Skin and pulmonary models using coated bentonite particles for the study of the inflammation evoked by Paracoccidioides brasiliensis antigens in previously immunized mice

Bentonite particles coated with polysaccharide antigen or crude soluble antigen of Paracoccidioides brasiliensis were injected intradermally or intravenously in mice. In control animals that were not pre-immunized with P. brasiliensis antigens, coated and uncoated bentonite caused minimal and nonspecific inflammation around the cutaneous injection site or around the bentonite thrombi in small lung vessels after intravenous injection. However, in mice previously immunized with P. brasiliensis antigens, the coated bentonite particles boosted the humoral and cellular immune responses to P. brasiliensis and evoked intense inflammatory reactions. Twelve days after intradermal injection, the inflammatory reaction around the bentonite was rich in neutrophils, macrophages, lymphocytes and plasma cells associated with young granulation tissue. In intravenously injected mice, the pulmonary inflammation was maximal at day 2, and was characterized by a florid neutrophilic and macrophagic cellular infiltration around bentonite thrombi; in some foci, there was incipient organization to mature granuloma. However, in both models, there was no formation of epithelioid granulomata, demonstrating that in paracoccidioidomycosis cellular immunity alone, without the presence of intact micro-organisms, may not be enough for the development of this type of granuloma.

Evaluation of Paracoccidioides brasiliensis exoantigen in the detection of delayed dermal hypersensitivity in experimental and human paracoccidioidomycosis

The exoantigen of Paracoccidioides brasiliensis standardized by Camargo et al. [1] (AgR) was used to evaluate the in vivo and in vitro cell immune response of experimental animals and of patients with paracoccidioidomycosis (PBM). Fava Netto antigen (AgF) was tested in parallel as a control antigen. The study was conducted with mice and guinea pigs infected with P. brasiliensis or immunized with its fungal antigens, on patients with PBM and on their respective control groups. The cell immune response was analysed by skin tests, and by the macrophage and leucocyte migration inhibition tests (MMIT and LMIT) in the animals and in the patients, respectively. The skin test with AgR as paracoccidioidin was positive in infected or immunized mice and guinea pigs and negative in control animals. The skin tests with AgR (24 h) showed 96.7% positivity in patients with PBM and were negative in control individuals. Histopathological study of the in vivo tests in the different experimental models was consistent with a delayed hypersensitivity response (DHR). Immunohistochemical study of the skin tests of PBM patients demonstrated a predominance of T lymphocytes, confirming the nature of a DHR to the fungal antigens. The in vitro cell immune response showed variable results for the various experimental models, i.e. significant rates of MMIT in immunized mice, a tendency to positivity in infected guinea pigs, and the absence of migration inhibition in PBM patients. Taken together, the data indicate that the AgR is efficient as paracoccidioidin in the evaluation of DHR in PBM, with an optimum time of reading the test of 24 h.

Skin test and tick immune status in susceptible and resistant cattle in Brazil

A cutaneous hypersensitivity test (CHT) was used to correlate host resistance to ticks and type of reaction elicited to unfed larval extract-ULE of the cattle tick Boophilus microplus in European and Indian cattle. Twenty calves were separated into four groups of five animals each: naïve or preinfested Indian or European cattle. CHT was induced by intradermal inoculation of 0.1 ml of ULE cattle tick B. microplus (50 μg protein) in the calf ear. Ear thickness was measured using calipers before and 10 min, 1, 2, 6, 18, 24, 48, 72, 96, and 144 h postinoculation (PI). Preinfested European calves showed only an immediate type reaction with maximum response (75% increase in ear thickness) at 10 min PI. On the other hand, preinfested Indian calves presented an immediate response with maximum reaction (70% increase in ear thickness) between 10 min and one hour PI, and a delayed type reaction at 72 h PI (60% increase in ear thickness). These results point out the crucial role of the cellular immune response of cattle in the expression of resistance to cattle tick B. microplus. Skin test might be useful in the ranking of cattle according to the susceptibility/resistance to ticks.

Amblyomma cajennense ticks induce immediate hypersensitivity in horses and donkeys

Since host immune reaction to ticks interferes with tick-borne pathogen transmission, it is important to recognize naturally occurring tick-host immune relationships to better understand the epidemiology of such infectious diseases. Amblyomma cajennense is an important tick-borne disease vector in the Neotropical region and horses maintain it in domestic environments. In the present work intradermal testing of A. cajennense tick exposed horses and donkeys using crude tick antigens was used to evaluate the type of hypersensitivity induced by infestations. Animals sensitized by A. cajennense infestation displayed an immediate hypersensitivity reaction at the antigen inoculation site. Foals sensitized with experimental infestations and field sensitized horses presented the most intense reactions (40% of ear thickness increase). Field sensitized donkeys presented less intense reaction reaching no more than 22% of mean thickness increase. Control horses (non-sensitized) had the least intense reaction, with a peak of no more 12% of increase. The presence of a prominent immediate hypersensitivity in equids sensitized experimentally or by field infestations indicates that A. cajennense ticks induce in this host an immune response that is associated with IgE production and which is known to be inappropriate against intracellular pathogens. Differences observed between horses and donkeys are discussed.

Hypersensitivity induced in dogs by nymphal extract of Amblyomma cajennense ticks (Acari:Ixodidae)

In general, hosts develop resistance to ticks after repeated infestations; nevertheless, several studies on naturally occurring host-tick interactions were unable to detect resistance of hosts to ticks even after repeated infestations. The purpose of this investigation was to study the type of cutaneous hypersensitivity to unfed nymphal extract of A. cajennense in dogs, which, unlike guinea pigs, do not develop resistance. A first, but no second, peak in skin reaction was observed, suggesting that cellular immunity is an important mechanism of resistance to ticks. This may partially explain why guinea pigs, but not dogs, develop resistance against ticks.

A cross-sectional study on canine Leishmania (L.) infantum chagasi infection in Amazonian Brazil ratifies a higher prevalence of specific IgG-antibody response than delayed-type hypersensitivity in symptomatic and asymptomatic dogs

This was a cross-sectional study which analyzed the prevalence and the clinical and immunological spectrum of canine Leishmania (L.) infantum chagasi infection in a cohort of 320 mongrel dogs living in an endemic area of American visceral leishmaniasis in the Amazonian Brazil by using, mainly, the indirect fluorescence antibody test (IFAT-IgG) and the delayed-type hypersensitivity (DTH), and the parasite research by the popliteal lymph node aspiration. The IFAT and DTH reactivity recognized three different immune response profiles: (1) IFAT((+))/DTH(-) (107 dogs), (2) IFAT((-))/DTH(+) (18 dogs), and (3) IFAT((+))/DTH(+) (13 dogs), providing an overall prevalence of infection of 43 % (138/320). Thus, the specific prevalence of IFAT ((+)) /DTH ((-)) 33.4 % (107/320) was higher than those of IFAT ((-)) /DTH ((+)) 5.6 % (18/320) and IFAT ((+)) /DTH ((+)) 4.0 % (13/320). Moreover, the frequency of these profiles among 138 infected dogs showed that the IFAT ((+)) /DTH ((-)) rate of 77.5 % (107/138) was also higher than those of 13.0 % (18/138) of IFAT ((-)) /DTH ((+)) and 9.5 % (13/138) of IFAT ((+)) /DTH ((+)) rates. The frequency of asymptomatic dogs (76 %-105) was higher than those of symptomatic (16.6 %-23) and oligosymptomatic ones (7.4 %-10). A total of 16 (11.6 %) L. (L.) i. chagasi isolates were obtained from infected dogs, all from the IFAT ((+)) /DTH ((-)) profile: 41 % (9/22) from symptomatic, 33.3 % (3/9) from oligosymptomatic, and 5.2 % (4/76) from asymptomatic dogs. These findings strongly suggested that despite the higher frequency of asymptomatic dogs (76 %-105), the majority (72.4 %-76) was characterized by the IFAT ((+)) /DTH ((-)) profile with a doubtful immunogenetic resistance against infection.

In vitro detection of cytotoxic T and NK cells in peripheral blood of patients with various drug-induced skin diseases

BACKGROUND: Cytotoxic cells are involved in most forms of drug-induced skin diseases. Till now, no in vitro test addressed this aspect of drug-allergic responses. Our report evaluates whether drug-induced cytotoxic cells can be detected in peripheral blood of nonacute patients with different forms of drug hypersensitivity, and also whether in vitro detection of these cells could be helpful in drug-allergy diagnosis. METHODS: GranzymeB enzyme-linked immunosorbent spot-forming (ELISPOT) and cell surface expression of the degranulation marker CD107a were evaluated on peripheral blood mononuclear cells from 12 drug-allergic patients in remission state and 16 drug-exposed healthy controls. RESULTS: In 10/12 allergic patients culprit but not irrelevant drug elicited granzymeB release after 48-72 h stimulation. It was clearly positive in patients with high proliferative response to the drug, measured in lymphocyte transformation tests. In patients, who showed moderate or low proliferation and low drug-response in granzymeB ELISPOT, overnight preincubation with interleukin (IL)-7/IL-15 enhanced drug-specific granzymeB release and allowed to clearly identify the offending agent. CD107a staining was positive on CD4+/CD3+, CD8+/CD3+ T cells as well as CD56+/CD3- natural killer cells. None of the drug-exposed healthy donors reacted to the tested drugs and allergic patients reacted only to the offending, but not to tolerated drugs. CONCLUSION: GranzymeB ELISPOT is a highly specific in vitro method to detect drug-reacting cytotoxic cells in peripheral blood of drug-allergic patients even several years after disease manifestation. Together with IL-7/IL-15 preincubation, it may be helpful in indentifying the offending drug even in some patients with weak proliferative drug-response.

Establishment of diagnostic criteria for feline nonflea-induced hypersensitivity dermatitis

Hypersensitivity dermatitides (HD) are commonly seen in cats, and they are usually caused by environmental, food and/or flea allergens. Affected cats normally present with one of the following clinical reaction patterns: head and neck excoriations, usually symmetrical self-induced alopecia, eosinophilic skin lesions or miliary dermatitis. Importantly, none of these clinical presentations is considered to be pathognomonic for HD skin diseases, and the diagnosis of HD is usually based on the exclusion of other pruritic diseases and on a positive response to therapy. The objectives of this study were to propose sets of criteria for the diagnosis of nonflea-induced HD (NFHD). We recruited 501 cats with pruritus and skin lesions and compared clinical parameters between cats with NFHD (encompassing those with nonflea, nonfood HD and those with food HD), flea HD and other pruritic conditions. Using simulated annealing techniques, we established two sets of proposed criteria for the following two different clinical situations: (i) the diagnosis of NFHD in a population of pruritic cats; and (ii) the diagnosis of NFHD after exclusion of cats with flea HD. These criteria sets were associated with good sensitivity and specificity and may be useful for homogeneity of enrolment in clinical trials and to evaluate the probability of diagnosis of NFHD in clinical practice. Finally, these criteria were not useful to differentiate cats with NFHD from those with food HD.

Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

Drug hypersensitivity

Drug hypersensitivity represents an immune-mediated reaction to a drug. Although several drug hypersensitivity reactions are confined to the skin and rather mild, some may be life threatening and also involve further organs such as liver, kidney and bone marrow. The exact pathogenesis of many drug hypersensitivity reactions is still obscure. In this review the concepts on how small molecular drugs can activate the immune system are discussed and the hapten, prohapten and p-i concept are explained. Furthermore, the classification of drug hypersensitivity reactions and some common and severe clinical manifestations of drug-induced T cell mediated reactions are presented.

A comparative study of the expression of cytotoxic proteins in allergic contact dermatitis and psoriasis: spongiotic skin lesions in allergic contact dermatitis are highly infiltrated by T cells expressing perforin and granzyme B

Recent reports indicate that cytotoxic T cells are critically involved in contact hypersensitivity reactions in animals. In this study we sought to investigate the in vivo expression of cytotoxic granule proteins in the elicitation phase of allergic contact dermatitis in humans. Skin biopsy specimens were obtained from patients with allergic contact dermatitis (n = 8) and psoriasis (n = 6) and from controls with normal skin (n = 6). Expression of perforin and granzyme B was investigated by in situ hybridization and immunohistochemistry. In contrast to normal skin and psoriasis, a significant enhancement of perforin and granzyme B gene expression and immunoreactivity was observed in the mononuclear cell infiltrate of allergic contact dermatitis. Immunoreactivity for perforin and granzyme B was mainly found in the cytoplasm of lymphocytic cells, which were located in the dense perivascular infiltrate as well as at sites of marked spongiosis in the epidermis. Double immunostaining revealed that both CD4+ and CD8+ T cells are capable of expressing perforin and granzyme B. In conclusion, our data suggest that T-cell-mediated mechanisms involving cytotoxic granule proteins may elicit epidermal cell injury in vivo and thereby strongly contribute to the development of allergic contact dermatitis in humans.

Dermatophagoides farinae-specific immunotherapy in atopic dogs with hypersensitivity to multiple allergens: a randomised, double blind, placebo-controlled study

A randomised, placebo-controlled, double blind study was conducted on 25 dogs that had atopic dermatitis, together with skin test reactivity and elevated serum IgE to Dermatophagoides farinae (Df) and at least one additional allergen. Dogs were treated with either a Df-restricted immunotherapy solution (n=14) or a placebo (n=11) and evaluated 6 weeks and 3, 5, 7 and 9 months after the initiation of treatment using a clinical scoring system (SASSAD) and pruritus analogue scale scores. The Df-restricted solution and the placebo had an equal effect on both pruritus and the skin manifestations (P>0.05). The results of this study indicate that in dogs with atopic dermatitis based on hypersensitivity to environmental allergens in addition to D. farinae, Df-restricted immunotherapy is insufficient to control the disease. Consequently, a solution for allergen-specific immunotherapy should remain customised.

Type I Hypersensitivity in Lambs With Coughing Syndrome

Lambs from two flocks with a chronic respiratory disease characterized by paroxysmal cough and rectal prolapses were tested for their skin reactivity to Mycoplasma ovipneumoniae (MO) and M. arginini (MA) antigens (Ags). There was a marked, immediate skin reaction to intradermal injection of MO Ag in many of the tested lambs. Some of these positive lambs also reacted to MA Ag. Phosphate buffered saline (PBS) used as a negative control gave no skin reaction in any of the tested animals. In addition, simultaneous serological tests revealed low antibody levels against MO and MA. However, both agents could be routinely isolated from nasal swabs of the affected lambs. There is reason to suspect that an immediate type hypersensitivity to MO Ag and perhaps to other allergens that develops in association with the mycoplasmal infection contributes to this coughing syndrome.