Correlation between in vitro and in vivo data on food digestion. What can we predict with static in vitro digestion models?

During the last decade, there has been a growing interest in understanding food's digestive fate in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore, simple in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments. Thus, it is no surprise that these models are increasingly used by the scientific community, although their various limitations to fully mirror the complexity of the digestive tract. Therefore, the objective of this article was to call upon the collective experiences of scientists involved in Infogest (an international network on food digestion) to review and reflect on the applications of in vitro digestion models, the parameters assessed in such studies and the physiological relevance of the data generated when compared to in vivo data. The authors provide a comprehensive review in vitro and in vivo digestion studies investigating the digestion of macronutrients (i.e., proteins, lipids, and carbohydrates) as well as studies of the bioaccessibility and bioavailability of micronutrients and phytochemicals. The main conclusion is that evidences show that despite the simplicity of in vitro models they are often very useful in predicting outcomes of the digestion in vivo. However, this has relies on the complexity of in vitro models and their tuning toward answering specific questions related to human digestion physiology, which leaves a vast room for future studies and improvements.

A major susceptibility locus for leprosy in India maps to chromosome 10p13.

Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is prevalent in India, where about half of the world's estimated 800,000 cases occur. A role for the genetics of the host in variable susceptibility to leprosy has been indicated by familial clustering, twin studies, complex segregation analyses and human leukocyte antigen (HLA) association studies. We report here a genetic linkage scan of the genomes of 224 families from South India, containing 245 independent affected sibpairs with leprosy, mainly of the paucibacillary type. In a two-stage genome screen using 396 microsatellite markers, we found significant linkage (maximum lod score (MLS) = 4.09, P < 2x10-5) on chromosome 10p13 for a series of neighboring microsatellite markers, providing evidence for a major locus for this prevalent infectious disease. Thus, despite the polygenic nature of infectious disease susceptibility, some major, non-HLA-linked loci exist that may be mapped through obtainable numbers of affected sibling pairs.

Production of transgenic calves expressing an shRNA targeting myostatin.

Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.

Development of a standardized low-dose double-blind placebo-controlled challenge vehicle for the EuroPrevall project.

BACKGROUND: Double-blind placebo-controlled food challenge (DBPCFC) is the gold standard for diagnosing food allergy. Standardized materials and protocols are essential for comparing DBPCFC results for multicentre studies such as EuroPrevall. This required the development and piloting of a standardized vehicle and low-dose protocol for confirming food allergy and determination of minimum eliciting doses (MEDs). METHODS: A low-dose DBPCFC protocol was developed, with eight titrated protein doses from 3 µg to 1 g. This was delivered using a simple, microbiologically stable food base incorporating allergenic food ingredients manufactured at three sites and centrally distributed to clinical centres. Allergen blinding was assessed by a professional sensory testing panel using a triangle test. Homogeneity and allergen content were confirmed by ELISA and clinical efficacy was assessed in a pilot study, using celeriac and hazelnut as exemplars. RESULTS: Celeriac and hazelnut ingredients were sufficiently blinded in the dessert. The dessert meals were successfully piloted with hazelnut in allergy clinics in Spain, the Netherlands and Italy and with celeriac and hazelnut in Zurich. The challenges elicited a range of subjective and objective reactions ranging in severity from mild itching of the oral mucosa to bronchospasm. CONCLUSIONS: A standardized challenge vehicle proven to sufficiently blind processed, powdered hazelnut and celeriac ingredients and that can be reproducibly manufactured has been developed. This pilot study shows that the vehicle is promising for the confirmation of food allergy and determination of MEDs in adults and children with body weight >28.8 kg (approximately 7-11 years old).

Detection of Borrelia and Babesia species in Haemaphysalis punctata ticks sampled in Southern England.

The distribution and population size of the red sheep tick (Haemaphysalis punctata) are increasing in Northern Europe, and in the United Kingdom reports of human biting by this species have increased in recent years. To assess the risk of tick-borne disease (TBD) transmission to humans and livestock by H. punctata, ticks sampled from sites in Southern England were screened using PCR for either Borrelia species or piroplasms over a three year period, 2018-2020. A total of 302 H. punctata were collected from eight locations. From these, two Babesia species associated with TBD infections in livestock, Babesia major and Babesia motasi, and the human pathogen Borrelia miyamotoi were detected, predominantly from a single location in Sussex. Consequently, the range expansion of this tick across Southern England may impact public and livestock health.

A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells.

The two main challenges facing retroviral transgenesis are variable expression and epigenetic silencing. Although modern lentiviral vectors incorporate several elements to increase transgene expression and reduce position effect variegation and silencing, therapeutic research in stem cells, as well as production of transgenic animals, is still hampered by these two key problems. On the basis of recent studies demonstrating the chromatin insulating properties of divergent promoters, we sought to develop a bidirectional lentiviral vector with which to conduct RNA interference (RNAi)-based genetic screens in embryonic and extraembryonic stem cells. To this end, we designed and tested a series of synthetic bidirectional promoters, combining the mouse phosphoglycerate kinase 1 (Pgk1) promoter with other strong mammalian and viral promoters. Here, we demonstrate that a back-to-back configuration of the mouse Pgk1 and human eukaryotic translation elongation factor 1 alpha 1 promoters provided a substantive increase in both transgene expression and RNAi-based transcript depletion as compared with previous designs and other promoter combinations. Using this vector, we were able to achieve stable and robust depletion of a transfected luciferase reporter, as well as an endogenous non-coding RNA. The described constructs are an improved transgene delivery system capable of conducting RNAi screens in stem cells at single copy.

Intracellular movement of green fluorescent protein-tagged phosphatidylinositol 3-kinase in response to growth factor receptor signaling.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein-tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein-tagged fragments of p85 we show that binding to the receptor requires the NH(2)-terminal part of the protein as well as its SH2 domains.

Mutant p53 but not hepatitis B virus X protein is present in hepatitis B virus-related human hepatocellular carcinoma.

Hepatitis B virus (HBV) X protein is thought to play an important role in the development of hepatocellular carcinoma. Recent studies on a transgenic mouse tumor model suggest that HBV X protein may contribute to transformation by binding to and inactivating the cellular growth suppressor protein p53. We have studied 31 hepatocellular carcinoma tissues from Chinese patients for the possible occurrence of such interactions. Although most of the samples contained markers of HBV infection, including free and/or integrated HBV DNA, there was no detectable expression of HBV X protein by Western blot, immunoprecipitation, or histochemical staining. There was also no evidence of HBV X protein associated with p53 immunoprecipitated from the tumors. These observations suggest that, in naturally occurring human hepatocellular carcinoma, such interactions are uncommon and, therefore, unlikely to be of relevance in the latter stages of tumor development. On the other hand, 29 of 31 (93%) samples contained mutated forms of p53, as determined by various antibodies that detect wild-type or mutant p53 or both, and by the association of heat shock protein 70 with immunoprecipitated p53. These results show that conformationally altered p53 protein is present in tumors at a much higher frequency than is suggested by the presence of known mutations in the gene. This mutant p53 is functionally inactive, as suggested by the lack of expression of the p53-induced M(r) 21,000 Cip1/Waf1 protein in the tumors. Because this inactivation of p53 was not correlated with the expression of HBV X protein, any interaction of HBV X protein with p53 may be relevant only during acute infection. Such an interaction could serve to relax cell growth control at a time when virus replication requires hepatocyte destruction to be balanced by regeneration.

The mouse Na+-K+-ATPase gamma-subunit gene (Fxyd2) encodes three developmentally regulated transcripts.

The Na(+)-K(+)-ATPase is understood to function as a hetero-oligomer of alpha- and beta-subunits, but a third subunit, gamma, has been proposed to influence the enzyme's catalytic function. Recently, two variants of the gamma-subunit have been described in kidney, raising the possibility of multiple gamma-subunits with diverse functions. We now report the cloning and sequencing of the mouse gamma-subunit gene (Fxyd2). Analysis of the structure of the gene shows that it encodes three mRNAs that have distinct NH(2)-terminal (extracellular) encoding sequences but common transmembrane and COOH-terminal-encoding sequences resulting from differential splicing and, probably, alternate promoter usage. The three mRNAs have tissue-specific expression patterns. The existence of three different extracellular domains of the gamma-variants and how they may interact with the sodium pump to alter its cation transport properties must now be taken into account for future understanding of the modulation of the Na(+)-K(+)-ATPase by its gamma-subunit.

Pluripotential liver stem cells: facultative stem cells located in the biliary tree.

The ability of the liver to regenerate after parenchymal damage is usually accomplished by the ephemeral entry of normally proliferatively quiescent (G0) hepatocytes into the cell cycle. However, when hepatocyte regeneration is defective, arborizing ductules which are continuous with the biliary tree, proliferate and migrate into the surrounding parenchyma. In man these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals similar ductal cells are usually called oval cells, and their association with defective regeneration has led to the belief that these cells represent a progenitor cell population. Oval cells are thought to take over the burden of regenerative growth after substantial hepatocyte loss, suggesting that they are the progeny of facultative stem cells. The liver is not, however, generally considered as a stem cell-fed hierarchy, although this is disputed by others. Despite this, the subject of oval cells has aroused intense interest as these cells may represent a target population for hepatic carcinogens, and they may be useful vehicles for ex vivo gene therapy. This review proposes that the liver does harbour stem cells which are located throughout the biliary epithelium, and that oval cells represent the progeny of these stem cells and function as an amplification compartment for the generation of 'new' hepatocytes. This is a conditional process which only occurs when the regenerative capacity of hepatocytes is overwhelmed and thus, unlike the intestinal epithelium, the liver is not behaving as a classical continually renewing stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the potential to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.

Catecholamine stimulation of testosterone production via cyclic AMP in mouse Leydig cells in monolayer culture.

The effects of the beta-adrenergic hormone agonist, isoproterenol, on testosterone and cyclic AMP production in mouse Leydig cells in culture have been investigated. It was found that isoproterenol increased testosterone production on days 1, 2 and 3 of culture but not in freshly cultured cells. Cyclic AMP production was however increased on all days of culture. In subsequent studies carried out on day 2 of culture the amounts of testosterone formed during incubation with isoproterenol were 20-90% of those obtained with maximum stimulating levels of luteinizing hormone. The amounts of cyclic AMP formed were extremely low compared with those obtained with luteinizing hormone (22 +/- 5.3 and 2320 +/- 100 pmoles/10(6) cells/2 h respectively). Isoproterenol (10(-8) -10(-7) M) gave a significant increase in testosterone production and reached a maximum with 10(-6) M. Similar dose-response curves for cyclic AMP production were obtained. The stimulation of cyclic AMP and testosterone by isoproterenol was highly dependent on the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. Propranolol blocked, in a dose-dependent manner, both isoproterenol-stimulated cyclic AMP and testosterone production. In the presence of excess luteinizing hormone no additional effects of isoproterenol were detected. Epinephrine also stimulated testosterone production. It is concluded that catecholamines stimulate testosterone production in mouse Leydig cells in monolayer culture and that this effect if mediated by cyclic AMP.

Reactive biliary epithelium: the product of a pluripotential stem cell compartment?

Liver parenchymal cells (hepatocytes) have a low rate of turnover, but can nevertheless mount a rapid and efficient regenerative response. However, in some cases of extreme hepatotoxicity hepatocyte proliferation is restricted or even abolished, and instead biliary epithelial cells, commonly referred to as ductular oval cells, migrate into the periportal and midzonal parenchyma. Initially these cells behave as authentic biliary epithelium with expression of the biliary cytokeratin intermediate filaments, but then show hepatocytic traits such as alpha fetoprotein and albumin synthesis. Thereafter these biliary ducts rapidly vanish to be replaced by either small hepatocytes or intestinal-type cells. The proliferation and differentiation of oval cells is probably strongly influenced by paracrine signalling from liver stellate cells. Oval cells appear to be the progeny of facultative pluripotential stem cells which have the lineage potential of uncommitted gastrointestinal stem cells; these stem cells are likely to be located in the cholangioles and small interlobular bile ducts. Oval cells thus constitute an important reserve compartment for hepatocytes when hepatocyte regeneration is compromised.

Exsanguinating hemoptysis.

Massive hemoptysis (600 ml in 24 hours) results in a mortality of more than 50%. We have performed 74 pulmonary resections in patients with massive hemoptysis in the last 15 years, with a mortality of 13%. The mortality correlated with the rate and the amount of recorded blood loss before the operation. From this experience, we have identified a subgroup of patients with such massive hemoptysis that life was threatened by exsanguination. Twenty-four of our patients lost more than 1,000 ml of blood, at a rate of at least 150 ml an hour, before the pulmonary resection was performed. The bleeding site was always identified by bronchoscopy. All patients were treated by resection of the bleeding lung parenchyma. Several methods were used to avoid the patient's drowning in his own blood during the operation. In five patients, a double-lumen endotracheal tube was used: Two died of suffocation during the procedure and another died of respiratory and liver failure. In four patients, single-lung ventilation with an endotracheal tube in the left main bronchus was used: All four survived. In another 10 patients a bronchial blocker (No. 9 Fogarty balloon venous catheter) was used to stop bleeding. Two patients died of renal failure and gastrointestinal bleeding, respectively, but none had aspiration problems. In five additional patients, a regular endotracheal tube was used: One patient died of massive aspiration. Our experience indicates that bleeding from the left lung and right lower lobe should be controlled by intubation of the left bronchus. Patients with exsanguinating hemoptysis should be treated, when possible, by pulmonary resection. A survival rate of 75% was obtained in our patients.

Validation of self-reported hearing loss. The Blue Mountains Hearing Study.

PURPOSE: Large-scale epidemiological studies have often used self-report to estimate prevalence of age-related hearing loss. However, few large population-based studies have validated self-report against measured hearing loss. Our study aimed to assess the performance of a single question and a brief hearing handicap questionnaire in identifying individuals with hearing loss, against the gold standard of pure-tone audiometry. METHODS: We examined 2015 residents, aged 55-99 years, living in the west of Sydney, Australia, who participated in the Blue Mountains Hearing Study during 1997-1999. Audiologists administered a comprehensive questionnaire, including the question: 'Do you feel you have a hearing loss?' The Shortened Hearing Handicap Inventory for Elderly (HHIE-S) was also administered during the hearing examination, which included pure-tone audiometry. The single question and HHIE-S were compared with measured losses at levels >25, >40 and >60 decibels hearing level (dBHL) to indicate mild, moderate and marked hearing impairment, for pure-tone averages (PTA) of responses to 500, 1000, 2000 and 4000 Hz. RESULTS: The single question yielded reasonable sensitivity and specificity for hearing impairment, and was minimally affected by age and gender. HHIE-S scores >8 had lower sensitivity but higher specificity and positive predictive value. The HHIE-S performed slightly better in younger than older subjects and performed better for moderate hearing impairment. CONCLUSIONS: In this older population with a high prevalence of hearing loss (39.4%), both a question about hearing and the HHIE-S appeared sufficiently sensitive and specific to provide reasonable estimates of hearing loss prevalence. Both could be recommended for use in epidemiological studies that aim to assess the magnitude of the burden caused by age-related sensory impairment but cannot measure hearing loss by audiometry.

Infection of total hip prostheses by Peptococcus magnus: an immunofluorescence and ELISA study of two cases.

In two cases of infected total hip replacements, Peptococcus magnus was isolated in pure culture from the implant when it was removed. Fluorescent antibody and ELISA studies have shown that both patients developed an antibody response to this anaerobic coccus soon after the replacement operation. These results suggest that the organism is a true infective agent, which was probably responsible for the failure of the arthroplasty operation.

Cloning of the gene conferring resistance to mupirocin in Staphylococcus aureus.

A plasmid known to be associated with mupirocin resistance of Staphylococcus aureus has been isolated and a restriction enzyme map constructed. An EcoRI fragment of 4.05 kb from this plasmid has been cloned into an Escherichia coli-Staphylococcus aureus shuttle vector and shown to carry the gene for resistance to mupirocin. The DNA sequence of a small section of the gene has been determined and the derived amino acid sequence compared with a data bank. The amino acid sequence is identical for eight amino acids with the sequence of isoleucyl tRNA synthetase of E. coli. This finding adds to the evidence that mupirocin resistance is the result of a modified isoleucyl tRNA synthetase.

Effect of nonnicotine pharmacotherapy on smoking behavior.

Smoking-related disease is the single biggest preventable cause of morbidity and mortality in the United States, yet approximately 25% of Americans continue to smoke. Various dosage forms of nicotine replacement therapy increase smoking quit rates relative to placebo, but they generally do not result in 1-year quit rates of over 20%. To increase these rates, a number of nonnicotine agents have been investigated. Drugs that modulate noradrenergic neurotransmission (bupropion, nortriptyline, moclobemide) are more effective than those affecting serotonin (selective serotonin reuptake inhibitors, buspirone, ondansetron) or other neurotransmitters.

Does fetal heart rate predict subsequent heart rate in childhood?

An investigation to determine whether there is any relationship between extremes of fetal heart rate during labour and subsequent heart rate at the age of 10 was carried out using data from the 1970 cohort of British Births. In 11,000 nationally representative children it was found that low fetal heart rate (below 120 beats/min) was associated with a heart rate at age 10 which was significantly lower than in those children whose fetal heart rate had remained between 120 and 160 beats/min (P less than 0.01). This relationship could not be explained by fetal asphyxiation, maternal antenatal hypotension or the method of pain relief during labour. There was no equivalent relationship with high fetal heart rate during labour. This could imply that some fetuses with low heart rates are not exhibiting fetal distress but have an inherent tendency to relatively slow heart rates.

A simple method for the preparation of liposomes for pharmaceutical applications: characterization of the liposomes.

A new method for the preparation of liposomes is described that avoids the use of pharmaceutically unacceptable solvents and energy-expensive procedures such as sonication. The method is based on the initial formation of a proliposome mixture containing lipid, ethanol and water, which is converted to lipsomes by a simple dilution step. Measurements using 6-carboxyfluorescein as a marker indicate that water-soluble drugs can be trapped with extremely high efficiency (65-80% depending on lipid composition). The structural organization of the proliposome mixture and the final liposomes were characterized using electron microscopy and 31P-NMR.

Neonatal viability evaluation by Apgar score in puppies delivered by cesarean section in two brachycephalic breeds (English and French bulldog).

This study tried to define neonatal viability after cesarean section in brachycephalic breeds and the efficacy of an adapted Apgar test to assess newborn survival. Data from 44 cesarean sections and 302 puppies were included. Before surgery (59-61 days after ovulation), an ultrasound evaluation defined the fetal biparietal diameter (BPD). Immediately after the uterine delivery, the pups were evaluated to detect birth defects and then, a modified Apgar score (range: 0-10) was used to define neonatal health at 5min (Apgar 1) and 60min (Apgar 2) after neonatal delivery; puppies were classified into three categories: critical neonates (score: 0-3), moderate viability neonates (score: 4-6) and normal viability neonates (score: 7-10). Mean (±SEM) value of BPD was 30.8±0.1mm and 28.9±0.1mm in English and French Bull-Dog fetus, respectively. The incidence of spontaneous neonatal mortality (4.98%, 14/281) and birth defects (6.95%) were not influenced by the sex; however, congenital anomalies and neonatal mortality were higher (p<0.01) in those litters with a greater number of neonates. In Apgar 1, the percentage of critical neonates, moderate viability neonates and normal viability neonates were 20.5%, 46.3% and 33.1% respectively; sixty minutes after birth, the critical neonates only represented 10.3% of the total puppies. Almost all neonates (238/239) showing moderate or normal viability at Apgar 1, survived for the first 24h after birth. The results of the study showed a direct relationship (p<0.01) between the Apgar score and neonatal viability. Therefore, the routine performance of the Apgar score would appear to be essential in the assessment of the status of brachycephalic breed puppies.