A collaborative evaluation of the cytotoxicity of two surfactants by using the human corneal epithelial cell line and the WST-1 test.

This study was undertaken to investigate the use of the in vitro test WST-1, an assay of cell proliferation and viability, for a preliminary safety evaluation of topical ophthalmic preparations. The cytotoxicity of two surfactants, benzalkonium chloride (BAC) and polyoxyethylene-20-stearyl ether (Brij78, PSE) was independently investigated in four laboratories in the EU by using an immortalized human corneal epithelial (HCE) cell line. The HCE cells were exposed to BAC and PSE for 5 min, 15 min, and 1 hour, and the results of the HCE-WST-1 tests were collected and compared. After one-hour exposure, the EC(50) values in BAC-treated cells in the presence of serum ranged between 0.0650 +/- 0.0284 (mean +/- SD) mM, and those in the absence of serum 0.0296 +/- 0.0081 mM. The corresponding values for PSE were 0.0581 +/-.0300 mM and 0.0228 +/-.0063 mM. There were variations in the results between different laboratories, with coefficients of variation ranging from 31 to 121%, mean 58%. The use of one-hour exposure time is to be preferred, and the elimination of serum in the culture medium is recommended to avoid both underestimation of toxic effects and variability of the test results.     

Determination of the Confidence Interval of the Relative Standard Deviation Using Convolution

Coefficient of variation is a widely used measure of dispersion and is important in comparing variables with different units or average values. In pharmaceutical industry, it is termed as the relative standard deviation (RSD) and is used widely to describe blend concentration variability, finished dose variability, dissolution q point variability, etc. Although theoretical formula and simulation methods for the estimation of the RSD confidence interval have been developed in previous literature, they are not well known, and they are either too complex to apply easily or require intensive computation. As a result, the statistical reliability of RSD estimates are rarely evaluated, which increases process risk as well as consumer risk. In this paper, we introduce a novel convolution numerical method for the quick and straightforward estimation of RSD confidence intervals. A standard statistical distribution group is developed, denoted as the Chi-on-Mu-square distribution, which is similar to the widely used Chi-square distribution. Results indicate the Chi-square distribution itself can be a good approximation in the RSD confidence interval calculation, especially when small RSD is expected or large number of samples is involved. The effect of deviations from the normal distribution populations is also discussed.     

Spectrum of Disease Severity in Patients With X-Linked Retinitis Pigmentosa Due to RPGR Mutations

PurposeThe purpose of this study was to perform a detailed longitudinal phenotyping of X-linked retinitis pigmentosa (RP) caused by mutations in the RPGR gene during a long follow-up period.MethodsAn Italian cohort of 48 male patients (from 31 unrelated families) with RPGR-associated RP was clinically assessed at a single center (mean follow-up = 6.5 years), including measurements of best-corrected visual acuity (BCVA), Goldmann visual field (GVF), optical coherence tomography (OCT), fundus autofluorescence (FAF), microperimetry, and full-field electroretinography (ERG).ResultsPatients (29.6 ± 15.2 years) showed a mean BCVA of 0.6 ± 0.7 logMAR, mostly with myopic refraction (79.2%). Thirty patients (62.5%) presented a typical RP fundus, while the remaining sine pigmento RP. Over the follow-up, BCVA significantly declined at a mean rate of 0.025 logMAR/year. Typical RP and high myopia were associated with a significantly faster decline of BCVA. Blindness was driven primarily by GVF loss. ERG responses with a rod-cone pattern of dysfunction were detectable in patients (50%) that were significantly younger and more frequently presented sine pigmento RP. Thirteen patients (27.1%) had macular abnormalities without cystoid macular edema. Patients (50%) with a perimacular hyper-FAF ring were significantly younger, had a higher BCVA and a better-preserved ellipsoid zone band than those with markedly decreased FAF. Patients harboring pathogenic variants in exons 1 to 14 showed a milder phenotype compared to those with ORF15 mutations.ConclusionsOur monocentric, longitudinal retrospective study revealed a spectrum disease progression in male patients with RPGR-associated RP. Slow disease progression correlated with sine pigmento RP, absence of high myopia, and mutations in RPGR exons 1 to 14.     

Mice with Inactivation of Aryl Hydrocarbon Receptor-Interacting Protein (Aip) Display Complete Penetrance of Pituitary Adenomas with Aberrant ARNT Expression

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been shown to predispose to pituitary adenoma predisposition, a condition characterized by growth hormone (GH)-secreting pituitary tumors. To study AIP-mediated tumorigenesis, we generated an Aip mouse model. Heterozygous mice developed normally but were prone to pituitary adenomas, in particular to those secreting GH. A complete loss of AIP was detected in these lesions, and full penetrance was reached at the age of 15 months. No excess of any other tumor type was found. Ki-67 analysis indicated that Aip-deficient tumors have higher proliferation rates compared with Aip-proficient tumors, suggesting a more aggressive disease. Similar to human AIP-deficient pituitary adenomas, immunohistochemical studies showed that expression of aryl hydrocarbon receptor nuclear translocator 1 or 2 (ARNT or ARNT2) protein was lost in the mouse tumors, suggesting that mechanisms of AIP-related tumorigenesis involve aberrant ARNT function. The Aip^+/− mouse appears to be an excellent model for the respective human disease phenotype. This model constitutes a tool to further study AIP-associated pituitary tumorigenesis and may be potentially valuable in efforts to develop therapeutic strategies to treat pituitary adenomas.Pituitary adenomas are common, benign, monoclonal neoplasms of the anterior pituitary gland. They account for approximately 15% of intracranial tumors. Approximately two thirds produce pituitary hormones in excess; among these, prolactin (PRL) and growth hormone (GH)-oversecreting adenomas are the most common. The significant morbidity associated with these lesions arises from the adverse effects of the hypersecretion, such as acromegaly or gigantism in the case of GH secreting adenomas, and/or local compressive effects.¹Mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene have been identified as an underlying cause in human pituitary adenoma predisposition (OMIM 102200), characterized mainly by GH secreting adenomas (somatotropinomas), although susceptibility to PRL (prolactinomas), adrenocorticotropic hormone (ACTH), and nonsecreting adenomas is also part of the disease phenotype.^2–5 So far AIP mutations have not been associated with any other tumor types.^6–8 Typically, pituitary adenoma predisposition patients have a young age at disease onset but do not necessarily display a strong family history of pituitary adenomas. AIP mutation positive tumors seem to be larger and may have a worse response to somatostatin analogs as compared to sporadic tumors.^4,5,9Inactivating germline mutations, loss of the normal allele in tumors, as well as recent functional evidence implicate the tumor suppressor role of the AIP gene.^2,5,10 Many of the proteins known to interact with AIP can be linked to tumorigenesis. The best known function of AIP is to stabilize aryl hydrocarbon receptor (AHR) (also known as dioxin receptor) in a cytoplasmic chaperone complex together with heat shock protein 90 (HSP90) and p23. The binding of dioxins or dioxin-like chemicals leads to shuttling of AHR to the nucleus, where it forms a complex with the aryl hydrocarbon receptor nuclear translocator 1 [ARNT, also known as hypoxia-inducible factor (HIF)-1β]. The AHR/ARNT heterodimer regulates the expression of several xenobiotic metabolizing enzymes.¹¹ ARNT is also required by HIF-1α in the nucleus. ARNT/HIF-1α heterodimer regulates several genes involved in tumorigenesis under hypoxia, and HIF-1α is present in many tumors, contributing to angiogenesis, proliferation, metastasis, and resistance to radiation therapy.¹² Thus, the ubiquitously expressed ARNT is an essential partner in the physiological response to chemical toxicants and hypoxia. ARNT participates also in the regulation of estrogen receptor signaling.^13,14 Interestingly, it has been shown that ARNT2, an ARNT homolog, can compensate the lack of ARNT and form a functional complex with HIF-1α under hypoxia. However, it seems that ARNT2 is not capable of cooperating with AHR in the activation of xenobiotic responsive element–dependent genes.¹⁵ ARNT2 was initially classified as being expressed in neural tissue and the kidney.¹⁶ While much is known about the function of ARNT, the expression pattern and dimerization partners of ARNT2 are less clear. Recently, we showed that expression of ARNT is significantly reduced in human AIP-deficient pituitary adenomas, suggesting a link between AIP and AHR/ARNT signaling in pituitary tumorigenesis.¹⁰ However, further studies are needed to unravel the mechanism by which AIP exerts its tumor suppressive action in the pituitary.Mouse models have been used to study various aspects of pituitary development, function, and disease. A recently published Aip (Ara9) mouse model revealed that homozygous germline Aip mutations are embryonic lethal and homozygous mutant embryos die due to various cardiovascular malformations. In addition, most mice with reduced Aip expression showed failure of liver vein inclusion resulting in reduced liver size.^17,18 However, possible tumor predisposition was not approached in either study.Here we report a novel conventional Aip knockout mouse, which models the tumor susceptibility caused by human AIP germline mutations. We show that Aip^+/− mice are extremely prone to pituitary tumors, in particular GH secreting adenomas. In addition, we provide evidence that Aip-associated tumors present a more aggressive disease profile and that Aip deficiency in pituitary tumors has a striking effect on the presence of ARNT and ARNT2 proteins, the heterodimerization partners of HIF-1α. Overall, these results indicate that the Aip mouse model provides an excellent model for the human phenotype and suggest that mechanisms of AIP related tumorigenesis involve aberrant ARNT function.     

Using Compartment Modeling to Investigate Mixing Behavior of a Continuous Mixer

In this paper, the development of a compartment model to simulate mixing within a continuous blender is reported. The main benefit of the method is that it can generate extensive modeling predictions in very short computational time. The model can also be used to explore the effect of sampling parameters on estimated mixing performance, a topic that has been central to pharmaceutical manufacturing for the past 15 years and that remains a central issue in the PAT initiative. However, this method requires more input than conventional particle dynamics methods. Thus, we investigate the effects of modeling parameters on mixing performance to develop general guidance needed to adapt this modeling framework to any continuous process. An experimental technique based on longitudinal sampling is used to examine the content uniformity of the blend along the continuous mixer. The model compares favorably with continuous mixing experiments, capture the effects of feeding rate variability, active product ingredient concentration, and blender processing angle, while effectively capturing and making explicit the effect of sampling parameters such as number of samples and sample size. The modeling approach provides a convenient tool for process design.     

The effects of implant length and diameter prior to and after osseointegration: a 2-D finite element analysis

A two-dimensional finite element analysis was used to evaluate the effects of implant length and diameter on the stress distribution of a single-implant supported crown and the strain distribution of its surrounding bone prior to and after the phase of osseointegration. The effect of length was investigated using implants with a diameter of 3.75 mm and lengths of 8 mm, 10 mm, 12 mm, and 14 mm. The effect of diameter was investigated using implants with a length of 10 mm and diameters of 3 mm, 3.75 mm, 4.5 mm, and 5mm. The phase prior to osseointegration was simulated by assuming a coefficient of friction for the interface between the implant and the surrounding bone, while the phase after osseointegration was simulated by assuming a fixed bond on the interface between the implant and the surrounding bone. The FEA results indicated a tendency towards stress reduction on the implant, both prior to and after osseointegration, when the length was increased. However, the calculated stresses on the implant were lower after the phase of osseointegration. Although no specific correlation could be seen regarding the influence of implant diameter, the calculated stresses on the implant were again lower after the phase of osseointegration. For all these cases, the maximum stress concentration occurred at the abutment-implant interface. As far as bone tissue was concerned, there was a tendency towards strain reduction, before and after osseointegration, when the length of the implant was increased from 10 mm up to 14 mm. This tendency was not manifested for the range of 8 to 10 mm. The effect of implant diameter on bone tissue was not clear. It appears that implants of a diameter more than 5 mm are not preferable for immediate loading. Finally, it seems that cortical bone is not influenced by the phase of osseointegration, while trabecular bone is highly affected.     

Interferon gamma-inducible protein 10: a predictive marker of successful treatment response in hepatitis C virus/HIV-coinfected patients

BACKGROUND: Elevated pretreatment interferon (IFN) gamma-inducible protein 10 (IP-10/CXCL10) levels are a marker of treatment nonresponse in hepatitis C virus (HCV)-monoinfected patients. We undertook this study to determine if IP-10 is a marker of treatment outcome in HCV/HIV-coinfected patients. METHODS: Nineteen HCV/HIV-coinfected patients were treated with weight-based pegylated (PEG) IFNalpha-2b (1.5 microg/kg) once weekly plus weight-based ribavirin (1000 or 1200 mg) daily for up to 48 weeks. Plasma IP-10, monokine induced by IFNgamma/CXCL9 (Mig), and IFN-inducible T-cell alpha-chemoattractant/CXCL11 (I-TAC) levels were measured by enzyme-linked immunosorbent assay on samples obtained frequently during the first 3 PEG-IFN doses and throughout treatment. RESULTS: Median pretreatment plasma IP-10 (interquartile range [IQR]) levels were significantly lower in virological responders (n=6) at 217 (IQR: 181-301) pg/mL compared with nonresponders (n=13) at 900 (IQR: 628-2048) pg/mL (P=0.002), whereas pretreatment Mig and I-TAC levels did not differ significantly. Plasma IP-10 levels of 400 pg/mL before treatment and on days 7 and 14 could be used to identify likely coinfected PEG-IFN/ribavirin nonresponders. PEG-IFN-induced elevations in IP-10 were greater in virological responders than in nonresponders (approximately 10-fold vs. approximately 4-fold) after the first PEG-IFN dose. CONCLUSIONS: IP-10 may be a biomarker of HCV treatment outcome in difficult-to-treat HCV/HIV-coinfected patients.     

The perioperative infusion of low doses of magnesium sulfate reduces analgesic requirements in patients undergoing abdominal hernioplasty

Magnesium sulfate, which is an antagonist of the N-methyl- -aspartate (NMDA) receptor and a physiological calcium channel blocker, has analgesic properties in a number of acute and chronic pain conditions. The aim of our study was to evaluate whether intravenous administration of low doses of magnesium during anaesthesia would reduce analgesic requirements intraoperatively and in the early postoperative period.In a prospective, randomized, double-blinded and placebo-controlled study, 42 patients undergoing abdominal hernioplasty in balanced general anaesthesia, with sevoflurane, N₂O, fentanyl and rocuronium, received intravenously, either magnesium sulfate 10%, a bolus of 30 mg kg⁻¹ before start of surgery and an infusion of 6 mg ( kg⁻¹ h⁻¹) over the entire operation period, or isotonic sodium chloride (control group). Intra- and postoperative analgesia were achieved with fentanyl.Our results showed that fentanyl consumption intra—and postoperatively was significantly less in the magnesium group and with no adverse events. The requirements of muscle relaxants did not differ significantly between the groups. We conclude that magnesium sulfate in lower bolus and infusion doses than those used in previous studies is an effective adjuvant for perioperative analgesic management.