Adjuvant effect of liposomes in the autoimmune response to rat male accessory glands

Wistar rats submitted to 3 intravenous (i.v.) or intraperitoneal (i.p.) immunizations with saline extract of rat male accessory glands (RAG) associated to liposomes developed a delayed type hypersensitivity (DTH) response to RAG after the first immunization, a remission state after the second immunization and a specific DTH response after the third injection. In a further study we transferred spleen mononuclear (SpM) cells from i.p. immunized rats taken 10 days after the second immunization (DTH negative) to normal or immunized recipients 24 h before or 10 days after the first immunization with RAG liposomes, respectively. The DTH response was reduced only in recipients previously immunized. Besides, it was possible to show that the transfer of SpM cells present when the response increased after the third injection in i.p. immunized donors reduced the suppression observed after the second injection. Rat accessory gland biopsies taken 10 days after the last immunization showed in nearly all cases mast cells, plasma cells and eosinophils with scarce lymphoid elements and increased acinar desquamation. This kind of infiltration had characteristics of cutaneous basophil hypersensitivity.     

IMMUNOPATHOLOGICAL CHARACTERIZATION OF THE DELAYED HYPERSENSITIVITY SKIN REACTION TO CARRIER PROTEIN

Characteristics of the allergic skin reaction to free carrier protein in sensitized guinea pig with hapten-carrier conjugate have been studied. The animals sensitized with a low dose (4 μg) of dinitrophenylated bovine gamma globulin with Freund's complete adjuvant demonstrated a dermatitis when challenged intradermally with a small dose (10 μg) of bovine gamma globulin en the 10th or 14th day. The dermatitis was grossly and histologically similar to the classical form of delayed-type hypersensitivity skin reaction except lesser induration and less abundant neutrophil infiltration in the former. This reaction was clearly different from cutaneous basophil hypersensitivity because of the long lasting time course and fewer basophil infiltration in the former. No humoral antibody to carrier protein was detected in the sera of the sensitized animals by passive cutaneous anaphylaxis or by immunodiffusion in agar plates, and the state of the hypersensitivity could be passively transferred with peritoneal exudate cells and spleen cells. These observations indicated that the skin reaction to carrier protein might be of primarily delayed-type hypersensitivity. The vascular permeability change at the reaction sites was observed as a single phase delayed response and preceded the maximum erythema by about ten hours.     

Dissociation of erythema and basophil accumulation in Jones-Mote type hypersensitivity in the guinea-pig.

Relationship between delayed-in-onset erythema and infiltrating leucocytes in the skin reaction site of Jones-Mote type hypersensitivity was observed in guinea-pigs. Guinea-pigs were immunized with formalin-fixed (F-SRBC) or native form (N-SRBC) of sheep red blood cells in incomplete Freund''s adjuvant (IFA). Elicitation was carried out with intradermal injection of F-SRBC or N-SRBC in saline 1 or 3 weeks after immunization. One week after immunization with F-SRBC or N-SRBC in IFA, erythema accompanied by basophil infiltration was detected, but antibody production was negative. Erythema reached a peak at 24 h, but basophil infiltration reached a peak at 48 h or later. In the skin reaction site elicited with N-SRBC, high levels of basophil infiltration were detected persistently even after erythema had disappeared. Three weeks after immunization, low titres of PCA (passive cutaneous anaphylaxis) were detected in guinea-pigs immunized with F-SRBC in IFA. At that time erythematous skin reaction was detected, but the level of basophil infiltration was lower than that of 1 week after immunization. In guinea-pigs immunized with N-SRBC in IFA, skin reaction of the Arthus type accompanied by haemorrhage was observed at the site elicited with N-SRBC.     

Contact sensitivity to oxazolone in the chicken: evidence for Arthus type hypersensitivity of the cutaneous reaction.

Cutaneous hypersensitivity reaction can be induced in chickens by skin painting with oxazolone, 33 mg/Kg of body weight (KBW). The B cell contribution to the generation of the cutaneous reaction has been a matter of controversy. In an attempt to characterize this reaction we placed special interest on the possibility that the nature of this reaction could be Arthus type hypersensitivity. From the kinetics study on the cutaneous hypersensitivity after challenge with oxazolonated egg-albumin (EA-OX) it was excluded that the nature of this reaction could be delayed type hypersensitivity. Immune sera transfer experiments demonstrated that the cutaneous reaction was antibody dependent. Serum anti-oxazolone antibody titers in sensitized chickens were assayed by antiglobulin haemagglutination, using oxazolone coupled sheep erythrocytes (OX-SRBC). High titres of IgG were found in contact sensitized chickens. Furthermore this cutaneous reaction was characterized by neutrophils, inflammatory edema, rare thrombotic occlusion of small venules and on absence of monocytes. The utilization of complete Freunds' adjuvant (CFA) given at sensitization demonstrated that CFA enhanced oxazolone antibodies in the sera of immunized chickens without a correlated increase in the intensity of the cutaneous reaction to EA-OX. Animals sensitized to oxazolone (33 mg/KBW) without CFA and challenged intravenously seven days later with oxazolone coupled to autologous chicken red blood cells (OX-CRBC) died from anaphylactic shock; instead animals with the same treatment but with CFA given at sensitization did not die from anaphylactic shock. Taken collectively it was concluded that the cutaneous reaction to oxazolone in the chicken can be categorized as Arthus hypersensitivity. The relationship between cutaneous Arthus reaction and anaphylactic shock in chickens sensitized to oxazolone is discussed.     

Carrier requirement for development of acute experimental hypersensitivity pneumonitis in the rabbit

Fluorescein isothiocyanate (FITC) conjugated to protein carriers was used to explore carrier dependence in an established rabbit model of acute hypersensitivity pneumonitis (HSP). Rabbits were immunized via toepads with either FITC-ovalbumin (OA) or FITC-human gamma-globulin (HGG) in complete Freund's adjuvant, and were aerosol challenged with homologous or heterologous conjugates 30 days later. Only those rabbits challenged with the homologous carrier developed acute HSP, despite the presence of comparable levels of anti-FITC antibodies in the sera of all groups. These findings indicate a strict carrier dependence in the pathogenesis of HSP in this model and provide further evidence that the mechanism of inflammation depends upon a cellular immune response.     

Contact Sensitivity to Oxazolone in the Chicken: Evidence for Arthus Type Hypersensitivity of the Cutaneous Reaction

Cutaneous hypersensitivity reaction can be induced in chickens by skin painting with oxazolone, 33 mg/Kg of body weight (KBW). The B cell contribution to the generation of the cutaneous reaction has been a matter of controversy. In an attempt to characterize this reaction we placed special interest on the possibility that the nature of this reaction could be Arthus type hypersensitivity. From the kinetics study on the cutaneous hypersensitivity after challenge with oxazolonated egg-albumin (EA-OX) it was excluded that the nature of this reaction could be delayed type hypersensitivity. Immune sera transfer experiments demonstrated that the cutaneous reaction was antibody dependent. Serum anti-oxazolone antibody titers in sensitized chickens were assayed by antiglobulin haemagglutination, using oxazolone coupled sheep erythrocytes (DX-SRBC). High titres of IgG were found in contact sensitized chickens. Furthermore this cutaneous reaction was characterized by neutrophils, inflammatory edema, rare thrombotic occlusion of small venules and on absence of monocytes. The utilization of complete Freunds' adjuvant (CFA) given at sensitization demonstrated that CFA enhanced oxazolone antibodies in the sera of immunized chickens without a correlated increase in the intensity of the cutaneous reaction to EA-OX. Animals sensitized to oxazolone (33 mg/KBW) without CFA and challenged intravenously seven days later with oxazolone coupled to autologous chicken red blood cells (OX-CRBC) died from anaphylactic shock; instead animals with the same treatment but with CFA given at sensitization did not die from anaphylactic shock. Taken collectively it was concluded that the cutaneous reaction to oxazolone in the chicken can be categorized as Arthus hypersensitivity. The relationship between cutaneous Arthus reaction and anaphylactic shock in chickens sensitized to oxazolone is discussed.     

Relationship between the tuberculin-type and Jones-Mote-type hypersensitivities: suppression of basophil infiltration by mycobacterial adjuvant

Guinea-pigs immunized with bovine gammaglobulin (BGG) in incomplete Freund's adjuvant (IFA) showed the typical Jones-Mote-type hypersensitivity (JMH) reaction when tested 5 days later. This is characterized by prominent basophil infiltration. After pretreatment with complete Freund's adjuvant (CFA) 16 days before immunization with BGG in IFA, the lesions resembled the JMH reaction macroscopically in their evolution over time and in the absence of a positive macrophage migration inhibition (MIT) test. However, histologically, the lesions resembled classical tuberculin-type hypersensitivity with prominent mononuclear cell infiltration without any basophils. The pretreated animals, which failed to show basophil infiltration, were able to transfer JMH reactions with basophil infiltration into normal animals. In contrast, pretreatment of recipients with CFA or Corynebacterium parvum prevented the passive transfer of the characteristic effect on the JMH reaction when given shortly before skin testing. We postulate that macrophages activated by CFA may play an important role in regulating basophil infiltration in the effector phase of the delayed hypersensitivity reaction.     

Contact Sensitivity to Oxazolone in the Chicken: Evidence for Arthus Type Hypersensitivity of the Cutaneous Reaction

Abstract

Cutaneous hypersensitivity reaction can be induced in chickens by skin painting with oxazolone, 33 mg/Kg of body weight (KBW). The B cell contribution to the generation of the cutaneous reaction has been a matter of controversy. In an attempt to characterize this reaction we placed special interest on the possibility that the nature of this reaction could be Arthus type hypersensitivity. From the kinetics study on the cutaneous hypersensitivity after challenge with oxazolonated egg-albumin (EA-OX) it was excluded that the nature of this reaction could be delayed type hypersensitivity. Immune sera transfer experiments demonstrated that the cutaneous reaction was antibody dependent. Serum anti-oxazolone antibody titers in sensitized chickens were assayed by antiglobulin haemagglutination, using oxazolone coupled sheep erythrocytes (DX-SRBC). High titres of IgG were found in contact sensitized chickens. Furthermore this cutaneous reaction was characterized by neutrophils, inflammatory edema, rare thrombotic occlusion of small venules and on absence of monocytes. The utilization of complete Freunds' adjuvant (CFA) given at sensitization demonstrated that CFA enhanced oxazolone antibodies in the sera of immunized chickens without a correlated increase in the intensity of the cutaneous reaction to EA-OX. Animals sensitized to oxazolone (33 mg/KBW) without CFA and challenged intravenously seven days later with oxazolone coupled to autologous chicken red blood cells (OX-CRBC) died from anaphylactic shock; instead animals with the same treatment but with CFA given at sensitization did not die from anaphylactic shock. Taken collectively it was concluded that the cutaneous reaction to oxazolone in the chicken can be categorized as Arthus hypersensitivity. The relationship between cutaneous Arthus reaction and anaphylactic shock in chickens sensitized to oxazolone is discussed.     

A reliable method for reconstituting thymectomized, lethally irradiated guinea pigs with bone marrow cells

We developed a reliable method for reconstituting thymectomized, lethally irradiated guinea pigs. Injection of 2.5 − 10 × 10⁷ syngeneic bone marrow cells into adult thymectomized, lethally irradiated guinea pigs produced survival of 46–100% of treated animals. Gentamycin sulfate (5 mg/kg of body weight) for 10 days was required for optimal results. Acidified drinking water (pH 2.5) appeared to be required for optimal results. Thymectomized, lethally irradiated, bone marrow reconstituted (‘B’) guinea pigs had impaired ability to develop delayed cutaneous hypersensitivity to mycobacterial antigens and cutaneous basophil hypersensitivity to keyhole limpet hemocyanin; proliferative responses to phytohemmagglutinin were impaired.     

Delayed-type hypersensitivity measured as an increase of ear thickness in guinea pigs.

Determination of swelling at an intracutaneous test site in the pinna of the ear of guinea pigs immunized with protein antigens in complete Freund's adjuvant was found to be a more sensitive assay of delayed-type hypersensitivity than the measurement of flank skin erythema and induration. In fact, 100 times less specific antigen was needed to detect 24-hour reactivity in the pinna of the ear. Reactivity early after sensitization (cutaneous basophil hypersensitivity), however, was best revealed as an erythematous lesion of the flank skin 24 h after testing.     

The effect of cyclosporine on immunization with tetanus and keyhole limpet hemocyanin (KLH) in humans

Eleven patients with chronic uveitis treated with Cyclosporine were immunized with keyhole limpet hemocyanin (KLH) and tetanus toxoid. Delayed cutaneous hypersensitivity responses, lymphocyte blastogenic responses, and antibody production were compared with those of similarly immunized control individuals. A significant decrease in delayed cutaneous hypersensitivity (P<0.001 for KLH andP<0.01 for tetanus toxoid) was observed. No significant differences in blastogenic or antibody responses were noted. These findings demonstrate that the majority of the Cyclosporine-treated patients had intact T cell-dependent antigen responses as measured by both proliferative response and antibody production to primary and secondary antigenic challenges but that other immune functions such as delayed cutaneous hypersensitivity are affected by therapeutic doses of systemic Cyclosporine.     

Zymosan-induced experimental hypersensitivity pneumonitis in rabbits.

An experimental model of hypersensitivity pneumonitis is presented. New Zealand white rabbits, previously immunized against yeast-derived zymosan, reacted to intratracheal challenge developing extensive pneumonitis. The lesions healed in a few weeks. Control animals challenged with inert particulate material (latex beads) or suspending fluid (PBS-Mg++) did not show pulmonary inflammation. Nonimmunized rabbits developed only transient pneumonitis after zymosan challenge. This reaction was clearly different from that seen in the group of immunized animals. The model reveals that biologically active substances such as zymosan, which is able to activate the alternate pathway of complement and mononuclear phagocytes, requires an active immune state in order to cause significant tissue damage. Isolated exposure to this kind of substance may not be sufficient to cause lung disease.     

Cutaneous basophil hypersensitivity and inhibited macrophage migration in guinea-pigs with schistosomiasis

In guinea-pigs infected with schistosomes, delayed cutaneous reactions rich in basophils (CBH) were found to characterize skin test responses to schistosome egg antigens. In addition, strong contact hypersensitivity-like skin eruptions with large basophil infiltrates resulted from skin penetration challenge by live cercariae (larvae) in these animals. Oedema and diminished basophil granule staining were noted around schistosomula which had penetrated the skin of sensitized animals. CBH responses to egg antigens and to live cercarial challenges were also noted after immunization with a single injection of dead cercariae.

Using peritoneal exudates from guinea-pigs immunized with dead cercariae or infected with schistosomes, direct macrophage migration inhibition with schistosome antigens was found only in animals with infections. Thus, CBH correlated with intradermal exposure to schistosome cercarial antigens, while MMI correlated with live infections. It is suggested that cutaneous basophil responses may play a role in protection from re-infection with schistosomes, and that dead cercarial vaccines might stimulate this beneficial response, without immunizing for potentially harmful granulomatous hypersensitivity.

     

Immediate moxifloxacin hypersensitivity: Is there more than currently meets the eye?

Immediate drug hypersensitivity reactions (IDHR) to moxifloxacin constitute a pathomechanistic conundrum and a diagnostic challenge. Our objective was to study whether simultaneous phenotyping and quantification of histamine release might add to our knowledge about the basophil activation properties of moxifloxacin and constitute a reliable diagnostic aid. Fifteen patients with an IDHR to moxifloxacin and nine moxifloxacin challenged controls were selected. All had a basophil activation test (BAT) with moxifloxacin. Flow cytometric analysis of basophil responses implied labeling for CD63, CD203c, and intracellular histamine. Unlike tolerant challenged controls, basophilic upregulation of CD203c in response to moxifloxacin was observed in seven of 15 patients. Only two of these seven patients demonstrated appearance of CD63 and release of histamine. In the remainder eight patients, no basophil responses were demonstrable. In conclusion, immediate hypersensitivity to moxifloxacin might involve mechanisms difficult to capture by traditional CD63‐/CD203c‐based BAT. Deciphering the complexity of quinolone IDHR seems mandatory.     

Enhancement of Pseudomonas aeruginosa lung clearance after local immunization with a temperature-sensitive mutant.

We investigated the capacity of the temperature-sensitive mutant strain A/10/25 of Pseudomonas aeruginosa (ts-Psa) to induce enhancement of lung defenses against wild type P. aeruginosa (wt-Psa). Mice of the DBA/2J inbred strain were immunized by aerosolization with a single dose of 2 x 10(5) to 4 x 10(5) CFU of ts-Psa and were challenged 7, 14, and 21 days later with wt-Psa. The uncleared bacteria ratio was determined 4 h after aerosol exposure; significant enhancement in lung clearance of wt-Psa (P less than 0.01) was evident as early as 7 days after immunization and detectable for at least 21 days. Aerosol immunization with Staphylococcus aureus did not enhance lung clearance of wt-Psa; however, slight but significant enhancement in S. aureus clearance was observed in mice immunized 7 days before with ts-Psa. No enhancement of S. aureus clearance was seen in ts-Psa immunized animals after 14 and 21 days. Analysis of the cell composition of lung lavage fluids revealed a transient cell response characterized by rapid increase in the absolute number of polymorphonuclear leukocytes, followed later by an increase in alveolar macrophages. The characteristics of lung lavages returned to base-line values 6 days after aerosol immunization, and a second exposure to a ts-Psa aerosol produced a response of similar magnitude and quality. We conclude that aerosol immunization with a temperature-sensitive mutant of P. aeruginosa enhances specific pulmonary defense mechanisms against the parental pathogen in mice.     

Delayed-type hypersensitivity in mice immunized with Trypanosoma rhodesiense antigens.

When mice were immunized intravenously, subcutaneously, or by the footpad route with formaldehyde-killed Trypanosoma rhodesiense, delayed-type hypersensitivity was elicited by the use of frozen-thawed trypanosomal antigen. The delayed footpad swelling technique was used to measure delayed hypersensitivity. Hypersensitivity induction was dose dependent (greater than or equal to 10(6) formaldehyde-treated T. rhodesiense) and was affected by the route of immunization. The footpad route induced higher levels of hypersensitivity than other routes of immunization. Mice immunized with a single dose of formaldehyde-treated antigen and challenged with live T. rhodesiense did not survive. Yet, mice immunized subcutaneously with formaldehyde-treated antigen and then injected with frozen-thawed antigen and challenged 28 days after immunization survived. The results suggest that T-cell activation, manifested by delayed hypersensitivity responses, was a necessary component in the protective response, perhaps functioning in a helper cell capacity.     

Cell-mediated and Humoral Immune Responses to Cartilage Antigenic Components

Cell-mediated immunity and antibody production to cartilage antigens were studied in rabbits. From day 3 of immunization an inhibition of leucocyte migration was observed in animals immunized with collagen-free fractions of bovine nasal or human rib cartilage. Delayed cutaneous hypersensitivity was elicited between 5 and 6 days and the circulating antibodies were demonstrated by passive haemagglutination on day 9. No significant correlation was observed between the antibody production and the cell-mediated immunity. Cell-mediated immune responses to cartilage antigens and to purified protein derivative were distinctly different, but the two antigens influenced one another when they were administered together. The 'species common' antigen of connective tissues may be primarily responsible for the early immune reactions.     

Omalizumab-induced reductions in mast cell Fce psilon RI expression and function

BACKGROUND: By design, omalizumab binds free IgE in the circulation and prevents its attachment to the surface of mast cells and basophils, thereby preventing them from responding to allergens. Previously, omalizumab rapidly reduced free IgE levels, as well as basophil high-affinity IgE receptors, leading to significant reductions in basophil mediator response to allergen. It is assumed that tissue mast cells are similarly altered in their Fc epsilon RI density and function. OBJECTIVE: We examined the phenotypic shift of skin mast cells in parallel to that of blood basophils in 3 subjects infused with omalizumab. METHODS: Three subjects with allergic rhinitis underwent intradermal skin test titration with house dust mite antigen at days 0, 7, 70, and 196 of omalizumab treatment. As control subjects, 5 untreated subjects with allergic rhinitis were evaluated at similar time points. All subjects underwent skin biopsy 18 to 24 hours later at the site of allergen injection. Biopsy specimens were characterized by means of immunohistochemisty for tryptase and Fc epsilon RI alpha immunoreactivity, as well other markers (CD3, CD45RO, CD68, cutaneous lymphocyte antigen, and major basic protein). RESULTS: Omalizumab recipients, but not control subjects, demonstrated reductions in Fc epsilon RI alpha immunoreactivity at days 70 and 196 in parallel with reductions in the acute wheal response to allergen. However, no reductions in tryptase-positive cells were noted at these time points. CONCLUSION: Reductions in free IgE levels by omalizumab leads to a rapid reduction in basophil Fc epsilon RI receptor expression. In contrast, the time course for the decrease of Fc epsilon RI expression in skin mast cells is slower and associated with decreased acute allergen wheal size.     

Nonspecific and Candida-specific immune responses in mice suppressed by chronic administration of anti-mu

CBA/J mice were immunosuppressed by repeated administration of goat antibody specific for mu chain of immunoglobulin M (IgM) and tested for nonspecific and Candida albicans-specific immune responses. Immunosuppression was demonstrated by a dramatic reduction in the number of antibody-forming cells in the spleens of anti-mu-treated mice when immunized with sheep erythrocytes, by greatly reduced in vitro responsiveness of both spleen and lymph node lymphocytes from anti-mu-treated mice to lipopolysaccharide, and by a large reduction in the number of splenic IgM-positive cells. T cell function, on the other hand, appeared to be relatively unaltered in anti-mu-treated animals, in the cytotoxic T lymphocyte activity against an allogeneic target was similar in splenocyte cultures from anti-mu- and mock-treated animals, and splenic and lymph node lymphocytes proliferated in response to concanavalin A in a lymphocyte stimulation assay. Moreover, Candida-specific delayed hypersensitivity to two different Candida antigens, one cell wall-derived (GP) and the other cell membrane-derived (BEX), was of comparable intensity in immunosuppressed and normal animals. When anti-mu- and mock-treated mice were immunized by the cutaneous inoculation of viable C. albicans blastospores and then challenged intravenously to assess the development of protective immunity, only mock-treated animals, male and female, had significant (p less than or equal to 0.05) protective responses demonstrable by reduction in the number of colony-forming units cultured from their kidneys 28 days after intravenous challenge. If consideration was given to the number of animals which had cleared Candida completely from the kidney, however, there appeared to be protective responses operative in the female anti-mu-treated animals as well. Neither anti-mu-treated males nor females, when immunized and challenged with C. albicans, produced Candida-specific antibody detectable by counterimmunoelectrophoresis, whereas all immunized and challenged mock-treated animals produced antibody. The data are consistent with the hypothesis that anti-mu treatment has little effect on multiple cellular immune functions, including those specific for C. albicans, and the combination of antibody, cell-mediated immunity and innate defenses are responsible for solid systemic defense against the fungus.