The renal handling of hemoglobin. I. Glomerular filtration.

The glomerular filtration of hemoglobin (alpha(2)beta(2)) was studied under conditions in which its dissociation into alphabeta dimers was experimentally altered. Rats receiving hemoglobin treated with the sulfhydryl reagent bis(N-maleimidomethyl) ether (BME) showed a much lower renal excretion and prolonged plasma survival as compared with animals injected with untreated hemoglobin. Plasma disappearance was also prolonged in dogs receiving BME hemoglobin. Gel filtration data indicated that under physiological conditions, BME hemoglobin had impaired subunit dissociation. In addition, BME hemoglobin showed a very high oxygen affinity and a decreased rate of auto-oxidation. Glomerular filtration was enhanced under conditions which favor the dissociation of hemoglobin into dimers. Cat hemoglobin, which forms subunits much more extensively than canine hemoglobin, was excreted more readily by the rat kidney. The renal uptake of (59)Fe hemoglobin injected intra-arterially into rabbits varied inversely with the concentration of the injected dose.

Animal models of autoimmune hemolytic disease.

The dog is the principal known animal that develops spontaneous AIHA in the same confusing way as does man. All other occurrences of AIHA in animals are secondary to a variety of immune complex disorders. A recent review has more than adequately covered those conditions. The truly interesting point is that in all the animals studied except the dog, a viral etiology has been found in those conditions in which AIHA is expressed. Lastly, an area that has not been adequately exploited, and may well be an experimental direction to follow in the future, is the observation that graft-versus-host models could well provide significant new knowledge of the mechanisms involved in the recognition of self and subsequent cell destruction.

Cyclosporine pharmacokinetics in normal and pancreatectomized dogs.

Oral and i.v. cyclosporine (Cs) pharmacokinetics determined from radioimmunoassay (RIA) data were compared in normal and pancreatectomized dogs. An altered pharmacokinetics of Cs was observed in the pancreatectomized dogs that include: a 170% larger central compartment volume; a 34% greater total-body clearance; and lower steady-state average serum concentrations relative to the normals. Even though there were marked intersubject variations, both groups displayed a triexponential decline in Cs serum concentrations and disposition kinetics. Following 7 daily oral doses of commercial cyclosporine (CsA) (20 mg/kg) the Cs serum trough concentrations of the pancreatectomized dogs were consistently below 100 ng/ml, while those of the normal dogs were above 400 ng/ml. No alteration of CsA oral absorption was noted following pancreatectomy. This study suggests that frequent serum Cs concentration monitoring, with appropriate dosage adjustments, even in normals, is necessary to assure adequate drug levels. More significantly, the CsA dosage for pancreatectomized dogs should be several times greater to maintain serum concentrations comparable to normal dogs.

Absorption and elution of anti-DLA-A and B antibodies with acetone-dried dog spleen powder.

Acetone-dried powders were prepared from the spleens of DLA-serotyped dogs and assayed for their specific absorptive properties for anti-DLA-A and B antisera. The specific activity of two different antisera (anti-DLA-A9 and anti-DLA-B13) was readily absorbed with acetone-dried powders prepared from the spleens of dogs of the corresponding DLA types. This specific activity was recovered by the elution of the powder used for the serum absorption. The same method was used to narrow the specificity of a highly polyspecific antiserum. The comparison of the absorption properties of acetone-dried spleen powder versus fresh spleen cells shows that the treatment with acetone does not modify qualitatively and quantitatively the DLA specificities.

Effect of prior third-party blood transfusions on canine renal allograft survival.

The relationships between immune reactivity after blood transfusions, subsequent kidney allograft survival, and donor selection were studied in dogs. Animals with a high as well as low serological immune reactivity toward antigens contained in blood transfusion were observed. Genetic control of this reactivity or a linkage of this property to DLA, sex, or red blood cell markers inheritance was not apparent in the four beagle families studied. The two recipients with the lowest immune reactivity scores were also found to be the longest survivors after a DLA-mismatched kidney graft. Seven other recipients with higher scores rejected their DLA-mismatched kidneys as rapidly as did untransfused animals. Kidney graft survival was decreased in some recipients of DLA-identical kidneys (n = 5), presumably through sensitization for minor histocompatibility antigens. A normal or an increased survival time of DLA-identical kidneys was found in the remaining animals (n = 6). The majority of these recipients appeared to have a higher than average reactivity in two-stage microcytotoxicity testing. This might have been attributable to the presence of enhancing antibodies. Further studies in preclinical animal models are needed to define the optimal transfusion policy for human patients awaiting a kidney graft.

Joint report of the Third International Workshop of Canine Immunogenetics. II. Analysis of the serological typing of cells.

Using a standardized microlymphocytotoxicity assay, seven international laboratories evaluated 144 anti-dog lymphocyte antigen (DLA) sera in 319 mixed breed and 152 Beagle dogs. The workshop confirmed the serological definitions for DLA-A2, A3, A9; DLA-B4, B5, B6, B13; DLA-C11(Cwl); and C12(Cw2). Two new specificities were assigned to the DLA-A locus (Aw14 and Aw15) in only the mixed breed dogs. A third specificity (Cw3), was assigned to the DLA-C locus. The antigen and gene frequencies of these alleles differed between the two groups of dogs, but the frequencies of the "blank" were similar in both groups. Future international collaborations will be necessary to definite more completely the polymorphisms of the major histocompatibility complex (MHC) of the dog. Those efforts will benefit from the standard serological test established in this workshop.

A comparative study of histological conditions suitable for both immunofluorescence and in situ hybridization in the detection of Herpesvirus and its antigens in chicken tissues.

Our objective was to identify an optimal single set of conditions for use in both indirect immunofluorescence assays (IFA) and in situ hybridization (ISH) to detect viral proteins and nucleic acids in avian lymphoid and neural tissues. Various fixatives were evaluated for use with IFA to detect turkey Herpesvirus (HVT) glycoprotein B (gB) and ISH to identify HVT mRNA in chicken tissues. A precipitating fixative (acetone) was compared to crosslinking fixatives [buffered glutaraldehyde-picric acid (BGPA), 10% formalin, and 4% paraformaldehyde] for both IFA and ISH using spleen, thymus, bursa, sciatic plexus, and brachial plexus of 28-day-old chickens. Four percent paraformaldehyde was found to be the optimal fixative for preservation of all chicken tissues examined with both IFA and ISH. Glass slide preparation, incubation temperatures, and tissue processing were each individually evaluated for ISH and IFA. Silylated slides provided the best retention of tissue sections for both procedures. For IFA, 37 degrees C was the ideal incubation temperature tested, whereas the optimal incubation temperature tested for ISH was 47 degrees C. Of the blocking agents compared, Evans blue dye prevented background fluorescence to a greater extent than either calf serum or bovine serum albumin. These findings provide a technical basis for investigations into various aspects of the molecular pathology of avian diseases.

Further characterization of HLA homozygous typing cell lines at the LMP2 polymorphic codon 60 by an ARMS typing method.

LMP2 is a subunit of the 20S proteasome within the cellular cytosolic compartment that is thought to cleave proteins into approximately 9 amino acid long oligopeptides. It is hypothesized that changes in the low molecular mass protease (LMP) gene sequence may alter the activity or specificity in which the LMP genes cleave peptides. Currently, the typing method for LMP2 involves polymerase chain reaction (PCR), restriction enzyme digestion, and gel electrophoresis. To help reduce the cost and cumbersomeness of this method, a new typing method was adapted for the LMP2 gene. To establish this new amplification refractory mutation system (ARMS) typing method, primers have been defined, amplification conditions optimized, and control cell lines sequenced to validate testing parameters. Results are listed for selected 10th and 11th International Histocompatibility Workshop homozygous cell lines.

Peripheral lymphocyte function in dogs with Brucella canis infection.

Peripheral lymphocyte function in dogs with Brucella canis infection was evaluated using in vitro lymphocyte stimulation with the mitogens PHA, Con A and PWM, and killed Brucella canis organisms. Bitches with naturally occurring Brucella canis infection were compared to negative controls. There was no difference in the response to Con A and PWM between these two groups. Lymphocytes from infected dogs were less responsive (p less than .05) to PHA than were lymphocytes from controls. There was a significant (p less than .005) difference in response to Brucella canis antigen between the two groups. Lymphocytes from infected dogs were stimulated by Brucella canis antigen, whereas those from controls did not respond.

In vitro maintenance of Eperythrozoon suis.

In vitro maintenance of Eperythrozoon suis was attempted using a Petri dish erythrocyte culture system. In preliminary experiments, the optimal conditions for maintaining E. suis attachment to erythrocytes during incubation were anticoagulation with heparin or citrate solution, incubation with 5 or 10% CO2 at 37 degrees C, and incubation with reduced or non-reduced Eagle's minimum essential medium. Using heparin, a CO2 incubator and reduced Eagle's medium (rEM), E. suis metabolic activity was evaluated by measuring glucose consumption, and lactate and pyruvate production. Glucose consumption and lactate production were measurable while pyruvate production was not detected. Erythrocyte integrity was improved by the addition of inosine although no effect was observed on maintenance of E. suis attachment to erythrocytes or the rate of glucose consumption. To determine whether the glucose consumption observed in culture was due to E. suis glycolytic activity or enhanced erythrocyte glycolytic activity, the effect of E. suis killing by EDTA addition to medium was evaluated using rEM containing inosine (rEMI). Glucose consumption decreased proportionally with the decline in the percentage of parasitized erythrocytes induced by EDTA, indicating that glucose consumption was due to E. suis. In a subsequent experiment, the effect of different types of serum (pig or fetal calf serum) and different gaseous environments (5% CO2 incubator or candle jar) were evaluated using rEMI. Glucose consumption by E. suis was significantly increased by the addition of fetal calf serum; however, no difference in the maintenance of E. suis attachment to erythrocytes and in E. suis glycolytic activity was observed between a 5% CO2 incubator and a candle jar. Finally, the effect of medium refreshment (rEMI containing fetal calf serum) was evaluated. Maintenance of E. suis parasitism on erythrocytes and E. suis glycolytic activity were significantly improved by frequent medium refreshment. The maintenance system developed enabled successful metabolic radiolabeling of E. suis for protein/antigen analysis.

Identification and localization of glycoprotein B expression in lymphoid tissues of chickens infected with turkey herpesvirus.

One-day-old chickens were inoculated with turkey herpesvirus (HVT). Using an indirect immunofluorescence assay with a monoclonal antibody against HVT glycoprotein B (gB), we determined the course of productive HVT infection in peripheral blood mononuclear cells (PBMCs), spleen, thymus, and bursa. PBMCs were examined from days 4 through 35 postinfection (PI). The spleen, thymus, and bursa were examined from 21 through 70 days PI. Although productive infection in PBMCs was detected at 4 to 12 days PI, it ended by 14 days PI. Splenic cells expressed gB at 21, 28, 35, and 70 days PI, whereas the thymus was positive for gB expression at 21 and 35 days PI. The bursa was never positive for gB expression. At 21, 28, 35, and 70 days PI, plaque formation after co-cultivation of PBMCs with chicken embryo fibroblasts indicated the presence of HVT in infected chickens by co-cultivation assays. On the basis of indirect immunofluorescence assay, gB expression in the spleen and thymus indicates a productive HVT infection in chickens.

Latent turkey herpesvirus infection in lymphoid, nervous, and feather tissues of chickens.

In earlier studies, we found that a late gene product, glycoprotein B (gB) was highly expressed in lymphoid tissues of chickens inoculated with turkey herpesvirus (HVT). The objectives of the present study were twofold. First, we wanted to expand on our previous research and determine if gB expression declines or disappears during later time periods of HVT infection. Second, we wanted to correlate gB expression with presence of HVT, i.e. if gB expression is absent, can HVT still be detected? Fifteen 1-day-old chicks were inoculated by intraperitoneal inoculation with 2000 plaque forming units of strain FC126 HVT. Thymus, spleen, bursa, brachial plexus, sciatic plexus, and feather tips were harvested at 21, 28, 35, 70, and 105 days postinoculation (PI). Brachial plexus and sciatic plexus were examined at 21, 28, and 35 days PI, and feather tips were examined at 21 and 28 days PI. An indirect immunofluorescence assay was used to detect HVT gB expression, and an in situ hybridization assay was used to detect HVT. At 21 days PI, gB expression was present in the thymus, spleen, and bursa. At 28 and 35 days PI, gB expression was detected in the thymus and spleen. At 70 days PI, gB expression was detected only in the spleen, and at 105 days PI, gB expression was not detected in any of the lymphoid tissue (thymus, spleen, or bursa). gB expression was not detected in the brachial plexus, sciatic plexus, or feather tips at any of the five time points. The bursa contained HVT only at 21 and 28 days PI. However, HVT was demonstrated in all other tissues from 21 to 105 days PI. Progression from a productive HVT infection to a latent HVT infection results in the loss of gB expression. Throughout this progression, a region of the HVT genome can be detected by appropriate methods.

Effects of vitamin E and selenium on immune responses of peripheral blood, colostrum, and milk leukocytes of sows.

This study was designed to assess how dietary vitamin E (E) and (or) selenium (Se) concentrations affect immune responses of gestating and peripartum sows. Multiparous sows (24), assigned to one of four groups at breeding, were fed ensiled, shelled corn-soybean meal-based diets without supplemental E or Se (-E-Se), with .3 mg of Se/kg (-E+Se), with 60 IU of E/kg (+E-Se), or with both supplemental E and Se (+E+Se) during gestation and to d 4 of lactation. Blood was obtained on 0, 30, 60, and 90 d of gestation and at parturition for serum E and Se assays. Lymphocytes and polymorphonuclear cells (PMN) were isolated from the blood, colostrum, and 4-d milk samples for immune studies. Compared with the control (+E+Se) diet, the -E-Se diet reduced (P < .05) the serum tocopherol and Se concentrations, the mitogenic responses of lymphocytes of peripheral blood (PBL) and colostrum (CL), the phagocytic activity of blood and colostral PMN, and the microbicidal activity of blood, colostral, and milk PMN. The -E+Se diet reduced (P < .05) the serum tocopherol concentrations, the mitogenic responses of PBL and CL, and the phagocytic activity of PBL. The +E-Se diet reduced (P < .05) serum Se concentrations and the phagocytic activity of PMN. The data indicated that E restriction depressed PBL and PMN immune functions, whereas Se restriction depressed mainly PMN function.

Breed differences in the phenotype and gene frequencies in canine D blood group system.

The D system of canine blood groups was studied in 3,191 dogs of many different breeds. The frequencies of the D system phenotypes and genes were measured. These frequencies varied considerably between the breeds native to Japan. The frequency of the D1 phenotype was higher in breeds native to Japan than in those of non-Japanese origin. Conversely, non-Japanese breeds generally had the D2 phenotype. The dogs described as mongrel in Japan had D system frequencies intermediate between native Japanese and non-Japanese breeds. One of the most interesting findings was that in the Afghan bound the frequency of the D1 gene (0.3333) was the same as in the Shiba, though only the Shiba was native to the Japanese isles. Another Japanese breed was the Tosa, and its D1 gene frequency was 0.063, a value even lower than that for the non-Japanese Maltese (0.097).

Determination of protein concentrations and their molecular weight in tears from cats with normal corneas and cats with corneal sequestrum.

Protein concentration was determined, using the Bradford technique, in tears from cats with normal corneas and from cats with corneal sequestrum. Tears from the former group contained 5.81 +/- 2.29 mg of protein/ml; those from corneal sequestrum-affected cats contained 6.21 +/- 2.21 mg/ml. Difference between the 2 values was not significant. Molecular weight determination was made, using 4 to 20% sodium dodecyl sulfate-polyacrylamide gels. Molecular mass of proteins ranged from 263 to 14 kDa. There was no detectable difference in the band patterns for the 2 groups.

Isolation of viable canine intestinal lymphocytes.

A modification of a previously reported enzymatic technique was used to isolate 60% to 90% pure populations of viable lymphocytes from canine small intestinal mucosa. Mitogenesis assays were then simultaneously performed on these and isolated peripheral blood lymphocytes from the same dogs. A technique to isolate intraepithelial lymphocytes was not successful. Intestinal mucosal lymphocytes were simultaneously purified by 2 different techniques (suspensions B and C), which produced cell populations that responded similarly to phytohemagglutinin (PHA), concanavalin A, and pokeweed mitogens. The peripheral blood lymphocytes demonstrated greater response to PHA and concanavalin A mitogens than did the intestinal mucosal lymphocytes (P less than 0.05). Furthermore, peripheral blood lymphocytes had a significantly higher response to PHA than to pokeweed mitogen (P less than 0.05). The impure preparation of intraepithelial lymphocytes (suspension A) did not show any evidence of reactivity to mitogens. Further studies are needed to determine whether the EDTA or collagenase had any effect upon the intestinal mucosal lymphocyte responsiveness. The present study demonstrated that viable mucosal lymphocytes can be isolated from the canine intestine and that they can maintain their responsiveness to mitogens.

Comparison of electrophoretograms of normal canine serum and plasma and of serum and plasma of hemolyzed specimens.

Comparison of the electrophoretograms of normal canine plasma and serum revealed a greater concentration of the beta3-fraction in plasma due to the presence of fibrinogen. When serum or plasma of hemolyzed canine blood was analyzed electrophoretically, there were slurring of the beta-globulin peaks due to the presence of free hemoglobin and increases in alpha2-globulins due to the formation of the haptoglobin-hemoglobin complex.

Prevalence of autoantibodies to thyroglobulin, thyroxine, or triiodothyronine and relationship of autoantibodies and serum concentrations of iodothyronines in dogs.

Assays were developed to detect and measure autoantibodies (AA) to thyroglobulin (Tg) and to the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). An ELISA to detect AA to Tg was developed, using purified canine Tg as the antigen and goat anti-canine IgG conjugated with alkaline phosphatase as the second antibody. A highly charged agarose electrophoresis assay was used for determination of AA to T4 and T3. Sera from dogs (n = 119) with clinical signs consistent with hypothyroidism were tested for AA to Tg, T4, and T3. Autoantibodies to at least 1 of the 3 thyroid antigens were detected in 58 of the 119 (48.7%) sera tested. Autoantibodies to Tg were detected more frequently in samples with low serum concentrations of thyroid hormones than in samples with normal concentrations. The presence of AA to T4, T3, or both was not significantly associated with low thyroid hormone concentrations, but this lack of association may have been attributable to binding of AA in the measurement of thyroid hormones by radioimmunoassay.

Isolation of thyroid peroxidase and lack of autoantibodies to the enzyme in dogs with autoimmune thyroid disease.

Fifty serum samples from dogs with clinical signs of hypothyroidism and autoantibodies (AA) to thyroglobulin (Tg), thyroxine, or triiodothyronine were screened for AA to thyroid peroxidase (TPO). Thyroid peroxidase is the antigen against which microsomal AA are formed in human beings with lymphocytic thyroiditis. The TPO was isolated from canine thyroid tissue, using a modification of the procedure for purifying porcine TPO. The enzyme was solubilized from the membrane, using a deoxycholate-trypsin solution, followed by ammonium sulfate precipitation and diethylaminoethyl Sephadex chromatography. Activity of TPO was determined, using an iodide oxidation assay and a guaiacol assay. A monoclonal antibody to canine Tg, coupled to an immunoaffinity column, was used to eliminate the contaminating Tg from the TPO preparation. Using the TPO preparation as an antigen, an ELISA was performed on 10 serum samples and immunoblot assays were performed on 50 canine sera. Autoantibodies to TPO were not found in any of the sera. Assays also were performed, using purified porcine and human TPO and evidence of cross-reactivity with canine TPO was not identified. The absence of AA to TPO in dogs suggests a different pathogenesis for autoimmune thyroid disease in dogs than that hypothesized for lymphocytic thyroiditis in human beings.

Immunologic profiles of cats with persistent, naturally acquired feline leukemia virus infection.

Several immunologic responses were measured in 13 healthy cats with naturally acquired, persistent feline leukemia virus (FeLV) viremia from 4 multiple-cat households and were compared with responses from 28 of their healthy, non-FeLV-viremic housemates. Significant differences (P = less than 0.05) were not observed between results of FeLV-viremic and nonviremic cats for peripheral blood leukocyte or lymphocyte count, percentage of peripheral blood mononuclear cells able to form rosettes with guinea pig RBC or with antibody- and complement-coated sheep RBC, lymphocyte proliferative response to concanavalin A or pokeweed mitogen, or serum immunoglobulin G concentration. Seemingly, persistent FeLV viremia, when naturally acquired, may exist for some time without lymphopenia or a marked loss of mitogen-induced lymphocyte proliferation.