Isolation and partial characterization of immunologically reactive fractions from chitinase digested cell wall of Trichophyton mentagrophytes

Summary Cell wall preparations ofTrichophyton mentagrophytes were digested with chitinase following which various fractions were isolated by ultrafiltration and Sephadex gel filtration. All fractions isolated contained both polysaccharide and peptide material. A correlation was seen between those fractions capable of eliciting immediate and delayed skin reactions in sensitized guinea pigs and those capable of stimulating thein vitro proliferation of lymphocytes taken from sensitized guinea pigs. These immunologically active fractions also developed precipitin lines with antiserum taken from sensitized animals. A low molecular weight fraction was found to be completely reactive immunologically (UM_2(a)), and appeared to have a molecular weight in the range of 2,000–4,000 as assessed by ultrafiltration and gel filtration studies.This work was supported by grant number 3411 from the Medical Research Council of Canada.A. Kh. Al-Rammahy was supported by a scholarship from the Ministry of High Education, Iraq.     

Biochemical and Immunological Studies on Aspergillus

Two kinds of polysaccharides, glucan and glycopeptide (galactomannan-peptide), were obtained from Aspergillus fumigatus (IFO 5840) by extraction with 50% pyridine and were purified by fractional precipitation with acetone, by a column chromatography on Dowex-50 and DEAE-cellulose and by the gel filtration on Sephadex G-50 and G-200. The glycopeptide, designated APSK-66 fraction, showed both an Arthus and delayed type (tuberculin type) skin reactions in sensitized rabbits and guinea pigs. By treatment with proteolytic enzymes, the delayed type skin reactivity of APSK-66 fraction was reduced but the Arthus type skin reactivity was not affected. However, the Arthus type skin reactivity of APSK-66 fraction was completely lost by periodate oxidation, and the delayed type skin reactivity of APSK-66 fraction was retained. The APSK-66 fraction showed precipitation, complement fixation and passive hemagglutination reactions with rabbit antisera against A. fumigatus. Glucan, designated as APSK-33 fraction, did not show any immunological activity when tested in the present experiment. The chemical structures of the glucan and galactomannan were discussed.     

The cellular immune response to synthetic peptides containing sequences known to be heptenic in performic acid-oxidized ferredoxin from Clostridium pasteurianum

The NH₂-terminal heptapeptide and the COOH-terminal pentapeptide of performic acid-oxidized ferredoxin from Clostridium pasteurianum have been shown to encompass the two major haptenic regions of this molecule. These peptides were conjugated to succinylated bovine serum albumin (S-BSA) to yield an immunologically bivalent hapten-carrier conjugate (N + C-S-BSA). Peptides were also synthesized which contained the NH₂-terminal and COOH-terminal haptenic peptides linked by a bridge of five amino acids (N-5-C), these two peptides linked by 10 amino acids (N-10-C), and one containing two COOH-terminal peptides linked by 12 amino acids (C-12-C). The ability of these preparations to elicit various immunological responses was tested. In O-Fd-sensitized guinea pigs, N + C-S-BSA, N-5-C, and N-10-C elicited immediate and delayed skin reactions; N-5-C and N-10-C inhibited the migration of macrophages; N + C-S-BSA and N-10-C stimulated the proliferation of lymphocytes from these sensitized animals, however, N-5-C and C-12-C did not. In animals sensitized to N + C-S-BSA, skin reactions were elicited by O-Fd, S-BSA, and the NH₂-and COOH-terminal peptides alone. In these animals, lymphocyte proliferation was stimulated significantly by either O-FD or S-BSA. The N-5-C peptide was found to be nonimmunogenic by the schedule used here. However, the N-10-C peptide was found to be strongly immunogenic, and, in animals sensitized to N-10-C, skin reactions and MIF were elicited by N-10-C and 0-Fd, and lymphocyte proliferation was stimulated by N-10-C and O-Fd, but not by C-12-C. The implications of these results in relation to the bicellular mechanism of the immune response are discussed.     

Immunological comparison of basic encephalitogen and histone F2A1.

The extent of immunological cross-reaction between basic encephalitogen and histone F2A1 on both the humoral antibody level and on the cellular level has been established. The extent of humoral cross-reaction was tested by direct complement fixation employing both anti-histone F2A1 and antisera to basic encephalitogen, by inhibition of complement fixation, by radioimmunoassay and by passive cutaneous anaphylaxis. The data obtained failed to reveal immunological cross-reaction at the cellular level was tested by the lymphocyte stimulation technique in rabbits and guinea pigs, by inhibition of lymphocyte stimulation and by delayed hypersensitivity skin reactions. A slight but significant cross-reaction between the two proteins on the cellular level was detected by inhibition of lymphocyte stimulation and by the delayed hypersensitivity test. It is concluded that the immunological studies provide limited evidence that the two proteins share antigenic determinants.     

Experimental avian aspergilosis

One-day-old chicks were infected by aerosolized conidia ofAspergillus fumigatus orA. flavus. Plaques containing viable organisms were observed on the third day of infection. Although plaques persisted for several weeks, cultures could not be recovered from them after about ten days. Flourescent antibody methods revealed an immune response to both agents within three days of challenge. Precipitin titers againstAspergillus active protein (AAP) did not appear until the tenth day, and was not detected after seven weeks. Aspergillus polysaccharide (APS) failed to react antigenically in infected birds. AAP was separated by Sephadex chromatography into nine distinct fractions. One fraction from each species was associated with an immediate and one with a delayed type of skin response in wattles. A low molecular weight fraction which lacked skin reactivity was an effective antigen in precipitin tests. Except for a weak skin reaction toA. fumigatus APS in rabbits, skin tests with extracts and APP fractions correlated with wattle reactions in chicks.     

Physico-chemical properties of microbial antigens and the immuno-allergic tests]

Experiments were conducted on guinea pigs sensitized by the delayed and immediate types of allergy. Different antigens obtained from the strain of Brucella abortus BA-19 were used for sensitization and the resolving action. Comparison of the resolving properties of the corpuscular, soluble (ultrasound treated) antigens and purified protein fractions, polysaccharide and RNA was carried out in the skin reactions of the immediate and delayed type, passive skin anaphylaxis, acute anaphylactic shock, and the Schults-Dale test. Immediate reactions to the purified protein fraction were weaker than those to the whole soluble antigen, by which the animals were sensitized. Polysaccharide and the RNA-fractions proved to be inactive in the allergic reactions.     

Vitamin B6 deficiency and the lymphoid system. II. Effects of vitamin B6 deficiency in utero on the immunological competence of the offspring.

Results of this study indicated that an absence of vitamin B₆ from the diet of pregnant rats led to reduced immunological competence in the offspring. While the numbers of cells in the thoracic duct lymph (TDL) of rats approximately 3 months old and progeny of vitamin B₆-deficient mothers were nearly equivalent to control values, such cells had a reduced capacity to respond in the mixed lymphocyte and normal lymphocyte transfer reactions. It is suggested that this reduction may have reflected (i) an alteration in the capacity of TDL cells or their precursors to give rise to immunologically competent cells, (ii) a shift in the proportions of T and B cells in the TDL, and/or (iii) an absence or ineffectiveness of a humoral factor required for the development of immunologically competent lymphocytes.     

Immunological Abnormalities and Hydantoins

Various immunological features were measured in 20 patients with epilepsy who had received prolonged treatment with hydantoins. Immunoglobulin A (IgA) was shown to be absent or low in five patients, β₁C/A was low in 10 patients. Five patients showed negative reactions to skin tests, and two could not be sensitized to dinitrochlorobenzene. The corresponding features were normal in 14 control patients with epilepsy but without hydantoins. It is suggested that the hydantoins influence humoral immunity, whereas other immunosuppressant agents have been found to affect cellular immunity.     

Detection of hypersensitivity toLegionella pneumophila in guinea pigs by skin test

Cross-reacting antigens of three serogroups ofLegionella pneumophila differed serologically and immunologically from the serogroup-specific antigens. Intradermal injection of cross-reacting antigens into sensitized guinea pigs evoked skin hypersensitivity. Animals were sensitized either by injection of inactivatedL. pneumophila in adjuvant or by infection with live organisms. Skin reactions were measurable about 2–4 h after injection and continued to increase in intensity for the first 24 h, followed by a gradual decline over the next 48 h. Histological examination of skin reactions taken from test sites at 48 h revealed infiltration of mononuclear cells in and about the small subcutaneous blood vessels and throughout the dermis, compatible with a delayed-type reaction. The overall appearance and time course of the reaction resembled a combination of immediate and delayed types of hypersensitivity. Each cross-reacting antigen of the three serogroups evoked skin reactions in animals which had been sensitized to any of those serogroups, but was not reactive in nonsensitized animals. These observations indicate the possibility of detecting present or past infection ofL. pneumophila by skin tests.     

Nonantibody-mediated suppression of delayed hypersensitivity to human gamma-globulin in mice.

Development of delayed hypersensitivity (DHS) to human γ-globulin (HIgG) in mice was documented by histological analysis, by the kinetics of footpad swelling in animals exhibiting humoral or delayed responses, and by the failure of sera to transfer delayed reactions to normal, syngeneic recipients. Since cyclophosphamide (CY) treatment resulted in diminished humoral and augmented delayed reactions, we used this as a tool to explore the nature of the regulatory mechanisms which affect expression of this type of cell-mediated immunity. In order to evaluate the effect which the presence or absence of antigen-specific cells might exert on expression of DHS, we subjected mice to experimental regimes which would result in lymphocyte proliferation or depletion, respectively (see Bachvaroff, R., and Rapaport, F. T., Cell. Immunol. 15, 336, 1975). Cell proliferation was induced by injection of 80 μg of aqueous antigen on Day ?4; this was followed by sensitization with HIgG-CFA (Freund's adjuvant) on Day 0, and footpad challenge on Day 13. These mice exhibited strong humoral reactivity; three of six died of anaphylaxis following footpad challenge, and the remaining three showed a diminished delayed response. Similarly treated mice that, in addition, received 6 mg of CY 3 days after injection of aqueous antigen and, therefore, would have antigen-specific cells present showed greatly diminished humoral reactivity, due to B-cell depletion. However, they also exhibited a marked diminution in delayed responsiveness. The data clearly demonstrate that a nonantibody-mediated, possibly cell-directed, regulatory influence is exerted on DHS where cell proliferation has occurred. We next examined the impact which the depletion of proliferating cells would exert on the expression of DHS. Cell depletion was attempted by giving one injection of aqueous antigen (Day 0) early in a regime of chronic CY administration (Days ?1 through +3) ; antigen-induced proliferating cells would be susceptible to CY and, therefore, depleted under these conditions. The results show that mice receiving both aqueous antigen and CY have depressed humoral and markedly diminished delayed reactivity compared to animals that were injected with CY alone. Thus, the augmenting effect which CY exerts on DHS is abrogated by stimulation with aqueous antigen. One interpretation is that CY removes a regulatory cell population in the normal animal, thereby allowing enhanced expression of delayed responsiveness. Clearly, regulatory function cannot be attributed solely to bumoral antibody production.     

Dynamics of lymphocyte subpopulations, immunoglobulins and specific antibodies in acute brucellosis

The main lymphocyte subpopulations (T-, B-, T mu-, T gamma, and L-lymphocytes), serum immunoglobulins (A, M, G), and specific antibodies have been analyzed at different periods of the development of acute brucellosis. The heterogeneous pattern of changes in the main characteristics of both humoral and cell-mediated immunity has been revealed. A definite interrelation of cell-mediated and humoral reactions in the immunological process in brucellosis has been established.     

Immunological studies on a yeast peptido-galactomannan. Nature of antigenic determinants reacting with rabbit antisera and those involved delayed hypersensitivity in guinea pigs.

The antigenicity of the peptido-phosphogalactomannan (PPGM) of Cladosporium werneckii and the carbohydrate and peptide moieties isolated from it were studied in rabbits and guinea pigs. In rabbits, the antisera produced against whole C. werneckii cells reacted only with the carbohydrate portions of the antigen. The major portion of the antibody was directed towards the long phosphogalactomannan carbohydrate chains. O-Acetyl substituents were shown to play important roles in the determinants on these chains since selective removal of these groups eliminated their reactivity with antibody. A relatively small amount of the antibody response was directed towards the more numerous, short, mannose-containing chains. Concanavalin A, on the other hand, precipitated the polymer by virture of its reactivity with these units and not with the phosphogalactomannan chains.Delayed type (cell-mediated) immunity to C. werneckii was studied in guinea pigs. PPGM was able to elicit strong delayed skin-tests as was a peptide-rich fraction derived from PPGM by mild acid hydrolysis. Pure carbohydrate chains of PPGM were completely inactive. In contrast, two of the modified peptides derived from PPGM were able to elicit a response although they were less active than PPGM. It was concluded that the determinants responsible for delayed hypersensitivity reside in the peptide portion of the antigen but that the technique used to isolate the peptides (alkaline β-elimination) partially destroyed the determinants.     

Enhancement of lipase stability and productivity through chemical modification and its application to latex-based polymer emulsions

The primary focus of this research was to employ amino-group specific chemical modifications for improving the productivity and stability of two commercially produced lipases, Lipase-A from Candida antarctica (CAL_UM) and Greasex from Humicola lanuginosa (HLL_UM), for application in a latex-based paint formulation. The modified lipases showed higher percentage increase (benzoic anhydride-modified, HLL_BA, 150%; PEG-modified, HLL_PEG,162% at 75 °C) as well as higher absolute productivities 41, 50, 52 and 53 μmole substrate mg⁻¹ lipase for unmodified, CAL_PEG, HLL_PEG and HLL_BA, respectively at 37 °C. The half-lives of thermal inactivation for all modified variants were improved from 40 to 166% at 50, 60 and 70 °C relative to unmodified lipases. The higher thermal stability and catalytic efficiency (k_cat/K_m) with concomitant lower activity (k_cat) indicates that enhanced productivity is likely to be due to the modified enzymes being better able to resist thermal denaturation over the time course of the productivity experiments. Importantly, both lipases, CAL_BA (60%) and HLL_BA (55%) retained the highest activity in paint compared with CAL_UM (36%) and HLL_UM (39%) after 20 weeks incubation at 25 °C. The long term stability of the modified lipases illustrates their potential value for commercial paint and other industrial applications.     

Antibodies to guinea pig lymphokines. II. Suppression of delayed hypersensitivity reactions by a "second generation" goat antibody against guinea pig lymphokines.

A "second generation" antibody to a highly purified lymphocyte product was raised in a goat against material eluted from a rabbit anti-guinea pig lymphokine immunoadsorbent column. This anti-lymphokine serum, in constrast to anti-lymphocyte serum (ALS) did not appear to contain cytotoxic antibodies directed against membrane antigens on guinea pig lymph node lymphocytes. Furthermore, the anti-lymphokine serum did not inhibit the formation of spontaneous T rosettes nor significantly depress lymphocyte response to mitogens. The anti-lymphokine serum totally suppressed the delayed skin reactivity to PPD and contact sensitivity to DNCB when injected intradermally around the site of antigen challenge. By contrast, intradermally injected ALS did not appear to suppress the PPD response in sensitized guinea pigs. Intravenously and i.p. administered anti-lymphokine serum was somewhat less effective in suppressing the delayed skin response to PPD. The intradermal injection of the antiserum had no effect on nonspecific inflammation evoked by turpentine-olive oil or on the extravasation of circulating Evans blue evoked by intradermally injected histamine. Histologic examination of 24-hr DNCB-induced skin lesions from sensitized guinea pigs treated with intradermally injected anti-lymphokine serum showed marked reduction of mononuclear infiltration of the dermis and of epidermal lesions, as compared with skin sites taken from sensitized animals pretreated with normal goat serum. The anti-lymphokine serum injected i.v. also markedly reduced the perivascular infiltration of the dermis and subcutis in skin reaction sites from sensitized animals challenged with PPD. Intravenous treatment with ALS for 3 consecutive days caused extensive depletion of the paracortical areas of peripheral lymph nodes whereas treatment with normal serum and anti-lymphokine serum caused no such depletion. It is proposed that the anti-lymphokine serum is directed against activated lymphocyte products, one of them being MIF. These products are involved in the mediation of delayed hypersensitivity reactions. This is in marked contrast to ALS, the suppressive action of which appears to be central rather than peripheral.     

Skin Reaction and Macrophage Migration Inhibition Tests for Polysaccharides from Aspergillus fumigatus and Candida albicans

Guinea pigs sensitized with purified galactomannan from Aspergillus fumigatus and mannan from Candida albicans, each containing negligible quantities of nitrogen, were examined for their immunological responses against the corresponding polysaccharides with respect to the delayed-type skin reaction and the macrophage migration inhibition phenomenon. In both cases, the delayed-type skin reaction test and the macrophage migration inhibition test showed positive results. The reactivity was stronger in animals sensitized with polysaccharides in Freund's complete adjuvant than those sensitized with the same polysaccharides in Freund's incomplete adjuvant. Polysaccharides chemically modified by partial acid degradation or by periodate oxidation were found to be completely incapable of eliciting such immune responses. These results are also discussed in relation to the antigenic determinant of the polysaccharides in such immune responses and the precipitin reaction previously observed by us and other investigators.     

Suppression of in vitro lymphocyte responsiveness to purified protein derivative by measles virus. A reexploration of the phenomenon.

Clinical measles and measles vaccination have classically been associated with transient in vivo impairment of delayed hypersensitivity-type responses, especially skin test reactivity to purified protein derivative (PPD). In vitro data appeared to substantiate this in vivo observation by the demonstration of suppression of lymphocyte responsiveness to PPD by measles. Utilizing a measles preparation which has been recently demonstrated to elicit specific blastogenesis of sensitized human lymphocytes in vitro, we have reexplored the question of in vitro suppression of lymphocyte responsiveness to PPD by this virus. In contrast to previous reports, this study demonstrates that the addition of both measles and PPD to lymphocyte cultures can have a variable effect on lymphocyte responsiveness to PPD alone. This effect varies from marked inhibition to enhancement beyond a summation effect. The response is different for each lymphocyte donor and is dose related but cannot be predicted on the basis of combinations of high or low concentrations of either antigen. Purified, attenuated measles virus (Enders' strain), which uniformly suppressed in vitro lymphocyte reactivity when tested alone also demonstrated a significant dose related enhancement of the response to PPD alone. The present data suggest a reconsideration of the supposed importance of transient diminution of skin test reactivity during measles infection or immunization.     

Study of the immunotropic activity of aminoglycoside antibiotics

The action of some aminoglycoside antibiotics on the immune system was studied on both intact mice and the animals with immune deficiency caused by administration of cyclophosphamide. The following tests were used: local hemolysis (the Herne test), lymphocyte transformation (LT), delayed hypersensitivity to sheep red blood cells and the local graft versus host reaction (GVHR). Amikacin was shown to have no significant action on the activity of lymphocytes in the intact mice and stimulated both cellular (LT and GVHR) and humoral (the Herne test) immunity in the animals with lowered immunological reactivity. Sisomicin had no significant action on the immune system of the animals. Gentamicin suppressed the immune response only in the intact mice. Kanamycin and streptomycin induced inhibition of humoral and cellular immunity in both the intact mice and animals with immune deficiency. On the basis of the results it was concluded that gentamicin, amikacin and sisomicin may be used in the treatment of diseases developing in the presence of immune deficiency whereas streptomycin and kanamycin should be recommended when inhibition of the immunity is needed.     

Modulation of the immune response by passive antibodies. III. Effects of IgG1 and IgG2 anti-carrier antibodies.

The effects of IgG₁ and IgG₂ anti-carrier antibodies were studied on cellular and humoral reactions induced by immunization with a hapten-carrier complex. IgG₁ was shown to depress both delayed hypersensitivity reactions (DHR) to the carrier and anaphylaxis to the hapten whereas IgG₂ had no activity. A mixture of IgG₁ and IgG₂ depressed only DHR to the carrier. The modulating effects of passive anti-carrier antibodies were shown to depend on their immunoglobulin class and the concentration used.     

Eimeria stiedai: delayed hypersensitivity response in rabbit coccidiosis.

The development of delayed hypersensitivity (DH) in rabbits infected with Eimeria stiedai was shown by skin testing. A particulate antigen fraction was prepared by extraction of nonsporulated E. stiedai oocysts and found to be effective in producing dermal induration similar to that seen in a tuberculin reaction. The average diameter was 9 mm (range 7–11.0 mm, n = 26) with an average thickness of 0.4–0.5 mm for infected rabbits. All skin reactions were negative in noninfected animals (0–3.0 mm diameter and 0–0.2 mm thickness). Histological examination of dermal reactions revealed mononuclear cell infiltration within 48 hr with areas of necrobiosis. Skin reactivity was passively transferred to noninfected rabbits with lymphocyte suspensions and cell-free transfer factor but not with serum from infected skin-reactive animals. Delayed hypersensitivity was detected in 11 of 28 infected rabbits at 10 days, and by 20–30 days, 53 of the 55 animals tested showed positive skin reactions.     

Gag protein epitopes recognized by CD4(+) T-helper lymphocytes from equine infectious anemia virus-infected carrier horses

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.