Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. METHODS: We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. RESULTS: We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. CONCLUSIONS: The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).

Morquio B disease: From pathophysiology towards diagnosis.

Morquio B disease is an attenuated phenotype within the spectrum of beta galactosidase (GLB1) deficiencies. It is characterised by dysostosis multiplex, ligament laxity, mildly coarse facies and heart valve defects due to keratan sulphate accumulation, predominantly in the cartilage. Morquio B patients have normal neurological development, setting them apart from those with the more severe GM1 gangliosidosis. Morquio B disease, with an incidence of 1:250.000 to 1:1.000.000 live births, is very rare. Here we report the clinical-biochemical data of nine patients. High amounts of keratan sulfate were detected using LC-MS/MS in the patients' urinary samples, while electrophoresis, the standard procedure of qualitative glycosaminoglycans analysis, failed to identify this metabolite in any of the patients' samples. We performed molecular analyses at gene, gene expression and protein expression levels, for both isoforms of the GLB1 gene, lysosomal GLB1, and the cell-surface expressed Elastin Binding Protein. We characterised three novel GLB1 mutations [c.75 + 2 T > G, c.575A > G (p.Tyr192Cys) and c.2030 T > G (p.Val677Gly)] identified in three heterozygous patients. We also set up a copy number variation assay by quantitative PCR to evaluate the presence of deletions/ insertions in the GLB1 gene. We propose a diagnostic plan, setting out the specific clinical- biochemical and molecular features of Morquio B, in order to avoid misdiagnoses and improve patients' management.

Subcutaneous neurofibroma as a cause of lameness in a warmblood horse: Neurofibroma in a horse.

A neurofibroma was excised from the subcutis on the medial side of the left thigh of a 15-year-old Warmblood gelding, which had shown lameness of the left hind limb. No other source of lameness was found. Two weeks after surgery, the horse was sound at a lameness examination.

Serum creatinine during physiological perinatal dehydration may estimate individual nephron endowment.

It is well known that the nephron endowment of healthy subjects is highly variable and that individual nephron mass has potentially important implications both in health and disease. However, nephron count is technically impossible in living subjects. Based on the observation of an increase in serum creatinine (sCr) in otherwise healthy newborns with solitary kidney during the physiological perinatal dehydration, we hypothesized that perinatal sCr might be helpful in identifying healthy subjects with a reduced nephron mass. In the framework of a study on blood pressure in babies (NeoNeph), sCr of normal Caucasian neonates was determined 48-96 h after birth and their association with a family history of arterial hypertension (AH) was analyzed. SCr was determined in 182 normal newborns (90 males) at a mean of 61 ± 8 h after birth (range 46-82). Newborns with paternal AH had a higher mean sCr (0.97 + 0.28 mg/dL) then newborns without paternal AH (0.73 + 0.28 mg/dL; p = 0.006). No differences in mean sCr were found in relation with mother or grandparent's history of AH. CONCLUSION: The association between parental AH and high sCr during perinatal dehydration supports the hypothesis that the latter is a promising tool for identifying normal subjects with a reduced nephron mass with potential important implications in prevention and in understanding the individual outcome of renal and extrarenal diseases (including AH). What is Known: • Nephron endowment of healthy subjects is highly variable and individual nephron mass has potentially important implications both in health and disease however nephron count is not feasible in living subjects. What is New: • Serum creatinine during perinatal dehydration is a possible biomarker for identifying normal subjects with a reduced nephron mass.

Expanded newborn screening by mass spectrometry: New tests, future perspectives.

Tandem mass spectrometry (MS/MS) has become a leading technology used in clinical chemistry and has shown to be particularly sensitive and specific when used in newborn screening (NBS) tests. The success of tandem mass spectrometry is due to important advances in hardware, software and clinical applications during the last 25 years. MS/MS permits a very rapid measurement of many metabolites in different biological specimens by using filter paper spots or directly on biological fluids. Its use in NBS give us the chance to identify possible treatable metabolic disorders even when asymptomatic and the benefits gained by this type of screening is now recognized worldwide. Today the use of MS/MS for second-tier tests and confirmatory testing is promising especially in the early detection of new disorders such as some lysosomal storage disorders, ADA and PNP SCIDs, X-adrenoleucodistrophy (X-ALD), Wilson disease, guanidinoacetate methyltransferase deficiency (GAMT), and Duchenne muscular dystrophy. The new challenge for the future will be reducing the false positive rate by using second-tier tests, avoiding false negative results by using new specific biomarkers and introducing new treatable disorders in NBS programs.

Study protocol: safety and efficacy of propranolol 0.2% eye drops in newborns with a precocious stage of retinopathy of prematurity (DROP-ROP-0.2%): a multicenter, open-label, single arm, phase II trial.

BACKGROUND: Retinopathy of prematurity (ROP) still represents one of the leading causes of visual impairment in childhood. Systemic propranolol has proven to be effective in reducing ROP progression in preterm newborns, although safety was not sufficiently guaranteed. On the contrary, topical treatment with propranolol eye micro-drops at a concentration of 0.1% had an optimal safety profile in preterm newborns with ROP, but was not sufficiently effective in reducing the disease progression if administered at an advanced stage (during stage 2). The aim of the present protocol is to evaluate the safety and efficacy of propranolol 0.2% eye micro-drops in preterm newborns at a more precocious stage of ROP (stage 1). METHODS: A multicenter, open-label, phase II, clinical trial, planned according to the Simon optimal two-stage design, will be performed to analyze the safety and efficacy of propranolol 0.2% eye micro-drops in preterm newborns with stage 1 ROP. Preterm newborns with a gestational age of 23-32 weeks, with a stage 1 ROP will receive propranolol 0.2% eye micro-drops treatment until retinal vascularization has been completed, but for no longer than 90 days. Hemodynamic and respiratory parameters will be continuously monitored. Blood samplings checking metabolic, renal and liver functions, as well as electrocardiogram and echocardiogram, will be periodically performed to investigate treatment safety. Additionally, propranolol plasma levels will be measured at the steady state, on the 10th day of treatment. To assess the efficacy of topical treatment, the ROP progression from stage 1 ROP to stage 2 or 3 with plus will be evaluated by serial ophthalmologic examinations. DISCUSSION: Propranolol eye micro-drops could represent an ideal strategy in counteracting ROP, because it is definitely safer than oral administration, inexpensive and an easily affordable treatment. Establishing the optimal dosage and treatment schedule is to date a crucial issue. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02504944, registered on July 19, 2015, updated July 12, 2016. EudraCT Number 2014-005472-29.

Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno). METHODS: Dried blood spots (DBS) samples were extracted with 200 µL of methanol and 100 µL of water (by two steps). Internal standards were added at a final concentration of 10 µmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column. RESULTS: The assay was linear over a concentration range of 0.05-50 µmol/L (R2>0.999) for dGuo and 0.5-50 µmol/L (R2>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 µmol/L and 0.5 µmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified. CONCLUSIONS: The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.

Early skeletal outcomes after hematopoietic stem and progenitor cell gene therapy for Hurler syndrome.

Mucopolysaccharidosis type I Hurler (MPSIH) is characterized by severe and progressive skeletal dysplasia that is not fully addressed by allogeneic hematopoietic stem cell transplantation (HSCT). Autologous hematopoietic stem progenitor cell-gene therapy (HSPC-GT) provides superior metabolic correction in patients with MPSIH compared with HSCT; however, its ability to affect skeletal manifestations is unknown. Eight patients with MPSIH (mean age at treatment: 1.9 years) received lentiviral-based HSPC-GT in a phase 1/2 clinical trial (NCT03488394). Clinical (growth, measures of kyphosis and genu velgum), functional (motor function, joint range of motion), and radiological [acetabular index (AI), migration percentage (MP) in hip x-rays and MRIs and spine MRI score] parameters of skeletal dysplasia were evaluated at baseline and multiple time points up to 4 years after treatment. Specific skeletal measures were retrospectively compared with an external cohort of HSCT-treated patients. At a median follow-up of 3.78 years after HSPC-GT, all patients treated with HSPC-GT exhibited longitudinal growth within WHO reference ranges and a median height gain greater than that observed in patients treated with HSCT after 3-year follow-up. Patients receiving HSPC-GT experienced complete and earlier normalization of joint mobility compared with patients treated with HSCT. Mean AI and MP showed progressive decreases after HSPC-GT, suggesting a reduction in acetabular dysplasia. Typical spine alterations measured through a spine MRI score stabilized after HSPC-GT. Clinical, functional, and radiological measures suggested an early beneficial effect of HSPC-GT on MPSIH-typical skeletal features. Longer follow-up is needed to draw definitive conclusions on HSPC-GT's impact on MPSIH skeletal dysplasia.

Screening of lysosomal storage disorders: application of the online trapping-and-cleanup liquid chromatography/mass spectrometry method for mucopolysaccharidosis I.

In recent years, new treatments have become available to treat some lysosomal storage disorders (LSDs) and many studies suggest that there is a benefit with starting therapy early. Newborn screening should detect diseases early enough for prompt treatment. Some countries include additional conditions, such as some LSDs, into their newborn screening panels. Mucopolysaccharidosis Type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA) activity. Currently, enzyme replacement therapy (ERT) or bone marrow transplantation is available and this has raised a growing interest for the development of a newborn screening test. In 2009, we reported a new fast and simplified tandem mass spectrometry-based method for quantifying five enzyme activities on dried blood spots. Here, we describe the inclusion of IDUA activity determination for the simultaneous detection of six lysosomal storage diseases. We have defined reference normal ranges by testing 680 healthy newborns and 240 adults. The assay was checked through three confirmed MPS I patients whose IDUA activity was below the normal range. Reproducibility of the assays has been established by assessing the intra-day and inter-day assay imprecisions. This quick assay has been devised to be implemented in newborn screening by liquid chromatography tandem mass spectrometry.

Short-Term Survival and Postoperative Complications Rates in Horses Undergoing Colic Surgery: A Multicentre Study.

The occurrence of colic could be influenced by the characteristics of a population, geographical area, and feeding management. The aim of this study was to report the short-term postoperative complications and survival rates and to identify factors that might affect the outcome of horses that underwent colic surgery in three Italian surgical referral centres. Data of horses subjected to colic surgery in three referral centres (2018-2021) were analysed. Comparisons of the outcomes were performed using a Mann-Whitney or a Chi square test. Areas under the receiver operating characteristic (ROC) curve and multivariable logistic regression analysis were used for parameters that were significant in the previous univariate analysis. The goodness-of-fit of the model was assessed using the Akike information criterion (AIC). Significance was defined as p < 0.05, and odds ratios and 95% confidence intervals were calculated as percentages. A total of 451 horses were included. The survival rate was 68.5% of all of the horses that underwent colic surgery and 80% of the horses surviving anaesthesia. Age, BCS, PCV and TPP before and after surgery, amount of reflux, type of disease, type of lesion, duration of surgery, surgeon's experience, and amount of intra- and postoperative fluids administered influenced the probability of short-term survival. The multivariate analysis revealed that PCV at arrival, TPP after surgery, and BCS had the highest predictive power. This is the first multicentre study in Italy. The results of this study may help surgeons to inform owners regarding the prognosis of colic surgery.

Management of uroperitoneum through combination of conservative and surgical treatments in two colts.

Background: Ruptures of the urinary bladder and urachus are the most frequent cause of uroperitoneum in foals. Surgical correction is often the first treatment choice, however, nonsurgical methods, such as urine removal via urinary catheters and abdominal drains, have been successfully performed in foals. Case Description: Two foals were referred to the Equine Perinatology Unit for suspicion of uroperitoneum. The diagnosis was confirmed by hematobiochemical and ultrasound examinations, thus cystorrhaphy and cystoplasty were attempted. Surgeons found a lesion in the dorsocranial margin of the bladder (Case 1) and a tear in the pelvic urethra (Case 2); in the first case, the defect was routinely repaired, while the last lesion was impossible to repair due to its localization. A urinary catheter was left in place in both cases. Uroperitoneum recurred 72 hours after the surgery in both foals: a second surgical correction was not recommended due to the localization of the tears and conservative treatment, with the placement of a 32F chest tube in the most ventral part of the abdomen, was preferred. Abdominal drains were removed 5-7 days after surgery, while urinary catheters were left in place for up to 7-8 days. Colts' conditions improved during hospitalizations. Two months after bladder surgery, Case 1 was euthanized due to multiple adhesions between the small intestine and the abdominal wall. Case 2 was still alive one year postoperatively. Conclusion: Although it cannot be considered the first choice for the treatment of uroperitoneum in the foal, nonsurgical treatment was successful in both cases in the short-term follow-up. However, the prognosis should be cautious due to the risk of long-term complications. Conservative management may be used to manage bladder/urethral tears that cannot be solved by surgery.

Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol.

DNA methylation is a key regulator of gene transcription. Alterations in DNA methylation patterns are common in most cancers, occur early in carcinogenesis and can be detected in body fluids. Reliable and sensitive quantitative assays are required to improve the diagnostic role of methylation in the management of cancer patients. Here we present an optimized procedure, based on differential-high resolution melting analysis (D-HRMA), for the rapid and accurate quantification of methylated DNA. Two sets of primers are used in a single tube for the simultaneous amplification of the methylated (M) and unmethylated (Um) DNA sequences in D-HRMA. After HRM, differential fluorescence was calculated at the specific melting temperature after automatic subtraction of UM-DNA fluorescence. Quantification was calculated by interpolation on an external standard curve generated by serial dilutions of M-DNA. To optimize the protocol, nine primer sets were accurately selected on the basis of the number of CpG on promoters of hTERT and Bcl2 genes. The use of optimized D-HRMA allowed us to detect up to 0.025% M-DNA. D-HRMA results of DNA from 85 bladder cancers were comparable to those obtained with real time quantitative methylation specific PCR. In addition, D-HRMA appears suitable for rapid and efficient measurements in 'in vitro' experiments on methylation patterns after treatment with demethylating drugs.

The Comparison of Latero-Medial versus Dorso-Palmar/Plantar Drilling for Cartilage Removal in the Proximal Interphalangeal Joint.

The aims of the present study were to compare the percentages of articular cartilage removed using a lateral drilling approach of the proximal interphalangeal joint (PIPJ) and a dorsal drilling approach, and to assess the usefulness of digital fluoroscopy when performing a lateral drilling approach. Sixty cadaveric PIPJs were drilled using a surgical drill bit to remove the articular cartilage. The limbs were divided into three groups containing 10 forelimbs and 10 hindlimbs each. One group received the dorsal drilling approach, the second one received the lateral drilling approach and the last one received the lateral drilling approach under digital fluoroscopy guidance. The percentage of articular cartilage removed from each articular surface was assessed using Adobe Photoshop ® software. The percentages of removed cartilage turned out to be significantly higher with lateral approach, especially under fluoroscopic guidance, both in the forelimbs (p = 0.00712) and hindlimbs (p = 0.00962). In conclusion, the lateral drilling approach seems to be a minimally invasive technique with which to perform PIPJ arthrodesis, even more efficient than the previously reported dorsal approach.

Treatment of Septic Arthritis With Acoustic Cavitation and Lavage: A Case Report.

A 2-year-old Quarter Horse filly was admitted to our facility with a two-day history of trauma caused by barbed wire on the front face of the right front limb fetlock joint. A septic arthritis was confirmed with the synovial fluid examination. The cytologic evaluation of synovial fluid showed 101,000 leukocytes/mm3, 90% neutrophils, and a total protein concentration of 4 g/dL. It was, therefore, decided to perform an arthroscopic lavage using an ultrasonic device with the tip inserted inside the synovial cavity to exploit the phenomenon of acoustic cavitation. After the removal of the synovial pannus, the probe was left to vibrate in the area near the visible penetrating wound inside the joint until three liters of saline was finished. Further studies are needed for understanding and applying this technology in different equine fields and with different bacterial species. However, these first results are promising for the efficacy of this innovative procedure.

Development of Strategies to Decrease False Positive Results in Newborn Screening.

The expansion of national newborn screening (NBS) programmes has provided significant benefits in the diagnosis and early treatment of several rare, heritable conditions, preventing adverse health outcomes for most affected infants. New technological developments have enabled the implementation of testing panel covering over 50 disorders. Consequently, the increment of false positive rate has led to a high number of healthy infants recalled for expensive and often invasive additional testing, opening a debate about the harm-benefit ratio of the expanded newborn screening. The false-positive rate represents a challenge for healthcare providers working in NBS systems. Here, we give an overview on the most commonly used strategies for decreasing the adverse effects due to inconclusive screening results. The focus is on NBS performance improvement through the implementation of analytical methods, the application of new and more informative biomarkers, and by using post-analytical interpretive tools. These strategies, used as part of the NBS process, can to enhance the positive predictive value of the test and reduce the parental anxiety and healthcare costs related to the unnecessary tests and procedures.

Effect of Beta 3 Adrenoreceptor Modulation on Patency of the Ductus Arteriosus.

ß3-adrenoreceptor (ß3-AR), a G-protein coupled receptor, has peculiar regulatory properties in response to oxygen and widespread localization. ß3-AR is expressed in the most frequent neoplasms, also occurring in pregnant women, and its blockade reduces tumor growth, indicating ß3-AR-blockers as a promising alternative to antineoplastic drugs during pregnancy. However, ß3-AR involvement in prenatal morphogenesis and the consequences of its blockade for the fetus remain unknown. In this study, after the demonstrated expression of ß3-AR in endothelial and smooth muscle cells of ductus arteriosus (DA), C57BL/6 pregnant mice were acutely treated at 18.5 of gestational day (GD) with indomethacin or with the selective ß3-AR antagonist SR59230A, or chronically exposed to SR59230A from 15.5 to 18.5 GD. Six hours after the last treatment, fetuses were collected. Furthermore, newborn mice were treated straight after birth with BRL37344, a ß3-AR agonist, and sacrificed after 7 h. SR59230A, at the doses demonstrated effective in reducing cancer progression (10 and 20 mg/kg) in acute and chronic mode, did not induce fetal DA constriction and did not impair the DA ability to close after birth, whereas at the highest dose (40 mg/kg), it was shown to cause DA constriction and preterm-delivery. BRL37344 administered immediately after birth did not alter the physiological DA closure.

LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization.

Mucopolysaccharidoses are a group of lysosomal storage disorders (LSDs) characterized by the accumulation of glycosaminoglycans (GAGs). Recently, LC-MS/MS has been widely applied in GAGs analysis combined with different sample preparations for cleaving GAGs to disaccharide units. The aim of the present is paper is to present a new method for the simultaneous quantification of urinary dermatan sulfate (DS) and heparan sulfate (HS) by LC-MS/MS, after butanolysis reaction. Chromatographic separation was achieved with a gradient of acetonitrile and water in 0.1% formic acid on a Kinetex Biphenyl analytical column in 21 min. Calibration curves ranging from 0.78 to 50 µg/mL for HS and from 1.56 to 100 µg/mL for DS were prepared and the coefficient of determination (r2) was higher than 0.99 for both analytes. Intra-day and inter-day imprecisions and the bias for both compounds were <10.0%. Up to now, most analytical procedures for quantifying GAGs have not had a high level of reproducibility among laboratories, despite the availability of various techniques. The adoption of a new protocol incorporating the methods outlined in this paper could significantly improve the quality and reproducibility of MS results. A procedure using simple steps for preparing samples and reagents that are easily available on the market could promote the standardization of analytical procedures and increase the use of these measurements in clinical practice.

Development of a fast LC-MS/MS protocol for combined measurement of six LSDs on dried blood spot in a newborn screening program.

New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs. Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5 min. Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4 h is enough to achieve good reaction efficiency without any impact on sensitivity. The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.

ß3 -Adrenoceptor as a potential immuno-suppressor agent in melanoma.

BACKGROUND AND PURPOSE: Stress-related catecholamines have a role in cancer and ß-adrenoceptors; specifically, ß2 -adrenoceptors have been identified as new targets in treating melanoma. Recently, ß3 -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which ß3 -adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub-population homeostasis. Understanding the mechanisms of cancer immune-tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of ß3 -adrenoceptors in immune-tolerance regulation. EXPERIMENTAL APPROACH: A mouse model of melanoma in which syngeneic B16-F10 cells were injected in C57BL-6 mice was used to evaluate the effect of ß-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with ß-blockers (propranolol and SR59230A) and specific ß-adrenoceptor siRNAs targeting ß2 - or ß3 -adrenoceptors were used. KEY RESULTS: Only ß3 -, but not ß2 -adrenoceptors, were up-regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub-populations including Treg, MDSC, and NK. SR59230A and ß3 -adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub-populations in the tumour mass, blood, and spleen. SR59230A and ß3 -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes. CONCLUSIONS AND IMPLICATIONS: Our data suggest that ß3 -adrenoceptors are involved in immune-tolerance, which opens the way for new strategic therapies to overcome melanoma growth. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Propranolol 0.2% Eye Micro-Drops for Retinopathy of Prematurity: A Prospective Phase IIB Study.

Background: Oral propranolol reduces retinopathy of prematurity (ROP) progression, although not safely. Propranolol 0.1% eye micro-drops administered to newborns with stage 2 ROP are well-tolerated, but not sufficiently effective. Methods: A multi-center open-label trial was conducted to assess the safety and efficacy of propranolol 0.2% eye micro-drops in newborns with stage 1 ROP. The progression of the disease was evaluated with serial ophthalmologic examinations. Hemodynamic, respiratory, biochemical parameters, and propranolol plasma levels were monitored. Demographic and perinatal characteristics, co-morbidities and co-intervention incidences, together with ROP progression, were compared with a historical control group in the same centers participating in the trial. Results: Ninety-eight newborns were enrolled and compared with the historical control group. Populations were not perfectly homogeneous (as demonstrated by the differences in the Apgar score and the different incidence rate in surfactant administration and oxygen exposure). The progression to ROP stage 2 or 3 plus was significantly lower than the incidence expected on the basis of historical data (Risk Ratio 0.521, 95% CI 0.297- 0.916). No adverse effects related to propranolol were observed and the mean propranolol plasma level was significantly lower than the safety cut-off of 20 ng/mL. Unexpectedly, three newborns treated with oral propranolol before the appearance of ROP, showed a ROP that was unresponsive to propranolol eye micro-drops and required laser photocoagulation treatment. Conclusion: Propranolol 0.2% eye micro-drops were well-tolerated and appeared to reduce the ROP progression expected on the basis of a comparison with a historical control group. Propranolol administered too early appears to favor a more aggressive ROP, suggesting that a ß-adrenoreceptor blockade is only useful during the proliferative phase. Further randomized placebo-controlled trials are required to confirm the current results. Clinical Trial Registration  The trial was registered at ClinicalTrials.gov with Identifier NCT02504944 and with EudraCT Number 2014-005472-29.