Juliana Leoni

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.

Mitochondrial dysfunction and cellular stress progression after ultraviolet B irradiation in human keratinocytes.

Background: Ultraviolet (UV) radiation is the major environmental harmful factor that affects human skin. UVB radiation is known to be a potent inducer of reactive oxygen species (ROS) production and has also been associated with the generation of nitric oxide (NO), all of which have been implicated in various skin disorders. It is well known that mitochondria can also be affected by UVB, leading to alterations in their membrane structure and permeabilization with cytochrome c release, which consequently affects the cell function. However, the loss of keratinocyte mitochondrial function generated by UVB, as well as its kinetics, has not been characterized completely.

Skin damage and mitochondrial dysfunction after acute ultraviolet B irradiation: relationship with nitric oxide production

Background: Ultraviolet (UV) radiation is the main environmental carcinogen. It is able to induce injury in the keratinocytes, which triggers mechanisms in order to protect the skin against molecular alterations that may lead to the development of skin cancer. UVB is capable of producing genotoxic damage, directly or indirectly through reactive oxygen species, inducing DNA alterations and mutations. UVB radiation has also been associated with the generation of nitric oxide (NO), which is able to induce many physiological and physiopathological processes. The aim of the current study was to investigate the effect of UVB irradiation in hairless mice skin.

Time-course evaluation and treatment of skin inflammatory immune response after ultraviolet B irradiation.

Skin exposure to high doses of ultraviolet B (UVB) radiation generates a severe inflammatory skin response. In the present study we aim to investigate, using in vitro and in vivo models, the time-course of the inflammatory skin immune response after an acute exposure to UVB irradiation, as well as its modulation by a topical non-steroidal anti-inflammatory drug (NSAID) treatment, naproxen. PGE2 production and TNF-alpha levels increase in a post-irradiation time-dependent manner both in vivo and in vitro. This production pattern is also reflected in the iNOS expression levels in vivo and in the IL-6 levels in vitro. Changes observed in these mediators are correlated with histological alterations and dermal infiltration after the acute UVB irradiation. Naproxen treatment notably reduces PGE2 production and iNOS expression, reflecting the COX-NOS crosstalk already reported, although it causes an important increment in TNF-alpha synthesis in the epidermis of irradiated mice. Taken together, our data indicates that the epidermis is severely damaged by UVB radiation but then it is able to fully recover, and that the immune response is modulated by the NSAID treatment, since it is able to reduce the levels of some mediators as well as it can increase others.

Lipoteichoic acid from Lactobacillus rhamnosus GG as an oral photoprotective agent against UV-induced carcinogenesis

Probiotics are live micro-organisms that when administered in adequate amounts confer a health benefit on the host. Cell surface molecules of these micro-organisms are being studied in relation to their ability to interact with the host. The cell wall of lactobacilli possesses lipoteichoic acids (LTA) which are molecules with immunomodulatory properties. UV radiation (UVR) has been proposed as the main cause of skin cancer because of its mutagenic and immunosuppressive effects. Photoprotection with some nutrition interventions including probiotics has recently been shown. The aim of the present study was to investigate whether the oral administration of purified LTA from Lactobacillus rhamnosus GG can modulate the immune-suppressive effect of UVR and skin tumour development in female Crl:SKH-1-hrBR mice. For this purpose, two irradiation models were studied: (1) a chronic irradiation scheme consisting of daily irradiations during twenty consecutive days and (2) a long-term irradiation schedule, irradiating the animals three times per week, during 34 weeks for tumour development. The results showed that T-cells in the inguinal lymph node of LTA-treated mice produced higher levels of (1) interferon-γ and (2) a number of total, helper and cytotoxic T-cells compared with non-treated mice. Moreover, a significant delay in tumour appearance was found in LTA-treated mice. An increased IgA⁺ cell number was found in the small intestine together with a higher number of activated dendritic cells in the mesenteric lymph nodes. The latter results might be indicative of a direct effect of LTA in the gut, affecting the cutaneous immune system and restoring homeostasis through the gut-skin axis.

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 VHCDR3, VLCDR1, and VLCDR3 form the cavity, whereas the small pocket is defined essentially by residues from the hypervariable loops VHCDR1 and VHCDR2. This fact could explain the VH4-34 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 Å wide and 24 Å 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 CH1:CL domain. The structural analysis shows that the CH1-CL interface is mainly conserved during the isotype switch process from IgM to IgG1.

Alterations in Skin Immune Response Throughout Chronic UVB Irradiation—Skin Cancer Development and Prevention by Naproxen

Skin exposure to UVB radiation has been reported to produce both a significant inflammatory response and marked immunosuppression. This work was aimed to evaluate whether the response of murine skin to an acute UVB dose was modified by pre‐exposure to chronic UVB irradiation and by topical treatment with naproxen, a nonsteroidal anti‐inflammatory drug. Moreover, the effect of naproxen on the incidence of UV‐induced skin tumors was studied. Prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF‐α) levels were increased 96 h post‐UVB in acutely irradiated animals and both mediators were modified by topical naproxen application—PGE2 was decreased while TNF‐α was increased. Such inflammatory response was suppressed when mice were chronically irradiated. Naproxen application on chronically irradiated mice reduced the incidence of tumor lesions. Taken together, our data suggest that chronic UVB irradiation generates an immunosuppressive state that prevents skin cells from responding normally to an acute irradiation challenge, thus impairing the protective effect of TNF‐α against skin tumor development. Furthermore, reduction in the incidence of tumor lesions by naproxen may be due to its ability to increase TNF‐α levels as well as to decrease PGE2.

Development of ELISAs for the measurement of IgM and IgG subclasses in sera from llamas (Lama glama) and assessment of the humoral immune response against different antigens

Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.

Up-regulated expression of MICA and proinflammatory cytokines in skin biopsies from patients with seborrhoeic dermatitis

Seborrhoeic dermatitis is a disease of unknown etiopathogenesis that affects 5% of the population. In this study, we investigated expression of mRNA for IL-1 alpha, IL-6, IL-4, IFN-gamma, and the stress-inducible MICA molecule in skin biopsies from 12 patients with moderate to severe seborrhoeic dermatitis and 2 healthy volunteers by RT-PCR and hybridization with specific probes. Eight patients expressed INF-gamma, 2 expressed IL-6, 8 expressed IL-1 alpha, and 2 expressed IL-4 (1 with moderate disease). Eight patients expressed inflammatory cytokines (IL-1 alpha, IL-6, and/or IFN-gamma) in healthy skin. Higher cytokine mRNA in damaged vs healthy skin was also observed, suggesting the existence of an inflammation that predisposes healthy skin to develop overt disease. Up-regulated expression of MICA mRNA was observed in 8 patients. Although the pathogenesis of seborrhoeic dermatitis remains to be elucidated, expression of cytotoxicity-activating ligands (MICA), recruitment of NK cells, and a local pro-inflammatory microenvironment may facilitate the development of tissue injury.

Non-precipitating antibodies isolated by immunoadsorption

Abstract In previous papers we have eliminated several possibilities regarding the lack of formation of insoluble complexes when antigen was mixed with purified non-precipitating antibody from different mammalian species by immunoadsorption. From the results of this investigation it seems reasonable to assume that in the two antigen binding arms of non-precipitating antibody there exist combining sites which behave differently, expecially with large antigens. This assumption is based on the following: (1) the difference in K 0 values between anti-DNP sheep precipitating and non-precipitating IgG1 is no greater than 1 × 10 1 M −1 ; (2) the bi-modal characteristics of the Scatchard plots when different populations of non-precipitating antibody (isolated by affinity chromatography from the same serum sample) are analyzed; and (3) the formation of precipitating antibody upon random hybridization of non-precipitating antibody half-molecules. The foregoing should enable us to understand why non-precipitating antibody, which does not give rise to insoluble complexes with antigen, can become firmly bound and incorporated into the complexes formed by precipitating antibody.