Quantification and pharmacological characterization of dialysate levels of noradrenaline in the striatum of freely-moving rats: release from adrenergic terminals and modulation by α2-autoreceptors

Information concerning striatal levels of noradrenaline (NA) remains inconsistent. Here we have addressed this issue using a sensitive method of HPLC coupled to amperometric detection. The NA reuptake-inhibitor, reboxetine, selectively elevated levels of NA versus dopamine (DA), and NA levels were also selectively elevated by the α₂-adrenoceptor (AR) antagonist, atipamezole. The actions of atipamezole were mimicked by the preferential α_2A-AR antagonist, BRL44408, while JO-1 and prazosin, preferential antagonists at α_2C-ARs, caused less marked elevations in NA levels. In contrast to antagonists, the α₂-AR agonist, S18616, decreased NA levels and likewise suppressed those of DA. Unilateral lesions of the substantia nigra with 6-hydroxydopamine depleted DA levels without affecting those of NA. Further, the D₃/D₂ receptor agonist, quinelorane, decreased levels of DA without modifying those of NA. However, the D₃/D₂ receptor antagonists, haloperidol and raclopride, and the DA reuptake-inhibitor, GBR12935, elevated levels of both DA and NA. Levels of 5-HT (but not of NA or DA) were increased only by the 5-HT reuptake-inhibitor, citalopram. They were decreased by S18616 and prazosin, reflecting the inhibitory and excitatory influence of α₂- and α₁-ARs, respectively, upon serotonergic pathways. In conclusion, NA in the striatum is derived from adrenergic terminals. Its release is subject to tonic, inhibitory control by α₂-ARs, possibly involving both α_2A- and α_2C-AR subtypes, though their respective contribution requires clarification. A role of dopaminergic terminals in the reuptake of NA likely explains the elevation in its levels elicited by DA reuptake-inhibitors and D₃/D₂ receptor antagonists.     

Comparison of two target genes for detection and genotyping of Giardia lamblia in human feces by PCR and PCR-restriction fragment length polymorphism

A PCR assay targeting the tpi gene was developed to detect and to genotype Giardia lamblia in human feces. Our assay was specific and discriminated between G. lamblia assemblages A and B. G. lamblia cysts isolated from human feces were also analyzed with two previously described PCR-restriction fragment length polymorphism (RFLP) assays, which are based on the detection of tpi or gdh genes. These RFLP analyses distinguished groups I and II within assemblage A or groups III and IV within assemblage B. Among 26 fecal samples from patients with sporadic giardiasis diagnosed by hospital laboratories, the tpi gene was amplified from 25 (96%) with our PCR assay, whereas only 21 (81%) samples were positive when the gdh gene was targeted. Of the 25 positive samples, nine (36%) contained assemblage A and 16 (64%) contained assemblage B. Thus, RFLP analysis classified eight samples (32%) in assemblage A group II, eight (32%) in assemblage B group III, and five (20%) in assemblage B group IV. The group could not be specified for four samples. The tpi and gdh genes of G. lamblia assemblage B were amplified from 14 (93%) of 15 samples collected only from French soldiers coming back from the Ivory Coast. All of these contained assemblage B group III. The PCR method developed is sensitive, simple, and specific and shows that the tpi gene is well adapted for G. lamblia genotyping.     

Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis

Background

The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children.

Objective

We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model.

Methods

The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified.

Results

Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis.

Conclusion

We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

     

High-Density SNP genotyping defines 17 distinct haplotypes of the TNF block in the Caucasian population: implications for haplotype tagging

The region spanning the tumor necrosis factor (TNF) cluster in the human major histocompatibility complex (MHC) has been implicated in susceptibility to numerous immunopathological diseases, including type 1 diabetes mellitus and rheumatoid arthritis. However, strong linkage disequilibrium across the MHC has hampered the identification of the precise genes involved. In addition, the observation of "blocks" of DNA in the MHC within which recombination is very rare, limits the resolution that may be obtained by genotyping individual SNPs. Hence a greater understanding of the haplotypes of the block spanning the TNF cluster is necessary. To this end, we genotyped 32 human leukocyte antigen (HLA)-homozygous workshop cell lines and 300 healthy control samples for 19 coding and promoter region SNPs spanning 45 kb in the central MHC near the TNF genes. The workshop cell lines defined 11 SNP haplotypes that account for approximately 80% of the haplotypes observed in the 300 control individuals. Using the control individuals, we defined a further six haplotypes that account for an additional 10% of donors. We show that the 17 haplotypes of the "TNF block" can be identified using 15 SNPs.     

Current immunoassays and detection of antibodies elicited by Omicron SARS-CoV-2 infection

The present study aimed to determine whether current commercial immunoassays are adequate for detecting anti-Omicron antibodies. We analyzed the anti-SARS-CoV-2 antibody response of 23 unvaccinated individuals 1–2 months after an Omicron infection. All blood samples were tested with a live virus neutralization assay using a clinical Omicron BA.1 strain and four commercial SARS-CoV-2 immunoassays. We assessed three anti-Spike immunoassays (SARS-CoV-2 IgG II Quant [Abbott S], Wantaï anti-SARS-CoV-2 antibody ELISA [Wantaï], Elecsys Anti-SARS-CoV-2 S assay [Roche]) and one anti-Nucleocapsid immunoassay (Abbott SARS-CoV-2 IgG assay [Abbott N]). Omicron neutralizing antibodies were detected in all samples with the live virus neutralization assay. The detection rate of the Abbott S, Wantai, Roche, and Abbott N immunoassays were 65.2%, 69.6%, 86.9%, and 91.3%, respectively. The sensitivities of Abbott S and Wantai immunoassays were significantly lower than that of the live virus neutralization assay (p = 0.004, p = 0.009; Fisher's exact test). Antibody concentrations obtained with anti-S immunoassays were correlated with Omicron neutralizing antibody concentrations. These data provide clinical evidence of the loss of performance of some commercial immunoassays to detect antibodies elicited by Omicron infections. It highlights the need to optimize these assays by adapting antigens to the circulating SARS-CoV-2 strains.     

Primary Therapy of Waldenström Macroglobulinemia With Nucleoside Analogue–Based Therapy

Waldenström macroglobulinemia is a rare chronic lymphoproliferative disorder. Treatments are currently reserved for symptomatic patients and usually consist of nucleoside analogues (NAs), alkylating agents, bortezomib, and monoclonal antibodies, alone or in combination. Fludarabine and 2-chlorodeoxyadenosine (2-CdA) have been studied in first-line treatment of Waldenström macroglobulinemia (WM) since the end of the 1990s. In monotherapy, response rates vary between 36% and 94%. In a phase III trial, fludarabine in monotherapy was more efficient than chlorambucil for progression-free survival (PFS) (37.8 vs. 27.1 months), duration of response (DOR) (38.5 vs. 21.3 months) and overall survival (OS) (median not reached vs. 69.8 months), but the overall response rate (ORR) was similar (45.6% and 35.9%). NAs have been studied in combination with rituximab and/or alkylating agents for increasing the quality and duration of the response. Hematologic toxicities are a major concern, limiting the indication for NAs in first-line treatment to patients who are not candidates for autologous stem cell transplantation, those in need of rapid control of the disease, or those with poor prognostic factors.