Tests for Antibody- and Cell-Mediated Hypersensitivity to Trypanosome Antigens in Rabbits Infected with Trypanosoma congolense

Rabbits infected with Trypanosoma congolense were tested for immediate and delayed-type hypersensitivity responses to parasite antigens. Positive skin-test reactions were exclusively of the Arthus type, as shown by sequential histological analyses which revealed that more than 90% of the infiltrating cells at 24 hr postinjection were polymorphonuclear neutrophils. Skin reactions could be elicited in normal rabbits by intradermal injection of trypanosome antigen-antibody complexes. The absence of cell-mediated hypersensitivity to trypanosome antigens in infected rabbits was demonstrated by skin tests and by migration inhibitory factor and skin reactive factor tests. The role of the immediate-type skin reaction to trypanosome antigens in the pathology of infection and the possibility of its utilization for diagnosis are discussed.     

Delayed hypersensitivity to Trypanosoma cruzi in mice: specific suppressor cells in chronic infection.

In mice chronically infected with Trypanosoma cruzi there are antigen-specific suppressor cells which inhibit the development of delayed hypersensitivity (DTH) to T. cruzi antigen but not to an unrelated antigen, keyhole limpet haemocyanin (KLH). This was shown by comparing the 24 hr footpad reactivity, elicited by injection of soluble T. cruzi antigen, of infected mice with that of mice immunized with killed T. cruzi antigen after pretreatment with cyclophosphamide. In the latter, 24 hr footpad swelling represented a DTH reaction in that the cellular infiltrate was predominantly mononuclear and the reactivity could be transferred to normal recipients by lymphoid cells but not by serum. Chronically-infected mice also developed 24 hr footpad swelling but the fact that this was undiminished from an earlier 3 hr reaction and could not be transferred to normal recipients by either local or systemic injection of cells, as well as the histological features, all implied that it represented a prolonged Arthus reaction. The absence or minimal levels of specific DTH detectable in chronic T. cruzi infected mice was accompanied by the presence in their spleens of cells which specifically suppressed the generation of DTH to T. cruzi in normal mice. Suppressor cell activity was radioresistant (10 Gy/1000 rad) and T-cell mediated as defined by significantly decreased and increased suppression following anti-Thy 1.2 serum treatment and nylon wool fractionation, respectively. The ability of chronic T. cruzi mice to develop DTH to an unrelated antigen KLH was unimpaired.     

Host Defenses in Experimental Scrub Typhus: Delayed-Type Hypersensitivity Responses of Inbred Mice

Delayed-type hypersensitivity responses of inbred mice during the course of lethal and chronic infections with strains of Rickettsia tsutsugamushi were evaluated by using the influx of radiolabeled cells into antigen-injected ears. Congenic strains of C3H mice, which previously have been shown to be resistant (C3H/RV) or sensitive (C3H/HeDub) to lethal intraperitoneal infection with the Gilliam strain of rickettsiae, both expressed delayed-type hypersensitivity early in the course of infection (5 to 7 days). The sensitive C3H/HeDub mice, however, exhibited a marked decline in reactivity just before death. In contrast, reactivity of C3H/RV mice remained high through day 9 and declined slowly through day 15 after infection. Similar results were obtained when BALB/c mice were infected with either the Karp or the Gilliam strain of rickettsiae, which produce a lethal or nonlethal infection, respectively, in this strain of mice. Rechallenge of C3H/RV mice elicited a rapid increase in reactivity, suggesting a secondary memory response. To analyze delayed-type hypersensitivity during chronic infection, C3H/HeDub mice were immunized by subcutaneous infection with the Gilliam strain of R. tsutsugamushi, and both delayed-type hypersensitivity reactivity and resistance to intraperitoneal challenge were examined. Delayed-type hypersensitivity reactivity developed slowly and peaked at 21 days postimmunization, which correlated with resistance to intraperitoneal challenge. Delayed-type hypersensitivity reactivity declined thereafter, but resistance to intraperitoneal challenge remained through 28 days postimmunization. Delayed-type hypersensitivity reactivity increased after secondary challenge at 28 days, again suggesting antigen memory generated by primary immunization. Transfer of delayed-type hypersensitivity reactivity was accomplished by using immune thymus-derived splenic lymphocytes isolated with nylon-wool columns. Abrogation of the ability of immune spleen cells to transfer delayed-type hypersensitivity reactivity after treatment with anti-Thy 1.2 alloantiserum and complement further supported the view that delayed-type hypersensitivity responses to scrub typhus rickettsiae were mediated by thymus-derived lymphocytes.     

Staphylococcidal Capability of Rabbit Peritoneal Macrophages in Relation to Infection and Elicitation: Delayed-Type Hypersensitivity Without Increased Resistance

The staphylococcidal capability of populations of peritoneal macrophages in rabbits has been measured before and after repeated infections with Staphylococcus aureus. Such rabbits after infection showed delayed-type hypersensitivity to S. aureus antigens, but the staphylococcidal capability of the peritoneal macrophages was not increased. This result at the cellular level is in agreement with previous assessment in vivo of the consequences of staphylococcal infection. Pathways to cell-mediated resistance, with and without delayed-type hypersensitivity, are presented.     

Time course of contact hypersensitivity to DNFB and histologic findings in mice

This experiment pursued the time course of contact hypersensitivity to 2,4-dinitro-1-fluorobenzene (DNFB) and histologic changes of the cutaneous reaction in mice. The contact hypersensitivity reached a maximum 4 days after sensitization (96.9 +/- 6.7% vs. 22.7 +/- 1.3% in control) and persisted for 3 weeks. The cutaneous hypersensitivity reaction showed peak reactivity at 24 hr after challenge (96.2 +/- 4.7% vs. 11.5 +/- 1.7% in control), and persisted up to 96 hr (13.2 +/- 2.1%). Prime histologic changes observed in this experiment were the exocytosis of lymphoid cells and epidermal thickening which appeared at 20 hr after challenge. Edema, vasodilatation and increased mast cells were observed within the dermis at 4-8 hr. However, edema and vasodilatation disappeared gradually, but numbers of mast cell increased up to 96 hr. The dermal infiltrates were maximum at the 28-72 hr after challenge.     

Protease‐activated receptor 2 signalling promotes dendritic cell antigen transport and T‐cell activation in vivo

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c^high DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44⁺ CD62⁻) CD4⁺ and CD8⁺ T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC⁺ DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0·95% versus 0·47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.     

Delayed-type hypersensitivity reaction in the lungs of guinea pigs due to potassium dichromate

Potassium dichromate was inhaled by guinea pigs previously immunized by potassium dichromate until strong positive patch tests were obtained. No obvious respiratory changes were noted during and after inhalation. Histologically, however, mononuclear cells infiltrated the interstitial spaces in large areas of the lung, producing considerable thickening of the alveolar spaces in 24 to 48 hr after inhalation. Polymorphonuclear cells were predominant initially. These changes were similar to the delayed-type hypersensitivity reaction in the lung elicited by the inhalation of purified protein derivative (PPD) in the guinea pigs immunized by an injection of dry-killed tubercle bacilli. A less marked reaction was observed in guinea pigs passively sensitized with peritoneal exudate cells and lymph node cells. Consequently, the pulmonary changes were thought to be elicited by delayed-type hypersensitivity reaction due to a simple chemical. The clinical implications of delayed-type hypersensitivity reaction in the lung due to simple chemicals are discussed.     

Cell-mediated immunity and delayed-type hypersensitivity inPseudomonas aeruginosa-infected mice

In mice repeated systemic injections ofPseudomonas aeruginosa viable cells were able to induce a specific delayed-type hypersensitivity, which was evaluated as increase both in footpad swelling and in the weight of popliteal lymph nodes, after a challenge in the footpad. Unfractionated spleen cells or T lymphocyte-enriched spleen cells from sensitized donors were able to specifically transfer the delayed-type hypersensitivity to syngeneic recipients but failed to protect them against a lethal challenge withP. aeruginosa. In contrast, serum or B lymphocyte and macrophage-enriched spleen cells from the same donors were capable of transferring protective immunity but failed to induce any delayed-type hypersensitivity reaction in the recipients.These results clearly show that in systemicP. aeruginosa infections a dissociation between delayed-type hypersensitivity and acquired cellular resistance occurs.     

Modulation of delayed-type hypersensitivity during the time course of immune response to a protein antigen

Delayed-type hypersensitivity reactions elicited in the footpad of ovalbumin-sensitized mice after challenge with aggregated ovalbumin on day 4 or 8 of immunization are distinct. The former was characterized by a dense mononuclear infiltrate and, macroscopically, the reaction peaked at 48 hr after antigen challenge; the latter was preceded by immediate-type reactions, reached the maximum at 24 hr and faded drastically later. Histologically, oedema and a mixed granulocytic-lymphocytic infiltrate was found at this time-point. Immunoglobulin G1 (IgG1), IgG2a and IgE antibodies were detected only in plasma obtained after 8 days of immunization. Regarding the cytokines produced by draining lymph node cells after in vitro restimulation, interleukin-4 (IL-4) and IL-10 were predominant after 4 days and interferon-gamma and IL-2 after 8 days of immunization. These two types of delayed-type hypersensitivity (DTH) were used to study the influence of antibody-mediated responses on the inductive and effector phases of cell-mediated immunity. The effector phase of DTH was not affected by immediate-type reactions, as abrogation of these reactions by mediators' antagonists on day 8 or induction of passive reactions by transfer of immune serum on day 4 did not change the extent or kinetics of either type of DTH. Only transfer, before immunization, of whole or T-cell-enriched spleen cells, but not sera, from hyperimmunized donors (high antibody producers) abolished the induction of pure DTH in 4-day immunized recipient mice and changed their cytokine profile to a T helper 2 type. These results indicate that in a non-polarized immune response to a protein antigen there is initially a bias towards cell-mediated immunity, which is gradually dampened by the development of antibody-mediated immunity.     

Kinetoplast DNA minicircles are inherited from both parents in genetic crosses of Trypanosoma brucei

In the order Kinetoplastida, genetic exchange has been demonstrated only in the genus Trypanosoma. Analysis of kinetoplast DNA (kDNA) in genetic crosses of T. brucei has shown that whereas maxicircles are inherited uniparentally, minicircles are inherited from both parents. This result was confirmed for a new cross of T. b. brucei and T. b. rhodesiense by restriction enzyme digestion and Southern analysis of purified kDNA. By hybridisation with small minicircle-derived probes, we could demonstrate the presence of particular parental minicircles in the kDNA of hybrid progeny clones. All hybrid clones had inherited two minicircles from one parent despite two of the four clones having maxicircles from the other parent. The results suggest that rather than small-scale exchange of minicircles between parental networks, gross breakdown and reassembly of the minicircle network occurs during genetic exchange. Received: 26 October 1996 / Accepted: 12 December 1996     

Absence of Correlation Between Delayed-Type Hypersensitivity and Protection in Experimental Systemic Candidiasis in Immunized Mice

We found that in mice which had been immunized intraperitoneally with 2 × 10⁸ heat-killed Candida albicans cells there was a striking temporal relationship between resistance to systemic challenge with 10⁶ living C. albicans cells and a number of measurable cellular parameters of the host response. These included the emergence of delayed-type hypersensitivity and the development of granulocytosis. Since it had been shown in previous work that granulocytosis was associated with an increase in resistance when nonspecific immunostimulation was used, we performed experiments to induce delayed-type hypersensitivity without any measurable modification of the granulocyte population. Adoptive transfer of delayed-type hypersensitivity with spleen cells from immune and resistant donor mice did not produce any increase in resistance in normal recipients. When separate groups of mice were immunized intraperitoneally or subcutaneously with varying doses of heat-killed C. albicans, we found that doses of less than 10⁸ cells did induce significant delayed-type hypersensitivity without any increase in granulocytosis. In such mice, as well as in animals pretreated with immunomodulators before immunization with heat-killed C. albicans, the presence of cell-mediated immunity, as measured by the delayed-type hypersensitivity test, was not associated with an increase in resistance against systemic candidiasis. On the contrary, the results suggested that cell-mediated immunity was associated with an increase in the susceptibility of these mice. The same effect on candidiasis susceptibility was observed when animals were immunized with heat-killed filamentous C. albicans.     

Induction of suppressor T cells in delayed-type hypersensitivity to Mycobacterium bovis BCG in low-responder mice.

The induction of delayed-type hypersensitivity to Mycobacterium bovis BCG was specifically inhibited by suppressor T cells in C3H/He, a strain of mice which is a low responder to BCG. The existence of these suppressor cells was confirmed by an adoptive transfer of spleen cells of BCG-injected mice into cyclophosphamide-treated recipients. The suppressor cells appeared in the spleens of the mice 2 to 7 days after intravenous BCG injection. They were sensitive to anti-theta serum and complement and did not adhere to Sephadex G-10. A pretreatment of the mice with cyclophosphamide eliminated the suppression of delayed-type hypersensitivity. These suppressor cells effectively inhibited the induction of delayed-type hypersensitivity to BCG, but showed only weak effect on the expression of it.     

Differing role of dendritic cells and macrophages in the induction of delayed-type hypersensitivity responses to PPD.

In order to study the antigen-presenting cell (APC) requirements for the priming of delayed-type hypersensitivity (DTH), murine spleen cells were fractionated on bovine serum albumin gradients, pulsed in vitro with tuberculin, and then injected subcutaneously into normal mice. The other footpad was challenged with tuberculin between 24 hr and 7 days later and swelling was measured 2 hr and 18-24 hr after challenge. Optimum priming for 2-hr and 24-hr responses at 7 days was achieved by an injection of 5 X 10(5) tuberculin-pulsed intermediate-density Fc + ve cells. Time-course studies revealed that the 2-hr component could be elicited as early as 24 hr after injection of pulsed APC, while the 24-hr component became significant at 3 days. Elimination of T cells or B cells did not affect the response. Injection of pulsed APC into allogeneic mice primed the 2-hr but not the 24-hr component. Neither pulsed high-density cells (mostly T cells) nor pulsed dendritic cells (DC) primed mice for these responses. Failure to elicit DTH after injection of tuberculin-pulsed DC was due to their failure to prime the 2-hr component, which other authors have shown to be a prerequisite for the appearance of the later components. That DC did prime the MHC-restricted 24-hr component was demonstrated by protocols involving the use of both macrophages and DC as APC.     

Development of delayed-type hypersensitivity during Mycobacterium lepraemurium infection in mice.

Various preparations of Mycobacterium lepraemurium were used to elicit delayed-type hypersensitivity in the footpad of mice infected with this organism. With a sonicated preparation of the mycobacterium, a significant increase in footpad swelling was elicited in mice infected with M. lepraemurium 5 weeks previously, but not in BCG-infected animals or uninfected controls. This footpad reaction was shown to peak at 24 h and to be associated with an infiltration of mononuclear cells. The kinetics of footpad swelling, its association with lymphoproliferation, and its dependence on T lymphocytes were each examined. The results support the hypothesis that this is a delayed-type hypersensitivity reaction. The ability to transfer this reactivity to normal mice with cells but not serum offers further confirmation that this hypersensitivity is dependent on cell-mediated immunological mechanisms rather than humoral antibody. The relevance of this to the study of the immunological response of mice to murine leprosy is discussed.     

in vitro: Inhibition of the Formation of Hypersensitivity Pyrogen from Delayed Hypersensitive Cell Extract by Protease Inhibitors

When extracts from lymphoid tissue of rabbits or guinea pigs showing delayed hypersensitivity are allowed to react with the specific antigen in vitro, pyrogenic substances (the so-called hypersensitivity pyrogen—HSP) are formed. The effect of protease inhibitors on the HSP formation in vitro was studied.

Various protease inhibitors added to the cell extract before mixing it with antigen inhibited the formation of HSP; some control substances did not. Inhibitors added to the preformed HSP (i.e. to the mixture of cell extract with antigen) did not affect the pyrogenic activity of HSP. By adding the protease inhibitor at various time intervals after mixing the cell extract with the antigen, the reaction leading to HSP formation has been shown to last a few seconds at 37° and some minutes at 0–1°.

The significance of these results on the mechanism of delayed-type hypersensitivity is discussed.

     

Preliminary characterization of inflammatory infiltrates in response to Rickettsia prowazekii reinfection in man: immunohistology.

The local perivascular mononuclear cell inflammatory infiltrate (PVI) response to intradermal (ID) challenge with viable R. prowazekii was studied in a typhus-immune subject. An immunohistochemical method for specific mononuclear cell markers was used on skin punch biopsies taken 6, 24 and 48 hr after challenge. By 24 to 48 hr, the histologic findings were consistent with a delayed-type hypersensitivity reaction (DTHR). Both CD4+ and CD8+ T lymphocytes dominated the PVI at 48 h. CD8+ cells entered the PVI more rapidly than CD4+ cells. Local control of R. prowazekii challenge in an immune human subject was associated with recruitment into the PVI of T lymphocytes which are rich sources of gamma-interferon, and CD8+ T cells which are potentially cytotoxic for R. typhi-infected cells.     

Trans-sialidase from Trypanosoma brucei as a potential target for DNA vaccine development against African trypanosomiasis

African trypanosomiasis (AT), also known as sleeping sickness in humans and Nagana in animals, is a disease caused by the protozoan parasite Trypanosoma brucei. AT is an extremely debilitating disease in human, cattle, and wild animals, and the treatment is difficult with frequent relapses. This work shows that BALB-c mice immunized intramuscularly with a single dose (100 μg) of a plasmid DNA encoding the 5′-terminal region of the trans-sialidase (nTSA) gene of T. brucei brucei are able to produce IgG antibodies that bind to the bloodstream form of T. brucei-protein extract and recognize the recombinant nTSA protein, expressed in Escherichia coli. Furthermore, this DNA vaccination process was able to protect 60% of mice submitted to a challenge assay with the infective form of T. brucei brucei parasites. These results demonstrate that a DNA vaccine coding for trans-sialidase from T. brucei is potentially useful in the prophylaxis of AT.     

Dissociation between tumour resistance and delayed-type hypersensitivity to tumour-associated antigens in the mouse.

A comparison was made in mice between resistance to growth of a syngeneic methylcholanthrene-induced fibrosarcoma (Meth A) and specific delayed-type hypersensitivity to this tumour. Resistance could be induced in 90% of mice following a single injection of 10(4) Meth A cells. This resistance could be transferred by spleen cells but not serum. Delayed skin reactivity to ultrasonicated Meth A cells was found in both tumour-resistant and tumour-bearing mice and could be passively transferred by spleen cells and serum. These findings suggest a dissociation between tumour resistance and specific delayed-type hypersensitivity. It would appear that, in this system, delayed skin reactivity to Meth A tumour cells is mediated by humoral antibody despite a mononuclear cell infiltrate at 24 and 48 h and that this could be the reason for its lack of correlation with host resistance to tumour growth which is cell-mediated and not transferable by serum.     

Effects of alcohol consumption on the haematology and testis of mice experimentally infected with Trypanosoma brucei

Forty 6-week-old inbred albino mice were used to study the effect of alcohol consumption on haematology and testis of mice experimentally infected with Trypanosoma brucei (T. b. brucei). The forty mice were divided into four groups of 10 mice each. Groups 1 and 3 mice received graded levels of ethanol at 10, 20 and 30 % (v/v) in water for 1 week, 2 weeks and the rest of the experimental period, respectively. At day 29 of the experiment, mice in groups 1 and 2 (non-alcohol exposed) were infected intraperitoneally with 1.0?×?10⁶ T. brucei in PBS-diluted blood. Group 4 mice served as control and were neither given alcohol nor infected with trypanosomes. The animals in all the groups were given water and commercial diet ad libitum. The alcohol-exposed infected mice had significantly (P?<?0.01) higher levels of parasitaemia than the non-alcohol-exposed infected group. The control group had significantly (P?<?0.01) higher body weights, packed cell volume, red blood cell, white blood cell counts and testicular sperm reserve than the other groups. These parameters were significantly (P?<?0.01) lowest in the alcohol-exposed infected group when compared with the other groups. Microscopically, degeneration and necrosis of spermatogenic cells were observed in seminiferous tubules of the testes of alcohol-exposed and T. brucei-infected mice. Sections of testes of the mice exposed to alcohol alone and non-alcohol-exposed T. brucei-infected mice had seminiferous tubules with poor development of spermatogenic cells. It was concluded that alcohol exposure markedly increased the deleterious effects of T. brucei considering the increases in the haematological values and various lesions produced in testes of the experimental mice.     

A new model for investigations of T-cell functions in mice: differential immunosuppressive effects of two monoclonal anti-thy-1.2 antibodies

Two monoclonal anti-Thy-1.2 antibodies were investigated for their activity in eliminating T cells in vitro and in vivo. Both antibodies exert a complement-dependent cell cytotoxicity in vitro. Antibody B that belongs to the IgM class shows a 100-fold higher complement-dependent cytotoxic activity than antibody C, which is of IgG_2a class. However, administration of antibody C into Balb/c mice results in the elimination of T cells as determined by the failure of different T-cell functions. Within 24 hours after administration of antibody C, the reactivity of spleen or lymph-node cells to T-cell mitogens, the antibody response to the Tcell- dependent antigen SRBC and the SRBC-induced delayed-type hypersensitivity are completely abolished. These effects are dose-dependent in a dose range of 0.1–1.0 mg Ig protein per animal and affects only T cells in the peripheral lymphoid organs. The Thy-1.2-bearing cells residing in the thymus are not impaired by the treatment of the animals with this monoclonal antibody and are able to repopulate the peripheral lymphoid organs within 30 to 60 days.Investigations into the mode of action of the removal of peripheral T cells revealed that antibody-C-coated Thy-1.2-bearing cells are rapidly phagocytosed by macrophages, while antibody-B-coated Thy-1.2-bearing cells are not. This might be the reason for the differential in-vivo activities of the two monoclonal antibodies.A model with new qualities for the study of functions and the regeneration of T cells in vivo has been established.