Javier's main research interest is host-pathogen interactions and the basis of immune response against pathogens. The long-term objective of his research is to gain further insight into the understanding of the changes induced in the animal host after infection, by using experimental animal models, with special interest in transboundary and emerging diseases, principally Porcine Reproductive and Respiratory Syndrome in pigs and Mycobacterium spp. infection in cattle and wildlife species. Javier is also interested in the application of novel techniques to study the changes in the host after infection, such as laser-capture microdissection linked to qPCR and RNAseq and other molecular biology techniques. As a qualified veterinary pathologist, the investigation of the aetiology and pathogenesis of spontaneous diseases are always a nice challenge to accept. A true believer in the 3Rs, Javier is very interested in the development of animal and human disease models.
Member of the Veterinary Pathology Diagnostic Group
Fellow of the Royal College of Pathologists
Diplomate of the European College of Porcine Health Management
Member of the European Society of Veterinary Pathology
Member of the European Association of Porcine Health Management
Member of the Spanish Society of Veterinary Anatomic Pathology
Member of the Editorial Board of the Journal of Medical Microbiology Case Reports
Associate Editor (Pathology) of Frontiers in Veterinary Science
Find me on campus Room: 01 VSM 02
Monday to Friday 9.30 - 18.00, by appointment only
Background: Sheep have been traditionally considered as less susceptible to Mycobacterium bovis (Mbovis) infection than other domestic ruminants such as cattle and goats. However, there is increasing evidence for the role of this species as a domestic Mbovis reservoir, mostly when sheep share grazing fields with infected cattle and goats. Nevertheless, there is a lack of information about the pathogenesis and the immune response of Mbovis infection in sheep. The goals of this study were to characterize the granuloma stages produced by the natural infection of Mbovis in sheep, to compare them with other species and to identify possible differences in the sheep immune response. Samples from bronchial lymph nodes from twelve Mbovis-naturally infected sheep were used. Four immunohistochemical protocols for the specific detection of T-lymphocytes, B-lymphocytes, plasma cells and macrophages were performed to study the local immune reaction within the granulomas.
Results: Differences were observed in the predominant cell type present in each type of granuloma, as well as differences and similarities with the development of tuberculous granulomas in other species. Very low numbers of T-lymphocytes were observed in all granuloma types indicating that specific cellular immune response mediated by T-cells might not be of much importance in sheep in the early stages of infection, when macrophages are the predominant cell type within lesions. Plasma cells and mainly B lymphocytes increased considerably as the granuloma developed being attracted to the lesions in a shift towards a Th2 response against the increasing amounts of mycobacteria. Therefore, we have proposed that the granulomas could be defined as initial, developed and terminal.
Conclusions: Results showed that the study of the lymphoid tissue granulomata reinforces the view that the three different types of granuloma represent stages of lesion progression and suggest an explanation to the higher resistance of sheep based on a higher effective innate immune response to control tuberculosis infection.
Influenza is a major health threat, and a broadly protective influenza vaccine would be a significant advance. Signal Minus FLU (S-FLU) is a candidate broadly protective influenza vaccine that is limited to a single cycle of replication, which induces a strong cross-reactive T cell response but a minimal Ab response to hemagglutinin after intranasal or aerosol administration. We tested whether an H3N2 S-FLU can protect pigs and ferrets from heterosubtypic H1N1 influenza challenge. Aerosol administration of S-FLU to pigs induced lung tissue-resident memory T cells and reduced lung pathology but not the viral load. In contrast, in ferrets, S-FLU reduced viral replication and aerosol transmission. Our data show that S-FLU has different protective efficacy in pigs and ferrets, and that in the absence of Ab, lung T cell immunity can reduce disease severity without reducing challenge viral replication.
Influenza virus infection is a significant global health threat. Because of the lack of cross protective universal vaccines, short time window during which antivirals are effective and drug resistance, new therapeutic anti-influenza strategies are required. Broadly cross-protective antibodies that target conserved sites in the hemagglutinin (HA) stem region, have been proposed as therapeutic agents. FI6 is the first proven such monoclonal antibody to bind to H1-H16 and is protective in mice and ferrets. Multiple studies have shown that Fc-dependent mechanisms are essential for FI6 in vivo efficacy. Here we show that therapeutic administration of FI6 either intravenously or by aerosol to pigs did not reduce viral load in nasal swabs or broncho-alveolar lavage, but aerosol delivery of FI6 reduced gross pathology significantly. We demonstrate that pig Fc receptors do not bind human IgG1 and that FI6 did not mediate antibody dependent cytotoxicity (ADCC) with pig PBMC, confirming that ADCC is an important mechanism of protection by anti-stem antibodies in vivo. Enhanced respiratory disease, which has been associated in pigs with cross-reactive non-neutralising anti-HA antibodies, did not occur after FI6 administration. Our results also show that in vitro neutralizing antibody responses are not a robust correlate of protection for the control of influenza infection and pathology in a natural host model.
Objectives: To analyse the biological effects of a 1920 nm endovenous laser (EVL) on extra-fascial great saphenous vein (GSV) in vitro.
Methods: A 10 cm length of a large tributary bypassing a hypoplastic segment of the GSV (sometimes called an “extra-fascial GSV”) was obtained during routine varicose vein surgery. The length was treated in five sections with different LEEDs (0 (control), 20, 40, 60, and 80 J/cm) with a 1920 nm EVL at 4W power, in a novel in vitro treatment model. The biological effects were assessed by histological staining of the samples for haematoxylin and eosin (HE) and Martius Scarlet Blue (MSB), and by immunofluorescent detection of p-p53 and VCAM-1.
Results: Histological analysis showed significant structural damage at LEEDs above 60 J/cm, especially in the intima and media, with the treatment at 80 J/cm causing perforation of the vein wall. In addition, there was a significant increase in p-p53 expression in treated tissue at 60 and 80 J/cm.
Conclusions: Using this ex vivo model, the results indicate that in vitro treatment with a 1920 nm EVL, at or above an LEED of 60 J/cm and 4 W power, causes significant vein wall cell death reaching deep into the media by a combination of direct thermal damage and apoptosis. A wavelength of 1920 nm appears to be effective for the endovenous ablation of truncal veins.
Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of NAFLD and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE-/-) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model of NAFLD. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinylated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r=0.520, p<0.0001). Conclusions: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.
Many of the molecular and pathological features associated with human Alzheimer disease (AD) are mirrored in the naturally occurring age-associated neuropathology in the canine species. In aged dogs with declining learned behaviour and memory the severity of cognitive dysfunction parallels the progressive build up and location of Aβ in the brain. The main aim of this work was to study the biological behaviour of soluble oligomers isolated from an aged dog with cognitive dysfunction through investigating their interaction with a human cell line and synthetic Aβ peptides. We report that soluble oligomers were specifically detected in the dog’s blood and cerebrospinal fluid via anti-oligomer- and anti-Aβ specific binders. Importantly, our results reveal the potent neurotoxic effects of the dog’s cerebrospinal fluid on cell viability and the seeding efficiency of the cerebrospinal fluid-borne soluble oligomers on the thermodynamic activity and the aggregation kinetics of synthetic human Aβ. The value of further characterising the naturally occurring Alzheimer-like neuropathology in dogs using genetic and molecular tools is discussed.
The Mycobacterium tuberculosis complex (MTBC) is the collective term given to the group of bacteria that cause tuberculosis (TB) in mammals. It has been reported that M. tuberculosis H37Rv, a standard reference MTBC strain, is attenuated in cattle compared to Mycobacterium bovis. However, as M. tuberculosis H37Rv was isolated in the early 1930s, and genetic variants are known to exist, we sought to revisit this question of attenuation of M. tuberculosis for cattle by performing a bovine experimental infection with a recent M. tuberculosis isolate. Here we report infection of cattle using M. bovis AF2122/97, M. tuberculosis H37Rv, and M. tuberculosis BTB1558, the latter isolated in 2008 during a TB surveillance project in Ethiopian cattle. We show that both M. tuberculosis strains caused reduced gross and histopathology in cattle compared to M. bovis. Using M. tuberculosis H37Rv and M. bovis AF2122/97 as the extremes in terms of infection outcome, we used RNA-Seq analysis to explore differences in the peripheral response to infection as a route to identify biomarkers of progressive disease in contrast to a more quiescent, latent infection. Our work shows the attenuation of M. tuberculosis strains for cattle, and emphasizes the potential of the bovine model as a ‘One Health’ approach to inform human TB biomarker development and post-exposure vaccine development.
Cell motility and migration is a complex, multi-step, and multi-component process, intrinsic to progression and metastasis. Motility is dependent on the activity of integrin receptors and Rho-family GTPases resulting in the remodelling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumourigenesis largely depends on the expression of the small tumour antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumourigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we have described the action of MCPyV ST on the microtubule network and how this impacts on cell motility and migration. Here we demonstrate that MCPyV ST affects the actin cytoskeleton, to promote the formation of filopodia, through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activity of Rho-family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of β1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumour virus induces cell motility, which may ultimately lead to cancer metastasis and provides opportunities and strategies for targeted interventions for disseminated MCC.
Porcine reproductive and respiratory syndrome viruses (PRRSV) present a wide phenotypic and genetic diversity. Experimental infections have demonstrated viral replication, including highly pathogenic strains (HP-PRRSV), in primary lymphoid organs such as the thymus. However, studies of the bone marrow are scarce but necessary to help elucidate the immunobiology of PRRSV strains of differing virulence. In this study, whereas viral RNA was detected within the bone marrow of animals experimentally infected with both low virulent Lelystad (LV) and 215-06 PRRSV-1 strains and with the highly virulent SU1-bel strain, PRRSV positive cells were only occasionally detected in one SU1-bel infected animal. PRRSV RNA levels were associated to circulating virus with the highest levels detected in LV-infected pigs. At 3 dpi, a decrease in the proportion of haematopoietic tissue and number of erythroid cells in all infected groups was associated with an increase in TUNEL or cleaved caspase 3 labelling and higher counts of myeloid cells compared to control. The expression of IL-1α and IL-6 was elevated at the beginning of the infection in all infected animals. The expression of TNF-α was increased at the end of the study in all infected groups with respect to control. Different PRRSV-1 strains induced, presummably by indirect mechanisms and independently of viral load and strain virulence, moderate and sustained hypoplasia of erythroid cells and myeloid cell hyperplasia at early stages of infection. These changes were paralleled by a peak in the local expression of IL-1α, IL-6 and TNF-α in all infected groups.
Organ tissue damage is a key contributor to host morbidity and mortality following infection with microbial agents. Severe immune responses, excessive cellular recruitment and necrosis of cells all play a role in disease pathology. Understanding the pathogenesis of disease can aid in identifying potential new therapeutic targets or simply act as a diagnostic tool. Burkholderia pseudomallei is a gram-negative bacterium that can cause acute and chronic diseases. The BALB/c mouse has been shown to be highly susceptible to aerosol challenge with B. pseudomallei and hence acts as a good model to study the acute and potentially lethal form of the disease melioidosis. In our study, BALB/c mice were challenged and culled at pre-determined time points to generate a pathological time course of infection. Lung, liver and spleen were subjected to pathological and immunohistochemical analysis. The number and type of microscopic lesions within each organ, as well as the location and the mean percentage of neutrophils, B cells, T cells and Burkholderia capsule antigen within the lesions were all characterised during the time course. Neutrophils were determined as the key player in tissue pathology and generation of lesions, with B cells playing an insignificant role. This detailed pathological assessment increases our understanding of B. pseudomallei disease.
Background: Visceral leishmaniasis (VL) is a neglected tropical disease (NTD), caused by the intracellular protozoan parasites Leishmania donovani and Leishmania infantum. Symptomatic VL is considered fatal when left untreated. At present, there is no effective vaccine licensed for human use and available chemotherapies have limitations. Understanding the local immune mechanisms required for the control of infection is a key factor for developing effective vaccines and therapeutics.
Methods: We have investigated the development of the typical granulomatous lesions in the liver in experimental VL over time, together with the local immune responses. BALB/c mice were infected intravenously with a dose of 2 × 107 L. donovani amastigotes (MHOM/ET/67/HU3) and sacrificed at 15, 35 and 63 days post-infection (dpi). Histopathology and immunohistochemical techniques were used for the detection of Leishmania antigen, selected cell types including B and T lymphocytes, macrophages and neutrophils (CD45R-B220+, CD3+, F4/80+ and Ly-6G+) and iNOS.
Results: Granulomatous lesions were identified as early as 15 dpi in the livers of all infected animals. Three categories were used to classify liver granulomas (immature, mature and clear). Clear granulomas were exclusively detected from 35 dpi onwards. Kupffer cells (F4/80+) were predominant in immature granulomas, regardless of the dpi. Nonetheless, the highest expression was found 63 dpi. Positive staining for iNOS was mainly observed in the cytoplasm of fused Kupffer cells and the highest expression observed at 35 dpi. T cells (CD3+) and B cells (CD45R-B220+) were predominant in more advanced granuloma stages, probably related to the establishment of acquired immunity. Neutrophils (Ly-6G+) were predominantly observed in mature granulomas with the highest expression at 15 dpi. Neutrophils were lower in numbers compared to other cell types, particularly at later time points.
Conclusions: Our results reflect the role of macrophages during the early stage of infection and the establishment of a lymphocytic response to control the infection in more advanced stages.
The European badger (Meles meles) is a reservoir host of Mycobacterium bovis and responsible for a proportion of the tuberculosis (TB) cases seen in cattle in the United Kingdom and Republic of Ireland. An injectable preparation of the bacillus Calmette-Guérin (BCG) vaccine is licensed for use in badgers in the UK and its use forms part of the bovine TB eradication plans of England and Wales. However, there are practical limitations to the widespread application of an injectable vaccine for badgers and a research priority is the development of an oral vaccine deliverable to badgers in bait. Previous studies reported the successful vaccination of badgers with oral preparations of 108 colony forming units (CFU) of both Pasteur and Danish strains of BCG contained within a lipid matrix composed of triglycerides of fatty acids. Protection against TB in these studies was expressed as a reduction in the number and apparent progression of visible lesions, and reductions in the bacterial load and dissemination of infection. To reduce the cost of an oral vaccine and reduce the potential for environmental contamination with BCG, it is necessary to define the minimal efficacious dose of oral BCG for badgers. The objectives of the two studies reported here were to compare the efficacy of BCG Danish strain in a lipid matrix with unformulated BCG given orally, and to evaluate the efficacy of BCG Danish in a lipid matrix at a ten-fold lower dose than previously evaluated in badgers. In the first study, both BCG unformulated and in a lipid matrix reduced the number and apparent progression of visible lesions and the dissemination of infection from the lung. In the second study, vaccination with BCG in the lipid matrix at a ten-fold lower dose produced a similar outcome, but with greater intra-group variability than seen with the higher dose in the first study. Further research is needed before we are able to recommend a final dose of BCG for oral vaccination of badgers against TB or to know whether oral vaccination of wild badgers with BCG will significantly reduce transmission of the disease.
Cattle vaccination against bovine tuberculosis (bTB) has been proposed as a supplementary method to help control the incidences of this disease. Bacillus Calmette-Guérin (BCG) is currently the only viable candidate vaccine for immunisation of cattle against bTB, caused by Mycobacterium bovis (M. bovis). In an attempt to characterise the differences in the immune response following M. bovis infection between BCG vaccinated and non-vaccinated animals, a combination of gross pathology, histopathology and immunohistochemical (IHC) analyses was used. BCG vaccination was found to significantly reduce the number of gross and microscopic lesions present within the lungs and lymph nodes. Additionally, the microscopically visible bacterial load of stage III and IV granulomas was reduced. IHC using cell surfaces markers revealed the number of CD68+ (macrophages), CD3+ (T-lymphocytes) and WC1+ cells (γδ T-cells) to be significantly reduced in lymph node granulomas of BCG vaccinated animals, when compared to non-vaccinated animals. B lymphocytes (CD79a+) were significantly increased in BCG-vaccinated cattle for granulomas at stages II, III and IV. IHC staining for iNOS showed a higher expression in granulomas from BCG vaccinated animals compared to non-vaccinated animals for all stages, being statistically significant in stages I and IV. TGFβ expression decreased alongside the granuloma development in non-vaccinated animals, whereas BCG vaccinated animals showed a slight increase alongside lesion progression. IHC analysis of the cytokines IFN-γ and TNFα demonstrated significantly increased expression within the lymph node granulomas of BCG vaccinated cattle. This is suggestive of a protective role for IFNγ and TNFα in the response to M. bovis infection. Findings shown in this study suggest that the use of BCG vaccine, can reduce the number and severity of lesions , induce a different phenotypic response and increase the local expression of key cytokines related to protection.
Porcine reproductive and respiratory syndrome virus (PRRSV) exists as two distinct viruses, type 1 (PRRSV-1) and type 2 (PRRSV-2). Atrophy of the thymus in PRRSV-2 infected piglets has been associated with a loss of thymocytes. The present study aimed to evaluate the impact of PRRSV-1 strains of differing virulence on the thymus of infected piglets by analysing the histomorphometry, the presence of apoptotic cells and cells producing cytokines. Thymic samples were taken from animals experimentally infected (with LV, SU1-bel, and 215-06 strains) or mock inoculated animals at 3, 7 and 35 days post-infection (dpi) and processed for histopathological and immunohistochemical analyses. PRRSV antigen was detected in the thymus from 3dpi until the end of the study in all virus-infected animals with the highest numbers of infected cells detected in SU1-bel group. The histomorphometry analysis and counts of CD3+ thymocytes in the thymic cortex displayed significant differences between strains at different time-points (p ≤ 0.011), with SU1-bel group showing the most severe changes at 7dpi. Cell death displayed statistically significant increase in the cortex of all infected animals, with SU1-bel group showing the highest rate at 3 and 7dpi. The number of cells immunostained against IL-1α, TNF-α and IL-10 were predominantly detected in the medulla (p ≤ 0.01). An increase in the number of TNF-α and IL-10 positive cells was observed in LV and SU-1bel groups. Our results demonstrate that different PRRSV-1 strains induced depletion of the thymic cortex due to apoptosis of thymocytes and that the most severe depletion was associated with the highly virulent SU1-bel strain.
Varicose veins, often thought as a purely cosmetic issue, are a common symptom resulting from chronic venous insufficiency (CVI). CVI can result in serious fasciocutaneous and haematological complications. In 2013, NICE recommended that the treatment of varicose veins should preferably be carried out by endothermal ablation (ETA). The mechanism of action of ETA is still not fully understood. In 2004 it was suggested that successful treatment must involve transmural vein wall death (TMVD) in the target vein. Through the development of a novel in-vitro¬ model, using ex-vivo veins, the work carried out in this thesis has indicated that TMVD is reliant on a combination of thermal necrosis, followed by the upregulation of apoptosis. The novel model has been used to compare four different ETA techniques; endovenous laser ablation (EVLA) using an 810nm endovenous laser (EVL) with a jacketed fibre, EVLA using a 1470nm EVL with a jacketed fibre, EVLA using a 1470nm EVL with a radial fibre and Radiofrequency-induced Thermo Therapy (RFiTT). The comparisons indicate that treatment with the 810nm EVL is inferior at causing TMVD, in comparison to the 1470nm EVL. Treatment with a radial fibre, compared to a jacketed fibre, when using a 1470nm EVL, shows an improved damage profile that is much more homogenous with less overtreatment of target tissue. Comparisons between RFiTT and EVLA, indicate that treatment with RFiTT is as effective at causing TMVD as the 1470nm EVL with a radial fibre but with differences in the thermal damage profile. These results correlate well with reports from the current literature. However, this novel model has been able to show that the upregulation of apoptosis plays a pivotal role in TMVD after ETA treatment. The results also show that histology is often not sufficient to alone determine the difference between successful and inadequate treatment.
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