea pigs with 20 mug of ABA-T given intradermally in incomplete Freund's adjuvant (IFA) whereas this same dose of ABA-T induces ABA-specific immunity when administered in complete Freund's adjuvant. This tolerance was not reversible by administration of ABA-T and IFA in the presence of bacterial lipopolysaccharide, was generated before the formation of primed T cells, and persisted for at least 3 mo after initiation. Moreover, cell transfer studies performed herein demonstrate that the unresponsiveness resulting from administration of ABA-T in IFA reflects the activity of suppressor cells to induce and maintain a state of unresponsiveness could only be demonstrated in unprimed animals may indicate a severe limitation on the potential clinical usefulness of such an approach to regulation of the immune system.

Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.

We have previously reported that hapten specific antibodies are produced in guinea pigs immunized with certain N-substituted phosphatidylethanolamine derivatives (either free or incorporated into liposomal membranes) in complete Freund's adjuvant. In this paper, we describe the synthesis of mono(p-azobenzenearsonic acid)tyrosylphosphatidylethanolamine (ABA-Tyr-PE). Immunication with this compound (either free or present in liposomes) not only results in the formation of anti-azobenzenearsonyl antibodies, but also confers cellular immunity as manifested by delayed hypersensitivity reactions elicited by challenge with either azobenzenearsonyl-bovine serum albumin or sensitized liposomes. Thus, ABA-Tyr-PE immunized guinea pigs differ from those immunized with azobenzenearsonyl-bovine serum albumin which produce anti-bodies but do not reveal a delayed reaction. Moreover, the ABA-Tyr-PE immunized animals differ from those immunized with mono(p-azobenzenearsonic acid)tyrosine; this substance has been shown by other investigators to confer cellular immunity without antibody formation in guinea pigs. However, the deacylated homolog of ABA-Tyr-PE (i.e., mono(p-azobenzenearsonic acid)tyrosylglycerophosphorylethanolamine) has the same immunological properties as mono(p-azobenzenearsonic acid)tyrosine. These observations justify the further exploitation of liposomal model membranes as novel immunogens that are able to elicit both cell and humoral mediated immune responses.

Amino acid sequence analysis has been performed on three groups of heavy (H) chains of A/J mice. H chains derived from unimmunized animals were compared to anti-p-azophenylarsonate (anti-Ar) antibodies which were further subdivided into those possessing and those depleted of a cross-reacting idiotype (CRI). It was found that anti-Ar antibodies bearing the CRI are homogeneous through the first hypervariable region of the H chain. The same sequence was obtained for pooled antibody isolated from the ascites fluid of 18 A/J mice or from a single mouse. The H chains appear to belong to a minor V-H subgroup. In the first 30 positions Anti-Ar antibodies depleted of the CRI had the same sequence as those containing the CRI (with small amounts of heterogeneity at some positions), but contained a mixture of sequences in the first hypervariable region of the H chain. These studies indicate that antibodies with similar specificity and with identical framework sequences, but which differ in their hypervariable regions, contain different idiotypic determinants, and support the concept that the idiotypic determinants reside primarily within hypervariable regions.

Affinity maturation was studied by the analysis of the kinetics of the appearance of antibody subpopulations with different affinities during the immune response, using an hapten-inhibition ELISA. The immune response in KLH-Ar-immunized A/J mice was used as a model system. Five antibody subpopulations of different affinity (10(3)-10(7) M-1) could be detected, the relative concentrations of which changed during affinity maturation. The high-affinity antibody subpopulations did not represent the major fraction at any stage during affinity maturation. The appearance of the highest affinity subpopulation (10(7) M-1), despite exhibiting relative concentrations no higher than 12%, produced an important increase in average affinity. On the other hand, its disappearance at the end of the maturation process could explain the average affinity decrease observed at this stage. Our results indicate that affinity maturation cannot be explained by the dominance of high-affinity clones, as proposed by Siskind & Benacerraf (1969). The increase in affinity could rather be due to the progressive appearance of low percentages of high-affinity clones, which are not present in the primary response and never become dominant.

Radioactive tracers and immunofluorescence were employed to detect and quantitate fibrinogen/fibrin deposition in two types of cell-mediated hypersensitivity reactions in the guinea pig. Classic delayed hypersensitivity (DH) reactions to Old Tuberculin and to the azobenzenearsonate hapten were characterized by a progressive increase in the fibrinogen (125-I-HF) content which exceeded that of the albumin tracer (131-I-HSA) and paralleled the development of induration and erythema. Accumulation of 125-I-HF could be related both to increased vascular permeability to 125-I-HF and, more specifically, to retarded efflux of extra vascular 125-I-HF from tuberculin reaction sites. Warfarin inhibited 125-I-HF accumulation and the formation of urea-insoluble 125-I-HF (cross-linked fibrin) as well as induration in tuberculin reactions. Immunofluorescence studies revealed the site of Fib deposition to be extravascular, among the connective tissue fibers of the dermis, similar to that in DH reactions in man. In contrast, little 125-I-HF accumulated in cell-mediated reactions rich in basophils--cutaneous basophil hypersensitivity (CBH) reactions to keyhole limpet hemocyanin, ovalbumin, and dinitrochlorobenzene--due in part to less vascular leakage of macromolecules and to decreased formation of urea-insoluble fibrin. By immunofluorescence Fib deposits were found in CBH reactions in a pattern similar to that in DH reactions, but the intensity of staining was appreciably less. Thus, fibrin accumulation further distinguishes DH from CBH reactions and is very likely responsible for the induration characteristic of DH reactions.

The recurrence of allergic inflammation, as examined by exudate accumulation, infiltration of polymorphonuclear leukocytes into the exudate, edema formation, vascular permeability and prostaglandin E2 levels in the exudate, was induced by injecting the antigen, azobenzene arsonate-conjugated acetyl bovine serum albumin, into the capsule of the proliferative granulation tissue which had been formed by the first-time antigenic challenge injection into an air pouch in the dorsum of the sensitized rat. The recurrence of the allergic inflammation 4 and 24 h after the antigenic challenge was inhibited dose-dependently by treatment with cyclooxygenase inhibitors, suggesting the possible participation of cyclooxygenase products, especially prostaglandin E2. The difference in the allergic inflammatory responses induced by the first-time antigenic challenge and the second-time antigenic challenge was discussed from the viewpoint of chemical mediators.