Albumin-based nanoparticles as contrast medium for MRI: vascular imaging, tissue and cell interactions, and pharmacokinetics of second-generation nanoparticles.

This multidisciplinary study examined the pharmacokinetics of nanoparticles based on albumin-DTPA-gadolinium chelates, testing the hypothesis that these nanoparticles create a stronger vessel signal than conventional gadolinium-based contrast agents and exploring if they are safe for clinical use. Nanoparticles based on human serum albumin, bearing gadolinium and designed for use in magnetic resonance imaging, were used to generate magnet resonance images (MRI) of the vascular system in rats ("blood pool imaging"). At the low nanoparticle doses used for radionuclide imaging, nanoparticle-associated metals were cleared from the blood into the liver during the first 4 h after nanoparticle application. At the higher doses required for MRI, the liver became saturated and kidney and spleen acted as additional sinks for the metals, and accounted for most processing of the nanoparticles. The multiple components of the nanoparticles were cleared independently of one another. Albumin was detected in liver, spleen, and kidneys for up to 2 days after intravenous injection. Gadolinium was retained in the liver, kidneys, and spleen in significant concentrations for much longer. Gadolinium was present as significant fractions of initial dose for longer than 2 weeks after application, and gadolinium clearance was only complete after 6 weeks. Our analysis could not account quantitatively for the full dose of gadolinium that was applied, but numerous organs were found to contain gadolinium in the collagen of their connective tissues. Multiple lines of evidence indicated intracellular processing opening the DTPA chelates and leading to gadolinium long-term storage, in particular inside lysosomes. Turnover of the stored gadolinium was found to occur in soluble form in the kidneys, the liver, and the colon for up to 3 weeks after application. Gadolinium overload poses a significant hazard due to the high toxicity of free gadolinium ions. We discuss the relevance of our findings to gadolinium-deposition diseases.

[Voiding disorders in geriatric patients : Correlation with age-related functional impairments]. / Blasenentleerungsstörungen bei geriatrischen Patient(inn)en : Korrelation mit altersassoziierten funktionellen Defiziten.

INTRODUCTION: Voiding disorders are a common problem in elderly people. The highest incidence and prevalence occurs in female patients with a high level of dependency and cognitive impairments. AIM: The aim of the study was to define the correlation between the presence of voiding disorders and age-related functional deficits in hospitalized elderly patients within the framework of a comprehensive geriatric assessment. This is of utmost importance for planning adequate further diagnostic and therapeutic measures. METHODS: This study involved a retrospective cross-sectional assessment of data from 7487 hospitalized patients (74.1 % females, 25.9 % males, mean age 78.9 ± 7.2 years) evaluated by a multidimensional geriatric assessment. Items tested were symptoms of voiding disorders, activities of daily living, cognitive and emotional status, mobility, handgrip strength, need for walking aids, pain, nutritional status and visual function. Data were evaluated with respect to the prevalence of voiding disorders, patient functional status and to the relationship between these findings. RESULTS: Among all patients 4494 (60.0 %) presented with voiding disorders. Of these 95.8 % showed additional relevant functional deficits in three or more test items. Voiding disorders were positively correlated to patient age, dependency in activities of daily living and pain scores and were negatively correlated to cognitive and emotional status, mobility, hand grip strength, nutritional status and visual function. The functional deficits were associated with the severity of voiding disorders. Female patients were more often affected by voiding disorders as well as by functional impairments in comparison to male patients. DISCUSSION: The results showed that more than half of the elderly hospitalized patients suffered from voiding disorders. The high prevalence and correlation between the presence of voiding disorders and functional deficits indicate the need to clearly define and plan diagnostic and therapeutic measures (e. g. bladder diaries and bladder retraining) for these patients, taking the individual functional status into consideration. CONCLUSION: In older patients with voiding disorders, high levels of functional impairment should be considered before planning diagnostic and therapeutic measures in order to ensure the quality of implementation.

Urinary incontinence and poor functional status in fragility fracture patients: an underrecognized and underappreciated association.

PURPOSE/INTRODUCTION: Urinary incontinence (UI) affects some 20 % of community-dwelling older people and 30-60 % of people in institutional care. UI is known as an independent predictor of falls, and likely impacts fracture rates. The aim of the study was to measure the prevalence of UI in a typical fragility fracture population, to evaluate the relationship of UI with functional disability in the post-acute setting. METHODS: Our study is a retrospective cross-sectional study of patients admitted to rehabilitation setting after inpatient hospital management for a fragility fracture. We included all consecutively admitted fragility fracture patients aged over 65. All patients underwent standard clinical examination and Geriatric Assessment. We assessed UI using a two-stage process with a six-item UI screening questionnaire followed by an interview. RESULTS: 1,857 (80.7 % female) patients were available for analysis, mean age was 81.7 years. UI was identified in 59.2 % of all fragility fracture patients, and was more prevalent in females. Patients suffering from UI differed significantly in almost all measured functional and cognitive tests, with increased dependency/lower ADL scores, increased rates of immobility, and higher rates of cognitive dysfunction and depression. CONCLUSION: This study confirms the high prevalence of UI in older fragility fracture patients, and the association between UI and functional impairments. The diagnostic work-up and treatment of patients should be focused on the special needs of these older patients. More efforts are needed to increase awareness about prevalence and consequences of UI among older fragility fracture patients.

[Urinary incontinence in older multimorbid female patients. Screening and assessment]. / Harninkontinenz geriatrischer Patientinnen. Screening und Abklärung.

Symptoms of urinary incontinence are a common problem in elderly, multimorbid female patients. The first step in establishing optimal management is to screen patients routinely and address this pathology directly. Urinary incontinence is considered to be a geriatric syndrome. It develops as a consequence of many complex underlying and potentially synergistic factors related to ageing and disease. A multidimensional clinically based assessment of urinary incontinence should be performed using a defined multimodal method including geriatric assessment, medical history, targeted physical investigation, urinalysis, and measurement of post-void residual urine volume. The aim is to identify as many of the underlying factors and pathologies, followed by step-by-step treatment to alleviate or eliminate incontinence. With this approach, excellent therapeutic results can be achieved in practice, even in elderly patients.

Polypharmacy and incontinence.

Polypharmacy as well as urinary incontinence are common geriatric problems. Possible adverse drug effects remain a matter of concern in geriatric medicine and must be considered in urinary incontinence. The occurrence or aggravation of lower urinary tract symptoms might be caused by medication, especially when the symptom is newly diagnosed. On the other hand geriatric patients are at an increased risk for adverse effects of medications, commonly used for treatment of urinary incontinence. Especially antimuscarinic drugs reveal several complex anticholinergic adverse effects. Therefore, knowledge of inappropriate medication and of possible adverse drug effects is important in the diagnostic evaluation and therapeutic considerations to prevent a cascade of symptom-related medications.

Albumin-based nanoparticles as magnetic resonance contrast agents: II. Physicochemical characterisation of purified and standardised nanoparticles.

We are developing a nanoparticulate histochemical reagent designed for histochemistry in living animals (molecular imaging), which should finally be useful in clinical imaging applications. The iterative development procedure employed involves conceptual design of the reagent, synthesis and testing of the reagent, then redesign based on data from the testing; each cycle of testing and development generates a new generation of nanoparticles, and this report describes the synthesis and testing of the third generation. The nanoparticles are based on human serum albumin and the imaging modality selected is magnetic resonance imaging (MRI). Testing the second particle generation with newly introduced techniques revealed the presence of impurities in the final product, therefore we replaced dialysis with diafiltration. We introduced further testing methods including thin layer chromatography, arsenazo III as chromogenic assay for gadolinium, and several versions of polyacrylamide gel electrophoresis, for physicochemical characterisation of the nanoparticles and intermediate synthesis compounds. The high grade of chemical purity achieved by combined application of these methodologies allowed standardised particle sizes to be achieved (low dispersities), and accurate measurement of critical physicochemical parameters influencing particle size and imaging properties. Regression plots confirmed the high purity and standardisation. The good degree of quantitative physicochemical characterisation aided our understanding of the nanoparticles and allowed a conceptual model of them to be prepared. Toxicological screening demonstrated the extremely low toxicity of the particles. The high magnetic resonance relaxivities and enhanced mechanical stability of the particles make them an excellent platform for the further development of MRI molecular imaging.

Albumin-based nanoparticles as magnetic resonance contrast agents: I. Concept, first syntheses and characterisation.

To develop a platform for molecular magnetic resonance imaging, we prepared gadolinium-bearing albumin-polylactic acid nanoparticles in the size range 20-40 nm diameter. Iterative cycles of design and testing upscaled the synthesis procedures to gram amounts for physicochemical characterisation and for pharmacokinetic testing. Morphological analyses showed that the nanoparticles were spheroidal with rough surfaces. Particle sizes were measured by direct transmission electron microscopical measurements from negatively contrasted preparations, and by use of photon correlation spectroscopy; the two methods each documented nanoparticle sizes less than 100 nm and generally 10-40 nm diameter, though with significant intrabatch and interbatch variability. The particles' charge sufficed to hold them in suspension. HSA retained its tertiary structure in the particles. The nanoparticles were stable against turbulent flow conditions and against heat, though not against detergents. MRI imaging of liquid columns was possible at nanoparticle concentrations below 10 mg/ml. The particles were non-cytotoxic, non-thrombogenic and non-immunogenic in a range of assay systems developed for toxicity testing of nanoparticles. They were micellar prior to lyophilisation, but loosely structured aggregated masses after lyophilisation and subsequent resuspension. These nanoparticles provide a platform for further development, based on non-toxic materials of low immunogenicity already in clinical use, not expensive, and synthesized using methods which can be upscaled for industrial production.

In vivo investigation of thiomer-polyvinylpyrrolidon nanoparticles using magnetic resonance imaging.

This study focused on the investigation of the permeation enhancing effects of a stomach targeted, nanoparticulate drug delivery system. The polyacrylic acid-cysteine/polyvinylpyrrolidon nanoparticles were loaded with the magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid gadolinium(III)dihydrogen salt (Gd-DTPA). Average particle size was determined to be 130 nm and the optimum for stability was found to be below a pH of 4.5. In vitro permeation studies were performed on rat gastric mucosa and revealed an eightfold increase in Gd-DTPA uptake when incorporated in the nanoparticles compared to evaluation in the presence of unformulated polyacrylic acid-cysteine. In vivo investigations with rats were performed via the noninvasive MRI method in order to track the nanoparticles way through the gastrointestinal tract. When Gd-DTPA was administered orally as nanoparticulate suspension, an increased MRI signal in the urinary bladder was detected after 34 min, providing evidence for systemic uptake and renal elimination of the contrast agent. As control experiments with Gd-DTPA only or in combination with unformulated polyacrylic acid-cysteine revealed no MRI signal increase at all, the significant permeation enhancing effect could be identified based on the nanoparticulate formulation.

Evaluation of pelvic floor muscle function in a random group of adult women in Austria.

Despite an increasing clinical interest in female pelvic floor function, there is a lack of data with respect to the knowledge of average adult women about the physiological role of the pelvic floor and their ability to contract pelvic floor muscles (PFM) voluntarily. It was the aim of our study to evaluate the percentage of PFM dysfunction in adult women and the impact of risk factors, such as age, body mass index (BMI), number of children delivered, and the influence of previous PFM training. A total of 343 Austrian adult women (mean age, 41.2 +/- 14.6 years; range, 18-79 years), selected at random, were examined to test their ability to contract the PFM. The examination was carried out by three independent gynecologists during the course of a routine gynecological visit. The ability to contract the PFM voluntarily or involuntarily was assessed by digital intravaginal palpation with the patients in a supine position. The muscle strength was graded according to the Modified Oxford Grading Scale by Laycock. A high percentage (44.9%) of the women was not able to voluntarily perform a normal PFM contraction. In only 26.5%, an involuntary contraction of the pelvic floor was present before an increase in intra-abdominal pressure. The inability to contract the PFM did not correlate with women's age but revealed a weak relationship with the number of childbirths and the patient's BMI. A significant correlation was found between the Oxford Grading Scale rating and the patient's report about previous PFM training.

[Female geriatric patients with urinary incontinence symptoms and their control over pelvic floor muscles]. / Geriatrische Patientinnen mit Harninkontinenz-Symptomen und ihre Kontrolle über den Beckenboden.

UNLABELLED: Not only do anatomy and function of the pelvic floor play an important role as possible causes of female urinary incontinence, they are also crucial for its therapy. The aim of this case control study of female geriatric patients with symptoms of urinary incontinence was to determine the knowledge about their pelvic floor and to assess their ability to contract pelvic floor muscles voluntarily and reflexly. METHODS: A total of 377 female geriatric patients with symptoms of urinary incontinence were investigated in a Basis Assessment for Urinary incontinence. The ability to contract their pelvic floor muscles was examined by a digital vaginal palpation. The extent of the registered muscle strength was graded by the Modified Oxford Grading Scale by Laycock (1994). RESULTS: Of the patients, 65.5% were not aware of their pelvic floor and were not able to contract the pelvic floor muscles (Grade 0 to 1 by Laycock), 22% had an inaccurate knowledge and only performed an insufficient pelvic floor muscle contraction (Grade 2 by Laycock). Only 12.5% could contract their pelvic floor muscles properly (Grade 3 to 4 by Laycock). A subgroup of 83 patients had already absolved pelvic floor exercises in the past, 80 patients with conventional instructions, 3 patients with digital vaginal control. In this subgroup 54.2% of the patients were not able to contract the pelvic floor muscles (Grade 0 to 1 by Laycock) 25.3% only performed an insufficient contraction (Grade 2 by Laycock), while 20% were able to perform a sufficient and powerful contraction (Grade 3 to 4 by Laycock). The three patients in the past controlled by a digital vaginal palpation were part of this group and managed a pelvic floor muscle strength Grade 4 by Laycock. A high percentage of female geriatric patients with symptoms of urinary incontinence have a lack of understanding regarding the position and function of their pelvic floor. These results suggest that conventional pelvic floor muscle exercises without specific control are not an appropriate method to improve geriatric patients' ability to contract their pelvic floor muscles and to prevent urine leakage.

Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor.

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.

Cardiac troponin I to diagnose percutaneous transluminal coronary angioplasty-related myocardial injury.

The purposes of the present study were to evaluate cardiac troponin 1 (cTnl) in the diagnosis of percutaneous transluminal coronary angioplasty (PTCA)-related myocardial injury in comparison with cardiac troponin T (cTnT) and creatine kinase (CK) MB mass concentration, and to investigate the frequency of myocardial injury, as indicated by myocardial protein release, after clinically symptomless side-branch occlusion (SBO) which may occur in the proximity of the attempted stenosis. The final study population comprised 80 patients undergoing elective, single vessel PTCA. Blood samples were drawn before, 6, 24 and 48 h after PTCA. cTnI, cTnT and CKMB mass baseline values were within the reference intervals in all patients (cTnI < 0.1 microgram/l, cTnT < 0.2 microgram/l, CKMB < 5 micrograms/l). Two patients presented with primary failure of PTCA, and visually successful PTCA was performed in all remaining patients. Seven patients (four with SBO) subsequently developed acute myocardial infarction (AMI). Symptomless SBO occurred in 16 patients. In controls (n = 55) there were no significant increases in cTnI, cTnT, or CKMB concentrations compared with baseline values, and all markers stayed within their reference intervals. In half the patients with symptomless SBO (n = 8) all markers were slightly to moderately increased, in two additional patients only CKMB was elevated (cTnI: 0.1-1.0 microgram/l; cTnT: 0.25-0.81 microgram/l and CKMB: 7.9-25.6 micrograms/l). In the majority of patients with primary failure or AMI we found pronounced increases in all tested markers (cTnI: 0.2-12.0 micrograms/l; cTnT: 0.44-12.10 micrograms/l; CKMB: 19.2-423.0 micrograms/l). The results of this study indicate that cTnI is comparably useful to cTnT or CKMB mass for diagnosing myocardial injury in PTCA patients. From our results a preference for one of the tested parameters cannot be clearly derived. Post-procedural cTnI, cTnT, and CKMB mass values are not higher than baseline values in uncomplicated cases, whereas AMI after PTCA leads to pronounced marker increases. SBO, even when symptomless, leads frequently (in about half the patients) to slight marker increases.

Inhibitory effects of the nucleoside analogue gemcitabine on prostatic carcinoma cells.

Gemcitabine (2',2'difluoro-2'deoxycytidine, dFdC) is a synthetic antimetabolite of the cellular pyrimidine nucleotide metabolism. In a first series of in vitro experiments, the drug showed a strong effect on the proliferation and colony formation of the human androgen-sensitive tumor cell line LNCaP and the androgen-insensitive cell lines PC-3 and DU-145. Maximal inhibition occurred at a dFdC concentration as low as 30 nM. In contrast to the cell lines which were derived from metastatic lesions of prostate cancer patients, no inhibitory effects were found in normal primary prostatic epithelial cells at concentrations up to 100 nM. The effect of gemcitabine was reversed by co-administration of 10-100 microM of its natural analogue deoxycytidine. In view of a future clinical application of this anti-tumor drug in advanced prostatic carcinoma, we have compared the effect of gemcitabine on prostatic tumor cells with that on bone marrow granulopoietic-macrophage progenitor cells, because neutropenia is a common side effect of gemcitabine treatment. The time course of action on the two kinds of cells was markedly different. Colony formation of tumor cells was inhibited by two thirds at a gemcitabine concentration of about 3.5 nM. The same effect on granulopoietic-macrophagic progenitor cells required a concentration of 9 nM. Co-administration of deoxycytidine to gemcitabine-treated tumor cell cultures completely antagonized the effect of gemcitabine whereas addition of deoxycytidine after 48 hr of gemcitabine treatment could not prevent gemcitabine action on the tumor cells. In contrast, more than half of the granulopoietic-macrophagic progenitor cells could still be rescued by deoxycytidine administration after 48 hr. These findings and the marked difference in the susceptibility of neoplastic and normal prostatic cells suggest that gemcitabine is a promising substance which should be further evaluated as to its efficacy in the treatment of advanced prostatic carcinoma.

In vivo phosphorylation of histone H1 variants during the cell cycle.

In vivo phosphorylation of the five histone H1 variants H1a-H1e including H1(0) in NIH 3T3 mouse fibroblasts was examined during the cell cycle by using a combination of HPLC techniques and conventional AU gel electrophoresis. Phosphorylation starts during the late G1 phase and increases throughout the S phase. In the late S phase, the H1 variants exist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 (H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphorylated protein ranging between 35% and 75%, according to the particular subtype. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphorylation maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosphorylated molecules. Phosphorylation of mouse H1c, however, occurs stepwise during this transition. Phosphorylated mouse histone subtypes from cells in mitosis contain four phosphate groups in the case of H1a, H1c, and H1e and five in the case of H1b and H1d. Comparison of the mouse phosphorylation pattern to that in rat C-6 glioma cells showed differences for H1e and H1d. By comparing the different phosphorylation patterns of the individual H1 variants during the cell cycle, we were able to classify the H1 histones into subtypes with low (H1a, H1c, H1(0)) and high (H1b, H1d, H1e) phosphorylation levels.

Cardiac troponin T release in multiply injured patients.

Cardiac troponin T (cTnT), a new marker of myocardial tissue damage, was investigated in 32 consecutive multiply injured patients. cTnT, creatine kinase (CK) and CK isoenzyme MB (CK-MB) mass concentrations were measured immediately after admission, 12 and 24 h later and daily thereafter for 4 days. We found a moderate increase in cTnT in 22 patients (72 per cent; peaks: 0.6-5.1 micrograms/l). In only four of these 22 patients did the CK-MB mass/CK index indicate myocardial injury. ST-T alterations and arrhythmias did not occur significantly more frequently in patients with increased cTnT plasma concentrations or positive CK-MB mass/CK index. We found a moderate increase in cTnT in 72 per cent of all patients with multiple injuries, but we found no association between an increase in cTnT and the occurrence of electrocardiographic changes and arrhythmias.

G1- and S-phase synthesis of histone H1 subtypes from mouse NIH fibroblasts and rat C6 glioma cells.

The rates of synthesis of histone H1 subtypes in synchronized mouse NIH 3T3 fibroblasts were compared with those of rat C6 glioma cells during the G0, G1, and S phases by using a combination of HPLC techniques and conventional gel electrophoresis. In the mouse cell line, all H1 subtypes, H1a-H1e including histone H1(0), were detectable. In the rat cell line, however, no histone H1a was found. H1c and H1e from both cell lines show in the quiescent state a relatively high specific activity comparable with that of H1(0). After release from the G0/G1 block, the synthesis of H1(0) and likewise that of H1c and H1e increase for a short period. All H1 subtypes have their maximum specific activity at the same time after stimulation. The percentage of total H1 specific activity of H1a, H1b, and H1d increases, those of H1c and H1e remain relatively constant, and that of H1(0) decreases while cells cycle from the G0/G1 to the S phase. These findings support our assumption that H1 subtypes could be classified into three groups with common metabolic characteristics: one consists of H1a, H1b, and H1d; another of H1c and H1e; and a third of H1(0) histone. Moreover, the corresponding H1 subtypes from two different species seem to have similar specific activities during the G1 and S phases.