Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues.

In this contribution, we review the molecular and physiological barriers to oral delivery of peptides and nanoparticles. We discuss the opportunities and predictivity of various in vitro systems with special emphasis on human intestine in Ussing chambers. First, the molecular constraints to peptide absorption are discussed. Then the physiological barriers to peptide delivery are examined. These include the gastric and intestinal environment, the mucus barrier, tight junctions between epithelial cells, the enterocytes of the intestinal epithelium, and the subepithelial tissue. Recent data from human proteome studies are used to provide information about the protein expression profiles of the different physiological barriers to peptide and nanoparticle absorption. Strategies that have been employed to increase peptide absorption across each of the barriers are discussed. Special consideration is given to attempts at utilizing endogenous transcytotic pathways. To reliably translate in vitro data on peptide or nanoparticle permeability to the in vivo situation in a human subject, the in vitro experimental system needs to realistically capture the central aspects of the mentioned barriers. Therefore, characteristics of common in vitro cell culture systems are discussed and compared to those of human intestinal tissues. Attempts to use the cell and tissue models for in vitro-in vivo extrapolation are reviewed.

Diffuse reflectance spectroscopy of the human tympanic membrane in otitis media.

We have investigated if features in the diffuse reflectance spectra from in vivo spectroscopic measurements of the tympanic membrane could aid the diagnosis of otitis media in children. Diffuse reflectance spectroscopy, in the visible wavelength range, was used in 15 ears from children with otitis media with effusion before and after myringotomy and in 15 healthy ears as a reference. Two previously published erythema detection algorithms yielded numerical quantities of haemoglobin content. With a combination of the algorithms, induced erythema (after myringotomy) was distinguished from healthy ears using Student's t-test (p < 0.01). Otitis media with mucous effusion was distinguished from (1) otitis media with serous effusion, (2) induced erythema and (3) healthy ears, (p < 0.05) using Student's t-test for independent groups and the paired t-test for dependent groups. Our results imply that reflectance spectroscopy is a promising technique to be used for the diagnosis of otitis media.

Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization.

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast-like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC-1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular P, and Ca2+ uptake activities and expression of type II high-capacity Na+-Pi cotransporters. The odontoblast-like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5-7). Pi uptake declined to a basal level during proliferation and then was up-regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up-regulation coincided with a conspicuously increased glycosylation of NaPi-2a, indicating an activation of this Na+-Pi cotransporter. The study showed that MRPC-1 cells express an odontoblast-like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary.

A protocol for polymerase chain reaction detection of Enterococcus faecalis and Enterococcus faecium from the root canal.

AIM: The present study was set up to develop a protocol for detection of Enterococcus faecalis and Enterococcus faecium from the root canal. METHODOLOGY: A collection of type strains and clinical isolates ol E. faecalis and faecium was used. Specific polymerase chain reaction (PCR) primers targeted against the 16S/23S rDNA intergenic region were used and PCR reactions were set up. PCR products were run on TBE-agarose gel and analysed. The sensitivity of the PCR systems was studied using serial dilutions of (i) bacterial DNA and (ii) bacterial cells from E. faecalis. The specificity of the identification was tested against closely related species. RESULTS: All strains of E. faecalis and E. faecium produced identical amplicon profiles composed of two major bands corresponding to sizes of 320 and 420 bp. When amplifying DNA of higher purity, a third band of 600 bp became evident as well. Closely related species demonstrated single bands of various sizes and were easily distinguished from enterococci. The detection level of DNA from serial dilutions of DNA was 10(-13) g. The DNA extraction protocol from bacterial cell suspensions resulted in a detection level of 10 bacterial cells per sample. CONCLUSIONS The present study demonstrated a good potential for using PCR technology in the detection of F. faecalis and E. faecium from root canal samples. With a high specificity the methodology was able to detect 10 cells of E. faecalis.

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1.

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

[Acute abdomen calls for considerable care resources. Analysis of 3727 in-patients in the county of Stockholm during the first quarter of 1995]. / Akut buksjukdom kräver stora vårdresurser. Analys av 3,727 inlagda patienter första kvartalet 1995 i Stockholms län.

A total of 3,727 in-patients with acute abdominal symptoms were identified during the first quarter of 1995 at the surgical clinics of the nine hospitals with emergency departments in the county of Stockholm. The diagnoses were: non-specific abdominal pain 24%; cholecystitis 9%; appendicitis 8%; bowel obstruction 7%; intra-abdominal malignancy, diseases of the urinary tract and peptic ulcer 6% each; gastrointestinal hemorrhage, diverticulitis of the colon and pancreatitis 5% each; other diseases as a cause of abdominal symptoms, 19%. 1,601 operations were performed of which 47% were endoscopic procedures. The mean duration of hospital stay was 4.8 days. The length of stay increased significantly with age. The age-related relative frequency of hospitalization due to acute abdominal pain was also dramatically higher in the elderly cohorts. These facts and the prognosis of an 18% increase of inhabitants 50 years of age or older until 2010 in Greater Stockholm signal an increased need of hospital resources for this large group of patients in the coming years.

Endovascular treatment of atherosclerotic lower limb lesions using a PTFE-collared stent-graft.

PURPOSE: To assess the feasibility of a polytetrafluoroethylene-collared (PTFE) endoluminal graft in the treatment of lower limb atherosclerosis. METHODS: We designed an endograft using a 3-mm-diameter balloon-expandable PTFE graft with terminal Palmaz stents placed on the outside of the graft, folding the PTFE back over the stents to form a collar at each end. Under protocol, this device was implanted in 8 symptomatic patients with lower limb ischemia. The lesions, ranging from 4 to 20 cm long, were located in the superficial femoral artery (n = 5), femoropopliteal segment (n = 1), and common (n = 1) and external (n = 1) iliac arteries. The device required a 14-F introducer system. RESULTS: Graft lengths varied from 4 to 35 cm. Implantation was successful in all cases, but procedural complications occurred in 4 patients (2 access site hematomas, 1 leading to endograft occlusion; 1 arterial injury, and 1 distal thromboembolism). At a mean 14-month follow-up, 5 endografts were patent (2 after reintervention for restenosis or thrombosis). The common iliac endograft and 2 superficial femoral artery devices occluded after 3, 2, and 12 months, respectively. CONCLUSIONS: Although this endoluminal graft system is technically feasible and showed encouraging intermediate-term patency in a small pilot study, the early and late complications identified several shortcomings of this design, which needs refinement.

Na+/Ca2+ exchanger isoforms of rat odontoblasts and osteoblasts.

In odontoblasts as well as osteoblasts, a number of mechanisms for the inflow and extrusion of Ca2+ have been demonstrated. The entrance of Ca2+ ions into odontoblasts occurs mainly through voltage-gated calcium channels. Extrusion of Ca2+ is found to be an ATP-dependent process and, in addition, Na+/Ca2+-antiports exist, which are provoked by extracellular Na+. The aim of this study was to identify the Na+/Ca2+-antiport isoforms expressed in dentinogenically active rat incisor odontoblasts and to make a comparison with different osteoblastic cells. Using RT-PCR and RNAse protection assay, we demonstrated the expression of three different isoforms, NaCa 3, 7, and 10, of the NCX1-encoded antiport in odontoblasts and osteoblastic cells. When incubated in the presence of Na+, dissected rat incisor odontoblasts as well as the osteoblastic cells extruded Ca2+ ions, as detected by chlorotetracycline and Fura-2 fluorometry, thus supporting a physiological role for the detected isoform expression. Odontoblasts and rat calvarial osteoblasts, as well as osteoblast-like cell lines UMR-106.01 and Saos-2, were shown to exhibit identical phenotypes of Na+/Ca2+-antiport isoform expression, different from the expression patterns of other tissues. The significance of this specific expression pattern is unknown, but there is a possibility that it is in some way related to the unique demands on these cell types to produce mineralized connective tissue.

Regional variation in plasminogen activator inhibitor-1 expression in adipose tissue from obese individuals.

High plasma plasminogen activator inhibitor-1 (PAI-1) activity is a frequent finding in obesity and adipose tissue has recently been suggested to be a source of circulating PAI-1 in humans. In the present study, differences in adipose tissue gene expression and protein secretion rate of PAI-1 between subcutaneous and visceral adipose tissue was analysed in specimens obtained from 22 obese individuals. The secretion rate of PAI-1 was two-fold higher in subcutaneous adipose tissue than in visceral adipose tissue (292 +/- 50 vs 138 +/- 24 ng PAI-1/10(7) cells, P <0.05). In accordance with the secretion data, subcutaneous adipose tissue contained about three-fold higher levels of PAI-1 mRNA than visceral adipose tissue (2.43 +/- 0.37 vs 0.81 +/- 0.12 attomole PAI-1 mRNA/microg total RNA, P <0.00 ). PAI-1 secretion from subcutaneous but not from visceral adipose tissue correlated significantly with cell size (r = 0.43, P<0.05). In summary, subcutaneous adipose tissue secreted greater amounts of PAI-1 and had a higher PAI-1 gene expression than visceral adipose tissue from the same obese individuals. Bearing in mind that subcutaneous adipose tissue is the largest fat depot these finding may be important for the coagulation abnormalities associated with obesity.

Effect of carbon dioxide on cochlear blood flow in guinea pigs.

The influence of carbon dioxide (CO2) on cochlear blood flow (CBF), blood pressure (SBP) and skin blood flow (SBF) was studied in anaesthetized guinea pigs. A transient acute respiratory acidosis was produced by inhalation of CO2 and oxygen (O2) gas mixtures. The blood flows were measured by laser Doppler flowmetry (LDF). High CO2 increased CBF and SBP, and decreased SBF in a dose-dependent manner. The responses of CBF, SBP and SBF to high CO2 were reversible. Our results indicate that high CO2 (and low pH) dilates the smooth muscle of the blood vessels, resulting in an increase in CBF. CO2 also activates the sympathetic nervous system in the whole body, producing an increase in SBP. The distribution of alpha-adrenergic fibres receptors is abundant in skin and scarce in the cochlea. The constrictive effect on blood vessels is much greater in the skin than in the cochlea, thus our results showed a decrease in SBF during stimulation with higher CO2.

Nitrogenase activity in Alnus incana root nodules. Responses to O(2) and short-term N(2) deprivation.

O(2) and host-microsymbiont interactions are key factors affecting the physiology of N(2)-fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O(2) concentration, and short-term N(2) deprivation, intact nodulated roots were exposed to various O(2) pressures (pO(2)) and Ar:O(2) in a continuous flow-through system. Nitrogenase activity (H(2) production) occurred at a maximal rate at 20% O(2). Exposure to short-term N(2) deprivation in Ar:O(2) carried out at either 17%, 21%, or 25% O(2) caused a decline in the nitrogenase activity at 21% and 25% O(2) by 12% and 25%, respectively. At 21% O(2), nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N(2) fixation. Respiration (net CO(2) evolution) decreased in response to the N(2) deprivation at all pO(2) values and did not recover during the time in Ar:O(2). Increasing the pO(2) from 21% to 25% and decreasing the pO(2) from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O(2) limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O(2) inhibition of nitrogenase at 25% O(2). These results are consistent with a fixed O(2) diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O(2) and N(2) deprivation.

Comparative effects of omeprazole, amoxycillin plus metronidazole versus omeprazole, clarithromycin plus metronidazole on the oral, gastric and intestinal microflora in Helicobacter pylori-infected patients.

Fourteen patients with Helicobacter pylori infection were treated with 20 mg omeprazole, 1 g amoxycillin and 400 mg metronidazole bd for 7 days (OAM), and 16 patients were treated with 20 mg omeprazole, 250 mg clarithromycin and 400 mg metronidazole bd for 7 days (OCM). Saliva, gastric biopsies and faecal samples were collected before, during (day 7) and 4 weeks after treatment in order to analyse alterations of the normal microflora and to determine antimicrobial susceptibility. Both treatment regimens resulted in marked quantitative and qualitative alterations. A selection of resistant streptococcal strains were noticed in both treatment groups, most apparent in the OCM group where a shift from susceptible to resistant strains was recorded. In the OAM group, six patients had overgrowth of resistant enterobacteriaceae during treatment compared with none in the OCM group, in the gastric microflora. The MICs for Enterococcus spp. and Enterobacteriaceae in faeces increased significantly during treatment in both groups. Nine patients in the OAM group became intestinally colonized by yeasts during treatment. The total anaerobic microflora was strongly suppressed in both treatment groups, although most pronounced in the OCM group, where the frequency of clarithromycin-resistant bacteroides strains increased from 2 to 76% during treatment, and remained at 59% 4 weeks post-treatment. Even if the treatment outcome was better in the OCM group (100%) than in the OAM group (71%), the amoxycillin-based treatment might be preferable from an ecological point of view, since the qualitative alterations in terms of emergence and persistence of resistant strains seemed to be most pronounced in the clarithromycin-treated group.

Symptom criteria do not distinguish between functional and organic dyspepsia.

DESIGN: Retrospective study. SETTING: Teaching hospital, Sweden. OBJECTIVE: To find out if various diagnostic criteria could distinguish organic from non-organic causes of dyspepsia. SUBJECTS: 635 patients previously interviewed by computer questionnaire. INTERVENTIONS: Upper gastrointestinal endoscopy, laboratory tests, clinical examination. MAIN OUTCOME MEASURE: Differentiation between organic and functional dyspepsia. RESULTS: 106 patients had functional dyspepsia. Of these 83 had ulcer-like dyspepsia, 76 motility-like dyspepsia, and 50 reflux-like dyspepsia. Eight patients had unspecified dyspepsia. CONCLUSIONS: There was a considerable overlap between different subgroups, and the criteria did not differentiate between organic and non-organic causes of dyspepsia though the symptom criteria in most cases showed an independent value in discriminating between different subgroups. The clinical usefulness of the criteria remains to be shown.

Essential dataset for ambulatory ear, nose, and throat care in general practice: an aid for quality assessment.

OBJECTIVE: To describe the documentation of care for the usual range of ear, nose, and throat (ENT) problems seen in primary care as a basis for developing a computerised information system to aid quality assessment. DESIGN: Descriptive study of the pattern of ENT problems and diagnoses and treatment as recorded in individual case notes. SETTING: The primary health care centre in Mjölby, Sweden. PATIENTS: Consultations for ENT problems from a 10% sample randomly selected from all consultations (n = 22,600) in one year. From this sample 375 consultations for ENT problems (16% of all consultations) by 272 patients were identified. MAIN MEASURES: The detailed documentation of each consultation was retrieved from the individual records and compared with the data required for a computer based information system designed to help in quality management. RESULTS: Although the overall picture gained from the data retrieved from the notes suggested that ENT care was probably adequate, the recorded details were limited. The written case notes were insufficient when compared with the details required for a computerised system based on an essential dataset designed to allow assessment of diagnostic accuracy and appropriateness of treatment of ENT problems in primary care. CONCLUSION: There is a gap between the amount and the type of information needed for accurate and useful quality assessment and that which is normally included in case notes. More detailed information is needed if general practitioners' notes are to be used for regular quality assessment of ENT problems but that would mean more time spent on keeping notes. This would be difficult to justify. IMPLICATIONS: The routine information systems used at this primary healthcare centre did not produce sufficient documentation for quality assessment of ENT care. This dilemma might be resolved by specially designed desktop computer software accessed through an essential dataset.

Prognostic significance of DNA ploidy in mucoepidermoid carcinoma.

Thirty-four mucoepidermoid carcinomas were studied retrospectively with regard to histological and clinical parameters. In 28 of the tumors DNA patterns were also assessed using flow cytometry. Twenty-two of the 28 tumors (79%) were DNA diploid and 6 (21%) DNA aneuploid. Two tumors (7%) showed intratumoral DNA as indicated by different stemlines in specimens investigated from different parts of the tumor. DNA ploidy correlated significantly with cervical lymph node status (P < 0.01), but not with tumor size or histological grade. The mean S-phase value was 2.7% and was significantly higher in aneuploid samples than in diploid ones (P < 0.05). The recurrence rate was significantly lower for patients with stage I and II tumor compared with those with stage III and IV disease (P < 0.01). Five aneuploid tumors showed significantly higher recurrence rates (5/6) than the diploid ones (1/22) (P < 0.01). In univariate analysis for survival, only N stage tumor (P < 0.05) and tumor DNA ploidy (P < 0.0003) had significant prognostic influence. Thus, DNA ploidy seems to be a valuable parameter for evaluating the biological behavior of mucoepidermoid carcinomas of the salivary glands.

Cyanide detoxification in rats exposed to acetonitrile and fed a low protein diet.

Different neurological syndromes have been associated with exposure to cyanide. Dietary cyanide exposure from cassava roots combined with a low intake of the sulfur amino acids necessary for cyanide detoxification has been implicated in the causation of konzo, an upper motoneuron disease identified in Africa. We have investigated the effect of a low protein diet on the capacity for cyanide detoxification. Rats were fed normal chow containing 18% protein or a low protein diet with 5% protein. To expose rats to cyanide the drinking water was supplemented with 40 or 80 mM acetonitrile (CH3CN) for up to 4 weeks. Weight gain was monitored and 24-hr urines were collected for analyses of total sulfur, inorganic sulfate, thiocyanate, and 2-aminothiolazine-4-carboxylic acid (ATC). Blood was collected for analyses of cyanide and cyanate. Rats on a normal diet grew throughout the experiment, while those on a low protein diet initially lost weight and then stabilized at a constant weight. Rats exposed to acetonitrile all progressively lost weight, those on a low protein diet at the highest rate. Signs of neurological damage were not observed. Rats not exposed to acetonitrile excreted < 0.2% of sulfur as thiocyanate and those on a low protein diet reduced their total sulfur excretion to one-third that of rats of the normal diet. Rats on the normal diet did not change total sulfur excretion during exposure to acetonitrile, although thiocyanate now contributed more than two-thirds of excreted sulfur. Rats on a low protein diet exposed to acetonitrile increased both total sulfur and thiocyanate excretion to the levels of rats on a normal diet. Rats exposed to acetonitrile had manyfold increases of circulating concentrations of cyanide and cyanate and of urinary excretion of ATC. There was a positive correlation between blood cyanide concentrations and the plasma concentration of cyanate. It is concluded that the rat has a high capacity for detoxification of cyanide. During adaptation to a low protein intake, sulfur is conserved but cyanide detoxification is still possible at the cost of extensive protein catabolism. It is thus possible that subclinical cyanide exposure could interfere with normal growth and development. The observation of a relationship between circulating cyanide on the one hand and circulating cyanate and urinary excretion of ATC on the other highlights the possibility that cyanide metabolites may mediate neurotoxic effects of cyanide.

Holographic digital data storage in a photorefractive polymer.

We report high-contrast storage of 64-kbit digital data pages in a photorefractive polymer material. Singlepage writing, reading, and erasing operations were demonstrated with a dual-function-dopant polymeric material having a dark lifetime of several days. Data were reconstructed without error by use of several simple readout algorithms.

Exclusion of a primary gene defect at the HLA locus in familial idiopathic dilated cardiomyopathy.

Case control studies have reported associations between specific HLA class II antigens and idiopathic dilated cardiomyopathy (DCM), suggesting that genetically regulated immune response factors may be involved in the pathogenesis of this disease. In this study, families with DCM were used to test the hypothesis that a heritable gene defect in the HLA region is the primary genetic determinant for a subset of cases. Twelve families with DCM were identified. By formal segregation analysis, the inheritance of the disease was most consistent with an autosomal dominant gene defect with incomplete penetrance. Genotyping was performed with five highly polymorphic linked dinucleotide repeat markers that span the HLA locus. Linkage analysis was used to determine whether or not these genetic markers cosegregated with the disease phenotype. Genetic linkage between the disease phenotype and a 21 cM region spanning the HLA was excluded (lod score < or = -2) in at least 60% of our families. These results indicate that a gene defect in the HLA locus region is not the primary genetic determinant of DCM in a series of familial cases. However, our data do not exclude the possibility that HLA regulated immune response factors may have a modifying effect on disease penetrance and expression.