Delayed Hypersensitivity Reactions Provoked by Ribosomes from Acid-Fast Bacilli I. Ribosomal Isolation, Characterization, Delayed Hypersensitivity, and Specificity

Ribosomes and ribosomal subunits of Mycobacterium bovis (strain BCG) and M. smegmatis have been isolated and employed as skin test antigens in guinea pigs sensitized with homologous or heterologous organisms. Ribosomes and ribosomal subunits were found to be potent antigens for skin test purposes, and the 30S subunits were found to be more specific and active than the 50S subunits.     

Delayed Hypersensitivity Reactions Provoked by Ribosomes from Acid-Fast Bacilli: Physical Characteristics and Immunological Aspects of Core Ribosomal Proteins from Mycobacterium smegmatis

Ribosomal subunits from Mycobacterium smegmatis were analyzed by using sedimentation velocity, sedimentation equilibrium, and acrylamide gel electrophoresis experiments. These s(20.w) values for the subunits are 48.7S and 28.1S. The molecular weight of the 49S subunit is about 1.65 x 10(6), and that of the 28S subunit is 7.8 x 10(5). Both subunits contain about 37% protein and 63% ribonucleic acid. A protein-deficient particle having an s(20.w) value of 15.7S contains about 11% protein and 89% ribonucleic acid. Skin tests showed all subunits and proteins to be active as agents in provoking delayed hypersensitivity, but the 16S protein-deficient particle, as well as the proteins derived from it, was more specific than the subunits themselves.     

In Vitro Correlates of Delayed Hypersensitivity in Man: Ambiguity of Polymorphonuclear Neutrophils as Indicator Cells in Leukocyte Migration Test

Delayed cutaneous hypersensitivity (DCH) of 12 normal adult subjects to purified protein derivative (PPD) of Mycobacterium tuberculosis, streptococcal streptokinase-streptodornase (SK-SD), and Candida albicans Dermatophytin O (DO) was assayed in vivo by skin testing and compared with such in vitro correlates of cellular immunity as lymphocyte transformation (LT) and inhibition of leukocyte migration (ILM) from microcapillary tubes or in agarose gel. LT was shown to be the best in vitro correlate of specific lymphocyte sensitization with all antigens. In the ILM assays, PPD showed good correlation with in vivo DCH and in vitro LT; SK-SD showed partial correlation; DO showed no correlation, not being active in any of the ILM tests. Cell distribution and morphology of stained migration patterns, ILM tests performed on separated populations of lymphocytes and polymorphonuclear leukocytes (PMN), as well as the ability of test antigens to stimulate PMN cells to reduce nitroblue-tetrazolium dye, indicated that in ILM tests mononuclear cells were not inhibited in their migration, whereas migration of PMN cells appeared to depend on their direct reaction with the test antigens.     

Lymphoid Cells in Delayed Hypersensitivity II. In Vitro Phagocytosis and Cellular Immunity

Guinea pigs which develop delayed hypersensitivity, as measured by skin test, after intraperitoneal infection with Candida albicans have mononuclear cells (macrophages and lymphocytes) which show migration inhibition in vitro in the presence of soluble antigen. Macrophages from such animals phagocytose in vitro fewer numbers of C. albicans per cell than those of normal animals, and lower numbers of macrophages are phagocytic in vitro. Nevertheless, such sensitized animals have lower numbers of pathogens in their tissues than the controls, after both groups have been challenged. Thus, the result of macrophages from sensitized guinea pigs being less mobile, less phagocytic, and more destructive than those from normal animals may be a more limited spread of the infectious agent throughout the tissues; the animal would seem more resistant.     

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.     

Human Schistosomiasis: Modulation of In Vitro Granulomatous Hypersensitivity and Lymphocyte Proliferative Response by Macrophages Undergoing Differentiation

The mechanisms associated with the modulation of immune response in the chronic phase of human schistosomiasis mansoni infection are complex and involve many cell types. In the present paper the authors demonstrate that antigenic stimulation of peripheral blood mononuclear cells (PBMC) from chronic-intestinal schistosomiasis mansoni patients with polyacrylamide beads (PB) conjugated to Schistosoma mansoni soluble egg antigens (PB-SEA) or adult worm antigen preparation (PB-SWAP) were able to induce a statistically significant increase on the in vitro multinucleated giant cell (MGC) formation after the 15th day in culture. A correlation between an increase in the number of MGC and a decrease in in vitro granuloma formation index to PB-SEA and PB-SWAP was observed. Moreover, the authors demonstrated a down-regulation of lymphocyte proliferative responses to S. mansoni antigens, during the differentiation pathway of monocytes towards MGC formation, due to a decrease in the antigen-presenting capacity of these cells. These phenomena also correlate with a concomitant decrease in the expression of HLA-DR and CD54 adhesion molecules on the surface of MGC. The results suggest that differentiation of monocytes to MGC may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs.     

Corticosteroids, Serum, and Phagocytosis: In Vitro and In Vivo Studies

The phagocytic-bactericidal capacity (PBC) of human polymorphonuclear leukocytes (PMNs) for strains of Klebsiella (K) and of Enterococcus (E) was unaffected in vitro by the presence of 100 mug of either hydrocortisone (HC) or of methylprednisolone (MP) per ml in the medium. At higher concentrations (500 to 2,000 mug/ml) both compounds impaired PBC-K and PBC-E, but the latter was less sensitive to steroid-induced inhibition. In addition to interfering with intracellular killing of both organisms by PMNs, 2,000 mug of HC per ml also inhibited ingestion of E, although not of K. Steroid-induced inhibition of PBC-K in vitro was completely abolished by increasing the concentration of serum used as opsonin. The PBC-K of human PMNs obtained 30 min after intravenous injection of 1 g of MP was unimpaired in vitro in the presence of 10 to 90% simultaneously obtained autologous serum containing 42 mug of MP per ml. These findings suggest that short-term, high-dosage administration of MP is unlikely to produce clinically significant impairment of intraleukocytic bacterial killing.     

Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) or with DT-anti-toxin precipitate, both in Freund's complete adjuvant (FCA). DT-induced inhibition of peritoneal cell migration was equally strong in both groups and increased with time after immunization. Some guinea-pigs immunized with DT-antitoxin precipitate were boosted with DT or DT-antitoxin precipitate, both in FCA. This increased the antibody titre but did not affect 24-hour skin reactivity or migration inhibition. Migration inhibition did not correlate with anti-DT passive haemagglutinins, haemolysins, or cytophilic antibody on peritoneal cells, but did correlate with 24-hour skin reactivity.     

Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) in Freund's complete adjuvant (FCA) and boosted three times with DT in saline. Boosting increased anti-DT passive haemagglutinins, haemolysins, and cytophilic antibody on the surface of peritoneal exudate cells, but skin reactions to DT became negative and peritoneal cell migration was no longer inhibited by DT. When normal peritoneal cells were incubated in the sera of boosted animals, their migration was not inhibited by antigen, although they had cytophilic antibody on their surface as shown by rosette formation.     

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.     

In Vivo Splenic Clearance Correlates with In Vitro Deformability of Red Blood Cells from Plasmodium yoelii-Infected Mice

Recent experimental and clinical studies suggest a crucial role of mechanical splenic filtration in the host''s defense against malaria parasites. Subtle changes in red blood cell (RBC) deformability, caused by infection or drug treatment, could influence the pathophysiological outcome. However, in vitro deformability measurements have not been directly linked in vivo with the splenic clearance of RBCs. In this study, mice infected with malaria-inducing Plasmodium yoelii revealed that chloroquine treatment could lead to significant alterations to RBC deformability and increase clearance of both infected and uninfected RBCs in vivo. These results have clear implications for the mechanism of human malarial anemia, a severe pathological condition affecting malaria patients.     

Cell-Mediated Immunity in Humans During Viral Infection: Dermal Hypersensitivity and In Vitro Lymphocyte Proliferation During Mild Viral Respiratory Infections

In vitro lymphocyte proliferation in response to phytohemagglutinin and streptokinase-streptodornase and delayed dermal hypersensitivity to several antigens were assessed in patients with mild viral upper respiratory infections. The response to phytohemagglutinin in 18 patients was not diminished during the viral infection. Deoxyribonucleic acid synthesis induced by streptokinase-streptodornase remained unaffected by the viral illness in five patients, and skin test reactivity was not depressed during the viral infection. Thus, it appeared that localized viral upper respiratory infections were not associated with suppression of systemic cell-mediated immunity.     

Immunogenicity and In Vitro and In Vivo Protective Effects of Antibodies Targeting a Recombinant Form of the Streptococcus mutans P1 Surface Protein

Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1_39–512), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1_39–512 in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1_39–512 preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1_39–512-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1_39–512 antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1_39–512 as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.     

Differential Modulation of Contact Hypersensitivity and Delayed Hypersensitivity Reactions by Topical Application of 12-0-Tetradecanoylphorbol-13-Acetate

Ear/footpad swelling following sensitization and challenge with 2,4-dinitrofluoro-benzene (DNFB)/allogenic splenocytes (AS) were used to monitor the effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on contact hypersensitivity (CHS) and delayed hypersensitivity (DHS) reactions, respectively. Topical treatment of dorsal or ventral SENCAR mouse skin 4x with 2 μg of TPA prior to sensitization of dorsal skin with DNFB suppressed attempts to induce CHS by susequent challenge with DNFB. the adoptive transfer of splenocytes isolated from mice pretreated on the dorsum with TPA prior to dorsal sensitization with DNFB inhibited the development of CHS to DNFB in recipient mice. Conversely, topical treatment with TPA prior to s.c. sensitization with AS neither suppressed subsequent attempts to induce DHS, nor resulted in the generation of a splenocyte population capable of suppressing DHS reactions in adoptive transfer studies. Thus, promoting doses of topically applied TPA has differential effects on CHS and DHS reactions.     

In Vitro Elicitation of Intestinal Immune Responses in Teleost Fish: Evidence for a Type IV Hypersensitivity Reaction in Rainbow Trout

In fish the gut immune system has been the subject of few investigations until now. Here, we provide novel morphological and immunological data on the gut isolated from rainbow trout Salmo gairdneri. The pyloric (P) and terminal (T) segments of trout gut, when morphologically examined, evidenced lymphocytes and macrophages (MØ) loosely dispersed in the intestinal mucosa and in the lamina propria in the absence of typical Peyer's patches-like structures. Furthermore, incubation of P and T sections with Candida albicans (Ca) and functional analysis of supernatants generated some interesting results. In fact, active supernatants, when compared with controls, exhibited cytokine-like activities attributable to the presence of interferon (IFN)-γ and migration inhibiting factor (MIF), respectively. In particular, IFN-γ-like activity gave rise to an enhancement of Ca phagocytosis by MØ, whereas MIF inhibited MØ migration in agarose. Taken together, these in vitro data suggest that the gut-associated lymphoreticular tissue in fish possesses the appropriate armamentarium to mount a type IV hypersensitivity response when challenged by microbial antigens.