Detection of Salivary Tryptase Levels in Children following Oral Food Challenges.

BACKGROUND: Oral food challenge (OFC) is commonly used to diagnose food allergy. This test is time and resource intensive, and conclusions are not always unequivocal as this relies on the interpretation of symptoms. Therefore, an objective marker would improve the accuracy of the diagnostic workup of food allergy. OBJECTIVES: The aim of this study was to investigate whether tryptase can be detected in saliva of children following OFC. METHOD: Children from 3 to 18 years of age were eligible for inclusion if an OFC for peanut or tree nut had been recommended. Saliva samples were collected prior to the first dose and 5, 10, and 15 min following the last administered dose during OFC. Assay precision, spike-and-recovery, and assessment of lower limit of detection of the tryptase immunoassay were examined before analysis of tryptase in saliva was performed. RESULTS: A total of 30 children were included (median age 8 years, 63.3% male, 53.3% positive OFC outcome). Tryptase was detected in saliva samples. The mean of the change in baseline tryptase value to each saliva collecting time point was significantly different in patients with a positive OFC outcome compared to a negative outcome (p < 0.01). CONCLUSIONS: This study showed that tryptase can be detected in saliva of children following OFC. Increased levels of tryptase compared to baseline were found if the OFC outcome was positive, suggesting that measuring tryptase in saliva may be useful in the diagnosis of food allergy. Further research is needed to evaluate the potential association between tryptase levels and symptoms.

Polymorphic mononuclear neutrophils CD64 index for diagnosis of sepsis in postoperative surgical patients and critically ill patients.

BACKGROUND: Surface neutrophil CD64 expression is upregulated in patients with bacterial infection. As it was suggested that the CD64 index could be used to detect sepsis in hospitalized patients, we questioned whether the CD64 index could discriminate between septic patients and postoperative surgical patients, defined as systemic inflammatory response syndrome (SIRS), both admitted at the intensive care unit (ICU). Furthermore, we wondered whether the CD64 index was an improved diagnostic compared to standard assays used at the laboratory. For this, outclinic (OC) patients were included as controls. METHODS: The Leuko64™ assay was used to determine the CD64 index in residual EDTA blood samples from selected septic patients (n=25), SIRS patients (n=19), and OC patients (n=24). Additionally, WBC count, neutrophilic and eosinophilic granulocyte count, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured simultaneously. RESULTS: The CD64 index was higher in septic patients compared to both the SIRS and OC group (p<0.0001). In addition, the WBC count, neutrophil count, ESR and CRP were also higher in septic patients than the OC group (p<0.0001). However, only the WBC count, eosinopenia, and ESR were comparable between the SIRS and the sepsis group and proved to be discriminative to the OC group (p<0.05). The CD64 index demonstrated higher sensitivity and specificity than CRP, WBC count, neutrophilic and eosinophilic granulocyte count, and ESR. CONCLUSIONS: A high CD64 index was found in septic intensive care patients, while a low CD64 index was observed in OC and SIRS patients, demonstrating that the CD64 index can be used for routine diagnostics in the ICU setting.

Migration of allosensitizing donor myeloid dendritic cells into recipients after liver transplantation.

It is thought, but there is no evidence, that myeloid dendritic cells (MDCs) of donor origin migrate into the recipient after clinical organ transplantation and sensitize the recipient's immune system by the direct presentation of donor allo-antigens. Here we show prominent MDC chimerism in the recipient's circulation early after clinical liver transplantation (LTx) but not after renal transplantation (RTx). MDCs that detach from human liver grafts produce large amounts of pro-inflammatory [tumor necrosis factor alpha and interleukin 6 (IL-6)] and anti-inflammatory (IL-10) cytokines upon activation with various stimuli, express higher levels of toll-like receptor 4 than blood or splenic MDCs, and are sensitive to stimulation with a physiological concentration of lipopolysaccharide (LPS). Upon stimulation with LPS, MDCs detaching from liver grafts prime allogeneic T cell proliferation and production of interferon gamma but not of IL-10. Soluble factors secreted by liver graft MDCs amplify allogeneic T helper 1 responses. In conclusion, after clinical LTx, but not after RTx, prominent numbers of donor-derived MDCs migrate into the recipient's circulation. MDCs detaching from liver grafts produce pro-inflammatory and anti-inflammatory cytokines and are capable of stimulating allogeneic T helper 1 responses, and this suggests that MDC chimerism after clinical LTx may contribute to liver graft rejection rather than acceptance.

Non-HLA T-cell reactivity during the first year after HLA-identical living-related kidney transplantation.

BACKGROUND: It has been reported that donor-reactive T-cell responses may decrease during the first year after HLA-mismatched organ transplantation. We wondered whether donor-reactive T-cell responses directed to minor histocompatibility antigens (mHAgs) or other non-HLA antigens also decrease after HLA-identical living-related (LR) kidney transplantation. METHODS: We studied donor-reactive T-cell responses by IFN-gamma and granzyme B (GrB) Elispot assays in 15 HLA-identical LR kidney transplant recipients before, six months and one yr after transplantation. Third-party reactivity was used as control. Patient and donor peripheral blood mononuclear cells were typed for 11 known mHAgs. RESULTS: During the study period, 60% and 36% of the patients demonstrated donor-reactive IFN-gamma and GrB producing cells (pc), respectively. The number of donor-reactive IFN-gamma and GrB pc was significantly lower than the number of third-party reactive IFN-gamma and GrB pc. After transplantation, donor-reactivity and third-party reactivity were comparable to pre-transplant values. No relation was found in mHAg mismatches between donor and recipient and donor-reactive T-cell response. CONCLUSIONS: Donor-reactivity could be detected before and after HLA-identical LR kidney transplantation, but was not related with the number of mHAg mismatches, and did not decrease after transplantation.

Donor-reactive cytokine profiles after HLA-identical living-related kidney transplantation.

BACKGROUND: After HLA-identical living-related (LR) kidney transplantation, only non-HLA antigen mismatches between donor and recipient may exist. We questioned whether donor-reactive responses against non-HLA antigens could be found after HLA-identical LR kidney transplantation, and wondered whether donor reactivity in the HLA-identical setting was different from the HLA-mismatched setting during immunological quiescence. Healthy individuals served as controls. METHODS: Elispot assays were performed to determine the number of alloreactive IFN-gamma-producing cells (pc), IL-10 pc, granzyme B (GrB) pc and IL-13 pc from peripheral blood mononuclear cells (PBMC) of HLA-identical, HLA-mismatched LR kidney transplant recipients and healthy individuals. RESULTS: The frequency of alloreactive IFN-gamma pc, IL-13 pc and GrB pc was higher in healthy individuals compared to both transplant patient groups. In the HLA-identical group, significantly higher numbers of donor-reactive IL-10 pc were found compared to their autologous control. These frequencies were also higher compared to the HLA-mismatched and healthy control group. The number of donor-reactive GrB pc was higher in the HLA-mismatched group than in the HLA-identical group. Donor-reactive IFN-gamma pc and IL-13 pc were comparable in both transplant groups. CONCLUSIONS: In recipients of HLA-identical LR kidney transplant, high donor-reactive IL-10 pc, in combination with low donor-reactive IFN-gamma pc, IL-13 pc and GrB pc, suggests active downregulation of reactivity against non-HLA molecules.

Peripheral blood manipulation significantly affects the result of dendritic cell monitoring.

It has been postulated that the plasmacytoid/myeloid dendritic cell ratio (pDC/mDC) reflects immune reactivity, and can therefore be used to monitor transplant recipients. We investigated the influence of Ficoll-Paque separation and PBMC cryopreservation on the pDC/mDC ratio and the expression of maturation markers, e.g. chemokine receptors (CKRs) CCR7, CXCR4, and CCR5, in comparison to fresh blood cells. Fractions of pDCs and mDCs, and CKR expression were measured by flow cytometry in fresh blood, in Ficoll-isolated PBMCs and in cryopreserved PBMCs from healthy individuals and kidney transplant recipients. Ficoll-isolation of PBMCs resulted in higher pDC/mDC ratios in both groups compared to fresh blood cells resulting from a relatively large increase in pDCs compared to mDCs. The pDC/mDC ratio increased further after cryopreservation of PBMCs from kidney transplant recipients. Ficoll-isolation and cryopreservation of PBMCs affected the proportion of mDCs and pDCs positive for CKRs, and their expression levels resulting in a more mature phenotype. In conclusion, the pDC/mDC ratio and pDC or mDC maturation status based on CKR expression, is dependent on manipulation of PBMCs. Therefore, fresh blood is preferable for monitoring purposes in transplant patients, as only these cells reflect the in vivo immune-status of patients accurately.

Successful tapering of immunosuppression to low-dose monotherapy steroids after living-related human leukocyte antigen-identical renal transplantation.

BACKGROUND: Living-related (LR) human leukocyte antigen (HLA)-identical renal transplant (RTx) recipients often receive standard immunosuppression, despite the absence of mismatched major HLA-antigens and the known complications of long-term use of immunosuppression. No data are available on the need for immunosuppression for these specific patients. We wondered whether their immunosuppressive load could be radically reduced. METHOD: Between November 1982 and November 2005, 83 LR HLA-identical RTx were performed in our center. Their unadjusted graft survival was 74% at 10 years. In 29 patients (median time after transplantation 5.6 [range 1.0-21.4] years) with stable uncompromised renal function, we tapered their immunosuppression from triple or dual therapy to prednisolone 5 mg/day. Follow up on prednisolone monotherapy was at least 24 months. RESULTS: In 27 of 29 patients reduction of immunosuppression to prednisolone monotherapy was uneventful. One patient, using dual therapy, developed JC-virus nephropathy resulting in graft loss. One refused further discontinuation of his medication. Four (15%) of the 27 patients on monotherapy developed biopsy-proven recurrence of their original disease. Only one of them showed a transient decline in renal function. One additional patient developed minor proteinuria and a rise in serum creatinine level, as a result of chronic urinary tract infections. The remaining 23 of 27 patients (85%) had an uneventful follow up during 24 months prednisolone monotherapy. CONCLUSION: We conclude that HLA-identical LR RTx recipients who are at least 1 year after transplantation might be treated with low-dose steroid monotherapy. Close surveillance of patients for recurrence of their original disease is recommended to allow for potential early therapeutic intervention.

T-cell reactivity during tapering of immunosuppression to low-dose monotherapy prednisolone in HLA-identical living-related renal transplant recipients.

BACKGROUND: In many transplant centers, human leukocyte antigen (HLA)-identical living-related (LR) renal transplant recipients receive standard maintenance immunosuppression from 1 year after transplantation. We questioned whether discontinuation of azathioprine (AZA) or mycophenolate mofetil (MMF) influenced T-cell reactivity, circulating dendritic cell (DC) subsets numbers and their maturation status. METHODS: Twenty-nine HLA-identical LR renal transplant recipients were withdrawn from AZA or MMF. Thereafter, the patients received only prednisolone. T-cell reactivity was determined by interferon-gamma (n=23), interleukin (IL)-10 (n=16), and granzyme B (n=10) Elispot assays. Circulating DC subset numbers and their maturation status determined by CCR2, CCR5, CCR7, and CD83 expression were measured by flow cytometry (n=12). RESULTS: The number of donor, third-party, and tetanus toxoid-reactive interferon-gamma and granzyme-B producing cells was not affected after withdrawal of immunosuppression. Discontinuation of AZA or MMF resulted in significant increased numbers of third-party (P=0.003) and tetanus toxoid-reactive (P=0.008) IL-10 producing cells, and a trend in higher numbers of donor-reactive IL-10 producing cells (P=0.06). No effect was found on the number of circulating DC subsets, but DC was shifted toward a more mature phenotype. CONCLUSIONS: In HLA-identical LR renal transplant recipients, therapy with AZA and MMF suppress the IL-10 production and the maturation of DC. This suggests that these immunosuppressants may hinder suppression of immune responses in general, including allogeneic responses.

Stable T-cell reactivity after successful tapering of azathioprine in HLA-identical living-related kidney transplant recipients despite minor histocompatibility antigen mismatches.

BACKGROUND: Human leukocyte antigen (HLA)-identical living-related (LR) kidney transplant recipients often receive the standard regimen of immunosuppression. We wondered whether these patients should be exposed to the side effects of these drugs any longer. Safe tapering of immunosuppression should not result in rejection and high donor-directed T-cell responses. In the present study, we investigated the effect of tapering azathioprine (AZA) on T-cell reactivity. METHODS: Fifteen HLA-identical LR kidney transplant recipients receiving a median of 150 mg/day AZA and 5-10 mg/day prednisone were tapered to a median of 50 mg/day AZA. Donor-, third-party and tetanus toxoid (TET)-reactivity were determined in interferon (IFN)-gamma and interleukin (IL)-13 Elispot assays, which reflect the T-helper (Th)1 and T-helper (Th)2 response. RESULTS: After the tapering of AZA, none of the patients developed acute rejection and the renal function remained stable, even at 1-year follow-up. The frequency of donor-specific IFN-gamma and IL-13 producing cells (pc) was low. Tapering of AZA did not influence the frequency of both IFN-gamma and IL-13 pc. Also, the reactivity against third-party cells and TET remained unchanged. CONCLUSIONS: The AZA-dose can be safely reduced in recipients of an HLA-identical LR kidney transplant without affecting kidney function and without increasing T-cell responses directed against donor or other antigens.