Encapsulation-induced aggregation and loss in activity of gamma-chymotrypsin and their prevention.

Development of alternative procedures to the commonly employed water-in-oil-in-water technique to encapsulate proteins in polymers is needed due to protein stability issues. Herein the model protein gamma-chymotrypsin has been encapsulated in poly(D,L-lactic-co-glycolic)acid (PLGA) microspheres using the solid-in-oil-in-water (s/o/w) encapsulation technique. The model protein was chosen because it has a measurable biological activity and its unfolding is irreversible. The latter make the protein an excellent sensor for unfolding events in the encapsulation procedure. While lyophilization did not cause any irreversible aggregation or loss in activity, encapsulation of the lyophilized enzyme by the s/o/w technique proved detrimental to its integrity. Specifically, 34% of the encapsulated protein was aggregated and the specific activity of enzyme released within 24 h was reduced to ca. 50% of that prior to encapsulation. FTIR spectra demonstrated substantial encapsulation-induced perturbations of the secondary structure of gamma-chymotrypsin. To achieve stabilization of gamma-chymotrypsin during encapsulation, excipients were employed during the initial lyophilization process. When gamma-chymotrypsin was co-lyophilized with poly(ethylene glycol) (PEG) the formation of non-covalent aggregates inside the microspheres decreased significantly to 8%. FTIR data showed that PEG prevented encapsulation-induced structural perturbations. In contrast, the amount of aggregates remained high (34%) when gamma-chymotrypsin was co-lyophilized with trehalose. No additional non-soluble aggregates were formed during 1 week of in vitro release. Furthermore, the amount of non-soluble aggregates in the microspheres after encapsulation correlated with the amount of non-released protein. Therefore in vitro release did not cause aggregation. Similar results were found with respect to the retention of the specific enzyme activity where PEG afforded excellent stability.     

From cure to palliation: staff communication, documentation, and transfer of patient

In the transition from curative treatment to palliative care of a general end-of-life patient population, the internal communication of the acute care staff seems to be less than optimal. The communication had reference to the dialogue within the staff both before and after the decision to concentrate on palliative care, and possible transfer of the patient. This survey of Swedish nurses and physicians showed that most of 780 respondents wanted more internal communication, and a more individualized procedure of decision-making. All staff should be informed about the decision made but full agreement was not seen as realistic. The largest difference of opinion between nurses and physicians concerned the involvement of nurses in the decision-making about the transition. A uniform documentation of the decision to transfer care focus was the ideal. Approximately every fourth patient in acute care is transferred to receive palliative care. Only approximately half of the respondents had any training in palliative care and the majority wanted more training. There seems to be a need for more palliative care training, perhaps somewhat different for each specialty. Furthermore, a common language to enable nurses and physicians to communicate more easily may improve the transition process.     

Sonographically guided small-bore chest tubes and sonographic monitoring for rapid sclerotherapy of recurrent malignant pleural effusions.

OBJECTIVE: To evaluate the role of sonographically guided small-bore chest catheters and sonographically based monitoring of fluid evacuation in rapid sclerotherapy of malignant pleural effusions. METHODS: In 50 patients with recurrent malignant pleural effusions, a 9F catheter was inserted into the pleural space under sonographic guidance. When sonography documented complete fluid evacuation, bleomycin (0.75 mg/kg) was injected via the tube. Fluid drainage was monitored for 12 hours; if fluid output was less than 100 mL, the pleural catheter was removed; otherwise, a second dose of bleomycin was administered after 24 hours. If loculations or fluid reaccumulations due to tube malfunctioning were detected, they were evacuated by sonographically guided thoracentesis, and bleomycin (1.5 mg/100 mL of fluid) was injected through the thoracentesis needle. All patients were monitored for fluid recurrence with thoracic sonography. RESULTS: Twenty-nine patients received 1 dose of bleomycin, and 21 received 2 doses. In 11 patients with residual loculations, sonographically guided thoracentesis was performed, and bleomycin was injected into the loculations. In 29 patients, pleurodesis was completed within 24 hours; in 21, it was completed within 48 hours. The 30-day response was 84%; the long-term response was 60%. No complications or serious side effects were observed. CONCLUSIONS: Rapid pleurodesis can be accomplished within 24 to 48 hours, with good short- and long-term responses. Thoracic sonography plays a pivotal role. It guides placement of the pleural catheter and is valuable in the monitoring of fluid evacuation for determining the right time for sclerosing agent administration and in the detection and treatment of loculations or residual pleural fluid due to tube malfunctioning.     

Quantitation of Antibiotics Using High-Pressure Liquid Chromatography: Tetracycline

A method for quantitative determination of tetracycline in serum, using high-pressure liquid chromatography, is reported. After extraction of the drug from serum, using methanol-trichloroacetic acid solution, tetracycline was separated by reverse-phase chromatography. Quantitation of tetracycline was based on a linear relationship between peak heights in the chromatograms and known concentrations of the drug in the original serum samples. Serum tetracycline concentrations as low as 0.3 μg/ml could be accurately measured. Serum samples obtained after a single intravenous injection of tetracycline to three human volunteers and six dogs were assayed by microbiological and chemical assays. Correlation coefficients of 0.95 and 0.97, respectively, were found. This chemical method is rapid (less than 30 min), accurate, sensitive, and reproducible, and it seems feasible for routine clinical use.     

Perceived source of anabolic-androgenic steroids and the construal of users’ personality

Users obtain anabolic-androgenic steroids (AAS) from various sources and it is unclear if observers’ perception of these sources has an effect on their construal of users’ personality. We investigated the influence of observers’ perceived source of AAS on their construal of users’ personality. A total of 283 individuals (209 females; mean age = 26.84 years) recruited online were randomized to three independent experimental scenarios or vignettes differing only in the source of AAS used by the protagonist: the Internet (n = 91), trainer/coach (n = 91), and physician/doctor (n = 101). Participants in all three conditions rated their protagonist on the NEO Five-Factor Inventory. Multivariate analysis of variance results indicated no significant between-group differences on personality ratings of the protagonists. It is evident that observers’ perceived source of AAS does not influence their construal of users’ personality.     

Circadian Control of the Daily Plasma Glucose Rhythm: An Interplay of GABA and Glutamate

The mammalian biological clock, located in the hypothalamic suprachiasmatic nuclei (SCN), imposes its temporal structure on the organism via neural and endocrine outputs. To further investigate SCN control of the autonomic nervous system we focused in the present study on the daily rhythm in plasma glucose concentrations. The hypothalamic paraventricular nucleus (PVN) is an important target area of biological clock output and harbors the pre-autonomic neurons that control peripheral sympathetic and parasympathetic activity. Using local administration of GABA and glutamate receptor (ant)agonists in the PVN at different times of the light/dark-cycle we investigated whether daily changes in the activity of autonomic nervous system contribute to the control of plasma glucose and plasma insulin concentrations. Activation of neuronal activity in the PVN of non-feeding animals, either by administering a glutamatergic agonist or a GABAergic antagonist, induced hyperglycemia. The effect of the GABA-antagonist was time dependent, causing increased plasma glucose concentrations only when administered during the light period. The absence of a hyperglycemic effect of the GABA-antagonist in SCN-ablated animals provided further evidence for a daily change in GABAergic input from the SCN to the PVN. On the other hand, feeding-induced plasma glucose and insulin responses were suppressed by inhibition of PVN neuronal activity only during the dark period. These results indicate that the pre-autonomic neurons in the PVN are controlled by an interplay of inhibitory and excitatory inputs. Liver-dedicated sympathetic pre-autonomic neurons (responsible for hepatic glucose production) and pancreas-dedicated pre-autonomic parasympathetic neurons (responsible for insulin release) are controlled by inhibitory GABAergic contacts that are mainly active during the light period. Both sympathetic and parasympathetic pre-autonomic PVN neurons also receive excitatory inputs, either from the biological clock (sympathetic pre-autonomic neurons) or from non-clock areas (para-sympathetic pre-autonomic neurons), but the timing information is mainly provided by the GABAergic outputs of the biological clock.     

Parenting stress in mothers with cystic fibrosis*

Aim: To assess the parenting experience of mothers with cystic fibrosis (CF) and to compare with normative data. Methods: Cross-sectional study with a validated generic parental stress questionnaire (PSQ). This PSQ differentiates four components of parental stress: main factor “parental stress”, compounding factor “role restrictions”, protective factors “support from spouse”, and “social support”. Cut-off scores categorise results as “normal”, “borderline” or “concerning”. Sample: Seventy-three women were informed by their local CF centre. Of these, 36 enrolled and had a first-born child aged 1–12 years (consistent with reference values of the PSQ). Of these, 31 (86%) returned the PSQ. Mean age of mothers was 32.6 years?±?6.9 years, mean age of first-born child was 5.2 years?±?3.4 years. Most of the mothers had one biological child, five women had two children and one had three children. Results: Parental stress scores were normally distributed, the same applies for contributing factors and for the two protective factors. Favourable scores were twice as frequent as concerning scores. Mothers of younger children scored slightly better than mothers of school-aged children. Conclusion: In line with the only comparable study, mothers with CF seem to be a remarkably resilient group who mostly cope well with parental stress even in the face of a progressive, chronic disease requiring time-consuming treatment.

• Implications for Rehabilitation

• Today, motherhood is increasingly becoming an option in fertile women with cystic fibrosis.

• The additional burden of parenting seems to be rewarded by fulfilling essential personal goals.

• CF clinics should routinely address a possible wish for a child and to discuss it, openly.

     

Activity limitation in rheumatoid arthritis correlates with reduced grip force regardless of sex: the Swedish TIRA project

OBJECTIVE: To evaluate activity limitations 3 years after diagnosis of early rheumatoid arthritis (RA) in relation to grip force and sex. METHODS: A total of 217 patients, 153 women and 64 men, with recent-onset RA were included. Activity limitations were reported using the Health Assessment Questionnaire (HAQ) and the Evaluation of Daily Activities Questionnaire (EDAQ). The relationships between activity limitations versus grip force (measured by the Grippit), walking speed, functional impairment, grip ability, pain, plasma C-reactive protein, the 28-joint disease activity score and its components, the physician's global assessment of disease activity, and sex were analyzed by partial least squares (PLS). RESULTS: Women had significantly lower grip force and more activity limitations (HAQ and EDAQ) than men. The PLS analyses demonstrated that grip force was the strongest regressor of activity limitation, closely followed by walking speed. However, within subgroups based on grip force (group 1 = grip force <114 N, group 2 = 116-206 N, group 3 = 214-321 N, group 4 = grip force >328 N) and including sexes, women and men had corresponding degrees of activity limitation as reported by the HAQ and EDAQ. CONCLUSION: Our results indicate that the more pronounced activity limitations seen in women with RA, as compared with men, may be explained by lower grip force rather than sex.     

Inhibition of IFN-alpha signaling by a PKC- and protein tyrosine phosphatase SHP-2-dependent pathway

Cytokine signaling by the Jak-STAT pathway is subject to complex negative regulation that limits the amplitude and duration of signal transduction. Inhibition of signaling also mediates negative crosstalk, whereby factors with opposing biological activities crossinhibit each other's function. Here, we investigated a rapidly inducible mechanism that inhibited Jak-STAT activation by IFN-alpha, a cytokine that is important for antiviral responses, growth control, and modulation of immune responses. IFN-alpha-induced signaling and gene activation were inhibited by ligation of Fc receptors and Toll-like receptors 7 and 8 in a PKCbeta-dependent manner. Neither PKCbeta nor PKCdelta influenced responses of cells treated with IFN-alpha alone. Inhibition of IFN-alpha signaling correlated with suppression of IFN-alpha-dependent antiviral responses. PKC-mediated inhibition did not require de novo gene expression but involved the recruitment of PKCbeta to the IFN-alpha receptor and interaction with protein tyrosine phosphatase SHP-2, resulting in augmented phosphatase activity. PKC-mediated inhibition of IFN-alpha signaling was abolished in SHP-2-deficient cells, demonstrating a pivotal role for SHP-2 in this inhibitory pathway. Together, our data describe a rapidly inducible, direct mechanism of inhibition of Jak-STAT signaling mediated by a PKCbeta-SHP-2 signaling pathway.