Upregulation of phagocytosis and candidicidal activity of macrophages exposed to the immunostimulant,acemannan

Previous studies by these investigators have shown that mannosylated bovine serum albumin (m-BSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans by resident murine peritoneal macrophages (Mø). Upregulation of the above MØ functions was associated with binding of m-BSA to the MØ-mannose receptor. The present study was done to determine if the immunostimulant, acemannan prepared from aloe vera, could stimulate MØ in a similar manner. Resident peritoneal MØ collected from C57BL/6 mice were exposed to acemannan for 10min. The RB was measured using chemi-luminescence and demonstrated approximately a two-fold increase above the media controls. In studies involving phagocytosis, MØ were exposed to acemannan, washed and exposed to Candida at a ratio of 1:5. The percent phagocytosis and Candida killing were determined using fluorescence microscopy. There was a marked increase in phagocytosis in the treated cultures (45%) compared to controls (25%). Macrophages exposed to acemannan for 10 min resulted in ca 38% killing of Candida albicans compared with 0–5% killing in controls. If MØ were incubated with acemannan for 60 min, 98% of the yeast were killed compared to 0–5% in the controls. The results of the present study indicate that short term exposure of MØ to acemannan upregulates the RB, phagocytosis and candidicidal activity. Further studies are needed to clarify the potential use of this immunostimulant as an anti-fungal agent.     

Effect of hydrocortisone on immune system of the lizard,Chalcidesocellatus II. Differential action on T and B lymphocytes

Lymphocytes of thymus, spleen, peripheral blood (PB) and bone marrow (BM) collected from adult lizards, $\text{Chalcides}$$\text{ocellatus}$ were cultured for 24 hr in the presence of 10^?3M hydrocortisone acetate (HC) in order to assess the effect of in vitro HC on lizard T and B cell viability. The results indicated that HC induced stepwise, time-dependent mortality of the majority of thymocytes carrying T cell specific antigen(s) (TSA), 30–50% of T cells of spleen, PB and BM, and of a proportion of splenic B lymphocytes. Administration of 1 mg/g body weight HC to adult $\text{Ch}$. $\text{ocellatus}$ lead to depletion of all TSA⁺ thymocytes. In contrast, T lymphocytes in the peripheral lymphoid compartments revealed both sensitivity and resistance to HC; similarly, B lymphocytes constituted susceptible and resistant subpopulations.     

Effect of hydrocortisone on immune system of the lizard,Chalcidesocellatus I. Response of lymphoid tissues and cells to in vivo and in vitro hydrocortisone

A single dose of 1 mg/g body weight of hydrocortisone acetate (HC) administered intraperitoneally to adult lizards, $\text{Chalcides}$$\text{ocellatus}$ induced rapidly a reduction of about 85% of thymic lymphocytes. Histological evidence indicated that cortical, as well as, medullary thymocytes are sensitive to HC exposure. Around 40–50% of lymphocytes in peripheral blood (PB) and spleen were depleted at 3–7 days post-HC injection; such depletion durated about 4 weeks for PB but was rather temporary in spleen. Increase in number of bone marrow (BM) lymphocytes was negligible and transient and could by no way account for the dramatic cell losses in the different lymphoid tissues. The findings thus suggested that HC-mediated lymphocyte depletion in lizards is not attributable to redistribution between the different lymphoid compartments but rather to destruction. In direct conformation, lymphocytes were readily lysed in vitro by 10^?3M HC, thymocytes being more vulnerable > PB> spleen >BM lymphocytes.     

Ontogeny of the transplantation immunity of mice for rejecting ascitic allogeneic tumors

Ontogeny of the murine transplantation immunity for rejecting ascitic allogeneic tumors (chemically-induced RG lymphoma and L1210 leukemia) as a model of $\text{in vivo}$ cytotoxic T cell immunity was studied. challenge by 10⁶ to 10⁷ allogeneic tumor cells per 20 g body weight (b.w.) of the mouse was fatal to 1–3 day-old mice, whereas 7–30 day-old mice rejected the tumor. In newborn mice however some yet undetermined mechanism worked to temporally depress the initial tumor growth. Injection of low (10⁶ cells per 20 g b.w.) to moderate (10⁷) doses of semiallogeneic spleen cells into newborn mice prepared for second set rejection of the tumor carrying the same alloantigens as the spleen cells, although injection of high dose (3 × 10⁸) cells reduced the tumor rejecting immunity. This second set rejection occurred even against the allogeneic tumor inoculated as early as 3 days old, if the mice had been primed with the alloantigens at birth. It appears therefore that newborn and early suckling mice are protected from tumor invasion by cytotoxic immunity more powerfully than expected from earlier $\text{in vitro}$ works.     

A study of plaque formation with avian RNA tumor viruses.

Avian leukosis viruses of subgroups B, D, F, and one strain of reticuloendotheliosis virus (REV) are able to make plaques in several lines of chick embryo fibroblasts. Moreover, plaque formation with some strains of subgroup A is obtained only in chicken fibroblasts of $\text{C}\text{C}$ phenotype. One exception is RAV-3 which produces plaque in line 6 ($\text{C}\text{E}$) only. Several important steps for the reproducibility of the assay are described. Of all the vital stains tested, only neutral red incorporated into the agar overlay is capable of inducing the formation of plaques. Several lysosome stabilizers and labilizers are examined and their role in the mechanism of plaque formation is discussed.     

B cell activation in chickens induced by Staphylococcusaureus

Conventional mammalian polyclonal B cell activators were evaluated for activity in chicken spleen and peripheral blood lymphocyte (PBL) cultures. Although lipopolysaccharide was found to have a marginal influence on proliferation, two strains of the bacterium $\text{Staphylococcus}$$\text{aureus}$ (Cowan I and Wood 46 strains) induced moderate proliferation in both spleen and PBL cultures. In spleen cell cultures the proliferating cell population was identified as the B cell. The mitogenic response required the presence of adherent cells since their removal eliminated the response. Evidence of $\text{in vitro}$ polyclonal immunoglobulin synthesis could not be obtained. However, when administered intravenously, $\text{S.}$$\text{aureus}$ induced polyclonal immunoglobulin synthesis.     

Mitogenic responses of frog lymphocytes to crude and purified preparations of bacterial lipopolysaccharide (LPS)

We have compared $\text{in}$$\text{vitro}$ mitogenic responses of frog ($\text{Xenopus}$$\text{laevis}$ and $\text{Rana}$$\text{pipiens}$) lymphocytes to various preparations of lipopolysaccharide (LPS). Commercial LPS prepared from $\text{E}$. $\text{coli}$ (phenol extraction) and from $\text{S}$. $\text{abortus-equi}$ (phenol and TCA extraction procedures) was mitogenic for frog lymphocytes. After reextraction of these LPS preparations with phenol-water, the remaining LPS was either considerably less mitogenic or not mitogenic. Purified $\text{E}$. $\text{coli}$ 055:B5 LPS, prepared by phenol water extraction, enzyme treatment and column chromatography, was not mitogenic. Frog cells proliferated poorly or not at all with all concentrations of reextracted or purified LPS tested (0.5–400 ug/ml) and at all culture periods examined (days 1–7). All LPS preparations used were mitogenic for CAF₁ mouse lymphocytes, whereas reextracted and purified LPS preparations were not mitogenic for lymphocytes from C3H/HeJ cells. $\text{Xenopus}$ were also not susceptible to toxicity induced by parenterally administered LPS in concentrations which killed CAF₁ mice.     

Immune function in congenital osteopetrosis: Defective lymphocyte function in microphthalmic mice

Osteopetrosis is a prominent feature of a congenital mutation described in microphthalmic mice and is thought to be due to defective osteoclast function which causes a generalized lack of bone resorption. Reversal of defective bone resorption in osteopetrotic mutants has been achieved by hematopoietic cell trans-plantations; and conversely, defective bone resorption has been transferred to normals by hematopoietic cells from osteopetrotic littermates. This suggested that osteopetrotic mutants might also demonstrate defective immune functions which could in turn be related to the lack of normal bone resorption. To that end, aspects of $\text{in}$$\text{vitro}$ lymphocyte function in microphthalmic mice were compared to those of their phenotypically normal littermates. There was a significant diminution in the proliferative response of splenocytes to mitogens in microphthalmic mice. Microphthalmic splenocytes also were less responsive in an $\text{in}$$\text{vitro}$ assay which measured the capacity to form antibody forming cells.     

Cell-mediated lysis of murine target cells by nonimmune salmonid lymphoid preparations

Lymphoid preparations from nonimmune rainbow and brook trout were found to lyse murine tumor cells (EL-4 &; P815Y) $\text{in}$$\text{vitro}$ in an 18 hr ⁵¹Cr-release assay conducted at 16–18°C. Lysis was proportional to the effector: target cell ratio, required direct cell to cell contact, and was not depleted by the removal of nylon wool adherent cells. Lymphoid populations from peripheral blood, the thymus, and the anterior kidney, but not the spleen, were active in the cytotoxicity assay. Individual fish varied considerably in their ability to lyse one or both target cells. These data and the results of unlabelled target cell inhibition studies suggest that the reaction is selective if not specific. The addition of PHA to the reaction mixture resulted in markedly enhanced cytotoxic reactivity. In the presence of PHA lysis was readily detectable at 4 hr. The data demonstrate that nonimmune Salmonids possess a cytolytic effector cell population which has considerable cytotoxic potential and may represent a heterogeneous “natural killer cell” population.     

Tryptophan-induced stimulation of hepatic ornithine decarboxylase activity in the rat

The effect of a single tube feeding of l-tryptophan on hepatic ornithine decarboxylase (ODC) activity in rats was investigated. The levels of ODC activity in the livers of control and experimental rats were assayed in vitro by measuring the release of ¹⁴CO₂ from DL-[1-¹⁴C]ornithine. Single tube feedings of varying levels of l-tryptophan ($\text{2.5–30 mg}\text{100 g}$ body wt) to overnight-fasted rats 1 hr before sacrifice exhibited increases in the hepatic ODC activities. l-Tryptophan ($\text{30 mg}\text{100 g}$ body wt) tube fed to overnight-fasted rats $\text{1}\text{6}$ to 12 hr before sacrifice induced hepatic ODC activities which were significantly elevated beginning at 1 hr and peaking at 2 hr (6.5-fold increase over controls). In vitro [¹⁴C]leucine incorporation into protein using hepatic microsomes of tryptophan-treated rats was significantly increased at 1 hr in comparison with that of controls. The tryptophan-induced stimulation of hepatic ODC activity was not affected by prior adrenalectomy but was abolished by pretreatment with cycloheximide. These studies demonstrate that a single feeding of l-tryptophan can significantly enhance in the rat the activity of ODC, a key enzyme in the biosynthesis of polyamines.     

Studies on lectins of amago (Oncorhyncusrhodurus) I. Amago ova lectin and its receptor on homologous macrophages

A lectin was found in the ova of amago, a Japanese trout ($\text{Oncorhyncus}$$\text{rhodurus}$), which agglutinates rabbit, rat and human B-type ery-throcytes. The hemagglutination was specifically inhibited by monosaccharides, L-rhamnose, D-galactose, and their C₂ and C₄ analogs, and p-nitrophenyl-α-D-galactoside and melibiose, indicating a binding specificity for α-L-ramnosyl or α-D-galactosyl type sugar moiety. To study its interaction with homologous cells, amago peritoneal macrophages were isolated from corn starch-stimulated peritoneal exudates. The lectin-rabbit erythrocyte complexes were found to adhere onto the macrophages harvested on the 4th day or later after the stimulation, but not to those obtained within 3 days; the latter macrophages acquired the complex-binding capacity when cultured for 3 to 4 days $\text{in}$$\text{vitro}$. These findings indicated that a lectin receptor is expressed on peritoneal macrophages after inflammatory stimulation. Similar lectin receptor-bearing macrophage-like-cells were also detected during $\text{in}$$\text{vitro}$ amago head kidney culture. This suggested that the inflammatory induced peritoneal macrophages may be differentiated from the head kidney macrophage precursor cells and during this process the ova lectin receptors also become expressed.     

Infection of chick cells by subgroup E viruses.

Studies were carried out to determine whether there is a restriction of replication of the endogenous chicken leukosis virus Rous associated virus type O (RAV-0) in chick embryo fibroblasts (CEF) beyond that imposed by the known cell surface barrier. Following either Sendai virus mediated fusion of RAV-O with surface resistant CEF ($\text{C}\text{E}$ cells), or infection of CEF lacking the surface barrier ($\text{C}\text{O}$ cells), a quantitative 10³- to 10⁴-fold restriction in replication was noted in comparison with RAV-60, a recombinant leukosis virus bearing the same subgroup E envelope as RAV-0 The failure of this internal restriction to operate against subgroup E leukosis viruses was investigated in detail in a series of cloned subgroup E sarcoma virus recombinants isolated following mixed infection with RAV-0 and Prague strain of Rous sarcoma virus subgroup C (PR-C) (i.e., two factor crosses) or following PR-C infection of chf⁺ CEF. RNA from one of these PR-E clones was shown to contain a nearly full complement of RAV-0 specific and RSV specific nucleotide sequences, but that isolate and six others were all free of the restriction observed with RAV-0 replication. One clone may be subject to some restriction. Thus some genetic function(s) of RAV-0 lost or inoperative in recombinant viruses appears important for this restriction. Since RAV-0 can replicate to a “normal” titer in some CEF from particular lines of chickens (line 7, line 100 × 7, and line 15), but apparently not in embryo culture from chicken flocks used in these studies, some cell function(s) also appears important for this restriction.     

Characteristics of a nucleoproteinaceous subviral entity resulting from partial degradation of southern bean mosaic virus

Virions of southern bean mosaic virus (SBMV) degrade to a discrete (47–50 S) subviral entity (SVE) when treated with a buffer (pH 7.5 or 9.1) containing EDTA and bentonite. SVE comprises of intact viral RNA and about $\text{1}\text{3}$ as much capsid protein as virions. Treatment of SVE with sodium dodecyl sulfate or poly(G) released infectious SBMV-RNA. Unlike SBMV, SVE is unstable in vitro and sensitive to ribonuclease attack. SVE particles are of variable size (diameter, 15–25 nm), appear “loose” or deformed and lack a discrete outline. We propose that ca.$\text{2}\text{3}$ of the total SBMV coat protein complement constitutes a rigid “surface” capsid which encloses a “loose” nucleoproteinaceous complex of the remaining coat protein and the entire viral genome.     

In vitro demonstration of serological cross-reactivity between Ichthyphthirius multifiliis foquet and Tetrahymena pyriformis lwoff

The antigenic relation between $\text{Tetrahymena pyriformis}$ (Ehrenberg, 1830) Lwoff 1947 and $\text{Icthyophthirius multifiliis}$ Fouquet 1876 was investigated using various $\text{in vitro}$ immunologic tests. Tomites of $\text{I. multifiliis}$ exposed to dilutions of rabbit anti-tetrahymena serum were immobilized in 24 hours. $\text{Tetrahymena}$ in this test system were agglutinated at low dilutions and immobilized at higher dilutions. Using an indirect fluorescent antibody test, both organisms showed fluorescence when treated with either rabbit anti-tetrahymena serum or rabbit anti-tomite serum. In heterologous crosses, titers of 1:1024 were obtaibed using the passive hemagglutination test. These results indicate that an antigenic relationship does exist between these two organisms, cross-reacting antigens appear to be localized on the cilia.     

Antigen clearance in trout

$\text{In}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ experiments were performed on isolated areas of skin and gills of brown ($\text{Salmo}$$\text{trutta}$) and rainbow trout ($\text{S.gairdneri}$) subjected to hyperosomotic infiltration (HI) of viable bacteria. The route of antigen entry was shown to be the gills. After HI, live bacteria were found mainly in the spleen (Spl) and anterior kidney (AK). By 16–24 h the bacterial counts had returned to the lower numbers present in liver and muscle. When keyhole limpet haemocyanin (KLH) was injected into $\text{S.trutta}$, the time course of its detection in the Spl and AK depended upon the route of injection. KLH was present in both organs as early as 0.5 h and persisted up to 36 to 46 days. An immunoperoxidase technique proved to be sensitive in the detection of antigen in fish tissues.     

Thrombosis and atherosclerosis: the organization of pulmonary thromboemboli in the pig. Individual phospholipids, fatty acid composition of lecithin, sphingomyelin, esterified cholesterol, and 3 H-cholesterol specific activity

The organization of pulmonary thromboemboli in 8 to 12-week-old pigs was studied at intervals from 6 hr to 4 weeks. Thrombi were prepared in vitro in Chandler rotating loops.The predominant phospholipids of organizing emboli at all time periods were lecithin (45–57%) and sphingomyelin (15–24%). The content of all phospholipids decreased from 6 hr to 5 days. From 5 days to 4 weeks, there was no statistically significant change in the content of any phospholipid. The predominant fatty acids of organizing emboli were present in the order, 16:0 > 18:1 > 18:0 in lecithin, and 16:0 > 18:0 > 18:1 in both sphingomyelin and esterified cholesterol.Spontaneous aortic fatty streaks and fibrous plaques of 5- to $\text{7}\text{1}\text{2}\text{-}\text{year-old}$ pigs contained greater amounts of lecithin and particularly sphingomyelin than the organizing emboli. The predominant fatty acids of fibrous plaques were present in the order, 18:0 > 18:1 > 16:0 in lecithin, 16:0 > 18:0 > 20:0 in sphingomyelin, and 18:2 > 18:1 > 16:0 in esterified cholesterol.Intravenous injection of ³H-cholesterol in autologous plasma resulted in significant amounts of both labeled free and esterified cholesterol in the emboli at all periods of organization.This study indicates that the organization of pulmonary thromboemboli into fibro-fatty plaques is not associated with phospholipid and fatty acid profiles which evolve toward those of spontaneous atherosclerotic plaques.     

The invitro generation of an antibacterial activity from the fat body and hemolymph of non-immunized larvae of Galleriamellonella

A state of immunity in $\text{Galleria}$$\text{mellonella}$ against the pathogen $\text{Pseudomonas}$$\text{aeruginosa}$ is known to be induced by the injection of lipopolysaccharide (LPS), isolated from the homologous organism. An $\text{in}$$\text{vitro}$ mixture of the LPS and whole or cell-free hemolymph from non-immunized larvae is not antibacterial. $\text{In}$$\text{vitro}$ mixtures of fat body and cell-free hemolymph from non-immunized larvae, incubated at 25°C for 20 hours generated a proteinaceous antibacterial activity. The generation of this activity was enhanced by the presence in the incubation mixture of LPS and/or hemocytes from non-immunized larvae. It is suggested that LPS causes the release of a hemocyte factor(s) which acts in conjunction with or directly on the fat body resulting in an enhanced production of antibacterial factors.     

Lectins and antibodies as molecular probes of molluscan hemocyte surface membranes

A variety of carbohydrate-specific lectins and antigen-specific antibodies have been employed to assess the occurrence and topographic distribution of surface membrane determinants on hemocytes of two stocks of the snail, $\text{Biomphalaria}$$\text{glabrata}$, which differ in their susceptibilities to larval blood fluke ($\text{Schistosoma}$$\text{mansoni}$) infections. Using fluorescence labeling methods, several “classes” of hemocyte membrane components have been identified. These include (1) macromolecules, primarily glycoproteins, possessing reactive sites for concanavalin A and $\text{Ricinus}$$\text{communis}$ agglutinin, (2) snail hemolymph or hemolymph-like determinants, (3) fibronectin-like membrane components, and (4) surface determinants with structural similarities to murine Thy-1 antigen. No qualitative differences in surface molecular composition were observed between hemocytes of the two $\text{B}$. $\text{glabrata}$ stocks, although the broad reaction specificity of most of the probe reagents employed preclude the detection of small or minor differences in hemocyte membrane structure. Results of these investigations indicate that snail hemocytes possess a complex array of surface membrane components some of which appear to be shared with higher vertebrate species.