Lymphocyte blastogenic response to ovalbumin in a model for canine allergy

Lymphocyte stimulation tests (LST) were performed in five dogs sensitised with ovalbumin (OVA) and seven healthy dogs. In addition, all five OVA-sensitised and two control dogs were tested after two in vivo provocations with OVA-containing eye drops. The isolated cells were suspended in culture media containing OVA and were cultured for up to 12 days. Proliferation was measured as reduction in 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity by flow cytometry on days 0, 3, 6, 9 and 12. A cell proliferation index (CPI) for each day and the area under the curve (AUC) of the CPI was calculated for each dog. All OVA-sensitised dogs demonstrated increased erythema after conjunctival OVA application. The presence of OVA-specific lymphocytes was demonstrated in 2/5 OVA-sensitised dogs before and 4/5 after in vivo provocation. Using the AUC, the difference between OVA-sensitised and control dogs was significant in all three LST before in vivo provocation (P<0.05) and borderline significant (P=0.053) in 2/3 LST after provocation. The most significant difference in CPI was observed after 9 days of culture (P=0.001). This pilot study indicates that the LST allows detection of rare antigen specific memory T-cells in dogs previously sensitised to, but not concurrently undergoing challenge by a specific antigen.

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.

T cell-mediated hypersensitivity to quinolones: mechanisms and cross-reactivity

BACKGROUND: Quinolones are widely used, broad spectrum antibiotics that can induce immediate- and delayed-type hypersensitivity reactions, presumably either IgE or T cell mediated, in about 2-3% of treated patients. OBJECTIVE: To better understand how T cells interact with quinolones, we analysed six patients with delayed hypersensitivity reactions to ciprofloxacin (CPFX), norfloxacin (NRFX) or moxifloxacin (MXFX). METHODS: We confirmed the involvement of T cells in vivo by patch test and in vitro by means of the lymphocyte proliferation test (LTT). The nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones were investigated through the generation and analysis (flow cytometry and proliferation assays) of quinolone-specific T cell clones (TCC). RESULTS: The LTT confirmed the involvement of T cells because peripheral blood mononuclear cells (PBMC) mounted an enhanced in vitro proliferative response to CPFX and/or NRFX or MXFX in all patients. Patch tests were positive after 24 and 48 h in three out of the six patients. From two patients, CPFX- and MXFX-specific CD4(+)/CD8(+) T cell receptor (TCR) alphabeta(+) TCC were generated to investigate the nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones. The use of eight different quinolones as antigens (Ag) revealed three patterns of cross-reactivity: clones exclusively reacting with the eliciting drug, clones with a limited cross-reactivity and clones showing a broad cross-reactivity. The TCC recognized quinolones directly without need of processing and without covalent association with the major histocompatability complex (MHC)-peptide complex, as glutaraldehyde-fixed Ag-presenting cells (APC) could present the drug and washing quinolone-pulsed APC removed the drug, abrogating the reactivity of quinolone-specific TCC. CONCLUSION: Our data show that T cells are involved in delayed immune reactions to quinolones and that cross-reactivity among the different quinolones is frequent.

Lymphocyte transformation by insulin and insulin-zinc suspensions

The lymphocyte transformation response to the mitogen phytohaemagglutinin (PHA) was determined in 15 well controlled insulin-dependent diabetics (IDD) with a history of insulin allergy or an acute insulin allergy. There was no significant difference in the PHA response of IDD and normal subjects matched in respect of age and sex. The response of peripheral blood lymphocytes to insulin (Actrapid) and an insulin zinc suspension (Monotard) was also determined. Fifty-three percent of IDD gave a positive reaction to Actrapid. Monotard produced positive reactions both in IDD and normal subjects. In normal subjects, a close correlation between the stimulation indices of Monotard and PHA was found (r = 0 . 966) suggesting that these stimulations depend on a common parameter namely, the reactivity to mitogens.

The MHC-haplotype influences primary, but not memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes of the caprine arthritis encephalitis virus Gag protein

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.

Involvement of drug-specific T cells in acute drug-induced interstitial nephritis

Drug-induced interstitial nephritis can be caused by a plethora of drugs and is characterized by a sudden impairment of renal function, mild proteinuria, and sterile pyuria. For investigation of the possible pathomechanism of this disease, drug-specific T cells were analyzed, their function was characterized, and these in vitro findings were correlated to histopathologic changes that were observed in kidney biopsy specimens. Peripheral blood mononuclear cells from three patients showed a proliferative response to only one of the administered drugs, namely flucloxacillin, penicillin G, and disulfiram, respectively. The in vitro analysis of the flucloxacillin-reactive cells showed an oligoclonal immune response with an outgrowth of T cells bearing the T cell receptor Vbeta9 and Vbeta21.3. Moreover, flucloxacillin-specific T cell clones could be generated from peripheral blood, they expressed CD4 and the alphabeta-T cell receptor, and showed a heterogeneous cytokine secretion pattern with no clear commitment to either a Th1- or Th2-type response. The immunohistochemistry of kidney biopsies of these patients revealed cell infiltrations that consisted mostly of T cells (CD4+ and/or CD8+). An augmented presence of IL-5, eosinophils, neutrophils, CD68+ cells, and IL-12 was observed. In agreement with negative cytotoxicity assays, no cytotoxicity-related molecules such as Fas and perforin were detected by immunohistochemistry. The data indicate that drug-specific T cells are activated locally and orchestrate a local inflammation via secretion of various cytokines, the type of which depends on the cytokine pattern secreted and which probably is responsible for the renal damage.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice.

BACKGROUND The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4+CD25+ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. METHODS/FINDINGS Key parameters for infection outcome in E. multilocularis-infected fgl2-/- (AE-fgl2-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4+CD25+ Treg suspensions were incubated with CD4+ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. CONCLUSIONS FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.

Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats

Objective: Central to the process of osseointegration is the recruitment of mesenchymal progenitor cells to the healing site, their proliferation and differentiation to bone synthesising osteoblasts. The process is under the control of pro-inflammatory cytokines and growth factors. The aim of this study was to monitor these key stages of osseointegration and the signalling milieu during bone healing around implants placed in healthy and diabetic bone. Methods: Implants were placed into the sockets of incisors extracted from the mandibles of normal Wistar and diabetic Goto-Kakizaki rats. Mandibles 1-12 weeks post-insertion of the implant were examined by histochemistry and immunocytochemistry to localise the presence of Stro-1- positive mesenchymal progenitor cells, proliferating cellular nuclear antigen proliferative cells, osteopontin and osteocalcin, macrophages, pro-inflammatory cytokines interleukin (IL)-1 , IL-6, tumour necrosis factor (TNF)- and tumour growth factor (TGF)- 1. Image analysis provided a semi-quantification of positively expressing cells. Results: Histological staining identified a delay in the formation of mineralised bone around implants placed in diabetic animals. Within the diabetic bone, the migration of Stro-1 mesenchymal cells in the healing tissue appeared to be unaffected. However, in the diabetic healing bone, the onset of cell proliferation and osteoblast differentiation were delayed and subsequently prolonged compared with normal bone. Similar patterns of change were observed in diabetic bone for the presence of IL-1 , TNF- , macrophages and TGF- 1. Conclusion: The observed alterations in the extracellular presence of pro-inflammatory cytokines, macrophages and growth factors within diabetic tissues that correlate to changes in the signalling milieu, may affect the proliferation and differentiation of mesenchymal progenitor cells in the osseointegration process. To cite this article: Colombo JS, Balani D, Sloan AJ, St Crean J, Okazaki J, Waddington RJ. Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats Clin. Oral Impl. Res22, 2011; 578-586 doi: 10.1111/j.1600-0501.2010.01992.x.

Life cycle complexity, environmental change and the emerging status of salmonid proliferative kidney disease

P>1. Proliferative kidney disease (PKD) is a disease of salmonid fish caused by the endoparasitic myxozoan, Tetracapsuloides bryosalmonae, which uses freshwater bryozoans as primary hosts. Clinical PKD is characterised by a temperature-dependent proliferative and inflammatory response to parasite stages in the kidney.;2. Evidence that PKD is an emerging disease includes outbreaks in new regions, declines in Swiss brown trout populations and the adoption of expensive practices by fish farms to reduce heavy losses. Disease-related mortality in wild fish populations is almost certainly underestimated because of e.g. oversight, scavenging by wild animals, misdiagnosis and fish stocking.;3. PKD prevalences are spatially and temporally variable, range from 0 to 90-100% and are typically highest in juvenile fish.;4. Laboratory and field studies demonstrate that (i) increasing temperatures enhance disease prevalence, severity and distribution and PKD-related mortality; (ii) eutrophication may promote outbreaks. Both bryozoans and T. bryosalmonae stages in bryozoans undergo temperature- and nutrient-driven proliferation.;5. Tetracapsuloides bryosalmonae is likely to achieve persistent infection of highly clonal bryozoan hosts through vertical transmission, low virulence and host condition-dependent cycling between covert and overt infections. Exploitation of fish hosts entails massive proliferation and spore production by stages that escape the immune response. Many aspects of the parasite's life cycle remain obscure. If infectious stages are produced in all hosts then the complex life cycle includes multiple transmission routes.;6. Patterns of disease outbreaks suggest that background, subclinical infections exist under normal environmental conditions. When conditions change, outbreaks may then occur in regions where infection was hitherto unsuspected.;7. Environmental change is likely to cause PKD outbreaks in more northerly regions as warmer temperatures promote disease development, enhance bryozoan biomass and increase spore production, but may also reduce the geographical range of this unique multihost-parasite system. Coevolutionary dynamics resulting from host-parasite interactions that maximise fitness in previous environments may pose problems for sustainability, particularly in view of extensive declines in salmonid populations and degradation of many freshwater habitats.

Enhancement of Drug-Specific Lymphocyte Proliferation Using CD25-Depleted CD3(+) Effector Cells

Background: The lymphocyte transformation test (LTT) is used for in vitro diagnosis of drug hypersensitivity reactions. While its specificity is over 90%, sensitivity is limited and depends on the type of reaction, drug and possibly time interval between the event and analysis. Removal of regulatory T cells (Treg/CD25(hi)) from in vitro stimulated cell cultures was previously reported to be a promising method to increase the sensitivity of proliferation tests. Objective: The aim of this investigation is to evaluate the effect of removal of regulatory T cells on the sensitivity of the LTT. Methods: Patients with well-documented drug hypersensitivity were recruited. Peripheral blood mononuclear cells, isolated CD3(+) and CD3(+) T cells depleted of the CD25(hi) fraction were used as effector cells in the LTT. Irrelevant drugs were also included to determine specificity. (3)H-thymidine incorporation was utilized as the detection system and results were expressed as a stimulation index (SI). Results: SIs of 7/11 LTTs were reduced after a mean time interval of 10.5 months (LTT 1 vs. LTT 2). Removal of the CD25(hi) fraction, which was FOXP3(+) and had a suppressive effect on drug-induced proliferation, resulted in an increased response to the relevant drugs. Sensitivity was increased from 25 to 82.35% with dramatically enhanced SI (2.05 to 6.02). Specificity was not affected. Conclusion: Removal of Treg/CD25(hi) cells can increase the frequency and strengths of drug-specific proliferation without affecting specificity. This approach might be useful in certain drug hypersensitivity reactions with borderline responses or long time interval since the hypersensitivity reaction. © 2014 S. Karger AG, Basel.

Allopurinol hypersensitivity is primarily mediated by dose-dependent oxypurinol-specific T cell response

BACKGROUND Allopurinol is a main cause of severe cutaneous adverse reactions (SCAR). How allopurinol induces hypersensitivity remains unknown. Pre-disposing factors are the presence of the HLA-B*58:01 allele, renal failure and possibly the dose taken. OBJECTIVE Using an in vitro model, we sought to decipher the relationship among allopurinol metabolism, HLA-B*58:01 phenotype and drug concentrations in stimulating drug-specific T cells. METHODS Lymphocyte transformation test (LTT) results of patients who had developed allopurinol hypersensitivity were analysed. We generated allopurinol or oxypurinol-specific T cell lines (ALP/OXP-TCLs) from allopurinol naïve HLA-B*58:01(+) and HLA-B*58:01(-) individuals using various drug concentrations. Their reactivity patterns were analysed by flow cytometry and (51) Cr release assay. RESULTS Allopurinol allergic patients are primarily sensitized to oxypurinol in a dose-dependent manner. TCL induction data show that both the presence of HLA-B*58:01 allele and high concentration of drug are important for the generation of drug-specific T cells. The predominance of oxypurinol-specific lymphocyte response in allopurinol allergic patients can be explained by the rapid conversion of allopurinol to oxypurinol in vivo rather than to its intrinsic immunogenicity. OXP-TCLs do not recognize allopurinol and vice versa. Finally, functional avidity of ALP/OXP-TCL is dependent on both the induction dose and HLA-B*58:01 status. CONCLUSIONS AND CLINICAL RELEVANCE This study establishes the important synergistic role of drug concentration and HLA-B*58:01 allele in the allopurinol or oxypurinol-specific T cell responses. Despite the prevailing dogma that Type B adverse drug reactions are dose independent, allopurinol hypersensitivity is primarily driven by oxypurinol-specific T cell response in a dose-dependent manner, particular in the presence of HLA-B*58:01 allele.

In vitro drug causality assessment in Stevens-Johnson syndrome - alternatives for lymphocyte transformation test

BACKGROUND Patients with Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) are often exposed simultaneously to a few potentially culprit drugs. However, both the standard lymphocyte transformation tests (LTT) with proliferation as the assay end-point as well as skin tests, if done, are often negative. OBJECTIVE As provocation tests are considered too dangerous, there is an urgent need to identify the relevant drug in SJS/TEN and to improve sensitivity of tests able to identify the causative drug. METHODS Fifteen patients with SJS/TEN with the ALDEN score ≥ 6 and 18 drug-exposed controls were included. Peripheral blood mononuclear cells (PBMC) were isolated and cultured under defined conditions with drugs. LTT was compared to the following end-points: cytokine levels in cell culture supernatant, number of granzyme B secreting cells by ELISpot and intracellular staining for granulysin and IFNγ in CD3(+) CD4(+), CD3(+) CD8(+) and NKp46(+) cells. To further enhance sensitivity, the effect of IL-7/IL-15 pre-incubation of PBMC was evaluated. RESULTS Lymphocyte transformation tests was positive in only 4/15 patients (sensitivity 27%, CI: 8-55%). Similarly, with granzyme B-ELISpot culprit drugs were positive in 5/15 patients (sensitivity 33%, CI: 12-62%). The expression of granulysin was significantly induced in NKp46(+) and CD3(+) CD4(+) cells (sensitivity 40%, CI: 16-68% and 53%, CI: 27-79% respectively). Cytokine production could be demonstrated in 38%, CI: 14-68% and 43%, CI: 18-71% of patients for IL-2 and IL-5, respectively, and in 55%, CI: 23-83% for IFNγ. Pre-incubation with IL-7/IL-15 enhanced drug-specific response only in a few patients. Specificities of tested assays were in the range of 95 (CI: 80-99%)-100% (CI: 90-100%). CONCLUSIONS AND CLINICAL RELEVANCE Granulysin expression in CD3(+) CD4(+) , Granzyme B-ELISpot and IFNγ production considered together provided a sensitivity of 80% (CI: 52-96%) and specificity of 95% (80-99%). Thus, this study demonstrated that combining different assays may be a feasible approach to identify the causative drug of SJS/TEN reactions; however, confirmation on another group of patients is necessary.

Pathophysiology of proliferative vitreoretinopathy in retinal detachment

Because proliferative vitreoretinopathy cannot be effectively treated, its prevention is indispensable for the success of surgery for retinal detachment. The elaboration of preventive and therapeutic strategies depends upon the identification of patients who are genetically predisposed to develop the disease, as well as upon an understanding of the biological process involved and the role of local factors, such as the status of the uveovascular barrier. Detachment of the retina or vitreous activates glia to release cytokines and ATP, which not only protect the neuroretina but also promote inflammation, retinal ischemia, cell proliferation, and tissue remodeling. The vitreal microenvironment favors cellular de-differentiation and proliferation of cells with nonspecific nutritional requirements. This may render a pharmacological inhibition of their growth difficult without causing damage to the pharmacologically vulnerable neuroretina. Moreover, reattachment of the retina relies upon the local induction of a controlled wound-healing response involving macrophages and proliferating glia. Hence, the functional outcome of proliferative vitreoretinopathy will be determined by the equilibrium established between protective and destructive repair mechanisms, which will be influenced by the location and the degree of damage to the photoreceptor cells that is induced by peri-retinal gliosis.

Hypertrophy of infected Peyer’s patches arises from global, interferon-receptor, and CD69-independent shutdown of lymphocyte egress

Lymphoid organ hypertrophy is a hallmark of localized infection. During the inflammatory response, massive changes in lymphocyte recirculation and turnover boost lymphoid organ cellularity. Intriguingly, the exact nature of these changes remains undefined to date. Here, we report that hypertrophy of Salmonella-infected Peyer's patches (PPs) ensues from a global "shutdown" of lymphocyte egress, which traps recirculating lymphocytes in PPs. Surprisingly, infection-induced lymphocyte sequestration did not require previously proposed mediators of lymphoid organ shutdown including type I interferon receptor and CD69. In contrast, following T-cell receptor-mediated priming, CD69 was essential to selectively block CD4+ effector T-cell egress. Our findings segregate two distinct lymphocyte sequestration mechanisms, which differentially rely on intrinsic modulation of lymphocyte egress capacity and inflammation-induced changes in the lymphoid organ environment.