Immunological Unresponsiveness in Guinea Pigs: I. Immunological Unresponsiveness to Heterologous Serum Proteins

A state of immunological unresponsiveness to bovine serum albumin (BSA) or human gamma globulin (HGG) can be produced in guinea pigs by contact with these proteins either before and soon after birth, or even soon after birth alone. As judged by failure of treated animals to show immune elimination of the antigen, or anaphylaxis on reinjection, the animals were completely unresponsive when tested up to 5 months after birth.

Administration of 6-mercaptopurine to guinea pigs, even in lethal doses, did not prevent the development of antibodies to HGG.

After administration of BSA or HGG, labelled with ¹³¹I, to pregnant guinea pigs, their offspring at term contained significant amounts of the labelled proteins. HGG was catabolized in new-born guinea pigs with the same half-life as in adults.

     

The Prevention of Delayed Hypersensitivity to Homologous Serum and Transplantation Antigens in Guinea Pigs

The injection of normal guinea-pig serum in Freund's complete adjuvant into guinea pigs other than the serum donor led to the development of a long-lasting, delayed hypersensitivity. Serum alone, without adjuvant, had no sensitizing capacity. Circulating antibodies to the allotypic antigens could not be detected.

Injection of as much as 8 ml. of serum into sensitized animals did not achieve desensitization. However, the intravenous injection of the same amount of serum prevented normal guinea pigs from becoming sensitized to the same antigen for over 100 days. This unresponsiveness was interpreted to be due to an interference by the serum with the process of sensitization.

This method of producing unresponsiveness was applied to the homograft reaction: guinea pigs, given a series of intravenous injections of spleen extracts containing transplantation antigens, could not be subsequently sensitized by the injection of spleen cells from the same donors. However, immunization provided by skin grafting could probably break through this unresponsiveness: guinea pigs, judged to be unresponsive by the intradermal injection of spleen cells before skin grafting, all developed an intense cutaneous hypersensitivity after they had rejected the graft.

     

Cell-Free Passive Transfer of Delayed Hypersensitivity to Chemicals in Guinea Pigs

Delayed-type cutaneous reactivity to 2,4-dinitrochlorobenzene and 2,4-dinitrofluorobenzene in guinea pigs was transferred passively by fluids in which leukocytes from sensitive animals were incubated. Cells from peritoneal exudates, lymph nodes, and alveolar washings were employed. The cell-free transfer material was dialyzable, of small molecular size, and stable to 56 C for 30 min and -65 C for at least 9 weeks. It gave a ratio at 280 to 260 nm of 0.71. The relationships between temperature, pH, and cellular release of the transfer material were studied.     

Immunological Unresponsiveness to Protein Antigens in Rabbits Exposed to X-Irradiation or 6-Mercaptopurine Treatment

Immune unresponsiveness to HSA and BSA was induced in adult rabbits exposed to 550 R. total-body irradiation. Immunogenic doses of antigen, 20–75 mg., were found to be tolerogenic in this system. The degree of unresponsiveness was found to be a function of the time interval between X-irradiation and the beginning of antigen administration. The unresponsive animals retained the circulating antigen in quantities detectable by the gel-diffusion technique. These results were compared with the state of unresponsiveness induced by means of 6-mercaptopurine, and similar parameters were found to be operative. Some aspects of the significance of the ratio of antigen to competent cells, in the induction of immune unresponsiveness, are discussed.     

The Passive Transfer of Delayed Hypersensitivity in Guinea Pigs by the Transfusion of Isotopically-Labelled Lymphoid Cells

Delayed hypersensitivity to tuberculin and contact sensitivity to picryl chloride were transferred passively to normal guinea pigs by the intravenous injection of spleen, lymph node and peripheral blood mononuclear cells labelled in vitro with ⁵¹Cr or in vivo with ³²P or ³H-thymidine. No significant difference was found between the number of injected cells arriving at the passive lesions and actively induced lesions in controls, after 24 hours. The proportion of labelled cells in the exudate was the same as that in the peripheral blood and they showed a random distribution throughout the lesion.

The fate of labelled cells in the body was followed, especially their role of elimination from the lungs and peripheral blood.

It was concluded that no special affinity of the transferred cells for antigen could be demonstrated after 24 hours.

     

Adjuvant Action of Bacterial Lipopolysaccharide in Induction of Delayed-Type Hypersensitivity to Protein Antigens. II. Relationships of Intensity of the Action to that of Other Immunological Activities

Various kinds of lipopolysaccharides (LPS) isolated from different bacterial strains exhibited more or less an adjuvant action in induction of delayed-type footpad response to ovalbumin (OA) in mice when antigen was injected subcutaneously together with LPS. There was a wide diversity in strength of the adjuvant action of LPS, and the action of the 03 antigen isolated from the culture supernatant fluid of Klebsiella was the most potent of eleven kinds of LPS tested. Besides this substance, LPS extracted from Escherichia coli 08, 09 and 0128, and Salmonella typhosa exhibited relatively strong action. The difference in strength of the action of the high and low active LPS in induction of delayed-type hypersensitivity could be observed at any doses of OA and LPS. The strength of the adjuvant activity of LPS in induction of delayed-type hypersensitivity roughly correlated with that of their adjuvant activity in augmenting immunological memory for the secondary antibody response to OA and also their ability to enlarge the regional lymph node, whereas it did not apparently correlate with that of their activity to stimulate B cells polyclonally and their ability to enlarge the spleen.     

Adjuvant Activity of 6-Amino-6-Deoxy-Muramyldipeptides and Their Acylamino Derivatives on the Induction of Delayed Hypersensitivity to Azobenzenearsonate-N-Acetyl-l-Tyrosine in Guinea Pigs

The 6-amino-6-deoxy-N-acetylmuramyldipeptides and their 6-acylamino derivatives were shown to be active as adjuvants on the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-l-tyrosine in guinea pigs. However, 6-acylamino-6-deoxy-N-(acyl)muramyldipeptides were inactive as adjuvants.     

Delayed Hypersensitivity to Toxoplasma and Unrelated Antigens in Toxoplasma-Infected Mice: Induction and Elicitation of Delayed-Type Hypersensitivity by Antigen-Pulsed Macrophages

Delayed-type hypersensitivity (DTH) to Toxoplasma and unrelated antigens in Toxoplasma-infected BALB/c mice was investigated by the radioisotopic uptake method of Vadas et al. (Int. Arch. Allergy Appl. Immunol. 49: 670-692, 1975). DTH became positive on day 30 of infection and remained positive during chronic infection. The expression of DTH in mice infected with the relatively avirulent C37 strain of the parasite paralleled the Toxoplasma antibody response as detected by the Sabin-Feldman dye test. Mice sensitized with Toxoplasma, keyhole limpet hemocyanin, or sheep erythrocytes during the acute or chronic phase of Toxoplasma infection showed a DTH reaction similar to that of uninfected sensitized controls. No parasite antigens could be detected by immunofluorescence techniques on the surface of Toxoplasma-infected cells. When killed organisms were added to the cell cultures, specks of fluorescence appeared on cells containing intracellular parasites as well as on cells without intracellular organisms. That the antigens may be present in or on macrophages in a form readily recognizable by T cells is suggested by experiments in which we demonstrated that injection of uninfected normal macrophages pulsed with Toxoplasma-soluble antigens into the ears of chronically infected mice elicited a DTH reaction comparable to that observed when 10⁶ Formalin-fixed tachyzoites were used as the test antigen. When macrophages pulsed with Toxoplasma antigen were used in attempts to induce DTH in naive uninfected mice, the intensity of the reaction was similar to that observed in infected mice.     

Skin Testing of Guinea Pigs and Footpad Testing of Mice With a New Antigen for Detecting Delayed Hypersensitivity to Cryptococcus neoformans

This study was undertaken to evaluate the potential of a cryptococcal culture filtrate antigen, cryptococcin C184, for detecting delayed hypersensitivity in Cryptococcus neoformans-injected animals. The antigen was tested on guinea pigs which had received saline or C. neoformans and on animals sensitized to Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans, or Sporothrix schenckii. A delayed-type hypersensitivity response was elicited by cryptococcin C184 in C. neoformans-injected guinea pigs, whereas no indurations or erythemas were seen at 48 h after skin testing of saline controls or heterologously sensitized guinea pigs. Besides being specific for Cryptococcus, the antigen showed a high degree of sensitivity and was reproducible. Footpad tests were conducted with the antigen on mice which had previously received either 10(5) viable C. neoformans cells or saline. Delayed hypersensitivity was indicated in the C. neoformans-injected mice by the increase in thickness of antigen-injected footpads when compared with the saline-injected footpads. In control mice, antigen- and saline-injected footpads were comparable in thickness 24 h after injection. Mice sensitized to B. dermatitidis were footpad tested with C184, and no cross-reactivity was demonstrated.     

The Immunomodulatory Effect of Yeast Glucan on Delayed Hypersensitivity

Abstract

The effect of yeast β-1,3-glucan as an immunopotentiator of delayed type-hypersensitivity reactions (DHR) was studied. Delayed-type-hypersensitivity reactions in mice sensitized intraperitoneally (IP) with sheep red blood cells (SRBC) and pretreated three days previously with glucan given IP were significantly increased. However, mice sensitized IP with SRBC three days after the subcutaneous (SC) administration of glucan showed depressed DHR. Glucan given at the same site but not at distance strongly potentiated the DHR induced by SC sensitization with SRBC. Subcutaneous injection of glucan and SRBC given together also resulted in a sustained DHR which persisted twelve days after sensitization when DHR of control mice had waned.     

The Immunomodulatory Effect of Yeast Glucan on Delayed Hypersensitivity

The effect of yeast β-1,3-glucan as an immunopotentiator of delayed type-hypersensitivity reactions (DHR) was studied. Delayed-type-hypersensitivity reactions in mice sensitized intraperitoneally (IP) with sheep red blood cells (SRBC) and pretreated three days previously with glucan given IP were significantly increased. However, mice sensitized IP with SRBC three days after the subcutaneous (SC) administration of glucan showed depressed DHR. Glucan given at the same site but not at distance strongly potentiated the DHR induced by SC sensitization with SRBC. Subcutaneous injection of glucan and SRBC given together also resulted in a sustained DHR which persisted twelve days after sensitization when DHR of control mice had waned.     

In Vivo and In Vitro Studies of Delayed-Type Hypersensitivity to Toxoplasma gondii in Guinea Pigs

Delayed-type hypersensitivity develops late in the course of human toxoplasmosis, and a positive skin test is of some value for implicating chronic or eliminating acute forms of toxoplasmosis as a cause of disease. Toxoplasma-infected guinea pigs were studied to determine the onset and development of delayed-type hypersensitivity. Both the toxoplasmin skin test and the in vitro macrophage migration inhibition technique indicated that delayed hypersensitivity to toxoplasma antigen existed as early as 1 week after infection. The mechanism responsible for the observed inhibition of macrophage migration in vitro appeared to be an inhibitory factor(s) released from sensitized lymphoid cells in the presence of antigen.     

Adjuvant Activity of N-Acetyl Muramyl Dipeptides for the Induction of Delayed-Type Hypersensitivity to Azobenzenearsonate-N-Acetyl-l-Tyrosine in Guinea Pigs

The adjuvant activity of 23 kinds of synthetic N-acetyl muramyl dipeptide analogs on the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-l-tyrosine was examined in guinea pigs.     

A Comparison of Four Methods for Measuring Cutaneous Delayed-Type Hypersensitivity Reactions to Protein Antigens in the Mouse

Delayed-type hypersensitivity was induced in mice by injecting keyhole limpet haemocyanin or human serum albumin in Freund's complete adjuvant. Four techniques for assessing specific skin reactions in these immunized animals were compared: increase in thickness of the footpad; increase in thickness of the ear; arrival of 51Cr-labelled syngeneic lymph node cells at the challenge site in the ear; and accumulation of (125I)IUdR at the challenge site in the ear. The most sensitive and reliable test for detecting strong and weak reactions proved to be measurement of antigen-induced thickening in the ear.     

Mechanism of Intensification by Complete Freund's Adjuvant of the Acquisition of Delayed Hypersensitivity in the Guinea Pig

In the guinea pig, delayed administration of complete Freund's adjuvant (CFA) to skin sites previously primed with a cellular antigen leads to a marked intensification of the acquired sensitivity to that antigen. This effect has been demonstrated with diverse cellular antigens including foreign erythrocytes (both fresh and formaldehyde treated), HeLa cells and dead bacteria, Thus, when a borderline-sensitizing quantity of cells is given intradermally on Day 0, followed by adjuvant into the same site on Day 4, there results a marked intensification of the delayed hypersensitivity reaction to Day 6 testing with the priming cells in the adjuvant treated animals. It appears that CFA intensified acquisition of delayed hypersensitivity to cellular antigens, as well as to soluble protein antigens and contact allergens, involves two steps: 1) initial interaction of antigen with immunocompetent cell, 2) subsequent stimulation by adjuvant of immunocommitted cells (probably by enhancing their clonalization). It appears that the CFA effect takes place primarily in lymph nodes regional to the sensitization site.     

Mechanism of Intensification by Complete Freund's Adjuvant of the Acquisition of Delayed Hypersensitivity in the Guinea Pig

In the guinea pig, delayed administration of complete Freund's adjuvant (CFA) to skin sites previously primed with a cellular antigen leads to a marked intensification of the acquired sensitivity to that antigen. This effect has been demonstrated with diverse cellular antigens including foreign erythrocytes (both fresh and formaldehyde treated), HeLa cells and dead bacteria, Thus, when a borderline-sensitizing quantity of cells is given intradermally on Day 0, followed by adjuvant into the same site on Day 4, there results a marked intensification of the delayed hypersensitivity reaction to Day 6 testing with the priming cells in the adjuvant treated animals. It appears that CFA intensified acquisition of delayed hypersensitivity to cellular antigens, as well as to soluble protein antigens and contact allergens, involves two steps: 1) initial interaction of antigen with immunocompetent cell, 2) subsequent stimulation by adjuvant of immunocommitted cells (probably by enhancing their clonalization). It appears that the CFA effect takes place primarily in lymph nodes regional to the sensitization site.     

CRM197对豚鼠血脑屏障通透性的影响

目的研究白喉毒素的无毒突变体CRM197对豚鼠血脑屏障通透性的影响和作用机制。方法给予豚鼠不同浓度的CRM197后,采用伊文氏兰法检测血脑屏障通透性的变化;荧光显微镜观察给予示踪剂伊文氏兰和荧光素钠后血脑屏障完整性的改变;透射电镜观察血脑屏障中血管内皮细胞间紧密连接等结构的变化。结果随着给予CRM197浓度的增加,血脑屏障对伊文氏兰的通透性逐渐增大,其中100,300,500μg/kgCRM197剂量组血脑屏障的通透性显著高于对照组(P<0.01)。给予300μg/kgCRM197后30min,未见有伊文氏兰-白蛋白渗漏到脑血管外周。透射电镜观察,不同浓度的CRM197作用后30min血脑屏障的完整性无明显改变,随着CRM197浓度的增大,脑微血管内皮细胞中吞饮小泡的数量有一定程度的增加,未见紧密连接明显开放。结论CRM197以剂量依赖的方式增加血脑屏障的通透性,其机制主要与胞吞转运增加有关,血脑屏障的完整性不受影响。