A novel interferometric method for the study of the viscoelastic properties of ultra-thin polymer films determined from nanobubble inflation.

Glass formation and glassy behavior remain as the important areas of investigation in soft matter physics with many aspects which are still not completely understood, especially at the nanometer size-scale. In the present work, we show an extension of the "nanobubble inflation" method developed by O'Connell and McKenna [Rev. Sci. Instrum. 78, 013901 (2007)] which uses an interferometric method to measure the topography of a large array of 5 µm sized nanometer thick films subjected to constant inflation pressures during which the bubbles grow or creep with time. The interferometric method offers the possibility of making measurements on multiple bubbles at once as well as having the advantage over the AFM methods of O'Connell and McKenna of being a true non-contact method. Here we demonstrate the method using ultra-thin films of both poly(vinyl acetate) (PVAc) and polystyrene (PS) and discuss the capabilities of the method relative to the AFM method, its advantages and disadvantages. Furthermore we show that the results from experiments on PVAc are consistent with the prior work on PVAc, while high stress results with PS show signs of a new non-linear response regime that may be related to the plasticity of the ultra-thin film.

Isoelectric focusing of IgA and IgM in composite acrylamide-agarose gels.

A convenient method for isoelectric focusing of intact polymeric IgA and IgM is described. This technique employed composite gels containing 1.0% acrylamide and 0.75% agarose which exhibited minimal electroendosmotic properties. The spectrotypes obtained with mouse IgA myeloma proteins, a human IgA myeloma and rabbit secretory IgA preparations were compared in three gel systems: 5% acrylamide, 0.8% agarose and the composite gel. With respect to resolution of component bands, the composite gel was superior to the other two systems. Hapten binding studies with MOPC-315 IgA and a rabbit secretory IgA anti-DNP antibody indicated that the focused IgA molecules retained their binding site integrity in the composite gel. The pI ranges obtained with microscale sucrose isoelectric focusing and composite gel system showed good correspondence, with the latter exhibiting enhanced resolution. Studies with MOPC-104E IgM revealed improved resolution in the composite gel when compared to the agarose system. Comparison of pI ranges for IgA and IgM immunoglobulins obtained in the present study with those reported previously suggest that IgA spectrotypes are confined to an acidic pI range (3.4--6.4), whereas IgM spectrotypes are not (4.3--8.8).

Selective interactions of lymphocytes with neonatal and adult lacrimal gland tissues.

The lacrimal gland is a functional part of the mucosal immune system and is populated by lymphoid cells that begin to appear early in neonatal development. To define the events controlling the accumulation of these cells, an in vitro adherence assay was used to investigate the interactions of lymphocyte populations with neonatal and adult lacrimal tissue. It was found that (1) lymphocytes adherence to neonatal lacrimal tissue is significantly enhanced over that seen in adults; (2) the increased binding is caused, in part, by adherence to nonacinar structures in neonatal tissue; (3) binding to both neonatal and adult lacrimal gland tissue is an active process that is observed only with viable lymphocytes; (4) lymphocyte adherence to neonatal and adult lacrimal gland tissues is differentially affected by metabolic and cytoskeletal inhibitors; and (5) attachment appears to require the presence of Ca++, a lymphocyte surface protein, and may involve target tissue carbohydrate recognition. These findings suggest that the initial accumulation of lymphocytes in the neonatal lacrimal gland results from a generalized enhanced binding capacity of the developing tissue and that the preferential binding of certain populations (thoracic duct and mesenteric lymph node lymphocytes) to acinar cells maintains the pool in the adult lacrimal gland.

Lymphocyte adhesive interactions with lacrimal gland acinar epithelial cells in primary culture.

PURPOSE: In lacrimal glands, cell-cell interactions control the localization of lymphocyte populations that play a role in immune defense at the ocular surface. This study describes lymphocyte adhesive interactions with cultured lacrimal gland acinar epithelial cells. METHODS: Primary cultures of lacrimal gland epithelial cells were used as targets for in vitro lymphocyte binding assays. The relative adherence of lymphocyte populations was determined. Various physiologically active agents and putative ligand analogs were tested for their effect in the binding assay. RESULTS: Thoracic duct lymphocytes (TDL) bound to cultured lacrimal acinar epithelial cells in greater numbers than did thymocytes (54 cells/mm2 versus 8 cells/mm2). B cells showed preferential adherence compared with T cells (75% sIg+, 14% W3/13+). Thoracic duct lymphocyte binding required intact metabolic and membrane-cytoskeletal function and was inhibited by treating the lymphocytes with sodium azide, formaldehyde, or cytochalasin B (23%, 12%, and 10% of control binding, respectively). Further, adherence was dependent on divalent cations. Ethylenediaminetetraacetic acid-mediated inhibition (42% of untreated) was restored by replacing calcium (89%) but not magnesium (41%). Lymphocyte adherence was inhibited in the presence of fucoidin or phosphomannan polysaccharides (36% and 48% of control binding, respectively). Fibronectin peptides, which are involved in certain types of integrin-mediated adherence, had no effect in this system. Lacrimal culture supernatants contained a factor that was inhibitory for TDL adherence (more than 50% inhibition when concentrated 5 or 10 times). CONCLUSIONS: Thoracic duct lymphocyte adherence to cultured lacrimal gland acinar epithelial cells shows good correlation with previously reported adherence to lacrimal gland frozen sections. Further, lacrimal cell culture supernatants contain soluble factors that inhibit TDL adherence to epithelial cells. These findings suggest that the lacrimal molecules involved in lymphocyte localization are shed and that lacrimal epithelial cell cultures will be useful for ligand isolation and characterization.

Inductive pathways leading to rat tear IgA antibody responses.

PURPOSE: To define the inductive pathways leading to rat tear IgA antibody responses. METHODS: Fluoresceinated dinitrophenylated bovine serum albumin was encapsulated in poly(lactide-co-glycolide) microparticles and was administered by intranasal, ocular topical, or gastrointestinal routes. Histologic methods were used to determine the microparticles' ability to access tissues associated with mucosal inductive pathways. Rats were immunized with microencapsulated antigen by intranasal or ocular topical routes. Tear IgA and serum IgG antibody concentrations were assessed by radioimmunoassay. The frequency of antibody-secreting cells in tissues, postulated to function in tear IgA induction, was measured by enzyme-linked immunospot assay. RESULTS: Although uptake of microencapsulated antigen was greatest at the site of delivery, ocular topical administration resulted in antigen uptake in the conjunctiva and in nasal-associated lymphoid tissue. Intranasal immunization resulted in earlier and significantly higher tear IgA and serum IgG antibody responses and in higher frequencies of antibody-secreting cells in corresponding draining cervical lymph nodes and lacrimal glands than did ocular topical immunization. CONCLUSIONS: Nasal-associated lymphoid tissue functions as a primary inductive site for tear IgA antibody responses by contributing triggered IgA-committed B cells to the lacrimal gland.

Secretory IgA inhibits Pseudomonas aeruginosa binding to cornea and protects against keratitis.

PURPOSE: To determine whether secretory IgA (SIgA) antibody inhibits Pseudomonas aeruginosa binding to cornea in vitro and if boosting SIgA antibody in tears using heat-killed P. aeruginosa as an immunizing antigen is protective in vivo in experimentally induced bacterial keratitis in the mouse. METHODS: SIgA, immunoglobulin-G, immunoglobulin-M, and an undiluted crude human milk preparation were tested in vitro for their ability to inhibit P. aeruginosa binding to the scarified corneas of adult (6 weeks to 6 months of age) mice by topical application of each before similar delivery of the bacterial inoculum. Scanning electron microscopy (scanning EM) was used to quantitate bacterial adherence. In vivo mice were immunized topically with heat-killed P. aeruginosa or sham immunized by application of a similar volume of phosphate-buffered saline (PBS). Tears were collected from both groups of mice and levels of immunoglobulins (Igs) measured by enzyme-linked immunosorbent assay (ELISA). After the second immunization, the same two groups were challenged ocularly with 5.0 x 10(7) colony forming units P. aeruginosa and the response to infection graded. RESULTS: In vitro, after a 30-minute preincubation with Igs, SIgA (250 micrograms/ml) significantly decreased P. aeruginosa binding to cornea in vitro when compared to the number of bacteria bound in PBS control specimens, and binding reduction was concentration dependent. In vivo, 15 days after a second ocular topical immunization, tear SIgA was elevated significantly and was specific for P. aeruginosa when measured by ELISA. In vivo, corneal disease response grades in the heat-killed antigen immunized mice also were significantly less severe when compared to sham-immunized mice. CONCLUSIONS: SIgA significantly inhibits binding of P. aeruginosa to the wounded mouse cornea in vitro, and inhibition is concentration dependent. In vivo, specific antipseudomonal SIgA in mouse tears can be elicited by topical ocular immunization with heat-killed P. aeruginosa, and a significant number of immunized animals with elevated levels of SIgA in their tears exhibited less severe ocular disease after bacterial challenge.

Parameters influencing the expression of IgA antibodies in tears.

Rats were immunized repeatedly with dinitrophenylated type III pneumococcal vaccine by the intravenous (IV), subcutaneous (SC), gastrointestinal (GI), or ocular/topical (OT) routes at biweekly intervals. IgA anti-DNP antibodies were measured in serum, tears, saliva, bronchial, and intestinal washings, obtained 7 days after the third and sixth immunizations, using a solid phase radioimmunoassay. The GI route most effective at eliciting and maintaining IgA antibody responses in tears. The OT group displayed markedly diminished IgA response frequencies and antibody levels in tears following prolonged immunization. These data show that repeated central mucosal (gastrointestinal associated lymphoid tissue) stimulation maintains a local IgA response in tears, while continued topical antigen stimulation does not. Isoelectric focusing was used to probe the spectral complexity of the IgA antibodies of individual animals undergoing GI and OT immunization. The reduction of spectral complexity and the decreased responses following OT immunization appear to reflect a diminution of IgA antibody producing cells in the lacrimal gland. The concomitant changes in spectral components and maintenance of responsiveness of the GI group suggests that central mucosal site stimulation provides the lacrimal compartment with a continuous but variable population of IgA antibody producing cells.

The increase in vivo by electric currents of the 99mTc-methylene diphosphonate uptake and cyclic AMP content of the bone of the cat mandible.

Technetium-99-methylene diphosphonate (99mTc-MDP) bone images were obtained from live cats whose mandibles had been stimulated non-invasively and unilaterally with electric current (12 +/- 2 microA) for 1 week. The images were obtained using a gamma camera interfaced to a digital computer for quantitative comparison of the mandible. Radionuclide uptake by the stimulated sides increased by 59 +/- 26 per cent when compared with the contralateral control sites. Immunohistochemical staining of mandibular sections for adenosine 3',5'-monophosphate (cyclic AMP) showed that periosteal osteoblasts opposite the electrodes were intensely stained. Thus 99mTc-MDP scintigraphy is a sensitive non-invasive technique for determining in vivo enhanced bone remodelling activity. The immunohistochemistry indicates that the stimulation of bone cells was limited to surfaces adjacent to both cathode and anode.

Development of T cell lineages in rat lacrimal glands.

PURPOSE: The purpose of this study was to examine T cell development in rat lacrimal glands, determine whether the thymus is the source of immature T cells in this tissue and compare lacrimal gland T lymphocytes with other T cell subpopulations. METHODS: Mononuclear cells were isolated from lacrimal glands of normal or thymectomized female Fischer 344 rats and stained for flow cytometric analysis. RESULTS: The lacrimal gland T lymphocyte population included large percentages of cells with an activated phenotype and also subpopulations of immature, naive and memory T cells. The numbers of immature (Thy-1(+)) lacrimal gland T cells were unchanged following short-term adult thymectomy. In comparison, spleen had large percentages of naive T cells, only a small subpopulation of activated T cells, and similar percentages of immature (Thy-1(+)) T cells, which were nearly eliminated after thymectomy. Lacrimal gland T cells had small subpopulations of TCRgammadelta(+) and CD8alphaalpha( +) T cells, a large subpopulation of NKT cells and many integrin alphaEbeta7( +) T cells. CONCLUSIONS: Lacrimal gland T cells are composed of a variety of subpopulations whose composition is distinct from splenocytes. The marked reduction of immature splenic T cell percentages eleven days after adult thymectomy indicates that these cells were mostly derived from thymic precursors. In contrast, the unchanged percentages of immature lacrimal gland T cells following thymectomy indicate that they may have an extrathymic source. These studies provide a foundation for further investigation into the cellular basis of lacrimal gland immunobiology.

Antibody production in rats following ocular-topical or gastrointestinal immunization: kinetics of local and systemic antibody production.

The kinetics of the anti-DNP antibody response to DNP-pneumococcus appearing in tears and bile (IgA) and serum (IgM and IgG) was examined in rats after the application of antigen either via the ocular-topical (OT) or gastrointestinal (GI) routes. It was found that IgA responses were obtained each time in tears after either OT or GI antigen doses given monthly for three months, but that the OT route gave rise to consistently higher tear antibody titres than the GI route of immunization. Comparable IgA responses were found in bile using either route. In serum a small primary IgM response was consistently obtained but the main antibody found was IgG, the timing and degree of response being about the same for both routes. When the adjuvants Avridine in liposomes or MTP-PE were added along with the antigen it was found that with either immunization route, the tear IgA response was much reduced compared to when no adjuvant was used; the serum IgG response was marginally increased when adjuvant was added. The effects of binding anti-DNP monoclonal IgA or IgG1 to antigen before immunizing via the OT route was also studied. It was found that the presence of immunoglobulin of either isotype in the complex caused an increase in the serum IgG response, but that the tear IgA response was diminished in rats receiving IgA/antigen complexes compared with those receiving IgG/antigen or antigen alone.

A comparison of lymphocyte subset distribution in rat lacrimal glands with cells from tissues of mucosal and non-mucosal origin.

The subset distribution of lymphocyte populations isolated from rat lacrimal glands (LG) was compared with those from tissues of both mucosal and non-mucosal origin. In spleen (SPL), mesenteric (MLN) and cervical lymph node (CLN) populations the percentages of cells bearing W3/13 (pan T) and OX19 (pan T) were greater than the percentages obtained for cells bearing the OX7 (Thy-1) marker. In contrast, for lacrimal (LG), salivary (SG) and mammary gland (MG) populations, cells bearing OX7 predominated over those bearing the W3/13 and OX19 markers, with the exception of day 1 post-partum MG tissue which displayed equal numbers of OX7 and OX19 cells. Except for MG, in which OX8 (T non-helper) predominated over W3/25 (T helper) populations, the proportions of these two subsets in the other tissues were generally similar. Analysis of SPL and LG cells for coexpression of OX7 with OX19 or L chain indicated that significant percentages of OX7 bearing cells also expressed T or B cell markers. However, the higher values noted for the OX7 population in LG were not attributable to increased numbers of cells coexpressing pan T or B cell markers. These findings show that lymphocyte subset distribution in LG and other glandular mucosal tissues is distinct from that of non-mucosal tissues, in that mucosal tissues contain a predominance of cells bearing the Thy-1 (OX7) phenotype.

The effect of immunization route and sequence of stimulation on the induction of IgA antibodies in tears.

The capacity of ocular/topical (OT) or gastrointestinal (GI) immunization routes alone or sequentially to elicit and maintain tear IgA antibody responses was assessed in the rat model. Seven days after each biweekly immunization with dinitrophenylated type-III pneumococcal vaccine, the IgA antibody levels in serum, saliva and tears were measured. All groups generally lacked serum IgA responses and eventually possessed similar salivary response frequencies with OT, OT/GI and GI groups showing a tendency for increased salivary IgA antibody levels. Tear IgA antibody responses in all groups were comparable after the third immunization. Subsequently the OT group displayed a gradual reduction in response frequency with a significant drop in IgA levels after the sixth immunization. The OT/GI group maintained tear IgA response frequencies while displaying a significant increase in tear IgA antibody levels; the GI and GI/OT groups maintained tear IgA antibody responses. These data demonstrate that the immunization route and sequence of stimulation have a marked impact on the expression of IgA anti-DNP antibodies in tears.

Sensory information and geographical orientation in healthy subjects.

The effect of sensory input on the performance of a geographical orientation task of children at two different ages (kindergartners and fourth graders) and adults was determined. I investigated the ability of subjects to point accurately to the starting position after experiencing identical routes under three sensory conditions. In Condition 1, subjects were led walking through routes and could see the walls and ceiling of the test room (visual, somatosensory, and vestibular information). In Condition 2, subjects were led walking with vision occluded (somatosensory and vestibular information). In Condition 3, subjects were pushed in a wheelchair with vision occluded (primarily vestibular information). As more sensory information was available, subjects maintained their orientation better to their starting position, and accuracy improved with age. This quantitative analysis of geographical orientation may be appropriate for future clinical studies of neurologically impaired adults and children.

Assessment and treatment of the child with mental retardation. Guidelines for the public school therapist.

Physical therapists employed in public schools may be responsible for the evaluation and treatment of not only physically handicapped children, but also children who have moderate to severe motor disabilities secondary to mental retardation. The purpose of this article is to suggest appropriate assessment and treatment techniques for these children. General principles of intervention based on neurophysiologic treatment approaches, particularly sensory integration, are described. Examples of specific assessment and treatment strategies are given for visual, auditory, tactile, olfactory-gustatory, proprioceptive-kinesthetic, and vestibular functions. In addition, self-stimulatory behaviors, tests of motor and reflex development, problems in muscle tone and strength, and variations in gait patterns are discussed.

Norm-referenced and criterion-referenced tests. Use in pediatrics and application to task analysis of motor skill.

The purposes of this article are 1) to compare the similarities and differences between norm-referenced and criterion-referenced tests and 2) to summarize how each should be used in the assessment of developmental performance in children. Specific developmental assessments, the populations they address, and the information they provide are described briefly. The need for additional criterion-referenced tests in physical therapy is discussed, and an example of how task analysis can be applied to movement or motor skills in the development of a criterion-referenced test is provided. Physical therapists can enhance the credibility of their assessments by appropriate use of norm-referenced and criterion-referenced tests.

Effect of state on nystagmus duration in the Southern California Postrotary Nystagmus Test.

The purpose of this study was to determine the effect of state on nystagmus duration following administration of the Southern California Postrotary Nystagmus Test (SCPNT). Twenty-four normal children, 12 first-graders and 12 fourth-graders, were administered with SCPNT under three conditions: aroused, relaxed, and alert (standard instructions). Six boys and six girls in each grade participated. First-grade boys and fourth-grade girls demonstrated a statistically significant response decline in the aroused conditions, and a further decline in the relaxed condition as compared to the alert condition. Further analyses demonstrated that the first-grade boys accounted for most of the variance. Possible reasons for the results obtained are discussed, and suggestions are made for future research.