Diagnosing and Monitoring Celiac Patients with Selective IgA Deficiency: Still an Open Issue

Digestive Diseases and Sciences - Although, the association between celiac disease (CD) and selective immunoglobulin A deficiency (SIgAD) has been known for more than fifty years, the procedures...     

A straightforward synthesis of phenyl boronic acid (PBA) containing BODIPY dyes: new functional and modular fluorescent tools for the tethering of the glycan domain of antibodies

We report here on the efficient and straightforward synthesis of a series of modular and functional PBA-BODIPY dyes 1–4. They are an outstanding example of the efficient merge of the versatility of the 3,5-dichloro-BODIPY derivatives and the receptor-like ability of the PBA moiety. The potential bioanalytical applicability of these tools was assessed by measuring the binding to glycan chains of antibodies by a Quartz Crystal Microbalance (QCM).

PBA-BODIPY dyes as functional and modular fluorescent probes for the tethering of the glycan domain of mAbs.

High performance organic fluorophores (e.g. large molar extinction coefficient, high quantum yield, high photo-stability, large Stokes shift) represent a booming research topic, with a wide range of applications ranging from materials to life sciences.^1,2 In this framework, modular and functional probes have become highly sought-after and researchers'' attention is manly focused on the fine tuning of optical properties and new functionalities for highly versatile tethering. However, despite the huge amount of effort in this field, only few fluorophores meet all these theoretical claims.³ Moreover, the availability of convenient and few steps synthetic protocols associated with a good overall yield is one of the major bottlenecks that needs to be addressed for ensuring the successful wide applicability of these probes.⁴ A brilliant example of a versatile fluorophore is the UV-absorbing dye 4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene, known as BODIPY,⁵ which displays high quantum yield, tunable photo-physical properties and excellent photo-stability.^5,6 Strategic structural modifications of BODIPY''s molecular architecture offer an unparalleled opportunity to tune its spectroscopic features, and diverse successful functional and bioactive BODIPY-like probes have been proposed and some commercialized, so far.⁷ 3,5-Dichloro-BODIPY dyes are one of the most recent synthetic analogues whose potential versatility has been demonstrated by Dehaen and Boens.⁸ They showed that appropriate substituents at 3,5 positions of the pyrrole shifted the excitation/emission bands of the corresponding substituted BODIPY derivatives. Thus, the 3,5-dichloro-BODIPY dyes give access, by nucleophilic substitution, to a variety of symmetrical and non-symmetrical BODIPY with several applications including labeling, sensing, energy transfer cassette.^1e,8,9 Then, 3,5-dichloro-BODIPY derivatives have been directly employed for the selective detection, in in vitro models, of sulphur containing metabolites.¹⁰ Recently, the valuable properties of other BODIPY dyes have been used for saccharides detection by phenyl boronic acid (PBA) moiety introduced at the meso position of the BODIPY core.¹¹ Notably, the dynamic covalent interaction between boronic acid and saccharides has been studied since the pioneering work of Lorand et al.¹² and PBA ability to bind 1,2 and 1,3-cis-diols motifs of carbohydrates has been used for the development of synthetic ‘boron-lectins’,¹³ and lately for the fishing of glycoproteins from complex mixtures, for the site-oriented immobilization of antibodies and for biorthogonal conjugations.¹⁴ However, the few examples of PBA-BODIPY probes have been reported to date¹¹ and they are confined to 3,5-dimethyl-BODIPY derivatives that require specific protocols for the further functionalization.In this context, we report here a straightforward synthetic route to obtain the functional and modular PBA-containing dye 1 and the subsequent late-stage diversification of the architecture of 1. Therefore, we obtained a small family of PBA-BODIPY derivatives with a ‘traffic light’ emission range (Fig. 1) for which we have studied the optical properties in different solvents and in physiological media. Thus, the present approach aims at the outstanding merging of the versatility of the 3,5-dichloro-BODIPY dyes with the presence of a functional PBA at the meso position of the BODIPY core. Finally, we show an example of the formation of the boronate esters between the PBA-BODIPY 4 and the glycan chains of an antibody, i.e. anti-streptavidin monoclonal antibody, by using Quartz Crystal Microbalance.Open in a separate windowFig. 1(A) Structure of PBA-BODIPY derivatives 1–4. (B) Solution (1.0 mg mL⁻¹ in MeOH) of PBA-BODIPY derivatives (from the left to the right PDA-BODIPY 1, 4, 2, 3) (a) under white light and (b) under UV-light (λ_ex = 364 nm).The BODIPY-core is traditionally accessible following a few steps synthetic strategy,^8a,15 however, the main concerns are related to the low overall yields of some synthetic steps and to the small scale availability of BODIPY derivatives due to the unstable nature of pyrrole derivatives and/or of some synthetic intermediates.^15a,16 In this paper, the boron dipyrromethane dye 1 (Scheme 1) was prepared following the synthetic strategy usually reported for the synthesis of 3,5-dichloro-BODIPY dyes¹⁵ by using the commercially available pyrrole-based derivatives and p-substituted benzaldehyde as starting materials.Open in a separate windowScheme 1Synthesis of the PBA-BODIPY 1–4. (a) CF₃COOH, 0 °C → r.t., 15 minutes, 74%; (b) NCS, THF, −78 °C → −20 °C, 18 h; (c) DDQ, CH₂Cl₂, r.t., 16 h, 75% over two steps; (d) BF₃Et₂O, CH₂Cl₂, r.t., 2 h, 75%; (e) aniline, CH₂Cl₂, 60 °C, 48 h, 58%; (f) aniline, 140 °C, 0.5 h; (g) ethylendiamine, r.t., 45 minutes, 44%.At first, the meso-PBA substitute dipyrromethane 5 was prepared by acid-catalyzed condensation^8a,15a,b of commercially available 4-formylbenzeneboronic acid 6 with neat excess (25 eq.) pyrrole (Scheme 1). In general, the purification of dipyrromethane derivatives is not trivial,^15a,b,16 depending on substituents at the meso position, as previously reported.^8a,15b A mixture of side products, have been identified and fully characterized,^15b and some improvements on the purification steps have been reported^15a,b switching from flash chromatography to a bulb-to-bulb distillation followed by recrystallization protocols (yield 27–68%).^15b Here, we set up, a straightforward crystallization protocol for the isolation of the pure dipyrromethane 5 (74%) avoiding the low yielding flash chromatography and tedious bulb-to-bulb distillations elsewhere described.^15b We have also examined the effect related to the workup of the reaction media (pyrrole : 6, 25 : 1 ratio, catalytic TFA) on the yield of this synthetic step, by using diverse protocols to refine the formation of 5, observing an higher yield (74% vs. 50%) and an easier removal of side products,^15b by the addition of trimethylamine (see ESI^†) to neutralize the TFA, and the subsequent concentration to dryness of the reaction mixture. Notably, the use of pure 5 is critical for the overall yield of the two following synthetic steps. The chlorination of 5 (Scheme 1) with N-chlorosuccinimide (NCS) followed by oxidation with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) afforded the 3,5-disubstututed dipyrromethene 7 (75% over two steps). Finally, 7 was reacted with boron trifluoride diethyl etherate (BF₃–OEt₂) and N,N′-diisopropylethylamine (DIPEA) to give the green emitting PBA-BODIPY 1 (75%). Dye 1 is amenable to structural modifications at the 3,5 positions of the pyrrole moiety so we prepared a small family of dyes which showed the spectral shift in the absorption and emission bands depending on the substitution pattern (Scheme 1). To demonstrate the versatility of PBA-BODIPY 1 we selected aniline as nucleophile already studied in this kind of reactions.^9e,f Thus, the PBA-BODIPY 1 was reacted with an excess of aniline in dichloromethane to give PBA-BODIPY 2 (58%) as pink solid, according to earlier investigations showing that one of the two chlorine atoms is more prone to be involved in the nucleophilic substitution.^8,9 Therefore, the di-substitute PBA-BODIPY 3 has been obtained from 3,5-dichloro-BODIPY 1 by using more stringent experimental conditions (neat excess of aniline, 140 °C) as previous reported.^9f Finally, mono-substitute PBA-BODIPY 4 was prepared in order to have a functional group on the BODIPY-core useful for further functionalization of the dye for the following studies. Thus, 1 was reacted with a neat excess of 1,2-diaminoethane to afford the yellow emitting PBA-BODIPY 4 (44%).The spectroscopic properties of the PBA-BODIPY dyes 1–4 (Fig. 2, and S1–S11^†) were studied in methanol, dichloromethane, water and physiological PBS buffer (pH = 7.4). The spectra reported in the left side of Fig. 2, show the absorption pattern of known PBA-BODIPY derivatives 1–4.^8,9,17 In all tested media, the main absorption band attributed to the 0–0 band of the strong S₀ → S₁ transition is located around 510 nm for PBA-BODIPY 1 and 2 (Fig. 2A, and C) and 590 nm for PBA-BODIPY 3 (Fig. 2E). For PBA-BODIPY 4 (Fig. 2G) the S₀ → S₁ transition is not the main absorption and is observed around 490 nm. The data are summarized in Fig. 2, the normalized fluorescence spectra of PBA-BODIPY 1–4 are also reported. A reduced solvatochromic effect is observed as both the S₀ → S₁ absorption and the emission bands are slightly red-shifted in dichloromethane than in more polar media (e.g. MeOH or H₂O). The broader full width at half maximum as well as the shape of the absorption and fluorescence bands of asymmetric PBA-BODIPY dyes 2 and 4 are consistent with previously reported amino-containing dyes and they were attributed to the diverse contributes of the proposed Lewis structural formulas.^9b The molar extinction coefficient (ε) was assessed in the four different solvents, showing that the highest hyperchromic effect is observed in MeOH (†). Finally, the Stokes shift values result moderately low for the symmetric derivatives 1 and 3, while higher value are found for dyes 2 and 4, where symmetry is reduced. The S₁ → S₀ fluorescence quantum yields (Φ_f) of 1–4 have been measured in MeOH using Rhodamine 6G as standard reference for 1, 2 and 4 and DODCl for 3 (see ESI^†) in the same solvent. High values of Φ_f have been found for 1 and 3, 0.16 and 0.22 respectively, suggesting that the symmetric presence of bulky substituents on the indacene moiety can stiffen the molecule, promoting radiative decay processes towards the ground state. Moreover, the relatively small Stokes shift observed for both compounds point at a molecular structure that is not much affected by the electronic excitation. On the opposite, the large Stokes shifts measured for the dyes 2 and 4, indicate a non negligible molecular structure variations going from S₀ to S₁. The less rigid skeleton of these dyes can endorse thermal relaxation, lowering the respective Φ_f, as shown in Open in a separate windowFig. 2Normalized absorbance and fluorescence spectra of PBA-BODIPY 1–4 in MeOH (black line) physiological PBS buffer pH = 7.4 (green line), H₂O (blue line) and CH₂Cl₂ (red line). (A and B) PBA-BODIPY 1; (C and D) PBA-BODIPY 2; (E and F) PBA-BODIPY 3; (G and H) PBA-BODIPY 4.Absorption and fluorescence emission spectral data of PBA-BODIPY 1–4

Evaluation of Two Different Protocols for Peripheral Blood Stem Cell Collection with the Fresenius AS 104 Blood Cell Separator

Objectives: Reconstitution of hematopoiesis by means of peripheral blood stem cells is a valid alternative to autologous bone marrow transplantation. The aim of this investigation was to increase the efficiency of collection of circulating blood progenitor cells and to obtain a purer product for transplant. Methods: We carried out leukapheresis procedures with the Fresenius AS 104 blood cell separator, using two different protocols, the previously used PBSC-LYM and a new mononuclear cell collection program. Results: Both programs were highly effective in collecting mononuclear cells (MNC) and CD34+ cells. Some differences were found, especially regarding MNC yield and efficiencies. There are remarkable differences in the efficiency of collection of CD34+ cells (62.38% with the new program as opposed to 31.69% with the older one). Linear regression analysis showed a negative correlation between blood volume processed and MNC efficiency only for the PBSC-LYM program. Differences were also observed in the degree of inverse correlation existing in both programs between patients' white blood cell precount and MNC collection efficiency. The inverse correlation was stronger for the PBSC-LYM program. Seven patients with solid tumors and hematologic malignancies received high dose chemotherapy and were subsequently transplanted with peripheral blood stem cells collected using the new protocol. All patients obtained a complete and stable engraftment with the reinfusion product collected with one or two leukapheresis procedures. Conclusions: High efficiencies and yields were observed in the new protocol for MNC and CD34+ cells. These were able to effect rapid and complete bone marrow recovery after myeloablative chemotherapy.     

Interferon–ribavirin therapy induces serum antibodies determining ‘rods and rings’ pattern in hepatitis C patients

A cytoplasmic antigen associated to inosine‐5'‐monophosphatedehydrogenase 2 eliciting specific antibodies (antirods and rings, RR) has been identified in patients with chronic hepatitis C who were exposed to pegylated interferon (PI) and ribavirin (RBV). The significance of anti‐RR in these patients merits to be investigated. Sera from 88 chronic hepatitis C virus (HCV)‐infected patients undergoing PI‐RBV therapy were analysed for the presence of RR pattern by indirect immunofluorescence on HEp‐2 substrate (Inova Diagnostics, San Diego, CA, USA). Anti‐RR antibodies developed de novo in 32 patients independently of any demographic and virological feature, but with a significant association with cumulative exposure to PI‐RBV (P = 0.0089; chi‐square test). RR pattern was significantly more frequent in relapsers than in patients achieving sustained virological response (56% vs 30%; P = 0.0282, chi‐square test). Anti‐RR titre ranged from 1:80 to 1:1280, but significantly declined following treatment cessation. Anti‐RR develop de novo in a substantial proportion of patients exposed to PI‐RBV in relation to the duration of treatment exposure. Further investigations are necessary to unravel the mechanisms leading to the formation of these autoantibodies.     

Prognostic value of chromosomal imbalances,gene mutations,and BAP1 expression in uveal melanoma

Uveal melanoma (UM) exhibits recurring chromosomal abnormalities and gene driver mutations, which are related to tumor evolution/progression. Almost half of the patients with UM develop distant metastases, predominantly to the liver, and so far there are no effective adjuvant therapies. An accurate UM genetic profile could assess the individual patient's metastatic risk, and provide the basis to determine an individualized targeted therapeutic strategy for each UM patient. To investigate the presence of specific chromosomal and gene alterations, BAP1 protein expression, and their relationship with distant progression free survival (DPFS), we analyzed tumor samples from 63 UM patients (40 men and 23 women, with a median age of 64 years), who underwent eye enucleation by a single cancer ophthalmologist from December 2005 to June 2016. UM samples were screened for the presence of losses/gains in chromosomes 1p, 3, 6p, and 8q, and for mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX. BAP1 protein expression was detected by immunohistochemistry (IHC). Multivariate analysis showed that the presence of monosomy 3, 8q gain, and loss of BAP1 protein were significantly associated to DPFS, while BAP1 gene mutation was not, mainly due to the presence of metastatic UM cases with negative BAP1 IHC and no BAP1 mutation detected by Sanger sequencing. Loss of BAP1 protein expression and monosomy 3 represent the strongest predictors of metastases, and may have important implications for implementation of patient surveillance, properly designed clinical trials enrollment, and adjuvant therapy.