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.

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.

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.

Cambio del isotipo en la respuesta a antígenos T independientes por co-inoculación con antígenos T dependientes / Isotype switch in the response against T independent antigens by co-inoculation with T dependent antigens

Un paradigma clásico de la inmulogía plantea que para que ocurra cambio de isotipo en los anticuerpos es condición sine qua non la presentación del antígeno a un linfócito T colaborador por parte de una célula presentadora de antígenos. En el presente trabajo se diseñó un modelo animal, ratones BALB/c, de respuesta inmune frente a dos antígenos típicos. Se utilizo dextrán como antígeno T independiente (AgTI) y seroalbúmina bovina (SAB) como antígeno T dependiente (AgTD), y se estúdio la respuesta, analizando los isotipos de los anticuerpos específicos producidos. Los resultados obtenidos muestran que la respuesta a dextrán en presencia de SAB ocurre con cambio de isotipo (swith), essencialmente de IgM a IgG. Estos experimentos sugieren que la SAB genera un entorno bioquímico inductor de cambio de isotipo tanto en supropia via de procesamiento como en del dextrán. Los resultados señalan que la asociación exclusiva de los AgTDs con las respuestas em las que ocurre cambio de isotipo es incorrecta. Considerando el modelo propuesto resulta poco probable encontrar in vivo y en forma espontânea casos en los que los AgTIs ingreses al organismo aislados; en cambio, es mucho más probable que el ingreso ocurra conjuntamente con AgTDs, y en consecuencia ocurra cambio de isotipo.

Cambio del isotipo en la respuesta a antígenos T independientes por co-inoculación con antígenos T dependientes / Isotype switch in the response against T independent antigens by co-inoculation with T dependent antigens

Un paradigma clásico de la inmulogía plantea que para que ocurra cambio de isotipo en los anticuerpos es condición sine qua non la presentación del antígeno a un linfócito T colaborador por parte de una célula presentadora de antígenos. En el presente trabajo se diseñó un modelo animal, ratones BALB/c, de respuesta inmune frente a dos antígenos típicos. Se utilizo dextrán como antígeno T independiente (AgTI) y seroalbúmina bovina (SAB) como antígeno T dependiente (AgTD), y se estúdio la respuesta, analizando los isotipos de los anticuerpos específicos producidos. Los resultados obtenidos muestran que la respuesta a dextrán en presencia de SAB ocurre con cambio de isotipo (swith), essencialmente de IgM a IgG. Estos experimentos sugieren que la SAB genera un entorno bioquímico inductor de cambio de isotipo tanto en supropia via de procesamiento como en del dextrán. Los resultados señalan que la asociación exclusiva de los AgTDs con las respuestas em las que ocurre cambio de isotipo es incorrecta. Considerando el modelo propuesto resulta poco probable encontrar in vivo y en forma espontÔnea casos en los que los AgTIs ingreses al organismo aislados; en cambio, es mucho más probable que el ingreso ocurra conjuntamente con AgTDs, y en consecuencia ocurra cambio de isotipo. (AU)

Evaluación de diferentes procedimientos de detección de anticuerpos anti Ro/SS-A 52 kDa y anti-Ro/SS-A 60 kDa en síndrome de Sjögren primario / Evaluation of the different procedures in the detection of anti RO/SS-A 52 kDa and anti-RO/SS-A 60 kDa antibodies in primary Sjögren's syndrome

Un grupo de pacientes con Síndrome de Sjögren primario fue analizado en el presente trabajo con el fin de evaluar diversas técnicas para la determinación de autoanticuerpos anti-Ro/SS-A de 52 y 60 kDa. Las técnicas evaluadas fueron: doble inmunodifusión en geles de agarosa utilizando como antígeno un extracto de bazo humano; enzimoinmuno análisis (ELISA) y Western Blot ambos realizados utilizando antígenos proteicos recombinantes (Ro/SS-A 52 y 60 kDa en ensayos individuales) como antígenos. Si bien las técnicas de ELISA y Western Blot poseen una mayor sensibilidad que la inmunodifusión, en ciertos casos esta última no puede ser completamente reemplazada por las primeras. Hay que tener en cuenta la naturaleza del antígeno utilizado para poder sacar conclusiones. Los antígenos recombinantes pueden mantener los epitopes lineales y conformacionales de las moléculas nativas, pero carecen de la interacción con las demás moléculas formadoras del complejo. En cambio, en el extracto antigénico utilizado en la inmunodifusión se podrían mantener epitopes formados por la interacción entre las distintas moléculas del complejo RoRNP. Nuestros resultados indican que, a pesar de su mayor sensibilidad, las técnicas de ELISA y Western Blot no serían capaces de desplazar totalmente a la inmunodifusión en geles (AU)

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.

Three-dimensional structure of the Fab from a human IgM cold agglutinin.

Cold agglutinins (CAs) are IgM autoantibodies characterized by their ability to agglutinate in vitro RBC at low temperatures. These autoantibodies cause hemolytic anemia in patients with CA disease. Many diverse Ags are recognized by CAs, most frequently those belonging to the I/i system. These are oligosaccharides composed of repeated units of N:-acetyllactosamine, expressed on RBC. The three-dimensional structure of the Fab of KAU, a human monoclonal IgM CA with anti-I activity, was determined. The KAU combining site shows an extended cavity and a neighboring pocket. Residues from the hypervariable loops V(H)CDR3, V(L)CDR1, and V(L)CDR3 form the cavity, whereas the small pocket is defined essentially by residues from the hypervariable loops V(H)CDR1 and V(H)CDR2. This fact could explain the V(H)4-34 germline gene restriction among CA. The KAU combining site topography is consistent with one that binds a polysaccharide. The combining site overall dimensions are 15 A wide and 24 A long. Conservation of key binding site residues among anti-I/i CAs indicates that this is a common feature of this family of autoantibodies. We also describe the first high resolution structure of the human IgM C(H)1:C(L) domain. The structural analysis shows that the C(H)1-C(L) interface is mainly conserved during the isotype switch process from IgM to IgG1.

Antibodies from patients with psoriasis recognize N-acetylglucosamine terminals in glycoproteins from Pityrosporum ovale.

We have previously reported the finding of circulating antibodies recognizing two proteins of 100 and 120 kD (PO100 and PO120) from Pityrosporum ovale in patients with psoriasis. These antibodies were specific, since they were not detected in normal sera nor in other diseases linked to P. ovale such as seborrhoeic dermatitis or pityriasis versicolor. The present study aimed at further characterizing the specificity of these antibodies. Enzyme-labelled lectins were used to determine the carbohydrate composition of PO120 and PO100. BSII, a lectin that recognizes terminal N-acetylglucosamine (GlcNAc), showed the same banding pattern as sera from patients. Reactivity against these proteins was inhibited after mild oxidation of the carbohydrate moieties of the extract. Treatment of the extracts with lyticase altered the immune reactivity against the PO120 band as seen in Western blot assays. PO100 was not detected after lyticase digestion. Digestion with lysozyme did not alter the immune reactivity of the PO100 and PO120 bands, although the protein pattern in SDS-PAGE was modified. To examine the relevance of anti-GlcNAc antibodies in the immune response to P. ovale in psoriasis, we performed a binding inhibition ELISA. Psoriatic sera that were positive in the ELISA against a heat-denatured extract of P. ovale were rendered negative only by pre-incubation with GlcNAc in a concentration-dependent manner. Our results are indicative that the antibody response to PO100 and PO120 in patients with psoriasis is directed towards terminal GlcNAc residues.

The frequent mutation Gly/Asp in CDR1H may determine a cross-reactive idiotope in anti-I cold agglutinins.

Variable domains (VH) of all known anti i/I cold agglutinin (CA) heavy chains are codified by the VH4-21 gene. While anti-i CAs are the expression of gene rearrangement without mutations represented by amino acid changes, anti-1 CAs present, among others, a frequent somatic mutation of Gly by Asp at position 31. The hydropathy profile calculated for the CDR1H (position 30 to position 35), as well as some adjacent positions of the heavy chain belonging to anti-i and anti-I antibodies, showed the conformational changes accompanying the replacement of Gly by Asp. A MoAb (LP91), which had been obtained in BALB/c mice immunized with a Fabmu fragment from a monoclonal IgMkappaIIIb anti-I CA (protein KAU), proved capable of inhibiting human adult erythrocyte cryoagglutination by anti-I CAs but not that of fetal erythrocytes by anti-i CAs. Western blot analysis disclosed that such MoAbs recognized a sequential epitope located in the Fd fragment of all anti-I CAs employed in this study. With the purpose of checking whether Asp(31) was involved in the epitope recognized by the MoAb, two peptides, D and G, were synthesized which mimicked the CDR1H structure of anti-I and anti-i, respectively; the MoAb only reacted with peptide D by ELISA. Subsequent experimental results indicate that the Gly/Asp mutation could be associated with the diverse specificity presented by these autoantibodies, a change determining a characteristic epitope/idiotope, recognized by LP91 in the CDR1H.

Characterization of the lipase activity of Malassezia furfur.

Enzymes capable of metabolizing lipids are essential for the growth of Malassezia furfur in vitro and in vivo. We designed a series of experiments to characterize the lipolytic system in this yeast. The optimal pH of the lipase system was 7.5 Lipase activity was detected in soluble and insoluble saline cell extracts and in supernatant from the cultures. Esterase activity screened in samples separated by native polyacrylamide gels showed that it was restricted to one band of low mobility. An FPLC analysis of the soluble saline extract demonstrated that the lipase activity was present in three major peaks with different protein composition as revealed by SDS-PAGE. The enzymatic activity and cell growth were first induced and later inhibited by increasing concentrations of polyethylene-sorbitan-monooleate (Tween-80). The characterization of the lipolytic system (e.g. its induction by substrate and the effect of pH and/or different cations) could help to explain the increment in the number of M. furfur infections related to alterations of surface lipids in the skin such as seborrheic dermatitis.

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.

Antibodies to proteins from Pityrosporum ovale in the sera from patients with psoriasis.

In order to analyse the humoral immune response to the commensal yeast Pityrosporum ovale, we developed a western immunoblot technique with a salt soluble extract of P. ovale cytoplasm. In the present study, we tested sera from patients with psoriasis (n = 15), seborrhoeic dermatitis (n = 10), pityriasis versicolor (n = 8), and normal controls (n = 10). Seventy-three per cent (11/15) of the patients with psoriasis showed specific reactivity with a protein derived from P. ovale of estimated molecular mass 120 kDa, and 46% (7/15) of the cases recognized a 100-kDa protein. Sera from pityriasis versicolor and normal donors showed nonspecific reactivity with several bands of lower molecular weight. To characterize the location of the 100 and 120-kDa proteins, we performed a lyticase digestion of the cell wall, and analysed the soluble digested products by western blotting. The sera from psoriasis patients detected several bands in the range 100-120 kDa. The finding of the immunoreactive 120-kDa protein in this fraction suggests its location at the space between cell wall and membrane (periplasmic space). As a control, we performed an extraction of the cytoplasmic proteins of the dimorphic yeast Candida albicans. C. albicans showed a different pattern of banding in SDS-PAGE. Immunoblots with C. albicans did not allow the detection of any related band. A smear was observed in the high molecular weight range consistent with the presence of lipopolysaccharides. The role of the immune response in infection by P. ovale has not yet been fully explored.(ABSTRACT TRUNCATED AT 250 WORDS)

[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)

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.

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.

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.

Hereditary angioedema: therapeutic effect of danazol on C4 and C1 esterase inhibitors.

Hereditary angioedema (HAE) is an inherited deficiency of C1 esterase inhibitor (C1 inh). The two types of genetic C1 inh deficiency are type I, which is quantitative, and type II, which is functional. For the purpose of the present study, four HAE patients were selected. None of them had received any androgenic therapy. The group included three type I and one type II cases. All patients that entered the protocol received danazol, 400 mg/day for 14 days. The complement system was evaluated by monitoring C4, hemolytic complement 50% (CH50), circulating immune complexes (CIC), and antigenic and functional C1 inh during the study. The level of complement factors at the beginning and at the end of this period demonstrated a statistically significant increase in C4 and CH50 and the disappearance of CIC, while C1 inh remained unmodified. These results suggest that the therapeutic effect of danazol may have two mechanisms of action: (1) promotion of C4 synthesis by anabolic effect resulting in an improvement of the complement system with the disappearance of CIC and (2) a minor increase in C1 inh level primarily due to the lack of its consumption.

Binding of cystatin C to C4: the importance of sense-antisense peptides in their interaction.

Hydropathic anticomplementarity of amino acids indicates that peptides derived from complementary DNA strands may form amphiphilic structures and bind one another. By using this concept, we have found that the antisense peptide Ser-Tyr-Asp-Leu complementary to the segment Gln-Ile-Val-Ala-Gly (residues 55-59) in cystatin C (an inhibitor of cysteine proteases) is located at positions 611-614 of the beta chain of human C4, the fourth component of complement. Here we describe and characterize the specific interaction between cystatin C and C4 by ligand affinity chromatography and ELISA. Interaction between the two native proteins was mimicked on replacement of one of them with the corresponding sense-antisense peptide coupled to a carrier protein, and the binding was inhibited by these synthetic peptides in solution. Through the interaction with C4, cystatin C may play a regulatory role in complement activation that might be of particular importance at tissue sites where both proteins are produced by macrophages.