Biomedical subjects
Publications and source records attributed to D L Dawe.
Sera from 14 dogs with active pemphigus foliaceus were tested for the presence of circulating antiepithelial antibodies. These antibodies could not be detected in the dogs with indirect immunofluorescent staining or in vitro complement-staining methods.
Channel catfish demonstrate a shift in the tissue distribution of nonspecific cytotoxic cells (NCC) when infected with the protozoan parasite, Ichthyophthirius multifiliis. NCC, isolated from head kidney (HK) tissues (hemopoietic organ) or peripheral blood leukocytes, were assessed for cytotoxic activity against NC-37 (a transformed mammalian cell line). NCC activity from HK tissue of moribund I. multifiliis-infected fish was depressed compared to HK-NCC activity in uninfected or I. multifillis-immune fish. The activity of NCC, isolated from the peripheral blood of moribund I. multifiliis-infected fish was significantly greater than the NCC activity in peripheral blood from either immune or uninfected fish. Chromium-51 release assays were combined with effector and target conjugate assays to determine killing capacity (Vmax) and affinity (Km) for target cells of peripheral blood NCC from moribund I. multifiliis-infected and uninfected fish. These experiments indicated that the peripheral blood from the moribund infected fish contained an increased percentage of active NCC with increased killing capacity and target cell affinity compared to peripheral blood NCC activity of uninfected fish.
Nonspecific cytotoxic cells (NCC) obtained from channel catfish (Ictalurus punctatus) kill Tetrahymena pyriformis, an opportunistic parasite in fish. Based upon this fact, a new mechanism for nonspecific cellular anti-parasitic immunity in fish is proposed. Optimum in vitro conditions for NCC killing of deciliated T. pyriformis were first obtained. Lysis of T. pyriformis by NCC occurred by 10 hr of cocultivation of effector and target cells. During this time period, 50 to 60% cytotoxicity occurred. Fish anti-T. pyriformis serum enhanced NCC killing of T. pyriformis either by prolonging immobilization (after the cilia regeneration period) or by delaying cilia regeneration. Shared antigenic determinants between T. pyriformis, Ichthyophthirius multifiliis, and NC-37 target cells were demonstrated by binding-depletion experiments. For these studies, NCC were depleted from anterior kidney cells (the hemopoetic organ in fish) by preincubating formalin-treated T. pyriformis, I. multifiliis, or viable NC-37 target cells with NCC for 3 hr. Conjugates of effector and target cells were removed by overlaying on fetal bovine serum. Unconjugated fish anterior kidney cells were tested for cytotoxic activity against NC-37 or T. pyriformis target cells. Cold target inhibition experiments by using a 4-hr 51chromium cytotoxicity assay also demonstrated these shared antigenic determinants. Target-specific antisera, used to mediate the killing of T. pyriformis by NCC, were required only for immobilizing the targets, and did not function in an antibody-dependent cell-mediated (ADCC)-like mechanism. Scanning electron micrographs of NCC-T. pyriformis conjugates additionally demonstrated NCC binding to both cilia and cell surface determinants.
The etiology of keratoconjunctivitis sicca (KCS) in 62 dogs was evaluated, using immunologic techniques. Using direct fluorescent antibody testing, autoantibodies within the lacrimal, salivary, or pancreatic glands were detected in 5 of 8 dogs tested. Circulating antibodies to the nictitating membrane gland, main lacrimal gland, parotid salivary gland, or mandibular salivary gland were detected using indirect fluorescent antibodies in 9 of 31, 3 of 31, 5 of 31, and 5 of 31 sera, respectively. Using radial immunodiffusion, hyper-gamma-globulinemia was detected in 21 of 30 dogs with KCS. Antinuclear antibodies, primarily in a nucleolar pattern, were demonstrated in 20 of 50 dogs with KCS. Lymphocytic infiltrates were evident in 5 of 9 labial salivary biopsies, 2 of 4 parotid gland specimens, 2 of 4 mandibular gland specimens, and 2 of 3 thyroid gland specimens taken from dogs with KCS. Autoimmune diseases had been previously documented in 4 of 62 dogs. Twenty-five of the 62 dogs (40%) had concurrent problems indicative of an underlying immunologic disorder.
Two techniques, leukoagglutination and indirect immunofluorescence, were adapted to test for the presence of antineutrophil antibody in cat serum. The leukoagglutination test was analogous to an indirect Coombs' test. The test was performed on freshly isolated cat blood neutrophils, with the test results read from stained smears (Wright's stain) made from sedimented antiserum-treated neutrophils. A positive test response was indicated by agglutinated neutrophils on the stained smear. The indirect immunofluorescence test was performed by incubating paraformaldehyde-fixed cat blood neutrophils with test serum, after which the neutrophils were stained with fluorescein isothiocyanate-tagged antiglobulin. A positive test response was a ring of fluorescence surrounding the cells, as viewed through a UV microscope. Serum samples (n = 55) from clinically neutropenic cats were tested for the presence of antineutrophil antibody by the indirect immunofluorescence technique. Ten positive-control sera (rabbit anti-cat neutrophil serum) and 10 negative-control sera (normal cat serum) were included. Only the positive control sera exhibited neutrophil fluorescence, indicative of antineutrophil antibody. None of the 55 samples of clinical origin showed any appreciable fluorescence.
Rabbit anti-cat neutrophil serum was injected intraperitoneally into cats to study its effects on blood neutrophil numbers, on development of neutrophils in bone marrow, and on the fate of circulating and developing neutrophils. There was a significant difference (P less than 0.05) in curves of blood neutrophil numbers between antineutrophil serum (ANS)- and normal rabbit serum (NRS)-injected cats; neutrophil counts tended to decrease in ANS-injected cats, whereas a transient increase in counts occurred in NRS-injected cats. Significant left shifts (P less than 0.05) were present in ANS-injected cats, but absent in NRS-injected cats. Toxic morphologic changes were noted in blood neutrophils in all ANS-injected cats. Significant bone marrow changes (P less than 0.05) occurred in ANS-injected cats, but were absent in NRS-injected cats. Myelocyte percentages of the granulocyte marrow population increased during the time that segmented neutrophil percentages decreased. In ANS-injected cats, the percentage of cells in the mitotic pool (myeloblasts, promyelocytes, myelocytes) significantly increased (P less than 0.05), with a corresponding significant decrease (P less than 0.05) in the postmitotic pool (metamyelocytes, bands, segmented neutrophils). Aspirated bone marrow smears (Wright's stain) revealed marrow macrophages containing phagocytized neutrophil bands and segmented neutrophils. Sections of liver obtained after cats were necropsied revealed neutrophil phagocytosis by Kupffer's cells, but neutrophil phagocytosis was not demonstrated in other tissues examined.
Nonspecific cytotoxic cells (NCC) obtained from the head (anterior) kidney of fish (Ictalurus punctatus) lyse human transformed B-cell targets. Lysis depended on direct cell-cell contact. Fish size, age, environmental holding temperatures, and lytic reaction conditions such as osmolality and optimum effector:target cell ratios were optimized. Experiments to characterize optimum kinetics demonstrated highly efficient killing after two hours incubation. This rapid cytolysis was further studied by determining NCC activity against appropriately labeled target cells after 30, 60, 90 and 120 minutes of cocultivation. At 160:1 (E:T) greater than 40% of the 5 hour percent specific release value was produced after 30 minutes. After 90 minutes, more than 90% of total percent specific release was observed. At least one mechanism of regulation of NCC killing was described. In the presence of normal (homologous or heterologous) catfish serum (CFS), essentially no NCC activity was observed. This suppression was reversible by preincubation in 10% fetal bovine serum (FBS). NCC "activation" by preincubation in 10% FBS was time-dependent (at least four hours was required to generate significant lysis). NCC activation could be reversed by treating potentially lytic cells with supernatants containing dissociated CFS. In addition, reversible activation could be demonstrated by treating potentially lytic effector cells with CFS to produce suppression. Regulation occurred at the effector cell level because treated target cells did not suppress NCC activity. These data demonstrate a population of nonspecific effector cytolytic cells that potentially represent a phylogenetic precursor to mammalian natural killer cells.
Fish nonspecific cytotoxic cells (NCC) lyse various transformed human B-cells (NC-37, P3HR-1) and erythroblastoid cells (K562) as well as mouse YAC-1 and P815 cells. Highest NCC activity was found in the anterior (head) kidney, but spleen cells and peripheral blood leucocytes (PBL) also demonstrated cytolytic abilities. Lysis of chromium-51 labeled target cells occurred rapidly and optimum cytolysis developed at either 16 degrees C or 26 degrees C incubation temperatures. Preincubation at temperatures of 4 degrees C or 37 degrees C for 4 hours reduced NCC cytotoxicity. Although catfish (Ictalurus punctatus) are extensively outbred, interfish NCC activities did not significantly vary at the optimum E:T ratios (160). The NCC target antigen specificities were partially determined by cold target inhibition (CTI) studies. YAC-1 and K562 did not produce significant CTI, however. These studies demonstrated the presence of a highly active cytotoxic cell which is widely distributed in fish lymphoreticular tissue. NCC kill divergent kinds of transformed cell types, and the target cell specificity for human transformed B-cells is different from the NCC target cell antigens on other human (K562) and on mouse (YAC-1 and P815) cells.
Nonspecific cytotoxic cells (NCC) from the catfish (Ictalurus punctatus) may comprise a population of cells that are responsible for cellular immunity in the fish. NCC kill a wide variety of transformed target cells, and previous studies have indicated that NCC share properties with mammalian natural killer cells. In the present study, many biophysical and biochemical properties of NCC were defined. NCC were nylon wool nonadherent and adherent. NCC activity was also enriched in plastic nonadherent cells. NCC were nonphagocytic (for carbonyl iron), and they did not bind to Sephadex G-10. Characterization of NCC by density gradient centrifugation indicated that they comprise a relatively homogenous population of cytolytic cells that band at 45.5% Percoll. Moderate to high doses (500-2500 R) of X-irradiation produced a stimulatory effect on NCC lysis of labeled target cells. Additional studies indicated that a soluble suppressor protein in catfish serum (CFS) regulated NCC activity. This S. aureus protein A binding component isolated from CFS suppressed NCC activity. Analysis by SDS-PAGE indicated that the soluble regulatory protein had properties similar to immunoglobulin. These data indicate that NCC share some biophysical properties with mammalian natural killer cells. In addition, NCC appear to be under partial cell regulation by a radiation sensitive suppressor cell and also by a soluble regulator serum immunoglobulin component.
Nonspecific cytotoxic cells (NCC) from fish (Ictalurus punctatus) were treated with different concentrations of retinolacetate and poly I:C. Both in vitro and in vivo experiments were conducted. Retinolacetate significantly increased NCC activity against chromium-51 labeled human B-cell lymphoma target cells (NC-37). Preincubation (treatment prior to adding the labeled target cells) of NCC for 4 to 8 h in 10(-3) to 10(-12) M concentration of retinolacetate produced significant increases in NCC activity compared to treatment during the killing assay only. Similar experiments with different concentrations of poly I:C had no NCC augmenting effects when tested by adding poly I:C either during preincubation periods or during the cytotoxicity assay. Retinolacetate probably produces positive modulation of cytotoxicity by increasing the killing effectiveness of individual NCC, rather than recruiting larger numbers of cytolytic cells. In vitro studies were also conducted by injecting catfish (i.p.) with 1 X, 3X and 5X the daily recommended vitamin A dosages and determining NCC activity after 24, 48 and 72 h treatment. The 1X dose significantly increased NCC activity at 72 h. This increase was not transient because NCC activity after 33-37 days' treatment was significantly higher than controls in the 1X, 2X and 3X groups. Intraperitoneal injections of fish with poly I:C produced no significant increases in NCC activity at 24 or 72 h post-inoculation.
The chicken thymus-specific antigen (T1) was localized within certain cell types of the thymus using an indirect immunofluorescent (FA) test. One of the cell types was similar to small mononuclear cells, whereas the other was much larger and irregular in shape and probably of the recticuloepithelial type. The small cells occurred in clumps mostly at the cortico-medullary junction, although single cells were found scattered in both the medulla and cortex. The large cell was restricted to the medulla and often was surrounded by the smaller cells. The antigen was not detected in frozen tissue sections from bursa, spleen or liver which suggests that the cells expressing the antigen are restricted to the thymus. Using the same antiserum, T1 was localized in thymus sections of other avian species. Soluble extracts prepared from the thymus of various avian and mammalian species were tested for T1 by immunoelectrophoresis (IE) and passive hemagglutination inhibition (HI) using anti-T1 serum. T1 was found in all avian species tested, but was not found in mammalian thymuses. The amount of T1 was quantitated in chickens of various ages by radial immunodiffusion (RID). The concentration of T1 increased linearly with age, and ranged from 32 micrograms/mg protein at 1 day of age to 45.6 micrograms/mg protein at 20 weeks of age. The total amount of T1 present reached maximal levels at 12 weeks of age and then declined during the period of thymus involution.
Sera from 50 dogs with keratoconjunctivitis sicca were examined for rheumatoid factor. In 17 (34%) of dogs tested, rheumatoid factor was found at titers equal to or greater than 1:16. Fifteen dogs (30%) had probable concurrent immunologic disorders. Ten dogs (20%) had xerostomia, as indicated by dry oral mucosa, marked periodontitis, and loss of teeth.
Twenty-five satisfactory and 25 unsatisfactory potential breeder bulls were examined with a modified Shulman spermagglutination test for the presence of spermagglutinating antibodies in their serum and seminal fluid. This was done to determine whether antibodies to spermatozoa were a contributing factor to classification of the bull as an unsatisfactory potential breeder. No correlation was found between classification as an unsatisfactory potential breeder and the presence of spermagglutinating antibodies. In addition, indirect fluorescent antibody tests were performed to detect antisperm antibodies in the serum and seminal fluid of the bulls. Again, no correlation was found between antisperm antibodies and breeding soundness classification. Finally, there was no correlation between the age of the bulls and occurrences of spermagglutination antibodies in serum or seminal fluid.
The effects of surgery (ovario-hysterectomy) and anesthesia on phytohemagglutinin-induced lymphocyte blastogenesis were studied in vitro in 12 dogs. Four dogs had depressed lymphocyte blastogenic responses after surgery. This suppression was transient with normal blastogenic responses occurring in cells from all dogs 24 hours after surgery. Seemingly, T-lymphocyte function may be depressed, only transiently, after surgery.
The acute effects of cyclophosphamide, vincristine, and L-asparaginase on the humoral and cell-mediated immune responses in dogs were evaluated. Dogs were given a single dose of cyclophosphamide (10 mg/kg of body weight, IV), a single dose of vincristine (0.025 mg/kg, IV), a single dose of L-asparaginase (400 U/kg, IV), or a combination of these drugs at the aforementioned dosages administered simultaneously. The total number of leukocytes decreased significantly (P = less than 0.05) on posttreatment day 3 in the cyclophosphamide-treated dogs. Antibody responses and lymphocyte blastogenesis were not affected. A transient lymphopenia on posttreatment day 1 was the only response seen in the vincristine-treated dogs. Antibody responses to sheep RBC and lymphocyte blastogenesis were suppressed in the L-asparaginase-treated dogs. L-Asparaginase did not affect the antibody response to bovine serum albumin or peripheral blood leukocyte counts. Suppressed antibody responses to sheep RBC and to bovine serum albumin, depressed lymphocyte blastogenesis, and decreased numbers of all peripheral blood leukocytes were seen in dogs given the 3 drugs in combination.
Neutrophilic movement was studied in dogs with atopy, 3 bacterial pyoderma syndromes, flea allergy dermatitis, and generalized demodicosis. Random neutrophil movement was decreased in generalized demodicosis, and this deficit correlated with the extent of body surface skin lesions. Neutrophilic chemotactic movement was decreased in 2 bacterial pyoderma syndromes: bacterial pyoderma, and staphyloccocal pyoderma. Although serum samples from diseased dogs showed no significant differences in chemoattractive qualities from normal pooled canine serum, control canine neutrophils incubated in demodectic dog serum showed decreased chemotactic responsiveness. It was postulated that demodectic dog serum had an inhibitor or deactivator of neutrophil chemotaxis.
A genus-specific enzyme-linked immunosorbent assay (ELISA) was applied to the detection of antileptospiral antibodies in swine sera. Results of the ELISA directly correlated with the results of the microscopic agglutination test. The ELISA had a sensitivity of 100%, specificity of 86.4%, and predictive accuracy of 91.3%. Because the ELISA is a simple and extremely accurate test, it lends itself as a diagnostic screening test for large numbers of samples.
Occult heartworm infection, ie, infection with adult Dirofilaria immitis in the absence of circulating microfilariae, was detected in dogs with prepatent infection, unisexual heartworm infection, drug-induced sterility of adult heartworms, and an immune-mediated infection. In the immune-mediate occult infection, female heartworms contained microfilariae that were antigenically similar to circulating microfilariae but were in a privileged site inasmuch as the intrauterine microfilariae were not coated with circulating antimicrofilarial immunoglobulin G. Transplantation of these female worm into heart-worm-negative dogs resulted in microfilaremia.