Primary Invasive Cutaneous Fusariosis in Patients with STAT3 Hyper-IgE Syndrome.

Dominant negative (DN) mutations in signal transducer and activator of transcription 3 (STAT3) are known to cause hyper-IgE syndrome, a rare primary immunodeficiency. STAT3 DN patients are prone to develop fungal infections, including chronic mucocutaneous candidiasis due to impaired IL-17-mediated immunity, and pulmonary aspergillosis. Despite having preserved phagocyte functions, STAT3 DN patients present connective tissue abnormalities and a defect in the immunological skin barrier. Fusarium species are ubiquitous molds, whose potential to infect humans depends on the host's innate and cellular immune status. Our aim was to describe four STAT3 DN patients with fusariosis confined to the skin. Medical records were reviewed and summarized. Four patients, aged 4, 11, 30, and 33 years, presented with chronic skin lesions which started in the extremities. Two patients had remote lesions, and none had systemic involvement. Skin biopsies showed mycelial threads with deep inflammatory-occasionally granulomatous-infiltrates, reaching the dermis; cultures grew Fusarium solani. Response to treatment was heterogeneous, often requiring multimodal therapies, including topical antifungal preparations. In this work, we describe primary invasive cutaneous fusariosis as a syndromic entity in four STAT3 DN patients.

Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies.

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.

Dissimilar Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Distribution of Erlotinib to the Retina and Brain in Humans and Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [11C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 µg) of [11C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg, n = 17) to saturate erlotinib transport. In addition, transport of [3H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [11C]erlotinib total volume of distribution (VT) in the human retina by -25 ± 8% (p ≤ 0.05) and -41 ± 16% (p ≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [11C]erlotinib VT in the human brain (+20 ± 16%, p ≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [11C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.

Physicochemical Stability Study of Oral Suspension Containing Ruxolitinib in Children with Steroid-Refractory Acute Graft-Versus-Host Disease.

Ruxolitinib, used in children with steroid-refractory acute graft-versus-host (GVH) disease, is currently commercially available only as a tablet adult dosage. For the paediatric population, an oral liquid would be an adapted dosage formulation. The aim of this study was to develop ruxolitinib compounded oral suspensions at 2 mg/mL by using commercial tablets in available aqueous vehicle (Inorpha) and to measure its stability at both room temperature and under refrigeration. Chemical stability of suspensions containing ruxolitinib was evaluated for 60 days based on pH, degradation, and drug content. Physical stability of the drug suspension was evaluated by visual aspect and odour. The remaining ruxolitinib concentration of the suspension was at least 95% of the initial concentration after 60 days at both temperatures. The pH, colour, and odour of the suspensions throughout the study remained unchanged with respect to the initial time point.

TSPO PET Imaging as a Potent Non-Invasive Biomarker for Diffuse Intrinsic Pontine Glioma in a Patient-Derived Orthotopic Rat Model.

Diffuse intrinsic pontine gliomas (DIPG), the first cause of cerebral pediatric cancer death, will greatly benefit from specific and non-invasive biomarkers for patient follow-up and monitoring of drug efficacy. Since biopsies are challenging for brain tumors, molecular imaging may be a technique of choice to target and follow tumor evolution. So far, MR remains the imaging technique of reference for DIPG, although it often fails to define the extent of tumors, an essential parameter for therapeutic efficacy assessment. Thanks to its high sensitivity, positron emission tomography (PET) offers a unique way to target specific biomarkers in vivo. We demonstrated in a patient-derived orthotopic xenograft (PDOX) model in the rat that the translocator protein of 18 kDa (TSPO) may be a promising biomarker for monitoring DIPG tumors. We studied the distribution of 18F-DPA-714, a TSPO radioligand, in rats inoculated with HSJD-DIPG-007 cells. The primary DIPG human cell line HSJD-DIPG-007 highly represents this pediatric tumor, displaying the most prevalent DIPG mutations, H3F3A (K27M) and ACVR1 (R206H). Kinetic modeling and parametric imaging using the brain 18F-DPA-714 PET data enabled specific delineation of the DIPG tumor area, which is crucial for radiotherapy dose management.

Stability and Formulation of Erlotinib in Skin Creams.

Recent studies have highlighted the benefit of repurposing oral erlotinib (ERL) treatment in some rare skin diseases such as Olmsted syndrome. The use of a topical ERL skin treatment instead of the currently available ERL tablets may be appealing to treat skin disorders while reducing adverse systemic effects and exposure. A method to prepare 0.2% ERL cream, without resorting to a pure active pharmaceutical ingredient, was developed and the formulation was optimized to improve ERL stability over time. Erlotinib extraction from tablets was incomplete with Transcutol, whereas dimethyl sulfoxide (DMSO) allowed 100% erlotinib recovery. During preliminary studies, ERL was shown to be sensitive to oxidation and acidic pH in solution and when added to selected creams (i.e., Excipial, Nourivan Antiox, Pentravan, and Versatile). The results also showed that use of DMSO (5% v/w), neutral pH, as well as a topical agent containing antioxidant substances (Nourivan Antiox) were key factors to maintain the initial erlotinib concentration. The proposed ERL cream formulation at neutral pH contains a homogeneous amount of ERL and is stable for at least 42 days at room temperature in Nourivan cream with antioxidant properties.

The role of brain barriers in the neurokinetics and pharmacodynamics of lithium.

Lithium (Li) is the most widely used mood stabilizer in treating patients with bipolar disorder. However, more than half of the patients do not or partially respond to Li therapy, despite serum Li concentrations in the serum therapeutic range. The exact mechanisms underlying the pharmacokinetic-pharmacodynamic (PK-PD) relationships of lithium are still poorly understood and alteration in the brain pharmacokinetics of lithium may be one of the mechanisms explaining the variability in the clinical response to Li. Brain barriers such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a crucial role in controlling blood-to-brain and brain-to-blood exchanges of various molecules including central nervous system (CNS) drugs. Recent in vivo studies by nuclear resonance spectroscopy revealed heterogenous brain distribution of Li in human that were not always correlated with serum concentrations, suggesting regional and variable transport mechanisms of Li through the brain barriers. Moreover, alteration in the functionality and integrity of brain barriers is reported in various CNS diseases, as a cause or a consequence and in this regard, Li by itself is known to modulate BBB properties such as the expression and activity of various transporters, metabolizing enzymes, and the specialized tight junction proteins on BBB. In this review, we will focus on recent knowledge into the role of the brain barriers as key-element in the Li neuropharmacokinetics which might improve the understanding of PK-PD of Li and its interindividual variability in drug response.

Formulation and stability study of hydroxychloroquine sulfate oral suspensions.

Hydroxychloroquine is an antimalarial drug indicated in the treatment of acute attacks of malaria due to Plasmodium vivax, P. malariae, P. ovale, and susceptible strains of P. falciparum. It is also used for the treatment of rheumatoid arthritis, discoid and systemic lupus erythematosus, and more recently proposed in COVID-19 therapy. Hydroxychloroquine is only available in tablets which are not easy to administer for pediatric and geriatric patients, and patients unable to swallow such as patients found in intensive care units. The aim of this work was to develop and optimize a ready to use liquid hydroxychloroquine formulation and to carry out the corresponding chemical and microbiological stability studies. The formulation was evaluated for ease of preparation, physical properties, and palatability. Its stability was performed at ambient temperature and under refrigeration. After 6 months of stability testing, the results showed no pH change, no drug loss, no microbial development, and no visual change. The formulation, employing excipients in a range that EMA has recommended, showed chemical and microbiological stability for at least 6 months even in the worst storage conditions.

Stability of Pentobarbital Hydrogel for Rectal Administration in Pediatric Procedural Sedation.

Purpose: Pentobarbital is a sedative agent to limit children motion during computed tomography or magnetic resonance imaging (MRI) and ensures the successful completion of the imaging procedure. However, data on rectal drug formulation and its stability in practice are not available. The aim of this study was to formulate and evaluate the stability of a ready-to-use rectal pentobarbital gel. Methods: The formulation consisted of a hydrated gel containing 25 mg/mL of pentobarbital sodium, packaged in 10-mL amber glass bottles and stored at either 22°C to 25°C or 2°C to 8°C. At each predetermined time point, samples were taken for visual inspection, pH measurement, and analysis by a validated stability-indicating high-performance liquid chromatography (HPLC) method. The viscosity parameters of the hydrogel formulation were assessed. Results: The freshly prepared rectal formulations appeared clear, colorless, and particular-free with pH readings of 9.75 to 9.83. Over the 90 days of the study period, there was no significant change in appearance or pH values for all stability samples. The HPLC results confirmed the chemical stability when stored at 2°C to 8°C or at 22°C to 25°C. Conclusion: Pentobarbital hydrogel 25 mg/mL are stable chemically at least 90 days and can be administered to children for an effective and fast sedation.

Maternal ABVD chemotherapy for Hodgkin lymphoma in a dichorionic diamniotic pregnancy: a case report.

BACKGROUND: Hodgkin lymphoma (HL) is the most common hematological malignancy during pregnancy. The first-line treatment for HL in pregnancy is the standard ABVD regimen without any drug and/or dose adjustment. However, data on chemotherapy during twin pregnancies are sparse, and a better understanding of the mechanisms involved in exposure to and the toxic effects of anticancer drugs in the fetuses is needed. CASE PRESENTATION: A 41-year-old dichorionic diamniotic pregnant patient was given ABVD treatment for HL at a gestational age of 28 weeks and 3 days. The patient received 2 cycles of chemotherapy with a 15-day therapeutic window including an actual 25 mg/m2 dose of doxorubicin per cycle. Unlike the female twin, the male twin presented four days after birth a left cardiac dysfunction. Doxorubicin cardiotoxicity in the male newborn was also supported by high blood levels of troponin. At one month of age, echocardiography findings were normal. We investigated literature data on physiological aspects of pregnancy that may influence doxorubicin pharmacokinetics, and pharmacodynamic and pharmacokinetic data on the use of doxorubicin in pregnancy. We detailed the role of the transporters in doxorubicin placenta distribution, and tried to understand why only one fetus was affected. CONCLUSIONS: Fetal safety depends at least on maternal doxorubicin pharmacokinetics.Because of drug interactions (i.e. drug metabolism and drug transport), care should always be taken to avoid maternal pharmacokinetic variability. The toxic effects were discrepant between the dizygotic twins, suggesting additional fetus-specific pharmacokinetic/pharmacodynamic factors in doxorubicin toxicity.

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.

A virtual centralized cytotoxic preparation unit simulation to evaluate the pharmacy staff knowledge.

BACKGROUND: The risk of medication errors related to drug preparation unit cannot be totally avoided because of human interference. The aim of this study is to investigate the background and knowledge of the pharmacy staff by replicating the cytotoxic preparation unit with potential errors. METHODS: A 10-m2 room was provided to duplicate the centralized chemotherapy unit with three areas reproducing virtually the equipment preparation bench, the isolator, and the dispensing bench. The 14 situations selected by experts were integrated to each corresponded area. For each participant, a form was given and answers were analyzed by two independent experts. Statistical processing data were performed using GraphPad Prism® software. RESULTS: A total of 19 professionals participated in error simulation workshop over a one-month period. The overall rate of correct responses was 58 ± 19%. In five situations, correct responses rate was lower than 50%: wrong drug batch related to the preparation sheet (40%), inappropriate sterilizing conditions (15%), the time on the preparation sheet provides an expired expiry date for melphalan preparation (45%), a maximum drug dose exceeded (25%), the dispensing form not corresponds to the preparation sheet and final product label (30%). The rate of correct responses was 45 ± 25% for professionals not specifically dedicated to chemotherapy preparation. The overall satisfaction workshop rate was 8.7 ± 1.0 out of 10. CONCLUSION: This study showed the importance of training programs to sensitize personal staff to the risks of chemotherapy preparation and prevent errors.

Safety of intrathecal route: focus to methylprednisolone acetate (Depo-Medrol) use.

PURPOSE: Complications of the intrathecal route may cause potential toxicity related to the medical device and properties of the administered drug and/or excipient. A description of clinical and histological effects of polyethylene glycol and miripirium after Depo-Medrol injection, and the adverse reactions of particulate methylprednisolone acetate was conducted. The safety of the intrathecal route with excipients, label and off-label drugs is discussed. METHODS: A bibliographic search in Medline, Google, and Cochrane database from 1940 to June 2016 was performed. The keywords included 'intrathecal methylprednisolone acetate', 'miripirium', 'myristyl-gamma-picolinium', 'side effects', 'intrathecal Depo-Medrol', 'polyethylene glycol', and 'intrathecal devices' used individually or in combination. RESULTS: Adverse reactions have been reported with this intrathecal administration route such as arachnoiditis, bladder dysfunction, headache, meningitis. Some pharmaceutical excipients have been associated with specific toxicity issues and with allergic and anaphylaxis reactions. Additives of methylprednisolone acetate formulations such as polyethylene glycol and miripirium chloride can be neurotoxic when injected intrathecally. Polyethylene glycol-an antimicrobial agent widely used in pharmaceutical drugs-has been associated with cardiovascular, hepatic, respiratory, and CNS toxicity. CONCLUSIONS: Intrathecal methylprednisolone acetate (Depo-Medrol) therapy seems not fully safe due to reported adverse events. The use of other forms of corticosteroid therapy free from excipients should be emphasized such as soluble methylprednisolone sodium succinate.

Sodium Transporters Are Involved in Lithium Influx in Brain Endothelial Cells.

Variability in drug response to lithium (Li+) is poorly understood and significant, as only 40% of patients with bipolar disorder highly respond to Li+. Li+ can be transported by sodium (Na+) transporters in kidney tubules or red blood cells, but its transport has not been investigated at the blood-brain barrier (BBB). Inhibition and/or transcriptomic strategies for Na+ transporters such as NHE (SLC9), NBC (SLC4), and NKCC (SLC12) were used to assess their role on Li+ transport in human brain endothelial cells. Na+-free buffer was also used to examine Na+/Li+ countertransport (NLCT) activity. The BBB permeability of Li+ evaluated in the rat was 2% that of diazepam, a high passive diffusion lipophilic compound. Gene expression of several Na+ transporters was determined in hCMEC/D3 cells, human hematopoietic stem-cell-derived BBB models (HBLEC), and human primary brain microvascular endothelial cells (hPBMECs) and showed the following rank order with close expression profile: NHE1 > NKCC1 > NHE5 > NBCn1, while NHE2-4, NBCn2, and NBCe1-2 were barely detected. Li+ influx in hCMEC/D3 cells was increased in Na+-free buffer by 3.3-fold, while depletion of chloride or bicarbonate had no effect. DMA (NHE inhibitor), DIDS (anionic carriers inhibitor), and bumetanide (NKCC inhibitor) decreased Li+ uptake significantly in hCMEC/D3 by 52, 51, and 47%, respectively, while S0859 (NBC inhibitor) increased Li+ influx 2.3-fold. Zoniporide (NHE1 inhibitor) and siRNA against NHE1 had no effect on Li+ influx in hCMEC/D3 cells. Our study shows that NHE1 and/or NHE5, NBCn1, and NKCC1 may play a significant role in the transport of Li+ through the plasma membrane of brain endothelial cells.

[18F]FEPPA a TSPO Radioligand: Optimized Radiosynthesis and Evaluation as a PET Radiotracer for Brain Inflammation in a Peripheral LPS-Injected Mouse Model.

[18F]FEPPA is a specific ligand for the translocator protein of 18 kDa (TSPO) used as a positron emission tomography (PET) biomarker for glial activation and neuroinflammation. [18F]FEPPA radiosynthesis was optimized to assess in a mouse model the cerebral inflammation induced by an intraperitoneal injection of Salmonella enterica serovar Typhimurium lipopolysaccharides (LPS; 5 mg/kg) 24 h before PET imaging. [18F]FEPPA was synthesized by nucleophilic substitution (90 °C, 10 min) with tosylated precursor, followed by improved semi-preparative HPLC purification (retention time 14 min). [18F]FEPPA radiosynthesis were carried out in 55 min (from EOB). The non-decay corrected radiochemical yield were 34 ± 2% (n = 17), and the radiochemical purity greater than 99%, with a molar activity of 198 ± 125 GBq/µmol at the end of synthesis. Western blot analysis demonstrated a 2.2-fold increase in TSPO brain expression in the LPS treated mice compared to controls. This was consistent with the significant increase of [18F]FEPPA brain total volume of distribution (VT) estimated with pharmacokinetic modelling. In conclusion, [18F]FEPPA radiosynthesis was implemented with high yields. The new purification/formulation with only class 3 solvents is more suitable for in vivo studies.

Acute Morphine Exposure Increases the Brain Distribution of [18F]DPA-714, a PET Biomarker of Glial Activation in Nonhuman Primates.

Background: The neuroinflammatory response to morphine exposure modulates its antinociceptive effects, tolerance, and dependence. Positron emission tomography radioligands for translocator protein-18kDa such as [18F]DPA-714 are noninvasive biomarkers of glial activation, a hallmark of neuroinflammation. Methods: [18F]DPA-714 positron emission tomography imaging was performed in 5 baboons at baseline and 2 hours after i.m. morphine injection (1 mg/kg). Brain kinetics and metabolite-corrected input function were measured to estimate [18F]DPA-714 brain distribution. Results: Morphine significantly increased [18F]DPA-714 brain distribution by a 1.3 factor (P<.05; paired t test). The effect was not restricted to opioid receptor-rich regions. Differences in baseline [18F]DPA-714 binding were observed among baboons. The response to morphine predominated in animals with the highest baseline uptake. Conclusions: [18F]DPA-714 positron emission tomography imaging may be useful to noninvasively investigate the brain immune component of morphine pharmacology. Correlation between baseline brain distribution and subsequent response to morphine exposure suggest a role for priming parameters in controlling the neuroinflammatory properties of opioids.

Stability study of oral pediatric idebenone suspensions.

Adapted forms for administration to infants are limited. The proposed study was performed to propose oral liquid formulations of idebenone in Ora-Plus and either Ora-Sweet or Ora-Sweet SF, Ora-Blend, Ora-Blend SF and Inorpha. Each formulation was stored in 30 ml amber glass bottle at 5 or 25 °C for 90 days. Idebenone contents in these suspensions, determined by a stability-indicating high-performance liquid chromatography method, remained stable at least 90 days in Inorpha when stored at the two temperatures. In Ora-Blend, the stability was estimated at 14 days and in other suspensions at 20 days at the two temperatures. After 90 days storage, the pH of Ora-Plus and Ora-Sweet or Ora-Sweet SF changed between -0.10 and -0.25 units. For others suspensions, the pH changes were not significant (< -0.09 unit). No change was observed in color, odor or visual microbiology. To conclude, we recommended the use of idebenone in Inorpha vehicle stable for at least 90 days at 25 °C.

Immune quiescence of the brain is set by astroglial connexin 43.

In the normal brain, immune cell trafficking and immune responses are strictly controlled and limited. This unique homeostatic equilibrium, also called brain immune quiescence, is crucial to maintaining proper brain functions and is altered in various pathological processes, from chronic immunopathological disorders to cognitive and psychiatric impairments. To date, the precise nature of factors regulating the brain/immune system interrelationship is poorly understood. In the present study, we demonstrate that one of these regulating factors is Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the blood-brain barrier (BBB) interface. We show that, by setting the activated state of cerebral endothelium, astroglial Cx43 controls immune recruitment as well as antigen presentation mechanisms in the mouse brain. Consequently, in the absence of astroglial Cx43, recruited immune cells elaborate a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein of the brain. Altogether, our results demonstrate that Cx43 is a new astroglial factor promoting the immune quiescence of the brain.

Immunoregulation at the gliovascular unit in the healthy brain: A focus on Connexin 43.

In the brain, immune cell infiltration is normally kept at a very low level and a unique microenvironment strictly restricts immune reactions and inflammation. Even in such quiescent environment, a constant immune surveillance is at work allowing the brain to rapidly react to threats. To date, knowledge about the factors regulating the brain-immune system interrelationship in healthy conditions remains elusive. Interestingly, astrocytes, the most abundant glial cells in the brain, may participate in many aspects of this unique homeostasis, in particular due to their close interaction with the brain vascular system and expression of a specific molecular repertoire. Indeed, astrocytes maintain the blood-brain barrier (BBB) integrity, interact with immune cells, and participate in the regulation of intracerebral liquid movements. We recently showed that Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the BBB interface, is an immunoregulating factor. The absence of astroglial Cx43 leads to a transient endothelial activation, a continuous immune recruitment as well as the development of a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein expressed by astrocytes. In this review, we propose to gather current knowledge on how astrocytes may influence the immune system in the healthy brain, focusing on their roles at the gliovascular interface. We will also consider pathological situations involving astrocyte-specific autoimmunities. Finally, we will discuss the specific role of astroglial Cx43 and the physiological consequences of immune regulations taking place on inflammation, cognition and behavior in the absence of Cx43.

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor.

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 106 s-1.M-1 range, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.