Kuru and its contribution to medicine

The solution of kuru led us to the solution of Creutzfeldt–Jakob disease and to the elucidation, in humans and other species, of previously unknown mechanisms of infection. These require very close three-dimensional matching, which determines infectious nucleant or prion activity. Evidence for nucleation processes is found widely in the organic and inorganic worlds and in the interactions between them: in the formation of amyloid fibrils; in the biochemistry of silicon; in cave formations deep in the Earth; and in outer space. Kuru in its location in Papua New Guinea has also led to an understanding of the cultural achievements of the Palaeo-Melanesians, with deep roots in human history

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use

Reflections on amyloidosis in Papua New Guinea

The amyloidoses comprise a heterogeneous group of diseases in which 1 out of more than 25 human proteins aggregates into characteristic beta-sheet fibrils with some unique properties. Aggregation is nucleation dependent. Among the known amyloid-forming constituents is the prion protein, well known for its ability to transmit misfolding and disease from one individual to another. There is increasing evidence that other amyloid forms also may be transmissible but only if certain prerequisites are fulfilled. One of these forms is systemic AA-amyloidosis in which an acute-phase reactant, serum AA, is over-expressed and, possibly after cleavage, aggregates into amyloid fibrils, causing disease. In a mouse model, this disorder can easily be transmitted from one animal to another both by intravenous and oral routes. Also, synthetic amyloid-like fibrils made from defined small peptides have this property, indicating a prion-like transmission mechanism. Even some fibrils occurring in the environment can transmit AA-amyloidosis in the murine model. AA-amyloidosis is particularly common in certain areas of Papua New Guinea, probably due to the endemicity of malaria and perhaps genetic predisposition. Now, when kuru is disappearing, more interest should be focused on the potentially lethal systemic AA-amyloidosis

Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17

The effect of milk production level on host resistance of dairy cows, as assessed by the severity of experimental Escherichia coli mastitis

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4',6'-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting

Inclusion of linseed and linseed expeller meal in piglet diets affects intestinal gene expression profiles

Linseed as a feed ingredient contains a number of constituents, such as mucilage, gel forming polysaccharides, structural carbohydrates and ¿-3 fatty acids, which could have functional properties in relation to maintaining and supporting gastro-intestinal health in post weaning piglets. Intestinal gene expression was studied in weanling piglets fed diets containing either or not linseed or linseed expeller meal. Piglets were either or not orally challenged with an enterotoxigenic strain of E. coli K88. Pairwise comparisons identified several mRNAs expressed significantly different, in particular between treatments C¿ (control, not challenged) and LS¿ (linseed, not challenged) and LS+ (linseed, challenged) and LSM+ (linseed expeller meal, challenged). The intestinal micro-array technique is a promising tool to study the relationship between nutrition and feeding and gut tissue responses under defined conditions

Plasma intestinal fatty acid binding protein (I-FABP) concentrations increase following intestinal ischemia in pigs

Intestinal fatty acid binding protein (I-FABP) is an intracellular epithelial protein in the intestinal mucosa of many animals. IFABP appears in the circulation following epithelial damage, and in humans, is proven to be a parameter for damage to the mucosa. In this paper, an ELISA test designed for human I-FABP analysis was used to assay pig blood samples. The test recognized I-FABP cloned from pig small intestine and expressed in Escherichia coli. Furthermore, in our experimental model of (low flow) intestinal ischemia and reperfusion a significant rise in plasma I-FABP concentrations 15-30 min after clamping of the mesenteric artery was demonstrated. This is the first report that in pigs circulating I-FABP is a useful marker for (mild) intestinal injury, and could possibly be used to monitor (intestinal) health in clinical practice. (C) 2004 Elsevier Ltd. All rights reserved