Sagittal, vertical and transversal dimensions of the maxillary complex in patients with ectopic maxillary canines.

OBJECTIVES: To analyse the craniofacial maxillary complex in cases with labially and palatally located ectopic canines, subgrouped accordingly: Group I: no deviations in the dentition; Group IIa: deviations in the maxillary incisors only; Group IIb: deviations in the dentition in general. SETTING AND SAMPLE POPULATION: Sixty nine patients (mean age 13 years 6 months) with palatally or labially located ectopic canines. MATERIAL AND METHODS: Profile radiographs and dental casts were analysed. The patients were subgrouped according to a previous registration of dental deviations registered radiographically. Maxillary cross-arch transversal width was analysed on dental casts. Sagittal and vertical dimensions were registered cephalometrically on profile radiographs. RESULTS: In the patient sample the maxillary cross-arch transversal width (from first maxillary molar left to first maxillary molar right), was significantly larger than the normal mean (0.65 mm, 95% Cl: 0.02-1.28, p = 0.043). The sagittal length N-S was significantly shorter (-0.97, 95% Cl:-1.72-(-)0.22, p = 0.002). The vertical length ANS-N length was also significantly shorter (-0.79, 95% Cl:-1.65-(-)0.02, p = 0.047). The remaining variables were non-significant. Tests for interaction between groups (I, IIa and IIb) and palatal/labial ectopic location did not demonstrate significance. CONCLUSION: In patients with ectopic maxillary canines, the maxillary complex is shorter sagittally as well as vertically, while it is wider transversally.

Differences between dentitions with palatally and labially located maxillary canines observed in incisor width, dental morphology and space conditions.

AIM: To analyze the interrelationship between incisor width, deviations in the dentition and available space in the dental arch in palatally and labially located maxillary ectopic canine cases. MATERIALS AND METHODS: Size: On dental casts from 69 patients (mean age 13 years 6 months) the mesiodistal widths of each premolar, canine and incisor were measured and compared with normal standards. Dental deviations: Based on panoramic radiographs from the same patients the dentitions were grouped accordingly: Group I: normal morphology; Group IIa: deviations in the dentition within the maxillary incisors only; Group IIb: deviations in the dentition in general. Descriptive statistics for the tooth sizes and dental deviations were presented by the mean and 95% confidence limits for the mean and the p-value for the T-statistic. Space: Space was expresses by subtracting the total tooth sizes of incisors, canines and premolars from the length of the arch segments. RESULTS: Size of lateral maxillary incisor: The widths of the lateral incisors were significantly different in groups I, IIa and IIb (p=0.016) and in cases with labially located ectopic canines on average 0.65 (95% CI:0.25-1.05, p=0.0019) broader than lateral incisors in cases with palatally located ectopic canines. Space: Least available space was observed in cases with labially located canines. The linear model did show a difference between palatally and labially located ectopic canines (p=0.03). Space related to deviations in the dentition: When space in the dental arch was related to dental deviations (groups I, IIa and IIb), the cases in group IIb with palatally located canines had significantly more space compared with I and IIa. CONCLUSION: Two subgroups of palatally located ectopic maxillary canine cases based on registration of space, incisor width and deviations in the morphology of the dentition were identified.

Antisense properties of peptide nucleic acid.

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose phosphate backbone has been replaced by a pseudo-peptide polymer to which the nucleobases are linked. PNA-oligomers can be synthesized in relatively large amounts, are highly stable in biological environments, and bind complementary DNA and RNA targets with remarkably high affinity and specificity. Thus PNA possesses many of the properties desired for a good antisense agent. Until recently, limited uptake of PNA into cells has been the major obstacle for applying PNA as an antisense agent in cell cultures and in vivo. Here, the antisense properties of PNA in vitro and in vivo will be reviewed. In particular, we will focus on recent observations indicating that PNA equipped with or without various uptake moieties may function as an efficient and gene-specific inhibitor of translation in Escherichia coli and in certain mammalian cell types.

Structural isomers of bis-PNA bound to a target in duplex DNA.

Upon binding of a decamer bis-PNA (H-Lys-TTCCTCTCTT-(eg1)(3)-TTCTCTCCTT-LysNH(2)) to a complementary target in a double-stranded DNA fragment, three distinct complexes were detected by gel mobility shift analysis. Using in situ chemical probing techniques (KMnO(4) and DMS) it was found that all three complexes represent bona fide sequence-specific PNA binding to the designated target, but the complexes were structurally different. One complex that preferentially formed at higher PNA concentrations contains two bis-PNA molecules per DNA target, whereas the other two complexes are genuine triplex invasion clamped structures. However, these two latter complexes differ by the path relative to the DNA target of the flexible ethylene-glycol linker connecting the two PNA oligomers that comprise a bis-PNA. We distinguish between one in which the linker wraps around the non-target DNA strand, thus making this strand part of the triplex invasion complex and another complex that encompass the target strand only. The implications of these results are discussed in terms of DNA targeting by synthetic ligands.

Cooperative strand displacement by peptide nucleic acid (PNA).

BACKGROUND: Synthetic homopyrimidine peptide nucleic acids (PNAs) can bind complementary targets in double-stranded DNA, generating strand-displacement complexes, and so offering an opportunity to modulate specific gene expression. Several issues remain to be addressed before these attributes can be exploited in vivo, however. RESULTS: The kinetics of the interaction between a homopyrimidine PNA and a complementary homopurine target on double-stranded DNA were analyzed in the presence or absence of a preformed strand-displacement complex proximal to the target. The complex was established under low salt conditions by the binding of a different homopyrimidine PNA to a target situated adjacent to the first PNA target. These two targets were placed next to each other on opposite strands at distances of 0, 2, 4 and 8 base pairs apart. The presence of a preformed strand-displacement complex near the target accelerates the binding of PNA to double-stranded DNA in a salt-dependent manner. The influence of salt on the binding rates was also examined. The binding rate is increased by a factor of 1 x exp(70[NaCl]), that is, 16-fold at 40 mM NaCl and more than 10(4)-fold if extrapolated to 140 mM NaCl. This effect is significantly reduced if the two targets are 2 base pairs apart and completely absent if the distance is 4 base pairs or more. CONCLUSIONS: The perturbation of the DNA helix imposed by a PNA strand-displacement complex only propagates a few base pairs. It is therefore possible to target sites in the immediate vicinity of strand invasion complexes specifically. The results presented have implications for the mechanism of strand displacement and for the application of PNA in a genomic context.

Use of magnetic beads for Gram staining of bacteria in aqueous suspension.

A Gram staining technique was developed using monodisperse magnetic beads in concentrating bacteria in suspension for downstream application. The technique does not require heat fixation of organisms, electrical power, or a microscope. Gram-negative and Gram-positive bacteria were identified macroscopically based on the colour of the suspension. The bacteria concentrated on magnetic beads may also be identified microscopically.

Organochlorines in top predators at Svalbard--occurrence, levels and effects.

Alarmingly high polychlorinated biphenyl (PCB) levels have been found in the top predators such as glaucous gull (Larus hyperboreus) and polar bear (Ursus maritimus) at Svalbard [Gabrielsen, G.W., Skaare, J.U., Polder, A., Bakken, V., 1995. Chlorinated hydrocarbons in glaucous gull (Larus hyperboreus). Sci. Total Environ. 160/161, 337-346; Bernhoft, A., Skaare, J.U., Wiig, O., 1997. Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Pollut. 95, 159-175; Henriksen, E.O., Gabrielsen, G.W., Trudeau, S., Wolkers, H., Sagerup, K., Skaare, J.U., 1999. Organochlorines and possible biochemical effects in glaucous gull (Larus hyperboreus) from Bear Island, the Barents Sea. Arch. Environ. Contam. Toxicol. (in press). ]. Studies of the possible toxic effects, particularly on the immune system and reproduction, of the very high PCB levels in these species are currently being investigated. Data obtained in the field (f.i. reproductive success in polar bears and intestinal nematodes in glaucous gulls), as well as levels of various biochemical and physiological parameters (f.i. thyroid hormones, retinol, EROD activity, CYP1A, IgG), have been coupled with the PCB levels [Skaare, J.U., Wiig, O., Bernhoft, A., 1994. Klorerte organiske miljogifter; Nivâer og effekter i isbjorn. Norwegian Polar Institute Reportseries no. 86, 1-23 (in Norwegian); Bernhoft, A., Skaare, J.U., Wiig, O., 1997. Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Pollut. 95, 159-175; Bernhoft, A., Skaare, J.U., Wiig, O., Derocher, A.E., Larsen, H.J., 2000. Possible immunotoxic effects of organochlorines in polar bears (Ursus maritimus) at Svalbard (in press); Henriksen, E.O., Gabrielsen, G.W., Skaare, J.U., Skjegstad, N., Jensen, B.M., 1998a. Relationship between PCB levels, hepatic EROD activity and plasma retinol in glaucous gull, Larus hyperboreus. Marine Environ. Res. 46, 45-49; Henriksen, E.O., Gabrielsen, G.W., Trudeau, S., Wolkers, H., Sagerup, K., Skaare, J.U. , 1999. Organochlorines and possible biochemical effects in glaucous gull (Larus hyperboreus) from Bear Island, the Barents Sea. Arch. Environ. Contam. Toxicol. (in press); Sagerup, K., Gabrielsen, G.W., Skorping, A., Skaare, J.U., 1998. Association between PCB concentrations and intestinal nematodes in glaucou gulls, Larus hyperboreus, from Bear Island. Organohalogen compounds 39, 449-451; Skaare, J.U., Wiig, O., Bernhoft, A., 1994. Klorerte organiske miljogifter; Nivâer og effekter i isbjorn. Norwegian Polar Institute Reportseries no. 86, 1-23. (in Norwegian)].

Kinetics of IL-2 receptor expression on lymphocyte subsets from goats infected with Mycobacterium avium subsp. paratuberculosis after specific in vitro stimulation.

Quantification of surface IL-2R expression on activated lymphocytes by flow cytometry have recently been reported to be useful in measuring cellular immunity against Mycobacterium avium subsp. paratuberculosis in goats (Whist et al., 2000, Vet. Immunol. Immunopathol. 73, 207-218). To characterise the phenotype of the peripheral lymphocytes expressing IL-2R after in vitro stimulation with purified protein derivative (PPD) from M. a. paratuberculosis, cells were processed for dual or triple colour analysis by flow cytometry (CD4 and IL-2R or CD8, gammadelta-TcR and IL-2R). To distinguish the response of antigen-specific T cells from non-specific stimulation, we performed a time-course study of proliferating cells in a group of M. a. paratuberculosis-infected animals and a control group. Following in vitro stimulation with PPD of whole blood for three different periods of time, IL-2R expression was detected mainly not only in gammadelta-T cells, but also in CD4+ and CD8+ T cells. We found a specific response of gammadelta-T cells from infected animals after 24h of stimulation. Following 120h of stimulation, however, gammadelta-T cells from control animals up-regulated IL-2R to the same level as those from infected animals, indicating either a non-specific stimulation or activation due to a first line of defence against Mycobacterium antigens. The CD4+ cells showed a specific response to PPD stimulation at all three time points. A minor population of antigen reactive gammadelta+ cells also expressed CD8. The proliferative responses differed between alphabeta and gammadelta-T cells; the IL-2R+ alphabeta T cell population mainly comprised proliferating cells, while the gammadelta+ population showed less expansion.

Possible association of antibody responses to human serum albumin and (T,G)-A--L with the bovine major histocompatibility complex (BoLA).

Antibody responses to human serum albumin (HSA) and (T,G)-A--L were determined in 130 young bulls in Norway and the BoLA types of the bulls were defined. Significant associations of some BoLA antigens with immune responsiveness were shown, indicating the likely existence of an immune response (Ir) region linked to the BoLA class I antigens. High response to HSA seems to be a dominant trait. BoLA w2 showed an association with low response to HSA. This may reflect the effect of a specific MHC-associated immune suppressor gene.

Functions of neutrophils in sheep experimentally infected with Ehrlichia phagocytophila.

Ehrlichia phagocytophila infection in sheep is characterized by persistent neutropaenia, indicative of decreased phagocytic capacity. This predisposes infected animals to other infections. A whole blood flow cytometrical method was used to document the degree and extent of reduced phagocytic and respiratory burst activity in phagocytes during an experimental infection with E. phagocytophila, and monitored until 56 days post-infection. Six sheep at 5 months of age were inoculated with an intravenous injection of infected blood. Six age-matched sheep were used as controls. A period of reduced respiratory burst lasting up to Day 17 post-infection was recorded. The population of cells showing phagocytic activity without respiratory burst was larger in the infected animals compared to controls up to Day 45 post-infection.

The use of interleukin-2 receptor expression as a marker of cell-mediated immunity in goats experimentally infected with Mycobacterium avium ssp. paratuberculosis.

The purpose of the present work was to demonstrate cell-mediated immune response to paratuberculosis in experimentally infected animals, using quantification of interleukin-2 receptor (IL-2R) expression on activated lymphocytes by means of in vitro stimulation with Mycobacterium avium ssp. paratuberculosis-derived purified protein derivative (PPDp). A whole-blood technique was developed, and optimal conditions for quantification of IL-2R expression on caprine lymphocytes, using monoclonal antibodies (anti-bovine IL-2R-alpha) and low cytometrical analysis, were determined. Different PPDp-antigen concentrations and incubation times were compared. The whole-blood method was also compared to the more traditional IL-2R assay using peripheral blood mononuclear cultures (Hesketh et al., 1993). Cross-reactivity to Mycobacterium avium was studied at different mycobacteria-PPD concentrations. An immune response could be demonstrated in animals infected with Mycobacterium avium ssp. paratuberculosis. We found that a PPDp concentration of 10microgml(-1) together with an incubation time of 72h, gave the best results using the whole-blood method. The whole-blood method eliminates many laborious steps involved in lymphocyte separation, and the effects of all the constituents of blood are expressed in a way which corresponds more to in vivo conditions. The risk of selecting subpopulations of lymphocytes during cell separation is avoided.

Genetic variation in the humoral immune response in Atlantic salmon (Salmo salar) against Aeromonas salmonicida A-layer.

Antibody responses to Aeromonas salmonicida A-layer were analysed in family material of Atlantic salmon (Salmo salar), consisting of 791 fish belonging to 34 full-sib groups within 12 paternal half-sib groups. The fish were immunized twice and blood samples were collected three times. Significant increase in antibody titre from first to second, and from second to third sampling, was observed. Genetic variation in antibody titres was observed at the three samplings with estimated heritabilities ranging from 0.16 to 0.20, and a significant heritability estimate was recorded in the antibody response after the second immunization (h2 = 0.16). Moderate to high genetic (r = 0.5-0.72) and phenotypic (r = 0.29-0.57) correlations were found between the titre values at different samplings, and between selected titres and titre increases. Production parameters, such as mean slaughter weight and mean survival rate were obtained for fish which were reared commercially in the sea, and which belonged to the same full-sib groups as those analysed for A. salmonicida A-layer antibodies. No significant correlation between the mean antibody titre to A. salmonicida A-layer in this study and mean slaughter weight and survival rate in full-sib family material in the sea was observed.

Genetic variation in the humoral immune response against Vibrio salmonicida and in antibody titre against Vibrio anguillarum and total IgM in Atlantic salmon (Salmo salar).

Total IgM level and antibody titre to Vibrio anguillarum O-antigen after bath-vaccination, and specific antibody response to V. salmonicida O-antigen at three different samplings were analysed in family material of Atlantic salmon (Salmo salar), consisting of 791 fish belonging to 34 maternal full-sib groups within 12 paternal half-sib groups. The fish were immunized twice, and blood samples collected three times. After the third blood sampling, the fish were challenged with V. anguillarum. Medium to low genetic variation was recorded in total IgM and in the antibody titres against V. anguillarum O-antigen and V. salmonicida O-antigen, with heritability estimates of 0.12, 0.18 and from 0.03 to 0.12, respectively. Moderate to high genetic and phenotypic correlations were found between the V. salmonicida O-antigen titres at different samplings. Genetic and phenotypic correlations between the initial titres were moderate to low. The effect of different immune traits, including Aeromonas salmonicida A-layer titres (previously described), on the ability to survive the challenge was examined. The likelihood of surviving the challenge was affected positively by the A. salmonicida A-layer titre at the second sampling, and almost significantly affected by the initial V. anguillarum O-antigen titre. Production traits, such as mean slaughter weight and mean proportion of survivors in a corresponding full-sib material, were obtained in the sea-rearing period. No significant full-sib correlation between immune parameters and production traits was detected.

Modulation of leukocyte populations and immune responses in sheep experimentally infected with Anaplasma (formerly Ehrlichia) phagocytophilum.

Anaplasma phagocytophilum infection in sheep is characterized by an immune suppression as indicated by impaired antibody response, reduced lymphocyte response and reduced oxidative burst. The effect of A. phagocytophilum infection on leucocyte populations, especially lymphocytes, was therefore investigated in six sheep experimentally infected with A. phagocytophilum, and compared with leucocyte populations from control animals.To investigate the ability of the infection to interfere with the cellular and humoral responses to specific antigens, the animals were vaccinated with commercial vaccines at the time of experimental infection, and monitored for 56 days. There were reduced percentages of gammadelta T-cells and CD4+ T-cells in peripheral blood of infected animals throughout the study period, and these cell populations showed a down-regulation of CD25 expression; while there was a relative increase in CD8+ T-cells. The reduction in CD25+ gammadelta T-cells involved a subpopulation of WC1+ gammadelta T-cells. During the first 2 weeks of the study there were reduced percentages of B-cells and leukocytes expressing MHC II and CD11b, though this decrease changed to a relative increase later in the study. The relative reductions in leucocyte populations corresponded with the observed leucopenia during the first 3 weeks post-infection, which involved lymphocyte, neutrophil and eosinophil subsets [Vet. Immunol. Immunopathol. 86 (2002) 183]. There was a reduced expression of CD11b and CD14 on granulocytes during the first 2 weeks of the study, which corresponded with the previously reported leucopenia involving neutrophils and eosinophils. Antibody responses to vaccines, lymphocyte in vitro proliferative responses to antigens and mitogens, and in vitro IFN-gamma responses to antigens were reduced up to 4 weeks after infection.

Subclinical paratuberculosis in goats following experimental infection. An immunological and microbiological study.

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5-8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls. Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-gamma assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15-20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI. Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-gamma assay. The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of gammadelta T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.

Possible immunotoxic effects of organochlorines in polar bears (Ursus maritimus) at Svalbard.

Associations between immunoglobulin G (IgG) levels and the organochlorine contaminants (OCs) polychlorinated biphenyls (PCBs), chlordanes, 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene (DDE), hexachlorobenzene (HCB), and hexachlorocyclohexanes (HCHs) in blood plasma from polar bears caught at Svalbard were determined. The blood samples were collected from free-living polar bears of different age and sex between 1991 and 1994. The IgC concentration increased with age and was significantly higher in males than in females. IgG was negatively correlated with sigmaPCB level and with three individual PCB congeners, IUPAC numbers 99, 194, and 206. HCB was also negatively correlated with IgG. The significant negative OC correlation with IgG levels may indicate an immunotoxic effect.

Sequencing strategy of mitochondrial HV1 and HV2 DNA with length heteroplasmy.

We describe a method to obtain reliable mitochondrial DNA (mtDNA) sequences downstream of the homopolymeric stretches with length heteroplasmy in the sequencing direction. The method is based on the use of junction primers that bind to a part of the homopolymeric stretch and the first 2-4 bases downstream of the homopolymeric region. This junction primer method gave clear and unambiguous results using samples from 21 individuals with length heteroplasmy in the hypervariable regions HV1, HV2 or both. The method is of special value for forensic casework, because sequencing of both strands of an mtDNA region is preferable in order to reduce ambiguities in sequence determination.

Experimental pestivirus infections in newborn goat kids.

Two experiments were carried out in which 37 healthy newborn goat kids were inoculated with a non-cytopathic ovine (BDV) or a cytopathic bovine pestivirus (BVDV) by intramuscular or intracerebral injection. No kids showed signs of disease or gross lesions which could be attributed to these viruses, but inoculated kids had lower mean growth rates than the controls. Significant histological changes in the CNS of 14 kids were restricted largely to the white matter and consisted mainly of hypercellular foci comprising microglial/histiocytic cells and mild perivascular infiltration by mononuclear cells. Varying degrees of infiltration of the myocardium by lymphocytes and plasma cells were observed. All kids remained negative for neutralizing antibodies against pestivirus until 2 to 3 weeks after infection. Titres increased during the following weeks. Pestiviruses were recovered from kids necropsied 10 days after inoculation, but not from any kids killed 20 days after inoculation or later. Non-cytopathic virus was isolated from various tissues of four kids that had received BDV and three kids that had been given BVDV. Cytopathic viruses were not recovered from any kids. Mean white blood cell counts in all kids were within the normal range at 4 and 8 weeks after inoculation. The lymphocyte response to stimulation by phytohaemagglutinin was significantly increased on both sampling occasions in the BDV-inoculated kids, while in the BVDV-inoculated animals, a similar increase was seen only at 8 weeks.

A whole blood method for measuring mitogen-induced transformation of sheep lymphocytes.

A simple and reproducible micromethod for determination of in vitro mitogenic responses of sheep peripheral blood lymphocytes is described. The test uses (i) whole blood diluted in RPMI 1640 medium to give a cell count of 0.5 x 106-1 x 106 lymphocytes/ml, (ii) mitogens in the range of 5-20 micrograms of phytohaemagglutinin/ml, 20-80 micrograms of lipopolysaccharide/ml or 20-80 microliters of poke weed mitogen/ml, and (iii) a stimulation time of 42 to 90 h. A considerable variation in mitogenic response was observed both between animals and on different occasions in the same animal. Because of the large periodic variation it was suggested that the test should be repeated using blood drawn at different times in order to determine the mitogenic response of an animal.

Distribution of T and B lymphocytes in jejunal and ileocaecal Peyer's patches of lambs.

Jejunal (JPPs) and ileocaecal (IPP) Peyer's patches in lambs were studied by employing the indirect and the avidin-biotin-peroxidase methods, using antibodies to sheep IgM and thymocytes. Thymocyte sera absorbed with IPP germinal centre cells showed specificity for T cells in the thymus, lymph nodes and Peyer's patches. Whereas JPPs had large interfollicular T cell areas, IPP mainly contained B cells, although small triangular T cell areas were found at the apex of the follicles. Some T cells were also found in the dome and corona region of JPPs and IPP. While germinal centres of JPPs had about 40 per cent of IgM-positive cells, IPP germinal centres contained about 80 per cent of such cells. Negative or weak IgM-positive cells were seen in the peripheral zone of IPP germinal centres, contrasting with surface IgM-positive cells in the central zone, and indicating a centripetal migration of maturing lymphocytes.