Induction of interferon and host resistance in vivo by double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid

Summary Several double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid (poly IGC) were found to possess interferon inducing activity stronger than poly ICin vivo, Their activity increased in parallel with increase in the ratio of guanine base to hypoxanthine base in these copolymers as far as double-strand formation was observed with polyribocytidylic acid. Many other combinations of copolyribonucleotide with homopolyribonucleotide were also investigated, and several of them were found to induce interferon. However, the interferon inducing effects of these combinations including complementary base-pairings of hypoxanthine and cytosine increased in parallel with the length of the base-pairings, thus approaching to that of poly IC. It is, therefore, supposed that the activity of poly IG C is somewhat different from poly IC and that those of other combinations owe to the essential structure of poly IC. Furthermore, kinetics of interferon induction, cross tolerance to reinduction, and antiviral effectsin vivo of poly IGC and poly IC were studied.     

Biochemical characteristics,phage patterns,and O1 factor analysis ofEscherichia coli O1∶K1∶H7∶F11 and O1∶K1∶H−∶F9 strains isolated from patients with urinary tract infections

Biochemical characteristics, O1 antigen factors and phage patterns were examined in 35 urinary O1K1 isolates ofEscherichia coli different in H and F antigen. Fermentation of dulcitol, decarboxylation of ornithine, requirement for nicotinamide, and determination of O1 factor d allowed maximum differentiation. On the basis of these tests the strains could be divided into two major groups which are obviously of different clonal origin. Members of clone 1 represented by serotypes O1K1H7F11 (12 strains) and O1K1H^–F11 (5 strains) were characterized by positive biochemical reactions and absence of O1 antigen factor d. Negative biochemical tests and presence of O1 antigen factor d were shown by strains of clone 2 which were of serotypes O1K1H^–F9 (14 strains) and O1K1H^–F^– (3 strains). Phage patterns are less well correlated with clonal assignment. However, strains of clone 2 were not susceptible to K1-specific phage D and were non-typable with another set of 13 phages.     

Simple laboratory procedures for the evaluation of topically-active anti-inflammatory drugs

A simple laboratory test for the examination of topically-active steroids and non-steroids is described. The method(s) involves the application of 0.05 ml of 5% croton oil in ethanol or 0.1% H₂SO₄5% croton oil in ethanol to ears of rats. Irritants are allowed to stand at room temperature for 3–4 days. Sulfuric acid, applied alone in cottonseed oil or ethanol, is not inflammagenic but exacerbates the response to croton oil. The resulting inflammation is greater than that which can be elicited by greater concentrations of croton oil. Benzenesulfonic acid acts similarly when added to croton oil. The more intense inflammation elicited by the mixture is less sensitive to non-specific drugs.Drugs appearing of interest in preliminary tests, are applied subsequently at equivalent dosages by local application, by application at distant sites and by giving orally to rats with inflammed ears. This routine procedure helps decide if a drug is a topically-active drug exclusively and is acting by only one method of application and/or by two or more; hence, having amixed anti-inflammatory effect.The ear weights of untreated controls (5–10/group) are substracted from irritated ears and the increased weights recorded as milligrams edema. Hydrocortisone at varying concentrations is used routinely as a positive anti-inflammatory control in all routine assays. Differences of inflammatory responses to 5% croton oil and to 0.1% H₂SO₄5% croton oil, as well as individual responses to different drugs given in various ways, are described.Hundreds of compounds (steroidal and non-steroidal) have been tested by the use of these methods.     

Activity of the Leukocyte Nadph Oxidase in Whole Neutrophils and Cell-Free Neutrophil Preparations Stimulated with Long-Chain Polyunsaturated Fatty Acids

Fish oils are known for their anti-inflammatory effects. In this paper we investigated the influence of eicosapentaenoic acid and docosahexaenoic acid (-3 fatty acids), as well as docosapentaenoic acid, a metabolic product of -3 fatty acid metabolism, on O^– ₂-production catalyzed by the NADPH oxidase in whole neutrophils and in a cell-free system consisting of neutrophil membranes and cytosol. As a standard we used arachidonic acid (an -6 fatty acid) found in a high proportion in the Western diet, and known as an effective activator of the oxidase in both systems. Our data show that with -3 fatty acids, the O^– ₂-production in both systems is reduced as compared to the effect of arachidonic acid. The effects are more pronounced with increasing carbon chain length and increasing numbers of double bonds. Our results suggest another mechanism besides the inhibition of eicosanoid and cytokine production to explain the beneficial effects of fish oils in reducing inflammation.     

Effects of sonication on physicochemical and biological properties of synthetic double-stranded polyribonucleotides as interferon inducer

Summary Two kinds of synthetic double-stranded polyribonucleotides, poly IGC¹ and poly I:C and their constituent single-stranded polymers were subjected to sonication. Sonication of both poly IGC and poly IC resulted in decreases in viscosity, molecular size and heterogeneity in the size distribution. In poly IGC, whose average sedimentation constant was larger than or around 11 S, these changes were accompanied with enhancements of interferon inducing activity in rabbits and mice and antiviral activity in mice, and moreover with a decrease in the systemic toxicity in mice. In poly IC, however, such an enhancement in the interferon inducing activity was observed only when its molecular size corresponded to that of poly IGC. Previous sonication of poly C of the relatively large molecular size (> 10 S) has also been shown effective, to a certain extent, in obtaining double-stranded RNA of smaller size distribution with increased interferon inducing activity and lowered toxicity. It has been shown that these changes induced by sonication were based on the breakage of phosphodiester bonds of both double and single-stranded polyribonucleotides. On the basis of the analyses of the correlations between molecular sizes and the biological activities, it has been suggested that, while toxicity decreases always when the molecular size becomes smaller, the optimal size of the double-stranded polyribonucleotide complexes for interferon production ranges roughly from 10 S (9.1×10⁵ daltons) to 5 S (1.2 ×10⁵ daltons).     

The influence of Poly I∶C on the course of infection in mice inoculated with West Nile Virus

Summary In mice infected intraperitoneally with a hundred per cent lethal dose of West Nile virus a significant reduction in mortality was found if treatment with the complex of synthetic polyriboinosinic and polycytidylic acids (Poly IC) was given four hours prior to or twenty hours after virus challenge.Treatment induced large amounts of circulating interferon a few hours after inoculation. Only a slight difference in maximum viraemia in the various groups was found, but viraemia developed later in the mice given Poly IC a few hours before virus injection. Infection of the brain developed later in the groups treated with Poly IC.Using various doses of West Nile virus almost the same mortality was found in the group given a lethal virus dose but treated with Poly IC and the group receiving sublethal virus dose and no Poly IC treatment. Maximum of viraemia was high in the former group, while in the latter group it was significantly lower. Therefore it is supposed that Poly IC in these experiments did not protect through an interferon mediated suppression of the viraemia but rather through an effect of the interferon exerted directly upon the target organ. A reduction of circulating HI antibodies was found in the group on which Poly IC had the most pronounced effect.     

Characterization of the cellular response of spleen cells in BALB/c mice inoculated with Mycoplasma pneumoniae or the P1 protein

BALB/c mice were intranasally infected or intraperitoneally inoculated with Mycoplasma pneumoniae whole cells or were immunized with the isolated adhesin (P1 protein). Spleen cells were isolated and tested in vitro for proliferation activity after stimulation with the P1 protein and sonicated M. pneumoniae whole antigen preparations. In frequency analysis experiments the P1 protein-specific proliferative response of spleen lymphocytes increased from 1/11494 in mice immunized once to 1/3246 in eightfold-inoculated mice, demonstrating that the P1 protein is a prominent immunogen of M. pneumoniae cells. Depletion experiments showed that T and B cells are activated in a 21 relation. Fluorescence-activated cells sorting analysis revealed a shift of the CD4/CD8 ratio from 21 in control mice up to 31 in M. pneumoniae-, and to 3.41 in P1 protein-immunized mice, as well as an increase in interleukin 2 receptor-bearing cells and macrophage cell populations. The results indicate that this animal model is appropriate to study host-M. pneumoniae interactions and vaccination schedules.     

On the influence of apomorphine on the toxicity of adrenalin and phenamine

Summary Experiments on albino mice were used to determine the average lethal, doses of apomorphine, adrenalin, phenamine, and mixtures of apomorphine with adrenalin (13 and 31) and apomorphine with phenamine (13 and 31). The obtained values of average lethal doses for the mixtures were compared with those calculated according to these formulas: (1/LD_{50 M})=(P_a/LD_{50 a})+(P_b/LD_{50 b}), where LD_{50 M} is the average lethal dose of the mixture. LD_{50 a} and LD_{50 b} are the average lethal doses of its components and p_a and p_b are portions of the components in a mixture (p_a+p_b=1). This formula determines the relationship between the average lethal doses of a mixture and its components in case the latter's action is additive. It was shown that the effect of the combined action of apomorphine with adrenalin was considerably in excess of the additive effect whereas the effect of the combined action of apomorphine with phenamine was significantly less than the additive effect. These results were discussed from the viewpoint of the importance of pyrocatechol-O-methyltransferase in the inactivation of pyrocatecholamines.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 51, No. 5, pp. 70–72, May, 1966Deceased.     

Effect of adjuvant arthritis on the disposition of acebutolol enantiomers in rats

Disease states such as arthritis may interact with the kinetics of -blockers. Acebutolol (AC) is a chiral -blocker which is available as a racemate. The beneficial properties of AC, however, is attributed mainly to theS-(+)-enantiomer. The disposition of AC enantiomers and their active, chiral metabolites, diacetolol (DC) were examined after oral administration to healthy and adjuvant-induced arthritic (AA) female Sprague-Dawley rats. Arthritis was induced by tail base injection ofMycobacterium butyricum. Swelling of hind and forepaws were apparent in 10–16 days in AA but not controls. Control and AA rats were sacrificed at 0.5, 1, 2, 4, 6 and 8 h after a 25 mg/kg oral AC dose and blood was collected (n=6). Significant three to tenfold increases in the initial plasma concentrations (0.5–2h) of AC were observed in AA. Enantiomers were equally affected, thus ACSR ratio was not changed. Higher plasma concentrations of the metabolite were only significant at 2h. The ratio of DCAC, however, was unaffected by AA. The DCSR ratio was significantly decreased at 0.5 and 1 h in AA. The limited protein binding of AC (10%) was neither stereoselective nor affected by AA. Reduced intrinsic clearance in AA may be responsible for these observations.     

Decreased tissue immunoregulatory index in rheumatoid synovitis,determined by in-situ T-cell phenotyping with monoclonal antibodies

Conclusion The phenotypic tissue immunoregulatory index in rheumatoid synovitis is locally different and varies significantly between given areas. Ectopic lymphoid follicles show normal to high T4T8 ratios, whereas within the sublining perivascular and diffuse infiltrate T4T8 ratios are decreased. Thus the frequency reported immunoregulatory indices of eluted cells can't be representative for an immunoregulatory tissue disequilibrium in rheumatoid arthritis. An elevated T4T8 ratio in eluted cells may correlate with a follicular B-cell hyperplasia in rheumatoid synovitis (low local activity), a low T4T8 ratio may be related to rheumatoid synovitis with diffuse sublining T-cell infiltrates (high local activity). Our results are in principal accordance with the findings of Kurosaka and Ziff [15] and Meijer et al. [13]. They also found different T-cell distributions in synovial tissue depending on the histopathological localization.For these topographical reasons, immunoregulatory indices determined in eluted cells from tissue samples cannot be representative for the immunopathological reactions at the synovial membrane tissue level.The decrease in the phenotypic tissue immunoregulatory index in the rheumatoid synovium from deeper to superficial areas may be explained as a compensatory influx of peripheral, functionally defect T8-cytotoxic/suppressor cells (see Fig. 2).     

The effect of vitamin C on the absorption of glycine and chlorides by the intestine

Summary The effect of ascorbic acid on the regulation of absorption of glycine was studied in experiments on 3 dogs with chronic fistulae of the intestine and colon. It was established that ascorbic acid, administered in definite doses, improved absorption of glycine in the intestine and of sodium chloride in the colon. 32 and 22 are the optimal ratios of glycine and sodium chloride to the vitamin. In the presence of the above ratio absorption of glycine in the intestines increases by 24–28%, while absorption of chlorides in the colon increases by 18%.Presented by Active Member Acad. Med. Sci. USSR P. S. Kupalov     

The influence of magnesium ion and ascorbic acid on the erythrocytic schizogony of Plasmodium vivax

Asexual erythrocytic stages of Plasmodium vivax have been cultivated for one schizogonic cycle to investigate parasite requirements for metal ions and vitamins. Waymouth and RPMI 1640 (GIBCO) media were used in varying proportions resulting in varying concentrations of organic salts, vitamins, and growth factors. A 13 mixture gave the highest percentage (62.6%) of parasite development from the amoeboid forms to mature segmenters after 44 h of cultivation in a candle jar atmosphere at 38.5 C. Nevertheless, the total parasite count was significantly higher (P<0.50) in the mixture which had a Waymouth: RPMI ratio of 12. Differentiation of schizonts to merozoites as well as parasite counts could be further enhanced by the addition of magnesium chloride to a final concentration of 1.8 mM magnesium ions. The minimal requirement for ascorbic acid which was studied in Science Mahidol (SCMI 612) medium appeared to vary among isolates. For example, all parasite population of four isolates tested declined proportionally with the decrease in concentration of ascorbic acid, the critical point being 3 g/ml medium. However, two isolates used in this study could no longer differentiate to segmenters when the ascorbic acid concentration of the medium was less than 6 g/ml.     

Bone metastasizing renal tumour of childhood

Summary The histopathological spectrum of a large series of a recently described tumour entity is presented. Seven diagnostic features which may be encountered are described and their frequency discussed. The most striking clinical feature was the marked male preponderance (MF=7.61). It is suggested that an appreciation of the full histopathological spectrum is necessary to ensure adequate diagnosis.Consultant Pathologist and Honorary LecturerLecturer in Pathology     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

Ether phospholipids in control and 20∶4-depleted rat PMN: Additional evidence for a 1-0-alkyl-2-20∶4-sn-glycerol-3-phosphocholine specific phospholipase A2

Earlier studies from our laboratory have shown that PAF and LTB₄ biosynthesis are inhibited in rat PMN depleted of its 203 (JBC. '86,261, 7592). To test whether these cells contain sufficient 1-O-alkyl-2-acyl-GPC to support PAF synthesis, phosphotidyl choline was isolated from these 204-depleted cells, fractionated into different subclasses and their fatty acid composition determined. These results were compared with those obtained with control PMN. Both control and 204-depleted PMN contained significantly large amounts of alkylacyl-GPC and diacyl-GPC. Small amounts (4%) of alkenylacyl-GPC were also present. The amount of 203 in 204-depleted cells was more or less equal to the amount of 204 in control cells. About 62% of PC-bond 204 in control PMN and about 56% of PC-bound 203 in the 204-depleted PMN was found associated with the alkylacyl species. These results show that both control and 204 depleted PMN have ample precursor substrates to support PAF biosynthesis and these substrates are enriched with 204 in control cells and with 203 in 204-depleted cells. These findings are consistant with the existance of a highly specific phospholipase A₂ capable of distinguising 204 from 203 containing phospholipids.     

Effect of molsidomine on ex vivo platelet aggregation and plasma guanosine 3′∶5′-cyclic monophosphate levels in healthy volunteers

Summary To find out whether 3-morpholino-sydnonimine (SIN 1), the active metabolite of molsidomine, exerts its antiaggregatory effects not only in vitro but also in vivo, we tested ex vivo aggregation before and after intravenous application of molsidomine in healthy volunteers. We also measured plasma levels of guanosine 35-cyclic monophosphate (cyclic GMP) as SIN 1, the bioactive metabolite of molsidomine, becomes effective via activation of soluble guanylate cyclase. In eight out of ten subjects molsidomine had an inhibitory effect on platelet aggregation and a higher threshold concentration of platelet-activating factor was required after molsidomine application to induce irreversible aggregation. Despite the effect on platelets, plasma cyclic GMP levels did not increase. These results suggest that the nitric oxide-containing SIN 1 inhibits platelet aggregation not only in vitro but also in vivo and that this property can be a beneficial effect in antianginal therapy.Abbreviations Cyclic GMP guanosine 35-cyclic monophosphate - NO nitric oxide - PAF platelet-activating factor - PRP platelet-rich plasma - SIN 1 3-morpholino-sydnonimine     

Symmetry, broken symmetry, and handedness in bimanual coordination dynamics

Summary The symmetrical dynamics of 11 rhythmic bimanual coordination may be specified by an order parameter equation involving the relative phase between rhythmic components, and an interlimb coupling which determines the relative attractiveness of in-phase and anti-phase patterns. Symmetry breaking of these dynamics can occur via the difference in the natural frequencies, , of the left and right rhythmic components, or by the intrinsic asymmetrical dynamics of the body. The latter is captured by additional terms that render the symmetrical coupling slightly anisotropic. A major prediction resulting from this step is that although =0, as the frequency of coordination is increased, the asymmetrical coupling will increase and the symmetrical coupling will decrease. This results in a greater left-limb bias in left-handers and right-limb bias in right-handers. This increased handedness prediction was confirmed in an experiment in which 20 left-handed and 20 right-handed individuals performed 1 1 coordination with hand-held rigid pendulums. Manipulations of left and right pendulum lengths controlled , and the coupled frequency was determined by a metronome. Also confirmed was the prediction that the small shift in equilibria from in-phase and anti-phase due to the intrinsic asymmetry should be amplified in left-handers when > 0 and in right-handers when < 0. Further, the bias in left-handers was more consistent than the bias in right-handers, and a subgroup of right-handers was identified who performed similarly to left-handers. The coordination dynamics of functional asymmetry provides insights into the elementary synergy between the limbs, the dynamical mechanism that modulates it, and the nature of the asymmetry in left-handed and right-handed individuals.     

Complement-fixing antibodies to human cytomegalovirus induced early nuclear antigens in mononucleosis

Early nuclear antigen (CMNA) induced by human cytomegalovirus (HCMV) was extracted from infected human embryonic fibroblast cells and purified by ds DNA Sephadex chromatography. The purified antigen was added to acid-fixed preparations from human embryonic fibroblasts and these in vitro converted nuclei were exposed to human sera to estimate the antibodies to CMNA by anti-complement immunofluorescence staining. Serial serum samples were obtained from eight patients suffering from acute HCMV infection (mononucleosis), from nine patients with Epstein-Barr virus (EBV) mononucleosis, and from 20 healthy persons who had been shown to possess antibodies to HCMV late antigens. Acute HCMV infection is characterized by the presence of anti-CMNA antibodies at high titer (116–132) together with the elevated level of IgG antibodies to anti-HCMV-late antigens. During convalescence the anti-CMNA titer decreased to a lower level which was maintained for long period.The anti-CMNA antibodies were also regularly detected in sera of persons possessing antibodies to HCMV late antigens but without any sign of acute HCMV infection. It is concluded that antibodies to CMNA are not transitory and their presence even at high titer (116–132) in a serum sample cannot be taken as a presumptive evidence of acute virus infection.This method seems to be valuable for measuring antibodies to nuclear DNA-binding antigens induced by other viruses.     

Degeneration of two of nine types of synapses in the putamen after center median coagulation in the cat

Summary The cat putamen contains the identical nine types of synapses and the same proportion of axo-dendritic (or axo-somatic) synapses as described for the fundus striati. However, type III (cortico-striatal) (3116%) and type V (axon-collateral) (131%) occur much more frequently and type I (nigro-striatal) much less frequently (1434%) in the putamen than in the fundus striati. Of the axo-spinous synapses only type IV, with densely arranged small round vesicles and interrupted, asymmetric contact, shows a dark degeneration after center median lesions, mainly in the parvocellular part. Of the six axo-dendritic (or axo-somatic) synapses, only type VII, with densely packed small round vesicles and asymmetric contact, is degenerated after the same lesion in the center median nucleus. However, after such lesions type VII synapses are much more frequently degenerated in the putamen than those of type IV.Dedicated to Prof. Dr. Hartwig Kuhlenbeck, Philadelphia, USA on the occasion of his 80th birthdayDr. J. W. Chung on leave of absence from the Department of Anatomy, Catholic Medical College, Seoul, Korea     

Secretin and VIP-stimulated adenylate cyclase from rat heart

Cardiac adenylate cyclase activity was normal in 3 weeks-old spontaneously hypertensive rats of the Wistar-Okamoto substrain. The hormone-sensitive adenylate cyclase activity was reduced in 10 weeks-old or older animals, and secretin- and VIP-activations were definitely more impaired (by 64% and 69%, respectively) than isoproterenol- and glucagonactivation (17% and 22%, respectively). By contrast, the fluoride- and p[NH]ppG-stimulations of the enzyme were unaffected. These alterations in the adenylate cyclase system coupled to secretin and VIP appeared specific to the heart as the isolated pancreatic acinar cells from spontaneously hypertensive animals responded normally to secretin, as a liver particulate fraction responded normally to secretin and VIP, and both brain synaptic membranes and a particulate fraction of anterior pituitary to VIP.Abbreviations VIP vasoactive intestinal peptide - cyclic AMP cyclic adenosine 35-monophosphate - p[NH]ppG guanosine 5-(, -imido)triphosphate - EGTA ethylene-glycol-bis-(2-amino-ether)-N,N,N,N-tetraacetic acid - IBMX 3-isobutyl-1-methylxanthine