Changes in the immunological indices after splenectomy and the reimplantation of splenic fragments in an experiment

Splenectomized guinea-pigs underwent autotransplantation of splenic fragments into the greater omentum or mesentery of small intestine. Twenty-five to thirty days after operation the animals were infected with S. aureus and then were examined over time before and after infection at different times. Measurements were taken of T lymphocytes, B lymphocytes, lymphocytes with staphylococcal receptors, as well as of the content of neutrophils with IgG Fc-fragment and complement receptors. It was established that in guinea-pigs subjected to splenectomy followed by autotransplantation of decapsulated splenic fragments, the experimental generalized staphylococcal infection took a milder course, which was manifested by an increase in the above immunologic characteristics as compared to animals undergoing splenectomy alone.     

Antigen-stimulated rosette formation by T lymphocytes in experimental allergic encephalomyelitis

Peripheral blood lymphocytes form rosettes in the presence of heterologous etythrocytes. Spontaneous or active rosette formation has been reported to be a measure of circulating and immunologically functional thymus-dependent lymphocytes. The present study utilizes the rosette assay to measure changes in the circulating T cells of guinea pigs sensitized with encephalitogenic myelin basic protein (BP) or with nonencephalitogenic peptide S42 known to induce cellular transformation in experimental allergic encephalomyelitis, a cell-mediated disorder of the central nervous system. The results show a significant depression in the number of active but not in the total number of rosette-forming T lymphocytes from the peripheral blood of antigen-sensitized animals. This reduction, which was not related to the encephalitogenic property of the BP, was readiiy reversible by incubating lymphocytes with the sensitizing antigen but not with histone. Under these conditions, lymphocytes from unsensitized control animals were unresponsive to stimulation by any of the antigens used. The antigenstimulated rosette assay described in this report provides a specific assay for sensitization to basic protein in BP-related demyelinating diseases.     

Inhibition of secondary cartilage of the intermaxillary suture in Sprague-Dawley rats following the enucleation of maxillary molars

A single craniofacial suture can undergo several morphologic transformations during its development. From 3 to 7 weeks of age, the intermaxillary suture of the rat is synchondrotic in character, featuring secondary cartilage; at later times, this suture is syndesmotic in character, featuring a fibrous tissue interface. Since intermittent mechanical stimulation has been reported to initiate secondary cartilage formation, a study was done to determine if the functioning dentition were responsible for secondary cartilage formation in the intermaxillary suture of the rat. Twenty-two female Sprague-Dawley rats were used. At 3 weeks of age, prior to eruption, the maxillary molars were enucleated from nine animals. Body weights were recorded weekly. Animals were sacrificed weekly from 4 to 7 weeks of age. One hour prior to sacrifice, each rat was injected with [35S]sulfate at a dosage of 2 microCi/g body weight. The tissues were evaluated by light microscopy and autoradiography. In the experimental group, the midpalatal suture did not undergo the normal synchondrotic transformation. Instead, this suture remained fibrous with negligible metachromatic staining. In the control animals, the peak period of [35S]sulfate incorporation was 4 weeks of age and was five times greater than in the experimental group. The primary stimulus for the initiation of secondary cartilage formation in the midpalatal suture of the rat was molar function. Also, functioning molars were found to be important in the maintenance of the palatal bone.     

A comprehensive analysis of heat shock protein synthesis in human peripheral lymphocytes: the effect of penicillin/streptomycin.

A reliable experimental procedure is described for the simultaneous characterisation of a comprehensive range of heat shock proteins (hsps) in human peripheral lymphocytes. In this system, a mild heat shock from 37 to 42 degrees C for 1 h induced the synthesis of hsps 105, 90, 70, 60, 57, 47, 40, 27 and 16. Densitometric analyses of 35[S]-methionine labelled protein gels indicated that levels of these hsps peaked at 3 to 4 h, following post-heat shock recovery at 37 degrees C. The presence of penicillin and streptomycin in the cell culture medium, appeared to have little effect on the kinetics of hsp synthesis. The present method can be used for relatively small blood samples and its relative ease of application and reproducibility make it appropriate for screening the expression of hsps in human lymphocytes from a range of individuals.     

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Abstract Two groups of 4 cows were vaccinated subcutaneously with live Streptococcus uberis strain 0140J or a surface extract derived from the same strain, at 14 days prior to the cessation of lactation (drying off) and at calving. Both groups also received an intramammary administration of the surface extract 7 days after drying off. A third group of unvaccinated animals acted as controls. Following intramammary challenge of two quarters per cow with the vaccine strain, all quarters on control cows and those vaccinated only with surface extract developed clinical mastitis. However, only 12.5% of challenged quarters on cows which were vaccinated with live bacteria developed clinical mastitis. In addition, the numbers of bacteria in the milk following challenge were 10⁵ times higher from the control and extract vaccinated cows than those which received live vaccine. Serum levels of S. uberis specific IgG₂ were elevated in the animals vaccinated with the live organism when compared to that of either extract-vaccinates or controls, whilst S. uberis specific levels of IgG₁ and IgM were similar in all groups throughout the experiment. Specific antibody levels in milk were unaffected by vaccination. Despite increased levels of IgG₂, no increase in opsonic activity was detected in any serum or milk samples. Peripheral blood lymphocytes from animals vaccinated with live organisms showed a considerable increase in proliferative response to S. uberis antigen in vitro when compared with lymphocytes from control and extract-vaccinated animals. These results suggest that neutrophils and specific opsonising antibody may not form the major defence against infection with S. uberis .     

Dietary gangliosides positively modulate the percentages of Th1 and Th2 lymphocyte subsets in small intestine of mice at weaning.

We studied the influence of dietary gangliosides on the number of spontaneous cytokine-secreting cells from two intestinal lymphocyte populations: lamina propria lymphocytes and Peyer's patches lymphocytes in Balb/c mice for 28 days after weaning. Weanling mice were separated into two groups, designated as Control and BG. The Control group was fed with a semipurified diet without gangliosides and the BG group was fed with the semipurified diet supplemented with 47 mg/kg of a mixture of bovine brain gangliosides. Intestinal lymphocytes were isolated from mice killed at 3, 7, 14 and 28 days after weaning, and the percentages of spontaneous Th1 as well as Th2 cytokine-secreting lymphocytes were determined using the ELISPOT assay. The BG group animals showed an earlier development in the number of cytokine-secreting cells, which appeared one week later in Control animals. In addition, mice fed with the ganglioside-supplemented diet showed a significantly higher number of Th1 and Th2 cytokine-secreting lymphocytes than Control mice in lamina propria and Peyer's patches lymphocytes at the end of the experimental period (28 days). Our results suggest that dietary gangliosides influence the maturation process of the intestinal immune system that take place during weaning.     

Occurrence of atypical lymphocytes following intravenous injection of Candida albicans in mice

The occurrence of atypical lymphocytes has been observed in the course of experimental acute infection withCandida albicans in mice. In animals injected intravenously with 2.5 × 10⁶ ofCandida albicans cells, an increased number of monocytes was seen in 24 hours. Monocytes showed toxic vacuolisation in most instances in protoplasm and sometimes in the nuclei. Only a few atypical lymphocytes could be seen at that time. In the following days the number of monocytes diminished and the number of atypical lymphocytes increased. After four days atypical lymphocytes constituted frequently over 20 % of white cells. The autopsy of sacrificed or dead animals with the presence of such elevated percentages of atypical lymphocytes showed enlargement of cervical lymphnodes in all animals. In mice infected with 1.4 × 10³ ofCandida albicans cells, no level higher than 12% of atypical lymphocytes was seen. Pictures were returning to normal with only a few atypical lymphocytes present among the animals which survived for two months after infection withCandida albicans.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper. Memorial Funds and the Roon Foundation.     

The effect of neonatal rat graft-vs-host disease (GVHD) on Fc receptor lymphocytes.

The level of Fc receptor rosette-forming lymphocytes (Fc-RFL) was examined in spleen and lymph node cell suspension from neonatal DA and BN rats inoculated within 24 hr of birth with either allogeneic L (experimental) or syngeneic (control) lymphoid cells. In addition, these levels were compared to fetal and neonatal animals that received no injection. The indicator cells (EA) were sheep erythrocytes sensitized with one-half concentration of the highest dilution of rabbit anti-sheep erythrocyte IgG(A) which agglutinated an equal amount of 1% suspension of E. Care was taken to exclude scoring macrophages by injecting colloidal carbon at least 1 hr before killing the test animals. The spleen of 19-day DA fetal rats exhibited a level of 19.3% Fc-RFL, similar to that of animals having received adult syngeneic cells at birth (40.0%) by day 7. Thereafter the level of Fc-RFL did not vary appreciably between these two groups. However, as early as 2 days after inoculation there was a significantly greater number of Fc-RFL in the spleen of experimental DA neonates compared to controls. The lymph nodes of experimental animals did not exhibit a significantly greater number of Fc-RFL until day 6 with both tissue compartments peaking at day 10 and remaining significantly higher than controls until death. In neonatal BN animals significantly higher levels of Fc-RFL in experimental animals were not evident as early and peaked later (day 12) in both tissue compartments but again these differences remained until death. Cytotoxic alloantisera demonstrated that on days 6, 10, and 12 most, if not all, of the Fc-RFL were host in origion in both DA and BN GVHD, with a very significant host plasma cell response in such GVHD animals. One-micron tissue section revealed the presence of a great number of plasma cell especially prominent in the medulla of lymph nodes with the cortex of lymph nodes and white pulp of the spleen markedly depleted of lymphocytes indicative of cytotoxicity.     

Some aspects of the metabolism of sulfate-S35 and calcium-45 in the metaphyses of immature rats: influence of beta-estradiol benzoate

Weanling rats were given 2 mg. of 17-beta-estradiol benzoate at weekly intervals for 4 weeks. Twenty-four hours after each intraperitoneal injection of the estrogen 100 microc. of S(35)-sulfate or 11 microc. of Ca(45) was similarly injected. The animals were sacrificed 24 hours after the last dose of isotopes. An effect of estradiol benzoate on calcium metabolism was deduced from the observation that the concentration of calcium in some tissues of the treated rats was higher than the concentration in the tissues of untreated rats. Alkaline extracts of the distal metaphyses of femurs from the estradiol-treated and from control rats, given S(35)-sulfate, were shown by chromatography on an anion exchange resin to contain from 9 to 22 per cent of the S(35) as inorganic sulfate. From similar bone samples, 6 to 21 per cent of the S(35) was removed by decalcification with sodium versenate. Most of the remaining S(35) was associated with uronic acid and hexosamine; on paper chromatograms and paper electrophoretograms S(35) was shown to be part of material which migrated and was metachromatic in the same way as purified chondroitin sulfate. Autoradiograms of the proximal ends of tibiae from the animals given estradiol benzoate showed that both the S(35) and Ca(45) were deposited in the metaphyses in strata. The arrangement of the strata of S(35), however, was different from the arrangement of the strata of Ca(45). This difference in arrangement is interpreted as indicating that most of the S(35) in the metaphysis was derived from the chondroitin sulfate of the cartilage plate which the metaphysis had replaced.     

Effect of antenatal ethanol exposures on the function of the hemato-encephalic barrier in animals

Blood-brain barrier permeability for P32, H3-valine, H3-tyrosine, S35-methionine was investigated in rat offsprings, 20 and 60 days old, that had been antenatally exposed to ethanol. In experimental 20-day-old rat offsprings P32 incorporation in brain structures was lower than in the controls. In mature rats that had been exposed to antenatal ethanol, a different degree decrease in labelled amino acid penetration and alterations in P32 penetration into different brain structures were found. Significant differences in stress-induced changes of blood-brain barrier permeability were observed in experimental and control animals.     

Cysteine and methionine-precursors of methyl sulphone metabolites of 2,4',5-trichlorobiphenyl in mice

In order to account for the origin of the sulphur atom in methyl sulphones derived from polychlorinated biphenyls (PCB), groups of mice were treated with 2,4'-5-trichlorobiphenyl and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. The radioactive substances extracted from lung tissue were characterized by partition between hexane and sulphuric acid, thin-layer radiochromatography (TLRC), gas chromatography (GC) and mass fragmentography (MF). In lung extracts of both experimental groups sulphuric acid soluble metabolites were present: Their Rf-values on TLRC were identifical with that of 4-methylsulphonyl-2,4'-5-trichlorobiphenyl and their identity was confirmed by GC and GC-MF, which also indicated the presence of a minor sulphone isomer. The study shows that both cysteine and methionine could function as donors of the sulphur atom in the methyl sulphone metabolites derived from PCB.     

Vitamin A and the biosynthesis of sulphated mucopolysaccharides. Experiments with rats and cultured neoplastic mast cells

1. The uptake and incorporation of [(35)S]sulphate into mucopolysaccharides by colon and duodenum in vitro are unaffected by the vitamin A status of the animals. 2. Uptake and incorporation in vivo are unaffected at 4hr. after injection of [(35)S]sulphate, but at later times are decreased in some tissues of vitamin A-deficient animals. 3. The rate of removal of (35)S from blood, its rate of appearance in urine, the plasma concentration of sulphate and the uronic acid content of several tissues are not significantly altered in vitamin A deficiency. 4. These results, and direct measurement of (35)S in mucopolysaccharides at various times after injection of [(35)S]sulphate, suggest that the synthesis of mucopolysaccharides is unaffected but that their turnover is increased in vitamin A deficiency. 5. Neither the growth rate of, nor the incorporation of [(35)S]sulphate into heparin by, P815Y and HC cultured neoplastic mast cells is decreased when the horse serum necessary for growth is treated with ultraviolet light or is replaced by serum from vitamin A-deficient rats. 6. The addition of citral is no more toxic to growth rate or to incorporation of (35)S than is the addition of vitamin A itself. 7. It is concluded that neoplastic mast cells in culture do not require vitamin A for growth or for the synthesis of heparin. 8. None of these results is compatible with the view that vitamin A or a derivative is directly involved in the biosynthesis of sulphated mucopolysaccharides.     

Natural cell-mediated cytotoxicity among A-bomb survivors residing in the United States

Natural cell-mediated cytotoxicity (NCMC) by lymphocytes from Japanese atomic bomb survivors now living in the United States was measured. Seventy-one individuals were exposed to an estimated '0.00' Gy ('0 rads') (S0 group) and 58 to greater than '0.00 Gy' (S+ group) at the time of the bomb. Of this 58, 51 (88 per cent) received less than 0.50 Gy and 30 (52 per cent) received less than 0.10 Gy. NCMC was measured against 51Cr-labelled K562 target cells. Activity by lymphocytes from S+ group donors was significantly greater than that for the S0 group (p = 0.028 by the stratified Wilcoxon rank-sum test). This difference between the S+ and S0 populations was detected 35 years after exposure to the bomb. It is therefore feasible and important to examine appropriate biologic parameters to elucidate the effects of low doses of radiation in humans.     

Heparin clearance during activation of the anticoagulation system in animals

Retarded heparin-35S clearance from the blood stream during activation of the blood anticoagulation system was observed. Under these conditions the half-life of heparin-35S increased from 1.6 h in control animals to 2.35 h in the animals after intravenous injection of a threshold dose of thrombin causing activation of the anticoagulation system. Retarded heparin-35S clearance from the blood stream during activation of the blood anticoagulation system is accompanied by increase of its absorption in the liver and in the auricular atrii.     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

The effect of different sample collection methods on rabbit blood parameters

Nowadays great deal of research is physiological field is conducted on experimental animals and there is a lot of criticism from the wide public on methods used. Therefore, recently there is a lot of effort focused on the welfare of the animals. Main aim of this study is to determine the effect of experimental sample collection method on the selected parameters of stress. In the experiment two sample collections of rabbit blood from marginal ear vein were realized – first using standard method with one person fixing the animal and other collecting the blood using gently fixating the animal. In the second groups experimental method of inserting the experimental animal into a sack and further collection in dark was realized. During the experiment the levels of cortisol – main stress indicator in organism and other health parameters of animals including mineral profile and haematological parameters were observed. Our results show no significant changes in levels of cortisol but also a decreasing tendency in the sample from the second (dark) collection. Haematological parameters were generally in the reference values and any significant changes except levels of lymphocytes and percent of lymphocytes which shown significant increase in the second collection period were found. Also the levels of mean corpuscular haemoglobin and percent of neutrophils unveiled a significant decrease in values. Values of mineral profile parameters have indicated no significant changes except the levels of phosphorus. Based on the result we can state that the experimental sample collection has no effect on blood parameters of the animals but we spectated a statistically insignificant decrease in the levels of cortisol which can suggest that the dark collection is possibly less stressful to the animals.     

Immunomodulating action of interferon in congenital and experimental thymus-dependent immunodeficiency

A study of immunomodulating action of mouse alpha/beta interferon (IFN) was performed in conditions of congenital and experimental thymus-dependent immune deficiency. The action of IFN was assessed in vivo, with IFN injected intraperitoneally. It was shown that IFN did not change killer effect of lymphocytes in the reaction of stem cell inactivation in "nude" mice, but enhanced antibody and rosette formation in the spleen of these animals. In addition, IFN did not change these parameters in the spleen of HRS mice with abnormal differentiation of T lymphocytes, but enhanced antibody and rosette formation in the spleen of mice, thymectomized in old age. It is concluded that the normal function of T-system is essential for the action of IFN.     

Correlation between active rosette formation and delayed cutaneous hypersensitivity in experimental allergic encephalomyelitis

The effect of encephalitogenic myelin basic protein, BP, on active rosette-forming T cells (ARFC) was compared to that of nonencephalitogenic peptide S42, a synthetic analogue of the tryptophan region of BP. Depression of ARFC by these antigens was reversible within 24 h after a second dose of the antigen into the skin, or after in vitro incubation of lymphocytes with the sensitizing antigen (Ag-ARFC). The ratio of Ag-ARFC to ARFC rose with time following the sensitization but fell shortly before the clinical onset of experimental allergic encephalomyelitis in animals sensitized with BP. In contrast, the Ag-ARFC/ARFC ratios for animals sensitized with peptide S42 reached plateau levels from which they did not drop. The kinetics of the Ag-ARFC/ARFC responses paralleled those for delayed-type skin hypersensitivity (DTH) in the respective animals. The DTH responses rose following sensitization and fell shortly after the appearance of clinical signs of EAE. The results of this study provide in vitro and in vivo evidence for sensitization to myelin basic protein, and focus attention on the ARFC as a measure for an immunologically active cell population which may be quantitated by antigenic stimulation.Abbreviations used in this report EAE experimental allergic encephalomyelitis - DTH delayed-type skin hypersensitivity - ARFC active rosette-forming T cells - Ag-ARFC antigen-stimulated active rosette-forming T cells - TRFC total rosette-forming T cells     

Chromosomal aberrations in bone marrow and peripheral blood cells from the same rats

Spontaneous cytogenetic aberrations were analyzed in bone-marrow cells and cultured peripheral lymphocytes from the same animals. No significant differences in the total number of cells with aberrations or total aberrations were detected between the bone-marrow cells and cultured lymphocytes. It was concluded that short-term culture does not contribute significantly to in vivo aberration yield within the experimental conditions used.     

Social isolation-induced decreases in both the abundance of neuroactive steroids and GABA(A) receptor function in rat brain

The effects of social isolation on behavior, neuroactive steroid concentrations, and GABA(A) receptor function were investigated in rats. Animals isolated for 30 days immediately after weaning exhibited an anxiety-like behavioral profile in the elevated plus-maze and Vogel conflict tests. This behavior was associated with marked decreases in the cerebrocortical, hippocampal, and plasma concentrations of pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone compared with those apparent for group-housed rats; in contrast, the plasma concentration of corticosterone was increased in the isolated animals. Acute footshock stress induced greater percentage increases in the cortical concentrations of neuroactive steroids in isolated rats than in group-housed rats. Social isolation also reduced brain GABA(A) receptor function, as evaluated by measuring both GABA-evoked Cl(-) currents in XENOPUS: oocytes expressing the rat receptors and tert-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding to rat brain membranes. Whereas the amplitude of GABA-induced Cl(-) currents did not differ significantly between group-housed and isolated animals, the potentiation of these currents by diazepam was reduced at cortical or hippocampal GABA(A) receptors from isolated rats compared with that apparent at receptors from group-housed animals. Moreover, the inhibitory effect of ethyl-beta-carboline-3-carboxylate, a negative allosteric modulator of GABA(A) receptors, on these currents was greater at cortical GABA(A) receptors from socially isolated animals than at those from group-housed rats. Finally, social isolation increased the extent of [(35)S]TBPS binding to both cortical and hippocampal membranes. The results further suggest a psychological role for neurosteroids and GABA(A) receptors in the modulation of emotional behavior and mood.