The effects of sym. Triazinones on developmental stages ofEimeria tenella,E. maxima andE. acervulina: A light and electron microscopical study

The development of three chicken coccidia (Eimeria tenella, E. acervulina andE. maxima) was studied by means of light and electron microscopy. One group of chickens infected with 6000–20000 oocysts received a single dose of 5 mg/kg body weight (comparable to approx. 25 ppm in the feed) Bay Vg 7183 or Bay Vi 9142 orally (on day 3 or 4 p.i.), whereas others received two doses (on days 3 and 4 or on days 4 and 5 p.i.). The animals were killed on days 3, 4, 5, 6 and 7 p.i. and parts of the mucosa were dissected from the caecum (E. tenella), the ileum (E. maxima) and the duodenum (E. acervulina). Significant damage was observed in comparison to the controls, affecting nearly all of the parasites in those animals that had been treated twice, whereas some of the parasites remained microscopically unchanged after only one treatment. In general, the perinuclear space, mitochondria and the endoplasmatic reticulum were found to be considerably enlarged. Nuclear divisions were disturbed in schizonts and microgamonts, thus resulting in a greatly reduced production of parasites. The most important damage occurring in the macrogamonts concerned the wall-forming bodies II. As they burst, the formation of intact oocyst-walls was hindered, even if fertilization had taken place.Bay Vg 7183; Bay Vi 9142     

Ultrastructural observations of host-cell invasion by sporozoites ofEimeria papillata in vivo

Scanning and transmission electron microscopy were used to study the invasion of mouse small-intestinal epithelium by sporozoites ofEimeria papillata. Some mice received oocysts by gavage and others received either sporocysts or sporozoites by direct injection into the small intestine. The highest concentration of invaded cells were found in ligated intestinal tissues studied at 5–45 min after the inoculation of sporozoites. Sporozoites actively invaded anterior end first, which resulted in extensive damage to the host cell. Such cells showed disrupted microvilli; protuberances of cytoplasm into the lumen, apparently the result of a disrupted plasma membrane; vacuolization of the cytoplasm; and damage to the mitochondria. These damaged cells were rapidly vacated as the sporozoite moved laterally into one or more adjacent intact host cells without entering the lumen. It is suggested that the host cell initially entered from the lumen becomes so severely traumatized that the parasite of necessity enters an adjacent cell as a prelude to further development. Various aspects of host-cell invasion by coccidia and malarial parasites are reviewed.     

In vivo evaluation of anticoccidial effect of antibody fragments expressed in pea (Pasum sativum) on Eimeria tenella sporozoites

Coccidiosis in chicken causes great economic losses. The increasing resistance of Eimeria species to anticoccidials has induced the search for alternative methods of control. In vivo antibody neutralization assay was conducted to study the inhibitory effect of nine antibody fragments (Ab1–Ab9) on Eimeria tenella sporozoites. These anti-E. tenella antibody fragments were expressed in pea plant (Pasum sativum). To assess the inhibitory effect on parasite reproduction, the in vivo antibody neutralization assay was done by retrograde infection of chicken with sporozoites previously incubated with antibody fragments. The pre-incubated sporozoites with the examined antibody fragments displayed a reduced ability to reproduce following intracloacal application to chicken (especially Ab1, Ab3, Ab5, and Ab9). Other antibody fragments (Ab2, Ab4, Ab6, Ab7, and Ab8) were less capable to reduce sporozoite infectivity and reproduction.     

The action of polyether ionophorous antibiotics (monensin,salinomycin, lasalocid) on developmental stages ofEimeria tenella (Coccidia,Sporozoa) in vivo and in vitro: Study by light and electron microscopy

The effect of three polyether antibiotics (monensin, salinomycin, lasalocid) on developmental stages ofEimeria tenella (Coccidia, Sporozoa) was studied in vivo and in vitro by means of light and electron microscopy. It was found that these three drugs act against free merozoites, which are destroyed by bursting of the cell border (i.e. pellicle), endoplasmic reticulum and internal organelles even after very short exposure times (20 min) in media containing 1 ppm, 10 ppm or 100 ppm of these drugs. Sporozoites, however, survived these drug concentrations during an exposure time of 30 min (this would be sufficient to penetrate host cells and start development). Intracellular stages, which were situated in a parasitophorous vacuole within an intact host cell, were not attacked, apparently because these drugs are almost incapable of penetrating host cells. On the other hand, parasites (such as differentiated schizonts, gamonts) located within degenerating host cells showed slight disintegration, which did not necessarily led to their death. From these results it becomes clear why these polyether antibiotics have to be fed daily. Doses of 70 ppm salinomycin, 125 ppm monensin and 125 ppm lasalocid were found to bring about an equivalent protective effect against an infection with 40,000Eimeria tenella oocysts.     

Ultrastructural evaluation of the effects of diclazuril on the endogenous stages ofEimeria maxima andE. brunetti in experimentally inoculated chickens

The ultrastructural morphology of the different endogenous stages ofEimeria maxima andE. brunetti was evaluated after oral treatment of inoculated chickens with a single dose of 5 mg/kg diclazuril. The drug induced no ultrastructural change in the growth or differentiation of the various schizont stages of bothEimeria spp. InE. maxima, the micromorphological appearance of micro- and macrogamonts developing from the blast form to maturation also remained unaffected by drug treatment. However, in all fertilized macrogamonts the normal pattern of oocyst wall establishment was completely disturbed, resulting in the formation of an abnormally thickened, incomplete oocyst wall and the necrosis of the zygote. InE. brunetti, the growth and nuclear division during microgametogenesis were not affected but differentiation was clearly abnormal. In comparison with the controls, this abnormal differentiation was characterized by a less extensive enlargement of the parasite surface area, aberrant morphological configurations of condensed heterochromatin, intracytoplasmic flagella formation, and glycogen accumulation. Finally, the complete degeneration of all microgamonts ensued. The growth and differentiation leading to mature macrogamonts was not disturbed; however, subsequent oocyst wall formation was largely precluded and the macrogamonts proceeded to degenerate completely. We conclude that diclazuril treatment primarily affected particular stages in the sexual development of bothEimeria spp., resulting in the complete eradication of these coccidian species.     

Passive protection of chickens against Eimeria tenella infection by monoclonal antibody.

Monoclonal antibodies reactive with the surface of Eimeria tenella sporozoites were produced in mice. This paper concerns one of these monoclonal antibodies, designated 1073.10, which agglutinated sporozoites in vitro and lysed the parasite in the presence of complement. This treatment neutralized sporozoite infections when the treated parasites were injected into the ceca of normal chickens. Passive transfer of ammonium sulfate-precipitated 1073.10 ascites fluid into 2- to 3-day-old or 3-week-old chickens conferred protection against challenge infection with E. tenella. These studies show that serum antibody may play a role in immunity to coccidiosis and that the sporozoite surface epitope recognized by 1073.10 is a possible vaccine candidate antigen.     

Development by genetic recombination of a line ofEimeria tenella resistant to robenidine,decoquinate and amprolium

A line ofEimeria tenella (H) resistant to robenidine, decoquinate and amprolium has been produced by genetic recombination. It was not possible to obtain a cross between this line and lines resistant to clopidol or arprinocid and halofuginone. Recombination between lines resistant to arprinocid and halofuginone resulted in a loss of pathogenicity.     

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.     

Crossreactivity with sporozoites, exoerythrocytic forms and blood schizonts of Plasmodium berghei in indirect fluorescent antibody tests with sera of rats immunized with sporozoites or infected blood.

IFA studies are reported using plasmodial antigens from three different stages of the life cycle of Plasmodium berghei: sporozoites (SP); exoerythrocytic schizonts in rat liver (EEF); and parasitized rat erythrocytes (SCH = schizonts). Two series of specific sera were applied: sera from adult rats with a blood-induced infection (series A) and sera from rats immunized against sporozoites by mosquito bites and protected against parasitaemia by chloroquine (series B). In series A antibody titres with all three antigens were seen, but those with SCH were generally the highest. Superinfection with parasitized rat blood did not change the titre. In series B sera, collected from rats after a single exposure to infected mosquitoes, showed only titres with SP from day 3 onwards, but after a second exposure titres to all three antigens developed. Crossreactivity with the heterologous antigens in series B was clearly less than in series A. Anti-P. berghei sporozoite antibodies did not crossreact with P. vivax sporozoites. Rats of series A were resistant to a challenge of parasitized blood and could also inhibit the development of sporozoites. Rats of series B were protected against a challenge of sporozoites but not of infected blood. The results are discussed.     

In vivo suppression of T-dependent antibody responses by treatment with a monoclonal anti-L3T4 antibody

Minute amounts of the anti-L3T4 antibody designated GK1.5 were found to deeply suppress in vivo antibody responses to T-dependent antigens. Primary responses to sheep erythrocytes were completely inhibited even when GK1.5 was administered up to 6 days before or 4 days after the antigen. Secondary responses to potent immunogens like sheep erythrocytes or keyhole limpet hemocyanin were also completely abolished by a single injection of GK1.5 just before the boost. This treatment had no effect on T-independent reactions such as the polyclonal activation of B lymphocytes with lipopolysaccharide or the anti-2,4,6-trinitrophenyl (TNP) response elicited by injection of TNP-Ficoll.     

In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa

The development of an anti-bacterial drug in the form of a monoclonal antibody (mAb) targeting an exposed virulence factor, represents an innovative therapeutic strategy. Consequently, a fully human IgG1 mAb (LST-007) targeting Pseudomonas aeruginosa (PA) flagellin type b was recombinantly expressed and characterized in vitro and in an infection model driven by a multidrug resistant (MDR) PA strain. LST-007 demonstrated a highly specific binding towards whole PA bacteria harboring flagellin type b and its recombinant counterpart, with a K(D) of 7.4x10(-10) M. In bioactivity assays, LST-007 or titers of Cmax sera derived from pharmacokinetic studies, markedly attenuated PA motility in an equipotent manner. In vivo, parenteral LST-007 (20 mg/kg) given as a single or double-dosing paradigm post-infection, afforded survival (up to 75% at Day 7) in a lethal model of pneumonia driven by the intratracheal (i.t.) instillation of an LD(80) of the MDR PA isolate. This protective effect was markedly superior to that of imipenem (30% survival at Day 7) and totally devoid with an irrelevant, human isotype mAb. These data lay credence that LST-007 may be a valuable adjunct to the limited list of anti-bacterials that can tackle MDR PA strains, thereby warranting its continued development for eventual clinical evaluation.     

In vitro measurement and screening of monoclonal antibody affinity using fluorescence photobleaching.

Antibody screening is a routine in vitro assay in monoclonal antibody development and production. We have recently adapted the fluorescence photobleaching method to quantify antibody mass transport and binding parameters in bulk solution (Kaufman and Jain, 1990, 1991). The present study uses this in vitro method to screen a series of monoclonal antibodies (IgG) developed against the rabbit VX2 carcinoma tumor line. These experiments indicate that the three antibodies recognize distinct epitopes on the tumor, with equilibrium binding constants of 1.3 +/- 0.5, 5.1 +/- 3.6 and 2.0 +/- 1.1 x 10(7) M-1 for the antibodies RVC-184, RVC-626 and RVC-779, respectively. The antibody diffusion coefficient revealed no dependent upon protein concentration or antigen bead volume fraction within the ranges investigated. It was demonstrated experimentally that the interactions conformed to a reaction limited binding model of fluorescence recovery, that the system was at equilibrium, and that non-specific binding due to the fluorescein probe was not significant. Once the non-reactive fraction of antibody is determined, this photobleaching technique does not require perturbation or physical separation of the unbound species. As such, it has many potential applications including in vivo investigation of binding parameters.     

Inhibition of tumor growth in vitro and in vivo by a monoclonal antibody against human chorionic gonadotropin beta

Human chorionic gonadotropin (hCG) beta-subunit (hCGbeta) has been detected in a wide variety of tumors and implicated in tumor maintenance and progression. To better facilitate the investigation of the expression and biological roles of hCGbeta, we generated a set of monoclonal antibodies (mAbs) against hCGbeta by the approach of DNA immunization. All the generated mAbs worked well in detecting native hCGbeta antigen, while some of them were surprisingly found to exhibit potential cytotoxicity to tumor cells in our preliminary experiments. Here, one of those cytotoxic anti-hCGbeta mAb 6H1 was evaluated in detail for its anti-tumor efficacy in vitro and in vivo. 6H1 showed high binding specificity to hCGbeta, which was analyzed by Western blot and ELISA as well as indirect immunofluorescence assay. Treatment with 6H1 inhibited the growth of a panel of hCGbeta-expressing tumor cell lines (HeLa, HL-60, HepG2, SMMC-7721, PC-3) in vitro. Moreover, 6H1 significantly delayed the growth of HeLa-borne tumors in nude mice and prolonged the survival of tumor-bearing mice. The anti-tumor effect of 6H1 was associated with the induction of apoptosis, which was estimated by Hoechst 33258 staining, DNA ladder assay and flow cytometry. Collectively, 6H1 revealed potent anti-tumor activity in vitro and in vivo and therefore may be an effective therapeutic candidate for immuno-intervention of cancers that ectopically express hCGbeta.     

In vivo and in vitro binding of iodinated monoclonal antibody A2B5 to RIN insulinoma cells

Monoclonal antibody A2B5 reacts with the cell surface of a series of amine precursor uptake decarboxylation (APUD) cells and their tumors in many vertebrate species including chicken, rat, mouse, and man. We have studied the in vivo and in vitro binding of iodinated monoclonal antibody A2B5 to rat insulinoma cells. In vitro, radiolabeled A2B5 binds specifically to RINm5F insulinoma cells and the binding of 125I-A2B5 is inhibited by unlabeled A2B5 or a ganglioside extract of RINm5F cells. In vivo, scintigrams taken Day 0 to Day 5 after injection of 131I-labeled A2B5 showed a striking localization of 131I-A2B5 in transplanted RIN tumors grown in syngeneic rats. Other control radiolabeled monoclonal antibodies did not concentrate in the tumors. 131I-labeled A2B5 did not concentrate in other transplantable tumors (colon adenocarcinoma, osteosarcoma, renal cell carcinoma, and bladder transitional cell carcinoma) grown in nude mice. The tumor/blood ratio detected 5 days after antibody injection, was approximately two to 12 times higher in the insulinoma compared to other organs and only in the insulinoma did 131I-A2B5 show a higher concentration than control antibody 125I-P3X63.     

In vivo inhibition by a monoclonal antibody to CD4+ T cells of humoral and cellular immunity in sheep.

The ability of intravenously injected anti-CD4 and anti-CD8 monoclonal antibody (mAb) to deplete specific lymphocyte subsets in vivo and their effects on antibody responses to ovalbumin (OVA) and Brucella abortus, and skin reactivity to T-cell mitogens was examined in merino lambs. Repeated administration of anti-CD4 or anti-CD8 mAb caused a specific and sustained depletion of target cells from peripheral blood. Anti-CD4 mAb significantly inhibited the in vivo antibody response to OVA but had no effect on the antibody response to LPS of B. abortus. In contrast, antibody responses to both OVA and B. abortus lipopolysaccharides (LPS) remained unaffected in lambs depleted of their CD8+ T lymphocytes. These results confirm the T-cell dependence and independence of antibody responses to OVA and LPS, respectively. Skin reactions elicited by intradermal injections of phytohaemagglutinin (PHA) and concanavalin A (Con A) were also significantly suppressed in lambs depleted of their CD4+ T cells, but treatment with anti-CD8 mAb had no effect on skin responsiveness. Together, these results suggest that mAb can be extremely effective at selectively depleting lymphocyte subsets in vivo and can be used for studying various aspects of immunoregulation and immunity in sheep.     

A monoclonal antibody against rat platelets. I. Tissue distribution in vitro and in vivo.

In this study we describe a new monoclonal antibody (MoAb PL.1) against rat platelets. Immunohistology of various rat tissues showed staining of platelets, especially in the spleen, and staining of megakaryocytes in bone marrow and spleen red pulp. In the liver small platelet aggregates and endothelial cells were stained. After in-vivo administration of MoAb PL.1 an acute severe thrombocytopenia was observed. In general the distribution of the antibody and/or antibody-coated platelet aggregates showed the same pattern as after in-vitro incubation, i.e. staining of rat platelets and platelet aggregates in spleen red pulp, and staining of megakaryocytes in spleen and bone marrow. Platelet aggregates were observed in the liver and electron microscopy indicated that they were associated with Kupffer cells. Furthermore, liver endothelial cells were positively stained. Comparison of the molecular weight of the antigens recognized by this MoAb and by human anti-platelet MoAbs, as well as comparison of staining patterns of megakaryocytes indicated that MoAb PL.1 is probably directed to a GPIIb/IIIa complex analogue. Since MoAb PL.1 is of the non-complement-binding mouse IgG1 isotype, it can be used for studying clearance of platelet aggregates by Fc-receptors of the MPS. It also promises to be a useful tool in the study of platelet involvement in rats with experimental nephritis.     

In vivo human B-cell subset recovery after in vivo depletion with rituximab, anti-human CD20 monoclonal antibody

Rituximab, chimeric anti-human CD20 monoclonal antibody approved for B-cell lymphoma, depletes circulating B cells. Little data exist on its use for nonmalignant diseases or B-cell subset recovery after treatment. We hypothesized that rituximab may reduce panel reactive alloantibodies (PRA) in dialysis patients awaiting renal transplantation and performed a single-dose, dose-escalation phase 1 trial. Here we report changes in lymphocyte phenotypes in subjects treated with rituximab alone. Nine subjects, 44 +/- 10 years(Yr); (5 F, 4 M) received one dose (n=3) at 50, 150, or 375 mg/m(2) without other immunosuppression. Blood was collected before dosing and intervals thereafter. No significant changes in leukocytes, total or CD3(+) lymphocytes were noted. In all, there was CD19(+) depletion by day 2(D2) (12.0 +/- 5.6 cells/mm(3) vs. 181 +/- 137, D0; p<0.01) and CD20(+) (11.0 +/- 12.0 vs. 205 +/- 116, D0; p<0.01). At 6 months (mo), CD19(+) and CD20(+) remained low (51.1 +/- 42.2, p<0.05; 75.4 +/- 38.7, p<0.05, respectively) compared to D0. CD19(+)CD5(+) cells recovered more rapidly, returning to baseline by 6mo while B memory cells (CD19(+)CD27(+)) remained low (32.3 +/- 29.0) at 1Yr (7.5 +/- 4.5; p<0.001) and 2Yr (12.1 +/- 7.9; p<0.001) after treatment. We conclude that single dose rituximab ablates B cells in high PRA dialysis patients awaiting transplantation. B-cell ablation, particularly memory B cells, was long-lasting, lagging repopulation by CD5(+) B cells.     

In vitro anti-HIV-1 antibody production in subjects in different stages of HIV-1 infection.

We evaluated the in vitro antibody production from peripheral blood mononuclear cells (PBMC) against HIV-1 proteins in infected adults. Fifty-four HIV-1 infected patients (four recent seroconverters, 15 asymptomatics with a CD4 count higher than 500/microliters, 27 asymptomatics with a CD4 count between 200 and 500/microliters and eight symptomatic patients) were tested. PBMC were incubated in the presence or absence of 1% pokeweed mitogen (PWM) at 37 degrees C for 8 days. Western blot assay, p24 antigen ELISA and anti-p24 antibody ELISA were performed on serum and culture supernatants. Spontaneous production of anti-env antibody in culture supernatants was evidenced in all subjects. All the positive supernatants for anti-core antibodies (18/54) were derived from asymptomatic patients. PBMC from recent seroconverters and from symptomatic patients did not produce any anti-core antibody. Antibody production decreased after stimulation with PWM. The concentration of p24 antigen did not significantly increase in p24 positive supernatants following acidification (P = 0.1), suggesting that the inability to detect p24 antibody was not due to the anti-p24 antibody complexed to p24 antigen in culture supernatants. In vitro production of anti-p24 antibodies was significantly more frequent in asymptomatic subjects with high CD4+ cell counts (P = 0.02) and was absent in recent seroconverters. This last finding suggests that during the initial phases of the infection, anti-p24 antibody production may be restricted to cells residing in lymphoid organs. In addition, the lower percentage of anti-core antibody in people with low CD4+ cell counts is not merely a consequence of the binding of the antibody to an increased amount of antigen, but probably reflects an impaired production or a sequestration of producing cells in lymphoid tissue during the late stages of the infection.     

Selective depletion of rat neutrophils by in vivo administration of a monoclonal antibody

A monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo was developed by hybridization of mouse myeloma cells (P3-X63.Ag8.653) and spleen cells of BALB/c mice sensitized with peritoneal neutrophils of WKA/Hok rats. RP-3 reacted with rat neutrophils but not with lymphocytes, macrophages, natural killer cells, basophils, eosinophils, or tissues of various organs. The mitogenic responsiveness to concanavalin A (ConA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) of rats given RP-3 was not significantly different from that of normal rats. Administration of RP-3 into the peritoneal cavity of rats that had been kept under specific pathogen-free (SPF) or clean conditions induced selective depletion of circulating neutrophils to under 100/mm3 (0.5% WBC). The numbers of monocytes, lymphocytes, and platelets were not changed. Administration of 2 ml of RP-3 reduced blood neutrophils to under 100/mm3 for approximately 24 h, and administration of 1 ml caused depletion for approximately 12 h.