The predictive and prognostic significance of cell-free DNA concentration in melanoma.

BACKGROUND: Melanoma is the leading cause of skin cancer-related deaths worldwide. While there have been significant improvements in the treatment of advanced melanoma in the past decade, biomarker development lagged behind. OBJECTIVES: The majority of liquid biopsy biomarkers rely on the analyses of oncogenic mutations; however, about 20% of melanoma patients are wild type. Therefore, validation of universal predictive and prognostic biomarkers is urgently needed. METHODS: We analysed plasma samples in a discovery cohort (n = 20) and expansion cohort (n = 166) of metastatic melanoma patients and healthy donors (n = 116). Total plasma circulating cell-free DNA (cfDNA) concentrations were measured on the Qubit® platform using assays for single-(ss) and double (ds)-stranded DNA, DNA spectrophotometry and RNase P qPCR. We explored the diagnostic, predictive and prognostic potential of cfDNA concentration by bio-statistical methods and established a cfDNA threshold for risk stratification. RESULTS: Our selected best method was Qubit® dsDNA assay which quantified higher plasma cfDNA concentrations in melanoma patients than in healthy controls (AUC 72%). Measurement of baseline cfDNA concentration revealed that high cfDNA was associated with presence of metastases and higher AJCC stage (P < 0.05). Furthermore, high baseline cfDNA was an indicator of shorter overall survival in patients with oncogenic mutations (HR 2.12, P = 0.0008), and in wild-type patients (HR 5.55, P < 0.0001). CONCLUSIONS: We provide evidence that total cfDNA can be used as a biomarker for melanoma irrespective of the tumour genotype and can provide information on tumour load, risk of progression and risk of death.

Elevated baseline serum PD-1 or PD-L1 predicts poor outcome of PD-1 inhibition therapy in metastatic melanoma.

BACKGROUND: Programmed cell death protein 1 (PD-1) checkpoint inhibition has recently advanced to one of the most effective treatment strategies in melanoma. Nevertheless, a considerable proportion of patients show upfront therapy resistance and baseline predictive biomarkers of treatment outcome are scarce. In this study we quantified PD-1 and programmed death-ligand 1 (PD-L1) in baseline sera from melanoma patients in relation to therapy response and survival. PATIENTS AND METHODS: Sera taken at therapy baseline from a total of 222 metastatic melanoma patients (two retrospectively selected monocentric discovery cohorts, n = 130; one prospectively collected multicentric validation cohort, n = 92) and from 38 healthy controls were analyzed for PD-1 and PD-L1 concentration by sandwich enzyme-linked immunosorbent assay. RESULTS: Melanoma patients showed higher serum concentrations of PD-1 (P = 0.0054) and PD-L1 (P < 0.0001) than healthy controls. Elevated serum PD-1 and PD-L1 levels at treatment baseline were associated with an impaired best overall response (BOR) to anti-PD-1 (P = 0.014, P = 0.041), but not to BRAF inhibition therapy. Baseline PD-1 and PD-L1 serum levels correlated with progression-free (PFS; P = 0.0081, P = 0.053) and overall survival (OS; P = 0.055, P = 0.0062) in patients who received anti-PD-1 therapy, but not in patients treated with BRAF inhibitors. By combining both markers, we obtained a strong discrimination between favorable and poor outcome of anti-PD-1 therapy, with elevated baseline serum levels of PD-1 and/or PD-L1 associated with an impaired BOR (P = 0.037), PFS (P = 0.048), and OS (P = 0.0098). This PD-1/PD-L1 combination serum biomarker was confirmed in an independent multicenter validation set of serum samples prospectively collected at baseline of PD-1 inhibition (BOR, P = 0.019; PFS, P = 0.038; OS, P = 0.022). Multivariable Cox regression demonstrated serum PD-1/PD-L1 as an independent predictor of PFS (P = 0.010) and OS (P = 0.003) in patients treated with PD-1 inhibitors. CONCLUSION: Our findings indicate PD-1 and PD-L1 as useful serum biomarkers to predict the outcome of PD-1 inhibition therapy in melanoma patients and to select patients for PD-1-based versus BRAF-based therapy strategies.

Anti-HLA alloantibodies of the IgA isotype in re-transplant candidates part II: Correlation with graft survival.

We reported previously on the widespread occurrence of anti-HLA alloantibodies of the IgA isotype (anti-HLA IgA) in the sera of solid-organ re-transplantation (re-tx) candidates (Arnold et al., ). Specifically focussing on kidney re-tx patients, we now extended our earlier findings by examining the impact of the presence and donor specificity of anti-HLA IgA on graft survival. We observed frequent concurrence of anti-HLA IgA and anti-HLA IgG in 27% of our multicenter collective of 694 kidney re-tx patients. This subgroup displayed significantly reduced graft survival as evidenced by the median time to first dialysis after transplantation (TTD 77 months) compared to patients carrying either anti-HLA IgG or IgA (TTD 102 and 94 months, respectively). In addition, donor specificity of anti-HLA IgA had a significant negative impact on graft survival (TTD 74 months) in our study. Taken together, our data strongly indicate that presence of anti-HLA IgA, in particular in conjunction with anti-HLA-IgG, in sera of kidney re-tx patients is associated with negative transplantation outcome.

Humoral and Cellular Responses to a Single Dose of Fendrix in Renal Transplant Recipients with Non-response to Previous Hepatitis B Vaccination.

Approximately 70% of kidney transplant recipients are non-responders to conventional hepatitis B virus (HBV) vaccines. We examined whether Fendrix™, an HBV vaccine containing 3-O-desacyl-4'-monophosphoryl lipid A (MPL) as adjuvant, could induce HBV immunity in these patients and compared their vaccination efficacy with healthy controls tested previously by the same assays. We selected 35 kidney transplant recipients who had been vaccinated at least thrice against HBV but had never displayed anti-HBs antibodies. We re-assessed their anti-HBs antibody titres and further determined cellular HBV immunity by proliferation assay and interferon (IFN)-γ ELISpot. Seventeen recipients did neither display humoral nor cellular immunity and could be tested prior to and at month 1 after vaccination. Of note, HLA antigens associated with non-response to HBV vaccination (HLA-DRB1*03 and HLA-DQB1*02) were over-represented in these 17 recipients. At month 1 after a single vaccination with Fendrix™, we observed a significant increase in anti-HBs antibodies (P = 0.02). In seven of 17 recipients, we detected anti-HBs antibodies ≥10 IU/l (10-264), in four HBV-specific lymphocyte proliferation (stimulation index of 2.6-8.7) and in one specific IFN-γ responses (12 spots increment). The vaccination response to Fendrix™ was significantly higher (P = 0.035) than the response to HBVaxPro™ in young healthy controls. In summary, the results show that a single vaccination with Fendrix™ could already induce HBV-specific humoral and/or cellular responses in ten of 17 kidney transplant patients. Thus, Fendrix™ appears as an efficient vaccine in this patient cohort.

Diagnostics and treatment of a severe humoral rejection after liver transplantation: donor-specific antibodies as a still underestimated cause of graft failure.

BACKGROUND: Donor-specific antibodies (DSAs) are increasingly being considered a cause of complications after liver transplant (LT). However, neither monitoring of DSAs nor the appropriate therapeutic procedures for humoral graft damage are yet standardized. Here we report a case of DSA-positive humoral rejection after LT that was successfully treated with plasmapheresis and immunoglobulins. METHODS: Human leukocyte antigen (HLA)-specific DSAs were detected by Luminex bead assay. Patient characteristics, laboratory values, and data about the patient's general condition were documented from April 2013 to June 2015. CASE REPORT: Eighteen months after LT, a 54-year-old man experienced severe hepatopathy with rapidly increasing transaminase activity and total bilirubin levels. Histologic findings were inconclusive, demonstrating chronic cholestasis and minimal positive staining for C4 d. However, an analysis for anti-HLA antibodies detected DSAs against HLA class II molecules with high mean fluorescence intensity. The patient underwent 8 courses of plasmapheresis, resulting in sustained amelioration of his condition and decreases in bilirubin levels and transaminase activity. CONCLUSION: De novo DSAs can be responsible for graft failure after LT. Thus, procedures aimed at detecting DSAs are recommended, and regular monitoring of DSAs after LT is important for individualized risk management. Plasmapheresis is an efficient therapeutic procedure for DSA-associated graft failure.

Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions.

UNLABELLED: Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs. INTRODUCTION: Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation. METHODS: cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, ß-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies. RESULTS: In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition. CONCLUSIONS: The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.

Recruiting refugees and migrants as potential hematopoietic stem cell donors to serve patients of comparable ethnicities with rare human leucocyte antigen patterns - The BluStar.NRW project in North Western Germany.

Currently, approximately 19 million people with a migration background live in Germany. The majority of those descend from regions where the population has a genetically different distribution of HLA antigens when compared to the HLA frequencies usually found in North Western Europe. In case of severe haematological disorders of these individuals, allogeneic stem cell transplantation may be the treatment of choice. However, finding appropriate histocompatible hematopoietic stem cell donors continues to be a major challenge. If no matching sibling donors are available, there are only few suitable donors with a similar genetic background available in international blood stem cell donor registries. The "BluStar.NRW" project aimed to recruit new blood and hematopoietic stem cell donors with a migration background and to noticeably increase the number of suitable donors for patients within this group. Since December 2017, a total number of 9100 blood and stem cell donors with a migration background were recruited and typed for this project. HLA typing for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 was performed by Next Generation Sequencing. We assessed the proportion of rare alleles according to HLA frequency tables, as defined by a frequency of <1:1000. The rare HLA allele frequencies according to HLA frequency tables of the BluStar.NRW cohort were compared with a matched control donor cohort: Rare HLA-A, -B, -C, -DRB1 and -DQB1 alleles occurred three times more frequent than in the control group, but rare HLA-DPB1 alleles occurred more frequently in the control cohort. This difference was highly significant for all HLA alleles (p < 0.0001 for HLA-A, -B, -C, -DRB1, -DPB1; p = 0.0002 for HLA-DQB1). In addition, the distribution of rare alleles differed between the two groups. To date, 29 work-ups were initiated, 12 PBSC, one BM and three DLI were collected so far out of the BluStar.NRW cohort. The apheresis probability is twofold higher (0.18% vs. 0.07%) compared to the control group which clearly shows a serious medical need. However, 13 work-ups were cancelled in the BluStar.NRW donor cohort which represents an almost twice as higher cancellation rate (45% vs. 25%). This single registry analysis with a large sample cohort clearly indicates that hematopoietic stem cell donors with a migration background represent an adequate donor pool to serve patients of comparable ethnicity.

Inheritance of moyamoya disease in a Caucasian family.

BACKGROUND AND PURPOSE: Moyamoya disease is a very rare occlusive cerebrovascular disorder characterized by progressive stenosis or occlusion of the intracranial portion of the internal carotid artery and proximal cerebral arteries with an extensive network of fine collaterals. The aetiology and genetic susceptibility of moyamoya disease, especially in Caucasians, still remains unclear. METHODS AND RESULTS: We describe the cases of affected German father, daughter and son with juvenile stroke because of idiopathic moyamoya disease. The rare existing literature is reviewed and discussed. CONCLUSIONS: This is the first report on a father-to-child inheritance pattern in Caucasian patients with idiopathic Moyamoya disease (MMD). Our cases indicate possible genetic risk factors for the genesis of Caucasian Moyamoya disease.

Regenerative therapies in neonatology: clinical perspectives.

Regenerative therapy based on stem cells is applied as standard therapy in pediatric oncology. Furthermore, they are frequently used to treat immunodeficiency disorders of infants. For severe neonatal diseases, e. g. hypoxic-ischemic encephalopathy in term neonates or bronchopulmonary dysplasia in preterm infants, animal models have been established. According to some first preclinical results stem cell administration appears as a promising tool to improve the clinical outcome in high-risk infants. Provided the benefit of regenerative therapies can further be evaluated in appropriate preclinical neonate models, carefully controlled clinical trials to assess the significance of regenerative therapies, such as autologous stem cell administration, are indicated.

Lymphocyte function following radioiodine therapy in patients with thyroid carcinoma.

AIM: Since the nuclear disaster in Fukushima has raised great concern about the danger of radioactivity, we here addressed the question if the therapeutic use of iodine 131, the most frequently applied radionuclide, was harmful to immune function in patients. It was our aim to define for the first time in a clinical setting how radioiodine therapy alters anti-microbial immune responses. PATIENTS, METHODS: In 21 patients with thyroid carcinoma anti-microbial lymphocyte responses were assessed by lymphocyte transformation test and ELISpot - measuring lymphocyte proliferation and on a single cell level production of pro- and anti-inflammatory cytokines (interferon-γ and interleukin-10) - prior to therapy, at day 1 and day 7 post therapy. RESULTS: Proliferative lymphocyte responses and interferon-γ production after in vitro stimulation with microbial antigens were significantly (p < 0.05) increased at day 1 vs. pre therapy, and returned to pre therapy levels at day 7. On the contrary, at day 1 interleukin-10 production was significantly (p < 0.05) reduced. Thus, we observed a short-term increase in pro-inflammatory immune responses. However, T lymphocyte responses were in the range of healthy controls at all three time points. CONCLUSION: Thyroid carcinoma patients receiving radioiodine therapy do not display any sign of immunosuppression.

The new HLA-Cw*0524 allele increase the variability of position 49 in HLA-C.

The identification of the novel human leukocyte antigen (HLA)-Cw*0524, which was found in a potential stem cell donor, is described. The sequence of the new allele differs from HLA-Cw*0501 and HLA-Cw*0503 by a nonsynonymous amino acid (AA) exchanges at position 49.

DQB1*0323 is the result of a gene conversion event between a DQB1*06 and a DQB1*030303 allele.

A novel human leukocyte antigen-DQB1 allele, DQB1*0323, was identified in a volunteer hematopoietic stem cell donor. DQB1*0323 differs from the closely related allele DQB1*030303 in five nucleotide positions.

Early suppression of peripheral mononuclear blood cells in sepsis in response to stimulation with cytomegalovirus, OKT3, and pokeweed mitogen.

Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis (n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV (P = 0.01), OKT3 (P = 0.02), and PWM (P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points (P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis.NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.

Effect of ABO incompatibility on T-cell flow cytometry cross-match results prior to living donor kidney transplantation.

BACKGROUND: Due to its high sensitivity, the flow cytometry cross-match (FCXM) has been described as valuable tool for identifying an optimal donor. We here focused on the impact of ABO incompatibility on FCXM results. METHODS: We analyzed 29 ABO incompatible and 89 ABO compatible donor-recipient pairs (73 and 175 datasets, respectively) prior to living donor kidney transplantation. In all patients, lymphocytotoxic cross-matches for B and T cells were negative. RESULTS: Recipients with blood group O (A to O and B to O) displayed significantly (P < 0.05) higher T-FCXM results than those with blood group A and B (A to B, B to A and AB to A), respectively. Donor-specific T-FCXM responses (ΔMFI values) were significantly higher (P < 0.05) in ABO incompatible vs. compatible pairs (ABO incompatible recipients with blood group O: 32 ± 6; with blood group A: 19 ± 7; with blood group B: 7 ± 4; recipients with ABO compatibility: 3 ± 2, respectively, data represent mean ± SEM). Consistent with the T-FCXM results donor-specific isohemagglutinins (IgG titers) were significantly higher in recipients with blood group O vs. A, both prior to rituximab treatment and plasmapheresis/immune adsorption (P = 0.004) and immediately prior to transplantation, i.e., after rituximab and antibody-depleting therapies (P = 0.04). CONCLUSIONS: ABO incompatibility was associated with higher T-FCXM responses, especially in recipients with blood group O. This finding has major impact on the interpretation of flow cross-match results. Current cut-off values need to be reassessed in the ABO incompatible setting. © 2016 International Clinical Cytometry Society.

Primary central nervous system vasculitis and its mimicking diseases - clinical features, outcome, comorbidities and diagnostic results - A case control study.

OBJECTIVES: To compare clinical features and outcome, imaging characteristics, biopsy results and laboratory findings in a cohort of 69 patients with suspected or diagnosed primary central nervous system vasculitis (PCNSV) in adults; to identify risk factors and predictive features for PCNSV. PATIENTS AND METHODS: We performed a case-control-study including 69 patients referred with suspected PCNSV from whom 25 were confirmed by predetermined diagnostic criteria based on biopsy (72%) or angiography (28%). Forty-four patients turned out to have 15 distinct other diagnoses. Clinical and diagnostic data were compared between PCNSV and Non-PCNSV cohorts. RESULTS: Clinical presentation was not able to discriminate between PCNSV and its differential diagnoses. However, a worse clinical outcome was associated with PCNSV (p=0.005). Biopsy (p=0.004), contrast enhancement (p=0.000) or tumour-like mass lesion (p=0.008) in magnetic resonance imaging (MRI), intrathecal IgG increase (p=0.020), normal Duplex findings of cerebral arteries (p=0.022) and conventional angiography (p 0.010) were able to distinguish between the two cohorts. CONCLUSION: In a cohort of 69 patients with suspected PCNSV, a large number (64%) was misdiagnosed and partly received treatment, since mimicking diseases are very difficult to discriminate. Clinical presentation at manifestation does not help to differentiate PCNSV from its mimicking diseases. MRI and cerebrospinal fluid analysis are unlikely to be normal in PCNSV, though unspecific if pathological. Cerebral angiography and biopsy must complement other diagnostics when establishing the diagnosis in order to avoid misdiagnosis and mistreatment. CLINICAL TRIAL REGISTRATION: German clinical trials register: http://drks-neu.uniklinik-freiburg.de/drks_web/, Unique identifier: DRKS00005347.

Insulinoma cells contain an isoform of Ca2+/calmodulin-dependent protein kinase II delta associated with insulin secretion vesicles.

The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion. To determine which of the known subtypes (alpha, beta, gamma, delta) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the beta3-, gamma-, delta2- and delta6-subtypes in RINm5F insulinoma cells. None of the other 8 delta-subtypes was present. Antibodies generated against the bacterially expressed association domain of the delta2-subtype recognized the recombinant gamma and delta-subtypes. In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to the delta2-subtype expressed in NIH3T3 fibroblasts was detected. The delta2-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution was performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II delta by immune blots. The enzyme codistributed with insulin used as a marker for secretory granules but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lysosomes (arylsulfatase), or mitochondria (cytochrome c oxidase). CaM kinase II delta2 thus is identified as the subtype associated with insulin secretory granules and is likely to be involved in insulin secretion.

Effect of estradiol on insulin secreting INS-1 cells overexpressing estrogen receptors.

BACKGROUND: Estrogen has been shown to have profound effects on insulin and glucose metabolism in vivo. Indeed, estrogens were recently shown to modulate ion channel and secretory activities in endocrine cells. DESIGN AND METHODS: To investigate whether estrogenic influences are caused by direct effects on pancreatic beta-cells, we equipped INS-1 insulinoma cells with estrogen receptors and monitored insulin content and Ca(2+) fluxes as well as basal and stimulated insulin secretion upon different stimuli including glucose, the Ca(2+) ionophore ionomycin, the Ca(2+) channel agonist BayK8644, the protein kinase C activator TPA, and the adenylate cyclase activator forskolin. RESULTS AND CONCLUSION: Our data reveal that estradiol has no significant direct effect on proliferation rate, insulin content, basal and stimulated insulin output as well as Ca(2+) fluxes of insulin secreting cells in vitro, indicating that in vivo responses to estrogen on insulin and glucose metabolism result from indirect betacytotropic effects.

Use of the mouse intranasal test (MINT) to determine the allergenic potency of detergent enzymes: comparison to the guinea pig intratracheal (GPIT) test.

A mouse intranasal test (MINT) was developed to determine the relative allergenicity of detergent enzymes. In this simple method, various doses of the enzymes are administered in a detergent matrix, via intranasal instillation, on days 1, 3, and 10, with serum samples collected on day 15. The sera are then analyzed for enzyme specific IgG1 antibody by an antigen specific enzyme immunoassay. The protease enzyme Alcalase (protease Subtilisin Carlsberg) has been used as a benchmark enzyme for development and characterization of the model. The objective of the current studies was to obtain potency comparisons with other protease and nonprotease enzymes and to begin to assess the validity of the model by comparison with potency determinations obtained with the guinea pig intratracheal (GPIT) test. The range of potencies detected among several enzymes of different classes was approximately 60-fold (compared with Alcalase). Modification of the dosing regimen to permit slightly more extended dosing did not change the relative potency determination. Comparison of data from the MINT and GPIT methods indicate that both the mouse and the guinea pig recognize the bacterial amylase Termamyl and a fungal exocellulase as more potent than Alcalase, a serine protease (Subtilisin B) and a fungal alpha-amylase (Fungamyl) as less potent than Alcalase, and the serine protease, Savinase, as equivalent to Alcalase. Also, these data are in alignment with clinical data on the prevalence of occupational enzyme sensitization. Given the simplicity and low cost of the MINT method compared with the GPIT test, these results support continued development of the method as an alternative approach for assessing the allergenicity of enzymes.