Pig Spleen Research Articles

Flow Cytometric Analysis of Lymphocyte Populations in Guinea Pig Blood and Spleen

A panel of novel mouse monoclonal antibodies was used, in an indirect immunofluorescence procedure, to identify subsets of guinea pig lymphoid cells which were quantified by flow cytometric analysis. The staining characteristics of the antibodies were also examined by alkaline phosphatase-anti-alkaline phosphatase staining of cryostat sections of spleen and cytocentrifuge preparations of blood and spleen mononuclear cells. The values obtained for T and B cell populations were similar to those previously described using rosetting procedures, and the study also confirmed the high incidence of constitutive Ia antigen expression on guinea pig lymphocytes. Compared with animals receiving immunization (ovalbumin) alone, guinea pigs treated with ciclosporin A showed only minor changes in lymphocyte subsets, whereas those pretreated with cyclophosphamide revealed striking reductions in circulating T and B lymphocytes and in splenic B cells. Both ciclosporin A and cyclophosphamide markedly reduced Ia antigen expression on lymphocytes in blood, whilst cyclophosphamide also inhibited Ia antigen expression in the spleen.

Identification and partial purification of a protein binding to the human immunoglobulin kappa enhancer kappa E2 site.

We previously described a domain in the 5' half of the human immunoglobulin kappa enhancer which could bind nuclear proteins in vitro, as detected by a lambda exonuclease protection assay. A second more 3' binding domain in the enhancer has now been detected by a similar assay employing a different exonuclease, the T7 gene 6 exonuclease. Using this assay and starting with a pig spleen nuclear extract, we have purified 5000-fold a protein that binds to the 3' domain. In a DNase I footprint experiment the partially purified protein protects a 27 bp segment in the enhancer centered around the sequence CAGGTGGC, which corresponds to the kappa E2 sequence motif described in the mouse kappa enhancer. The protein, designated NF-kappa E2, also appears to bind at a position downstream of kappa E2, at or near the kappa E3 site. Proteins capable of binding at kappa E2 are found in several mammalian species and are expressed in both lymphoid and non-lymphoid tissues.

The development of a sensitive and specific radioreceptor assay for leukotriene B 4

To establish a simple and sensitive quantitation of leukotriene B 4 (LTB 4), we developed a radioreceptor assay (RRA) using a highly specific [ 3H] leukotriene B 4([ 3H]LTB 4) binding to a guinea pig spleen homogenate. The assay detected LTB 4 levels as low as 0.12 pmol per tube. Fifty percent inhibition of bound [ 3H]LTB 4 was obtained by 2.5 nM of unlabeled LTB 4. [ 3H]LTB 4 competition studies indicated that 20-hydroxy-LTB 4 was 8 times, 6-trans-LTB 4 was 640 times and 20-carboxy-LTB 4 was 1000 times less effective than LTB. The peptide leukotrienes C 4, D 4 and E 4 showed no effect on [ 3H]LTB 4 binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB 4. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB 4 released from human granulocytes stimulated with calcium ionophore A23187. The LTB 4 level was maximal at 10 min (156.8 ± 36.2 pmol/3×10 6 cells) and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B 4 is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive than other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B 4.

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, p I, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33–34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14–14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with p I4.38–4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.

Virulence for guinea pigs of tubercle bacilli isolated from the sputum of participants in the BCG trial, Chingleput district, South India

This study, conducted in Madras, India and in Madison, Wisconsin, USA, was concerned with the virulence of isolates of Mycobacterium tuberculosis obtained from the sputum of individuals living in the Chingleput district of south India. The following results were obtained. 1.1. The findings of Mitchison with respect to the predominance of low virulence for guinea pigs among isolates from persons living Madras, were confirmed on isolates from the sputum of residents of the Chingleput district.2.2. A high correlation was found between the log10 number of tubercle bacilli recovered from the spleen of guinea pigs infected intramuscularly with 1.0 mg of tubercle bacilli and the root index of virulence.3.3. A high correlation was found between the log10 number of tubercle bacilli recovered from the spleen of guinea pigs infected intramuscularly with 1.0 mg of tubercle bacilli and the number recovered from the spleen of guinea pigs infected by the respiratory route with 5–10 tubercle bacilli.4.4. Relatively low correlations were found between RIV and the susceptibility of isolates to thiophene-2 carboxylic acid hydrazide or to hydrogen peroxide.

Thymic hypoplasia, splenomegaly and immune depression in guinea pigs with neonatal cytomegalovirus infection

The effects of cytomegalovirus (CMV) infection on the spleen and thymus of neonatal guinea pigs were assessed. Guinea pigs with neonatally acquired CMV infection developed growth retardation, thymic hypoplasia and splenomegaly. Significant depletion of the T lymphocyte population occurred in the thymuses of these animals whereas inflammatory and immune proliferative responses were clearly evident in their spleens. Higher titers of infectious virus were recovered from the spleen than from the thymus. In addition, spleen cells from neonatally infected animals had significantly reduced proliferative responses to both the T-cell mitogen, concanavalin A, and the B-cell mitogen, lipopolysaccharide. Responses to concanavalin A were most severely impaired. These results point to the significant immunodepressive effect of acute CMV infection and to the dissimilar alterations induced by CMV in the spleen and thymus of acutely infected neonates.

Phosphorylation of human fibrinogen in vitro with protein kinase C: Characterization of the phosphorylated sites

Phosphorylation of human fibrinogen in vitro by incubation with [γ- 32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [ 32P]phosphate at serine residues located in the Aα-chain. In order to identify the residues that were phosphorylated, the Aα-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser 557, Ser 558, Ser 559, and Ser 599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the Aα-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [ 32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln 328 and Gln 366, in this portion of the Aα-chain take part in the cross-linking of fibrin.

E-Rosetting of Guinea Pig Lymphocytes with Lyophilized Rabbit Red Cells after Treatment with Papain and Glutaraldehyde

Guinea pig lymphoid cells were reacted to form spontaneous rosettes (E-rosettes) with rabbit red blood cells before and after treatment with papain or a very low concentration (0.01%) of glutaraldehyde alone or both. The papain-glutaraldehyde-treated cells showed as high ability as papain-treated cells to rosette with lymphoid cells prepared from thymus, spleen, lymph node and peripheral blood of guinea pigs. Moreover, papain-glutaraldehyde-treated red cells were lyophilized and stored for 1 year without any change in their ability to form E-rosettes with these lymphoid cells. Binding between lymphocytes and treated or nontreated red cells was completely inhibited by specific anti-guinea pig thymocyte serum. Furthermore, a lymphocyte population enriched for B cells from lymph nodes or leukemic cells of strain 2 guinea pigs (B cells) did not show any substantial E-rosettes with nontreated or treated red cells. Scanning electron microscopy revealed no surface alteration in the treated cells when compared to those in nontreated red cells.

繁殖豚の腸炭疽2例の病理学的観察

Intestinal anthrax was diagnosed in 2 pigs slaughtered. Local hemorrhage with pseudomembrane formation was found in the jejunum of the 2 pigs. Mesenteric lymph nodes enlarged. Carbuncles were observed on the surface of the spleen and reddish spots presented in the liver in pig No.1.Histologically, hemorrhagic-diphtheritic lesions were seen in the intestine of the two pigs. The intestinal wall was very thick in pig No.1. Hemorrhagic, fibrinous, necrotic, and inflammatory changes and the presence of bacilli were observed in the mesenteric lymph nodes and carbuncles of the spleen of pig No.1. In the liver focal necrosis and hemorrhage were noticed.Electron microscopically, two types of bacilli which had a capsule or spore were seen in the intestine of pig No.1. One was considered to be Bacillus anthracis, and the other Clostridium sp. Neither type was phagocytized by neutrophils or macrophages.

Histamine-releasing activity of lymphocyte supernatants of guinea pig spleen cell cultures

We demonstrated the production of a histamine releasing factor (HRF) by 24-h cultures of guinea pig spleen cells which were stimulated or not with specific antigen (ovalbumin, OA) or mitogen (phytohemagglutinins or concanavalin A). HRF induced the release of histamine from homologous mesenteric mast cells in a dose-dependent fashion. The HRF-induced histamine release was not high compared to the release induced by calcium ionophore A23187, but higher than that induced by compound sol48 80 , polymyxin B and con canavalin A. The mast cells from sensitized guinea pigs released histamine when challenged with OA. We found that HRF-induced histamine release was additive to that induced by antigen, when both agents were added simultaneously to sensitized mast cells. The phenomenon was most significant when a suboptimal dose of antigen was used. Moreover, we did not observe any differences in the magnitude of HRF-induced histamine release between the mast cells from nonsensitized and sensitized guinea pigs. The time course of histamine release induced by HRF was significantly slower than that with specific antigen (10 min and 45 sec, respectively). Our results may suggest that HRF acts on mast cells through a different not immunological mechanism.

Cytolysis of Junin infected target cells by immune guinea pig spleen cells

Spleen cells from guinea pigs infected with an attenuated strain of Junin virus (the causative agent of Argentine hemorrhagic fever) specifically lysed virus-infected syngeneic target cells in vitro. This activity was detected as early as 6 days after infection, reached a maximum on days 10–13, and persisted at lower levels, at least through day 30. Monoclonal antibody to guinea pig T cells had no effect on the activity. After B or T cell enrichment techniques, the cytolysis was found with the B cell fraction. Aggregated IgG blocked the cytolysis. These characteristics suggested lytic activity was mediated at least in part by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. Although some cytolysis could be detected by using exogenously added antiserum and normal spleen effector cells, such reconstruction showed less efficient killing than when spleen cells from Junin-infected guinea pigs were used. This apparent discrepancy was resolved when spleen cells from these infected animals exhibited enhanced activity in non-viral ADCC systems as well. The cytotoxic activity by spleen cells against Junin-infected targets was detected only with non-fatal Junin infections. Cytolysis could not be measured in spleen cell suspensions from guinea pigs lethally infected with Junin virus; i.e. adults infected with a virulent strain of Junin and baby guinea pigs or immunosuppressed adult animals infected with an attenuated strain. However, spleen cells from both the immunosuppressed, infected adults and the adult guinea pigs infected with a virulent strain of Junin were able to mediate cytotoxicity in a nonviral system (antibody-sensitized Vero cells). The development of spleen cell cytotoxicity by Junin-infected guinea pigs against Junin-infected target cells correlated with whether the infection was resolved or was lethal.

Monoclonal antibodies of African swine fever virus: antigenic differences among field virus isolates and viruses passaged in cell culture

An analysis of the binding properties of a collection of monoclonal antibodies to African swine fever virus particles showed that virus field isolates passaged in porcine macrophages changed antigenically more than a strain of a cell-adapted virus passaged in Vero cells. From seven clones isolated from the spleen of a field-infected pig, we found four clones that had the same antigenic properties, one clone that had large changes in proteins p150 and p27 and small changes in proteins p37 and p14, and two clones that had minor changes in proteins p150 and p27, respectively. An analysis of the binding properties of the monoclonal antibodies to 23 field isolates from Africa, Europe, and America showed that the African isolates differed among themselves more than the European and the American isolates; in this study we found changes in 8 of the 10 virus proteins tested. The most variable proteins in the African isolates were p150, p27, p14, and p12. In contrast to the African isolates, protein p12 from the non-African viruses did not change. The clustering of the field virus isolates in six antigenic homology groups indicated the existence of a complex variety of African swine fever virus serotypes.

Lactosamine type asialooligosaccharide recognition in NK cytotoxicity

Inhibition of pig NK cell activity by asialooligosaccharides (aOS) isolated from human serum glycoproteins was investigated. Triantennary aOS (aOSIII) of ceruloplasmin was found to be the most potent inhibitor up to the concentration 0.1 μg/ml, which is in agreement with its highly specific binding to NK-activity-enriched pig lymphocytes (with a morphology similar to human large granular lymphocytes (LGL)). Only lectins with the specificity to Gal(β1→4)GlcNAc or Gal(β1→3)GalNAc structures exhibited inhibition of NK cytotoxicity. F(ab) 2 fragments of rabbit antibodies against pig spleen membrane lectin cross-reacting with the pig liver membrane lectin completely inhibited NK activity when preincubated with the effectors or present in the incubation mixture during the assay. These data suggest that lectin receptors on cells of pig NK-activity-enriched fraction specific for aOSIII and antigenically related to membrane lectins isolated from pig spleen and liver, are involved in the NK recognition of several xenogeneic targets.

Relationship between blood iron parameters and trichinellosis in swine

Reduced levels of total iron binding capacity and unsaturated iron binding capacity were observed in the blood of trichinous and iron-injected trichinous pigs. No change was observed in their serum iron and saturation concentration levels. Also, reduced iron concentration levels were observed in the livers of trichinous pigs, while increased iron concentration levels were observed in the spleens of trichinous pigs and the livers and spleens of iron-injected pigs. No difference was found with regard to weight gains, number of larvae per gram of tissues, or histologic characteristics of ‘nurse cells’.

Chapter 16 Multiple co-existence of peptides and classical transmitters in peripheral autonomic and sensory neurons—functional and pharmacological implications

Publisher Summary This chapter focuses on some recent advances in studies on the co-existence of peptides with acetylcholine (Ach) and noradrenaline (NA) in certain autonomic nerves, as well as on the occurrence of multiple peptides in sensory nerves. Noradrenaline has for long been known to be the primary transmitter of peripheral sympathetic neurons with minor exceptions—for example, the sympathetic innervation of sweat glands, which is cholinergic. Immunohistochemical evidence has suggested that a member of the pancreatic polypeptide family—avian pancreatic polypeptide (APP)—has a widespread neuronal localization in mammals. Only a proportion of sympathetic NA nerves seems to contain the NPY-like peptide and these neurons innervate target organs such as blood vessels, the muscle of the heart, spleen, and vas deferens. Other populations of sympathetic neurons projecting to, for instance, exocrine elements in certain salivary glands or brown fat cells do not contain NPY, suggesting a chemical heterogeneity and a morphological basis for a functional differentiation of the sympathetic nerves. Sympathetic nerve stimulation is accompanied not only by release of NA but also of NPY-LI. Thus, by using the blood-perfused cat or pig spleen as an experimental model, it has been demonstrated by earlier studies that nerve stimulation causes a co-release of NA and NPY-LI. Intermittent stimulation with bursts of impulses at a high frequency enhances selectively the release of NPY-LI compared to NA.

Frequency- and reserpine-dependent chemical coding of sympathetic transmission: Differential release of noradrenaline and neuropeptide Y from pig spleen

The importance of impulse pattern and stimulation frequency for the release of noradrenaline (NA) and the coexisting peptide neuropeptide Y (NPY) in relation to vasoconstriction (perfusion-pressure increase) was studied in the blood-perfused pig spleen in vivo. Splenic nerve stimulation with intermittent bursts at high frequency (20 Hz) caused a several-fold larger release of NPY-like immunoreactivity (-LI) in relation to NA than a continuous stimulation at a low frequency (2 Hz), giving the same total number of impulses. α-Adrenoceptor blockade by phentolamine enhanced markedly both NA and NPY release, especially at low stimulation frequency, suggesting prejunctional adrenergic inhibition of release. Addition of propranolol unmasked a large remaining perfusion-pressure response to nerve stimulation. Reserpine treatment reduced the NA content of the spleen as well as the stimulation-evoked NA release by >90%. However, the perfusion-pressure increase in response to nerve stimulation was well maintained. A marked increase in the stimulation-evoked release of NPY-LI occurred after reserpine. Adrenoceptor blockade after reserpine treatment reduced only slightly the perfusion-pressure response in parallel with a decline in NPY output. NPY caused an adrenoceptor-resistant perfusion-pressure increase at plasma concentrations that were in the same range as the maximal increase during nerve stimulations. In conclusion, the present data suggest a frequency-dependent, chemical coding of sympathetic transmission with preferential release of the classical transmitter NA at low, continuous frequencies and release of NPY, mainly at high frequencies. Reserpine treatment enhances markedly NPY release, which may explain why the functional response is largely intact in spite of adrenoceptor blockade and marked NA depletion.

Chapter 17 On the possible roles of noradrenaline, adenosine 5′-triphosphate and neuropeptide Y as sympathetic cotransmitters in the mouse vas deferens

Publisher Summary This chapter focuses on one tissue: the mouse vas deferens. It discusses results that indicate that sympathetic neuromuscular transmission in this tissue is not mediated by noradrenaline (NA) alone but also by adenosine triphosphate (ATP) and neuropeptide Y (NPY), and it presents a working hypothesis concerning the possible pre- and postjunctional roles of these three putative co-mediators of the electrical and mechanical responses to sympathetic nerve stimulation. Several studies in the past have indicated that the vasa deferentia in various rodent species have a uniquely high content of noradrenaline, contained in the postganglionic sympathetic fibers forming a dense network around the smooth muscle cells. NPY seems to be stored in sympathetic nerves in large dense cored vesicles, together with NA and ATP. It has been found that in cat spleen, on electrical nerve stimulation, NPY may be secreted along with NA. The secretion of both substances is blocked by guanethidine, showing that both are released from sympathetic nerves. In pig spleen, it has been found that the relative proportions of secreted NA:NPY vary with the stimulation parameters. In earlier studies, bursts at high frequency increased the secretion of NPY relatively more than that of NA. Exogenous NPY has a number of marked direct and indirect pre- and postjunctional effects, which vary in intensity with the individual sympathetically innervated tissue. Thus, in some tissues and species, NPY is a potent vasoconstrictor per se, but it induces only a weak contraction of the (mouse) vas deferens. As per the observations based on studies in the past, in several tissues, NPY strongly enhances the smooth muscle contractile response to exogenous NA, as well as to ATP.

高速液体クロマトグラフィーによるマウス, ラット, モルモット, ブタ及びウシ脾臓中の<I>O</I>-Phosphoethanolamineの定量

O-Phosphoethanolamine (PEA) was determined by cation exchange high-performance liquid chromatography in trichloroacetic acid extracts of spleens. An in-stream fluorometric detection system by using o-phthalaldehyde permitted the rapid analysis of samples containing 11.3-680.4 nmol/ml of PEA.The PEA concentration in mouse, rat, guinea pig, porcine and cattle spleens was determined. Spleens studied were found to vary widely in their PEA concentration showing species differences.

Distribution of factor VIII mRNA and antigen in human liver and other tissues.

The cellular site of synthesis of factor VIII (FVIII:C; anti-haemophilic factor) has long been sought. Previous studies suggested the liver as a major site of synthesis, but extrahepatic sources such as spleen and lung have been implicated. Using an immunoradiometric assay (IRMA), we recently localized factor VIII antigen (FVIII:Ag, formerly FVIII:CAg), to whole perfused guinea pig liver and spleen, and to isolated hepatocytes, with lesser or trace amounts in other tissues. Using an immunohistological technique, Stel et al. detected FVIII:Ag in normal human liver sinusoidal endothelial cells, while Exner et al. detected FVIII:Ag by IRMA in extracts of human lymph nodes, lung, liver and spleen. The localization of antigen in tissues does not, however, distinguish sites of factor VIII synthesis from those of storage, and such experiments are subject to misinterpretation due to entrapment of plasma factor VIII in tissues. The recent cloning of the human factor VIII gene provides hybridization probes for the detection of factor VIII messenger RNA in cells, thus directly determining sites of synthesis. During complementary DNA cloning, we detected factor VIII mRNA in liver, and it has been localized by others in liver and placenta and in liver and kidney. In the present study, we detected factor VIII mRNA in isolated human hepatocytes, in spleen and in numerous tissues including lymph nodes and kidney, but not in white blood cells or cultured endothelial cells. We also found that the factor VIII, factor VII, factor IX and protein C antigens in liver are predominantly localized in hepatocytes, while very little von Willebrand factor antigen (vWF:Ag, formerly FVIIIR Ag) is detectable in this organ.

Deoxythymidine pools of human skin and guinea pig organs

The activity of antiviral nucleoside analogues like acyclovir is influenced by a number of cellular factors, one being the deoxythymidine (dThd) concentration. We therefore analysed the dThd concentration in human plasma and skin and in organs of guinea pig, the common experimental animal. High-performance liquid chromatography showed low amounts of dThd in human skin, 0.20–1.15 nmol g , whereas guinea pig skin and spleen had 20–30 nmol g and the concentration in guinea pig plasma was 10-times higher than in human plasma. These animals are therefore in this respect less suitable as accurate models for antiviral nucleoside activity in humans.