Alejandro Ferrari

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.

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.

Skin Exposure to Chronic But Not Acute UV Radiation Affects Peripheral T-Cell Function

Ultraviolet (UV) radiation (UVR) produces deleterious effects that may finally lead to carcinogenesis. These adverse effects include tissue inflammation, free radical formation with consequent oxidation of proteins and lipids, DNA damage, and immune function suppression. The aim of this study was to evaluate the effects of UVR at the local and systemic levels following acute (4 consecutive days with 0.5 minimal erythema dose [MED]) or chronic (20 consecutive days with 0.25 MED) exposure. Locally, histological alterations and epidermal T-cell populations were studied. Systemically, inguinal lymph-node and spleen T cells were analyzed with respect to proliferative response and cytokine production against a nonspecific mitogen. Lymph-node T-cell populations were also characterized. Our results indicated that while both acute and chronic UVR produced epidermal hyperplasia and a decrease in epidermal T-cell density, acute UVR increased T-cell proliferative response, while chronic UVR produced the opposite effect, shifting the cytokine production toward a Th2/Treg profile. Therefore, even though acute irradiation produced a direct effect on skin, it did not correlate with a marked modification of overall T-cell response, which is in contrast to marked effects in chronically irradiated animals. These findings may contribute to understanding the clinical relevance of occupational UVR exposure, typically related to outdoor activities, which is associated with nonmelanoma skin carcinogenesis.

Mitochondrial function evaluation in epidermal cells ex vivo after ultraviolet irradiation.

Abstract:  Ultraviolet radiation (UVR) effects on skin have been extensively studied. However, mitochondrial dysfunction and superoxide () production have only been studied using cell cultures, which are useful models, but do not consider the crosstalk between tissues or cellular differentiation. We aimed to evaluate the usefulness of fluorescent dyes to study skin ex vivo. Mitochondrial alterations were evaluated in epidermal cells isolated from UVR‐exposed mice. Furthermore, a combination of dyes and antibodies was tested to analyse specific skin cell types. UVR caused a decrease in the percentage of total cells with polarized mitochondria, but did not change the mitochondrial production. However, this production was increased significantly in cells. Furthermore, it was possible to evaluate the cellular damage produced to basal keratinocytes and Langerhans cells. The results show that fluorescent dyes – alone or in combination with antibodies – are useful to analyse cellular events that take place in whole organs.

Identification of Lama glama as Reservoirs for Acinetobacter lwoffii

South American Camelids have an increasing relevance in local economies, worldwide. These animals are bred for their meat, fur and as companion and therapy animals. Thus, their sanitary status should be well-established. According to the OIE (World Organization for Animal Health), respiratory infections mainly produced by Pasteurella spp. have been reported for camelids. It has been stated that this microorganism causes a mild disease, although many authors report it is an important cause of mortality among alpacas. Nevertheless, the incidence of infection by Pasteurella spp. in camelids still needs to be investigated. The aim of the present study was to analyze the occurrence of nasopharyngeal colonization of Lama glama by respiratory bacteria, and to assess the usefulness of serological tests for clinical diagnosis. The colonization was studied by culture techniques carried out with material taken by nasopharyngeal swabs. Bacterial isolates were first phenotypically characterized and then identified by MALDI/TOF-MS. The presence of specific serum antibodies was studied by ELISA and Western blot. In the present work Pasteurella spp. was not found. Nevertheless, we report for the first time, the colonization of L. glama by bacteria of the Acinetobacter lwoffii, at a reliable level in 19.4% of the animals. Acinetobacter species are found in different environmental sources, as well as vegetables, animals, and humans, and their role in infections has recently gained relevance. The results presented herein contribute to a better understanding of the respiratory microbiota in camelids, and increase the knowledge about environmental distribution of Acinetobacter non-baumanii species. Given that these respiratory bacteria might be the cause of infection among cattle, and even humans, this report highlights the need for further research.

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 β-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.

Biophysical characterisation of the recombinant human frataxin precursor

Friedreichs ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐TAT‐FXN1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐TAT‐FXN1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro: the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐TAT‐FXN1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6‐TAT‐FXN1‐210. Moreover, the results showed that His6‐TAT‐FXN1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1‐210, suggesting that the His6‐TAT extension does not interfere with the ubiquitination machinery.