Improving z-tracking accuracy in the two-photon single-particle tracking microscope.

Here, we present a method that can improve the z-tracking accuracy of the recently invented TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) microscope. This method utilizes a maximum likelihood estimator (MLE) to determine the particle's 3D position that maximizes the likelihood of the observed time-correlated photon count distribution. Our Monte Carlo simulations show that the MLE-based tracking scheme can improve the z-tracking accuracy of TSUNAMI microscope by 1.7 fold. In addition, MLE is also found to reduce the temporal correlation of the z-tracking error. Taking advantage of the smaller and less temporally correlated z-tracking error, we have precisely recovered the hybridization-melting kinetics of a DNA model system from thousands of short single-particle trajectories in silico. Our method can be generally applied to other 3D single-particle tracking techniques.

Surface decoration with gH625-membranotropic peptides as a method to escape the endo-lysosomal compartment and reduce nanoparticle toxicity.

The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.

Glial transforming growth factor (TGF)-beta isotypes in multiple sclerosis: differential glial expression of TGF-beta 1, 2 and 3 isotypes in multiple sclerosis.

We studied glial transforming growth factor (TGF)-beta isotype expression in 14 cases of multiple sclerosis. Acute active lesions exhibited selective TGF-beta 2 immunoreactivity of lesion encircling ramified microglia. In contrast, astrocytes within chronic active white matter lesions expressed all three isotypes. Chronic active lesions which extended into cortex exhibited selective cortical astrocyte TGF-beta 2 expression. This isotype was also selectively expressed by astrocytes in apparently normal white matter. A similar pattern of glial TGF-beta expression was seen in the pathological control, progressive multifocal leukoencephalopathy. The results suggest that TGF-beta cytokines are locally expressed in demyelination and that the beta 2 isotype may be uniquely regulated.

Differential expression of TGF-beta 1, 2 and 3 isotypes in Alzheimer's disease: a comparative immunohistochemical study with cerebral infarction, aged human and mouse control brains.

Based upon the hypothesis that growth regulatory and inflammatory mechanisms participate in the pathogenesis of Alzheimer's disease, we studied cases of Alzheimer's disease for immunoreactivity to each of the three mammalian transforming growth factor beta (TGF-beta) isotypes: TGF-beta 1, TGF-beta 2, TGF-beta 3. Results were compared with those seen in control brains and in a destructive pathological process, subacute infarction. In the cases of Alzheimer's disease, TGF-beta 1 immunoreactivity was limited to neuritic profiles within senile plaques. Neuronal neurofibrillary tangles, plaque neurites, microglia, astrocytes and macrophages expressed TGF-beta 2 immunoreactivity. TGF-beta 3 produced strikingly selective staining of Hirano bodies. In contrast, in cases with infarction, reactive astrocytes and macrophages were positive with all three antibodies. Ramified microglia labeled selectively, as in the Alzheimer brains, with the TGF-beta 2 antibody. Subtle generalized astrocyte and microglial immunoreactivity for TGF-beta 2 was seen in pathological and control brains. The localization of TGF-beta isotypes to the lesions of Alzheimer's disease supports the hypothesis that these cytokines may influence lesion expression. Their presence in reactive cells associated with cerebral infarction suggest that they may play a broader role in the pathogenesis of CNS disease.

A survey of dentists and the services they provide to disabled people in the Province of Manitoba.

Persons with disabilities occasionally report that dentists are reluctant to provide them with either dental care or necessary services. There is very little information about the kinds of services that Canadian dentists provide to disabled people. To answer this question, a mail survey, following Dillman's Total Design Method, was undertaken in Manitoba. Questionnaires were mailed to all dentists licensed to practise in Manitoba in 1990. A total of 342 surveys were returned, representing a 62 per cent response rate. In all, 259 (76 per cent) of respondents reported regularly providing dental care to disabled people of all ages who were afflicted by a broad range of disabling conditions. The private dental office was the primary location in which this dental care was provided, but 65 dentists (25 per cent) reported providing dental care in hospitals or other institutions such as nursing homes. Forty per cent of the dentists who reported treating disabled people were willing to accept referrals of new patients with disabilities. Dentists reported providing a full range of dental services to disabled people, and using similar modes of pain control for both disabled and non-disabled patients. The primary mechanism of payment was social assistance programs. Dentists reported that they received adequate compensation (defined as their usual and customary fee) in only 50 per cent of the cases in which they provided dental care to disabled patients. However, 27 per cent of the respondents reported always receiving their customary fee, and 52 per cent indicated that they would be willing to attend continuing education courses offered in the area of special patient care.(ABSTRACT TRUNCATED AT 250 WORDS)

The superficial basal lamina of ciliary processes: binding studies with IgG-immunogold and ferritins.

In this study, we employed two ultrastructurally visible probing systems, IgG-immunogold and ferritin molecules which differ by surface charge, to study binding activity of the aqueous face of the posterior ciliary processes in short term tissue baths. With these probes we demonstrated that the superficial basal lamina of the non-pigmented epithelium binds monomeric and heat aggregated IgG but not IgG F(ab')2. Binding was inhibited by preincubation with monosaccharides and NaCl suggesting that IgG binding was determined by lectin-like and electrostatic interactions. Anionic binding domains within the basal lamina, capable of exerting an electrostatic influence, were directly demonstrated by selective binding of cationic ferritin species. At high concentrations of cationic ferritin, anionic binding sites were saturated and tracer penetrated the basal lamina to reach intercellular spaces between non-pigmented epithelial cells. We concluded that the superficial basal lamina of the ciliary processes, which is bathed by the aqueous humor, may bind and immobilize IgG, IgG-opsonized antigens and accessible carbohydrate or cations on other molecules in this fluid. This binding may be important in the maintenance of normal aqueous humor composition and in the pathogenesis of infectious and immune-mediated ocular disease.

Localization of tissue inhibitor of matrix metalloproteinases in Alzheimer's disease and normal brain.

Based upon the hypothesis that metalloproteinases and their inhibitors might be involved in the pathogenesis of Alzheimer's disease, we studied brain samples of eight cases of Alzheimer's disease, six other pathological entities and three elderly controls for tissue inhibitor of metalloproteinase (TIMP) immunoreactivity. Specificity was supported by a loss of immunoreactivity following antigen preabsorption of antisera. Areas studied included ependyma, choroid plexus, frontoparietal, hippocampal and cerebellar cortex, n. basalis of Meynert, basal ganglia, midbrain, pons, and medulla. TIMP positivity was localized to neuritic senile plaques, neurofibrillary tangles and Purkinje cells. The pattern of TIMP plaque staining was similar to that observed with anti tau and SP18 antibodies. It differed from that observed with anti SP40, HAM 56 and GFAP antibodies. The selective localization of TIMP to the neuritic lesions of Alzheimer's disease in a codistribution with the amyloid precursor protein and abnormally phosphorylated and truncated tau supports a possible role for metalloproteinases and their inhibitors in the evolution of these lesions.

TGF-beta 2 and TGF-beta 3 immunoreactivity within the ciliary epithelium [corrected].

PURPOSE: To determine whether the ciliary epithelium exhibits immunoreactivity for antibodies to transforming growth factor beta (TGF-beta) 2 and TGF-beta 3. The hypothesis was that because the aqueous humor contains mainly biologically active TGF-beta 2, with little TGF-beta 1, the epithelium largely responsible for its composition would also contain this isoform of TGF-beta. The authors anticipated TGF-beta 3 immunoreactivity because TGF-beta 3 often co-localizes with TGF-beta 2. METHODS: The authors followed a standard immunohistochemical protocol using the avidin-biotin complex and newly available rabbit antibodies to synthetic peptide sequences of TGF-beta 2 and TGF-beta 3. Formalin-fixed, paraffin-embedded samples of freshly obtained rabbit and human autopsy eyes were studied. Specificity was supported by specific peptide absorption of antisera before tissue incubation. RESULTS: The pigmented and nonpigmented ciliary epithelia of rabbit and human eyes were stained by antibodies to both TGF-beta 2 and TGF beta-3, and the staining was inhibited by preabsorption of antibodies by peptides of TGF-beta 2 and TGF-beta 3. CONCLUSIONS: The authors conclude that the ciliary epithelium exhibits TGF-beta 2- and TGF-beta 3-like immunoreactivity that, based upon complementary work from other laboratories, is probably synthesized by this epithelium and is not simply absorbed by it from the aqueous humor.

Identification of Fc gamma RI, II and III on normal human brain ramified microglia and on microglia in senile plaques in Alzheimer's disease.

Using monoclonal antibodies to the three known human leukocyte IgG receptors, Fc gamma R, we examined the expression of Fc gamma R in normal brains and in Alzheimer's disease. We found Fc gamma RI, II and III immunoreactivity in senile plaques and on ramified microglia throughout the cortex and white matter of normal and Alzheimer's disease brains. Fc gamma RI expression was independently confirmed by a murine isotype binding study. These findings suggest that intrinsic Fc gamma R may play an important role in normal and disordered immune-related processes in the brain. They support the idea that microglia are brain macrophages.

Immunoglobulin G receptor-mediated phagocytosis by the pigmented epithelium of the ciliary processes.

The current ultrastructural study of cultured explants of bovine and rabbit posterior ciliary processes using immunoglobulin (Ig) G-opsonized red blood cells (E-IgG) showed specific IgG Fc receptor-mediated attachment, ingestion, and digestion of IgG-coated erythrocytes by the pigmented epithelium (PE). Nonpigmented epithelial cells (NPE) in the cultured explants and a transformed cell line of bovine NPE, with and without lymphokine stimulation, did not have IgG-receptor activity. The interaction between PE and E-IgG involved the extension of micropseudopods toward adherent E-IgG, the formation of a linear uniform cap of roughly 200 A between opposing cell membranes, the ingestion of E-IgG by PE into a membrane-lined compartment, and the disintegration of the ingested ligand into membranous debris. Disintegration of some surface-associated E-IgG was also observed and was consistent with the release of a lytic substance by the receptor-activated PE.

The effects of chronic serum sickness on albumin distribution and glucose utilization in rat brain.

The level of cerebrospinal fluid (CSF) protein is elevated in diseases and disease models that are associated with circulating immune complexes such as serum sickness. Circulatory immune complexes are known to deposit in the basal lamina of fenestrated capillaries and may, as a result, affect both capillary bed and parenchymal function. Since the brain has both fenestrated and unfenestrated capillaries and immune complexes deposit to a varying extent in the fenestrated capillaries in chronic serum sickness, cerebral capillary permeability to protein may be altered in some brain areas and lead to the elevation of CSF proteins. In addition various other cerebrovascular and metabolic functions may also be affected by this condition. In this study either radio-iodinated serum albumin (RISA) or 2-[14C]deoxyglucose (14C-2DG) was intravenously injected into control Wistar rats and Wistar rats with chronic serum sickness; subsequently the tissue levels of radioactivity were measured by quantitative autoradiography in 4 brain areas with fenestrated capillaries and 11 brain areas with unfenestrated capillaries. The 2-min distribution of RISA, which demarcates the volume of circulating plasma in perfused microvessels and is generally proportional to local plasma flow, was the same in control and experimental rats. The passage of RISA from blood into brain over 30 min was negligible in both groups; thus cerebral capillary permeability to albumin was not detectably increased in any of these 15 brain areas by chronic serum sickness. The rate of local cerebral glucose utilization, an indicator of local metabolic and neural activity, was calculated from the 14C-2DG data and was virtually identical in control and experimental rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies identify three IgG Fc receptors in normal human central nervous system.

Functional Fc receptors have been described in the central nervous system (CNS) in the subependymal periventricular regions, leptomeninges, including brain perivascular tissues, and choroid plexus. The distribution of this receptor activity suggests a role in protection of adjacent nervous tissue from IgG-opsonized antigens, including microorganisms. In this report, we have utilized monoclonal antibodies to human Fc gamma RI, II, and III; 32, IV.3, and 3G8, respectively, to immunohistochemically examine the distribution of these receptors in the CNS. Fc gamma RI was only occasionally present in the CNS where it was identified most often in the choroid plexus. Fc gamma RII was the predominant receptor in brain. It as consistently present in leptomeninges, including brain perivascular regions, arachnoid granulations, and choroid plexus stroma. Some samples of subependymal periventricular tissue also displayed Fc gamma RII. Fc gamma RIII was only identified in subependymal periventricular tissue but not in choroid plexus and arachnoid. These results demonstrate that regions of normal adult brain which produce cerebral spinal fluid (CSF) and border on CSF and vascular compartments display Fc gamma R heterogeneity consistent with that of blood monocytes and systemic macrophages.

Circumventricular organs in chronic serum sickness: a model for cerebral lupus.

The pathogenesis of the CNS manifestations of systemic lupus erythematosus (SLE) has been the subject of considerable investigation. The focus of many of these studies has concerned immune complex deposition within the choroid plexus (CP). Involvement of the other brain fenestrated vascular beds, the small, paraventricular circumventricular organs, has not been ascertained. For this purpose, chronic serum sickness, a good immunopathological experimental model of naturally occurring systemic immunological disorders such as SLE, was induced in Wistar rats by prolonged immunization with bovine serum albumin (BSA). The involvement of circumventricular vascular beds by immune deposits was ascertained immunohistochemically. The choroid plexus was found to be the most intensely involved circumventricular structure. Immune complex deposits were also present, in descending order of frequency, in the area postrema, subfornical organ, and pineal gland.