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Drug delivery using liposomes is a promising technology that has the potential to deliver drugs to the site of action. Stability of liposomal drug delivery system under physiological conditions plays a crucial role in achieving desired therapeutic efficacy. Stability of the liposomal delivery systems can be determined by performing certain in-vitro studies which have the ability to predict in-vivo stability of liposomes. The aim of our study was to evaluate current available in-vitro methods that are used in predicting stability of liposomal drug delivery systems to determine which one of them is a better predictor of in-vivo stability of liposomes. Our second aim was to evaluate the in-vitro stability of the pHsensitive liposomes (contains morpholine based pH-sensitive lipid MOR) that were developed by our group. Liposomes with different lipid compositions were prepared using thin film hydration method to perform protein adsorption study. Since serum proteins adsorb on to liposomes surface (which might include substantial amount of opsonins that can trigger immune response against liposomes) upon intravenous administration, in-vitro protein adsorption Study is used as an indicator of in-vivo circulation half-life of liposomal carrier. In-vitro protein adsorption study includes two major Steps, first step is separation of liposomes with adsorbed protein from unadsorbed serum proteins and the second step is to evaluate the adsorbed protein quantitatively as well as qualitatively. The separation of liposomes from serum was achieved through size exclusion chromatography (SEC) using sepharose CL-4B gel. From the results obtained and upon discussion with vendors of sepharose gel we concluded that SEC using sepharose cl-4b is not a suitable method to separate liposomes from serum, because the stationary phase can interact with liposomes and lipoproteins. The second in-vitro method studied was drug release from liposomes. Doxorubicin loaded liposomes with different lipid compositions were prepared to perform drug release assay. An indirect method (where drug retained inside liposomes was measured) developed by our group was used, which takes the advantage of interaction between dowex resin and doxorubicin. Upon comparing the drug release study data obtained with previously reported in-vivo data we observed that drug release assay has the potential to predict invivo stability of liposomes. From the in-vitro drug release study data it was observed that introduction of the pH-sensitive lipid compromised the stability of pegylated liposomes.

Human palatine tonsil lymphocytes, when compared to peripheral blood lymphocytes (PBL), were in an activated state even though there was no in vitro stimulation. When these tonsil lymphocytes were cultured in the absence of serum and polyclonal mitogens or antigens, the supernatant fluid often inhibited the proliferative response of target PBL to con A. The extent of this suppression ranged from 22% to 84%, and target cell viability was 90% or greater. There was no evidence for the presence of immunoglobulins or (alpha)2-macroglobulin in whole supernatant fluids. The suppressor was partially denatured at 80(DEGREES)C and was rendered completely inactive upon exposure to 100(DEGREES)C for 5 min. It was trypsin sensitive, and had an apparent molecular weight of 100,000 or greater. The protein adhered strongly to DE-52 cellulose, and the most active material eluted with 0.4-0.6 M NaCl. The suppressor was active in the pH range 5.0 (+OR-) 0.6 as demonstrated by isoelectric focusing. Occasionally, supernatant fluids comprised material which augmented the expected response of con A stimulated PBL. The augmentor was 30,000 in molecular weight and was eluted from DE-52 cellulose in the 0.15-0.25 M NaCl range. Nearly all supernatant preparations tested contained a mitogenic substance which stimulated naive allogeneic human PBL without the necessity of co-stimulation by a mitogen. The mitogenic factor (MF) behaved in a dose dependent fashion and was evidently different from the augmentor since the MF stimulated PBL independently of lectin co-stimulation.

Activated lymphocytes secrete many products including the lymphokine human lymphocyte mitogenic factor (HLMF). In preliminary experiments lymphocytes from peripheral blood and palatine tonsils were evaluated as possible sources of HLMF by evaluating their level of activation through screening their spontaneous and concanavalin A (con A)-induced blastogenic responses. Tonsil lymphocytes (TL) were found to have high spontaneous proliferation as compared to peripheral blood lymphocytes (PBL). Cells from both sources responded to con A by undergoing a typical blastogenic response. Because TL must be obtained septically, they are frequently cultured in the presence of the antimycotic agent, Amphotericin B (Am B). Since the primary and induced blastogenesis of TL were greatly inhibited by even low concentrations of Am B, those lymphocytes were considered unacceptable sources of HLMF. In contrast to TL the induced blastogenic responses of PBL were found to be augmented by concentrations of Am B less than 5 (mu)g/ml, but the drug appeared to provide no beneficial effect on the quantity of HLMF produced by the cells. HLMF appeared to be produced optimally in the first 48 hr of culture by 10('7) PBL/ml, cultured in Neuman-Tytell serumless medium which had been adjusted to 5 x 10('-5) M 2-mercaptoethanol, and 5-35 (mu)g con A/ml. Stability of the HLMF activity could best be maintained by immediate dialysis against 0.05 M NH(,4)HCO(,3) solution, followed by lyophilization and storage of the dried material at -80(DEGREES)C until use. Activity was retained at -80(DEGREES)C for greater than 3 months. The activity was diminished after exposure to 56(DEGREES)C for 30 min, and completely lost after treatment at 80(DEGREES)C for 10 min or 100(DEGREES)C for 5 min. HLMF was insensitive to trypsin and exposure to pH ranges 2-7. Separation of HLMF and con A blastogenic activities was accomplished by addition of ovalbumin followed by Bio-Gel P-100 column Chromatography. HLMF activity eluted in the 12,000-20,000 d and 30,000-50,000 d ranges. The lower molecular weight material was active in the pH range 3.4-4.6 as demonstrated by isoelectric focusing. The larger molecular weight fractions had a pI of 4.14 (+OR-) 0.97. HLMF activated T cells, B cells and unfractionated PBL in assay, with the T cell response being generally, but not always greater. The factor behaved in a dose dependent fashion when assayed against unfractionated PBL.

Macrophages play an important role in host antimicrobial immunity and in non-septic inflammatory reactions. Most studies on macrophage activation have focused on the roles of the T cell-produced cytokine, interferon-$\gamma$ (IFN$\gamma)$ and bacterial product, lipopolysaccharide (LPS). T cell-macrophage interaction is a critical step in initiating both specific and nonspecific immune responses to antigenic stimulation. The current study examines the role of cognate T cell-macrophage interaction in activation of macrophage effector functions and induction of macrophage early activation gene expression. Viable resting T$\sb{\rm H}$2 clone cells can activate IFN$\gamma$-primed macrophages to produce reactive nitrogen intermediates (RNI) or express cytostatic activity. The activating signal is mediated by cognate membrane contact between T cells and macrophages as evidenced by the ability of paraformaldehyde fixed anti-CD3-activated T$\sb{\rm H}$2 cells or plasma membranes isolated from the activated T cells to activate the IFN$\gamma$-primed macrophages. In contrast to the antigen-specific interaction of macrophages with viable resting T$\sb{\rm H}$2 cells, the activation of IFN$\gamma$-primed macrophages by fixed activated T$\sb{\rm H}$2 cells or by membranes from activated T$\sb{\rm H}$2 cells does not display antigen specificity. Fixed resting T$\sb{\rm H}$2 cells or plasma membranes isolated from the resting T cells can not activate the IFN$\gamma$-primed macrophages. Similar results are obtained with use of fresh splenic T cells to induce macrophage RNI production and cytostasis. Monoclonal antibody against CD4, which presumably blocks the interaction between CD4 (a co-receptor of T cell receptor) and class II MHC molecules on macrophages, inhibits significantly the activation of IFN$\gamma$-primed macrophages by viable resting T$\sb{\rm H}$2 cells but does not inhibit the ability of fixed activated T$\sb{\rm H}$2 cells to activate the macrophages. To examine the intracellular events in macrophages initiated by the cognate signaling, the expression of a panel of macrophage early activation genes, c-Myc, c-Fos, JE, IP10, D3, TNF$\alpha$ and IL-$\alpha$, are analyzed by dot blot hybridization. Plasma membranes from activated T$\sb{\rm H}$2 cells induce the expression of all these genes in macrophages stimulated for 1-4 hour. In contrast, the plasma membranes from resting T$\sb{\rm H}$2 cells are unable to induce the expression of most of the genes examined. These results suggest that the T cell-macrophage interaction involves reciprocal activation of both cells--an antigen specific activation of the T cells which results in the acquisition of T cell membrane components involved in antigen nonspecific activation of the macrophages. The nature of those T cell membrane components involved in cognate signaling of macrophage is currently being investigated.

Goal and Objectives: The goal of this study is to assess parental knowledge and experience regarding children's dental insurance, following enactment of the Affordable Care Act (ACA). The ACA emphasizes the importance of oral health, making pediatric dental services one of ten essential health benefits. With this major change in health care reform, parents should be equipped to make informed decisions regarding their children's dental care and insurance. Data from the first open enrollment period show lack of true mandate and subsequent lack of compliance to this pediatric dental objective. One of our aims was to assess uptake of pediatric dental benefits, type selected and effects on experience related to utilization, scheduling timeliness, and cost satisfaction. This study would allow dental professionals to better assist parents in navigating children's dental insurance and assist policymakers in improving access to care. Methods: The 28-question survey was administered to a national sample of 421 parents, of children ages 18 and younger. The states included were those operating through the Federally-Facilitated Marketplace (37 states). Data was collected by SurveyMonkey® via online survey and analyzed by an NSU statistical consultant. The data identified pediatric dental insurance status, source and type, parental knowledge of the marketplaces and pediatric dental benefits being an essential health benefit, issues in utilization, access to care, changes in providers, cost, satisfaction, and quality of care. Results: The majority of the sample respondents had incomes above the federal poverty level and had a Bachelor's degree or higher level of education. The majority of the sample respondents had pediatric dental benefits. For those who didn't, a major reason was due to plans being too expensive. The majority of the sample respondents selected pediatric dental benefits through their employer and selected a qualified health plan that included dental coverage (embedded plan) or a stand-alone dental plan. Regarding knowledge of the ACA, many didn't know that dental care for children is one of ten essential services covered. In reference to experience, the majority were satisfied with their child's pediatric dental benefits. There was a moderately strong association between having pediatric dental benefits and making a dental appointment (p=0.00, Cramer's V of 35%). Discussion: Results show that a majority of respondents had pediatric dental benefits. For those people who did not, cost still remains a main barrier. Since the majority didn't know that dental care for children is one of ten essential services, it appears that the public needs to be better informed about this major health insurance change. Regardless of whether one had pediatric dental benefits, pediatric dental services were used by the majority. This survey administered through SurveyMonkey ® identified some inherent limitations. The low sample size makes it difficult to generalize the results to the national population. This study, however, can assist in developing larger studies that will investigate the ACA's impact on families as they select a pediatric dental benefit plan.