Are Geriatrics-Focused Primary Care Clinics Better at Diagnosing Dementia Than Traditional Clinics? A Matched Cohort Study.

BACKGROUND: Dementia and mild cognitive impairment (MCI) are prevalent but underdiagnosed. OBJECTIVE: To compare new dementia/MCI diagnosis rates in geriatrics-focused primary care clinics and traditional primary care clinics. DESIGN: Secondary analysis of a prospective matched cohort study that spanned 2017-2021. PARTICIPANTS: Community-dwelling Veterans over 65 receiving primary care in a geriatrics-focused medical home (GeriPACT) or traditional primary care home (PACT) at one of 57 Veterans Affairs sites. We excluded individuals with a documented diagnosis of dementia or MCI in the year prior to enrollment. MAIN MEASURES: Diagnoses obtained from EHR. Cognitive status was assessed using modified Telephone Interview for Cognitive Status (mTICS) tool. KEY RESULTS: The 470 participants included in this analysis were predominantly white, non-Hispanic males with an average age of 80.3 years. 9.4% of participants received a diagnosis of dementia/MCI after 24 months: 11.5% in GeriPACT and 7.2% in PACT. Adjusted OR for dementia/MCI diagnosis based on GeriPACT exposure was 1.47 (95% CI 0.65-3.29). Low mTICS score (≤ 27) (OR 4.89, 95% CI 2.36-10.13) and marital status (married/partnered) (OR 1.89, CI 0.99-3.59) were independent predictors of dementia/MCI diagnosis. When stratified by cognitive status: diagnosis rates were 20.8% in GeriPACT and 16.7% in PACT among those who scored lower on the cognitive assessment (mTICS ≤ 27); 7.4% in GeriPACT and 3.6% in PACT among those who scored higher (mTICS > 27). The OR for new dementia/MCI diagnosis in GeriPACT was 1.19 (95% CI 0.49-2.91) among those with a low mTICS score and 1.85 (95% CI 0.70-4.88) among those with a higher mTICS score. CONCLUSIONS: Observed rates of new dementia/MCI diagnosis were higher in GeriPACT, but with considerable uncertainty around estimates. Geriatrics-focused primary care clinics may be a promising avenue for improving the detection of dementia in older adults, but further larger studies are needed to confirm this relationship.

Cover crop mixture diversity, biomass productivity, weed suppression, and stability.

The diversity-productivity, diversity-invasibility, and diversity-stability hypotheses propose that increasing species diversity should lead, respectively, to increased average biomass productivity, invasion resistance, and stability. We tested these three hypotheses in the context of cover crop mixtures, evaluating the effects of increasing cover crop mixture diversity on aboveground biomass, weed suppression, and biomass stability. Twenty to forty cover crop treatments were replicated three or four times at eleven sites using eighteen species representing three cover crop species each from six pre-defined functional groups: cool-season grasses, cool-season legumes, cool-season brassicas, warm-season grasses, warm-season legumes, and warm-season broadleaves. Each species was seeded as a pure stand, and the most diverse treatment contained all eighteen species. Remaining treatments included treatments representing intermediate levels of cover crop species and functional richness and a no cover crop control. Cover crop seeding dates ranged from late July to late September with both cover crop and weed aboveground biomass being sampled prior to winterkill. Stability was assessed by evaluating the variability in cover crop biomass for each treatment across plots within each site. While increasing cover crop mixture diversity was associated with increased average aboveground biomass, we assert that this was the result of the average biomass of the pure stands being drawn down by low biomass species rather than due to niche complementarity or increased resource use efficiency. At no site did the highest biomass mixture produce more than the highest biomass pure stand. Furthermore, while increases in cover crop mixture diversity were correlated with increases in weed suppression and biomass stability, we argue that this was largely the result of diversity co-varying with aboveground biomass, and that differences in aboveground biomass rather than differences in diversity drove the differences observed in weed suppression and stability.

Effect of interferon-ß1b on CXCR4-dependent chemotaxis in T cells from multiple sclerosis patients.

Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease triggered by infiltration of activated T cells into the central nervous system. Interferon (IFN)-ß is an established, safe and effective treatment for patients with relapsing-remitting MS (RRMS). The cytokine can inhibit leucocyte infiltration into the central nervous system; however, little is known about the precise molecular mechanisms. Previously, in vitro application of IFN-ß1b was shown to reduce CXCL12/CXCR4-mediated monocyte migration. Here, we analysed the effects of IFN-ß1b on CXCR4-dependent T cell function. In vitro exposure to IFN-ß1b (1000 U/ml) for 20 h reduced CXCR4-dependent chemotaxis of primary human T cells from healthy individuals and patients with RRMS. Investigating the IFN-ß1b/CXCR4 signalling pathways, we found no difference in phosphorylation of ZAP70, ERK1/2 and AKT despite an early induction of the negative regulator of G-protein signalling, RGS1 by IFN-ß1b. However, CXCR4 surface expression was reduced. Quantitative real time-PCR revealed a similar reduction in CXCR4-mRNA, and the requirement of several hours' exposure to IFN-ß1b supports a transcriptional regulation. Interestingly, T cells from MS patients showed a lower CXCR4 expression than T cells from healthy controls, which was not reduced further in patients under IFN-ß1b therapy. Furthermore, we observed no change in CXCL12-dependent chemotaxis in RRMS patients. Our results demonstrate clearly that IFN-ß1b can impair the functional response to CXCR4 by down-regulating its expression, but also points to the complex in vivo effects of IFN-ß1b therapy.

Racial differences in osteoarthritis pain and function: potential explanatory factors.

OBJECTIVE: This study examined factors underlying racial differences in pain and function among patients with hip and/or knee osteoarthritis (OA). METHODS: Participants were n=491 African Americans and Caucasians enrolled in a clinical trial of telephone-based OA self-management. Arthritis Impact Measurement Scales-2 (AIMS2) pain and function subscales were obtained at baseline. Potential explanatory variables included arthritis self-efficacy, AIMS2 affect subscale, problem- and emotion-focused pain coping, demographic characteristics, body mass index, self-reported health, joint(s) with OA, symptom duration, pain medication use, current exercise, and AIMS2 pain subscale (in models of function). Variables associated with both race and pain or function, and which reduced the association of race with pain or function by >or=10%, were included in final multivariable models. RESULTS: In simple linear regression models, African Americans had worse scores than Caucasians on AIMS2 pain (B=0.65, P=0.001) and function (B=0.59, P<0.001) subscales. In multivariable models race was no longer associated with pain (B=0.03, P=0.874) or function (B=0.07, P=0.509), indicating these associations were accounted for by other covariates. Variables associated with worse AIMS2 pain and function were: worse AIMS2 affect scores, greater emotion-focused coping, lower arthritis self-efficacy, and fair or poor self-reported health. AIMS2 pain scores were also significantly associated with AIMS2 function. CONCLUSION: Factors explaining racial differences in pain and function were largely psychological, including arthritis self-efficacy, affect, and use of emotion-focused coping. Self-management and psychological interventions can influence these factors, and greater dissemination among African Americans may be a key step toward reducing racial disparities in pain and function.

The National Registry of Veterans with amyotrophic lateral sclerosis.

BACKGROUND: The Department of Veterans Affairs (VA) Cooperative Studies Program has established a National Registry of Veterans with Amyotrophic Lateral Sclerosis (ALS). This article describes the objectives, methods, and sample involved in the registry. METHODS: United States military veterans with ALS were identified through national VA electronic medical record databases and nationwide publicity efforts for an enrollment period of 4 1/2 years. Diagnoses were confirmed by medical record reviews. Registrants were asked to participate in a DNA bank. Follow-up telephone interviews are conducted every 6 months to track participants' health status. RESULTS: As of September 30, 2007, 2,400 veterans had consented to participate in the registry, 2,068 were included after medical record review, 995 were still living and actively participating, and 1,573 consented to participate in the DNA bank. 979 participants had been enrolled in the registry for at least 1 year, 497 for at least 2 years, and 205 for at least 3 years. Fourteen studies have been approved to use registry data for epidemiological, observational, and interventional protocols. CONCLUSION: This registry has proven to be a successful model for identifying large numbers of patients with a relatively rare disease and enrolling them into multiple studies, including genetic protocols.

Systems biology of T cell activation.

T lymphocytes are central players in the adaptive immune response to pathogens. Cytotoxic T cells are able to identify and eliminate virally infected cells, while helper T cells support B lymphocyte-dependent antibody production as well as produce the cytokines that will determine whether a cell- or antibody-mediated immune response is required. The activation of T cells by pathogens is a complex process requiring multiple tightly regulated signaling pathways. Defects within this network, however, can cause severe and chronic disorders such as autoimmunity. Therefore, improving our understanding of how T cells discriminate between antigens and how these signals are organized to yield distinct immune responses is of importance as this may lead to the identification of novel drug targets and better therapeutic strategies.

Congestive heart failure patients' perceptions of quality of life: the integration of physical and psychosocial factors.

Congestive heart failure (CHF) lowers survival and worsens the quality of life (QOL) of over four million older Americans. Both clinicians and standardized instruments used to assess the QOL of patients with CHF focus primarily on physical symptoms rather than capturing the full range of psychosocial concerns. The purpose of this study was to gather descriptions of the components of QOL as understood by patients living with CHF. Focus groups were conducted with patients with known CHF, New York Heart Association (NYHA) class I-IV, and left ventricular fraction of <40%. Focus groups were audiotaped, transcribed, and reviewed for common and recurrent themes using the methods of constant comparisons. We conducted three focus groups (n = 15) stratified by NYHA stage with male patients ranging in age from 47-82 years of age. Five patients were classified with NYHA stage III/IV and ten with NYHA stage I/II. Thirty attributes of QOL were identified which fell into five broad domains: symptoms, role loss, affective response, coping, and social support. Expectedly, patients reported the importance of physical symptoms; however, participants also identified concern for family, the uncertainty of prognosis, and cognitive function as dimensions of QOL. Changes in patients' lives attributed to CHF were not always considered deficiencies; rather, methods of coping with CHF were identified as important attributes representing possible opportunities for personal growth. Clinicians must understand the full range of concerns affecting the QOL of their older patients with CHF. The findings suggest that psychosocial aspects and patient uncertainty about their prognosis are important components of QOL among CHF patients.

Occurrence of amyotrophic lateral sclerosis among Gulf War veterans.

BACKGROUND: In response to Gulf War veterans' concerns of high rates of ALS, this investigation sought to determine if Gulf War veterans have an elevated rate of ALS. METHODS: A nationwide epidemiologic case ascertainment study design was used to ascertain all occurrences of ALS for the 10-year period since August 1990 among active duty military and mobilized Reserves, including National Guard, who served during the Gulf War (August 2, 1990, through July 31, 1991). The diagnosis of ALS was confirmed by medical record review. Risk was assessed by the age-adjusted, average, annual 10-year cumulative incidence rate. RESULTS: Among approximately 2.5 million eligible military personnel, 107 confirmed cases of ALS were identified for an overall occurrence of 0.43 per 100,000 persons per year. A significant elevated risk of ALS occurred among all deployed personnel (RR = 1.92; 95% CL = 1.29, 2.84), deployed active duty military (RR = 2.15, 95% CL = 1.38, 3.36), deployed Air Force (RR = 2.68, 95% CL = 1.24, 5.78), and deployed Army (RR = 2.04; 95% CL = 1.10, 3.77) personnel. Elevated, but nonsignificant, risks were observed for deployed Reserves and National Guard (RR = 2.50; 95% CL = 0.88, 7.07), deployed Navy (RR = 1.48, 95% CL = 0.62, 3.57), and deployed Marine Corps (RR = 1.13; 95% CL = 0.27, 4.79) personnel. Overall, the attributable risk associated with deployment was 18% (95% CL = 4.9%, 29.4%). CONCLUSIONS: Military personnel who were deployed to the Gulf Region during the Gulf War period experienced a greater post-war risk of ALS than those who were not deployed to the Gulf.

VA Stroke Study: neurologist care is associated with increased testing but improved outcomes.

OBJECTIVE: VA Stroke Study (VASt) data were analyzed to determine whether neurologist management affected the process and outcome of care of patients with ischemic stroke. METHODS: VASt prospectively identified patients with stroke admitted to nine VA hospitals (April 1995 to March 1997). Demographics, stroke severity (Canadian Neurologic Score), stroke subtype (Trial of ORG 10172 in Acute Stroke Treatment [TOAST] classification), tests/procedures, and discharge status (independent, Rankin < or = 2, vs dead or dependent, Rankin 3 through 5) were compared between patients who were or were not cared for by a neurologist. RESULTS: Of 1,073 enrolled patients, 775 (neurologist care, n = 614; non-neurologist, n = 161) with ischemic stroke were admitted from home. Stroke severity (Canadian Neurologic Score 8.7 +/- 0.1 vs 8.4 +/- 0.2; p = 0.44), TOAST subtype (p = 0.55), and patient age (71.4 +/- 0.4 vs 72.4 +/- 0.7; p = 0.23) were similar for neurologists and non-neurologists. Neurologists more frequently obtained MRI (44% vs 16%; p < 0.001), transesophageal echocardiograms (12% vs 2%; p < 0.001), carotid ultrasounds (65% vs 57%; p = 0.05), cerebral angiography (8% vs 1%; p = 0.001), speech (35% vs 18%; p < 0.001), and occupational therapy (46% vs 33%; p = 0.005) evaluations. Brain CT, transthoracic echocardiogram, 24-hour ambulatory ECG use, and hospitalization durations (18.2 +/- 0.8 vs 19.7 +/- 4.1 days; p = 0.725) were similar. Neurologists' patients were less likely to be dead (5.6% vs 13.5%; OR = 0.38; 95% CI 0.22, 0.68; p = 0.001) and less likely to be dead or dependent (46.1% vs 57.1%; OR = 0.64; 95% CI 0.45, 0.92; p = 0.019) at the time of discharge. The benefit remained after controlling for stroke severity and comorbidity (OR = 0.63; 95% CI 0.42, 0.94; p = 0.025). CONCLUSION: Neurologist care was associated with more extensive testing, but similar lengths of hospitalization and improved outcomes.

Ten-year mortality for patients discharged after hospitalization for chest pain or other symptoms raising suspicion of acute myocardial infarction in relation to hospital discharge diagnosis.

AIM: To describe the 10-year prognosis for patients discharged after hospitalization for chest pain or other symptoms giving an initial suspicion of acute myocardial infarction (AMI) in relation to the final hospital diagnosis and furthermore to compare the outcome amongst these patients with the outcome amongst a sex-, age- and community-matched control population. METHODS: All patients who were hospitalized because of chest pain or other symptoms raising a suspicion of AMI and who were discharged alive from hospital. Patients were divided into three groups according to the final diagnosis: (1) confirmed or possible AMI, (2) confirmed or possible myocardial ischaemia and (3) other aetiology. Information on 10-year mortality was available in 3103 patients. A sex-, age- and community-matched control population (n=3221) was compared with the study population in terms of 10-year mortality. TIME OF SURVEY: 15 February 1986 to 9 November 1987. SETTING: Sahlgrenska University Hospital. RESULTS: Patients with confirmed or possible AMI (n=849) had a significantly higher mortality (59.4%) than patients with confirmed or possible myocardial ischaemia (n=1191) who had a mortality of 49.5% (P < 0.0001). The latter group had a higher mortality than patients with 'other aetiology' (n=1063) of whom 40.6% died (P < 0.0001). When comparing the prognosis for patients with AMI and myocardial ischaemia, there was a significant interaction with sex, with a more marked difference in women than in men. Amongst all patients, the 10-year mortality was 49.1 vs. 37.3% in the control group (P < 0.0001). CONCLUSION: The very long term prognosis was strongly associated with diagnosis amongst patients hospitalized and discharged alive because of chest pain or other symptoms raising suspicion of AMI. The absolute mortality difference between patients who were discharged from hospital with confirmed diagnosis of AMI and those whose symptoms were considered to have other aetiology than AMI or ischaemia was nearly 20%. However, the absolute mortality difference between the patients included in the survey and a control population was only 12%.

Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(I) mRNA.

Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein-RNA interactions in the 3'-UTR of the collagen alpha1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is alphaCP(2). Recombinant alphaCP(2) is sufficient for binding to the 3'-UTR of collagen alpha1(I). To characterize the binding affinity of and specificity for alphaCP(2), we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3'-UTR of collagen alpha1(I) as probe. The binding affinity of alphaCP(2) for the 3'-UTR sequence is approximately 2 nM in vitro and the wild-type 3' sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein-nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA-DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant alphaCP(2) to the wild-type 3' sequence, although the kinetics of binding were slower.

Fibrogenesis. III. Posttranscriptional regulation of type I collagen.

There are several independent metabolic steps that determine the level of a protein in eukaryotic cells. The steady-state level of the mRNA encoding the specific protein is determined by rate of transcription, percentage of transcripts that are ultimately processed and transported to the cytoplasm, and half-life of the mRNA in cytoplasm. The amount of protein that accumulates from a particular transcript is influenced not only by the amount of mRNA present in the cytoplasm but also by the rate of translation of the mRNA and stability of the protein product. There is compelling evidence that the steady-state level of many proteins is regulated at multiple steps, and when there is a large change in the amount of either mRNA or protein it is likely that multiple steps in the metabolism of the mRNA and protein have been altered. In the case of type I collagen production in the fibrotic liver, recent work has shown that there is regulation of multiple steps resulting in an approximately 70-fold increase in collagen production by the hepatic stellate cells. In addition to the well-documented relatively small effect on transcription, there are effects on processing/transport of the mRNA, translation of the mRNA, and stability of the mRNA. Large changes of protein levels are produced by altering the rates or efficiency of multiple steps. The molecular details of some of these posttranscriptional regulatory events are currently being elucidated. Here we review the various potential steps for regulation in the synthesis of a protein and discuss how the synthesis of type I collagen may be regulated in the fibrotic liver.

DAP12 and KAP10 (DAP10)-novel transmembrane adapter proteins of the CD3zeta family.

Transmembrane adapter proteins are molecules that associate with receptors and mediate intracellular signals following interaction of the receptor with its ligand. Many such molecules have been characterized in detail, particularly the small TM adapters of the CD3zeta class at the core of the T cell receptor. Recently, two new genetically linked members of this class of transmembrane adapters have been identified called DAP12 (KARAP) and KAP10 (DAP10), respectively. In this review, we discuss this new class of TM adapters using the wealth of knowledge concerning CD3zeta and FcRgamma to highlight similarities and differences with DAP12 and KAP10. In addition, novel receptor families which interact with these TM adapters have also been identified. The role of these receptors and their inhibitory isoforms are discussed.

Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a beta -adrenoreceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2.

To identify the signaling pathway that mediates the adrenergic stimulation of the expression of the gene for vascular endothelial growth factor (VEGF) during physiologically induced angiogenesis, we examined mouse brown adipocytes in primary culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced VEGF expression 3-fold, in a dose- and time-dependent manner (EC(50) approximately 90 nm). Also, the hypoxia-mimicking agent cobalt, as well as serum and phorbol ester, induced VEGF expression, but the effect of NE was additive to each of these factors, implying that a separate signaling mechanism for the NE-mediated induction was activated. The NE effect was abolished by propranolol and mimicked by isoprenaline or BRL-37344 and was thus mediated via beta-adrenoreceptors. The NE-induced VEGF expression was fully cAMP mediated, an effect which was inhibited by H-89 and thus was dependent on protein kinase A activity. Involvement of other adrenergic signaling pathways (alpha(1)-adrenoreceptors, Ca(2+), protein kinase C, alpha(2)-adrenoreceptors, and pertussis toxin-sensitive G(i)-proteins) was excluded. The specific inhibitor of Src tyrosine kinases, PP2, markedly reduced the stimulation by NE, which demonstrates that a cAMP-dependent Src-mediated pathway is positively connected to VEGF expression. However, inhibition of Erk1/2 MAP kinases by PD98059 was without effect. NE did not prolong VEGF mRNA half-life and its effect was thus transcriptional, and was independent of protein synthesis. These results demonstrate that adrenergic stimulation, through beta-adrenoreceptor/cAMP/protein kinase A signaling, recruits a pathway that branches off from the NE-activated Src-Erk1/2 cascade to enhance transcription of the VEGF gene.

Regulation of collagen alpha1(I) expression in hepatic stellate cells.

The regulation of collagen alpha1(I) expression in hepatic stellate cells (HSCs) occurs in a complex fashion that is just beginning to be determined. The presence of regulatory sequences in both the 5' and 3' regions of the mRNA appear to be critical to its regulation in HSCs and are involved in the increased expression of collagen in activated HSCs. The 3' UTR contains a C-rich site that binds alphaCP, a known RNA-binding protein that is responsible for the increased stability of the mRNA in activated HSCs. Given that alphaCP is present in both activated and quiescent HSCs, there must be a mechanism for modifying alpha(CP to bind to the RNA in activated but not quiescent HSCs. The 5' region contains an evolutionary conserved stem-loop region that encompasses the translation initiation codon. This stem-loop can bind protein(s) in activated HSCs in an RNA cap-dependent manner. Such binding, together with the binding of alphaCP to the 3' UTR, can facilitate translation of collagen alpha1(I) mRNA, resulting in increased mRNA steady-state levels and collagen synthesis. A role of alphaCP in activating translation initiation has also been demonstrated. These two mechanisms work together to upregulate collagen alpha1(I) production in activated but not quiescent HSCs.

Beta 3- and alpha1-adrenergic Erk1/2 activation is Src- but not Gi-mediated in Brown adipocytes.

A novel signaling pathway for mediation of beta(3)-adrenergic activation of the mitogen-activated protein kinases Erk1/2 (associated with proliferation, differentiation, and apoptosis) has recently been proposed, which implies mediation via constitutively coupled G(i)-proteins and Gbetagamma-subunits, distinct from the classical cAMP pathway of beta-adrenergic stimulation. To verify the significance of this pathway in cells in primary cultures that entopically express beta(3)-adrenoreceptors, we examined the functionality of this pathway in cultured brown adipocytes. Norepinephrine activated Erk1/2 via both beta(3) receptors and alpha(1) receptors but not via alpha(2) receptors. Forskolin induced Erk1/2 activation similarly to beta(3) activation, indicating cAMP-mediation; this induction could be inhibited with H89, implying protein kinase A mediation. The G(i)-pathway was functional in these cells, as pertussis toxin increased agonist-induced cAMP accumulation. However, pertussis toxin was unable to affect adrenergically induced Erk1/2 activation. Also, wortmannin was without effect, implying that Gbetagamma activation of the phosphatidylinositol 3-kinase pathway was not involved. PP1/2, which inhibits Src, abolished both beta(3)- and alpha(1)-induced Erk1/2 activation. Thus, the proposed novel G(i) pathway for beta(3) mediation is not universal, because it is not functional in the untransformed primary cell culture system with entopically expressed beta(3) receptors examined here. Here, the beta(3) signal is mediated classically via cAMP/protein kinase A. beta(3) and alpha(1) signals converge at Src, which thus mediates Erk1/2 activation in both pathways.

New aspects of hepatic fibrosis.

Hepatic stellate cells are the major source of extracellular matrix proteins in hepatic fibrosis, including Type I collagen. In response to liver injury, the hepatic stellate cells change from a quiescent to an activated phenotype. This activation process includes a phenotypic change to a myofibroblast-like cell, increased proliferation rate, loss of retinoid stores, increased production of extracellular matrix proteins, chemokines, and cytokines, and contractility. Ongoing studies are characterizing the genes that are differentially expressed in the quiescent and activated hepatic stellate cells. We have also investigated the regulation of Type I collagen expression, the cleavage of collagen propeptides, and the formation of collagen cross-links. Understanding these pathways may provide new insights into the molecular pathogenesis of hepatic fibrosis.

Analysis of film coating thickness and surface area of pharmaceutical pellets using fluorescence microscopy and image analysis.

A method is presented which enables geometrical characterisation of pharmaceutical pellets and their film coating. It provides a high level of details on the single pellet level. Image analysis was used to determine the coating thickness (h) applied on the pellets and the surface area (A) of the pellet cores. Different definitions of A and h are evaluated. Hierarchical analysis of variance was used to resolve different sources contributing to the total variance. The variance within pellets and the variance between pellets were found as significant sources of variation. Special emphasis was put on evaluation of A/h due to its influence on the release rate of an active drug substance from the pellet core. The pellet images were thus used to predict variations in the release rate using a mathematical model as a link between the image data and the release rate. General aspects of image analysis are discussed. The method would be useful in calibration of near infrared spectra to h in process analytical chemistry.

The 5' stem-loop regulates expression of collagen alpha1(I) mRNA in mouse fibroblasts cultured in a three-dimensional matrix.

The stability of collagen alpha1(I) mRNA is regulated by its 5' stem-loop, which binds a cytoplasmic protein in a cap-dependent manner, and its 3'-untranslated region (UTR), which binds alphaCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen alpha1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse-human collagen alpha1(I) mRNA with a wild-type 5' stem-loop decayed faster than the same mRNA with a mutated stem-loop. When the 5' stem-loop was placed in a heterologous mRNA, the mRNA accumulated to a lower level in cells grown in gel than in cells grown on plastic. This suggests that the 5' stem-loop down-regulates collagen alpha1(I) mRNA. Protein binding to the 5' stem-loop was reduced in cells grown in gel, which was associated with destabilization of the collagen alpha1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen alpha1(I) 5' stem-loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen alpha1(I) mRNA. Binding of alphaCP, a protein involved in stabilization of collagen alpha1(I) mRNA, was unchanged by the culture conditions.