Immunobiological role of llama heavy-chain antibodies against a bacterial beta-lactamase.

In 1993, a fraction of antibodies (Abs) devoid of L chain was found naturally occurring in the Camelidae. They were found to lack L chains, as well as the first constant heavy-chain domain (CH(1)) and therefore they were named "heavy-chain Abs" (HCAbs). Subsequent studies focused on the functional, structural and biochemical properties of recombinant variable fragments (rVHHs) of HCAbs. It was stated that rVHHs have an augmented capacity to interact with "partially hidden" epitopes, like enzymes active sites, and have an increased stability to thermal and chemical aggression. It has been suggested that these unconventional Abs could represent an evolutionary advantage, being more efficient than conventional Abs to inhibit microbial enzymes, and thus exerting a more protective immune response against pathogens. The present work focuses on the immunobiological role of HCAbs, in their capacity to inhibit microbial enzymes. Two animal models were selected, comprising a model for common vertebrates without HCAbs (rabbits), and a model for vertebrates with both conventional and unconventional Abs (Lama glama). A recombinant bacterial beta-lactamase (CTX-M-2) was selected as the microbial enzymatic antigen. After conventional immunization schedules, neither serum titers nor serum inhibitory capacity showed significant differences when rabbits and llamas were compared. These results indicate that the a priori assumption that the adaptive immune system of camelids could be better "prepared" to respond to bacterial enzymes because of the presence of HCAbs, is not always accurate. Furthermore, when the different llama antibody isotypes and subclasses were purified, it was demonstrated that the inhibitory capacity of total serum was due exclusively to IgG(1). HCAbs not only failed to inhibit CTX-M-2, but instead they activated its enzymatic activity. Altogether, these results indicate that the hypotheses extrapolated from the rVHHs properties need to be revised; the real role of HCAbs in vivo remains unknown, as well as their evolutionary cause.

Sunlight Effects on Immune System: Is There Something Else in addition to UV-Induced Immunosuppression?

Sunlight, composed of different types of radiation, including ultraviolet wavelengths, is an essential source of light and warmth for life on earth but has strong negative effects on human health, such as promoting the malignant transformation of skin cells and suppressing the ability of the human immune system to efficiently detect and attack malignant cells. UV-induced immunosuppression has been extensively studied since it was first described by Dr. Kripke and Dr. Fisher in the late 1970s. However, skin exposure to sunlight has not only this and other unfavorable effects, for example, mutagenesis and carcinogenesis, but also a positive one: the induction of Vitamin D synthesis, which performs several roles within the immune system in addition to favoring bone homeostasis. The impact of low levels of UV exposure on the immune system has not been fully reported yet, but it bears interesting differences with the suppressive effect of high levels of UV radiation, as shown by some recent studies. The aim of this article is to put some ideas in perspective and pose some questions within the field of photoimmunology based on established and new information, which may lead to new experimental approaches and, eventually, to a better understanding of the effects of sunlight on the human immune system.

The asymmetric IgG non-precipitating antibody. Localization of the oligosaccharide involved, by concanavalin A interaction.

Binding to Con A-Sepharose 4B of the low-affinity Fab fragment from sheep IgG1 anti-Dnp non-precipitating antibody was previously determined. This communication reports the results obtained when the concanavalin A interaction with Fd' fragments and L chains from non-precipitating and precipitating antibodies was examined. When Fd' fragments and L chains from non-precipitating and precipitating antibodies were tested as inhibitors of the binding of 125I-labelled concanavalin A to guinea pig erythrocytes, inhibition of such binding was only achieved by Fd' fragments from non-precipitating antibodies. Forty eight per cent of this Fd' fragment was bound by Con A-Sepharose 4B. From these results and our previous studies we conclude that mannose and/or glucose residues must be present and exposed at the Fd' fragment from the low-affinity Fab arm of sheep IgG1 anti-Dnp non-precipitating antibody. Glycosylation differences may explain the difference in precipitation behaviour of the 2 types of antibody.

Analysis of oligosaccharides involved in the asymmetrical glycosylation of IgG monoclonal antibodies.

The aim of this study is to identify the oligosaccharide residues involved in the asymmetric glycosylation of immunoglobulins. We have studied two anti-DNP monoclonal antibodies of the IgG1 isotype. Results show both qualitative and quantitative differences in the carbohydrates of both monoclonal antibodies and their fragments F(ab')2, Fab' and Fd. One of the antibodies -112D5-, which appears to be homogeneous in the Scatchard plot, has oligosaccharide residues in the L chain and in the Fd of one of the Fab'. On the other hand, 112B2 mAb, which also appears to be asymmetrically glycosylated, shows the bimodal curve characteristic of antibodies with different combining site affinities.

N-glycanase treatment of F(ab')2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity.

The aim of this study was to analyse four anti-DNP asymmetrically glycosylated monoclonal IgG3 antibodies (194/2, 194/5, 194/6 and 194/12) before and after carbohydrate manipulation. Microheterogeneity in the composition of the carbohydrate moiety involved in Fab' glycosylation was detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab' glycosylation produced a difference in the binding constants (Ka) in each paratope of two orders of magnitude, as determined by means of primary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BSA-DNP precipitation by the F(ab')2 fragments. The oxidation of the antibodies with sodium periodate caused the disappearance of the low affinity binding site as determined by fluorescence quenching. Furthermore, the enzymatic removal of the carbohydrate with N-glycanase determined the acquisition of precipitating activity by the F(ab')2 fragments.

Extracellular signal-regulated kinases and calcium channels are involved in the proliferative effect of bisphosphonates on osteoblastic cells in vitro.

Bisphosphonates (BPs) are analogues of pyrophosphate, which are widely used for the treatment of different pathologies associated with imbalances in bone turnover. Recent evidence suggested that cells of the osteoblastic lineage might be targets of the action of BPs. The objective of this work was to determine whether BPs induce proliferation of osteoblasts and whether this action involves activation of the extracellular signal-regulated kinases (ERKs). We have shown that three different BPs (olpadronate, pamidronate, and etidronate) induce proliferation in calvaria-derived osteoblasts and ROS 17/2.8 as measured by cell count and by [3H]thymidine uptake. Osteoblast proliferation induced by all BPs diminished to control levels in the presence of U0126, a specific inhibitor of the upstream kinase MEK 1 responsible for ERK phosphorylation. Consistent with this, BPs induced ERK activation as assessed by in-gel kinase assays. Phosphorylation of ERK1/2 was induced by the BPs olpadronate and pamidronate within 30 s, followed by rapid dephosphorylation, whereas etidronate induced phosphorylation of ERKs only after 90 s of incubation and returned to basal levels within 15-30 minutes. In addition, both BP-induced cell proliferation and ERK phosphorylation were reduced to basal levels in the presence of nifedipine, an L-type voltage-sensitive calcium channel (VSCC) inhibitor. These results show that BP-induced proliferation of osteoblastic cells is mediated by activation of ERKs and suggest that this effect requires influx of Ca2+ from the extracellular space through calcium channels.

Structural studies of sheep IgG1 precipitating and co-precipitating antibodies.

Descriptions have previously been given of the physiochemical and immunobiological behaviour of IgG co-precipitating antibodies, isolated from different animal species, compared with that of the precipitating ones belonging to the same immunoglobulin class. From those investigations it seems reasonable to assume that only one of the combining sites present in the two binding arms of the co-precipitating antibody molecule firmly binds the antigen. Consequently, the antibody does not form insoluble Ag-Ab complexes. The peptide maps, diagonal peptide maps and high voltage electrophoresis developed with peptides obtained after previous reduction and radioactive alkylation did not show differences between sheep IgG1 precipitating and co-precipitating antibodies. When diffused against rat anti-sheep IgG1 precipitating antibody serum, sheep IgG1 precipitating antibody gave a band of precipitation which was identical to the band given by sheep IgG1 co-precipitating antibody. Similar results were obtained when rat anti-sheep IgG1 co-precipitating antibody serum was used for precipitation. By immunodiffusion with cross absorbed sera no antigenic differences between the two antibodies could be demonstrated. The results obtained indicate that the different behaviour of precipitating and co-precipitating antibodies when interacting with antigen would probably not be a consequence of differences in the primary structures of their constant fragments. Both antibodies would belong to the same class, sub-class or type of immunoglobulin.

[Comparison of Orth-Evac and Solcotrans Plus devices for the autotransfusion of blood drained after total knee joint arthroplasty]. / Comparaison des dispositifs Orth-Evac et Solcotrans Plus pour l'autotransfusion du sang drainé après arthroplastie totale du genou.

Surgical wound blood which is ched through drains after total knee replacement surgery with a tourniquet may be returned to the patient using special collecting devices. This study aimed to compare two systems, Orth-Evac and Solcotrans Plus an to assess the safety of the reinfusion of non washed blood cells. It included 30 patients scheduled for total knee replacement surgery, free from tumoral or coagulation disease and allocated randomly in three groups of 10 each: the Orth-Evac group (OGr), the Solcotrans Plus group (SGr) and the Control group (CGr). The devices, not containing an anticoagulant, were connected to the deep suction drains in the operating room, after skin closure and before the tourniquet removal. The salvaged blood was reinfused in the subsequent six hours via a 40 microns filter. The volume of collected blood was measured and homologous blood was added as required, to maintain a hematocrit of 30%. A blood sample was obtained the day before surgery (D - 1), before reinfusion (D0), two hours later (D + 2h), one day later (D + 1), and from the collecting device before reinfusion. The statistical analysis used the Kruskal-Wallis test and Steel-Dwass procedure to confirm the difference between two groups. The three groups did not differ in age, weight, height and gender. The volume of salvaged and autotransfused blood was 925 +/- 156 mL in OGr and 605 +/- 178 mL in SGr respectively, transfusion of homologous blood was required in two patients of OGr, four of SGr and six of CGr. At D + 1, the hematocrit was comparable in all groups (OGr = 28%, SGr = 28.2% and CGr = 28.5%).(ABSTRACT TRUNCATED AT 250 WORDS)

[Hereditary angioedema: changes in serum levels of C4 in response to danazol]. / Angioedema hereditario: variaciones de los niveles séricos de C4 en respuesta a danazol.

12 patients (7 males and 5 females) suffering from the common form of HAE were included in the study protocol. All patients were older than 18 years. They were evaluated Cl INH, C4, Clq, Cls, C3, C5, C8, Bf, CH 50% and CIC. For the purpose of the study they were only considered Cl INH, C4, CH 50%, CIs and CIC levels. The rest of the complement components were among normal values. Data were recorded at day 0 and after 10 days on treatment with 400 mg/day of Danazol. Results were as follows: 50% of the patients had CIC when CH 50% values were below 120 U/ml., after treatment CIC disappeared and CH 50, Cls, C4 and Cl INH increased significatively. C4 seemed to increase more and quicker than Cl INH in terms of absolute values. We postulate that in the group of patients with CIC, the primary cause of the disease may be an alteration in C4 synthesis and that Cl INH may be lowered because of its consumption. We postulate that Danazol could act in these cases stimulating C4 synthesis independently or in accordance with Cl INH.

Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses.

Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a ß-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.

The primary structure of the Fab fragment of protein KAU, a monoclonal immunoglobulin M cold agglutinin.

The complete amino acid sequence of the Fab fragment of protein KAU, a human monoclonal cold agglutinin (IgMk) with anti-I activity, was determined. The light chain (L-chain) consists of 215 residues; the variable (V)L region belongs to the Hum/Kv325/kIIIb sub-subgroup that is preferentially selected in human IgM autoimmune response. The joining (J) region is encoded by the Jk4 gene, and the constant region (C)L domain expresses the km3 allotypic marker. The Fd fragment contains 232 amino acids, and 120 of them comprise the variable domain. The VH region corresponds to the VHIV subgroup and is closely related to the VHIV 2.1 gene isolated from genomic DNA expressed in peripheral blood of a healthy Caucasian. The complementary-determining region 1 has a unique amino acid (Asp) at position 31, and the complementary-determining region 3 codified by the diversity segment (D) gene, shows poor homology with other known D sequences. The joining segment with two unusual substitutions at the D-J junction is encoded by the JH4 gene. Thus, cold agglutinin KAU is an IgM, VkIIIb-Jk4-km3; VHIV-JH4-C mu.

The incomplete anti-Rh antibody agglutination mechanism of trypsinized ORh+ red cells.

The capacity for binding to trypsinized and non-trypsinized ORh+ red cells, of the IgG incomplete anti-Rh antibody and its F(ab')2 and Fc fragments has been investigated. An analysis has also been made of the capacity of non-specific human IgG, aggregated non-specific human IgG, human IgM (19S) and IgM (7S), and of fragments Fcgamma, Fcmu and Fc5mu to inhibit the agglutination of trypsinized ORh+ red cells by the IgG incomplete anti-Rh antibody. The results obtained indicate that these antibodies behave in a similar manner to that of nonprecipitating antibodies, and that the agglutination of trypsinized red cells seems to be a mixed reaction due to the interaction of an Fab fragment with its Rh antigenic determinant present in the surface of a red cell and the Fc of the same molecule with a receptor for Fc present in adjacent red cells. The trypsin treatment apparently results in the liberation of occult Fc receptors. It has also been demonstrated that in the agglutination of ORh+ red cells by IgG incomplete anti-Rh antibody in the presence of albumin, interaction must occur in some manner between the albumin and the Fc fragment since the F(ab')2 fragment does not give rise to agglutination under such conditions.

[Neuromuscular block control during and after anaesthesia (author's transl)]. / Contrôle de la curarisation au cours et au décours de l'anesthésie.

During many years the clinician's requirements for neuromuscular blocking drugs are satisfied by clinical investigations. Electromyographic recordings which are a satisfactory method are not useful in current practice. The use of nerve stimulators as Churchill Davidson apparatus modified for train of four impulses must reach the continental theatres, the response to neuromuscular blocking agents varying over a wide range. Five ways of stimulation can be used giving different informations, not only during anaesthesia, but so after particularly to specify the origin of some complications.

Structure of asymmetric non-precipitating antibody: presence of a carbohydrate residue in only one Fab region of the molecule.

The reactions between purified precipitating and non-precipitating anti-DNP sheep and rabbit antibodies and the antigens DNP-BSA and DNP-GABA-BSA have been studied by immunodiffusion, complement fixation and an inhibition test. Both antigens reacted identically with precipitating antibodies. On the contrary, non-precipitating antibodies did not precipitate and did not fix complement with DNP-BSA but were able to do so with DNP-GABA-BSA. A different behaviour with both antigens was also demonstrated by an inhibition test. The properties of these antibodies were also studied after treatment with endo-beta-N-acetylglucosaminidase H. Non-precipitating antibody was able to give precipitin bands in gel diffusion and to fix complement with DNP-BSA after treatment with the enzyme. The treated antibody was able to agglutinate sensitized erythrocytes. Studies by fluorescence quenching showed that the affinity for the ligand DNP-GABA was significantly increased after hydrolysis of the carbohydrate residue. The properties of precipitating antibody were not modified by the endoglycosidase. Affinity chromatography of the F(ab')2 and Fab fragments obtained from precipitating and non-precipitating antibodies was made with Con A-Sepharose. The Con A retained all the F(ab')2 and 50% of the Fab from non-precipitating antibody, which were subsequently eluted with alpha-methyl-D-mannoside. The fragments from precipitating antibody were not retained at all. It is concluded that the asymmetry of the non-precipitating antibody molecule is due to a carbohydrate moiety which is present in only one of the Fab regions. This carbohydrate affects the reaction between the combining site and the antigen, and renders the molecule functionally univalent.

Hereditary angioedema: polymorphism.

Four patients with hereditary angioedema were studied. All had low values of C4, C1 INH, (CH 50) complement activity and circulating immune complexes. This report takes into account the different aspects that this disease can present, often quite different from the classical description of hereditary angioedema.

Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis.

Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24.