Effect of insemination volume on uterine contractions and inflammatory response and on elimination of semen in the mare uterus-scintigraphic and ultrasonographic studies.

The effect of artificial insemination (AI) volume on uterine contractility and inflammation and on elimination of semen in the reproductive tract of mares was examined for 4 h after AI using two methods, scintigraphy and ultrasonography. The same doses were used in both methods: 2 and 100 ml of skim milk-extended frozen semen. In the scintigraphic study, the number of reproductively normal mares was four per group and in the ultrasonographic study five per group. For scintigraphy, the semen was radiolabelled with technetium-99m. The static scintigrams were acquired immediately before and 30, 60, 120, 180 and 240 min after AI. The activities in the vagina and uterus were calculated and the values for sperm that had been discharged from the mare were obtained by subtracting the counts for the uterus and vagina from the total radioactivity. The dynamic scintigrams were taken continuously for the first 30 min after AI and in 5-min periods immediately after having acquired the static scintigrams. The uterine contractions were counted. In the ultrasonographic study, the mares were scanned before AI and at 5, 10, 15, 20, 25, 30, 60, 120, 150, 180, and 240 min after AI, for at least 1 min each time. The examinations were videotaped and contractions counted per minute. More contractions were observed with the ultrasonographic method than with the scintigraphic method. No difference was present in the number of contractions between the groups, except in the ultrasonographic study at 4 h, when the mares inseminated with 100 ml showed more contractions than did the mares inseminated with 2 ml. The intraluminal fluid was sampled with a tampon and by uterine lavage 4 h after AI in the ultrasonographic study. The numbers of polymorphonuclear leukocytes and spermatozoa were counted, but the differences between the groups were not significant. Under our experimental conditions and with the number of mares examined, the volume of the AI dose had an insignificant effect on contractility - with the exception at 4 h - and inflammatory reaction and on semen elimination in the uterus.

Clinical significance of ossification of the cartilages of the front feet based on nuclear bone scintigraphy, radiography and lameness examinations in 21 Finnhorses.

REASONS FOR PERFORMING STUDY: Research on the clinical significance of ossification of the cartilages of the foot has been limited, despite the common nature of the condition and conflicting reports in previous literature. HYPOTHESIS: Some radiographic features in the ossification of the cartilages, such as incomplete fusion lines between separate centres of ossification and the ossified base, are of clinical significance. METHODS: The relationships between radiopharmaceutical uptake in bone phase nuclear scintigraphy at the heels (palmar processes of the distal phalanx, including ossification of the cartilages of the foot), radiographic extent and type of ossification of the cartilages and clinical lameness were evaluated retrospectively in 21 Finnhorses (age > or = 4 years) in a total of 36 front feet. RESULTS: No significant relationship between height of the ossifications and radiopharmaceutical uptake at the ipsilateral heels existed. Clearly separate centres of ossification were not associated with increased uptake. Moderately increased uptake was suspected to be associated with ossification of the adjacent cartilage in only one foot. Intense uptake was present unilaterally in 4 horses, at one medial and 3 lateral heels. In 2 of these horses, a unilateral palmar digital nerve block relieved the mild lameness; 2 horses had no obvious lameness but had a history of being stiff or having locomotion problems during high speed trot. At 2 of the lateral heels, an incomplete fusion line was present between a large separate centre of ossification and the base, and the third horse had a high sidebone with bony protrusions, suggestive of chronic entheseopathy in a narrow foot. At the medial heel, an oblique radiograph revealed a faint radiolucent line at the base of the ossification. In all cartilages with intense radiopharmaceutical uptake at the heel and/or lameness, the ossified part of the cartilage was wider and more irregular compared to other ossifications of the front feet of the individual. CONCLUSIONS: Increased radiopharmaceutical uptake, associated with a different radiographic appearance from that of other ossifications of the front feet, was a conclusive sign of clinical significance. Obscure locomotion problems were more commonly associated with ossification of the cartilages than true lameness. POTENTIAL RELEVANCE: This information is useful in lameness and prepurchase examinations and is likely also to be applicable to other coldblooded breeds used for athletic purposes.

Analysis of lapine cartilage matrix after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate.

OBJECTIVE: To study the short and long term effects of radiosynovectomy on articular cartilage in growing and mature rabbits. METHODS: The articular cartilage of the distal femurs of rabbits was examined four days, two months, and one year after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate ([(166)Ho]FHMA). Arthritic changes were evaluated from histological sections by conventional and polarised light microscopy, and glycosaminoglycan measurements using safranin O staining, digital densitometry, and uronic acid determination. Proteoglycan synthesis was studied by metabolic [(35)]sulphate labelling followed by autoradiography, and electrophoretic analysis of extracted proteoglycans. Northern analyses were performed to determine the mRNA levels of type II collagen, aggrecan, and Sox9 in cartilage samples. RESULTS: Radiosynovectomy had no major effect on the histological appearance of articular cartilage in mature rabbits, whereas more fibrillation was seen in [(166)Ho]FHMA radiosynovectomised knee joints of growing rabbits two months after treatment, but not after one year. Radiosynovectomy did not cause changes in the glycosaminoglycan content of cartilage or in the synthesis or chemical structure of proteoglycans. No radiosynovectomy related changes were seen in the mRNA levels of type II collagen, whereas a transient down regulation of aggrecan and Sox9 mRNA levels was seen in young rabbits two months after [(166)Ho]FHMA radiosynovectomy. CONCLUSIONS: [(166)Ho]FHMA radiosynovectomy caused no obvious chondrocyte damage or osteoarthritic changes in mature rabbits, but in growing rabbits some transient radiation induced effects were seen--for example, mild cartilage fibrillation and down regulation of cartilage-specific genes.

Synovial fluid studies in navicular disease.

The purpose of this study was to investigate biochemical changes in synovial fluid in navicular disease, and to establish if synovial fluid from the distal interphalangeal joint (DIP) could be used diagnostically to assess alterations in the synovial fluid of the navicular bursa. Cartilage oligomeric matrix protein (COMP), total glycosaminoglycans (GAG), hyaluronan (HA), metalloproteinases 2 and -9 (MMP-2 and MMP-9) and total protein (TP) levels were determined in synovial fluids obtained from 18 navicular bursae and 35 DIP -joints from animals suffering from navicular disease, and the same synovial structures in 16 joints of horses with no evidence of abnormalities involving the foot. To avoid dilution effects, GAG/COMP, HA/COMP, MMP-2/ COMP and MMP-9/COMP ratios were also calculated for different synovial cavities. There was a good correlation, for COMP, GAG, HA, MMP-2 and TP levels, between synovial fluid from the navicular bursa and fluid from the DIP -joint in healthy animals. However, in animals with navicular disease, only COMP levels showed no difference between the navicular bursal fluid and the DIP-joint fluid concentration. Thus, enabling the use of COMP to standardise other biochemical concentration measurements from the synovial joint fluids. In horses with navicular disease, there was a significantly lower absolute concentration of GAG, and a significantly lower GAG/COMP ratio, in the synovial fluid of the navicular bursa and the DIP-joint compared to synovial fluid from the same joints from healthy horses. In contrast, the absolute HA concentration and HA/ COMP, MMP-2/COMP and MMP-9/COMP ratios were higher in synovial fluid from the DIP-joint of horses with navicular disease, and MMP-2 and MMP-9 relative activity levels and MMP-2/COMP and MMP-9/ COMP ratios were increased in fluid from navicular bursae in horses with navicular disease when compared to a control group.

Transport of spermatozoa in the reproductive tracts of mares.

A scintigraphic method was developed to study sperm migration in the reproductive tracts of mares. Mares (n=5) and stallions (n=2) were used to test various steps of the procedure and three other mares and a stallion were used to study sperm transportation. A radiolabelling solution was prepared from 99mTc (Technetium-99m) and hexamethyl propylene amine oxime. The highest labelling of spermatozoa (57-72%) was obtained by incubation of the spermatozoa with the radiolabelling solution for 20 min at 20 degrees C. Radioactivity outside the spermatozoa was removed by centrifugation and by two subsequent washings with skimmed milk extender. The labelled spermatozoa were inseminated into the uterine bodies of detomidine-sedated mares undergoing oestrus and scintigraphic images were obtained for 4.5 h after insemination. Both dynamic (two pictures s(-1), 2 min multiple scans, 64 x 64 matrix) and static scans (20 s, 64 x 64 matrix) were used to image the mares. The attenuation of gamma radiation in the vagina and uterus was determined for each mare. Radioactive spermatozoa were found in the tips of the uterine horns for the first time at about 8-20 min after insemination. The frequency of uterine contractions varied considerably among mares and ranged from five to 65 contractions during the first 30 min after insemination. Within 1 h after insemination most of the sperm activity was in the uterus. At 2.5 h after insemination, most of the spermatozoa had left the uterus and were either in the vagina or had escaped into the vulva and been discharged. At 4.5 h after insemination there was hardly any sperm activity in the uterus and a small amount of activity only in the vagina; most spermatozoa had been eliminated from the mares.

Allotype-associated differences in concentrations of human IgA2.

Concentrations of immunoglobulin (Ig)A2 were determined in 176 Finnish blood donor sera. Their IgA, IgM, IgE, IgG1, IgG2, IgG3 and IgG4 concentrations and Gm allotypes had been determined earlier. The mean concentration of IgA2 was higher in individuals carrying the Gm(ax) allele (0.15 g/l) than in those negative for Gm(x) (0.103 g/l). The difference was statistically significant (P = 0.006). As > or = 70% of IgA was usually IgA1, its concentration could be calculated fairly reliably by subtracting the IgA2 value from the IgA value. The mean IgA1 concentration (2.03 g/l) seemed to be independent of the Gm allotypes.

Intrauterine fluid accumulation in oestrous mares.

Intrauterine fluid (IUF) was collected using a tampon from mid-oestrous mares (n = 57) with and without ultrasonically detectable accumulations of free intraluminal fluid. Bacteria were cultured and neutrophils counted from all samples (n = 57). Total protein concentration, trypsin-inhibitor capacity (TIC), and plasmin, beta-glucuronidase (B-Gase) and N-acetyl-beta-D-glucosaminidase (NAGase) activities were determined in 27 IUF samples. The motility of spermatozoa in the presence of IUF, IUF extended with Kenney's medium (1:1) and Kenney's medium alone was analysed in 9 samples using a Hamilton-Thorn motility analyser. Thirty-five mares were inseminated immediately after collection of IUF, and every second day until ovulation. Embryos were recovered nonsurgically 6 days after ovulation. After embryo transfer, fluid accumulations were recorded during oestrus and an endometrial biopsy specimen taken (n = 53). In the beginning of oestrus, fluid accumulations were detected in 39% (22/57) of mares, while on the day when IUF was collected, fluid accumulations were observed in 26% (15/57) of mares. The fluid was anechogenic, and in 80% of the mares located in the uterine body. None of the mares exhibited cytological or bacteriological evidence of acute endometritis. Total protein concentrations, TIC and B-Gase activities in IUF were statistically significantly lower in mares with fluid accumulations (n = 14) than in mares without fluid accumulations (n = 13) (p < 0.01). The addition of undiluted IUF to extended semen significantly reduced total and progressive motilities, path velocities and percentages of rapid spermatozoa (p < 0.05) in vitro. On endometrial biopsy, fibrosis was found to be more prominent (p = 0.025) in mares with fluid accumulations (n = 9) than in mares without (n = 44). It was concluded that anechogenic fluid accumulations during oestrus were associated with compositional changes in IUF. Although IUF had negative effects on spermatozoal motility in vitro, the presence of fluid accumulations at the time of insemination did not affect embryo recovery rates.

Extensive allelic sequence variation in the J region of the human immunoglobulin heavy chain gene locus.

During the initial stages of B lymphocyte differentiation heavy chain variable (VH), diversity (DH) and joining (JH) gene segments recombine to form a functional heavy chain variable region (VDJ) gene. Evidence for genetic polymorphism of the human JH gene segments has been obtained from mature rearranged VDJ sequences. We conducted an analysis of the published rearranged JH gene sequences and found that the JH alleles present in the two published germ-line JH region sequences were rare (approx. 2%) in the rearranged sequences. As an attempt to explain this discrepancy a 2.5-kb strech of DNA containing all the six heavy chain JH region genes and the most 3' DH gene segment, DHQ52, was amplified by the polymerase chain reaction from 39 individuals and analyzed for restriction fragment length polymorphism. Five new JH region haplotypes were found and sequenced. These new haplotypes contained the coding segment alleles that were frequent in antibody genes. Surprisingly, a high number of interallelic differencies in the non-coding sequence was found between the new and the two previously published haplotypes implying that the haplotypes had been separated early in evolution. In this respect the JH locus resembles HLA loci.

V-region-mediated binding of human Ig by protein A.

The Fab-mediated "alternative" binding of Ig by staphylococcal protein A is a marker of a set of VH genes (a subset of family VH3 in man). We typed 115 monoclonal human Ig as alternative binders or nonbinders. The proportion of binders varied depending on the isotype, 35% in IgM but only 11-13% in IgA1 and IgG3. It was 28% among lambda-chain-bearing but 16% among kappa-bearing monoclonal Ig. Independent estimates of the proportions bound were obtained by studying polyclonal Ig of 10 healthy adults. The proportions bound were close to those observed in the study of monoclonal Ig (the means were IgM 32%, IgA1 13%, IgA2 24%, IgG3 14%). A higher proportion of infant than adult Ig was bound by protein A. Also, the proportion was less isotype-dependent in infants than in adults. At the age of 4 mo, 47% of IgM was bound (mean of 10 children), the values of other isotypes were: IgA1 35%, IgA2 39%, and IgG3 38%. At the age of 14 mo the proportion of alternative binders had decreased but was still far from adult values. We propose that ontogenically early ("virgin") B cells, besides being rich in IgM and lambda-chain producers, are rich in producers of alternative binders. A subsequent selection reduces the proportion of these B cells so that in ontogenically most developed B cell populations, e.g., those producing IgA1 kappa, such cells make up only about 10% of the total.

Proportion of protein A bindable molecules in human IgM and IgA antibodies to seven antigens.

Human IgM or IgA antibodies to seven antigens (tetanus and diphtheria toxoids, and five bacterial polysaccharides) were studied by determining what proportion of these antibodies were bound by staphylococcal protein A. This alternative binding is a marker of VH genes of family 3. Each response was studied in an average of nine individuals. The binding proportion of antibodies to the two toxoids resembled that of total serum immunoglobulins; 13-14% of IgA and 40% of IgM antibodies were bound by protein A. All anti-polysaccharide antibodies had higher proportions of protein A bindable molecules than serum IgM or IgA indicating a bias for VH genes. This excess was high in antibodies to Haemophilus influenzae type b (Hib) and pneumococcal type 14 polysaccharides (> two-fold). It was moderate but statistically significant in antibodies to pneumococcal types 18C and 3 capsular polysaccharides and to C polysaccharide. All vaccinated Finns exhibited the VH3-preference in antibodies to Hib and type 14 polysaccharide.

Low concentrations of Gm allotypic subsets G3 mg and G1 mf in homozygotes and heterozygotes.

Serum concentrations of IgG3, IgG1, and of the Gm allotypic subsets of these two isotypes were measured in adult homozygotes and heterozygotes. Alleles G3 mb and G3 mst of the IgG3 locus, and alleles G1 ma and G1 max of the IgG1 locus were found to associate with a high concentration of the allotypic product. Alleles G3 mg (IgG3) and G1 mf (IgG1) were associated with a low concentration of the product. This was true regardless of the haplotype; for example, allele G3 mb was associated with a high concentration of the product in all haplotypes f;n+;b f;n-;b and fa;n+;b. One dose of allele G3 mg was associated with a characteristic mean concentration of the product (g-type IgG3). This rule was valid regardless of the other allele of the subject, thus, heterozygotes and G3 mg/g homozygotes had mean concentrations of 0.10 and 0.20 g/liter, respectively, of g-type IgG3. Products of the IgG1 alleles were also simply additive: one dose of allele G1 ma(x) or G1 mf was associated with mean concentrations of 3.63 and 2.84 g/liter, respectively, and two doses with twice these amounts. Only allele G3 mb did not completely follow this rule. We also studied the serum concentrations and the allotype distribution of 41 IgG1 and 31 IgG3 myeloma proteins. The results suggested that the allotype-associated differences in serum concentrations are caused by different numbers of B cells producing allotypic subsets of IgG1 or IgG3, not by different rates of synthesis per B cell.

Half-life of the maternal IgG1 allotype in infants.

The residence time of maternal IgG1 in the circulation of infants was measured by monitoring f-allotypic IgG1 or f-positive tetanus toxoid antibody in genetically G1mf-negative infants. G1ma-positive maternal tetanus toxoid antibody was similarly monitored in genetically a-negative infants. Blood samples were taken from infants at the age of 1-3 days, ca. 4 months, and ca. 6 months. An exponential decay at the same rate took place from age 1-3 days to 4 months and for the 2 subsequent months. The average concentration of the maternal IgG1 had dropped to ca. 10% of the 1- to 3-day value in 4 months and to ca. 3% in 6 months. The drop was due mainly to clearance but partly also to the weight increase of the child (doubling in 6 months). By correcting for the weight increase, we calculated that ca. 17 and 7% of the original maternal IgG1 was still present at ages 4 and 6 months, respectively. The average half-life of the maternal IgG1 was thus 48.4 days. The concentration of endogenous IgG1 in the cord blood was determined by studying a separate series of mother-newborn pairs. Assuming that cross-reactions of antiallotype reagents had no effect, the highest measured concentration of f-positive IgG1 in infants of f-negative mothers was 10 mg/L, half a percent of adult heterozygote values. Crossreaction may have played a role, however, and the value must be considered the upper limit of the true concentration.

The alternative binding site for protein A in the Fab fragment of immunoglobulins.

Twenty-six new human or murine monoclonal immunoglobulins (IgM, IgA, murine IgG1 or human IgG3) with a known V-region sequence were tested for alternative (non-Fc) binding to Staphylococcal protein A. Seven of them did not bind at all. Four immunoglobulins (all mouse IgG1) were bound but easily eluted (at pH 6). They were probably bound via the Fc part. All eleven were classified as negative for alternative binding. Fifteen immunoglobulins were found to bind more firmly; they came off the protein A column at pH 4-3 (alternative binders). Amino acid sequences of immunoglobulins that have been typed in the present work or earlier (25 binders and 26 non-binders) were compared. The light chain, the C region of the heavy chain and the D and JH segments look irrelevant for alternative binding. The N-terminal portion (amino acids 1-94) of the H chain probably forms the ligand of protein A. A peptide making the ligand cannot be reliably localized within this stretch but binder proteins had a high homology in residues 6-29. All mouse immunoglobulins expressing VH genes of families J606 or S107 were alternative binders; those expressing other families were non-binders.

The same few V genes account for a majority of oxazolone antibodies in most mouse strains.

The early primary anti-phenyloxazolone antibodies of 12 mouse strains were studied by determining proportions of two defined subsets id495 (the classical phOx idiotype) and id350. Id495-positive antibodies bear an H chain encoded by VHOx1 gene (family Q52) and an L chain usually coded for by VKOx1 but occasionally by other VK genes. Id350-positive antibodies are encoded by a VK gene VK45.1, and usually by a VH gene of the S107 family. All 12 strains (representing nine H-chain and four kappa-chain haplotypes) produced id350-positive anti-phOx antibodies. While id495 is the predominant major subset in the BALB/c response (originally studied), id350 seems to be the predominant subset of early anti-phOx antibodies in the mouse species. The combined proportion of the two subsets varied from ca. 50 to almost 100% of the total in all strains except C57BL.

Quantitation of human IgG subclass antibodies to Haemophilus influenzae type b capsular polysaccharide. Results of an international collaborative study using enzyme immunoassay methodology.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.

V genes of oxazolone antibodies in 10 strains of mice.

One pair of V genes (V kappa 45.1 and V11) code for a great portion of phenyloxazolone (anti-phOx) antibodies in 10 strains of mice. This combination replaces the first-known major combination VHOx1-V kappa Ox1 in some strains, and is important in most strains. C57BL/10 and SJL mice have an additional subset of antibodies encoded by genes V kappa 45.1 and V13 (a relative of V11). All three genes involved (V kappa 45.1, V11 and V13) have "allelic" variation. Four alleles of V11 were found, one in Igh haplotypes a, c and g, the second in haplotypes d, j and n, the third in b, and the fourth in f. The most distant alleles d, j, n and f had 10 nucleotide differences out of 429 determined (97.7% homology). Only one allele of the V13 gene was found from anti-phOx hybridomas but two others have been published. Three alleles of the V kappa 45.1 gene were found; one in NZB mice (Ig kappa haplotype b) another in CE (haplotype f), and the third in eight strains including representatives of three Ig kappa haplotypes (a, c and e). The three alleles had greater than 99.0% homology. The V11 and V13 genes that code for anti-phOx antibodies in C57BL/10 and SJL mice were different from the related genes found from the C57BL/10 germ line. C57BL/10 mice must have a chromosome bearing two V11 and two V13 genes. RF mice were found to have two V11 genes, and both code for anti-phOx antibodies. Our data show that the majority of antibodies in the anti-phOx response are encoded by the same restricted collection of V genes in most mouse strains. Antibody responses appear to be no less heritable than other functions of the body.

Gm allotypes influence the production of IgG3 but the effect is age-dependent.

Serum concentrations of IgG3 were found to be higher in Gm-f-positive (= b-positive) than in f-negative individuals except in young children. Young children aged 3-4 months had a mean concentration of 0.24 g/l of IgG3 regardless of allotype. The concentration gradually rose with age in f-positive individuals to a geometric mean of 0.56 g/l in adults but it remained essentially unchanged in f-negative people. A corresponding allotype effect was seen in influenza-specific antibody responses. While the total IgG response (mainly IgG1) was equally strong in f-positive and in f-negative patients, f-positive (= b-positive) patients produced more IgG3 antibodies than f-negative patients. The difference between geometric mean values of opposite homozygotes (f/f versus f-negative) was 2.3-fold (p = 0.0113). This finding indicates that the b-positive gamma-3 allele is more productive than the g-positive allele.

Allotype-associated differences in concentrations of human IgG subclasses.

The concentrations of seven immunoglobulin isotypes (IgA, IgE, IgM, IgG1, IgG2, IgG3, and IgG4) were measured in the sera of 207 Finnish blood donors, and they were allotyped with anti-Gm antibodies: anti-f, anti-a, anti-x, and anti-n. The above population could be divided into 12 phenotypes, and significant differences in isotype concentrations between different phenotypes were observed. They are best explained by postulating that the following alleles of different loci are associated with a high concentration of the product of the locus: a(x)-IgG1, n-IgG2, b-IgG3, and perhaps 4b-IgG4. The following concentration differences between the low and the high homozygotes were found: IgG1, 1.2-fold; IgG2, 1.5-fold; and IgG3, 2.6-fold. No significant allotype-associated differences in the concentrations of IgA, IgM, or IgE could be detected.