Yoghurt containing galacto-oligosaccharides, prunes and linseed reduces the severity of mild constipation in elderly subjects.

OBJECTIVE: Constipation is a common problem in the elderly. Dietary fibre is recommended for its treatment. The aim was to examine whether yoghurt containing galacto-oligosaccharides (GOS), prunes and linseed relieve constipation in elderly subjects. DESIGN: A randomized, double-blinded, cross-over study. SETTING: Free-living subjects. SUBJECTS: A group of 43 elderly subjects with self-reported constipation (mean age 76 years, range 61-92 years, 32 females, 11 males). INTERVENTIONS: The study consisted of a 2-week baseline period and 2, 3-week dietary interventions, with a 2-week wash-out period between the interventions. During the interventions, the subjects ingested, in random order, 260 g/day of either control yoghurt or test yoghurt containing GOS (12 g/day), prunes (12 g/day) and linseed (6 g/day). The use of laxatives was controlled and only allowed after 2 days without defecation. RESULTS: Defecation frequency was 5.7 times/week during the baseline period. During the test yoghurt period, defecation frequency was higher (8.0 vs 7.1 times/week, P=0.011), defecation was easier (on the scale 0-3, 1.3 vs 1.5, P=0.010), and there was a tendency towards softer stools (on the scale 0-3, 2.1 vs 2.2, P=0.059) compared with the control yoghurt period. The subjects felt that the test yoghurt relieved constipation more effectively than the control yoghurt (P=0.005). The sum of gastrointestinal symptoms did not differ between the interventions. The use of laxatives remained constant throughout the study. CONCLUSIONS: Daily intake of yoghurt containing GOS, prunes and linseed reduced the severity of constipation in elderly subjects with mild constipation. SPONSORSHIP: Valio Ltd, R&D.

Oral chronic ethanol administration to rodents by agar gel diet.

BACKGROUND: Chronic ethanol administration to rodents requires specially designed equipment and is labor intensive. Here we report a new procedure. METHOD: A commercial liquid diet preparation was made into a gel by addition of 0.5% agar. The gel, containing 5.3% ethanol, was offered in Falcon tubes equipped with a feeding opening. RESULTS: The gel consumption by C57/Bl mice resulted in high blood ethanol levels (average 43 mM). After 6 weeks, marked liver steatosis and significantly increased serum alanine aminotransferase levels had developed. CONCLUSIONS: Administration of ethanol in a nutritionally adequate gel provides a simple method for studies on chronic ethanol effects in rodents.

Poliovirus-specific intestinal antibody responses coincide with decline of poliovirus excretion.

Antibody responses to poliovirus type 3 were studied in fecal samples of 66 children immunized with 3 doses of enhanced-potency inactivated poliovirus vaccine (E-IPV), followed by 1 dose of monovalent oral poliovirus vaccine (OPV, type 3 Sabin). One fecal sample taken before OPV vaccination and 9 collected thereafter were tested for neutralizing antibodies by a microneutralization assay and for class-specific responses by heavy chain-capture radioimmunoassays. Both neutralizing antibody and IgA responses usually occurred during the second week and coincided with ceasing of virus excretion or a decrease in the excreted virus titer. Half of the vaccinees had received a trypsin-modified E-IPV, but their responses did not differ from those of children immunized with the regular E-IPV. These results are in agreement with the view that an intestinal antibody response, mainly consisting of IgA, may be involved in the ceasing of a primary poliovirus excretion.

Randomised, controlled trial with the trypsin-modified inactivated poliovirus vaccine: assessment of intestinal immunity with live challenge virus.

The enhanced potency inactivated poliovirus vaccine (E-IPV) was modified to contain trypsin-treated type 3 poliovirus (PV3), strain Saukett, as the type 3 component (TryIPV). This pilot vaccine was previously shown to redistribute the vaccine-induced antibody specificities in mice to mimic those seen in man after poliovirus infection. Groups of infants were then immunised with three doses of TryIPV or E-IPV in a randomised, double-blind trial. Six months after the third dose, at the age of 18 months, the children were challenged with one dose of oral monovalent type 3 poliovirus vaccine. Intestinal immunity was evaluated by assessing the length and extent of PV3 excretion through determination of PV3 titres in 9 successive faecal specimens (2-42 days after challenge). No significant difference in the length or extent of virus excretion was seen between the groups. The results indicate that TryIPV, under the conditions used, was no more potent than the regular E-IPV in inducing resistance to intestinal poliovirus infection.

Variability in the integrity of human enteroviruses exposed to various simulated in vivo environments.

We exposed representatives of different enteroviruses to treatments imitating various in vivo environments that they face during infection. Short-term treatment in trypsin or human intestinal fluid regularly resulted in a cleavage of the capsid protein VP1, and in some cases of other capsid proteins as well. Infectivity of the virus preparations was usually not affected but there were two exceptions. Coxsackievirus A9 retained its infectivity as tested in RD cells but showed reduced infectivity in GMK cells, as reported previously. More strikingly, the titre of echovirus 22 was decreased by 2 logs. Overnight incubation in intestinal fluid affected the infectivity of all tested viruses, including polioviruses and coxsackievirus B4. Echovirus 22 and, to a lesser extent, coxsackievirus A9 were also partially inactivated by faecal suspension. After 2 h exposure to pH 2 at 37 degrees C all tested viruses retained their infectivity, but after 24 h all were inactivated. We conclude that the stability of enteroviruses exposed to various natural environments varies significantly, and that echovirus 22, no more classified in the genus Enterovirus, appears relatively more sensitive in body fluids.

Immunogenicity of a pilot inactivated poliovirus vaccine with trypsin-treated type 3-component.

A modified trivalent enhanced potency IPV (E-IPV) containing trypsin-treated PV3/Saukett as the type 3-component (TryIPV) was evaluated in preclinical and Phase I and Phase II clinical trials with the regular E-IPV as a control. In Balb/c mice, TryIPV-induced antibodies targeted outside the trypsin-sensitive antigenic site, as expected. Immunogenicity of TryIPV in man, as judged by a booster response in neutralizing antibodies to type 1, 2 or 3 poliovirus, was as good as that of the regular E-IPV in both adults and children. In children the preimmunization titres of antibodies neutralizing the trypsin-treated PV3/Saukett were significantly lower than those for the untreated virus. Both vaccines induced a striking increase of antibody titres to both virus preparations. No significant harmful effects were observed. We conclude that the pilot vaccine is suitable for further evaluation.

Efficient RGD-independent entry process of coxsackievirus A9.

Previously we showed for coxsackievirus A9 (CAV-9) that specific interactions between the RGD motif of capsid protein VP1 and the alpha v beta 3 integrin are involved in virus binding and entry into green monkey kidney cells (GMK) and some other cell lines. The RGD-recognizing alpha v beta 3 integrin is known as the vitronectin receptor (VNR). During replication in the gut, CAV-9 like all other enteroviruses are exposed to host proteolytic enzymes, and we showed previously that the RGD-containing 15 amino acids long carboxy terminal extension of VP1 is cleaved off by trypsin. The trypsin-treated CAV-9 was still infectious, although at an apparently reduced level as assessed in GMK cells. This indicated that the virus was able to bypass the RGD-dependent entry and possibly use an alternative receptor. We have now found that in RD cells, a human rhabdomyosarcoma cell line, neither RGD-containing oligopeptides nor polyclonal antiserum to VNR are able to protect the cells from CAV-9 infection suggesting that the RGD motif is not involved in binding or entry of the virus into these cells. This result was further confirmed by demonstrating that, in RD cells, the trypsin-treated CAV-9 lacking the RGD-containing insert appeared to be as infectious as the untreated virus. The most striking difference between the virus receptors in the RD and the GMK cells was seen when the rate of virus uncoating was studied. For virus particles bound to the RD cells, the uncoating step started already at 18-20 degrees C and the process went on rapidly at 36 degrees C resulting in complete disintegration of cell-bound virions. In contrast, alpha v beta 3-bound virus particles in the GMK cells appeared to uncoat slowly even at 36 degrees C and during the 90 min observation period only a small, hardly visible fraction was found to be disintegrated. Trypsin-cleaved CAV-9 showed the rapid disintegration kinetics in GMK cells as well suggesting that these cells contain both types of receptor specificity. These results indicate that CAV-9 is able to use two different entry routes into host cells depending on the target cells and on phenotypic properties of the virus regulated by host proteases.

Selective cleavage by trypsin of the capsid protein VP1 of type 3 poliovirus results in improved sorting of cell bound virions.

A large proportion of host cell-bound virions of poliovirus type 1 strain Mahoney (PV1/M) is known to elute to the culture medium during incubation at 37 degrees C, and only a fraction of the virions remaining cell-associated will successfully uncoat and contribute to the new replication cycle. We found that while the proportion of inoculum type 3 poliovirus strain Saukett (PV3/S) bound to GMK cells was of the same order as that of PV1/M, the bound PV3/S virions uncoated much less efficiently, as judged by velocity sedimentation analysis of virion disintegration. Rather, the majority of the cell-associated PV3/S viruses remained apparently unaffected for several hours within an unidentified intracellular compartment. Incubation of PV3/S with intestinal trypsin is known to result in selective cleavage of the capsid protein VP1 and striking antigenic changes. Trypsin treatment of stock PV3/S preparations did not affect the infectivity titre or modify single-cycle progeny virus yields significantly. However, the fate of the cell-bound inoculum virus was profoundly altered. Trypsin-treated PV3/S virions (PV3/S-Try) attached to GMK cells less tightly than the untreated PV3/S virus or PV1/M, and a relatively larger proportion of the cell-bound virus eluted to the medium during subsequent incubation at 36 degrees C. However, the fraction of virions remaining cell-associated rapidly disintegrated suggesting efficient uncoating. In accordance with these observations, one step growth curves of PV3/S-Try in all cell lines tested showed lowered eclipse phase titres compared to those obtained with the untreated PV3/S inocula. Similar effects were also demonstrated for type 3 poliovirus strain Sabin while trypsin-sensitive strains of the other two serotypes of poliovirus remained unaffected in this sense. The putative biological significance of the altered sorting of cell-bound PV3/S-Try virions is not known. It might be related to the observations that sensitivity of type 3 poliovirus strains to trypsin is conserved in spite of the fact that the target site of trypsin action is flanked by highly variable motives in an immunodominant antigenic site.

Entry of coxsackievirus A9 into host cells: specific interactions with alpha v beta 3 integrin, the vitronectin receptor.

Attachment and entry of coxsackievirus A9 (CAV-9) to GMK cells were previously shown to be dependent on an arginine-glycine-aspartic acid (RGD) motif in the capsid protein VP1, suggesting integrins as candidate receptors for the virus. We have pursued the matter further and show that antibodies specific for the alpha v and/or beta 3 integrin subunits protect GMK cells from CAV-9 infection. Affinity purification of radioiodinated cell surface proteins using CAV-9 or virus-specific peptide (RRRGDL) columns confirmed that the alpha v beta 3 heterodimer, known as the vitronectin receptor, is recognized by the virus in GMK cells. Other proteins, of lower molecular weight (less than 40 kDa), were also bound to and specifically eluted from the columns, but their possible role in attachment and entry of CAV-9 remains to be elucidated by further studies. Of several other related viruses studied, only echovirus 22, which also has an RGD motif in the VP1 capsid protein, was found to compete for cell surface binding with CAV-9.

An immunodominant N-terminal region of VP1 protein of poliovirion that is buried in crystal structure can be exposed in solution.

According to previous X-ray crystallographic analyses of poliovirus structure, the amino terminal region of the capsid protein VP1 is inside the capsid shell. We have found that antibodies to synthetic peptides derived from an immunodominant part of this region readily precipitate intact infectious virions, indicating that in solution, the virus can assume a conformation that is partially different from that seen in the crystal structure. We propose that this part of VP1 is alternating between two conformations.

Persistence and class-specificity of neutralizing antibody response induced by trypsin-cleaved type 3 poliovirus in mice.

Antibody responses in Balb/c mice immunized with different preparations of type 3 poliovirus/Saukett were compared. Live and formalin-inactivated virus preparations, either as such or after selective cleavage by trypsin, were used. The latter treatment is known to yield surface-modified virus particles similar to those generated during natural infection in man. We have previously shown that these modified viruses are poorly neutralized by sera from persons immunized solely with inactivated poliovirus vaccine. Trypsin-cleaved virus-immunized mice produced antibodies capable of neutralizing both virus preparations. Surprisingly, a major part of these neutralizing antibodies 5 months after the third dose were still of the IgM class. In contrast, antibodies from mice immunized with untreated, intact virus were mainly of the IgG class and they readily neutralized the immunogen itself but were not usually able to neutralize the trypsin-cleaved virus. These results indicate that neutralizing antibodies induced in Balb/c mice by trypsin-modified type 3 poliovirus show an 'improved' distribution of antigenic site specificities resembling that seen in man after poliovirus infection.

RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases.

The recently reported nucleotide sequence of coxsackievirus A9 (CAV-9) showed that unlike other enteroviruses, CAV-9 has an insertion of about 17 amino acids at the C-terminal end of VP1 (K. H. Chang, P. Auvinen, T. Hyypiä, and G. Stanway, J. Gen. Virol. 70:3269-3280, 1989). This sequence includes the RGD (arginine-glycine-aspartic acid) motif which is known to be important in certain protein-protein interactions. We studied the inhibitory effect of RGD-containing peptides in the attachment of CAV-9 to African green monkey kidney cells. A peptide corresponding to the RRGDM sequence derived from the inserted segment of CAV-9 was found to block virus attachment effectively, and the inhibition was dose dependent. Substitution of glutamic acid for the homologous aspartic acid completely abolished the inhibitory effect, indicating great specificity of the action. During replication in the gut, all enteroviruses are exposed to host proteolytic enzymes. Exposure of CAV-9 to purified trypsin or human intestinal fluid resulted in selective cleavage of the VP1 capsid protein. Intact and trypsin-cleaved VP1 proteins gave identical N-terminal sequences, indicating that cleavage of VP1 takes place near the C terminus. Attachment of proteolytically cleaved infectious CAV-9 to green monkey kidney cells was not prevented by RGD-containing peptides, indicating that cleaved CAV-9 is able to bypass RGD-dependent entry. The altered receptor specificity of proteolytically cleaved viruses may have important consequences in the pathogenesis of enteric infections.

Viral antibodies in multiple sclerosis. A nationwide co-twin study.

Serum viral antibody titers against 21 viruses were studied in 19 of 23 same-sex twin pairs with multiple sclerosis derived from the Finnish Twin Cohort. Thorough neurologic examinations showed two monozygotic pairs to be concordant, whereas all dizygotic pairs were discordant. Special attention was given to measles, mumps, and rubella viruses, against which the antibody levels were determined with the complement fixation, hemagglutination inhibition, hemolysis-ingel, and enzyme immunoassay methods. Epstein-Barr virus antibody levels were determined by enzyme assay. In pairwise comparisons, the measles, mumps, and Epstein-Barr virus-IgG antibody levels were more often elevated in the patients with multiple sclerosis, compared with the healthy co-twins. The same antibody levels were more often above the median in the diseased twin, compared with the healthy twin, but the difference was not significant. No human T-cell lymphotropic virus type I antibodies were found in any of the individuals examined. The total IgG, IgA, and IgM levels did not differ between the diseased and healthy subjects. The HLA types, severity of the disease, and cell-mediated immunity parameters did not influence antibody levels.

Effect of acetylated derivatives of some sympathomimetic amines on the isolated auricles and tracheal chain of the guinea-pig.

The effects of acetylation of sympathomimetic amines, tyramine, amphetamine, ephedrine, phenylephrine, orciprenaline, and salbutamol, and their O- and N-acetyl derivatives and the effects of reserpine or physostigmine pretreatment on the isolated auricles and tracheal chain of guinea-pigs have been studied. All the parent drugs relaxed the tracheal chain and had a positive inotropic and chronotropic effect on the isolated auricles; only amphetamine, on the contrary, contracted the tracheal chain. O-acetylation of these sympathomimetic amines generally decreased less chronotropic than iontropic action on the isolated auricles. O-acetylation of tyramine however: actually increased the positive chronotropic activity of drug. As a rule, O-acetylation also decreased the beta-adrenergic effect of these compounds on the tracheal chain, but not so markedly as on the isolated auricles. N-acetylation generally abolished the adrenergic effects of these sympathomimetic amines on the isolated auricles and decreased those effects on the tracheal preparation. N,O-triacetylation of salbutamol abolished the stimulating effect of the parent drug on the auricles but increased the relaxant activity on the trachea. Physostigmine antagonized the effects of O-acetyltyramine and O-triacetylorciprenaline but not those of tyramine and orciprenaline on the trachea preparation. It is concluded that among the sympathomimetic amines acetylation may be utilized for the development of specific bronchodilators and O-acetylation for inducing drug latentiation.