Pharmacogenetics in critical care: association between CYP3A5 rs776746 A/G genotype and acetaminophen response in sepsis and septic shock.

BACKGROUND: Pharmacogenetics could represent a further resource to understand the interindividual heterogeneity of response of the host to sepsis and to provide a personalized approach to the critical care patient. METHODS: Secondary analysis of data from the prospective observational study NCT02750163, in 50 adult septic and septic shock patients treated with Acetaminophen (ACT) for pyrexia. We investigated the presence of two polymorphisms, located respectively in the genes UGT1A1 and CYP3A5, that encode for proteins related to the hepatic metabolism of ACT. The main dependent variables explored were plasmatic concentration of ACT, body temperature and hepatic parameters. RESULTS: 8% of the patients carried CYP3A5 rs776746 A/G genotypes and showed significantly higher plasma levels of ACT than GG wild type patients, and than patients with UGT1A1 rs8330 C/G genotypes. CONCLUSIONS: Identifying specific genotypes of response to ACT may be helpful to guide a more personalized titration of therapy in sepsis and septic shock. CYP3A5 might be a good biomarker for ACT metabolism; however further studies are needed to confirm this result. TRIAL REGISTRATION: NCT02750163.

Preconditioning in hypoxic-ischemic neonate mice triggers Na+-Ca2+ exchanger-dependent neurogenesis.

To identify alternative interventions in neonatal hypoxic-ischemic encephalopathy, researchers' attention has been focused to the study of endogenous neuroprotective strategies. Based on the preconditioning concept that a subthreshold insult may protect from a subsequent harmful event, we aimed at identifying a new preconditioning protocol able to enhance Ca2+-dependent neurogenesis in a mouse model of neonatal hypoxia ischemia (HI). To this purpose, we also investigated the role of the preconditioning-linked protein controlling ionic homeostasis, Na+/Ca2+ exchanger (NCX). Hypoxic Preconditioning (HPC) was reproduced by exposing P7 mice to 20' hypoxia. HI was induced by isolating and cutting the right common carotid artery. A significant reduction in ischemic damage was observed in mice subjected to 20' hypoxia followed,3 days later, by 60' HI, thus suggesting that 20' hypoxia functions as preconditioning stimulus. HPC promoted neuroblasts proliferation in the dentate gyrus mirrored by an increase of NCX1 and NCX3-positive cells and an improvement of behavioral motor performances in HI mice. An attenuation of HPC neuroprotection as well as a reduction in the expression of neurogenesis markers, including p57 and NeuroD1, was observed in preconditioned mice lacking NCX1 or NCX3. In summary, PC in neonatal mice triggers a neurogenic process linked to ionic homeostasis maintenance, regulated by NCX1 and NCX3.

Imaging in gynecological disease (11): clinical and ultrasound features of mucinous ovarian tumors.

OBJECTIVE: To describe the clinical and ultrasound findings in patients with mucinous ovarian tumors. METHODS: In this retrospective study, women with a histological diagnosis of mucinous ovarian tumor who had undergone preoperative ultrasound examination were identified from the database of a single ultrasound center. The histological examination was performed by the same pathologist in all cases, and the ultrasound appearance of the tumors was described using the terms and definitions of the International Ovarian Tumor Analysis group. RESULTS: We identified 123 women with a histological diagnosis of mucinous ovarian tumor, of whom 57 (46%) had benign cystadenoma, 34 (28%) had gastrointestinal (GI)-type borderline tumor, 10 (8%) had endocervical-type borderline tumor and 22 (18%) had GI-type invasive carcinoma. On ultrasound examination, 65% (37/57) of cystadenomas were multilocular, of which 59% had ≤ 10 locules, and 79% (27/34) of GI-type borderline tumors were multilocular, of which 89% had > 10 locules. Conversely, 60% (6/10) of endocervical-type borderline tumors had papillations. Eighty-two percent (18/22) of invasive masses contained solid components and 55% (12/22) were multilocular-solid cysts. Bilateral mucinous cystadenomas were found in two women (4% of women with benign tumors) and bilateral borderline tumors of endocervical type in two women (20% of women with borderline tumors of endocervical type). No woman had a bilateral GI-type borderline tumor or a bilateral invasive tumor. CONCLUSIONS: A multilocular cyst with 2-10 locules is representative of a benign cystadenoma, whereas a multilocular cyst with > 10 locules is indicative of a GI-type borderline tumor. Most invasive tumors of mucinous GI-type contain solid components, the most typical ultrasound appearance being that of a multilocular-solid tumor. Papillary projections are typical features of endocervical-type borderline tumors. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Incidence and predictive factors for complications after uterine leiomyoma embolization.

STUDY QUESTION: What is the risk of complications after uterine leiomyoma embolization and what are the factors associated with complications? SUMMARY ANSWER: The cumulative risk of complications after embolization is relatively low even in the long term, but submucosal leiomyoma location may increase the risk. WHAT IS KNOWN ALREADY: A broad spectrum of complications after leiomyoma embolization have been described with widely varying rates. There is uncertainty over the actual risk of complications and the factors associated with this risk. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study of 288 consecutive women undergoing leiomyoma embolization in the general gynaecology clinic of a university teaching hospital between January 2001 and December 2010. PARTICIPANTS/MATERIALS, SETTING, METHODS: Complications occurring after embolization were categorized as major or minor according to the severity of their impact on health, the level of care required and the outcome. Cumulative complication rates were estimated by survival analysis and log-rank tests according to baseline variables. Multivariable Cox proportional hazards analysis was performed to adjust for confounders. MAIN RESULTS AND THE ROLE OF CHANCE: There were 48 patients who experienced a complication at a median of 5 months (95% confidence interval, 4.1-11.4) after embolization. Complications were minor in 38 patients and major in 10 patients. The cumulative overall complication rate was 13% (95% CI, 9.0-17.0) at 6 months, 16% (95% CI, 11.0-20.0) at 1 year, 17% (95% CI, 12.0-22.0) at 3 years and 18% (95% CI, 12.9-22.8) at 5 years. The most frequent complication (19/48, 39.6%) was leiomyoma expulsion, which occurred spontaneously in 13 (68.4%) of these cases and required assistance in 6 (31.6%) cases. Eight (2.8%) patients underwent re-intervention, including six hysteroscopic myomectomies, one laparoscopic myomectomy and one hysteroscopic adhesiolysis, as a result of a complication. Submucosal leiomyoma location was the only baseline variable associated with an increased risk for complications [Hazard ratio (HR), 2.28, 95% CI, 1.24-4.18, P = 0.008]. LIMITATIONS, REASONS FOR CAUTION: Our population did not include women of African descent, who have been reported to be at higher risk of post-procedural complications compared with Causcasian women. If such women were involved in the study, higher morbidity rates might have been observed. WIDER IMPLICATIONS OF THE FINDINGS: Women with submucosal leiomyomas at the time of embolization are more likely to have post-procedural complications. This is important new information for counselling patients contemplating this therapeutic approach. STUDY FUNDING/COMPETING INTERESTS: The authors have no competing interests to declare. The study was not supported by any external grant.

Human leucocytes in asthenozoospermic patients: endothelial nitric oxide synthase expression.

In a basic study at the Andrology Unit, Department of Clinical and Molecular Sciences, Polytechnic University of Marche, Ancona, Italy, we evaluated the pattern of mRNA endothelial nitric oxide synthase (eNOS) expression in human blood leucocytes isolated from normozoospermic fertile and asthenozoospermic infertile men to elucidate any pathogenic involvement in sperm cell motility. Forty infertile men with idiopathic asthenozoospermia and 45 normozoospermic fertile donors, age-matched, were included. Semen parameters were evaluated, and expression analysis of mRNA was performed in human leucocytes using reverse transcription polymerase chain reaction. Sperm volume, count, motility and morphology were determined, and eNOS expression and Western blotting analyses were performed. A positive correlation was observed between the concentrations of NO and the percentage of immotile spermatozoa. The mRNA of eNOS was more expressed in peripheral blood leucocytes isolated from asthenozoospermic infertile men versus those of fertile normozoospermic men (7.46 ± 0.38 versus 7.06 ± 0.56, P = 0.0355). A significant up-regulation of eNOS gene in peripheral blood leucocytes was 1.52-fold higher than that of fertile donors. It is concluded that eNOS expression and activity are enhanced in blood leucocytes in men with idiopathic asthenozoospermia.

Short- and long-term consequences of prenatal exposure to the cannabinoid agonist WIN55,212-2 on rat glutamate transmission and cognitive functions.

The aim of the present review is to summarize integrated neurochemical, morphological and neurobehavioral evidence, in particular from our laboratory, which emphasize the short- and long-term consequences of prenatal exposure to the cannabinoid receptor agonist WIN55,212-2 on rat glutamate transmission and cognitive functions. The results obtained provide evidence that maternal exposure to WIN55,212-2 induces an impairment of cognitive capacities in the offspring. This impairment is associated with alterations of cortical and hippocampal glutamate outflow, cortical neuron morphology and hippocampal long-term potentiation. These findings are in line with clinical data showing that the consumption of marijuana by women during pregnancy has negative consequences on the cognitive functions of their children. Thus, although it is difficult and sometimes misleading to extrapolate findings obtained from animal models to humans, the possibility that an alteration of glutamate transmission might underlie, at least in part, some of the cognitive deficits affecting the offspring of marijuana users, is supported.

Safety of two cluster schedules for subcutaneous immunotherapy in allergic rhinitis or asthma patients sensitized to inhalant allergens.

BACKGROUND: Subcutaneous immunotherapy (SCIT) usually requires a long titration phase, which can be associated with various adverse events (AEs). OBJECTIVES: It was the aim of this study to determine the safety of 2 cluster regimens for SCIT in patients with allergic rhinitis, with or without mild or moderate allergic asthma, who were sensitized to grass and/or tree pollen, or house dust mites (HDM). PATIENTS AND METHODS: Adult patients were included in a European, open-label, prospective trial. Pollen-allergic patients received grass pollen, grass and olive pollen, or hazel, alder and birch pollen according to a 3-week titration cluster. HDM-allergic patients received HDM extract according to a 2-week titration cluster. The safety of the titration phase was assessed in terms of local and systemic AEs. RESULTS: The safety analysis included 157 patients: 110 received pollen and 47 HDM extract. During the cluster titration, 248 AE episodes were reported in the pollen group and 113 in the HDM group; these were mainly local reactions. Around one third of patients (30.9% pollen and 38.3% HDM) did not experience any AE. In most cases (67.1% of pollen and 71.1% of HDM patients), AEs did not lead to a change in titration schedule. No anaphylactic reaction or other serious life-threatening systemic AEs were reported. Only 2 patients in the HDM group discontinued treatment because of AEs. CONCLUSIONS: Rapid cluster titration was well tolerated in adults with allergic rhinitis, with or without mild to moderate allergic asthma, due to pollen or HDM. This short-titration, high-dose cluster regime may allow better patient compliance and cost savings.

Role of the mitochondrial sodium/calcium exchanger in neuronal physiology and in the pathogenesis of neurological diseases.

In neurons, as in other excitable cells, mitochondria extrude Ca(2+) ions from their matrix in exchange with cytosolic Na(+) ions. This exchange is mediated by a specific transporter located in the inner mitochondrial membrane, the mitochondrial Na(+)/Ca(2+) exchanger (NCX(mito)). The stoichiometry of NCX(mito)-operated Na(+)/Ca(2+) exchange has been the subject of a long controversy, but evidence of an electrogenic 3 Na(+)/1 Ca(2+) exchange is increasing. Although the molecular identity of NCX(mito) is still undetermined, data obtained in our laboratory suggest that besides the long-sought and as yet unfound mitochondrial-specific NCX, the three isoforms of plasmamembrane NCX can contribute to NCX(mito) in neurons and astrocytes. NCX(mito) has a role in controlling neuronal Ca(2+) homeostasis and neuronal bioenergetics. Indeed, by cycling the Ca(2+) ions captured by mitochondria back to the cytosol, NCX(mito) determines a shoulder in neuronal [Ca(2+)](c) responses to neurotransmitters and depolarizing stimuli which may then outlast stimulus duration. This persistent NCX(mito)-dependent Ca(2+) release has a role in post-tetanic potentiation, a form of short-term synaptic plasticity. By controlling [Ca(2+)](m) NCX(mito) regulates the activity of the Ca(2+)-sensitive enzymes pyruvate-, alpha-ketoglutarate- and isocitrate-dehydrogenases and affects the activity of the respiratory chain. Convincing experimental evidence suggests that supraphysiological activation of NCX(mito) contributes to neuronal cell death in the ischemic brain and, in epileptic neurons coping with seizure-induced ion overload, reduces the ability to reestablish normal ionic homeostasis. These data suggest that NCX(mito) could represent an important target for the development of new neurological drugs.

Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen.

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

A hybrid expressing genetically engineered major allergens of the Parietaria pollen as a tool for specific allergy vaccination.

BACKGROUND: Allergy is an immunological disorder affecting about 25% of the population living in the industrialized countries. Specific immunotherapy is the only treatment with a long-lasting relief of allergic symptoms and able to reduce the risk of developing new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis. METHODS: By means of DNA recombinant technology, we were able to design a head to tail dimer expressing disulphide bond variants of the major allergen of the Parietaria pollen. IgE binding activity was studied by Western blot, ELISA inhibition assays and the skin prick test. T cell recognition was studied by peripheral blood mononuclear cell proliferation. The immunogenicity of the hybrid was studied in a mouse model of sensitization. RESULTS: In vitro and in vivo analysis showed that the disruption of specific cysteine residues in both allergens caused a strong reduction in IgE binding activity of the PjEDcys hybrid. In addition,we were able to show that a reduction in the IgE epitope content profoundly reduced the anaphylactic activity of the hybrid (from 100 to 1,000 times less than wild-type allergens) without interfering with the T cell recognition. Sera from BALB/c mice immunized with the hybrid were able to bind the natural Parietaria allergens and to inhibit the binding of human IgE to wild-type Par j 1 and Par j 2 allergens up to 90%. CONCLUSION: Our results demonstrate that hybrid-expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and maintained T cell reactivity for induction of protective antibodies.

Pharmacoeconomics of subcutaneous allergen immunotherapy.

The current burden of allergic diseases, estimated by both direct and indirect costs, is very relevant. In fact the cost estimation for rhinitis amount globally to 4-10 billion dollars/year in the U.S. and to an average annual cost of 1089 euros per child/adolescent and 1543 euros per adult in Europe. The estimated annual costs in Northern America for asthma amounted to 14 billion dollars. Consequently, preventive strategies aimed at reducing the clinical severity of allergy are potentially able to reduce its costs. Among them, specific immunotherapy (SIT) joins to the preventive capacity the carryover effect once treatment is discontinued. A number of studies, mainly conducted in the US and Germany demonstrated a favourable cost-benefit balance. In the nineties, most surveys on patients with allergic rhinitis and asthma reported significant reductions of the direct and indirect costs in subjects treated with SIT compared to those treated with symptomatic drugs. This is fully confirmed in recent studies conducted in European countries: in Denmark the direct cost per patient/year of the standard care was more than halved following SIT; in Italy a study on Parietaria allergic patients demonstrated a significant difference in favor of SIT plus drug treatment for three years versus drug treatment alone, with a cost reduction starting from the 2nd year and increasing to 48% at the 3rd year, with a highly statistical significance which was maintained up to the 6th year, i.e. 3 years after stopping immunotherapy, corresponding to a net saving for each patient at the final evaluation of 623 euros per year; in France a cost/efficacy analysis comparing SIT and current symptomatic treatment in adults and children with dust mite and pollen allergy showed remarkable savings with SIT for both allergies in adults and children.

Economic evaluation of sublingual immunotherapy: an analysis of literature.

Allergic rhinitis and asthma constitute a global health problem because of their very high prevalence and the consequent burden of disease, concerning medical and economical issues. Among the treatments of allergy, specific immunotherapy has the capacity to favourably alter the natural history of the disease both during and after its performance and thus to reduce the direct and indirect costs of allergic rhinitis and asthma. A number of studies reported such cost reduction for traditional, subcutaneous immunotherapy and recent data demonstrate that also sublingual immunotherapy (SLIT) is associated to economic advantages and/or monetary savings, specifically in terms of reduction of disease economic burden. Only few formal economic assessments of SLIT have been carried out so far, this article will present and discuss the published studies addressed to this issue. The data obtained, although the number of studies is still limited, provide preliminary evidence supporting a SLIT effect on sparing costs for respiratory allergy.

Practice parameters for sublingual immunotherapy.

The efficacy and safety of sublingual immunotherapy (SLIT) are currently supported by clinical trials, meta-analysis and post-marketing surveys. Practice parameters for clinical use of SLIT are proposed here by a panel of Italian specialists, with reference to evidence based criteria. Indications to SLIT include allergic rhinoconjunctivitis, asthma, and isolated conjunctivitis (strength of recommendation: grade A). As to severity of the disease, SLIT is indicated in moderate/severe intermittent rhinitis, persistent rhinitis and mild to moderate asthma (grade D). SLIT may be safely prescribed also in children aged three to five years (grade B), and its use in subjects aged more than 60 years is not prevented when the indications and contraindication are ascertained (grade D). The choice of the allergen to be employed for SLIT should be made in accordance with the combination of clinical history and results of skin prick tests (grade D). Polysensitisation, i.e. the occurrence of multiple positive response does not exclude SLIT, which may be done with the clinically most important allergens (grade D). As to practical administration, co-seasonal, pre co-seasonal, and continuous schedules are available, being the latter recommended for perennial allergens or for pollens with particularly prolonged pollination, such as Parietaria (grade D). For pollens with relatively short pollination, such as grasses and trees (cypress, birch, alder, hazelnut, olive) the pre co-seasonal and perennial schedules are preferred (grade C). The build-up phases suggested by manufacturers can be safely used (grade A), but they can be modified according to the patient's tolerance (grade C). A duration of SLIT of 3-5 years is recommended to ensure a long-lasting clinical effect after the treatment has been terminated (grade C).

The presence of alexithymia investigated by the TAS-20 in chronic urticaria patients: a preliminary report.

OBJECTIVE: Chronic Idiopathic Urticaria (CIU), conventionally defined as the occurrence of widespread itchy weals lasting for at least six weeks, has a significant place among the dermatoses related to psychological factors. Emotions that cannot be expressed or elaborated, may be important in the etiology. The objective of this study was to evaluate the characteristics of patients with CIU and compare them in terms of alexithymia. METHODS: Forty consecutive subjects with chronic urticaria were recruited from an outpatient allergologic clinic. All of the subjects completed Toronto Alexithymia Scale, Rorschach Inkblot Test according with Comprehensive System modified by Maurizio Cuffaro administration rules and the Family Drawing Test by Corman. RESULTS: CIU patients had higher alexithymia levels (p < .05) on comparison to the normal population. There was also a positive correlation between CIU patients and the presence of depressant characteristics as evaluated by Rorschach Inkblot Test and Family Drawing Test. DISCUSSION: CIU, a severe and chronic dermatological problem, may be related to affect-regulation, particularly alexithymia. Individuals with CIU might benefit from learning how to regulate their emotions other than by medical treatments.

Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase.

The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na(+) ([Na(+)](e)), a condition that by reducing intracellular Ca(2+) ([Ca(2+)](I)) prevents Ca(2+)-dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as N(omega)-L-allyl-L-arginine (L-ALA), N(omega)-propyl-L-arginine (NPLA), and L-nitro-arginine-methyl-ester (L-NAME), respectively. The results demonstrated that the removal of [Na(+)](e) hampered the [Ca(2+)](i) increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na(+)](e). Furthermore, the absence of [Na(+)](e) in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L-ALA (200 nM), and L-NAME (500 microM) was able to effectively reduce NO(*) production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases.

The 2-oxopyrrolidinacetamide piracetam reduces infarct brain volume induced by permanent middle cerebral artery occlusion in male rats.

In this study, the temporal development of focal cerebral infarction induced by permanent middle cerebral artery occlusion (pMCAO) and the effects of piracetam, a derivative of gamma-aminobutyric acid widely used in clinical practice as a nootropic agent, on infarct area and volume were investigated. pMCAO caused a cerebral infarct whose size progressively increased after 3, 6, 9, and 24 h. Piracetam (125 mg/kg i.p.), administered 6, 9, and 22 h after pMCAO, did not reduce pMCAO-induced brain infarct area size detected at the 24th hour. By contrast, when this agent was administered at the doses of 250 and 500 mg/kg, it caused a marked reduction of the infarct area size. This reduction was observed in almost every brain slice affected by pMCAO, although statistical differences (p <0.05) were detected in slices located at 3-5.5 mm posterior to the anterior pole in animals treated with 250 mg/kg piracetam and in slices located at 3.5-5 mm in those receiving 500 mg/kg. When the mean total volumes of brain infarct resulting from pMCAO were calculated, it was observed that in animals which had received piracetam (250 or 500 mg/kg) infarction volume was markedly ( approximately 50%) and significantly (p <0.05) reduced in comparison with saline injected rats. Finally, piracetam (250 mg/kg administered i.p. 6, 9, and 22 h after the ischemic insult) significantly reduced brain infarct area evaluated 48 h and 7 days after pMCAO.

Hypoallergenic variants of the Parietaria judaica major allergen Par j 1: a member of the non-specific lipid transfer protein plant family.

BACKGROUND: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. METHODS: Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. RESULTS: The disruption of Cys14-Cys29 and Cys30-Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4-Cys52 bridge (PjC mutant) and the latter Cys50-Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4-Cys52 and Cys50-Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. CONCLUSION: Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.