Regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with progressing or spontaneously regressing canine transmissible venereal sarcoma (CTVS)

Summary We examined the regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with regressing or progressing canine transmissible venereal sarcomas (CTVS). Both regressor and progressor peripheral blood leukocytes (PBL), draining and nondraining lymph node cells (LNC), and splenic leukocytes were significantly responsive to CTVS antigen extract in tube LAI. In contrast, a significant decrease in basal glass adherence of progressor PBL, draining and nondraining LNC, and splenic leukocytes was observed. Normal glass adherence was restored to progressor leukocytes by extensive washing with warm serum-free media, while significant tube LAI responsiveness to CTVS antigen extract was maintained. Preincubation of regressor PBL and LNC with progressor sera in two-stage tube LAI decreased the basal glass adherence of treated leukocytes. This effect of progressor sera was heat labile, a characteristic of CTVS antigen. Collectively, these findings suggest that progessor leukocytes and progressor sera treated regressor leukocytes were activated by interaction with serum CTVS antigen and thus behaved in tube LAI as stimulated cells, even in the absence of CTVS antigen. Regressor but not progressor sera were shown to contain anti-CTVS IgG with specific arming activity for normal dog PBL, but not LNC in two-stage tube LAI. The nonadherent response of peripheral blood neutrophils in two-stage tube LAI was proportional to the concentration of arming IgG, whereas no change was observed in glass adherence of PBL. The results of this study define the role of progressor and regressor serum factors in the mechanism of tube LAI and demonstrate a relationship between leukocyte glass adherence and the clinical course of CTVS. These findings show that tube LAI is a simple and reproducible measure of active factors in the immune response to a tumor.This investigation was supported in part by grant, CA-23469, from the National Cancer Institute, DHHS, and is submitted as Scientific Contribution No. 1051, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06268 USA     

Amplification of lymph node cell tube leukocyte adherence inhibition (LAI) reactivity by leukocyte adherence inhibition factor (LAIF)

This investigation examines the immunologic basis for specific antigen-induced tube leukocyte adherence inhibition (LAI) reactivity of draining lymph node cells (LNC) from dogs with canine transmissible venereal sarcoma (CTVS). CTVS regressor LNC, macrophage-depleted LNC, and enriched T lymphocyte fractions, but not enriched B lymphocyte fractions, were specifically reactive to CTVS antigen extract in direct tube LAI. In addition, regressor LNC amplified tube LAI responses by generating supernatants with leukocyte adherence inhibition factor (LAIF) activity for normal dog indicator LNC and enriched peripheral blood mononuclear cells (PBMC) in an indirect tube LAI assay. However, macrophage-depleted LNC and enriched T lymphocyte fractions failed to generate supernatants with LAIF activity, suggesting that macrophage accessory cells play a central role in the amplification of tube LAI. Interestingly, CTVS regressor peripheral blood leukocytes (PBL) and PBMC, which were specifically reactive in direct tube LAI, also failed to generate supernatants with LAIF activity. These findings demonstrate a distinction between LAIF-mediated amplification and direct tube LAI reactivity, and suggest that leukocyte populations with differing cellular proportions and from different immunologic compartments may participate in tube LAI via different mechanisms.     

A mechanism of suppression of antitumor immunity (LAI reactivity) by surgery

Summary Antitumor immunity assayed by tube leukocyte adherence inhibition (LAI) is suppressed during and for up to 1–3 weeks after surgery. The results of this study suggest that when cortisol is elevated above physiologic levels by the stress of surgery this has a marked suppressive effect on the LAI-reactive cell. Cortisol added to the in vitro tube LAI assay had a two-phase effect. Cortisol at concentrations about two-fold above physiologic levels inhibited the sensitization of the peripheral blood leukocytes with cytophilic IgG antitumor antibody; however, if the cell was already armed, the cortisol did not negate its LAI reactivity. Higher concentrations of cortisol had a direct inhibitory effect on the LAI-reactive cells' ability to react with tumor antigen whether or not the cells were armed. In addition, the in vivo elevation of cortisol by the administration of cortisone acetate to LAI-positive patients inhibited the LAI reactivity of their leukocytes. Stress-induced elevation of cortisol by surgery may have an adverse effect on resistance to tumor growth, and the extent of the immunodepression may be one of the variables in the difference in survival of patients with cancers with similar degrees of invasion. Present address: 2911 Bayview Avenue, 206G, Willowdale, Ontario     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

Circulating antigens and antibodies in human and mouse dermatophytosis: use of monoclonal antibody reactive to phosphorylcholine-like epitopes

The presence of circulating antibodies in the sera of patients infected with either Trichophyton concentricum or Trichophyton rubrum, and in the sera of BALB/c mice chronically infected with Trichophyton quinckeanum, was determined by ELISA. High levels of antibody to dermatophyte cytoplasmic antigens were detected both in infected humans and in mice. Partial inhibition of this reaction was observed by pretreatment of the sera with the hapten phosphorylcholine (PC). Moreover, antibodies were shown to have some reactivity with PC when tested by ELISA against PC conjugated to bovine serum albumin. Significant levels of circulating antigen were detected in patients with T. concentricum and T. rubrum infections, but not in uninfected subjects, by an immunoradiometric assay using a monoclonal antibody, Tq-1, which reacts with the PC-like epitopes of dermatophytes. It is possible that this dermatophyte antigen may play a role in modulating the cell-mediated immune responses, which would appear to be defective in most patients with these chronic forms of dermatophytosis.     

Transfer factor in vitro abrogation of blocking factors and amplification of immunoreactivity directed to soluble tumor associated antigens in the leukocyte adherence inhibition assay

Summary 26 breast cancer patients with recurrent disease were studied in order to evaluate whether transfer factor (TF) could abrogate serum blocking factors (SBF) and transfer or amplify immune reactivity directed to tumor-associated antigens (TAA) by using the leukocyte adherence inhibition assay (LAI). When 11 leukocyte samples obtained from patients reactive to cancer extract were preincubated with autologous blocking sera in the presence of PBS, nonspecific TF, or specific TF, leukocytes from these patients gained reactivity against breast cancer extract in none out of 11, one out of 11, and nine out of 11 experiments, respectively. Specific TF was obtained from donors with positive LAI breast cancer extract. Mean percentage LAI differences between control extract and breast cancer extract were –4.5±2.6, 0.5±1.7, and 15.5±2.4. The results of specific TF were significantly different from those of nonspecific TF (P<0.005).When nine leukocyte samples obtained from patients unreactive to cancer extract were preincubated with autologous blocking sera in the presence of PBS, nonspecific TF, or specific TF, leukocytes from these patients gained reactivity against breast cancer extract in none out of nine, one out of nine, and seven out of nine experiments, respectively. Mean percentage LAI differences between control extract and breast cancer extract were –4.6±2.0, 0.6±2.3, and 11.8±3.5. The results of specific TF were again significantly different from those of nonspecific TF (P<0.005).When allogeneic blocking sera were utilized, similar results were obtained. When specific TF was administered in two doses, 1 week apart subcutaneously to six breast cancer patients who were unreactive to breast cancer extracts, four patients gained reactivity against breast cancer extracts.Our in vitro experiments support the presence of a specific component in transfer factor extract which can transfer or amplify cell-mediated reactivity with abrogation of SBF directed at TAA.Abbreviations BSS = Balanced Salt Solution - LAI = Leukocyte Adherence Inhibition - PBS = Phosphate Buffered Saline - RPMI = Roswell Park Memorial Institute - SBF = Serum Blocking Factors - TAA = Tumor Associated Antigen - TF = Transfer Factor     

Anti-idiotypic monoclonal antibody to a T-cell chronic lymphatic leukemia

Summary A murine anti-idiotypic monoclonal antibody (mAb), F1, (IgG_2a) was produced against the variable part of the T-cell receptor for antigen (T_i, /) on the tumor cells of a patient with T-cell chronic lymphatic leukemia (CD3⁺, 8⁺, 4^–). The molecular weight of the protein reactive with mAb F1, comodulation and coprecipitation with anti-CD3 antibody, and the restricted tumor-cell reactivity strongly support the anti-idiotypic nature of mAb F1. MAb F1 also stained 4% of peripheral blood lymphocytes of healthy donors. MAb F1 did not stimulate the tumor cells to DNA synthesis, but stimulated a fraction of the normal peripheral blood lymphocytes, mAb F1 did not mediate antibody-dependent cellular cytotoxicity or complement lysis to any significant degree in vitro. Three infusions of 1–10 mg anti-idiotypic mAb were given over a period of 4 weeks. The plasma half-life for mAb F1 was 3 h in the first 2 h after infusion and 44 h from 2 h to 120 h after infusion. After each treatment a rapid decrease of circulating tumor cells was seen. During the observation period an 80% reduction of the total circulating tumor cells was noted. After the second infusion, IgM and IgG antimouse antibodies were detected. Side-effects from therapy were fever, chills, nausea, vomiting, diarrhea, tachycardia, increase in systolic blood pressure and shortness of breath. Thus, in T-cell malignancies a major reduction of circulating tumor cells can be accomplished by low doses of anti-idiotypic mAb. Anti-idiotypic mAb might be a therapeutic agent of significant importance.     

Circulating melanoma-associated antigen detected by monoclonal antibody NKI/C-3

Summary NKI/C-3 and NKI/black-13 are monoclonal antibodies recognizing different epitopes on a melanoma-associated antigen that is preserved after fixation in formalin and embedding in paraffin in virtually all melanoma tissues. The antigen, a predominantly cytoplasmic vesicle membrane-bound heterogeneous glycoprotein of 25–110×10³ daltons, was shown to be a single 25×10³ dalton polypeptide when incorporation of N-linked carbohydrates was inhibited by tunicamycin. The antigen was measured in a double determinant enzyme immunoassay (DDEIA) using NKI/C-3 as catcher antibody. Results from in vitro experiments indicated that the antigen is actively shed from living cells. In sera from melanoma patients with a small tumor burden, the antigen concentrations were in the range of those of controls (0–22 U/ml). Significantly increased values (33–600 U/ml) were found in sera from patients with a moderate or large tumor burden. The antigen concentrations in sera from patients with multiple metastases of other tumors were within the range of controls. Several sera from patients with multiple metastases of colon, pancreatic, and stomach carcinoma, however, contained increased antigen concentrations (45–80 U/ml). These results correspond with the reactions of NKI/C-3 in tissue sections of some malignancies other than melanoma.During the follow-up of melanoma patients the concentrations of circulating antigen correlated with tumor progression. The predictive value of the NKI/C-3 assay was no better than determination of serum lactate dehydrogenase, alkaline phosphatase or gamma glutamyl transferase activity.     

Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with bacillus Calmette-Guérin

Summary The immune reactivity of patients with strongly recurrent superficial bladder cancer was followed after combined intravesical and intradermal bacillus Calmette-Guérin (BCG) immunotherapy. All patients in this study were previously treated without success with intravesical chemotherapy. The BCG treatment regimen consisted of weekly administrations with BCG (RIVM) for six consecutive weeks, both intravesically and intradermally. In this study, sera and peripheral blood leukocytes (PBL) of patients were tested serially. Besides BCG-antigen-specific reactions, e.g. skin reactivity to purified protein derivatives of Mycobacterium tuberculosis (PPD), antibody formation and antigen stimulation of PBL in vitro, non-antigen-specific immune reactivities were also measured, e.g. mitogen response and spontaneous cytotoxic activity of PBL. In addition the antibody response to bladder carcinoma antigens and the cytotoxic activity of PBL for the bladder carcinoma cell line T24 and the natural-killer-sensitive K562 cell line were investigated. The results obtained from the various assays were evaluated for their prognostic value in relation to the length of the tumor-free interval after the BCG treatment. Because sera and PBL were only obtained during the first 6 months after the BCG treatment, the immune reactivity was compared to the clinical results at that same time. At 6 months after therapy 12 out of 40 BCG-treated patients were tumor-free whereas 28 out of 40 showed a recurrence. Skin reactivity to tuberculin PPD was measured in 40 patients during a period of 3–6 months after therapy. Of patients who showed a recurrence of the tumor within 6 months, 48% of them showed a transient response or developed no response at all to PPD. In the group of patients with a longer tumor-free period (n=10), only one patient lost the response to tuberculin PPD. Although PBL of a limited number of patients were tested, it was observed that the cytotoxicity to the bladder carcinoma cell line T24, and the natural-killer-sensitive K562 cell line increased in a number of the patients (7 out of 14, and 9 out of 14 respectively). Reactivity of PBL to mitogens and subset distribution (ratio T-helper: T-suppressor/cytotoxic) were not influenced by the BCG treatment. Antibody response to mycobacterial antigen was detected in 9 out of 23 patients investigated. Of these 9 patients, 8 belonged to the group with a recurrence of the tumor within 6 months (n=17). There was no correlation between the skin reactivity and the antibody response to tuberculin PPD. Furthermore, none of the 25 patients showed an antibody response to bladder carcinoma antigens. Sera of bladder carcinoma patients (n=19) reduced the mitogen-induced proliferation of lymphocytes, compared to sera of healthy controls (n=13), indicating the presence of circulating suppressor factor(s). Our results indicate that the absence of a Mantoux conversion or the presence of transient reaction to tuberculin PPD were highly related (91%) to a relapse of the disease. On the other hand, the cytotoxic activity of PBL to T24 and K562 cell lines, or their reactivity to tuberculin PPD or mitogens, gives no predictive information about the clinical results (tumor-free interval) of the BCG therapy. Abbreviations used: BCG, bacillus Calmette-Guérin; NK, natural killer; PBL, peripheral blood leukocytes; PPD, purified protein derivative of Mycobacterium tuberculosis; ELISA, enzyme-linked immunosorbent assay     

Leukocyte migration inhibition and leukocyte adherence inhibition (LMI- and LAI-assay) in cancer patients by using preparations from human fetuses]

The sensitization of lymphocytes from patients with different tumors was tested against a 3 M KCl-extract of fetal tissue (3.--6. month) by the leukocyte adherence inhibition assay (LAI) and by the leukocyte migration inhibition assay (LMI). Sensitization was compared with the reactivity of controls without any detectable tumor. In the LAI assay the leukocytes of 13/15 patients and 2/12 controls showed an inhibition of the adherence. In the LMI-assay 11/17 tumor-bearing patients and 6/18 controls reacted positive in the presence of the antigen preparation. The two methods demonstrated that patients bearing tumors of different histology are sensitized to fetal antigens.     

Production of monoclonal antibodies against target schistosomal antigen secreted in the urine of Schistosoma mansoni-infected patients

This study was undertaken to develop an immunodiagnostic test of active human schistosomiasis mansoni using a monoclonal antibody which targets urinary schistosomal antigen. Polyclonal antisera raised in rabbits against the processed urine of Schistosoma mansoni-infected patients showed very high and significant reactivity with ES product of ova compared with other different S. mansoni antigens. The monoclonal antibody (4.23) was reactive with repetitive epitopes of S. mansoni soluble egg antigen and ES product of ova with molecular mass range of 65–23 kDa and 80–23 kDa, respectively. It recognised different stages of the parasite life-cycle, with no cross reaction with Fasciola or hydatid antigen. MAbs were characterised by isotyping, immunoelectrophoresis, SDS–PAGE and the enzyme-linked immunoelectrotransfer blot technique, ELISA, and their recognition of carbohydrate or protein antigenic epitopes by periodate oxidation and trichloroacetic acid treatment of the antigen. It was used for detection of circulating schistosomal antigen in an antigen capture antibody sandwich ELISA on sera and urines of 58 S. mansoni-infected patients, 17 S. haematobium-infected patients, 15 parasite-free negative healthy controls and sera from 13 schistosomiasis-free patients harbouring Fasciola or hydatid infections. The percentage sensitivity of the assay in the serum of S. mansoni-infected patients was 98.4% and in urine 94.8%. A positive correlation was found between the number of faecal S. mansoni eggs and the circulating antigen, both in serum and in urine. Antigen circulating in urine correlated with that in the sera of S. mansoni patients. These data provide a sensitive and non-invasive method almost comparable with the use of sera for immunodiagnosis of schistosomiasis and an indirect way to reflect the intensity of infection. Australian Society for Parasitology.     

The cellular mechanism of leukocyte adherence inhibition.

Human blood leukocytes from three subjects who had been contact sensitized to dinitrochlorobenzene were used in direct and indirect leukocyte-adherence-inhibition (LAI) reactions in an attempt to elucidate the cellular mechanism of reactivity. The leukocytes were separated and purified by standard procedures. In direct LAI, only T cells or populations containing T cells gave positive reactions (significantly reduced adherence) with the antigen. Supernatants from suitable leukocyte-antigen mixtures contained a soluble leukocyte-adherence-inhibition-factor (LAIF) that reduced the adherence of normal leukocytes. Only T cells or populations containing T cells were active in LAIF production; B cells, granulocytes, and monocytes were inactive. The cellular requirement for the action of preformed LAIF was not restricted: all major types of blood leukocytes were susceptible to its effect.     

Clinical and immunological effects of human recombinant interleukin-2 given by repetitive weekly infusion to normal dogs

Four normal adult dogs received two consecutive weekly cycles of human recombinant interleukin-2 (IL-2) by continuous infusion for 4 days/week. The dose of IL-2 given to each dog was 3×10⁶ units m^–2 day^–1. Toxicities consisted of mild vomiting, diarrhea, and lethargy to varying degrees in all the dogs. These side-effects were reversed when the treatment was discontinued. Fever, tachypnea, and weight gain were not seen. A marked lymphocytosis and eosinophilia developed in all dogs after completion of each course of IL-2 (resulting in a more than sevenfold increase in each cell type) and persisted for more than 1 month in some. Fresh peripheral blood lymphocytes (PBL) obtained during this lymphocytosis mediated enhanced in vitro lysis of a natural-killer-cell-sensitive canine tumor cell line (CTAC). The in vitro proliferative responses of these same PBL to IL-2 could be detected earlier, progressed faster, and involved more cells than PBL tested prior to IL-2 infusion. Thus, a relatively well-tolerated regime of IL-2 in dogs can induce dramatic increases in lymphocyte numbers and activation, which is associated with augmentation of their in vitro antitumor reactivity. The clinical effectiveness of this immunotherapeutic approach remains to be tested in tumor-bearing dogs where it could serve as a relevant large-animal model for immunotherapy of cancer with IL-2.This work was supported by grants from the University of Wisconsin Graduate School. University of Wisconsin School of Veterinary Medicine Companion Animal Fund, NIH CA-32685, CM-87290, and American Cancer Society CH-237     

Immuno-enzymatic evaluation of the recombinant TSSA-II protein of Trypanosoma cruzi in dogs and human sera: a tool for epidemiological studies

The rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruzi antibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected with T. cruzi VI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected with T. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence of T. cruzi circulating lineages in the region.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.     

Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

Early diagnosis of Schistosoma mansoni in mice using assays directed against cercarial antigens isolated by hydrophobic chromatography

Two diagnostic assays are described for the early diagnosis of acute schistosomiasis, using a defined cercarial antigen preparation obtained by hydrophobic chromatography. Circulating IgM antibodies against this antigen fraction could be detected by ELISA as early as 1 wk after exposure in experimentally infected mice; IgM levels against other antigens and IgG levels against all the preparations examined were not significantly elevated until approximately 4-5 wk postinfection. Circulating antigen was detected as early as 3 days after exposure by a competitive inhibition ELISA using rabbit serum prepared against the cercarial antigen; antigen levels in the serum of mice with a 100-worm infection were found to exceed 100 ng/ml. Studies using sera from infected humans indicate that the assay can also recognize chronic S. mansoni, S. haematobium or S. japonicum infections. In a very limited field study, the specificity of the circulating antigen assay with regard to other helminthic infections was found to be 85%; sensitivity 100%. Preliminary characterization of the relevant antigen indicates that it is a relatively hydrophobic polypeptide with a molecular weight of approximately 41,000 daltons. The implications of these findings with regard to the treatment of travelers or the conduct of seroepidemiological studies in endemic areas are discussed.     

Ostertagia ostertagi: Isolation and partial characterization of somatic and metabolic antigens

One metabolic (ES) and two somatic extracts (AS and MS) were prepared from Ostertagia ostertagi. Partial characterization of the three preparations was accomplished by sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. In immunoblots, AS showed a low number of bands that reacted weakly with sera collected from infected calves. MS reacted strongly with the homologous sera, and a sharp group of bands appeared from 12 to 14 kDa, increasing in intensity as the infection progressed. ES showed a group of strongly immunogenic bands in the range of 16–22 kDa. The three preparations were also tested for reactivity with specific antt-Ostertagia antibodies in an enzyme-linked immunosorbant assay (ELISA). The ELISA results showed that all three worm extracts contained antigen epitopes recognized by circulating antibody in sera taken from O. ostertagi-infected calves. The strongest response was seen when antibodies of the IgGl isotype were reacted with MS and ES. When sera taken from O. radiatum-infected calves were used both somatic extracts showed high levels of cross-reactivity.     

犬传染性肉瘤与原位移植性动物肿瘤模型

犬传染性肉瘤是犬类动物生殖系统自然发生的肿瘤,其具有独特的同种与异种移植特性。本文着重从肿瘤细胞的生物学特性探讨肿瘤模型的建立,该模型具有可靠的稳定性、模拟性及接种的多样性,能够在难以接种肿瘤的器官及大动物体内建立较为理想的原位移植性肿瘤模型,为临床及影像学科开展原位肿瘤方面的研究提供了较好的实验基础。