Subjective and objective nutritional assessment: nurses' role and the effect of cultural differences.

BACKGROUND: Even though the nutritional assessment of chronically ill patients has a significant effect on outcomes, nurses' time constraints in clinical encounters may make the process impractical. Also, cultural background has an effect on nutritional assessment. Patient nutritional self-assessment can ease some of the nurses' workload. OBJECTIVES: To compare tools for subjective and objective nutritional assessment and to examine cultural differences in nutritional assessment between Jews and Arabs living in Israel. METHODS: The research design was cross-sectional; data were collected from Jews and Arabs with chronic illnesses living in the community during their visit to a public health clinic. The admitting nurse performed an objective nutritional assessment (Mini Nutritional Assessment (MNA)) after the patients completed the Subjective Nutritional Assessment (SANS). The data were analyzed using descriptive statistics, Pearson's correlation coefficients were calculated to test the relationships between the variables, and independent student t-tests were used to compare the means and differences between groups. The diagnostic accuracy of the MNA and of the SANS was determined using the area under the curve (AUC) analysis of receiver operating characteristic (ROC) curves. The agreement between the MNA and SANS measurements was estimated by a Bland Altman plot. The level of significance employed throughout the analysis was 0.05. RESULTS: The sample was a convenience sample of 228 chronically ill patients, consisting of 121 Arabs and 107 Jews. A significant correlation was found between the subjective and objective nutritional assessments. The Bland-Altman plot demonstrated that the SANS and the MNA have a high level of agreement. Using the area under the curve (AUC) analysis of receiver operating characteristic (ROC) curves, showed an moderate diagnostic accuracy (73 % sensitivity and 30 % specificity). CONCLUSIONS: Since the patient-completed nutritional assessment requires minimal time investment by nurses and we found a significant correlation and evidence for the accuracy and agreement of the objective and subjective assessments, further studies should assess and validate the possibility of replacing the objective nutritional assessment by the subjective assessment. Cultural background has a significant effect on patients' nutritional self-assessment; hence, culture should be considered as part of the nutritional assessment.

Oral administration of live Shigella vaccine candidates in rhesus monkeys show no evidence of competition for colonization and immunogenicity between different serotypes.

Live oral monovalent Shigella flexneri 2a vaccine candidates as well as bivalent formulations with Shigella sonnei were evaluated in a rhesus monkey model for colonization and immunogenicity. Freshly harvested suspensions of S. flexneri 2a vaccine candidates WRSf2G12 and WRSf2G15 as well as S. sonnei vaccine candidate WRSs3 were nasogastrically administered to groups of rhesus monkeys, Macaca mulatta, either in a monovalent form or when combined with each other. The animals were monitored daily for physical well-being, stools were subjected to quantitative colony immunoblot assays for bacterial excretion and blood and stools were evaluated for humoral and mucosal immune responses. No clinical symptoms were noted in any group of animals and the vaccine candidates were excreted robustly for 48-72h without significant changes in either the magnitude or duration of excretion when given as a monovalent or as bivalent mixtures. Similarly, immunological interferences were not apparent in the magnitude of humoral and mucosal immune responses observed toward Shigella-specific antigens when monkeys were fed monovalent or bivalent formulations. These results predict that a multivalent live oral vaccine of more than one serotype can have a favorable outcome for protection against shigellosis.

Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection.

Shigella causes diarrhea and dysentery through contaminated food and water. Shigella sonnei live vaccine candidates WRSs2 and WRSs3 are attenuated principally by the loss of VirG(IcsA) that prevents bacterial spread within the colonic epithelium. In this respect they are similar to the clinically tested vaccine candidate WRSS1. However, WRSs2 and WRSs3 are further attenuated by loss of senA, senB and WRSs3 also lacks msbB2. As previously shown in cell culture assays and in small animal models, these additional gene deletions reduced the levels of enterotoxicity and endotoxicity of WRSs2 and WRSs3, potentially making them safer than WRSS1. However the behavior of these second-generation VirG(IcsA)-based vaccine candidates in eliciting an immune response in a gastrointestinal model of infection has not been evaluated. In this study, WRSs2 and WRSs3 were nasogastrically administered to rhesus monkeys that were evaluated for colonization, as well as for systemic and mucosal immune responses. Both vaccine candidates were safe in rhesus monkeys and behaved comparably to WRSS1 in bacterial excretion rates that demonstrated robust intestinal colonization. Furthermore, humoral and mucosal immune responses elicited against bacterial antigens appeared similar in all categories across all three strains indicating that the additional gene deletions did not compromise the immunogenicity of these vaccine candidates. Based on data from previous clinical trials with WRSS1, it is likely that, WRSs2 and WRSs3 will not only be safer in human volunteers but will generate comparable levels of systemic and mucosal immune responses that were achieved with WRSS1.

Characterization of WRSs2 and WRSs3, new second-generation virG(icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity.

Live, attenuated Shigella vaccine candidates, such as Shigella sonnei strain WRSS1, Shigella flexneri 2a strain SC602, and Shigella dysenteriae 1 strain WRSd1, are attenuated principally by the loss of the VirG(IcsA) protein. These candidates have proven to be safe and immunogenic in volunteer trials and in one study, efficacious against shigellosis. One drawback of these candidate vaccines has been the reactogenic symptoms of fever and diarrhea experienced by the volunteers, that increased in a dose-dependent manner. New, second-generation virG(icsA)-based S. sonnei vaccine candidates, WRSs2 and WRSs3, are expected to be less reactogenic while retaining the ability to generate protective levels of immunogenicity seen with WRSS1. Besides the loss of VirG(IcsA), WRSs2 and WRSs3 also lack plasmid-encoded enterotoxin ShET2-1 and its paralog ShET2-2. WRSs3 further lacks MsbB2 that reduces the endotoxicity of the lipid A portion of the bacterial LPS. Studies in cell cultures and in gnotobiotic piglets demonstrate that WRSs2 and WRSs3 have the potential to cause less diarrhea due to loss of ShET2-1 and ShET2-2 as well as alleviate febrile symptoms by loss of MsbB2. In guinea pigs, WRSs2 and WRSs3 were as safe, immunogenic and efficacious as WRSS1.

Calpastatin overexpression attenuates amyloid-beta-peptide toxicity in differentiated PC12 cells.

Amyloid beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). Abeta is toxic to neurons, possibly through causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca(2+). Calpain (Ca(2+)-dependent protease) and caspase have been implicated in AD. Previously, we used calpain and caspase pharmacological inhibitors to study effects of Abeta25-35 (sAbeta) on neuronal-like differentiated PC12 cells. We reported that sAbeta-treated cells exhibited calpain activation and protein degradation (due to both calpain and caspase-8). We have now found that overexpression of the calpain specific inhibitor calpastatin in differentiated PC12 cells significantly inhibited the sAbeta-induced calpain activation and decreased the protease activity. Calpastatin overexpression inhibited the sAbeta-promoted degradation of fodrin, protein kinase Cepsilon, beta-catenin (membrane structural proteins and proteins involved in signal transduction pathways), and prevented the sAbeta-induced alteration of neurite structure (manifested by varicosities). Overexpression of calpastatin also inhibited Ca(2+)-promoted calpain activation and protein degradation; this is consistent with the notion that the Abeta-induced increase in calpain activity results from a rise in cellular Ca(2+), provided the calpastatin level is not so high as to strongly inhibit calpain. Carrying out transfection without selection allowed the comparison in the same culture of calpastatin-overexpressing with non-overexpressing cells. In cultures transfected with green fluorescent protein (GFP)-calpastatin plasmid, calpastatin overexpression (indicated by GFP-labeling) led to inhibition in sAbeta-induced membrane propidium iodide (PI) permeability, whereas non-transfected, GFP-unlabeled cells exhibited PI permeability. Overall, the results demonstrate that the effects of Abeta-toxicity studied here were attenuated to a large extent by calpastatin overexpression, indicating that the protease calpain is involved in Abeta-toxicity (obviating a primary, direct role for caspases). Increased expression of calpastatin and/or decrease in calpain may serve as one of the means for ameliorating some of the early symptoms of AD.

Caspase-1 activity is required for neuronal differentiation of PC12 cells: cross-talk between the caspase and calpain systems.

Previously, we have found that caspase-1 activity is increased during myoblast differentiation to myotubes. Here we show that caspase-1 activity is required for PC12 differentiation to neuronal-like cells. Caspase-1 is shown to be activated (by immunoblotting and by assessing activity in cell extracts) in the PC12 cells following the initial stage of differentiation. The inhibition of caspase-1 arrests PC12 cells at an intermediate stage of differentiation and prevents neurite outgrowth in these cells; the inhibition is reversed upon the removal of the inhibitor. Calpastatin (calpain endogenous specific inhibitor, and a known caspase substrate) is diminished at the later stages of PC12 cell differentiation, and diminution is prevented by caspase-1 inhibition. The degradation of fodrin (a known caspase and calpain substrate) is found in the advanced stage of differentiation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in two distinct differentiation processes (myoblast fusion and neuronal differentiation of PC12 cells) indicates a function for this caspase in differentiation processes, and suggests some common mechanisms underlying caspase roles in such processes.

Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability.

Calpain (Ca(2+)-dependent intracellular protease)-induced proteolysis has been considered to play a role in myoblast fusion to myotubes. We found previously that calpastatin (the endogenous inhibitor of calpain) diminishes transiently during myoblast differentiation. To gain information about the regulation of calpain and calpastatin in differentiating myoblasts, we evaluated the stability and synthesis of calpain and calpastatin, and measured their mRNA levels in L8 myoblasts. We show here that mu-calpain and m-calpain are stable, long-lived proteins in both dividing and differentiating L8 myoblasts. Calpain is synthesized in differentiating myoblasts, and calpain mRNA levels do not change during differentiation. In contrast, calpastatin (though also a long-lived protein in myoblasts), is less stable in differentiating myoblasts than in the dividing cells, and its synthesis is inhibited upon initiation of differentiation. Inhibition of calpastatin synthesis is followed by a diminution in calpastatin mRNA levels. A similar calpastatin mRNA diminution is observed upon drug-induced inhibition of protein translation. On the other hand, transforming growth factor beta (which inhibits differentiation) allows calpastatin synthesis and prevents the diminution in calpastatin mRNA. The overall results suggest that at the onset of myoblast differentiation, calpastatin is regulated mainly at the level of translation and that an inhibition of calpastatin synthesis leads to the decrease in its mRNA stability. The existing calpastatin then diminishes, resulting in decreased calpastatin activity in the fusing myoblasts, allowing calpain activation and protein degradation required for fusion.

Association of calpain (Ca(2+)-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts.

Calpain isozymes (intracellular, Ca(2+)-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of mu-calpain and m-calpain (activated by microM and mM Ca(2+), respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca(2+)-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca(2+)levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested.

The calpain-calpastatin system and protein degradation in fusing myoblasts.

Calpain (Ca(2+)-activated cysteine protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously found that calpastatin (the endogenous inhibitor of calpain) diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing Ca(2+)-induced calpain activation at that stage. Here, we show that a limited degradation of some proteins occurs within the myoblasts undergoing fusion, but not in proliferating myoblasts. The protein degradation is observed at the stage when calpastatin is low. Protein degradation within the myoblasts and myoblast fusion are inhibited by EGTA, by the cysteine protease inhibitors calpeptin and E-64d and by calpastatin. The degradation appears to be selective for certain myoblast proteins. Integrin beta 1 subunit, talin and beta-tropomyosin are degraded in the fusing myoblasts, whereas alpha-actinin, beta-tubulin and alpha-tropomyosin are not. A similar pattern of degradation is observed in lysates of proliferating myoblasts when Ca2+ and excess calpain are added, a degradation that is inhibited by calpastatin. The results support the notion that degradation of certain proteins is required for myoblast fusion and that calpain participates in the fusion-associated protein degradation. Participation of calpain is made possible by a change in calpain/calpastatin ratio, i.e., by a diminution in calpastatin level from a high level in the proliferating myoblasts to a low level in the differentiating myoblasts. Degradation of certain proteins, known to be responsible for the stability of the membrane-skeleton organization and for the interaction of the cell with the extracellular matrix, would allow destabilization of the membrane and the creation of membrane fusion-potent regions.

Calpain and calpastatin in myoblast differentiation and fusion: effects of inhibitors.

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca(2+)-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-beta, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-beta and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of mu-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.

On the involvement of calpains in the degradation of the tumor suppressor protein p53.

A crude fraction that contains ubiquitin-protein ligases contains also a proteolytic activity of approximately 100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous approximately 250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N-myc, c-Fos and c-Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.

The role of calpastatin (the specific calpain inhibitor) in myoblast differentiation and fusion.

Using red cells as an experimental model, we previously showed that a limited degradation of certain membrane proteins by calpain (Ca2+-activated thiol protease) was a necessary prerequisite for cell fusion and that fusibility depended on the ratio of calpain to its endogenous inhibitor calpastatin. Here we show that fusion of rat L8 line myoblasts is accompanied by a dramatic change in the calpain/calpastatin ratio. The protein levels of mu-calpain and m-calpain increased only slightly during myoblast differentiation. In contrast, calpastatin diminished by a factor of 10 at the stages of myoblast alignment and start of fusion, allowing calpain activity to become apparent. Calpastatin reappeared at a late stage of myoblast fusion (myotube formation). The results indicate that calpastatin is regulated during myoblast differentiation, and that its diminution is important in determining the activity of the calpain required for myoblast fusion.

A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the cystic fibrosis transmembrane regulator delta F508 mutation.

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) stimulates 36Cl- efflux from pancreatic CFPAC-1 cells which bear the delta F508 genotype common to most cases of cystic fibrosis [Eidelman et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5562-5566]. By contrast, correction of the cystic fibrosis defect by retrovirus-mediated gene transfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF tracheal epithelial cell line IB3-1 bearing a delta F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CFTR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates 36Cl- efflux from cells expressing the CFTR (delta F508) gene product but not from 3T3 cells expressing the wild-type CFTR. In all three cell types expressing CFTR (delta F508), CPX was found to activate 36Cl- efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at higher CPX concentrations. Six CPX analogues, A1 receptor antagonists of affinity similar to that of CPX, were found to be much less effective than CPX at activating 36Cl- efflux from CFPAC-1 cells. These included 2-thio-CPX. CPT (8-cyclopentyl-1,3-dimethylxanthine),3,4-dehydro-CPX,3-F-CPX,3-1-CPX, and KW-3902 (8-noradamantyl-1,3-dipropylxanthine).(ABSTRACT TRUNCATED AT 250 WORDS)

Calpastatin in erythrocytes of young and old individuals.

To gain knowledge about the behaviour of calpastatin (the specific inhibitor of the Ca(2+)-dependent thiol protease calpain) in the intact cell, we analysed the inhibitor by specific antibodies and determined its activity in erythrocytes from individuals 20-34 years old (young) and 70-93 years old (old). Differences between old and young in the behaviour of erythrocyte calpastatin were observed. Erythrocytes of old individuals had lower amounts of calpastatin and less calpastatin activity than those of young ones. A difference between old and young was also found in the molecular-mass distribution of calpastatin subunits. Increasing the erythrocyte Ca2+ induced changes in calpastatin in young individuals, rendering it similar to calpastatin in cells of old individuals. When calpastatin (isolated from erythrocytes of a young individual) was added to erythrocyte membranes, the initial binding and subsequent association of calpastatin with the membrane were lower in old than in young individuals. We had previously found that calpain binding and activation were enhanced in erythrocyte membranes from old individuals, along with enhanced degradation of band 3 (a major erythrocyte transmembrane anion-transport protein). The overall results indicate an interaction of calpain with calpastatin in the intact cell. Enhanced activation of erythrocyte calpain and degradation of calpastatin occur under conditions of increased cellular Ca2+ and in cells of the aged.

Calpain (Ca(2+)-dependent thiol protease) in erythrocytes of young and old individuals.

Limited proteolysis by calpain (Ca(2+)-activated protease; EC 3.4.22.17) is believed to regulate the function of membrane enzymes and modify the behavior of membrane structural proteins. Calpain is activated by autolysis. The degradation of band 3 protein by mu-calpain is known to be enhanced in erythrocyte membranes from human individuals > 70 years old (old) as compared with that from individuals 20-30 years old (young). In the present study, monoclonal antibody to mu-calpain was used to study the behavior of calpain in erythrocytes of young and old individuals. Less calpain was found in erythrocyte cytosol and membranes from old than in those from young. Increasing the erythrocyte Ca2+ induced translocation of calpain to the cell membrane and autolysis of the enzyme. Alkylation of erythrocyte thiols also promoted translocation of calpain to the membrane, especially in the presence of Ca2+. When calpain was added to erythrocyte membranes, initial binding was greater and subsequent autolysis faster in old than in young individuals, possibly arising from alterations in cell membranes of old individuals. The enhanced calpain autolysis was accompanied by enhanced degradation of band 3 protein in the old. The results suggest that calpain in old individuals is translocated to the cell membrane and is activated by autolysis, resulting in degradation of certain membrane proteins and loss of calpain. Enhanced calpain-induced membrane proteolysis may play a role in abnormal cell destruction (e.g., shortening the life span of erythrocytes in the aged, neuronal degeneration, etc). The erythrocyte membrane provides a convenient model for the study of age-associated alterations in cell membranes and in calpain behavior.