The specificity of the combining site of the lectin from Vicia villosa seeds which reacts with cytotoxic T-lymphoblasts

The combining site of purified Vicia villosa lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin, which specifically hemagglutinated blood group A erythrocytes nevertheless precipitated to different extents with blood group A₁, A₂, H, B and precursor I substances from saliva and ovarian cysts, and with different blood group substances of the same specificity indicating an interaction of the lectin with a non-blood group determinant site. The agglutinating and precipitating activities of the lectin were not strongly affected by EDTA or bivalent cations. Precipitates of Vicia villosa lectin with certain blood group glycoproteins showed unusually high solubilities as compared to other lectin glycoprotein and antigen-antibody precipitates. Inhibition assays with various monosaccharides, glycosides and oligosaccharides indicate that the Vicia villosa lectin is specific for terminal, non-reducing, α-linked dGalNAc. Of the monosaceharides tested, methyl αdGalNAc, p-NO₂-phenyl αdGalNAc and dGalNAc were best. They were about 100 times better inhibitors than the corresponding dGal compounds. The most potent inhibitor was methyl αdGalNAc which was 10 times more active than dGalNAc and 18 times more potent than the β-anomer. Among disaccharides tested, dGalNAcαl → 3dGal was most active, about as potent as methyl αdGalNAc, twice as active as dGalNAcαl → 6dGal and 8 times more potent than $\text{d}\text{GalNAc}\text{αl → 6}\text{d}\text{GalNAc}$, indicating the importance of a subterminal $\text{α}\text{l}\text{→ 3-}\text{linked}\text{d}\text{Gal}$ in the binding. $\text{d}\text{GalNAc}\text{α}\text{l}\text{→ 3}\text{d}\text{Gal}\text{β}\text{l}\text{→ 3}\text{d}\text{GlcNAc}$ was 7 times less active and the blood group A determinant

was less than $\text{1}\text{40}$ as active as $\text{d}\text{GalNAc}\text{α}\text{l}\text{→ 3}\text{d}\text{Gal}$. These findings indicate that the combining site of the Vicia villosa lectin is at least as large as the disaccharide $\text{d}\text{GalNAc}\text{α}\text{l}\text{→ 3}\text{d}\text{Gal}$ and that the αl → 3 linkage is important in the binding. The unique nature of this site is consistent with its high specificity for a glycoprotein on cytotoxic T-lymphocytes.     

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.     

The modulation of peritoneal macrophage chemiluminescence by acute pleural inflammation,prostanoids and cyclo/lipoxygenase inhibitors

The chemiluminescent (CL) response of peritoneal macrophages was suppressed by induction 4 h earlier of an inflammatory reaction in the pleural cavity which was negated by prior administration of indomethacin, ketoprofen and BW 755C. These changes were accompanied by a concomitant rise in peritoneal PGI₂ levels which was abolished by drug pretreatment.In vitro treatment of normal peritoneal macrophages with PGI₂ inhibited their subsequent CL response. Indomethacin and ketoprofen produced elevated CL of macrophages obtained from untreated controlsin vitro which was blocked by the lipoxygenase inhibitor NDGA. BW 755C and NDGAin vitro strongly inhibited macrophage CL and partially inhibited CL in a cell-free system. Use of these drugsin vivo demonstrated that indomethacin and ketoprofen augmented the CL response of peritoneal macrophages while BW 755C had no effect. These results suggest the inflammatory processper se can modulate the functions of macrophages in parts of the body remote from the inflammatory site. Moreover this modulation may be under the control of the prostanoid system.     

Human peritoneal macrophages: Clinical models of inflammation and potential targets of antiinflammatory drugs

Human peritoneal macrophages have been obtained from patients with renal disease undergoing chronic peritoneal dialysis, patients with ascites and at laparoscopy. These macrophages in general have both morphological and enzymatic characteristics of activated macrophages, as judged by criteria derived from animal experiments. Human peritoneal macrophages produce a variety of eicosanoids, including leukotriene B₄ and leukotriene C₄. These cells are suitable for studies onin vitro andin vivo effects of drugs, and for investigation of changes in macrophage activity occurring in human diseases.     

Maturation of the lymphoid system. III. Induction of selective unresponsiveness by injection of a haptenated carbohydrate into neonatal mice

Neonatal treatment of A/J mice with DNP-Ficoll reduced or eliminated indirect anti-DNP PFC normally produced in response to adult challenge with DNP-keyhole limpet hemocyanin. The remaining direct anti-DNP PFC response was of low avidity. Spleen cells from neonatal A/J mice inhibited the $\text{in}$$\text{vitro}$ but not the $\text{in}$$\text{vivo}$ reponse of adult spleen cells to DNP-Ficoll.     

Motile behaviour of the plasmatocytes of Ephestiakühniellainvitro and invivo

The plasmatocytes of $\text{Ephestia}$$\text{k}\text{u}\text{?}\text{hniella}$ rapidly spread on a glass substratum $\text{in}$$\text{vitro}$. The characteristics of subsequent translocation are described. Migrating plasmatocytes, governed by contact inhibition of locomotion move from regions of high cell density to those of low. The same migratory behaviour seems to occur on the surface of glass beads implanted $\text{in}$$\text{vivo}$.     

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.     

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.     

Soluble peptidoglycan fragments stimulate antibacterial protein synthesis by fat body from larvae of Manduca sexta

Both hemocytes and fat body from larvae of $\text{Manduca}$$\text{sexta}$, which have been injected with inducers of antibacterial protein synthesis, contain immunoreactive lysozyme. However, fat body is a richer source and has been demonstrated to synthesize and release lysozyme and cecropin-like peptides (bactericidins) $\text{in}$$\text{vitro}$. Fat body secretion of lysozyme and bactericidins is stimulated by addition of soluble peptidoglycan fragments to culture medium. The rate of lysozyme secretion by fat body varies as a function of peptidoglycan inducer concentration. These data are consistent with the hypothesis that, $\text{in}$$\text{vivo}$, bacteria must be phagocytized and partially degraded (processed) by hemocytes to generate a signal (peptidoglycan) that subsequently induces antibacterial protein synthesis by fat body.     

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.     

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.     

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.     

Gustatory reaction time under variable stimulus parameters in human adults

Reaction times and perceived intensities for NaCl solutions were measured in sixteen human adults. Stimulus delivery was by means of a circular piece of solution-soaked filter paper held with forceps. The reaction time (T) decreased and the perceived intensity (S) increased with increasing the concentration (C) of NaCl solution applied to a fixed (78.5 mm²) tongue area. The comprehensive relations among T, S and C can be expressed by the following formulae; $\text{T = a +}\text{b}\text{log(}\text{C}\text{C}{}_0\text{)}$, $\text{T = p +}\text{q}\text{log(}\text{S}\text{S}{}_0\text{)}$, and logS = mlogC + logn, where a, b, p, q, m, n = constants, and S₀ indicates the perceived intensity at the threshold concentration (C₀). Meanwhile, the reaction time decreased and the perceived intensity increased with increasing the stimulated area (A) under a fixed (1.0 M) concentration of NaCl solution. The relations among T, S and A can also be expressed by the following equations; $\text{T = a′ +}\text{b′}\text{log(}\text{A}\text{A}{}_0\text{)}$, $\text{T = p′ +}\text{q′}\text{log(}\text{S}\text{S}{}_0\text{)}$, and logS = m′logA + logn′, where a′, b′, p′, q′, m′, n′ = constants, and A₀ indicates the threshold size of stimulated area.     

Histopathology and cell-mediated immune reactivity in halothane-associated lymphomagenesis and autoimmunity in BXSBMp and MRLMp mice

The present study examined whether halothane anesthesia could alter immunoregulation in autoimmune disease-prone $\text{BXSB}\text{Mp}$ and $\text{MRL}\text{Mp}$ mice. This was judged by histopathological examination of lymph nodes and by studies on mitogen-induced transformation of splenic lymphocytes. The development of lymphoma-like disease in BXSB males and the appearance of lymphoproliferation and systemic lupus erythematosus-like disease in $\text{MRL}\text{Mp}\text{lpr}\text{lpr}$ mice were revealed by histologic examination of lymph nodes and other tissues and by identification of immune complexes in kidney sections by immunofluorescence. Splenic lymphocytes from $\text{MRL}\text{Mp}$ +/+ mice exposed to halothane showed a 44% reduction in mean stimulation indices (MSI) compared to exposure to O₂. Likewise, the MSI of splenic lymphocytes from $\text{MRL}\text{Mp}\text{lpr}\text{lpr}$ mice exposed to halothane were reduced to 42% when compared with those from $\text{MRL}\text{Mp}\text{lpr}\text{lpr}$ mice subjected to O₂. In accord with the MRL results, halothane induced a 38% reduction in the stimulation indices of autoimmune and lymphoma-like disease-prone BXSB mice and a 27% decline in the lymphocyte proliferative responsiveness of autoimmune disease-insusceptible control $\text{C57B1}\text{6}$ mouse spleen cells, compared to their respective O₂ control values. Results demonstrate that halothane facilitated an earlier onset in loss of immunoregulation associated with development of autoimmunity and lymphomagenesis in genetically disease-prone mice receiving halothane than in those receiving oxygen.     

Increased release of hydrogen peroxide and superoxide anion from asbestos-primed macrophages

The ability of asbestos-elicited murine peritoneal macrophages to release superoxide anion and hydrogen peroxide, followingin vitro triggering, has been investigated. The asbestos-elicited macrophages produced increased levels of super oxide and hydrogen peroxide compared to control macrophages and similar levels to those produced byCorynebacterium parvum elicited macrophages. The supernatants from asbestos-elicited macrophages which had been triggered invitro were capable of impairing the ability of ₁protease inhibitor to inhibit elastase function. The catalase sensitivity of this effect showed it to be due to hydrogen peroxide.     

Peritoneal lavage fluid alters patterns of eicosanoid production in murine bone marrow-derived and peritoneal macrophages: Dependency on inflammatory state of the peritoneum

Murine resident macrophages produce an abundance of eicosanoids, whereas elicited macrophages produce lesser quantities of eicosanoids in general, and leukotriene C₄ (LTC₄) and prostacyclin (PGI₂) in particular. Macrophage precursors derived from bone marrow cells produce even smaller amounts. We postulated that these differences may be regulated by substances found in the microenvironment of the cell, which may alter arachidonate release from phospholipid and its subsequent metabolism to eicosanoids. To examine if inherent differences in phospholipid availability contributed to the observed differences in eicosanoid synthesis among these three groups of macrophages, we monitored uptake and release of arachidonic acid (AA) in resident and elicited peritoneal macrophages and in bone marrow-derived macrophages (BMDM). Although differences existed in the extent of arachidonate release (37% vs. 22% vs. 27% release), the differences were not enough to explain the much larger differences in eicosanoid production. We therefore determined whether the AA cascade enzymes, including phospholipase A₂ (PLA₂) were intact by adding exogenous AA to the three cell types. PGI₂ synthesis was not significantly increased in either elicited or BMDM. However, the enzymes necessary for LTC₄ production appeared intact in elicited cells but not in BMDM. To further characterize the differences in eicosanoid synthesis between resident and elicited peritoneal macrophages and BMDM, we determined if a variety of exogenous substances [growth factors, cytokines, and noninflammatory and inflammatory peritoneal lavage fluid (NPLF and IPLF)] could enhance the production of LTC₄ and PGI₂ in those macrophage groups. The addition of granulocyte-macrophage colony stimulating factor (GM-CSF) slightly increased LTC₄ production by BMDM and elicited macrophages. In contrast, NPLF increased the production of both LTC₄ and PGI₂ from BMDM, while IPLF had no effect. A similar effect of NPLF was seen on LTC₄ (but not PGI₂) production from elicited peritoneal cells, while IPLF decreased both LTC₄ and PGI₂ production from resident peritoneal macrophages. These studies indicate that substances found in the peritoneum of mice can enhance or diminish the production of LTC₄ and PGI₂ from the macrophage. This regulation appears to depend on the inflammatory state of the peritoneum.Supported by HL02254, HL44122, and CA50107.     

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.     

Serological characterization of humoral lectins from the freshwater prawn Macrobrachiumrosenbergii

We have detected and partially characterized multiple lectins present in the serum of the freshwater prawn $\text{Macrobrachium}$$\text{rosenbergii}$. Since agglutination of erythrocytes (RBC) is not abolished by treatment with $\text{Vibrio}$$\text{cholerae}$ neuraminidase (VCN), $\text{Macrobrachium}$ shows an agglutination pattern different from that of other sialic acid-specific lectins such as $\text{Limulus}$$\text{polyphemus}$ lectin. However, after absorption with primate and bird VCN-treated RBC, $\text{Macrobrachium}$ serum exhibits high titers with untreated and pronase-treated RBC and no agglutination of VCN-treated RBC, suggesting a typical sialic acid specific lectin agglutination profile. Hemagglutination-inhibition tests indicate that sialic acid containing compounds are the best inhibitors for $\text{Macrobrachium}$ lectins. Subterminal sugars and type of linkage are probably important for the lectin binding since bovine submaxillary mucin (containing mainly terminal NANA-α-2 6-GalNAc-) is a better inhibitor than fetuin (containing mainly terminal NANA-α-2→3-Gal-) and colominic acid (-NANA-α-2→8-NANA-) is a weak inhibitor.     

The chemokine receptor CCR6 is an important component of the innate immune response

In our initial studies we found that naïve CCR6‐deficient (CCR6^–/–) C57BL/6 mice possessed significantly lower number of both F4/80⁺ macrophages and dendritic cells (DC), but higher number of B cells in the peritoneal cavity, as compared to naïve wild type (WT) controls. Furthermore, peritoneal macrophages isolated from CCR6^–/– mice expressed significantly lower levels of inflammatory cytokines and nitric oxide following lipopolysaccharide (LPS)stimulation, as compared to WT macrophages. In a severe experimental peritonitis model induced by cecal ligation and puncture (CLP), CCR6^–/– mice were protected when compared with WT controls. At 24 h following the induction of peritonitis, CCR6^–/– mice exhibited significantly lower levels of inflammatory cytokines/chemokines in both the peritoneal cavity and blood. Interestingly, DC recruitment into the peritoneal cavity was impaired in CCR6^–/– mice during the evolution of CLP‐induced peritonitis. Peritoneal macrophages isolated from surviving CCR6^–/– mice 3 days after CLP‐induced peritonitis exhibited an enhanced LPS response compared with similarly treated WT peritoneal macrophages. These data illustrate that CCR6 deficiency alters the innate response via attenuating the hyperactive local and systemic inflammatory response during CLP‐induced peritonitis.