Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.     

Rat plasma 25-hydroxyvitamin D3 binding protein: An inhibitor of the 25-hydroxyvitamin D3-1 α-hydroxylase

An antibody was prepared from serum of rabbits injected with a pure inhibitor protein obtained from rat serum for chick renal 25-hydroxyvitamin D₃-1α-hydroxylase. The antibody was separated from the endogenous inhibitor in rabbit serum. The antibody shows a single precipitin line with the rat serum antigen and with crude calf serum. Furthermore, the antibody removes the 4.0 S 25-hydroxyvitamin D₃ binding protein from rat serum. The removal of the 25-hydroxyvitamin D₃ binding protein from rat serum with antibody brings about a proportionate removal of inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase. The pure inhibitor binds 25-hydroxyvitamin D₃, as demonstrated by sucrose density gradient sedimentation, and shows specificity of binding identical to the serum transport globulin for 25-hydroxyvitamin D₃. Thus, the previously reported inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase in rat preparations is the serum 25-hydroxyvitamin D₃ transport protein or some derivative thereof. The antibody added to rat renal mitochondrial preparations does increase the activity of the 1- and 24-hydroxylases slightly but not markedly.     

Mitogens and T-independent antigens stimulate T lymphocytes to secrete La antigens.

Previous reports from this laboratory suggest that certain I region-associated (Ia) antigens can be detected in normal mouse serum. It was found that, when mitogens are injected into mice, they produce substantial increases (up to 125-fold) in the levels of these Ia antigens in mouse serum. Similar increases were obtained when either T- or B-cell mitogens were injected. Furthermore, in vitro and in vivo studies demonstrated that the mitogens stimulated T cells to secrete Ia antigens. It appears likely, however, that the Ia antigens detected in these studies may differ from the conventional Ia glycoproteins found on the surface of B lymphocytes.All T-independent antigens tested also augmented the concentrations of Ia antigen in serum, the increases depending on the T-independent antigen injected and ranging from 3- to 125-fold. In contrast, T-dependent antigens, unless injected in large amounts, were unable to produce detectable changes in the serum levels of Ia antigen. These data indicate that an inverse relationship exists between the T dependence of an antigen and its ability to stimulate T cells to secrete Ia antigens. On the basis of this conclusion it is proposed that all antigens are T dependent and merely vary in the efficiency with which they activate T cells to release helper factors.     

Thymic control of secretory antibody responses in the rat.

The effect of neonatal thymectomy on salivary and serum antibody responses was studied in rats. Local immunization of thymectomized rats with a T-dependent antigen (DNPBGG) elicited negligible amounts of IgA anti-DNP antibody in saliva. In contrast, both normal and sham-thymectomized animals demonstrated substantial levels of salivary IgA antibody. All thymectomized rats locally injected with a T-independent antigen (DNP-Lys-Ficoll) exhibited salivary IgA antibody production. Salivary IgG antibodies were somewhat decreased in thymectomized rats injected with either antigen; however, the final effect of T cell deprivation on IgG synthesis was not as pronounced as on IgA synthesis. Serum IgA antibody was induced in control rats injected with DNPBGG, whereas this Ig class of antibody was absent in thymectomized rats. The results suggest that thymus-derived cells exert a regulatory influence on both serum and secretory IgA responses to antigens.     

Nude mice injected with DNA released by antigen stimulated human tlymphocytes produce specific antibodies expressing human characteristics

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.     

In vivo suppression of the primary immune response by a species of low density serum lipoprotein.

An apparent subspecies of normal human serum low density lipoprotein (LDL-In) has been identified with suppressive activity for early or facilitating events of human lymphocyte mitogen and allogenic cells stimulation in vitro. This report describes the effects of in vivo administration of LDL-In on the mouse anti-SRBC immune response. Human LDL-In is not species specific and was capable of suppressing the in vivo mouse anti-sheep erythrocyte (SRBC) hemagglutination response by 88% after the administration of 500 to 600 mug LDL-In IV, whereas human serum high density lipoproteins and fibrinogen had no effect. Maximal suppression occurred only when LDL-In was injected 24 to 48 hr before antigen administration. Simultaneous or subsequent injection of LDL-In had no effect. The activity of LDL-In was influenced by antigen dose and maximal at low antigen doses. The number of splenic plaque-forming cells was also reduced indicating a suppression of the clonal expansion of primary B cells to antibody-secreting cells rather than only suppression of antibody synthesis by differentiated B cells and their progeny. These observations suggest the hypothesis that endogenous LDL-In could play an important immunoregulatory role in the maintenance of immune homeostasis and the "natural" suppression of non-productive lymphocyte proliferation.     

Enhancement of the in vivo antibody response by an 8-derivatized guanine nucleoside

The capacity of the C8-substituted guanine ribonucleosides to enhance the in vivo humoral immune response to the protein antigen, human gamma globulin (HGG), in A/J mice was evaluated. It has been shown recently that the C8-substituted guanine ribonucleosides are a new class of potent adjuvant for humoral immune responses to the sheep erythrocyte antigen. The current studies extend these findings to HGG with 8-bromoguanosine (8BrGuo), a representative of this group of nucleosides. The adjuvant activity of 8BrGuo in this system is highly dose and time dependent. Although 8BrGuo enhanced responses when injected either early (Day 0) or late (Day 4 or 5) after immunization, its administration on Day 1 or 2 most often led to no enhancement, suggesting that 8BrGuo may act on two events separated by a resistant stage in an ongoing immune response. The plaque-forming cell (PFC) response to HGG was enhanced optimally at doses as low as 1 mg 8BrGuo/mouse administered either on the day of immunization or 4 days thereafter. In contrast, however, serum anti-HGG antibody concentration assayed by enzyme-linked immunosorbent assay (ELISA) was enhanced only at doses of 10 mg or more, injected on the day of immunization, but doses as low as 1 mg were effective on Day 4. 8BrGuo was also an effective adjuvant when injected after antigen administration in incomplete Freund's adjuvant or when administered by several different routes (intraperitoneal, subcutaneous, oral).     

The role of ferroxidase-II and a ferroxidase inhibitor in iron mobilization from tissue stores

Injection of ferroxidase-II into copper-deficient rabbits resulted in a rapid, large, increase in the serum iron concentration which was equivalent to the increase observed when ceruloplasmin was injected into the same animals. A recently discovered serum inhibitor of ferroxidase-II, was also shown to potently inhibit ceruloplasmin. Acceleration of iron mobilization from storage tissues by dietary manipulation or repetitive bleeding of rabbits leads to a large decrease in the serum content of the inhibitor and a corresponding increase in the total serum ferroxidase activity. These studies suggest that ferroxidase-II could serve as a viable, alternative mobilizer of iron from tissue stores and that the recently discovered serum ferroxidase inhibitor could participate in the regulation of the efflux of iron from tissue stores.     

Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector

Summary We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34°C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca⁺⁺-resistant cells did not proliferate at the nonpermissive temperature (40°C), indicating that they depended on T antigen for their proliferation. The temperaturesensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40°C. The serum/Ca⁺⁺-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca⁺⁺-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.     

Prevention by a platelet-derived factor (platelet factor 4) of induction of low dose tolerance to pneumococcal polysaccharides

It was previously shown that human or mouse serum, and platelet factor 4 (PF4) prepared from human platelet releasate, counteracts nonspecific immunosuppression induced in mice by injection of concanavalin A or syngeneic gamma-irradiated lymphoma cells. The present studies show that PF4 prepared from normal mouse or human serum by absorption to heparin-agarose and elution between 0.5 and 1.5 M NaCl is also active in this respect. The ability of PF4 to counteract antigen-specific suppression of the antibody response to pneumococcal polysaccharide (pps) was now studied. PF4 derived from human or mouse serum as well as recombinant PF4 interferes with induction of antigen-specific low dose tolerance when they are injected at the same time as a low dose (0.2 microgram) of type 14 pps 3 days before an optimal immunizing dose (25 micrograms). Furthermore, injection of platelet releasate at the time of an optimal primary immunizing dose of pps type 14 enhances the secondary response to killed bacteria injected 2 weeks later, but not the primary response itself. Both effects are interpreted as due to interference with antigen-specific suppressor cell induction during primary immunization. Injection of PF4 is much less effective in reversing low dose tolerance to an optimal immunizing dose (0.1 microgram) of type 3 pps induced by injection of 0.005 microgram of this antigen. Differences in the mechanism of tolerance induction for the two pps types that might be responsible for this are discussed.     

Elimination of I131-labelled heterologous antigen from the blood of chickens after total body X-irradiation

A study was made on the effect of ionizing radiation upon the rate of elimination of I¹³¹. labelled human serum albumin (HSA I¹³¹) from the blood and upon antibody formation in chickens irradited with 1,2 00R(i.e.with LD₅₀) and injected with antigen 30 min, 6 days or 14 days after irradiation. The elimination curve from unirradiated control birds followed the typical three-phase pattern. The effect of irradiation was most marked with chickens injected with antigen 6 days after irradiation, resulting in an extension of the second phase with practically no third phase at all. Exposure to irradiation 30 min prior to antigen administration resulted in an extension of the second phase by 2 days as compared to the controls, with the onset of the third phase occurring on day 7. Irradiation 14 days prior to antigen administration resulted in an extension of the second phase by 1 day as compared to the controls, with the onset of the third phase occurring on day 6. Elimination of HSA I¹³¹ in the second phase was more rapid than that of I¹³¹-labelled chicken serum albumin (CSA I¹³¹) no matter whether the chickens were irradiated or not. This suggests that the capacity of specific antigen uptake is not affected by irradiation. Antigen elimination curves from control irradiated groups given CSA I¹³¹ followed the same pattern as that found in control unirradiated birds injected with homologous antigen.     

Effect of treatment with methylprednisolone on duration of pseudopregnancy and on macrophages and T lymphocytes in rabbit corpora lutea.

The potential role of macrophages and T lymphocytes in the destruction of the corpus luteum at the end of the luteal phase was investigated by treating pseudopregnant rabbits with the immunosuppressant glucocorticoid methylprednisolone. Eleven specific pathogen-free New Zealand White rabbits were injected with pregnant mares' serum gonadotrophin (40 iu, i.m.), followed 2 days later by human chorionic gonadotrophin (40 iu, i.v.) to stimulate ovulation. The following day (day 1 of pseudo-pregnancy) all animals had an oestradiol-filled Silastic capsule implanted s.c., to ensure that oestradiol, the luteotrophic hormone in this species, would not be limiting. From day 10 of pseudopregnancy, three animals were injected with a low dose of methylprednisolone (2 mg kg-1 per day) until day 20. Three other animals were injected with a higher dose of methylprednisolone (20 mg kg-1 per day) from day 13 of pseudopregnancy until day 19. Five animals served as control, vehicle-injected animals. Blood samples were taken at intervals and assayed for progesterone. Immunofluorescence was used to stain luteal tissue for macrophages, T lymphocytes and class II antigens, and positive cells were counted under high-power magnification. Methylprednisolone treatment reduced (by about 70%), but did not eliminate, the macrophages in the regressing corpora lutea. In contrast, the high dose of methylprednisolone essentially eliminated T lymphocytes, and reduced (by about 90%) the number of cells expressing class II antigen in the luteal tissue. Despite the effects of methylprednisolone on these cells, serum progesterone profiles were not altered by treatment with methylprednisolone, and pseudopregnancy was of normal duration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine antigen H-2.7: In vitro phenotypic conversion of erythrocytes

The H-2.7 antigen in normal mouse serum can be passively adsorbed to H-2.7^– erythrocytes in 10 percent sucrose (low ionic strength) solution. This antigen can also be stripped off the H-2.7⁺ erythrocytes under the same conditions provided the H-2.7⁺normal serum is absent. The stripped red blood cells can regain the H-2.7 antigen upon reincubation with H-2.7⁺ normal serum. The attachment of the H-2.7 antigen to erythrocytes probably occurs via a specific receptor.Abbreviations used in this paper BSA bovine serum albumin - B10 C57BL/10Sn - HA hemagglutination - LIS low ionic strength solution - NMS normal mouse serum - PBS phosphate-buffered saline - PVP polyvinylpyrrolidone - RBCs red blood cells     

Serum trypsin inhibitor activity in rabbits immunized with mounting doses of bacterial antigen (Salmonella paratyphi B)

The author studied changes in serum trypsin inhibitor activity and in the leucocyte count in rabbits immunized withSalmonella paratyphi B vaccines in different concentrations. Changes in serum trypsin inhibitor activity followed a regular course. Six hours after administering the antigen it fell significantly, the degree of the decrease being correlated to the antigen concentration. After 24, 48, 72 and 96 hours it was significantly raised, the maximum increase being recorded after 72 hours; the increase was likewise correlated to the vaccine concentration. Pronounced changes were also recorded in the leucocyte count, but these did not follow a regular course and were not correlated to the amount of antigen administered.     

The interactions of the non-specific inhibitor of hyaluronidase with hyaluronidase and proteolytic enzymes in vivo

1. Following intravenous administration of testes hyaluronidase in rabbits and dogs, there is a decrease in the level of the non-specific inhibitor of hyaluronidase in serum. 2. If a large amount of hyaluronidase is injected, the inhibitor level is reduced to zero and hyaluronidase may be present in serum for some time after the injection. The hyaluronidase activity of such samples of serum increases when the serum is incubated with papain. 3. Hyaluronidase activity is found in the livers of the injected animals in large amounts and this activity is increased considerably when the homogenate of this tissue is incubated with papain. 4. Intravenous administration of several proteases or venom produces a decrease in the serum inhibitor level. Intravenous administration of streptokinase produces such a decrease in rabbits but not in dogs. 5. There is a correlation between the depletion of the inhibitor from the serum and the occurrence of a slow, persistent depression of blood pressure upon administration of proteolytic enzymes.     

Migration of T-lymphocyte precursors into the thymus and the factors affecting this process

Migration of FITC-labeled mouse bone marrow cells into the thymus was measured by flow cytometric analysis 3 hours after intravenous injection of cells into irradiated mice. The percentage of cells reaching the thymus diminished when the dose of injected cells increased. The dependence of the number or labeled cells in the thymus on the dose of injected cells was not linear. Pretreatment of cells with anti-SC-I serum, peanut lectin or H-2 incompatibility antigen abolished thymus migration, while treatment with anti-Thy-I serum, soybean lectin, trypsin or Thy-I-incompatibility antigen diminished cellular migration and treatment with neuraminidase enhanced it. It was concluded that the main type of migrating cells is SC-1+ precursors of T-lymphocytes. Penetration of these cells through the blood-thymus barrier is based on the recognition of their partly sialized surface glycoprotein receptors by membrane lectins of the blood-thymus barrier cells.     

IgG1 antibody-dependent mediator release after passive systemic sensitization of basophils arriving at cutaneous basophil hypersensitivity reactions

When antigen is injected into a 24-hr cutaneous basophil hypersensitivity (CBH) reaction of an actively sensitized guinea pig, local basophils degranulate and release histamine. This reaction is called cutaneous basophil anaphylaxis and may be antibody mediated. We now report passive sensitization of basophils at CBH sites by systemic transfer of anti-picryl immune serum. Keyhole limpet hemocyanin- (KLH) immunized animals were skin tested with KLH to elicit 24-hr CBH reactions at day 7. Anti-picryl serum was injected i.v. at various times. On day 7, blue dye was injected i.v., and then 24-hr CBH sites vs nearby normal skin were challenged with 0.1 microgram picryl-human serum albumin (Pic-HSA). An immediate increase in vascular permeability (blueing) was noted at normal skin sites due to systemic passive cutaneous anaphylaxis (PCA), and augmented blueing occurred at CBH sites compared with normal skin. Systemic passive sensitization of CBH sites occurred when antiserum was administered as little as 1 hr before challenge of CBH site. However, local administration of anti-picryl serum (as in a local PCA reaction) was not able to sensitize tissue basophils, whether antigen was administered locally or systemically. The serum factor that mediated cutaneous basophil anaphylaxis was heat-stable (56 degrees C X 4 hr) 7S IgG1 antibody. Electron microscopy of Pic-HSA-challenged CBH sites in animals that received IgG1 antibody showed that local basophils undergo anaphylactic degranulation by exocytosis. These studies suggest that basophils arriving at CBH reactions are sensitized for anaphylactic function by antibody that can be acquired in the circulation, but possibly not at the local site.     

Cyclic antibody formation to polyglycerophosphate in normal and injected rats.

Normal adult Sprague-Dawley rats were bled serially over a 30-week period and their sera were examined for antibodies to polyglycerophosphate (PGP) by a standardized passive hemolysis test. Levels of "natural" antibodies to PGP fluctuated during this period with a majority of animals exhibiting pronounced cycling of serum antibody levels, however, the individual cycles were not synchronized with each other. Feeding of radiolabeled Gram-positive bacilli to these animals and examination of lymphoid tissue by liquid scintillation counting revealed that the antigen persisted mainly in the mesenteric lymph nodes. A second group of rats was injected i.v. with radiolabeled Gram-positive bacilli and tissues were examined for plaque-forming cells (PFC) of PGP specificity, and the sera were examined by passive hemolysis. Cycling of both anti-PGP antibodies and PFC became synchronized in the injected animals with peaks of serum antibody evident at 16 and 28 days post-injection and splenic PFC peaks at 4 and 16 days post-injection. Cycling was also observed in the mesenteric lymph nodes and bone marrow Examination of lymphoid tissue from the rats injected i.v. revealed that antigen introduced by this route also perisisted mainly in the mesenteric lymph nodes, This report demonstrates individual cycling of natural responses to environmental antigen and to the same determinant in secondary responses, indicating its importance as a regulatory mechanism.     

Kinetics of the antibody response to type III pneumococcal polysaccharide (SSS-III). I. Use of 125I-labeled SSS-III to study serum antibody levels, as well as the distribution and excretion of antigen after immunization.

A simple method was described for the preparation of 125I-labeled type III neumococcal polysaccharide (SSS-III) with a high specific radioactivity which retained the physical and immunologic properties of native SSS-III. SSS-III was used to study the serum and tissue levels of antigen, as well as its excretion, after i.p. injection. When an optimally immunogenic dose (0.5 mug) of antigen was given, greater than 90% of the injected antigen was excreted during the first 3 days after injection; however, after day 3, the SSS-III which remained in each mouse was firmly bound to various tissues, and less than 5 ng SSS-III was released into the circulation daily. SSS-III was also used in a Farr test to measure serum antibody levels; the kinetics for the appearance of PFC/spleen and serum antibody levels were measured at 24-hr intervals after immunization with 0.5 mug of antigen. Maximum PFC/spleen were observed on day 4 after immunization whereas the peak serum antibody level was seen on day 5. The decay of serum antibody level from its maximum value was much slower than that of the PFC/spleen. The data describing the distribution of SSS-III in vivo and the measurement of serum antibody levels indicated that treadmill neutralization was not a factor in determining the serum antibody levels after immunization with an optimally immunogenic dose of SSS-III.     

Elimination of intestinally absorbed antigen into the bile by IgA

Polymeric serum IgA has been shown to transport antigens in the form of IgA immune complexes from the circulation into the bile. To test the hypothesis that this mechanism could aid in the clearance of intestinally absorbed antigens, mice were gastrically intubated with trinitrophenylated human serum albumin (TNP-HSA). After 3 hr, two groups were injected i.v. with either MOPC 315 (IgA, anti-TNP) or control ascitic fluid. One hour later, blood and bile samples were taken. Small amounts of antigen (0.0008% of the intubated dose) were detected in the serum of control animals by solid phase radioimmunoassay. In animals receiving anti-TNP IgA, serum levels were decreased significantly and intact antigen appeared in the bile, whereas no antigen was detectable in the bile of control mice. These results suggest that IgA-mediated hepatobiliary transport can function as one of the mechanisms for the elimination of intestinally absorbed antigens.