Two patients with ciguatera toxicity: a seafood poisoning in travellers to (sub) tropical areas.

Ciguatera toxicity is a type of seafood poisoning caused by the consumption of ciguatoxic reef fish. We describe two patients with characteristic gastrointestinal and neurological symptoms, both of whom had eaten local seafood. Although mortality is low, morbidity can be considerable due to debilitating symptoms. Most cases originate in the (sub)tropics but due to expanding tourism and fish exportation, it may be encountered in more temperate regions. Treatment is supportive, but some benefit from intravenous mannitol has been reported.

Benefits of flu vaccination for persons with diabetes mellitus: A review.

Diabetes mellitus imposes a significant and increasing burden on society, with major consequences for human health, welfare and the economy worldwide. Persons with diabetes mellitus are at increased risk of developing severe complications after influenza virus infection and guidelines advise vaccination. The present evidence for influenza vaccine effectiveness in persons with diabetes mellitus is mainly based on observational studies with clinical endpoints like hospitalization and death, indicating a beneficial reduction of morbidity and mortality. Further supportive evidence comes from serological studies, in which persons with diabetes mellitus usually develop similar antibody levels after vaccination as healthy people. Observational studies may be prone to selection bias, and serological studies may not completely mirror vaccine effectiveness in the field. Although more controlled trials in persons with diabetes mellitus with laboratory-confirmed, influenza-specific outcomes would be desirable to better estimate the effect of vaccination, the currently available data justify routine influenza vaccination in persons with diabetes mellitus. As in this risk group, the use of influenza vaccine is far below target worldwide, efforts should be made to increase vaccination coverage.

An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

Health care costs attributable to overweight calculated in a standardized way for three European countries.

This article presents a tool to calculate health care costs attributable to overweight in a comparable and standardized way. The purpose is to describe the methodological principles of the tool and to put it into use by calculating and comparing the costs attributable to overweight for The Netherlands, Germany and Czech Republic. The tool uses a top-down and prevalence-based approach, consisting of five steps. Step one identifies overweight-related diseases and age- and gender-specific relative risks. Included diseases are ischemic heart disease, stroke, hypertension, type 2 diabetes mellitus, colorectal cancer, postmenopausal breast cancer, endometrial cancer, kidney cancer and osteoarthritis. Step two consists of collecting data on the age- and gender-specific prevalence of these diseases. Step three uses the population-attributable prevalence to determine the part of the prevalence of these diseases that is attributable to overweight. Step four calculates the health care costs associated with these diseases. Step five calculates the costs of these diseases that are attributable to overweight. Overweight is responsible for 20-26% of the direct costs of included diseases, with sensitivity analyses varying this percentage between 15-31%. Percentage of costs attributable to obesity and preobesity is about the same. Diseases with the highest percentage of costs due to overweight are diabetes, endometrial cancer and osteoarthritis. Disease costs attributable to overweight as a percentage of total health care expenditures range from 2 to 4%. Data are consistent for all three countries, resulting in roughly a quarter of costs of included diseases being attributable to overweight.

Zika virus and the current outbreak: an overview.

Zika virus (ZIKV), a mosquito-borne flavivirus closely related to yellow fever virus and dengue virus, is currently causing a large outbreak in the Americas. Historically, ZIKV infection was considered a sporadic, relatively mild disease characterised by fever, maculopapular rash, conjunctivitis and often arthralgia. However, current observational studies suggest that ZIKV may cause more severe neurological sequelae such as Guillain-Barre syndrome, and birth defects, mainly microcephaly, in babies of whom the mother was infected with ZIKV during pregnancy. This article provides a clinically focussed overview of ZIKV, with emphasis on the current outbreak, clinical manifestations, diagnostic tools and caveats.

Targeting early events in T cell activation to construct improved vaccines.

Live, attenuated vaccines currently offer the best protection against virulent pathogens. Recent advances in Immunology and Molecular Biology provide an opportunity to design vaccines that will be more effective and safer than existing ones. Immunologists are rapidly developing the capacity to identify and construct the minimal immunogenic units from pathogens. The molecular signals required to fully activate antigen presenting cells (APCs) and responder T cells are becoming apparent. Improved vaccine delivery systems are being designed which will mimic the actions of pathogens in vivo. These vaccines will incorporate protective epitopes fused to immunoregulatory cytokines in chimeric proteins. They will be encapsulated in formulations which allow for the slow release of these chimeric proteins thereby inducing the memory T cells required for long-lived immunity. These vaccine formulations will target receptors present on the most active APCs. Here we discuss how these advances will allow us to rationally construct "virtual pathogens" which will provide improved protection against new and old microbial foes.

In vitro control of Mycobacterium bovis by macrophages.

Mycobacterium bovis is frequently seen inside macrophages in vivo. The outcome of M. bovis infection depends on T cell interactions with macrophages, however mycobacteria are thought to be relatively resistant to macrophage killing. Little is known about the immunological mechanisms which control intracellular growth of M. bovis, and in the absence of T cell help the organism is capable of intracellular survival and replication. We have investigated the role of macrophages in controlling growth of virulent M. bovis or M. bovis BCG in vitro. At a multiplicity of infection of 5:1, macrophages from a range of animal species including cattle, deer, possums, ferrets and mice restricted growth of BCG while M. bovis grew progressively. Inter-species variation in controlling growth of M. bovis by alveolar macrophages was observed. Pre-treatment of macrophages with interferon-gamma and lipopolysaccharide inhibited intracellular growth of M. bovis. Addition of freshly recruited macrophages further inhibited M. bovis, and intracellular growth was arrested by activated fresh macrophages. Our observations suggest that naïve macrophages can prevent BCG growth, while T cell activation in conjunction with freshly recruited macrophages is required for preventing growth of M. bovis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells.

Diindolylmethane (DIM) is an acid-catalyzed condensation product of indole-3-carbinol, a constituent of cruciferous vegetables, and is formed in the stomach. DIM alters estrogen metabolism and inhibits carcinogen-induced mammary tumor growth in rodents. DIM is a weak agonist for the aryl hydrocarbon (Ah) receptor and blocks the effects of estrogens via inhibitory Ah receptor-estrogen receptor cross-talk. DIM and various structural analogs were examined in H295R cells for effects on 3 cytochrome P450 (CYP) enzymes involved in estrogen synthesis and/or metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase activity was measured by conversion of 1 beta-(3)H-androstenedione to estrone and (3)H(2)O. H295R cells were exposed to the test chemicals dissolved in dimethyl sulfoxide for 24 h prior to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (0--30 nM) and DIM (0--10 microM) induced ethoxyresorufin-O-deethylase (EROD) activity, as a measure of CYP1A1 and possibly 1B1 activity, with EC(50) values of about 0.3 nM and 3 microM, respectively. DIM, but not TCDD, induced aromatase activity with an apparently maximal 2-fold increase at 10 microM; higher concentrations of DIM and many of its analogs were cytotoxic. TCDD (30 nM) significantly increased CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for CYP19. DIM (3 microM) significantly increased mRNA levels for all three CYPS: DIM analogs with substitutions on the 5 and 5' position (3 microM) induced aromatase and EROD activity, together with mRNA levels of CYP1A1, 1B1, and 19; analogs that were substituted on the central carbon of the methane group showed little or no inductive activity toward the CYPS: In conclusion, DIM and several of its analogs appear to induce CYPs via multiple yet distinct pathways in H295R human adrenocortical carcinoma cells.

In vitro responses of cervine macrophages to bacterial stimulants.

The function of cervine (deer) mononuclear phagocytes is poorly defined. In the present study, the potential of cervine macrophages to generate phagocytic and immunoregulatory responses following stimulation with bacterial products was investigated. Blood-derived macrophages of red deer were cultured in vitro with particulate stimulants (Mycobacterium bovis BCG and Staphylococcus aureus SAC) or soluble stimulants (M. bovis PPD and Escherichia coli LPS), prior to assessment of phagocytic responses, prostaglandin secretion and cytokine production. Particulate stimulants induced vigorous phagocytic responses (superoxide anion generation, lysosomal enzyme release), secretion of prostaglandin E2 and transcription of mRNA specific for the cytokines IL-1 beta, IL-10 and TNF alpha, while soluble products invoked weaker responses. These results are discussed in relation to the role of cervine mononuclear phagocytes in regulating and participating in inflammatory and immune processes relevant to bacterial challenge.

Macrophage function in deer.

Macrophage inflammatory and immune functions were characterised in red deer (cervus elaphus), for use as a model for natural infection with bovine tuberculosis. Highly enriched populations of deer macrophages were obtained from 14 day cultures of plastic-adherent peripheral blood mononuclear cells. Cervine macrophages produced superoxide anion in response to respiratory burst stimuli (serum-opsonised zymosan and phorbol myristic acetate), but nitric oxide production could not be detected under the conditions tested. The lysosomal enzymes acid phosphatase and lysozyme were detected at the intercellular and extracellular level. Stimulation with bacterial lipopolysaccharide extract (Escherichia coli LPS) enhanced the production of superoxide and acid phosphatase with a peak increase in activity observed after 2h. Production of interleukin 1 (IL-1) and tumour necrosis factor (TNF), determined using cytokine-sensitive cell lines and mRNA analysis (Northern blotting), indicated maximal secretion of both cytokines after 24 h stimulation with LPS, preceded by a peak in message accumulation at 2-6 h post-stimulation. Cervine macrophages stimulated proliferative responses in T cell-enriched lymphocyte populations derived from the peripheral blood of autologous animals that had been primed to mycobacterial antigens (Mycobacterium bovis Bacille Calmette-Guerin, BCG). Macrophages were able to stimulate responses after pulsing with particulate (BCG) or soluble (purified protein derivative) mycobacterial antigens. These results indicate that macrophage inflammatory and immune responses in red deer are similar to those in other mammalian species, and that macrophages may play an important role in resistance to mycobacterial infection.

The characterisation of a cervine immunoregulatory cytokine, interleukin 12.

The cloning, sequencing, and production of cervine interleukin-12 is described. The cervine IL-12 p35 subunit coding sequence is 666 bp long and has highest homology to bovine p35 (94%), followed by human (79%), then murine (57%). The cDNA codes for a 221 aa long protein with predicted molecular weight of 24,902 Da. The cervine p40 subunit has a coding sequence of 984 bp and shows 96% homology to bovine, 85% homology to human, and 65% homology to murine p40 respectively. Cervine p40 cDNA codes for a 327 aa long protein with a predicted molecular weight of 37,461. Both subunits were inserted into a recombinant baculovirus that was then used to produce cervine IL-12 in Trichoplusia ni cells. Interleukin-12 was secreted into the culture medium and was biologically active as measured by proliferation of mitogen sensitised peripheral blood lymphocytes and the induction of interferon-gamma transcription in peripheral blood lymphocytes.

The cloning and sequencing of cervine interleukin 10.

We report the cloning and sequencing of the cervine interleukin-10 gene. Specific cDNA was amplified by PCR using primers based on the bovine sequence. This was cloned into pGEM 5Zf and several clones were sequenced. The 762 nucleotide product coded for a 179 amino acid protein which was 86% homologous with its bovine and 77% homologous with its human counterparts. There is a strongly hydrophobic signal sequence consisting of the first 20 amino acids and a potential glycosylation site at amino acids 134-136. There are three regions, comprising 34% of the protein, which show complete homology between the cervine, bovine and human sequences. The transcription of the gene was shown by Northern Blotting where a single, 1.8kb, mRNA transcript was detected 4-8 hours after activation of peripheral blood mononuclear cells with mitogen.

Keeping venomous snakes in the Netherlands: a harmless hobby or a public health threat?

OBJECTIVE: To describe the incidence of venomous snakebites and the hospital treatment thereof (if any) amongst private individuals who keep venomous snakes as a hobby. STRUCTURE: Descriptive study. METHOD: Private keepers of venomous snakes were invited via the social media Facebook, Hyves, Twitter, Google Plus, Linked In and two large discussion forums to fill in an online questionnaire on a purely voluntary and anonymous basis. RESULTS: In the period from 1 September 2012 to 31 December 2012, 86 questionnaires were completed by individuals who keep venomous snakes as a hobby. One-third of the venomous snake keepers stated that they had at some point been bitten by a venomous snake. Out of those, two-thirds needed hospital treatment and one-third of those bitten required at least one, sometimes more, doses of antiserum. The chances of being bitten increased the more venomous snakes a person kept. An inventory of the collections of venomous snakes being kept further revealed that no antiserum exists for 16 of the species, including for the most commonly held venomous snake, the coral cobra. CONCLUSION: Keeping venomous snakes as a hobby is not without danger. Although in the majority of snakebite cases no antiserum had to be administered, there is nevertheless a significant risk of morbidity and sequelae. Preventing snakebites in the first place remains the most important safety measure since there are no antiserums available for a substantial number of venomous snakes.

DNA fusion vaccines incorporating IL-23 or RANTES for use in immunization against influenza.

The incorporation of RANTES or IL-23 into DNA vaccines may improve their immunogenicity by the recruitment and activation of dendritic cells. This may also select for a TH1 response counteracting the TH2 response which can predominate when a DNA vaccine is delivered by gene gun. We have immunized mice with various DNA constructs encoding APR/8/34 influenza virus hemagglutinin (HA), either fused to or separate from, IL-23 or RANTES using a gene gun. Those immunized with IL-23/HA fusion constructs and challenged with influenza 27 weeks post-vaccination, tended to have cleared more virus than those vaccinated with HA DNA. Mice immunized with the RANTES/HA fusion construct produced a mixed TH1/TH2 response whereas in HA-vaccinated mice, a TH2 response predominated. Immunization with a plasmid in which HA and RANTES were under the control of separate promoters, failed to generate a mixed TH1/TH2 response suggesting that enhanced antigen uptake via RANTES receptors may contribute to the mixed immune response generated to the fusion construct. Overall these findings provide further evidence that Type 1 cytokines or chemokines, fused to antigen in a DNA vaccine, can influence the nature and the longevity of the immune response and ultimately, its protective capacity.

The production and biological assessment of cervine interferon gamma.

Cervine interferon gamma (IFN-gamma) was cloned and expressed using an Escherichia coli expression system pET-32. The expressed protein contained a 6 histidine purification tag and an 11 kDa thioredoxin fusion partner 5' to the IFN-gamma molecule. The ability of IFN-gamma to inhibit the killing of Madin-Darby bovine kidney cells by Semliki forest virus was used as a measure of the bioactivity of the recombinant cervine IFN-gamma (rIFN-gamma). It was shown that the presence of the thioredoxin fusion partner 5' to the IFN-gamma molecule did not affect its biological activity. As in the mouse model, it was shown that cervine rIFN-gamma was able to down-regulate the transcription of interleukin 10 mRNA while up-regulating the transcription of interleukin 12 mRNA in lipopolysaccharide-sensitized, peripheral blood mononuclear cells. A prototype ELISA was tested for its ability to detect both recombinant and native IFN-gamma. The ELISA was able to detect rIFN-gamma at concentrations greater than 100 pg/ml. It was also used to detect native IFN-gamma produced by peripheral blood lymphocytes from Mycobacterium bovis infected or vaccinated deer after in vitro restimulation with antigen. The rIFN-gamma and the cervine IFN-gamma specific ELISA provide valuable tools with which to study important zoonotic infections in farmed and wild deer.

Influenza hemagglutinin peptides fused to interferon gamma and encapsulated in liposomes protects mice against influenza infection.

The immunogenicity of a peptide vaccine may be improved by fusing antigen to a cytokine and administering this chimeric protein in a particulate delivery system. We have investigated this using a vaccine comprising an immunodominant T cell epitope and a B cell epitope from influenza haemagglutinin (HATB) fused to interferon gamma and encapsulated in liposomes (HATB/IFN-gamma/lipo). Controls comprised groups receiving HATB/IFN-gamma mixed with liposomes, HATB incorporated in liposomes or heat inactivated PR8 influenza virus (HI PR8). IFN-gamma production in mice treated with HATB/IFN-gamma/lipo was significantly higher than in mice inoculated with either HATB/IFN-gamma mixed with liposomes or HATB incorporated in liposomes but less than HI PR8. Lung viral titres were significantly lower in mice treated with HATB/IFN-gamma/lipo compared with those treated with HATB/IFN-gamma mixed with liposomes. HI PR8-treated mice recorded a nil viral titre. There was no correlation between the level of antibody production and clearance of virus from the lungs. These data suggest that particulate delivery systems may be useful adjuncts to improve immune responses to chimeric proteins and to induce protection against disease.

IL-2 linked to a peptide from influenza hemagglutinin enhances T cell activation by affecting the antigen-presentation function of bone marrow-derived dendritic cells.

Chimeric proteins containing antigen linked to cytokines have shown some promise as vaccine candidates but little is known of their mechanism of action, particularly at the level of the antigen-presenting cell. We have investigated this using a chimeric protein in which an immunodominant T cell epitope from influenza hemagglutinin peptide (HA), recognized in the context of I-E(d), was fused to IL-2. Immature murine dendritic cells (DC) derived from bone marrow (BMDC) were used to present the chimeric protein to a T cell hybridoma with TCR specific for the HA peptide (A5 cell line). HA-IL-2 was found to induce significantly higher T cell activation than HA alone. Although the inclusion of IL-2 and HA separately did increase the response of A5 cells compared to HA alone, they were not as effective as the HA-IL-2 chimeric protein. When an antibody known to block IL-2 receptor alpha chain (CD25) was included, A5 activation was reduced, suggesting a role for the receptor in this process. Expression of CD25 on A5 cells was low during activation, implying that the effect was mediated by CD25(+) BMDC. Antigen uptake and processing of HA-IL-2 by BMDC was required since fixing BMDC, prior to antigen exposure, greatly reduced their ability to activate A5 cells. The function of CD25 on DC is currently unknown. Our results suggest this receptor may play a role in antigen uptake and subsequent T cell activation by receptor-mediated endocytosis of antigen attached to IL-2. This finding that may have implications for the development of a new generation of vaccines.

Vaccine protocols to optimise the protective efficacy of BCG.

SETTING: A deer model has been developed to study protection produced with BCG vaccination, against infection and the development of pathology, following experimental intratonsilar infection with virulent Mycobacterium bovis. OBJECTIVE: To determine how the dose of vaccine, the route of vaccination, the viability of the vaccine and exposure to glucocorticoids at the time of vaccination, may affect the protective efficacy of BCG vaccines. DESIGN: Deer were vaccinated with BCG and later challenged with virulent M. bovis via the tonsilar route. Protection against infection and development of disease was evaluated at necropsy six months after challenge with M. bovis, by histological examination and microbial culture. RESULTS: Significant protection against infection and disease were obtained following boosting with two low doses (5 x 10(4) cfu) or moderate doses (5 x 10(7) cfu) of live (freshly cultured and lyophilized) BCG. Inferior levels of protection were obtained with high dose (5 x 10(8) cfu) of live BCG. Similar levels of protection were found with vaccines given subcutaneously or via the tonsilar route. Killed vaccine in a mineral-oil adjuvant did not evoke protective immunity and treatment with dexamethasone prior to vaccination with live BCG ablated its efficacy. Protection against infection did not correlate with skin test delayed type hypersensitivity (DTH) or lymphocyte transformation to tuberculin. CONCLUSIONS: Two doses of live BCG gave significant protection against experimental infection and disease caused by virulent M. bovis. Single dose vaccine protected against disease but not infection. Vaccines administered at a dosage which did not evoke DTH, provided protection against tuberculosis infection and disease.

Bystander help within a polyepitope DNA vaccine improves immune responses to influenza antigens.

A polyepitope DNA vaccine has the potential to generate protective immune responses to a range of antigens in a single construct. We investigated whether it was possible to obtain responses to individual epitopes from different antigens, directly linked in a string, and whether the response to a given epitope was enhanced by adjacent epitopes within the construct. A polyepitope plasmid was created, which included three Th epitopes (influenza haemagglutinin, moth cytochrome c and ovalbumin), a Tc epitope (ovalbumin) and two B cell epitopes (haemagglutinin and ovalbumin). Mice were immunized with DNA by using a gene gun. Responses to the polyepitope DNA vaccine were compared with those to DNA vaccine comprising only the haemagglutinin Th and B epitopes (HAT(h)B) or with responses to the recombinant protein. These experiments showed that the polyepitope DNA vaccine induced greater antigen-specific responses to HAT(h)B peptide than the HAT(h)B DNA vaccine. Antigen-specific in vivo cytotoxic responses following polyepitope DNA vaccination were also clearly demonstrable. We conclude that a 'naked DNA' polyepitope vaccine generates specific responses to constituent epitopes and that adjacent irrelevant epitopes may enhance these responses.

An in vivo comparison of bacillus Calmette-Guérin (BCG) and cytokine-secreting BCG vaccines.

A recombinant bacillus Calmette-Guérin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)-2. Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL-2) and their immune responses were monitored over 3 months. Animals gained weight over this period and showed no signs of adverse reactions to either vaccine. Lymphocyte transformation responses did not differ significantly between the two groups. No antibody that was specific for BCG was detected in any animal. Intradermal skin-test responses to BCG antigens showed that the rBCG/IL-2 induced a smaller delayed-type hypersensitivity response than the normal BCG. Cytokine transcription was determined by reverse transcription-polymerase chain reaction (RT-PCR). While IL-2 and interferon-gamma (IFN-gamma) levels did not differ significantly between the two groups, the level of IL-4 was found to be lower in the group given rBCG/IL-2. This resulted in a strong interferon-gamma:IL-4 ratio, suggesting a skewing of the immune response towards a Type 1 response. The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used. At autopsy (3 months after vaccination) 99.99% of the organisms had been eliminated. The small number of organisms isolated from the draining lymph node of animals given rBCG/IL-2 were grown in antibiotic-containing media. They were shown to still contain the shuttle plasmid and to secrete biologically active IL-2, indicating that the plasmid was stably maintained despite the host's immune response and in the absence of antibiotic selection.