The effect of the extract of Crocus sativus and its constituent safranal, on lung pathology and lung inflammation of ovalbumin sensitized guinea-pigs

Different pharmacological effects of Crocus sativus have been demonstrated on guinea pig tracheal chains in previous studies. In the present study, the prophylactic effect of the extract of C. sativus and its constituent, safranal on lung pathology and total and differential white blood cells (WBC) of sensitized guinea pigs was examined. Guinea pigs were sensitized with injection and inhalation of ovalbumin (OA). One group of sensitized guinea pigs were given drinking water alone (group S) and three groups were given drinking water containing three concentrations of safranal (S+SA1, S+SA2 and S+SA3 groups), three groups, drinking water containing three concentrations of extract (S+CS1, S+CS2 and S+CS3 groups) and one group drinking water containing one concentration of dexamethasone (S+D group) (n=6, for all groups). The lung pathology was evaluated in control, non treated and treated sensitized groups. Total and differential WBC counts of lung lavage were also examined. All pathological indices in group S showed significant increased compared to control group (p<0.05 for lung congestion and p<0.001 for other groups). Total WBC number (p<0.001), eosinophyl percentage (p<0.001) in lung lavage and serum histamine levels (p<0.01) were also increased in sensitized animals compared to those of controls. Treatment of S animals with dexamethasone, all concentrations of the extract and safranal significantly improved lung pathological changes, most types of WBC and serum histamine levels compared to group S (p<0.05-0.001). Treatment of S group with first concentration of safranal also decreased total WBC. Treatment with safranal was more effective in improvement of most pathological changes, total and differential WBC count as well as serum histamine level (p<0.05-0.001). These results showed a preventive effect of the extract of C. sativus and its constituent safranal on lung inflammation of sensitized guinea pigs. The results also showed that the effect of the plant is perhaps due to its constituent safranal.     

Further study of the problem of specific cellular receptors in delayed hypersensitivity]

The work presents the materials obtained as a result of the further study of specific T lymphocyte receptors with the use of so-called receptor antisera, i. e. antisera against the lymphoid cells of mice sensitized with one of the two antigens (Macobacterium tuberculosis or bovine gamma globulin); thus these differed from control antisera against the lymphoid cells of intact mice. These mouse antisera reacted with the lymphoid cells of guinea pigs in experiments of delayed-type hypersensitivity transfer. The cells of sensitized guinea pigs lost their ability to transfer hypersensitivity if, prior to their injection into the recipient guinea pigs, these cells were treated with the above-mentioned mouse antisera, i. e. antisera against the lymphoid cells of mice had a blocking effect on the lymphoid cells of guinea pigs. The blocking action of the antisera proved to be specific: antisera against the lymphoid cells of mice sensitized to bovine gamma gloublin blocked the cells of guinea pigs, also sensitized to bovine gamma globulins, but did not block the cells sensitized to Mycobacterium tuberculosis. The control antisera, taken in the same doses as the factor antisera, did not show a blocking effect on the specific activity of lymphoid cells.     

Cytotoxic Michael-type amine adducts of alpha-methylene lactones alantolactone and isoalantolactone

Two series of cytotoxic (IC50, K562 cell line, 1-24 microM) alpha-aminomethyl substituted lactones 3 and 4 were prepared by stereoselective Michael-type addition of amines to alantolactone (1) and isoalantolactone (2). The lactones 1 and 2 and their amine adducts induce apoptosis and act as alkylating agents.     

O3-induced airway hyperresponsiveness to noncholinergic system and other stimuli.

The effect of O3 exposure (3 ppm, 1 h) on the in vivo and in vitro airway responsiveness, as well as the changes in cell contents in bronchoalveolar lavage (BAL) fluid, were evaluated 16-18 h after O3 exposure in sensitized and nonsensitized male guinea pigs. The sensitization procedure was performed through repeated inhalation of ovalbumin for 3 wk. Increase in pulmonary insufflation pressure produced by the excitatory nonadrenergic noncholinergic (eNANC) system, histamine, and antigen were assessed in in vivo conditions, whereas airway responsiveness to histamine and substance P was evaluated in in vitro conditions by use of tracheal chains with or without epithelium and lung parenchymal strips. We found that O3 exposure 1) increased the neutrophil content in BAL fluids in both sensitized and nonsensitized guinea pigs, 2) caused hyperresponsiveness to eNANC stimulation in nonsensitized guinea pigs (although combination of sensitization and O3 exposure paradoxically abolished the hyperresponsiveness to eNANC stimulation), 3) increased the in vivo bronchoconstrictor responses to histamine and antigen, 4) caused hyperresponsiveness to substance P in nonsensitized tracheae with or without epithelium and in sensitized tracheae with epithelium, 5) did not modify the responsiveness to histamine in tracheae with or without epithelium (and in addition, epithelium removal caused hyperresponsiveness to histamine even in those tracheae exposed to O3), and 6) produced hyperresponsiveness to histamine in lung parenchymal strips either from sensitized or nonsensitized guinea pigs.     

Induction of allergic reactions in guinea pigs with purified house dust mite allergens

To establish a guinea pig model for house dust mite allergy with purified mite allergens, we studied the immune response to two major mite allergens, native Der f 1 (nDer f 1) and recombinant Der f 2 (rDer f 2) and crude mite extract in Hartley guinea pigs. Animals were immunized with either mite extract, nDer f 1 or rDer f 2, four times at 2- to 3-week intervals. Then the guinea pigs were examined as to the status of sensitization to the sensitizing antigen. Intradermal injection of mite antigens to mite extract-, nDer f 1-, and rDer f 2-sensitized animals induced both immediate and late-phase cutaneous reactions. Allergic airway disease was also provoked by the intranasal instillation of rDer f 2 or mite extract. Anti-nDer f 1 and -rDer f 2 IgE as well as anti-mite extract IgE were produced in the sensitized guinea pigs and IgE titer for three mite antigens were comparable. We concluded that immunization of Hartley guinea pigs with nDer f 1 and rDer f 2 achieved sensitization to mite allergens, which was comparable to that obtained by the immunization with mite extract. A mite-allergic model suitable for immunological and pharmacological studies was established from rDer f 2-sensitized guinea pigs.     

Studies on the Tuberculin Reactivity of Tuberculin-Active Peptide

A comparative study of the tuberculin potency and reactivity to TAP and PPD-S was made in humans and guinea pigs. A comparison of reactions elicited by TAP and PPD-S was made in two groups of guinea pigs previously inoculated with killed bacilli or with living bacilli. The tuberculin reactions produced by TAP and PPD-S in human beings was studied in school children from rural districts. These children were divided randomly into three groups and each group was given a different amount of TAP and PPD-S. The results obtained are as follows: (1) While relative potency of TAP was relatively much weaker than PPD-S in guinea pigs sensitized with tubercle bacilli, in human beings the tuberculin potency of TAP was approximately 1/2 or 1/3 that of PPD-S. (2) There was a marked difference in the tuberculin reactivity to TAP and PPD-S between guinea pigs immunized with killed bacilli and guinea pigs immunized with living bacilli.     

Dermal and intravascular Langerhans cells at sites of passively induced allergic contact sensitivity.

In order to further define the role of Langerhans cells in contact allergic reactions, passive transfer studies were done in guinea pigs using 2,4-dinitro-1-chlorobenzene (DNCB)-sensitive donor cells. Langerhans cells were found in the lumen of dermal vessels resembling lymphatics at 2, 3, 15, and 48 hr after DNCB challenge. In contrast to the previously reported findings in actively sensitized guinea pigs, the changes involving Langerhans cells in passively sensitized guinea pigs were mainly noted in the dermis. These consisted of increased numbers of Langerhans cells and of mononuclear cells apposed to Langerhans cells 3 or more hours after challenge with DNCB. The increased Langerhans cell population in the dermis and the presence of Langerhans cells in dermal vessels in specifically challenged sites in adoptive immune reactions furnishes further support for a significant role of Langerhans cells in the interaction between antigen and sensitized cells.     

BN大鼠和豚鼠对卵清白蛋白致主动过敏反应的免疫学特性比较

目的 比较BN大鼠和豚鼠对卵清白蛋白(OVA)致敏前后机体免疫学特性的变化.方法 BN大鼠和豚鼠分别用OVA(每只1 mg)隔日致敏(i.p.),共5次;于末次致敏第10天以OVA(每只2 mg)激发致敏(i.v.);分别设正常对照组和OVA致敏组.于激发致敏后1h处死动物,分离腹腔肥大细胞、脾脏和骨髓,并制备脾脏和骨髓淋巴细胞.以annexin-V作为标志检测肥大细胞活性,同时以Fluo-3/AM标记胞内钙离子,检测钙离子水平;以PHA和LPS作为有丝分裂原,分别检测脾脏和骨髓T、B淋巴细胞活性.结果 ①致敏BN大鼠和豚鼠脾脏及骨髓T、B淋巴细胞活性均升高,其中骨髓淋巴细胞活性BN大鼠显著高于豚鼠,脾脏淋巴细胞活性两种属间差异无显著性;②致敏后,腹腔肥大细胞活性两种属间差异无显著性,但BN大鼠致敏后是致敏前的6倍,豚鼠是3倍;③肥大细胞内钙离子水平两种属致敏后均升高,豚鼠致敏前后钙离子水平具有统计学意义.结论 OVA致敏后,BN大鼠骨髓淋巴细胞活性明显高于豚鼠,豚鼠肥大细胞内钙离子较BN大鼠升高明显,肥大细胞活性两者无明显差异.因此,在实验中可以根据两种属在过敏反应中的特点以及具体的实验要求选择动物模型.     

So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Strain differences in susceptibility to experimental allergic encephalomyelitis and the immune response to the encephalitogenic determinant in inbred guinea pigs.

Strain differences in susceptibility to experimental allergic encephalomyelitis (EAE) in guinea pigs were correlated with the cellular immune response to the basic encephalitogenic protein (BE). The response to BE was determined in strains 2 and 13 guinea pigs in vivo by the delayed hypersensitivity skin test and in vitro by the lymphocyte transformation technique. The response to the intact BE of both heterologous (bovine) and homologous (guinea pig) origins was indistinguishable between the two strains. Guinea pigs sensitized with the guinea pig BE showed complete cross-reaction when tested with the bovine BE. On the other hand, there appears to be significant differences in the response to specific determinants on the molecule. Thus, only strain 13 and F₁ hybrids which are susceptible to EAE responded to the encephalitogenic nonapeptide (residue 114–122 of the BE molecule), whereas strain 2 guinea pigs which are resistant to EAE did not respond to this determinant.     

Passive transfer of cell-mediated immunity in xenogeneic animals

Cell-mediated immunity (delayed hypersensitivity) to 1-chloro-2,4-dinitrobenzene (DNCB) was passively transferred from sensitized guinea pigs to mice. Transfer was accomplished by subcutaneous injection of peritoneal exudate cells of sensitized guinea pigs.     

Muscarinic-receptor functioning in tracheas from normal and ovalbumin-sensitive guinea pigs

Responses to bilateral vagal nerve stimulation, to field stimulation, and to exogenous methacholine and histamine were compared in tracheas isolated from (a) saline injected (i.p.) and saline-aerosol exposed guinea pigs (control), (b) ovalbumin-sensitized and saline-aerosol exposed (sensitized) guinea pigs, and (c) ovalbumin-sensitized and 2% ovalbumin-aerosol exposed (challenged) guinea pigs. Tracheal pressor responses (cmH2O; 1 cmH2O = 98.1 Pa) to nerve and field stimulation, and maximal responses to methacholine and histamine were significantly increased in animals from group c compared with groups a and b. Dose-response lines in response to the two agonists, expressed as percent maximal contraction, did not differ among the groups. The M1 antagonist pirenzepine (0.1-10 nM) selectively reduced responses to nerve stimulation in all three groups. The M2 antagonist gallamine potentiated responses to nerve or field stimulation in all three groups. We conclude that M1, M2, and M3 muscarinic receptor functioning is similar in control and ovalbumin-sensitized guinea pigs. Changes in post-receptor transduction mechanisms may mediate the increased responsiveness noted in animals from group c.     

Effects of hyperoxia and allergic airway inflammation on cough reflex intensity in guinea pigs

Toxic influence of high oxygen concentration on pulmonary function and structures has been known for many years. However, the influence of high oxygen concentration breathing on defensive respiratory reflexes is still not clear. In our previous experiments, we found an inhibitory effect of 100 % oxygen breathing on cough reflex intensity in healthy guinea pigs. The present study was designed to detect the effects of hyperoxia on cough reflex in guinea pigs with allergic airway inflammation. In the first phase of our experiment, the animals were sensitized with ovalbumin. Thirty-two sensitized animals were used in two separate experiments according to oxygen concentration breathing: 100 % or 50 % oxygen for 60 h continuously. In each experiment, one group of animals was exposed to hyperoxia, another to ambient air. The cough reflex was induced both by aerosol of citric acid before sensitization, then in sensitized animals at 24 h and 60 h of exposition to oxygen/air in awake animals, and by mechanical stimulation of airway mucosa in anesthetized animals just after the end of the experiment. In contrast to 50 % oxygen, 100 % oxygen breathing leads to significant decrease in chemically induced cough in guinea pigs with allergic inflammation. No significant changes were present in cough induced by mechanical stimulation of airways.     

Adenosine induces a cholinergic tracheal reflex contraction in guinea pigs in vivo via an adenosine A1 receptor-dependent mechanism.

Adenosine induces dyspnea, cough, and airways obstruction in asthma, a phenomenon that also occurs in various sensitized animal models in which a neuronal involvement has been implicated. Although adenosine has been suggested to activate cholinergic nerves, the precise mechanism has not been established. In the present study, the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) induced a cholinergic reflex, causing tracheal smooth muscle contraction that was significantly inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 100 microg/kg) (P < 0.05) in anesthetized animals. Furthermore, the adenosine A(2) agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) induced a small reflex, whereas the A(3) selective agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) was without effect. The tracheal reflex induced by CPA was also inhibited by recurrent nerve ligation or muscarinic receptor blockade (P < 0.001), indicating that a cholinergic neuronal mechanism of action accounted for this response. The cholinergic reflex in response to aerosolized CPA was significantly greater in passively sensitized compared with naive guinea pigs (P < 0.01). Chronic capsaicin treatment, which inhibited sensory nerve function, failed to inhibit CPA-induced reflex tracheal contractions in passively sensitized guinea pigs, although the local anesthetic lidocaine inhibited CPA-induced tracheal contractions. The effects of CPA on the reflex response was not dependent on the release of histamine from tissue mast cells or endogenous prostaglandins as shown by the lack of effect of the histamine H(1) receptor antagonist pyrilamine (1 mg/kg) or the cyclooxygenase inhibitor meclofenamic acid (3 mg/kg), respectively. In conclusion, activation of pulmonary adenosine A(1) receptors can stimulate cholinergic reflexes, and these reflexes are increased in allergic guinea pigs.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Isolation of tuberculin peptides from tuberculin purified protein derivative (PPD).

Tuberculin purified protein derivative (PPD) obtained from the filtrate of Mycobacterium tuberculosis was hydrolysed with proteinase, trypsin, or chymotrypsin. Each hydrolysate consisted of a tuberculin peptides mixture (TPM). From each TPM 16 fractions were obtained by ion-exchange chromatography on Dowex 50W-X8 but only one fraction was isolated from each of the 16 fractions which showed tuberculin activity in guinea pigs sensitized with M. bovis (bcg) or M. tuberculosis. This fraction was designated "purified tuberculin peptide" (PTP). The PTP fraction from the proteinase hydrolysate (PTP-proteinase) was rechromatographed on Dowex 1-X2 and two tuberculin peptide fractions having molecular weights of 3200 and 12,000 were isolated. The potency of these two fractions was assessed in guinea pigs sensitized with M. bovis (BCG) and with M. tuberculosis and they were approximately 4 to 7 times more potent than either the international standaCG and of at least equal potency to either PPD-S or Connaught PPD in guinea pigs sensitized with either M. kansasii, M. scrofulaceum, M. intracellulare, or M. avium whereas very little if any cross-reactivity was elicited by these two fractions. This lack of response indicates that either fraction could be used as an aid to differentiate between sensitization due to M. tuberculosis or M. bovis and sensitization attributed to other mycobacteria.     

Identification and structure-activity relationship studies of 3-methylene-2-norbornanone as potent anti-proliferative agents presumably working through p53 mediated apoptosis

We have identified a novel series of alpha-methylene carbonyl compounds through structure-activity relationship (SAR) studies with high levels of anti-proliferative activities. The lead molecule, 3-methylene-2-norbornanone (3) showed potent activity (LC(50)=3-8 microM) against mutant p53 cell types and many fold selectivity (>13-29) over wild-type p53 cells. Further, compound 3 and its analogs showed refolding of mutant p53 protein comparable to their anti-proliferative activities suggesting possible interaction with mutant p53 protein.     

Non-specific antimicrobial cellular resistance in sensitized guinea pigs.

Specific delayed-type hypersensitivity was induced in guinea pigs with bovine albumin + complete Freund adjuvant, bovine gamma globulin + complete Freund adjuvant and BCG vaccine. The animals were subsequently tested for nonspecific antimicrobial resistance. Sensitized and control groups were challenged intraperitoneally with Listeria monocytogenes 2 hr after reinjection with the sensitizing antigen. The listeria content of the spleens was determined 1 or 5 days after the infection. The number of organisms recovered from the spleen one day after infection was significantly less in guinea pigs sensitized with bovine gamma globulin and BCG than in the control group; after 5 days no such difference was recorded. There was no difference between the bovine albumin sensitized and the control group 1 day after infection, while on the 5th postinfection day listeria counts were higher in the sensitized than in the control animals.     

Retention of 14C-Labeled Tuberculin Purified Protein Derivative in the Skin of Sensitized and Nonsensitized Animals

Tuberculin purified protein derivative labeled with (14)C ([(14)C]PPD) with a biological potency equivalent to the International Standard for tuberculin PPD was used to study the retention of tuberculin PPD in the skin of sensitized and nonsensitized animals. We found that [(14)C]PPD was almost entirely cleared from the skin test site during the first 18 to 24 h after injection and that when approximately 5% of the initial concentration of [(14)C]PPD was present in the skin test site, the size of the tuberculin skin reaction in sensitized guinea pigs was at its maximum. Furthermore, the addition of 5 or 50 mug of Tween 80 per ml to a solution of PPD did not change either the rate of clearance of PPD from the skin test sites of sensitized guinea pigs or the size of the tuberculin skin reactions. There was no difference in the rate of clearance of [(14)C]PPD from the skin test sites between sensitized and nonsensitized guinea pigs and between guinea pigs of different age. However, there was a significant difference in the rate of clearance of [(14)C]PPD between the guinea pig and the mouse. Finally, the percentage of [(14)C]PPD retained in the site of injection at 24 h was in the neighborhood of 5% of the initial concentration of the solution of PPD injected. The significance of these phenomena is discussed.     

Antiallergic/antiasthmatic effect of novel antiallergic hexapeptide-95/220 in various experimental models

The effects of newly synthesized antiallergic hexapeptide 95/220 was investigated on various allergic and asthmatic test models. This newly developed peptide was found to be more potent than clinically used drug disodium cromoglycate (DSCG). Hexapeptide 95/220 inhibited immediate hypersensitivity reactions such as passive cutaneous anaphylaxis (PCA) and mast cell degranulation in rats, antigen-induced bronchoconstriction in actively sensitized guinea pigs in dose dependent manner like DSCG. Antigen-induced contraction of guinea pig ileum was also markedly inhibited by this newly developed hexapeptide in the same fashion as ketotifen and DSCG did but at comparatively lower dose. Egg albumin-induced histamine release was also blocked by this hexapeptide from chopped lung tissues of sensitized guinea pigs. These results suggest that hexapeptide' 95/220 has potent inhibitory effect on immediate hypersensitivity reactions thereby inhibiting mediator release from mast cell. Moreover, this newly synthesized peptide is orally active and effective at lower doses as compared to standard drugs.