Activation of methotrexate-phenylalanine by monoclonal antibody--carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro.

Monoclonal antibody 4E3 directed against a glycosylated surface protein on human ovarian teratocarcinoma cells (CRL-1572 cell line) was conjugated to bovine carboxypeptidase A (CPA) using a 3400 Da polyethylene glycol chain bearing an N-hydroxysuccinimide group at both ends. The conjugate preparation was purified by fast protein liquid chromatography on a Superose 12/30 HR column. The 4E3-CPA conjugate was recovered in the third fraction by SDS-PAGE analysis. The specific binding of the 4E3-CPA conjugate to CRL-1572 cells was confirmed by a FACS analysis and the enzymatic activity of the conjugate remained while tested with hippuryl-L-phenylalanine. In vitro cytotoxic assays on CRL-1572 cells showed that the prodrug methotrexate-phenylalanine (MTX-Phe) alone was non-toxic (ID50 > 1000 ng ml-1) but was selectively converted to MTX when the cells were pretreated with 50 micrograms ml-1 4E3-CPA conjugate, which enhanced considerably the pharmacological activity of the prodrug with an ID50 of 70 ng ml-1. The co-culture assays with CRL-1572 and MRC-5 cells (human normal lung diploid fibroblast cell lines) demonstrated the specificity of the 4E3-CPA conjugate for the CRL-1572 cells since no cytotoxicity was observed on the MRC-5 cells. When both cell lines were mixed in ratios ranging from 1:10,000 to 1:5 (CRL-1572:MRC-5), the significant increase in the ID25 was correlated with the proportion of tumoral cells present in the cell inoculum. These results suggest that MTX-Phe combined with 4E3-CPA conjugate is a promising model for a more selective and localised anti-cancer chemotherapy based on the ADEPT concept.     

Human endogenous retrovirus (HERV)-W ENV and GAG proteins: Physiological expression in human brain and pathophysiological modulation in multiple sclerosis lesions

Antigen expression of a human endogenous retrovirus family, HERV-W, in normal human brain and multiple sclerosis lesions was studied by immunohistochemistry by three independent groups. The HERV-W multicopy family was identified in human DNA from the previously characterized multiple sclerosis-associated retroviral element (MSRV). A panel of antibodies against envelope (ENV) and capsid (GAG) antigens was tested. A physiological expression of GAG proteins in neuronal cells was observed in normal brain, whereas there was a striking accumulation of GAG antigen in axonal structures in demyelinated white matter from patients with MS. Prominent HERV-W GAG expression was also detected in endothelial cells of MS lesions from acute or actively demyelinating cases, a pattern not found in any control. A physiological expression of ENV proteins was detected in microglia in normal brain; however,a specific expression in macrophages was apparently restricted to early MS lesions. Thus, converging results from three groups confirm that GAG and ENV proteins encoded by the HERV-W multicopy gene family are expressed in cells of the central nervous system under normal conditions. Similar to HERV-W7q ENV (Syncitin), which is expressed in placenta and has been shown to have a physiological function in syncytio-trophoblast fusion, HERV-W GAG may thus also have a physiological function in human brain. This expression differs in MS lesions, which may either reflect differential regulation of inherited HERV-W copies, or expression of "infectious" MSRV copies. This is compatible with a pathophysiological role in MS, but also illustrates the ambivalence of such HERV antigens, which can be expressed in cell-specific patterns, under physiological or pathological conditions.     

RNA-binding proteins and neural development: a matter of targets and complexes

RNA-binding proteins control multiple steps of nuclear and cytoplasmic RNA processing including alternative splicing, stabilization, transport and translational repression of RNAs. Here we present existing evidence showing that RNA-binding proteins expressed in the nervous system are required in many steps of its development and play multiple roles during the life of a neuron. We describe emerging views based on recent studies strongly suggesting that RNA-binding proteins cooperate actively within neurons in large multifunctional complexes to regulate the flow of information encoded in ribonomes in a coordinated fashion.     

Dosage,duration and timing of nonsteroidal antiinflammatory drug use and risk of prostate cancer

Experimental studies suggest that NSAIDs could reduce prostate cancer risk. Results of observational studies on the relation between NSAIDs and prostate cancer risk have, however, been inconsistent. Moreover, none has addressed the issues of dosage, duration and timing of exposure. In a population-based, age-matched case-control study, we measured the association between prostate cancer risk and NSAIDs defined in terms of mean daily dose, cumulative duration and timing of exposure. Eight-year drug exposure history was obtained from the Quebec health insurance system database. Parallel analyses were performed for aspirin and NSAIDs other than aspirin. We controlled for detection bias and assessed the potential impact of protopathic bias. Analyses were performed with conditional logistic regression. Among the 2,221 cases and 11,105 controls, there was a negative trend between cumulative duration of aspirin use and prostate cancer risk (p = 0.0009). Also, exposure to a mean daily dose of aspirin of at least 80 mg, maintained throughout the entire 8 years of follow-up, was associated with an 18% reduction in prostate cancer risk (OR = 0.82, 95% CI 0.71-0.95). In more recent users of such a dose, the risk reduction was 7%. However, 1 year after the end of a 7-year regular aspirin exposure, no residual protective effect persisted. No association was observed between prostate cancer risk and exposure to NSAIDs other than aspirin. The results suggest that long-term and regular use of aspirin, at a dosage beneath that usually recommended for an anti-inflammatory effect, may prevent prostate cancer.     

Use of Zeolite for Removing Ammonia and Ammonia-Caused Toxicity in Marine Toxicity Identification Evaluations

Ammonia occurs in marine waters including effluents, receiving waters, and sediment interstitial waters. At sufficiently high concentrations, ammonia can be toxic to aquatic species. Toxicity identification evaluation (TIE) methods provide researchers with tools for identifying aquatic toxicants. For identifying ammonia toxicity, there are several possible methods including pH alteration and volatilization, Ulva lactuca addition, microbial degradation, and zeolite addition. Zeolite addition has been used successfully in freshwater systems to decrease ammonia concentrations and toxicity for several decades. However, zeolite in marine systems has been used less because ions in the seawater interfere with zeolites ability to adsorb ammonia. The objective of this study was to develop a zeolite method for removing ammonia from marine waters. To accomplish this objective, we performed a series of zeolite slurry and column chromatography studies to determine uptake rate and capacity and to evaluate the effects of salinity and pH on ammonia removal. We also assessed the interaction of zeolite with several toxic metals. Success of the methods was also evaluated by measuring toxicity to two marine species: the mysid Americamysis bahia and the amphipod Ampelisca abdita. Column chromatography proved to be effective at removing a wide range of ammonia concentrations under several experimental conditions. Conversely, the slurry method was inconsistent and variable in its overall performance in removing ammonia and cannot be recommended. The metals copper, lead, and zinc were removed by zeolite in both the slurry and column treatments. The zeolite column was successful in removing ammonia toxicity for both the mysid and the amphipod, whereas the slurry was less effective. This study demonstrated that zeolite column chromatography is a useful tool for conducting marine water TIEs to decrease ammonia concentrations and characterize toxicity.     

The place of premedication in pediatric practice

Behind the multiple arguments for and against the use of premedication, sedative drugs in children is a noble principle that of minimizing psychological trauma related to anesthesia and surgery. However, several confounding factors make it very difficult to reach didactic evidence-based conclusions. One of the key confounding issues is that the nature of expectations and responses for both parent and child vary greatly in different environments around the world. Studies applicable to one culture and to one hospital system (albeit multicultural) may not apply elsewhere. Moreover, the study of hospital-related distress begins at the start of the patient's journey and ends long after hospital discharge; it cannot be focused completely on just the moment of anesthetic induction. Taking an example from actual practice experience, the trauma caused by the actual giving of a premedication to a child who absolutely does not want it and may struggle may not be recorded in a study but could form a significant component of overall effect and later psychological pathology. Clearly, attitudes by health professionals and parents to the practice of routine pediatric premedication, vary considerably, often provoking strong opinions. In this pro–con article we highlight two very different approaches to premedication. It is hoped that this helps the reader to critically re-evaluate a practice, which was universal historically and now in many centers is more selective.     

Successful treatment of severe subcutaneous insulin resistance with inhaled insulin therapy

van Alfen‐van der Velden AAEM, Noordam C, de Galan BE, Hoorweg‐Nijman JJG, Voorhoeve PG, Westerlaken C. Successful treatment of severe subcutaneous insulin resistance with inhaled insulin therapy. The potential of inhaled insulin therapy for severe resistance to subcutaneous insulin was tested in a 7‐yr old boy with type 1 diabetes mellitus. The efficiency of 1 mg inhaled insulin (Exubera^®) was examined by a 4‐h euglycemic clamp study. During the clamp, the glucose infusion rate started to increase 25 min after inhalation and peaked 120 min after inhalation. Subsequently, a trial of inhaled insulin monotherapy was initiated consisting of pre‐meal inhalations and one inhalation during the night. Since glycemic control remained fair (HbA1c ～8.5%), this therapy was continued. Over the ensuing 18 months, mild keto‐acidosis occurred twice during gastro‐enteritis. Inhaled insulin was well tolerated and pulmonary function did not deteriorate. We conclude that severe resistance to subcutaneous insulin does not preclude sufficient absorption of insulin delivered by pulmonary.