Model simulation of the patient flow through a screening centre for diabetic retinopathy

PURPOSE: To construct a quantitative, flexible and simplified mathematical model of the patient flow through the Eye Clinic at the Steno Diabetes Centre (SDC) in order to enable rational dimensioning and assess the effects of modifications. METHODS: Patient data were drawn from the Eye Care database at the SDC. A simple patient flow model was constructed, allowing simultaneous adjustments of all variables, and the model was tested. Two scenarios were simulated: (1) adjusting the algorithm that assigns the follow-up intervals, and (2) increasing the population size to include all patients with diabetes in Copenhagen County. RESULTS: The model can describe the patient flow under steady state conditions, but is less precise in predicting transient changes with the present set-up. Accordingly all simulations were run for a substantial number of iterations. The two scenarios illustrate the usefulness of the model by calculating the required photographic examination capacity for the specific population, thereby allowing better estimations of future dimensioning of the organization. CONCLUSION: The study presents a patient flow model that can be used to illustrate the effects of proposed changes prior to their implementation, specifically with respect to the capacity of the system.     

The number of alveoli in the human lung

The number of alveoli is a key structural determinant of lung architecture. A design-based stereologic approach was used for the direct and unbiased estimation of alveolar number in the human lung. The principle is based on two-dimensional topology in three-dimensional space and is free of assumptions on the shape, size, or spatial orientation of alveoli. Alveolar number is estimated by counting their openings at the level of the free septal edges, where they form a two-dimensional network. Mathematically, the Euler number of this network is estimated using physical disectors at a light microscopic level. In six adult human lungs, the mean alveolar number was 480 million (range: 274-790 million; coefficient of variation: 37%). Alveolar number was closely related to total lung volume, with larger lungs having considerably more alveoli. The mean size of a single alveolus was rather constant with 4.2 x 10(6) microm3 (range: 3.3-4.8 x 10(6) microm3; coefficient of variation: 10%), irrespective of the lung size. One cubic millimeter lung parenchyma would then contain around 170 alveoli. The method proved to be very efficient and easy to apply in practice. Future applications will show this approach to be an important addition to design-based stereologic methods for the quantitative analysis of lung structure.     

Mapping immune reactivity toward Rv2653 and Rv2654: two novel low-molecular-mass antigens found specifically in the Mycobacterium tuberculosis complex

New tools are urgently needed for the detection of latent tuberculosis (TB). We evaluated the diagnostic potential of 2 novel Mycobacterium tuberculosis complex-specific candidate antigens (Rv2653 and Rv2654) and investigated T cell recognition during natural infection in humans and experimental infection in guinea pigs. Peripheral blood mononuclear cells stimulated with peptide pools covering the full length of Rv2654 induced interferon- gamma release in 10 of 19 patients with TB. Neither Rv2654 single peptides nor Rv2654 pools were recognized by bacille Calmette-Guerin-vaccinated donors. However, peptides from Rv2653 were recognized by both patients group. The cross-reactive epitope(s) in Rv2653 were located in a 36-amino acid stretch in the center of the molecule. Rv2654 also induced M. tuberculosis-specific skin-test responses in 3 of 4 aerosol-infected guinea pigs. Rv2654 is a strongly recognized T cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.     

Comparative analysis of different vaccine constructs expressing defined antigens from Mycobacterium tuberculosis

BACKGROUND: Studies of different vaccine constructs have demonstrated variable efficacy against Mycobacterium tuberculosis in animal models. Despite the fact that these vaccines have used one or another of a very small number of immunodominant antigens, a direct comparison of the relative efficacy of the antigens and delivery systems has been difficult, because the studies have used different parameters for assessment. METHODS: We compared the efficacies of the most commonly used vaccine constructs--adjuvanted protein, plasmid DNA, and live bacterial vectors--bearing the immunodominant secreted antigens early secreted antigen target-6 and antigen 85B, either alone or as a fusion protein. Mice were vaccinated with these constructs, and the effects of different delivery systems on protective efficacy (as assessed by survival studies and by monitoring bacterial load) and antigen-specific responses (including the contribution of CD4 and CD8 T cells to these responses) were assayed by various methods. RESULTS: The relative efficacy of different vaccines is dependent on the delivery system, the antigen, and the animal model. Likewise, the relative immunodominance of individual antigens in the fusion molecule is altered by the choice of delivery system. CONCLUSION: These results clearly demonstrate the importance of assessing vaccine function by use of multiple parameters and indicate which parameters are most reliable for assessing vaccine efficacy.     

Silent slipped capital femoral epiphysis in overweight and obese children and adolescents

Abnormal loading of the hip in obese children may lead to anatomic alterations and an increased prevalence of slipped capital femoral epiphysis (SCFE). The aims of this study were to examine the hip motion in obese children and adolescents and to estimate the prevalence of SCFE in a subgroup of patients characterized by pathological clinical examination and/or pain in the knee or hip joint. A total of 411 individuals (196 males), mean age 14.5?±?2.5?years (7.8-20.4), mean BMI of 32.9?±?5.6?kg/m(2) (20.3-51.5, z score +2.65) who were consecutively admitted for an inpatient weight loss program were included in the study. Twenty-six percent of the patients had load-dependent and 11.7?% had load-independent pain in the knee joint. A total of 9.3?% had load-dependent and 4.7?% had load-independent pain in the hip joint. Two patients (0.5?%) underwent surgical treatment of SCFE prior to entry. A total of 18.2?% of the patients showed a reduced range of motion for hip flexion (<90°) and 18.5?% a pathological decreased internal rotation (<10°). Radiological evaluation of the hips in the clinically conspicuous subgroup (n?=?54) revealed an abnormal head-neck ratio as a sign of prior silent slipped capital femoral epiphysis in 11 patients (20.4?% of the 54 patients, 2.7?% of total cohort). In conclusion, these data show a high prevalence of SCFE-like tilt deformities in a selected group of severely obese children. Mild deformation of the epiphysis at young age might be a major predisposing factor for the development of hip osteoarthritis in obese adults.     

KCNQ1OT1 hypomethylation: A Novel disguised genetic predisposition in sporadic pediatric adrenocortical tumors?

Pediatric adrenal tumors, other than neuroblastoma, are rare and can be associated with a genetic predisposition. In this report we describe two patients with an isolated and apparently sporadic adrenocortical tumor; one girl with a carcinoma, the other girl with an adenoma. In both patients genetic screening revealed hypomethylation of the KCNQ1OT1 gene, well-known for its association with the Beckwith-Wiedemann syndrome. This represents a likely novel genetic predisposition in patients with adrenocortical tumors without clear phenotypic features of the Beckwith-Wiedemann syndrome.     

Fragments of SLIT3 inhibit cellular migration

The repellent factor family of Slit molecules has been described as having a repulsive function in the developing nervous system on growing axons expressing the Roundabout (Robo) receptors. Recent studies determined the effects of Slit molecules on the migratory and invasive potential of several types of tumor cells but also on synovial fibroblasts (SFs) derived from rheumatoid arthritis (RA) patients. To optimize a potential therapeutic application we aimed at generatingfragments of Slit3 showing the same functional ability as the full-length molecule but having the advantage of a smaller size. Recombinant Slit3 proteins were expressed and analyzed by western blotting. Their activity was defined by functional assays such as migration assays with RASF and melanoma cells. Recombinant Slit3 containing only leucine rich repeat domain?2 (D2), the domain important for Robo binding and the minimal functional unit D2 dNC were both able to inhibit migration of RASFs as effectively as Slit3 with all 4?repeats. Collectively, our data showed that the ability of Slit3 to reduce the migratory activity of synovial cells from patients with RA and melanoma cells can be mimicked by small protein fragments derived from Slit3. Slit3 fragments may be helpful in therapeutic attempts; however, further studies are necessary in order to elucidate their activity in vivo.     

Three-dimensional vascularization of electrospun PCL/collagen-blend nanofibrous scaffolds in vivo

Nanofiber scaffolds have proven their various advantages for tissue engineering and have been analyzed extensively. However, to date the three-dimensional pattern of vascularization inside nanofibrous scaffolds is unknown. This study introduces a novel method to visualize and quantify vascularization of electrospun nanofibrous PCL/collagen scaffolds in 3D in vivo. Randomly spun PCL/collagen blend and parallel aligned PCL/collagen blend/PEO scaffolds were analyzed for numbers and patterns of sprouting vessels inside the constructs using microCT scans at different time points. The image data derived from the microCT scans was converted into three-dimensional vessel trees. The aligned scaffold showed a significantly smaller number of sprouting vessels but vascularization in the center of the constructs occurred considerably earlier than in the nonwoven scaffold. Thus, for the first time the actual pattern of vascularization in nanofibrous scaffolds can be visualized three-dimensionally. These results demonstrate that the 3D pattern of vessel trees could be an essential parameter to evaluate nanofiber scaffolds for their suitability for tissue engineering as well as in vivo applications in general.     

Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork

The most critical risk factor for optic nerve damage in cases of primary open-angle glaucoma (POAG) is an increased intraocular pressure (IOP) caused by a resistance to aqueous humor outflow in the trabecular meshwork (TM). The molecular pathogenesis of this increase in outflow resistance in POAG has not yet been identified, but it may involve transforming growth factor TGF-β2, which is found in higher amounts in the aqueous humor of patients with POAG. Connective tissue growth factor (CTGF) is a TGF-β2 target gene with high constitutive TM expression. In this study, we show that either adenoviral-mediated or transgenic CTGF overexpression in the mouse eye increases IOP and leads to optic nerve damage. CTGF induces TM fibronectin and α-SMA in animals, whereas actin stress fibers and contractility are both induced in cultured TM cells. Depletion of CTGF by RNA interference leads to a marked attenuation of the actin cytoskeleton. Rho kinase inhibitors cause a reversible decline in the IOP of CTGF-overexpressing mice to levels seen in control littermates. Overall, the effects of CTGF on IOP appear to be caused by a modification of the TM actin cytoskeleton. CTGF-overexpressing mice provide a model that mimics the essential functional and structural aspects of POAG and offer a molecular mechanism to explain the increase of its most critical risk factor.     

Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.