Biological Signatures of Vaccine Responses

The set of host- and pathogen-specific molecular features of a disease comprise its “signature”. We hypothesize that biological signatures enables distinctions between vaccinated vs. infected individuals. In our research, using porcine samples, protocols were developed that could also be used to identify biological signatures of human disease. Different classes of molecular features will be tested during this project, including indicators of basic immune capacity, which are being studied at this instance. These indicators of basic immune response such as porcine cytokines and antibodies were validated using Enzyme-linked immunosorbent assay (ELISA). This is an established method that detects antigens by their interaction with a specific antibody coupled to a polystyrene substrate. Serum from naïve and vaccinated pigs was tested for the presence of cytokines. We were able to differentiate the presence of porcine IL-6 in normal porcine serum with or without added porcine IL-6 by ELISA. In addition, four different cytokines were spotted on a grating-coupled surface plasmon resonance imaging system (GCSPRI) chip and antibody specific for IL-8 was run over the chip. Only the presence of IL-8 was detected; therefore, there was no cross-reactivity in this combination of antigens and antibodies. This system uses a multiplexed sensor chip to identify components of a sample run over it. The detection is accomplished by the change in refractive index caused by the interaction between the antibody spotted on the sensor chip and the antigen present in the sample. As the multiplexed GCSPRI is developed, we will need to optimize both sensitivity and specificity, minimizing the potential for cross-reactivity between individual analytes. The next step in this project is to increase the sensitivity of detection of the analytes. Currently, we are using two different antibodies (that recognize a different part of the antigen) to amplify the signal emitted by the interaction of antibody with its cognate antigen. The development of this sensor chip would not only allow to detect FMD virus, but also to differentiate between infected and vaccinated individuals, on location. Furthermore, the diagnosis of other diseases could be done with increased accuracy, and in less time due to the microarray approach.

Effects of breastfeeding and naturally acquired immunity on infection with {\it Giardia lamblia}: A study in a cohort of Mexican infants

Giardia lamblia is one of the most common causes of gastrointestinal tract infection among young children worldwide. Yet host protection against this parasite and the effect of infection with Giardia on infant growth are poorly understood. It was hypothesized that among young children, protection against infection with Giardia is afforded by breastfeeding and previous infection with the parasite and further, that infection with Giardia decreases growth velocity. From 4/88 to 4/90, 197 infants in a poor area of Mexico City were followed from 0 to 18 months of age, with stool specimens, symptoms and feeding status data collected weekly. A total of 6,031 stool specimens were tested for Giardia antigen by enzyme-linked immunosorbent assay. There were 1.0 Giardia infections per child-year; 25% were symptomatic and 54% lasted more than 1 month; 94 infants had 1, and 33 had 2 or more infections. Breastfeeding status was coded and analyzed for each child-week of follow up. 91% of study infants were breastfed from birth, 57% at 6 months and 38% at 12 months of age. Rate ratios for non-breastfeeding adjusted for confounding factors were calculated from stratified analyses and the Cox proportional hazards model. Not breastfeeding was a significant risk factor for first infection with Giardia vs. any breastfeeding (adjusted RR = 1.8; 1.1, 2.8) at all ages; a dose response was demonstrated by degree of breastfeeding. The adjusted rate ratio for non-breastfeeding vs. partial breastfeeding was 1.6 (1.03, 2.6) and for non-breastfeeding vs. complete breastfeeding was 4.7 (1.4, 15.9). Among Giardia infected infants, breastfeeding did not protect against diarrheal symptoms or shorten the duration of carriage. First and repeat infections with Giardia did not differ in duration or the percent symptomatic. The analysis of growth and Giardia infection was inconclusive but suggested that a history of Giardia infection might be associated with decreased weight velocity, while an immediate chronic infection might be associated with increased weight velocity. In summary, these data indicate that breastfeeding protects infants against infection with Giardia; provide no evidence of protection against repeat infections resulting from a prior infection and suggest but do not establish that a history of Giardia infection might be associated with decreased growth in young children. ^

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE Publication No.________Kiera Rycaj, B.S.Supervisory Professor: Feng Wang-Johanning, Ph.D., M.D. Ovarian cancer (OC) is the fourth most common cancer in women, and the most lethal gynecologic malignancy in the United States. Adequate screening methodologies are currently lacking and most women first present with either stage III or IV disease. To date, there has been no substantial decrease in death rates and the majorities of patients relapse and die from their disease despite response to first-line therapy. Several proteins, such as CA-125, are elevated in OC, but none has proven specific and sensitive enough to serve as a screening tool or for tumor cell recognition and lysis. It has been proposed that human endogenous retrovirus sequences (HERVs) may play a role in the etiology of certain cancers. In a previous study, we showed that HERV-K envelope (env) proteins are widely expressed in human invasive breast cancer (BC) and ductal carcinoma in situ (DCIS), and elicit both serologic and cell-mediated immune responses in BC patients. We also reported the expression of multiple HERV genes and proteins in OC cell lines and tissues. In this study, we strengthened our previous data by determining that HERV-K env mRNAs are expressed in 69% of primary OC tissues (n=29), but in only 24% of benign tissues (N=17). Immmunohistochemistry (IHC) staining revealed HERV-Kpositivecancer cells detected in endometrioid adenocarcinoma and serous adenocarcinoma but not in benign cyst or normal epithelium biopsies. Immunofluorescence staining (IFS) showed greater cell surface expression of HERV-K in OC samples compared to adjacent uninvolved samples. Enzyme-linked immunosorbent assay (ELISA) data confirmed that a humoral immune response is elicited against HERV-K in OC patients. T-cell responses against HERV-K in lymphocytes from OC patients stimulated with autologous HERV-K pulsed dendritic cells included induction of T-cell proliferation and IFN-γ production. HERV-K–specific cytolytic T cells induced greater specific lysis of OC target cells compared to benign and adjacent uninvolved target cells. Finally, upon T regulatory cell (T-reg) depletion, 64% of OC patients displayed an increase in the specific lysis of target cells expressing HERV-K env protein. These findings suggest that HERV-K env protein is a tumor-associated antigen capable of activating both T-cell and B-cell responses in OC patients, and has great potential in the development of immunotherapy regimens against OC.

Comparative virulence of enterotoxigenic non-Escherichia coli Enterobacteriaceae species from patients with travelers' diarrhea

Background: Family members of Enterobacteriaceae are found in small numbers associated with acute diarrhea. These species are sometimes mistaken for ETEC. ^ Methods: Forty-four non-E. coli species from travelers' diarrhea are compared to 30 strains of Escherichia coli (ETEC) and 30 strains of normal flora E. coli. Tissue culture supernatants were assayed by enzyme-linked immunosorbent assay for amounts of IL-8, IL-1, and IL-1ra. Amounts of heat-stable (ST) and heat-labile (LT) enterotoxins were assayed from cell culture supernatants by enzyme-linked immunoassay. PCR was use to determine which species was positive colonization factor antigens, CFA/I, CS3, and CS6. ^ Results: Normal flora E. coli significantly induced the production of more IL-8 than non- E. coli and ETEC. Normal E. coli also induced the production of more IL-1and IL-1ra than ETEC. Non-E. coli produced more ST than ETEC. A small percentage of enterotoxigenic non- E. coli gram negatives and ETEC were positive for CFA/I and CS6. None of the strains were positive for CS3. ^ Conclusions: Non-E. coli enterotoxigenic gram negatives were similar to ETEC in their virulence factors. Identification and further study of these non-E.coli strains is important for understanding their pathogenic role in acute diarrhea.^

Population-based cross -sectional survey on intestinal helminth infections with a focus on taeniasis among residents of El Paso, Texas and Ciudad Juarez, Mexico

Few studies have been conducted on the epidemiology of enteric infectious diseases of public health importance in communities along the United States-Mexico border, and these studies typically focus on bacterial and viral diseases. The epidemiology of intestinal helminth infections along the border has not recently been explored, and there are no published reports for El Paso and Ciudad Juarez, both of which are high traffic urban areas along the Texas-Mexico border. The purpose of this research project was to conduct a cross-sectional epidemiologic survey for enteric helminths of medical importance along the Texas-Mexico border region of El Paso and Ciudad Juarez and to evaluate risk factors for exposure to these parasites. In addition, an emphasis was placed on the zoonotic tapeworm, Taenia solium. This tapeworm is especially important in this region because of the increasing incidence of neurocysticercosis, a severe disease spread by carriers of intestinal T. solium. Fecal samples were collected from individuals of all ages in a population-based cross-sectional household survey and evaluated for the presence of helminth parasites using fecal flotations. In addition, a Taenia coproantigen enzyme linked immunosorbent assay (ELISA) was performed on each stool sample to identify tapeworm carriers. A standardized questionnaire was administered to identify risk factors and routes of exposure for enteric helminth infections with additional questions to assess risk factors specific for taeniasis. The actual prevalence of taeniasis along the Texas-Mexico border was unknown, and this is the first population-based study performed in this region. Flotations were performed on 395 samples and four (1%) were positive for helminths including Ascaris, hookworms and Taenia species. Immunodiagnostic testing demonstrated a prevalence of 2.9% (11/378) for taeniasis. Based on the case definition, a 3% (12/395) prevalence of taeniasis was detected in this area. In addition, statistical analyses indicate that residents of El Paso are 8.5 times more likely to be a tapeworm carrier compared to residents of Juarez (PR=8.5, 95% CI=2.35, 30.81). This finding has important implications in terms of planning effective health education campaigns to decrease the prevalence of enteric helminths in populations along the Texas-Mexico border. ^

(Table 2) Seroprevalence against Trichinella spp. in Ursus maritimus from Svalbard and Barents Sea pack ice

Blood samples of live-caught polar bears (Ursus maritimus) from Svalbard collected 1991-2000 (Period 1) and 2006-2008 (Period 2) and from the pack ice of the Barents Sea collected in Period 1, were assayed for antibodies against Trichinella spp. by ELISA. Of 54 cubs-of-the-year included in the Period 1 sample, 53 were seronegative, indicating that exposure to Trichinella infected meat is uncommon during the first months of life for polar bears in the Svalbard region. Of 30 mother-offspring pairs, 18 mothers were seropositive with seronegative offspring (n = 27), suggesting (1) that maternal antibodies had dropped to levels below detection limit by the time of capture in April (offspring approximately 4 months old), and (2) supporting experimental studies in other animal models showing that vertical transmission of Trichinella spp. is uncommon. Bear 1 year and older had higher prevalence in Svalbard (78%) than in the Barents Sea (51%). There was no temporal change in prevalence for bears from Svalbard during the time between the two periods. The prevalence increased with age in both sexes. A positive correlation was found between anti-Toxoplasma gondii and anti-Trichinella spp. antibodies.

Hyperspectral to multispectral imaging for detection of tree nuts and peanut traces in wheat flour

In current industrial environments there is an increasing need for practical and inexpensive quality control systems to detect the foreign food materials in powder food processing lines. This demand is especially important for the detection of product adulteration with traces of highly allergenic products, such as peanuts and tree nuts. Manufacturing industries dealing with the processing of multiple powder food products present a substantial risk for the contamination of powder foods with traces of tree nuts and other adulterants, which might result in unintentional ingestion of nuts by the sensitised population. Hence, the need for an in-line system to detect nut traces at the early stages of food manufacturing is of crucial importance. In this present work, a feasibility study of a spectral index for revealing adulteration of tree nut and peanut traces in wheat flour samples with hyperspectral images is reported. The main nuts responsible for allergenic reactions considered in this work were peanut, hazelnut and walnut. Enhanced contrast between nuts and wheat flour was obtained after the application of the index. Furthermore, the segmentation of these images by selecting different thresholds for different nut and flour mixtures allowed the identification of nut traces in the samples. Pixels identified as nuts were counted and compared with the actual percentage of peanut adulteration. As a result, the multispectral system was able to detect and provide good visualisation of tree nut and peanut trace levels down to 0.01% by weight. In this context, multispectral imaging could operate in conjuction with chemical procedures, such as Real Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay to save time, money and skilled labour on product quality control. This approach could enable not only a few selected samples to be assessed but also to extensively incorporate quality control surveyance on product processing lines.

Detection and quantification of peanut traces in wheat flour by near infrared hyperspectral imaging spectroscopy using principal-component analysis

The use of a common environment for processing different powder foods in the industry has increased the risk of finding peanut traces in powder foods. The analytical methods commonly used for detection of peanut such as enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) represent high specificity and sensitivity but are destructive and time-consuming, and require highly skilled experimenters. The feasibility of NIR hyperspectral imaging (HSI) is studied for the detection of peanut traces down to 0.01% by weight. A principal-component analysis (PCA) was carried out on a dataset of peanut and flour spectra. The obtained loadings were applied to the HSI images of adulterated wheat flour samples with peanut traces. As a result, HSI images were reduced to score images with enhanced contrast between peanut and flour particles. Finally, a threshold was fixed in score images to obtain a binary classification image, and the percentage of peanut adulteration was compared with the percentage of pixels identified as peanut particles. This study allowed the detection of traces of peanut down to 0.01% and quantification of peanut adulteration from 10% to 0.1% with a coefficient of determination (r2) of 0.946. These results show the feasibility of using HSI systems for the detection of peanut traces in conjunction with chemical procedures, such as RT-PCR and ELISA to facilitate enhanced quality-control surveillance on food-product processing lines.

Hyperspectral to multispectral imaging for detection of tree nuts and peanut traces in wheat flour.

In current industrial environments there is an increasing need for practical and inexpensive quality control systems to detect the foreign food materials in powder food processing lines. This demand is especially important for the detection of product adulteration with traces of highly allergenic products, such as peanuts and tree nuts. Manufacturing industries dealing with the processing of multiple powder food products present a substantial risk for the contamination of powder foods with traces of tree nuts and other adulterants, which might result in unintentional ingestion of nuts by the sensitised population. Hence, the need for an in-line system to detect nut traces at the early stages of food manufacturing is of crucial importance. In this present work, a feasibility study of a spectral index for revealing adulteration of tree nut and peanut traces in wheat flour samples with hyperspectral images is reported. The main nuts responsible for allergenic reactions considered in this work were peanut, hazelnut and walnut. Enhanced contrast between nuts and wheat flour was obtained after the application of the index. Furthermore, the segmentation of these images by selecting different thresholds for different nut and flour mixtures allowed the identification of nut traces in the samples. Pixels identified as nuts were counted and with the actual percentage of peanut adulteration. As a result, the multispectral system was able to detect and provide good visualisation of tree nut and peanut trace levels down to 0.01% by weight. In this context, multispectral imaging could operate in conjuction with chemical procedures, such as Real Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay to save time, money and skilled labour on product quality control. This approach could enable not only a few selected samples to be assessed but also to extensively incorporate quality control surveyance on product processing lines.

Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening

We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies. We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis. From an antibody library of 7 × 108 clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant Kd = 1 × 10–7 M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae)1 : III. Organization of Fucoglucuronogalactans within the Adhesive Stalks of Achnanthes longipes

Achnanthes longipes is a marine, biofouling diatom that adheres to surfaces via adhesive polymers extruded during motility or organized into structures called stalks that contain three distinct regions: the pad, shaft, and collar. Four monoclonal antibodies (AL.C1–AL.C4) and antibodies from two uncloned hybridomas (AL.E1 and AL.E2) were raised against the extracellular adhesives of A. longipes. Antibodies were screened against a hot-water-insoluble/hot-bicarbonate-soluble-fraction. The hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, ≥ 20,000,000 Mr; F2, ≅100,000 Mr; and F3, <10,000 Mr relative to dextran standards. The ≅100,000-Mr fraction consisted of highly sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and low-sulfate (approximately 2%) FGGs, whereas F1 was composed of O-linked FGG (F2)-polypeptide (F3) complexes. AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2 recognized carbohydrate complementary regions on FGGs, with antigenicity dependent on fucosyl-containing side chains. AL.C3 was unique in that it had a lower affinity for FGGs and did not label any portion of the shaft. Enzyme-linked immunosorbent assay and immunocytochemistry indicated that low-sulfate FGGs are expelled from pores surrounding the raphe terminus, creating the cylindrical outer layers of the shaft, and that highly sulfated FGGs are extruded from the raphe, forming the central core. Antibody-labeling patterns and other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility.

Proinsulin slows retinal degeneration and vision loss in the P23H rat model of retinitis pigmentosa

Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi−) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.

Synthesis, Modification, and Application of Siloxane Materials for Environmental Sensing

As human populations and resource consumption increase, it is increasingly important to monitor the quality of our environment. While laboratory instruments offer useful information, portable, easy to use sensors would allow environmental analysis to occur on-site, at lower cost, and with minimal operator training. We explore the synthesis, modification, and applications of modified polysiloxane in environmental sensing. Multiple methods of producing modified siloxanes were investigated. Oligomers were formed by using functionalized monomers, producing siloxane materials containing silicon hydride, methyl, and phenyl side chains. Silicon hydride-functionalized oligomers were further modified by hydrosilylation to incorporate methyl ester and naphthyl side chains. Modifications to the siloxane materials were also carried out using post-curing treatments. Methyl ester-functionalized siloxane was incorporated into the surface of a cured poly(dimethylsiloxane) film by siloxane equilibration. The materials containing methyl esters were hydrolyzed to reveal carboxylic acids, which could later be used for covalent protein immobilization. Finally, the siloxane surfaces were modified to incorporate antibodies by covalent, affinity, and adsorption-based attachment. These modifications were characterized by a variety of methods, including contact angle, attenuated total reflectance Fourier transform infrared spectroscopy, dye labels, and 1H nuclear magnetic resonance spectroscopy. The modified siloxane materials were employed in a variety of sensing schemes. Volatile organic compounds were detected using methyl, phenyl, and naphthyl-functionalized materials on a Fabry-Perot interferometer and a refractometer. The Fabry-Perot interferometer was found to detect the analytes upon siloxane extraction by deformation of the Bragg reflectors. The refractometer was used to determine that naphthyl-functionalized siloxanes had elevated refractive indices, rendering these materials more sensitive to some analytes. Antibody-modified siloxanes were used to detect biological analytes through a solid phase microextraction-mediated enzyme linked immunosorbent assay (SPME ELISA). The SPME ELISA was found to have higher analyte sensitivity compared to a conventional ELISA system. The detection scheme was used to detect Escherichia coli at 8500 CFU/mL. These results demonstrate the variety of methods that can be used to modify siloxanes and the wide range of applications of modified siloxanes has been demonstrated through chemical and biological sensing schemes.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Serum and salivary IgA antibody responses to Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans in orofacial granulomatosis and Crohn's disease

Orofacial granulomatosis (OFG) is a condition of unknown aetiology with histological and, in some cases, clinical association with Crohn's disease (CD). However, the exact relationship between OFG and CD remains uncertain. The aim of this study was to determine whether OFG could be distinguished immunologically from CD by comparing non-specific and specific aspects of humoral immunity in serum, whole saliva and parotid saliva in three groups of patients: (a) OFG only (n = 14), (b) those with both oral and gut CD (OFG + CD) (n = 12) and (c) CD without oral involvement (n = 22) and in healthy controls (n = 29). Non-specific immunoglobulin (IgA, SigA, IgA subclasses and IgG) levels and antibodies to whole cells of Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans were assayed by enzyme-linked immunosorbent assay (ELISA) in serum, whole saliva and parotid saliva. Serum IgA and IgA1 and IgA2 subclasses were raised in all patient groups (P < 0.01). Salivary IgA (and IgG) levels were raised in OFG and OFG + CD (P < 0.01) but not in the CD group. Parotid IgA was also raised in OFG and OFG + CD but not in CD. The findings suggest that serum IgA changes reflect mucosal inflammation anywhere in the GI tract but that salivary IgA changes reflect involvement of the oral cavity. Furthermore, the elevated levels of IgA in parotid saliva suggest involvement of the salivary glands in OFG. Serum IgA antibodies to S. cerevisiae were raised markedly in the two groups with gut disease while serum IgA (or IgG) antibodies to C. albicans were elevated significantly in all three patient groups (P < 0.02). No differences were found with antibodies to S. mutans. Whole saliva IgA antibodies to S. cerevisiae (and C. albicans) were raised in the groups with oral involvement. These findings suggest that raised serum IgA antibodies to S. cerevisiae may reflect gut inflammation while raised SIgA antibodies to S. cerevisiae or raised IgA or IgA2 levels in saliva reflect oral but not gut disease. Analysis of salivary IgA and IgA antibodies to S. cerevisiae as well as serum antibodies in patients presenting with OFG may allow prediction of gut involvement.