Tools for Etiologic Diagnosis of Drug-Induced Allergic Conditions.

Drug hypersensitivity reactions are a serious concern in clinical practice because they can be severe and result in lifelong sequelae. An accurate diagnosis and identification of the culprit drug is essential to prevent future reactions as well as for the identification of safe treatment alternatives. Nonetheless, the diagnosis can be challenging. In vivo and in vitro tests can be helpful, although none are conclusive; therefore, the tests are not usually performed in isolation but as part of a diagnostic algorithm. In addition, some in vitro tests are only available in research laboratories, and standardization has not been fully accomplished. Collaborating research is needed to improve drug hypersensitivity reaction diagnosis. In this review, we update the current available in vivo and in vitro tools with their pros and cons and propose an algorithm to integrate them into clinical practice.

Characterization of hypersensitivity reactions to polysulfone hemodialysis membranes.

BACKGROUND: In recent years, cases have been reported in which unexpected systemic hypersensitivity reactions occurred in patients dialyzed with polysulfone- or polyethersulfone-biocompatible membranes in the absence of other risk factors. The pathomechanisms involved in these reactions are largely unknown. OBJECTIVE: To characterize hypersensitivity reactions to polysulfone hemodialysis using clinical and laboratory data and to identify biomarkers suitable for endotype identification and diagnosis. METHODS: We prospectively collected data from 29 patients with suspected hypersensitivity reactions to polysulfone hemodialysis membranes. Clinical laboratory parameters such as tryptase, blood cell counts, and complement levels were recorded. Acute samples were obtained from 18 cases for the ex vivo assessment of basophil activation by flow cytometry analysis of CD63, CD203, and FcεRI cell membrane expression. Serum cytokines and anaphylatoxin concentrations were evaluated in 16 cases by Luminex and cytometric bead array analysis. RESULTS: Tryptase was elevated during the acute reaction in 4 cases. Evidence of basophil activation was obtained in 10 patients. Complement activation was found in only 2 cases. However, C5a serum levels tended to increase during the acute reaction in those patients with hypoxemia. Significantly higher serum levels of interleukin-6 were observed during the acute reactions to polysulfone hemodialysis (P = .0103). CONCLUSION: Based on biomarker analysis, various endotypes were identified, including type I-like (with the involvement of mast cells or basophils), complement, and cytokine (interleukin-6) release-related reactions, with some patients showing mixed reactions. Further research is needed to unravel the exact mechanisms involved in the activation of these cellular and molecular pathways.

Pre-transplant assessment of pp65-specific CD4 T cell responses identifies CMV-seropositive patients treated with rATG at risk of late onset infection.

Assessment of CMV-specific T cell immunity might be a useful tool in predicting CMV infection after solid organ transplantation. We have investigated CD4 and CD8 T-cell responses to CMV pp65 and IE-1 antigens in a prospective study of 28 CMV-seropositive kidney transplant recipients who were administered lymphocyte-depleting antibodies (Thymoglobulin®) as induction treatment and with universal prophylaxis for CMV infection. The response was analyzed by intracellular flow cytometry analysis of IFN-γ production in pretransplant samples and at 1, 6, 12 and 24 months post-transplant. Overall, only pretransplant CD4 T-cell responses to pp65 were significantly lower (p = .004) in patients with CMV replication post-transplant. ROC curve analysis showed that pre-transplant frequencies of pp65-specific CD4 + T cells below 0.10% could predict CMV infection with 75% sensitivity and 83.33% specificity (AUC: 0.847; 95% CI: 0.693-1.001; p = .0054) and seem to be mandatory for efficient control of CMV viral replication by the host immune system. In conclusion, the functional assessment of CMV-specific CD4 T-cell immunity pretransplant in seropositive patients may allow the identification of Thymoglobulin®-treated kidney transplant recipients at risk of developing CMV infection post-transplantation.

Incidence of Stevens-Johnson syndrome/toxic epidermal necrolysis among new users of different individual drugs in a European population: a case-population study.

PURPOSE: To estimate the specific incidences of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) among new users of drugs frequently reported to be associated with this serious event. METHODS: We performed a case-population approach, which combined data from a registry of SJS/TEN cases from the Madrid region (numerator) during the study period 2005-2015 and a primary healthcare database from the same catchment population. The proportion of new users of drugs estimated in the primary healthcare database was stratified by calendar year, sex and age (5-year bands), and then applied to the same strata of Madrid's population census to compute the number of new users (denominator). Incidences were re-estimated using only cases in which the concerned drug had a probable or very probable causal relationship. RESULTS: A total of 44 SJS/TEN cases aged > 14 years were registered during the study period. The highest SJS/TEN incidence was found for phenytoin with 68.9 per 100,000 new users (95% CI 27.7-141.9), followed by dexamethasone (5.48; 1.49-14.03), allopurinol (3.29; 1.07-7.67) and cotrimoxazole (3.19; 0.87-8.16). Considering only probable and very probable cases, the incidences hardly changed, except for dexamethasone, which was left without cases. Pantoprazole, levofloxacin and lorazepam showed incidences between 1 per 100,000 and 1 per 1,000,000 new users. Ibuprofen, amoxicillin-clavulanic acid, metamizole, amoxicillin, paracetamol and omeprazole showed incidences around 1 per one million new users. CONCLUSIONS: Phenytoin was the drug with the highest incidence of SJS/TEN, followed by allopurinol and cotrimoxazole. For the rest of the drugs, the estimated incidences were below 1 in 100,000 new users.

Active surveillance of severe cutaneous adverse reactions: A case-population approach using a registry and a health care database.

PURPOSE: The "case-population" design has been proposed for the surveillance of rare events like Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), wherein a registry of cases is combined with sales data from the source population in order to estimate crude odds ratios (ORs). A major drawback of this method is the inability to distinguish between new and non-new users of drugs, which for the study of hypersensitivity reactions is of utmost importance. METHODS: We have explored an approach in which the exposure to the drugs of interest in the source population is inferred from a primary health care database (BIFAP), which helped us to identify drug initiators among all users and additionally adjust for potential confounders. A total of 44 SJS/TEN cases from the Registry and 44 000 controls randomly sampled from BIFAP and matched with cases for index date were included. We estimated the adjusted ORs (AORs) and 95% confidence intervals (CI) of SJS/TEN associated with the new use of 13 drugs (for which we had at least two exposed cases) through a conditional logistic regression model. RESULTS: AORs (95% CI) were estimated for phenytoin, 4618 (434-49112); cotrimoxazole, 1142 (163-8015); allopurinol, 160 (36-709); dexamethasone, 38 (1.33-1077); ibuprofen, 33 (8.6-124); lorazepam, 27 (5.8-124); paracetamol, 13 (2.8-62); levofloxacine, 12 (1.24-120); amoxicillin, 6.9 (1.39-35); pantoprazole, 6.5 (0.10-420); metamizole, 6.3 (0.69-57); amoxicillin clavulanic acid, 4.2 (0.53-34); and omeprazole, 1.34 (0.06-31). The inclusion of non-new users dramatically decreased the AORs for all drugs. CONCLUSIONS: The case-population approach using a registry of cases and a primary health care database proved feasible and efficient for the active surveillance of SJS/TEN.

Significant HLA class I type associations with aromatic antiepileptic drug (AED)-induced SJS/TEN are different from those found for the same AED-induced DRESS in the Spanish population.

Aromatic antiepileptic drugs (AEDs) are among the drugs most frequently involved in severe cutaneous adverse reactions (SCARs), such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reactions with eosinophilia and systemic symptoms (DRESS). This study investigated the associations between the genetic polymorphisms of HLA class-I and AED-induced SCARs in the Spanish population. HLA class-I genotypes were determined in AED (phenytoin[PHT],lamotrigine[LTG],carbamazepine[CBZ],phenobarbital[PB])-induced SJS/TEN (n=15) or DRESS (n=12) cases included in the Spanish SCAR registry, PIELenRed. There were 3 control groups: (A)tolerant to a single AED, (B)tolerant to any AED, and (C)Spanish population controls. For SJS/TEN, concomitant HLA-A*02:01/Cw15:02 alleles were significantly associated with PHT-cases compared to control groups B and C [(B)odds ratio(OR):14.75, p=0.009;(C)OR:27.50, p<0.001], and were close to significance with respect to control group A (p=0.060). The genotype frequency of the HLA-B*38:01 was significantly associated with PHT-LTG-cases compared with the 3 groups of controls [(A)OR:12.86, p=0.012;(B)OR:13.81; p=0.002;(C)OR:14.35, p<0.001], and with LTG-cases [(A)OR:147.00, p=0.001;(B)OR:115.00, p<0.001;(C)OR:124.70, p<0.001]. We found the HLA-B*15:02 allele in a Spanish Romani patient with a CBZ-case. The HLA-A*11:01 was significantly associated with CBZ-cases [(A)OR:63.89, p=0.002;(B)OR:36.33, p=0.005;(C)OR:28.29, p=0.007]. For DRESS, the HLA-A*24:02 genotype frequency was statistically significant in the PHT-LTG-cases [(A)OR:22.56, p=0.003;(B)OR:23.50. p=0.001; (C)OR:33.25, p<0.001], and in the LTG-cases [(A),OR:49.00, p=0.015;(B)OR:27.77, p=0.005; (C)OR:34.53, p=0.002]. HLA-A*31:01 was significantly associated with the CBZ-cases [(A)OR:22.00, p=0.047;(B)OR:29.50, p=0.033;(C)OR:35.14, p=0.006]. In conclusion, we identified several significant genetic risk factors for the first time in the Spanish Caucasian population: HLA-A*02:01/Cw*15:02 combination as a risk factor for PHT-induced SJS/TEN, HLA-B*38:01 for LTG- and PHT- induced SJS/TEN, HLA-A*11:01 for CBZ-induced SJS/TEN, and HLA-A*24:02 for LTG- and PHT- induced DRESS. The strong association between HLA*31:01 and CBZ-DRESS in Europeans was confirmed in this study.

Eosinophilic drug reactions detected by a prospective pharmacovigilance programme in a tertiary hospital.

AIM: We conducted a prospective evaluation of all eosinophilic drug reactions (EDRs) through the Prospective Pharmacovigilance Program from Laboratory Signals at Hospital to find out the incidence and distribution of these entities in our hospital, their causative drugs, and predictors. METHODS: All peripheral eosinophilia >700 × 106  cells l-1 detected at admission or during hospitalisation, were prospectively monitored over 42 months. The spectrum of the localised or systemic manifestation of EDR, the incidence, the distribution of causative drugs, and the predictors were analysed. RESULTS: The incidence of EDR was 16.67 (95% Poisson confidence interval [CI]: 9.90-25.98) per 10 000 admissions. Of 274 cases of EDR, 154 (56.2%) cases in 148 patients were asymptomatic hypereosinophilia. In the remaining 120 (43.8%) cases, there was other involvement. Skin and soft tissue reactions were detected in 36 (13.1%) cases; visceral EDRs in 19(7.0%) cases; and drug-induced eosinophilic cutaneous and visceral manifestations were detected in the remaining 65 (23.7%) cases, 64 of which were potential drug reaction with eosinophilia and systemic symptoms (DRESS). After adjusting for age, sex, and hospitalisation wards, predictors of symptomatic eosinophilia were earlier onset of eosinophilia (hazard ratio [HR], 10.49; 95%CI: 3.13-35.16) higher eosinophil count (HR, 8.51; 95%CI: 3.28-22.08), and a delayed onset of corticosteroids (HR, 1.34; 95%CI: 1.01-1.73). A higher eosinophil count in patients with DRESS was significantly associated with greater impairment of liver function, prolonged hospitalisation, higher cumulative doses of corticosteroids, and if hypogammaglobinaemia was detected, a reactivation of human-herpesvirus 6 was subsequently detected. CONCLUSIONS: Half (53.3%, 64/120 cases) of symptomatic EDRs were potential DRESS. The main predictor of severity of EDR was an early severe eosinophilia.

Mechanisms Involved in Hypersensitivity Reactions to Polysulfone Hemodialysis Membranes.

Several cases of patients with anaphylactic or systemic hypersensitivity reactions to polysulfone (PS) hemodialysis (HD) membranes and tolerance to cellulose triacetate (CTA) membranes have recently been reported. To investigate the mechanisms involved in PS hypersensitivity, basophil, T cell, and complement activation were analyzed in acute-phase samples from two patients with systemic reactions to PS-based membranes. Basophil and T cell activation, as well as higher serum tryptase levels were detected in acute-phase samples compared with basal levels. Complement levels (C3 and C4) were decreased in acute-phase samples from PS-allergic patients to a higher extent than in samples from control donors taken at the same time points, indicating complement activation during the acute reactions. An experimental external circuit was established on pediatric membranes after rinsing with low or high priming volumes of saline solution, to analyze basophils, T cells, and complement activation in blood samples from 10 PS-allergic and 8 nonallergic HD patients upon contact with PS-based or CTA membranes. Predialysis and postdialysis samples were collected. Basophils from PS-allergic patients exhibited increased degranulation, and T cells showed significantly increased activation after contact with PS-based membranes primed with low volumes of saline. No activation was detected in leukocytes from nonallergic patients under the same experimental conditions. Membrane priming with high volumes of saline abrogated activation of basophils and T cells. However, basophils from allergic donors showed significantly higher responses to Fcεc stimulation after contact with PS membranes. Basophil degranulation and elevated serum tryptase levels in allergic patients during acute reactions support the systemic activation of mast cells and basophils during hypersensitivity reactions to PS-based membranes. A leachable component of the membranes might be responsible for cell activation in some patients.

TNF-α‒Mediated Keratinocyte Expression and Release of Matrix Metalloproteinase 9: Putative Mechanism of Pathogenesis in Stevens‒Johnson Syndrome/Toxic Epidermal Necrolysis.

Stevens‒Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are severe cutaneous adverse drug reactions characterized by widespread keratinocyte cell death and epidermal detachment. At present, there is little understanding of how the detachment occurs or how it is abrogated by the TNF-α inhibitor etanercept, an effective SJS/TEN treatment. RNA sequencing was used to identify upregulated transcripts in formalin-fixed paraffin-embedded SJS/TEN skin biopsies. Epidermal matrix metalloproteinase 9 (MMP9) expression was assessed by immunohistochemistry in skin biopsies and cultured human skin explants exposed to serum from patients with cutaneous adverse drug reactions. TNF-α‒induced MMP9 expression and activity and its abrogation by etanercept were determined using the HaCaT immortalized keratinocyte cell line. Epidermal MMP9 expression was significantly higher in SJS/TEN skin (70.6%) than in healthy control skin (0%) (P = 0.0098) and nonbullous skin reactions (10.7%) (P = 0.0002). SJS/TEN serum induced significant MMP9 expression and collagenase activity in healthy skin explants, which was reduced by etanercept. Etanercept was also able to negate the TNF-α‒induced MMP9 expression in the HaCaT cell line. Data suggest that elevated epidermal MMP9 expression and collagenase activity are a putative pathogenic mechanism in SJS/TEN, which is limited by etanercept. Modulation of MMP9 expression and activity represents, to our knowledge, a previously unreported therapeutic target for the treatment of SJS/TEN.

Coronavirus-19 disease risk and protective factors associated with HLA/KIR polymorphisms in Ecuadorian patients residing in Madrid.

BACKGROUND: Immigrants represented 21.8% of cases in a Spanish cohort of hospitalised patients with COVID-19, a proportion exceeding the percentage of immigrants in that area's total population. Among the ethnic-related genetic risk factors for COVID-19, human leukocyte antigen (HLA) genotypes in diverse populations might bias the response to SARS-CoV-2 infection and/or progression. Similarly, genetic differences in natural killer-activating and inhibitory receptors could play a role in the immune system's response to the viral infection. METHODS: We characterised HLA alleles and KIR genes in 52 Ecuadorian patients hospitalised for moderate and severe COVID-19 and 87 Ecuadorian controls from the general population living in the same area. RESULTS: There was a significantly increased frequency of the HLA-B*39 antigen and the activating KIR2DS4 receptor in the presence of its HLA-C*04 ligand in the COVID-19 group when compared with the control group. In contrast, there was a significant reduction in the frequency of carriers of KIR2DL1 and of the KIR3DL1/Bw4 receptor/ligand combination among COVID-19 group. On the other hand, HLA-A*24:02 and HLA-DRB1*09:01 alleles showed significantly lower frequencies specifically in the severe COVID-19 group. CONCLUSION: HLA-B*39 alleles might be genetic risk factors for developing COVID-19 in Ecuadorian individuals. In the presence of its ligand C*04, the natural killer-activating receptor KIR2DS4 might also increase the risk of developing COVID-19, while, in the presence of HLA-Bw4 alleles, the inhibitory receptor KIR3DL1 might play a protective role. Patients with COVID-19 who carry HLA-A*24:02 and HLA-DRB1*09:01 alleles might be protected against more severe forms of COVID-19.

Updates in SJS/TEN: collaboration, innovation, and community.

Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is a predominantly drug-induced disease, with a mortality rate of 15-20%, that engages the expertise of multiple disciplines: dermatology, allergy, immunology, clinical pharmacology, burn surgery, ophthalmology, urogynecology, and psychiatry. SJS/TEN has an incidence of 1-5/million persons per year in the United States, with even higher rates globally. One of the challenges of SJS/TEN has been developing the research infrastructure and coordination to answer questions capable of transforming clinical care and leading to improved patient outcomes. SJS/TEN 2021, the third research meeting of its kind, was held as a virtual meeting on August 28-29, 2021. The meeting brought together 428 international scientists, in addition to a community of 140 SJS/TEN survivors and family members. The goal of the meeting was to brainstorm strategies to support the continued growth of an international SJS/TEN research network, bridging science and the community. The community workshop section of the meeting focused on eight primary themes: mental health, eye care, SJS/TEN in children, non-drug induced SJS/TEN, long-term health complications, new advances in mechanisms and basic science, managing long-term scarring, considerations for skin of color, and COVID-19 vaccines. The meeting featured several important updates and identified areas of unmet research and clinical need that will be highlighted in this white paper.

Subepithelial collagen deposition, profibrogenic cytokine gene expression, and changes after prolonged fluticasone propionate treatment in adult eosinophilic esophagitis: a prospective study.

BACKGROUND: Recent research shows that both pediatric and adult patients with eosinophilic esophagitis (EoE) experience esophageal remodeling marked by increased collagen deposition in which TGF-ß plays an important role. However, limited data are available on the intensity and reversibility of fibrous remodeling in adults with EoE. OBJECTIVE: We sought to analyze differences in collagen deposition in the lamina propria (LP) and profibrogenic cytokine gene expression along with other changes induced by prolonged treatment with fluticasone propionate in adults with EoE. METHODS: Ten adults given consecutive diagnoses of EoE were studied prospectively. Deep esophageal biopsy specimens were obtained before and after 1 year of treatment with fluticasone propionate. Collagen deposition in the LP was assessed in tissue sections with the aid of the Masson trichrome technique. IL5, TGFB1, fibroblast growth factor 9 (FGF9), and CCL18 gene expression was quantified through real-time PCR. EoE results were compared among samples from 10 adult patients with gastroesophageal reflux disease and 10 control subjects with healthy esophagi. RESULTS: Patients with EoE showed a significant increase in subepithelial collagen deposition; this correlated positively with eosinophil density in the LP and the patient's age. Prolonged steroid treatment induced a nonsignificant reduction in subepithelial fibrosis, which remained significantly higher than in control subjects. Profibrogenic cytokine gene expression also increased in patients with EoE, with IL5 (P < .001), FGF9 (P = .005), and CCL18 (P = .008) all significantly upregulated. After 1 year of treatment, a reduction was observed in gene expression; for CCL18 expression, this decrease was statistically significant (P < .001). CONCLUSIONS: Esophageal remodeling is associated with upregulated gene expression of profibrogenic cytokines in adults with EoE. Prolonged treatment with fluticasone propionate leads to a nonsignificant reduction in subepithelial collagen deposition accompanied by downregulation of profibrogenic cytokine gene expression, with that of CCL18 being especially significant.

IL-15/IL-15Rα in SJS/TEN: Relevant Expression of IL15 and IL15RA in Affected Skin.

Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a life-threatening hypersensitivity reaction to medications characterized by keratinocyte apoptosis and skin detachment. IL-15 serum levels have been associated with severity and prognosis of SJS/TEN. We have measured IL-15 concentrations in serum and blister fluid (BF) from patients with SJS/TEN by ELISA and used quantitative RT-PCR to analyze the expression of IL15 and IL15RA (encoding for IL-15 Receptor-α chain) genes in peripheral blood and BF cells, including isolated monocytes, and in affected skin. A positive correlation was found between IL-15 serum levels and a percent of detached skin. BF concentrations were higher, but no correlation was found. Higher IL15 and IL15RA gene expression levels were found in skin-infiltrating blister fluid cells compared to peripheral mononuclear cells. Moreover, IL15RA transcripts were barely detected in healthy skin, being the highest expression levels found in samples from two SJS/TEN patients who did not survive. The cutaneous expression of IL-15Rα in SJS/TEN may provide an explanation to the tissue-specific immune cytotoxic response in this clinical entity, and the results suggest that the effects of IL-15 in SJS/TEN patients may be dependent on the expression of its private receptor IL-15Rα in affected skin.

LTT and HLA testing as diagnostic tools in Spanish vancomycin-induced DRESS cases: A case-control study.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe T-cell-mediated off-target adverse reaction. DRESS cases caused by vancomycin have often been reported. The HLA-A*32:01 allele has been associated with genetic susceptibility to vancomycin-induced DRESS in US citizens of European descent. We have analyzed the association of the HLA-A*32:01 allele in 14 Spanish DRESS cases in which vancomycin was suspected as the culprit drug, and the lymphocyte transformation test (LTT) as an in vitro assay to evaluate vancomycin sensitization. The results were compared to vancomycin-tolerant control donors. LTT was performed in 12 DRESS cases with PBMCs from resolution samples available and in a group of 12 tolerant donors. ROC curves determined that LTT is a suitable tool to identify patients sensitized to vancomycin (AUC = 0.9646; p < 0.0001). When a stimulation index >3 was regarded as a positive result, contingency tables determined 91% sensitivity, 91.67% specificity, 91% positive predictive value, and 91.67% negative predictive value (p = 0.0001, Fisher's exact test). The HLA A*32:01 allele was determined by an allele-specific PCR assay in 14 cases and 25 tolerant controls. Among the DRESS cases, five carriers were identified (35.7%), while it was detected in only one (4%) of the tolerant donors, [odds ratio (OR) = 13.33; 95% CI: 1.364-130.3; p = 0.016]. The strength of the association increased when only cases with positive LTT to vancomycin were considered (OR = 24.0; 95% CI: 2.28-252.6; p = 4.0 × 10-3). Our results confirm the association of the risk allele HLA-A*32:01 with vancomycin-induced DRESS in Spanish cases, and support LTT as a reliable tool to determine vancomycin sensitization.

Alternative activation of macrophages in human peritoneum: implications for peritoneal fibrosis.

BACKGROUND: Depending on the cytokine microenvironment, macrophages (Mϕ) can adopt a proinflammatory (M1) or a profibrotic (M2) phenotype characterized by the expression of cell surface proteins such as CD206 and CD163 and soluble factors such as CC chemokine ligand 18 (CCL18). A key role for Mϕ in fibrosis has been observed in diverse organ settings. We studied the Mϕ population in a human model of peritoneal dialysis in which continuous stress due to dialysis fluids and recurrent peritonitis represent a risk for peritoneal membrane dysfunction reflected as ultrafiltration failure (UFF) and peritoneal fibrosis. METHODS: We used flow cytometry and quantitative reverse transcription-polymerase chain reaction to analyse the phenotype of peritoneal effluent Mϕ and tested their ability to stimulate the proliferation of human fibroblasts. Mϕ from non-infected patients were compared with those from patients with active peritonitis. Cytokine production was evaluated by enzyme-linked immunosorbent assay (ELISA) in spent dialysates and cell culture supernatants. RESULTS: CD206(+) and CD163(+) M2 were found within peritoneal effluents by flow cytometry analysis, with increased frequencies of CD163(+) cells during peritonitis (P = 0.003). TGFB1, MMP9 and CCL18 messenger RNA (mRNA) levels in peritoneal macrophages (pMϕ) were similar to those found in M2 cells differentiated in vitro. The ability of pMϕ to stimulate fibroblast proliferation correlated with CCL18 mRNA levels (r = 0.924, P = 0.016). CCL18 production by pMϕ was confirmed by immunostaining of cytospin samples and ELISA. Moreover, CCL18 effluent concentrations correlated with decreased peritoneal function, which was evaluated as dialysate to plasma ratio of creatinine (r = 0.724, P < 0.0001), and were significantly higher in patients with UFF (P = 0.0025) and in those who later developed sclerosing peritonitis (P = 0.024). CONCLUSIONS: M2 may participate in human peritoneal fibrosis through the stimulation of fibroblast cell growth and CCL18 production as high concentrations of CCL18 are associated with functional deficiency and fibrosis of the peritoneal membrane.

CD94/NKG2C is a killer effector molecule in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis.

BACKGROUND: Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe, bullous cutaneous diseases with uncertain pathogenesis, although cytotoxic T cells seem to be involved. Natural killer (NK)-like activity has been found in blister infiltrates. Cytotoxic T lymphocytes (CTLs) with NK-like activity (NK-CTLs) have been shown to express T-cell receptors restricted by the HLA-Ib molecule HLA-E. Alternatively, the HLA-E-specific activating receptor CD94/NKG2C can trigger T-cell receptor-independent cytotoxicity in CTLs. OBJECTIVE: Our aim was to test whether HLA-E expression sensitizes keratinocytes to killing by CTLs with NK-like activity and to explore the expression of activating receptors specific for HLA-E in blister cytotoxic lymphocytes. METHODS: We used flow cytometry and immunohistochemistry to analyze HLA-E expression in keratinocytes from affected skin in patients with SJS, TEN, and other less severe drug-induced exanthemas. The expression of CD94/NKG2C was analyzed by means of flow cytometry in PBMCs and blister cells from patients. PBMCs and blister cells were analyzed for their ability to kill HLA-E-expressing cells. Involvement of CD94/NKG2C in triggering degranulation of cytolytic cells was explored by means of CD107a mobilization assays and standard cytotoxicity chromium release assays. RESULTS: We found that keratinocytes from affected skin expressed HLA-E and that cell-surface HLA-E sensitizes keratinocytes to killing by CD94/NKG2C(+) CTLs. Frequencies of CD94/NKG2C(+) peripheral blood T and NK cells were increased in patients with SJS and TEN during the acute phase. Moreover, activated blister T and NK lymphocytes expressed CD94/NKG2C and were able to degranulate in response to HLA-E(+) cells in an NKG2C-dependent manner. CONCLUSION: CD94/NKG2C might be involved in triggering cytotoxic lymphocytes in patients with SJS and TEN.

Role of Macrophages and Related Cytokines in Kidney Disease.

Inflammation is a key characteristic of kidney disease, but this immune response is two-faced. In the acute phase of kidney injury, there is an activation of the immune cells to fight against the insult, contributing to kidney repair and regeneration. However, in chronic kidney diseases (CKD), immune cells that infiltrate the kidney play a deleterious role, actively participating in disease progression, and contributing to nephron loss and fibrosis. Importantly, CKD is a chronic inflammatory disease. In early CKD stages, patients present sub-clinical inflammation, activation of immune circulating cells and therefore, anti-inflammatory strategies have been proposed as a common therapeutic target for renal diseases. Recent studies have highlighted the plasticity of immune cells and the complexity of their functions. Among immune cells, monocytes/macrophages play an important role in all steps of kidney injury. However, the phenotype characterization between human and mice immune cells showed different markers; therefore the extrapolation of experimental studies in mice could not reflect human renal diseases. Here we will review the current information about the characteristics of different macrophage phenotypes, mainly focused on macrophage-related cytokines, with special attention to the chemokine CCL18, and its murine functional homolog CCL8, and the macrophage marker CD163, and their role in kidney pathology.

HLA class I molecules regulate IFN-gamma production induced in NK cells by target cells, viral products, or immature dendritic cells through the inhibitory receptor ILT2/CD85j.

Recent advances support an important role for NK cells in determining immune responses beyond their cytolytic functions, which is supported by their capacity to secrete several cytokines and chemokines. In particular, NK-derived IFN-gamma has proven to be fundamental in shaping adaptive immune responses. Although the role of inhibitory NK receptors (iNKR) in the regulation of cytotoxicity has been widely explored, their involvement in the control of cytokine production has been scarcely analyzed. Specifically, no data are available referring to the role of the iNKR ILT2/CD85j in the regulation of IFN-gamma secretion by NK cells. Published data support a differential regulation of cytotoxicity and cytokine expression. Thus, formal proof of the involvement of HLA class I in regulating the production of cytokines through binding to ILT2/CD85j has been missing. We have determined the response of human NK-92 and primary human ILT2/CD85j(+) NK cells from healthy donors to target cells expressing or not HLA class I. We found specificities of HLA class I-mediated inhibition of IFN-gamma mRNA expression, protein production, and secretion consistent with the specific recognition by ILT2/CD85j. We also found inhibition of IFN-gamma production by ILT2/CD85j(+) T cells in response to superantigen stimulation. Furthermore, ligation of ILT2/CD85j inhibited the production of IFN-gamma in response to poly(I:C), and blocking of ILT2/CD85j-HLA class I interactions increased the secretion of IFN-gamma in NK/immature dendritic cell cocultures. The data support a role for self HLA class I in the regulation of IFN-gamma secretion at the mRNA and protein levels by interacting with the iNKR ILT2/CD85j.