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.

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.

Lymphocyte lineages at mucosal effector sites: rat salivary glands.

Development of T cell lineages and the role of the thymus as a source of immature T cells in parotid (PG) and submandibular salivary glands (SMG) were studied in Fischer 344 rats using the Thy-1/CD45RC/RT6 expression model. In addition, the phenotypes of salivary gland lymphocytes were compared with other conventional and extrathymic populations. PG mononuclear cells consisted of T cells (38%), B cells (29%), and NK cells (4%). SMG had 19% T cells, 7% B cells, 37% NK cells, and an unusual population of CD3(-)/RT6(+) cells. In comparison with lymph node (LN), both PG and SMG were enriched in immature (Thy-1(+)) and activated (Thy-1(-)/CD45RC(-)/RT6(-)) T cells. Unchanged percentages of Thy-1(+) T cells in PG and SMG following short-term adult thymectomy indicated that immature salivary gland T cells had an extrathymic source. In contrast, thymectomy eliminated LN recent thymic emigrants. SMG had T cells with characteristics of extrathymic populations, expressing TCRgammadelta(+) (28%), the CD8alphaalpha homodimer (11%), and NKR-P1A (66%). Many SMG T cells expressed integrin alpha(E)beta(7). PG T cells resembled those isolated from LN in respect to TCR and CD8 isoform usage, but were enriched in alpha(E)beta(7)(+) T cells and in NKT cells. Thus, salivary gland mononuclear cells are composed of a variety of subpopulations whose distributions differ between SMG and PG and are distinct from LN. These studies provide a basis for further investigation of regionalization in the mucosal immune network and are relevant to the design of vaccine regimens and intervention during pathological immune processes.

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.

Induction of secretory and serum antibody responses following oral administration of antigen with bioadhesive degradable starch microparticles.

Bioadhesive degradable starch microparticles were used to deliver antigen and immunoglobulin A (IgA)-enhancing cytokines to the oral mucosa. Degradable starch microparticle immunization groups consisted of rats dosed topically at the sublingual epithelium of the oral cavity, by subcutaneous injection in the vicinity of the major salivary glands or by oral intubation with degradable starch microparticles containing dinitrophenyl-bovine serum albumin +/- IL-5/IL-6 +/- penetration enhancer (alpha-lysophosphatidylcholine). Dinitrophenyl-bovine serum albumin was also adsorbed onto alum for salivary gland vicinity injection and administered to the oral cavity in soluble form. Animals were subjected to 3 immunization cycles, and sequential samples were assayed by radioimmunoassay for salivary IgA, tear IgA and serum IgG anti-dinitrophenyl antibodies after secondary and tertiary immunization. Salivary IgA responses were highest in degradable starch microparticle groups receiving penetration enhancer at 71 days post-secondary immunization and continued in one degradable starch microparticle((oral cavity) and two injected (salivary gland vicinity) groups for up to 88 days post-tertiary immunization. Long-term tear responses were also observed in degradable starch microparticle groups receiving penetration enhancer, but they dissipated before the salivary gland-alum responses following tertiary immunization. Serum IgG responses were most pronounced in salivary gland groups, but long-term low level responses were detectable in oral cavity groups receiving degradable starch microparticle formulations with penetration enhancer. Inclusion of IL-5 and IL-6 in oral cavity-delivered degradable starch microparticle formulations consistently enhanced tear IgA while only upregulating salivary IgA antibody responses at early time points post immunization. IL-5 and IL-6 did not enhance serum IgG antibodies in any group. These data indicate that bioadhesive degradable starch microparticles can be used as a vehicle to deliver antigen and cytokine signals to the oral cavity and, when delivered in combination with a penetration enhancer, can potentiate long-term salivary IgA responses.

Secretory immunoglobulin A blocks hypoxia-augmented bacterial passage across Madin-Darby canine kidney cell monolayers.

OBJECTIVE: To study the relative impact of previous hypoxic exposure and the addition of secretory immunoglobulin A (IgA) on bacterial translocation. DESIGN: In vitro randomized experimental study. MATERIALS AND METHODS: Transfected Madin-Darby canine kidney epithelial cells were grown as monolayers in a two-chamber tissue culture system. Stationary growth phase Escherichia coli M14 were inoculated in the apical chamber with medium or medium containing polymeric secretory IgA. Tissue culture dishes were then placed in a 21 or 5% O2 incubator environment for 90 minutes followed by a 21% O2 environment. Medium from the basal compartment was then obtained at timed intervals for bacterial culture. MEASUREMENT AND MAIN RESULTS: Bacterial translocation increased with time in co-culture. Previous hypoxic exposure augmented translocation across the monolayers. The addition of IgA blocked translocation under both normoxic and hypoxic conditions. CONCLUSION: Secretory IgA is important in mucosal defense under both normal and shock conditions.

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.

Lymphocyte adhesive interactions with cultured parotid salivary gland epithelial cells from rats.

Cellular interactions control lymphocyte localization within salivary gland tissues and contribute to the immune defense of oral surfaces. We examined lymphocyte adherence to cultured parotid cells using an in vitro assay and found good correlation with previously reported binding to parotid gland frozen sections. Thoracic duct lymphocytes (TDL) bound to parotid cells in greater numbers than thymocytes (74 vs 11 cells/mm2). B cells showed preferential adherence compared to T cells (75% vs 28%). TDL binding was inhibited by sodium azide or cytochalasin B (60% and 80%, respectively). EDTA inhibition (63%) was restored by replacing calcium (9%) but not magnesium (65%). Binding was inhibited by fucoidin or phosphomannan (approximately 70%). Fibronectin peptides had no effect. Culture supernatants were inhibitory for TDL adherence (60%), suggesting that molecules involved in lymphocyte localization may be shed and that parotid cell cultures will be useful for ligand isolation and characterization.

Nasal-associated lymphoid tissue is an inductive site for rat tear IgA antibody responses.

The role of nasal-associated lymphoid tissue (NALT) as a mucosal inductive site for tear IgA antibody responses was investigated in the rat model. Fluorescent microspheres were shown to access and be taken up by NALT after intranasal of ocular-topical administration, although fewer microspheres were found in the latter case. Tear IgA anti-DNP antibody responses to dinitrophenylated Streptococcus pneumoniae were 6 micrograms/ml at day 7, 10 micrograms/ml at day 10, and were still detectable on day 21 (5 micrograms/ml) following ocular or gastrointestinal immunization. Intranasal immunization induced tear IgA responses which were 1.7-fold higher at day 7 (10 micrograms/ml), peaked by day 10 (14 micrograms/ml) and were still 1.6-fold higher (8 micrograms/ml) at day 21 than responses of ocular or gastrointestinal groups. These findings suggest that intranasal immunization may be more effective than ocular or gastrointestinal administration in eliciting tear IgA antibody responses and, taken together with the microsphere data, indicate that NALT can serve as an inductive site for ocular mucosal IgA responses.

Phenotypic profiles of lymphocyte populations isolated from rat major salivary glands.

Marker expression was studied in rat lymphocyte populations isolated from parotid, submandibular and sublingual salivary glands. Comparative data were also obtained for lacrimal gland and spleen populations. Increased percentages of Thy-1+ cells were found in salivary gland populations when compared to spleen with the highest percentage noted for parotid gland. Thy-1 percentages in parotid gland were comparable to those obtained with lacrimal gland. Salivary gland sIg, CD5 and CD8 cell percentages were lower than those obtained for lacrimal gland and splenic populations. The percentages of CD4-bearing cells in submandibular and sublingual gland were lower than those found in parotid gland, lacrimal gland and spleen, and the CD4:CD8 ratios in parotid gland most closely approximated those in spleen. Increased percentages of Thy-1+ lymphocytes coexpressing sIg and CD5 were obtained for all salivary gland cell populations when compared to spleen; however, percentages of salivary gland cells bearing these 3 markers were lower than noted for lacrimal gland, which contained the highest percentage. With respect to adhesion molecules, lymph node homing receptor (LNHR) and Peyer's patch homing receptor (PPHR) bearing cells were found in all glandular populations with the percentages of LNHR+ exceeding PPHR+ lymphocytes. Homing receptor bearing populations were highest in spleen and were present in equal proportions. LFA-1 was expressed by all salivary gland cell populations in greater percentages than lacrimal gland, but lower than those in spleen. VLA-4 and CD44 expression was higher in parotid gland and spleen than in submandibular and lacrimal gland. These data show that the phenotypes of resident lymphocytes in salivary gland tissues differ from lacrimal gland and spleen, as well as each other, and indicate that Thy-1+ cells in glandular tissues bear both B and T cell markers. The adhesion molecule expression data suggest that these molecules may be utilized differently in various glandular tissues, potentially contributing to the variation in phenotypic profiles of resident glandular lymphocytes.

Preparation and characterization of a biodegradable microparticle antigen/cytokine delivery system.

Biodegradable microparticles (MPs) were prepared to contain dinitrophenylated bovine serum albumin (DNP-BSA) and the cytokines interleukin (IL)-5 and IL-6. The conformational integrity of these MPs was examined by scanning electron microscopy. DNP-BSA was evaluated, postentrapment, by a bicinchoninic acid assay, SDS-PAGE, isoelectric focusing, Western blotting, ELISA and spectrophotometric analysis. The bioactivity of the ILs postentrapment was measured using bioassays. Ocular-topical (OT) and intraperitoneal (IP) administration of loaded MPs induced serum IgG, tear IgA and vaginal wash (VW) IgA responses which persisted up to 45 days post secondary immunization (P2o). OT and IP delivery elicited serum IgG and tear IgA responses up to 140 days post tertiary immunization (P3o). VW IgA responses persisted up to 45 days and 140 days P3o OT and IP delivery, respectively. Overall, the inclusion of cytokines in antigen containing MPs enhanced tear IgA antibody levels following OT delivery P2o, while elevated VW IgA responses occurred following IP delivery P2o and P3o. These data demonstrate that antigen/cytokine loaded MPs can potentiate long-term mucosal antibody responses at both target and distal effector sites as well as elicit circulating antibodies.

The specificity of adhesive interactions between rat lymphocytes and salivary gland epithelia.

Salivary immune responses depend on localization of immunocytes in salivary glands. We tested effects of anti-adhesion molecule antibodies and several ligand analogs on in vitro adherence of rat thoracic duct lymphocytes (TDL) to parotid and submandibular gland sections. While TDL adherence to both tissues was markedly decreased by anti-L-selectin mAbs, binding ability after removal of L-selectin by chymotrypsin or PMA suggested that other adhesion systems were involved. Integrin involvement in parotid interactions was indicated by inhibitory effects of anti-HEBF(PP), LFA-1, ICAM-1, and alpha4 integrin antibodies as well as by the PMA-enhanced adherence. Anti-Thy-1 partially inhibited TDL binding to parotid gland, and anti-CD44 partially inhibited submandibular binding. The majority of salivary gland-bound TDL were sIg+ B cells. FACS analysis showed differences in parotid and submandibular endogenous lymphocyte adhesion molecule expression with greater percentages of L-selectin, HEBF(PP), alpha4 integrin, LFA-1, ICAM-1, CD44, and Thy-1-positive cells present in parotid gland. While precise roles of known or novel adhesion molecules in salivary gland lymphocyte retention are not clear, these data suggest that selectins (parotid, submandibular), integrins (parotid), Thy-1 (parotid), and CD-44 (submandibular), as well as other unidentified molecules, are involved.

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.

The effects of transforming growth factor-beta and interleukins 2, 5 and 6 on immunoglobulin production in cultured rat salivary gland tissues.

The effects of transforming growth factor-beta, alone, and in combination with selected interleukins on immunoglobulin production were investigated using an in vitro rat tissue fragment culture system with either parotid, submandibular or sublingual gland tissue. In the majority of culture groups, TGF-beta alone, or in combination with either interleukin 2 (IL-2), IL-5, IL-6 or IL-5 and IL-6 together, significantly increased immunoglobulin A (IgA) levels over those obtained in untreated cultures. The levels of IgG and IgM were generally not affected, with the exception of one sublingual and two submandibular groups(s), where cytokine administration up-regulated either IgG or IgM production. These data indicate that transforming growth factor-beta in combination with IL-2, IL-5, IL-6 or IL-5 and IL-6 can exert a stimulatory effect on IgA production in vitro, supporting a potential regulatory role for these cytokines in salivary gland IgA responses.

Regional differences in lymphocyte function following resuscitated hemorrhagic shock.

Although it is well known that hemorrhagic shock causes immunosuppression, there have been few attempts to define these changes in the various immune compartments. Accordingly, male rats were bled into severe hemorrhagic shock for 60 minutes (mean arterial pressure 35 +/- 5 mm Hg). Twenty-four hours following resuscitation, splenic, mesenteric, and peripheral lymphocytes were harvested for cell population analysis and mitogen stimulation assays. Cell marker analysis revealed no changes in B-cell or T-cell subpopulations in any immune compartment after shock. The splenic and peripheral lymphocytes showed marked depression of mitogen-induced stimulation after shock. In contrast, mesenteric lymphocyte responses to both T-cell and B-cell mitogens were not depressed after shock. Regional variability in mitogen responses after shock occur without change in B-cell or T-cell subpopulations in any immune compartment tested. The mechanism or mechanisms involved warrant further investigation.

In vitro adhesive interactions between rat lymphocytes and lacrimal gland acinar epithelium. Phenotype of adherent lymphocytes and involvement of adhesion molecules.

The selective interaction of trafficking lymphocytes with glandular epithelial cells is thought to link the lacrimal gland (LG) to the mucosal immune network. An in vitro binding assay was used to determine the phenotype of adherent cell populations and to study the involvement of lymphocyte adhesion molecules in the adherence process. Enriched B cell populations showed greater binding to LG epithelium than did enriched T cell populations. Direct phenotyping of adherent lymphocytes demonstrated that B lymphocytes (particularly IgA+ and IgG+ cells) were the predominant participants in LG binding. In vitro binding to LG acinar epithelium was inhibited by mAb with specificity for rat lymph node and Peyer's patch homing receptors and, to a lesser degree, by anti-VLA-4 and LFA-1 but not by anti-CD44 or Thy-1. In addition, the presence of rat lymph node and Peyer's patch homing receptors on endogenous LG lymphocytes was demonstrated by flow cytometry. These data show that B cells are the predominant population adhering to LG epithelium and suggest that lymphocyte homing receptors mediate the adherence process. These findings indicate that selective interactions between lymphocytes and the glandular epithelium contribute to the presence of IgA-producing cells in the LG.