Nanoparticle-encapsulated antioxidant improves placental mitochondrial function in a sexually dimorphic manner in a rat model of prenatal hypoxia.

Pregnancy complications associated with prenatal hypoxia lead to increased placental oxidative stress. Previous studies suggest that prenatal hypoxia can reduce mitochondrial respiratory capacity and mitochondrial fusion, which could lead to placental dysfunction and impaired fetal development. We developed a placenta-targeted treatment strategy using a mitochondrial antioxidant, MitoQ, encapsulated into nanoparticles (nMitoQ) to reduce placental oxidative stress and (indirectly) improve fetal outcomes. We hypothesized that, in a rat model of prenatal hypoxia, nMitoQ improves placental mitochondrial function and promotes mitochondrial fusion in both male and female placentae. Pregnant rats were treated with saline or nMitoQ on gestational day (GD) 15 and exposed to normoxia (21% O2 ) or hypoxia (11% O2 ) from GD15-21. On GD21, male and female placental labyrinth zones were collected for mitochondrial respirometry assessments, mitochondrial content, and markers of mitochondrial biogenesis, fusion and fission. Prenatal hypoxia reduced complex IV activity and fusion in male placentae, while nMitoQ improved complex IV activity in hypoxic male placentae. In female placentae, prenatal hypoxia decreased respiration through the S-pathway (complex II) and increased N-pathway (complex I) respiration, while nMitoQ increased fusion in hypoxic female placentae. No changes in mitochondrial content, biogenesis or fission were found. In conclusion, nMitoQ improved placental mitochondrial function in male and female placentae from fetuses exposed to prenatal hypoxia, which may contribute to improved placental function. However, the mechanisms (ie, changes in mitochondrial respiratory capacity and mitochondrial fusion) were distinct between the sexes. Treatment strategies targeted against placental oxidative stress could improve placental mitochondrial function in complicated pregnancies.

Placenta-targeted treatment in hypoxic dams improves maturation and growth of fetal cardiomyocytes in vitro via the release of placental factors.

NEW FINDINGS: What is the central question of this study? Does treatment of hypoxic dams with a placenta-targeted antioxidant prevent the release of placenta-derived factors that impair maturation or growth of fetal cardiomyocytes in vitro? What is the main finding and its importance? Factors released from hypoxic placentae impaired fetal cardiomyocyte maturation (induced terminal differentiation) and growth (increased cell size) in vitro, which was prevented by maternal treatment with a placenta-targeted antioxidant (nMitoQ). Moreover, there were no sex differences in the effects of placental factors on fetal cardiomyocyte maturation and growth. Overall, our data suggest that treatment targeted against placental oxidative stress could prevent fetal programming of cardiac diseases via the release of placental factors. ABSTRACT: Pregnancy complications associated with placental oxidative stress may impair fetal organ development through the release of placenta-derived factors into the fetal circulation. We assessed the effect of factors secreted from placentae previously exposed to prenatal hypoxia on fetal cardiomyocyte development and developed a treatment strategy that targets placental oxidative stress by encapsulating the antioxidant MitoQ into nanoparticles (nMitoQ). We used a rat model of prenatal hypoxia (gestational day (GD) 15-21), which was treated with saline or nMitoQ on GD15. On GD21, placentae were harvested, placed in culture, and conditioned medium (containing placenta-derived factors) was collected after 24 h. This conditioned medium was then added to cultured cardiomyocytes from control dam fetuses. Conditioned medium from prenatally hypoxic placentae increased the percentage of binucleated cardiomyocytes (marker of terminal differentiation) and the size of mononucleated and binucleated cardiomyocytes (sign of hypertrophy), effects that were prevented by nMitoQ treatment. Our data suggest that factors derived from placentae previously exposed to prenatal hypoxia lead to abnormal fetal cardiomyocyte development, and show that treatment against placental oxidative stress may prevent fetal programming of cardiac disease.

Sex-Specific Effects of Nanoparticle-Encapsulated MitoQ (nMitoQ) Delivery to the Placenta in a Rat Model of Fetal Hypoxia.

Pregnancy complications associated with chronic fetal hypoxia have been linked to the development of adult cardiovascular disease in the offspring. Prenatal hypoxia has been shown to increase placental oxidative stress and impair placental function in a sex-specific manner, thereby affecting fetal development. As oxidative stress is central to placental dysfunction, we developed a placenta-targeted treatment strategy using the antioxidant MitoQ encapsulated into nanoparticles (nMitoQ) to reduce placental oxidative/nitrosative stress and improve placental function without direct drug exposure to the fetus in order to avoid off-target effects during development. We hypothesized that, in a rat model of prenatal hypoxia, nMitoQ prevents hypoxia-induced placental oxidative/nitrosative stress, promotes angiogenesis, improves placental morphology, and ultimately improves fetal oxygenation. Additionally, we assessed whether there were sex differences in the effectiveness of nMitoQ treatment. Pregnant rats were intravenously injected with saline or nMitoQ (100 µl of 125 µM) on gestational day (GD) 15 and exposed to either normoxia (21% O2) or hypoxia (11% O2) from GD15 to 21. On GD21, placentae from both sexes were collected for detection of superoxide, nitrotyrosine, nitric oxide, CD31 (endothelial cell marker), and fetal blood spaces, Vegfa and Igf2 mRNA expression in the placental labyrinth zone. Prenatal hypoxia decreased male fetal weight, which was not changed by nMitoQ treatment; however, placental efficiency (fetal/placental weight ratio) decreased by hypoxia and was increased by nMitoQ in both males and females. nMitoQ treatment reduced the prenatal hypoxia-induced increase in placental superoxide levels in both male and female placentae but improved oxygenation in only female placentae. Nitrotyrosine levels were increased in hypoxic female placentae and were reduced by nMitoQ. Prenatal hypoxia reduced placental Vegfa and Igf2 expression in both sexes, while nMitoQ increased Vegfa and Igf2 expression only in hypoxic female placentae. In summary, our study suggests that nMitoQ treatment could be pursued as a potential preventative strategy against placental oxidative stress and programming of adult cardiovascular disease in offspring exposed to hypoxia in utero. However, sex differences need to be taken into account when developing therapeutic strategies to improve fetal development in complicated pregnancies, as nMitoQ treatment was more effective in placentae from females than males.

Conducting electrospun fibres with polyanionic grafts as highly selective, label-free, electrochemical biosensor with a low detection limit for non-Hodgkin lymphoma gene.

A highly selective, label-free sensor for the non-Hodgkin lymphoma gene, with an aM detection limit, utilizing electrochemical impedance spectroscopy (EIS) is presented. The sensor consists of a conducting electrospun fibre mat, surface-grafted with poly(acrylic acid) (PAA) brushes and a conducting polymer sensing element with covalently attached oligonucleotide probes. The sensor was fabricated from electrospun NBR rubber, embedded with poly(3,4-ethylenedioxythiophene) (PEDOT), followed by grafting poly(acrylic acid) brushes and then electrochemically polymerizing a conducting polymer monomer with ssDNA probe sequence pre-attached. The resulting non-Hodgkin lymphoma gene sensor showed a detection limit of 1aM (1 × 10-18mol/L), more than 400 folds lower compared to a thin-film analogue. The sensor presented extraordinary selectivity, with only 1%, 2.7% and 4.6% of the signal recorded for the fully non-complimentary, T-A and G-C base mismatch oligonucleotide sequences, respectively. We suggest that such greatly enhanced selectivity is due to the presence of negatively charged carboxylic acid moieties from PAA grafts that electrostatically repel the non-complementary and mismatch DNA sequences, overcoming the non-specific binding.

Histological and Ultrastructural Studies on the Conjunctiva of the Barred Owl (Strix varia).

This report is the first characterization of the histology and ultrastructure of the barred owl conjunctiva. The inferior eyelid was dominated by a large disk-shaped plate covered by a non-keratinized stratified squamous or cuboidal epithelium of variable thickness. The apical surface of the plate epithelium varied from flat to long microvilli or even short cytoplasmic extensions similar to those seen in the third eyelid. All specimens had a few goblet cells filled with mucous secretory granules in the plate region. The underlying connective tissue was a dense fibroelastic stroma. Eosinophils were surprisingly common in the epithelial layer and underlying connective tissue in the plate and more distal orbital mucosal region. The orbital mucosa contained goblet cells with heterogeneous glycosylation patterns. The leading edge and marginal plait of the third eyelid are designed to collect fluid and particulate matter as they sweep across the surface of the eye. The palpebral conjunctival surface of the third eyelid was covered by an approximately five-cell-deep stratified squamous epithelium without goblet cells. The bulbar surface of the third eyelid was a bilayer of epithelial cells whose superficial cells have elaborate cytoplasmic tapering extensions reaching out 25 µm. Narrow cytofilia radiated outwards up to an additional 15-20 µm from the cytoplasmic extensions. Lectin labeling demonstrated heterogeneous glycosylation of the apical membrane specializations but only small amounts of glycoprotein-filled secretory granules in the third eyelid.

Electrospun rubber fibre mats with electrochemically controllable pore sizes.

Electroactive, elastomeric, microfiber mats that show controllable pore size variation upon electrochemical stimulation are produced from semi-interpenetrating polymer networks (s-IPNs). This type of porous, elastomeric scaffolds that are mechanically dynamic under electrochemical stimuli could find new applications in stretchable electronics, (bio)filtration, soft robotics and stimulation of biological cells. These microfiber mats are prepared in two simple steps. Firstly, a mixture of high molecular weight nitrile butadiene rubber (NBR) and cross-linking agent, poly(ethylene glycol)dimethylacrylate are electrospun with in situ cross-linking. Secondly, a conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) is embedded into the electrospun fibres by oxidative chemical polymerization of EDOT-swollen microfiber mats. This two-step process affords robust, highly flexible and conductive s-IPN microfiber mats. The microfiber mat undergoes a controllable pore size variation upon applying an electrochemical stimulus in the form of a reduction-oxidation cycle to the mats in an electrolyte. The maximum average pore size variation, measured in situ using confocal microscopy, is 25%, achieved in 1 M lithium bis-trifluoromethanesulfonimide (LiTFSI) in propylene carbonate (PC) for a potential step between +0.6 V and -0.5 V (vs. Ag wire). These mats also show pore size variation in a biologically compatible solution, phosphate buffered saline.

The pharmacologic assessment of a novel lymphocyte function-associated antigen-1 antagonist (SAR 1118) for the treatment of keratoconjunctivitis sicca in dogs.

PURPOSE: Keratoconjunctivitis sicca (KCS) is characterized by inflammation and decreased production of tears containing increased levels of cytokines. The release occurs in the setting of conjunctival and lacrimal gland inflammation, potentially mediated by the interaction between lymphocyte function-associated antigen (LFA)-1, a cell surface protein found on lymphocytes, and its cognate ligand intercellular adhesion molecule (ICAM)-1. SAR 1118 is a novel LFA-1 antagonist and may be an effective therapeutic agent for the treatment of KCS. The following studies were performed to assess the in vitro activity of SAR 1118 and to evaluate the clinical efficacy of topical SAR 1118 for the treatment of idiopathic canine KCS. METHOD: Pharmacodynamics were assessed by measuring the ability of SAR 1118 to inhibit Jurkat T-cell binding with recombinant human ICAM-1 and to inhibit cytokine release from human peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin B. For the assessment of clinical efficacy, 10 dogs diagnosed with idiopathic KCS were treated with SAR 1118 1% topical ophthalmic solution three times daily for 12 weeks. Schirmer's tear test (STT) was used to measure tear production. RESULTS: SAR 1118 demonstrated concentration-dependent inhibition of Jurkat T-cell attachment, inhibition of lymphocyte activation, and release of inflammatory cytokines, particularly the Th1, Th2, and Th17 T-cell cytokines IFN-γ, IL-2, and IL-17F, respectively. Mean STT values increased from 3.4 mm during week 1 to 5.8 mm at week 12 (P < 0.025). No SAR 1118-related adverse events were observed. CONCLUSIONS: SAR 1118 appears to be an effective anti-inflammatory treatment for KCS. Additional studies are warranted to establish the efficacy of SAR 1118 for the treatment of KCS in humans.

M cell-targeted ocular immunization: effect on immunoglobulins in tears, feces, and serum.

PURPOSE: This study investigates whether antigen-sampling M cells, present in the follicle-associated epithelium (FAE) above organized conjunctiva-associated lymphoid tissue in rabbits, bind and retro-transport secretory IgA (sIgA) from the tear film. The hypothesis that IgA-mediated uptake of antigens promotes local and systemic production of immunoglobulins was tested. METHODS: sIgA binding and retro-translocation by M cells was characterized by immunocytochemistry. Immunoglobulin concentrations in tears, feces and serum were measured using enzyme-linked immunoassays (ELISA) after topical and systemic immunization with either goat IgG anti-rabbit IgA or nonspecific goat IgG. RESULTS: Endogenous sIgA was found associated with the apical membrane of conjunctival M cells. Exogenous anti-IgA immunoglobulins were translocated across M cells. Significant levels of sIgA against goat IgG were present in tears of pre-immune animals. Topical application of either goat IgG specific for rabbit IgA or nonspecific goat IgG led to similar increases in antigen-specific IgA in tear, feces, and serum. The antigen-specific IgG response in tears mirrored the serum response for both immunogens consistent with transudation of this immunoglobulin. The IgM response in tears and serum was weak for both immunogens. Systemic immunization did not sustain or enhance the local mucosal IgA responses. CONCLUSIONS: Conjunctival M cells bind and translocate sIgA from the tear film. Topical conjunctival immunization leads to generation of antigen-specific immunoglobulins from both local and distant mucosae and in serum. Natural antibodies, present in the tear film before immunization, may have contributed to similar immune responses to goat anti-rabbit IgA and nonspecific goat IgG.

Developmental changes in conjunctiva-associated lymphoid tissue of the rabbit.

PURPOSE: Organized conjunctiva-associated lymphoid tissue (O-CALT) is constantly exposed to environmental antigens and plays a central role in the common mucosal immune system. This study was undertaken to investigate whether O-CALT in rabbit lymphoid tissue, as in other lymphoid tissue in other mammals, changes with age. METHODS: Fluorescence stereomicroscopy was used to measure the number and size of conjunctival follicles stained with propidium iodide in rabbits ranging in age from 2 days to 57 months. To assess the function of M cells, an antigen-sampling cell type found in the follicle-associated epithelium, transcytosis of fluorescent latex beads was evaluated with confocal microscopy. RESULTS: O-CALT was not present in rabbits at birth, but appeared less than 24 hours after eyes opened at approximately day 11. The number of follicles increased with age until adolescence (2-4 months), when the number stabilized through early adulthood (17-20 months). In aged rabbits (47-57 months), there was a dramatic decline in the number of follicles. This disappearance was most pronounced in the superior conjunctiva. Average follicle diameter increased with age, except in the superior conjunctiva of aged rabbits, where the few remaining follicles were generally smaller. The uptake of latex beads showed that M-cell function was similar in all age groups. CONCLUSIONS: Age-related changes in rabbit O-CALT are similar to those that have been reported for the human conjunctiva. Preferential uptake of latex beads by follicle-associated epithelium indicates that the presence and function of M cells are not affected by aging. The lower level of O-CALT in young and elderly animals, however, would be expected to decrease their ocular mucosal immune responses.

Bacterial transcytosis across conjunctival M cells.

PURPOSE: Antigen-sampling M cells have been identified in conjunctival tissue overlying lymphoid follicles in rabbits and guinea pigs. Conjunctival M cells in the guinea pig display alpha(2-3) sialic acid on their surfaces, as evinced by selective labeling by Maackia amurensis leukoagglutinin (MAL)-I. Haemophilus influenzae strains OM12, which expresses the HMW1 adhesin for alpha(2-3) sialic acid, and Rd KW20, which lacks HMW1, were used to test the hypothesis that conjunctival M cells translocate large microbes. METHODS: Fluorescein-labeled bacteria were instilled into the conjunctival sac for up to 130 minutes. Confocal laser scanning microscopy and electron microscopy were used to visualize bacterial distribution. RESULTS: M cells, but not nonfollicular epithelial cells in the palpebral region, selectively bound and translocated bacteria. By 66 minutes, 423 +/- 165 bacteria/mm(2) of follicle-associated epithelial (FAE) surface were found in three-dimensional reconstructions extending 15.4 mum below the surface. By 127 minutes, the number of bacteria increased to 579 +/- 44/mm(2) of FAE surface and they had moved 50% deeper into the follicle. Coadministration with MAL-I reduced OM12 transport by 61%. Similarly, Rd KW20 uptake was 71% less at 63 minutes and 58% less at 121 minutes, indicating that OM12 uptake is at least partially mediated by binding to alpha(2-3) sialic acid. CONCLUSIONS: Conjunctival M cells are a port of entry for large microbes and may play a role in initiation of mucosal immune responses against commensal or transient ocular bacterial species and may allow the entry of pathogens.

Genetic segregation of airway disease traits despite redundancy of calcium-activated chloride channel family members.

Complex airway diseases such as asthma and chronic obstructive pulmonary disease exhibit stereotyped traits (especially airway hyperreactivity and mucous cell metaplasia) that are variably expressed in each patient. Here, we used a mouse model for virus-induced long-term expression of these traits to determine whether individual traits can be genetically segregated and thereby linked to separate determinants. We showed that an F2 intercross population derived from susceptible and nonsusceptible mouse strains can manifest individual phenotypic extremes that exhibit one or the other disease trait. Functional genomic analysis of these extremes further indicated that a member of the calcium-activated chloride channel (CLCA) gene family designated mClca3 was inducible with mucous cell metaplasia but not airway hyperreactivity. In confirmation of this finding, we found that mClca3 gene transfer to mouse airway epithelium was sufficient to induce mucous cell metaplasia but not airway hyperreactivity. However, newly developed mClca3(-/-) mice exhibited the same degree of mucous cell metaplasia and airway hyperreactivity as wild-type mice. Bioinformatic analysis of the Clca locus led to the identification of mClca5, and gene transfer indicated that mClca5 also selectively drives mucous cell metaplasia. Thus, in addition to the capacity of CLCA family members to exhibit diverse functional activities, there is also preserved function so that more than one family member mediates mucous cell metaplasia. Nonetheless, Clca expression appears to be a selective determinant of mucous cell metaplasia so that shared homologies between CLCA family members may still represent a useful target for focused therapeutic intervention in hypersecretory airway disease.

Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana.

Pollen-pistil interactions are crucial for controlling plant mating. For example, S-RNase-based self-incompatibility prevents inbreeding in diverse angiosperm species. S-RNases are thought to function as specific cytotoxins that inhibit pollen that has an S-haplotype that matches one of those in the pistil. Thus, pollen and pistil factors interact to prevent mating between closely related individuals. Other pistil factors, such as HT-B, 4936-factor and the 120 kDa glycoprotein, are also required for pollen rejection but do not contribute to S-haplotype-specificity per se. Here we show that S-RNase is taken up and sorted to a vacuolar compartment in the pollen tubes. Antibodies to the 120 kDa glycoprotein label the compartment membrane. When the pistil does not express HT-B or 4936-factor, S-RNase remains sequestered, unable to cause rejection. Similarly, in wild-type pistils, compatible pollen tubes degrade HT-B and sequester S-RNase. We suggest that S-RNase trafficking and the stability of HT-B are central to S-specific pollen rejection.

Gastrostomy and jejunostomy placement: the urban hospital perspective pertinent to nursing home care.

OBJECTIVES: In nursing home settings, providers often think that most percutaneous endoscopic gastrostomy (PEG) tubes are placed in older people, some perhaps inappropriately. We sought to describe the relationships between patient age and the indications for, the decision making behind, and the outcomes of gastrostomy and jejunostomy placement in an urban hospital to give perspective to those of us working in long-term care settings. DESIGN: Retrospective, observational study. SETTING: Urban hospital. PARTICIPANTS: Two hundred thirty-nine inpatients who underwent gastrostomy or jejunostomy (G/J) placement. MEASUREMENTS: Hospital records were reviewed for patient demographics, disease process, decision making, and short-term outcomes associated with G/J placement. Mortality at 30 days and 1 year was obtained by a search of the National Death Index. The prevalence of these variables in those aged 65 years and older was compared to the prevalence in those younger than 65 with associations calculated both unadjusted and adjusted for gender, place of residence, underlying condition, and Charlson comorbidity index. RESULTS: Patients who were aged 65 years and older were more likely to be female with more comorbid illnesses and were more likely to have had a stroke that precipitated their difficulty eating. They were more likely to have been referred by a medical specialist, to have been seen by a speech pathologist, and to have had their procedure without general anesthesia. The older patients had a shorter mean hospital length of stay with fewer complications but had higher mortality rates at 30 days and 1 year. CONCLUSION: Patient age was associated with gender and type of disease process and may have influenced the decisions made during the hospital stay. Despite a higher burden of chronic illness, older patient age was not associated with adverse short-term outcomes but was associated with higher mortality rates after discharge.

M cells in the follicle-associated epithelium of the rabbit conjunctiva preferentially bind and translocate latex beads.

PURPOSE: This study investigates the presence of M cells in the rabbit conjunctiva. Resolving whether the conjunctiva contains M cells is important, because at other mucosal sites, these antigen sampling cells are known to initiate the mucosal immune response and to act as a site of entry for opportunistic pathogens. METHODS: Fluorescent 0.2-microm polystyrene latex beads were either instilled into the conjunctival sac for 20 to 120 minutes in vivo or applied to flat mounts in vitro. Specimens were assessed by epi-fluorescence stereomicroscopy, widefield fluorescence microscopy, confocal scanning laser microscopy, and transmission and scanning electron microscopy. RESULTS: Latex beads preferentially bound to a subpopulation of cells in the epithelium overlying mucosal lymphoid follicles in the fornix region. At 4 degrees C, the beads were associated with the apical surface of cells that had longer, more irregular microvilli than the surrounding epithelial cells. Within 20 minutes of an in vivo exposure, latex beads were internalized by the follicle-associated epithelial cells and with time moved into the underlying follicle region. After 120 minutes of in vivo exposure, latex beads could be found in cervical lymph nodes. CONCLUSIONS: This study demonstrates that the follicle-associated epithelium of the rabbit conjunctiva contains a cell with morphologic characteristics and the ability to bind and translocate latex beads, which make it indistinguishable from antigen sampling M cells in the rabbit cecum and tonsils. Consistent with its hypothesized antigen sampling role, beads that have been translocated by this cell are rapidly transferred to cervical lymph nodes.

Conjunctival M cells selectively bind and translocate Maackia amurensis leukoagglutinin.

Antigen-sampling M cells are found in the follicle-associated epithelium above organized lymphoid tissue in many mucosae. They play a key role in initiating the mucosal immune response and act as a site of entry for opportunistic pathogens. This study investigates the presence of M cells in the Guinea pig conjunctiva. Maackia amurensis leukoagglutinin I and II (MAL-I and MAL-II) were identified as potential conjunctival M cell markers based on a screening of 12 lectins and 5 carbohydrate epitope antibodies on aldehyde-fixed follicles. Biotinylated or fluorescein-conjugated MAL-I was then instilled into conjunctival sacs in vivo for 15-60 min. Specimens were assessed by epi-fluorescence stereomicroscopy, confocal scanning laser microscopy and transmission and scanning electron microscopy (TEM and SEM). Selective labelling of a subset of epithelial cells overlying lymphoid follicles was observed following in vivo exposure to MAL-I. MAL-I labelling was restricted to cells with sparse, irregular microvilli. Cells preferentially labelled with MAL-I were found to internalize the lectin during a 60 min in vivo exposure. MAL-I was transcytosed to basolateral membranes of cells filled with intracellular vesicles during a 45 min in vivo incubation. This study demonstrates that the Guinea pig conjunctiva contains a cell with morphological and functional characteristics of antigen-sampling M cells.

A simple, low-cost device for processing and embedding tissues at sub-zero temperatures.

A simple apparatus to maintain tissues samples at sub-zero temperatures during dehydration, infiltration, and polymerization is described. The device uses a conventional siphon-type carbon dioxide gas cylinder to maintain an aluminum block at temperatures as low as -35 degrees C for over 15 hours/cylinder.

Targeted delivery of antiprotease to the epithelial surface of human tracheal xenografts.

The cystic fibrosis (CF) lung is uniquely susceptible to Pseudomonas aeruginosa, and infection with this organism incites an intense, compartmentalized inflammatory response that leads to chronic airway obstruction and bronchiectasis. Neutrophils migrate into the airway, and released neutrophil elastase contributes to the progression of the lung disease characteristic of CF. We have developed a strategy that permits the delivery of antiproteases to the inaccessible CF airways by targeting the respiratory epithelium via the human polymeric immunoglobulin receptor (hpIgR). A fusion protein consisting of a single-chain Fv directed against secretory component, the extracellular portion of the pIgR, linked to human alpha1-antitrypsin is effectively ferried across human tracheal xenografts and delivers the antiprotease to the apical surface to a much greater extent than occurs by passive diffusion of human alpha1-antitrypsin alone. Targeted antiprotease delivery paralleled hpIgR expression in the respiratory epithelium in vivo and was not increased by escalating dose, so airway penetration was receptor-dependent, not dose-dependent. Thus, this approach provides us with the ability to deliver therapeutics, like antiproteases, specifically to the lumenal surface of the respiratory epithelium, within the airway surface fluid, where it will be in highest concentration at this site.

Apolipoprotein B is synthesized in selected human non-hepatic cell lines but not processed into mature lipoprotein.

We studied apolipoprotein B100 (apoB) metabolism in a series of non-hepatic cell lines (HT29 colon adenocarcinoma, HeLa cervical epithelioid carcinoma, and 1321N1J astrocytoma human cell lines) and in the human hepatoma cell line HepG2. ApoB mRNA was detected by reverse transcription polymerase chain reaction in each non-hepatic cell line. ApoB was detected in HepG2 cells by immunoprecipitation, Western blotting, and immunocytochemistry using a polyclonal anti-human low-density lipoprotein (LDL) antibody, an anti-human apoB peptide antibody, and several monoclonal anti-apoB antibodies. ApoB was identified in the three non-hepatic cell lines by each method using the anti-apoB peptide and monoclonal antibodies, but not with the anti-LDL antibody. Immunocytochemistry indicated that epitopes of apoB were evident throughout the endoplasmic reticulum, and gel mobility of newly labeled apoB and immunoblot with anti-ubiquitin showed that apoB was highly ubiquinated in non-hepatic cells. The observations that apoB is synthesized in non-hepatic cell lines but never recognized by the anti-LDL antibody suggests that apoB is not processed into a nascent lipoprotein in these cells. Immunocytochemical localization of apoB epitopes at many locations throughout non-hepatic cells raises the exciting possibility that apoB can be used for other purposes in these cells.

Morphological evidence of M cells in healthy canine conjunctiva-associated lymphoid tissue.

PURPOSE: To characterize the follicle-associated epithelium (FAE) and organized lymphoid nodules from dog nictitating membranes to determine if canine conjunctiva-associated lymphoid tissue (CALT) contains M cells analogous to those described in other regions of mucosa-associated lymphoid tissue (MALT). METHODS: Nictitan lymphoid follicles from 15 healthy dogs (30 eyes) were harvested immediately post-mortem. Twelve follicles from each nictitating membrane were isolated. Four follicles from each eye of 10 dogs were examined by light microscopy, transmission electron microscopy and scanning electron microscopy. Five of the 10 dogs were treated with a heat-killed staphylococcal topical suspension immediately prior to euthanasia. Nictitan follicles from five other dogs were processed for immunohistochemistry to characterize follicle lymphocyte populations. RESULTS: The FAE overlying CALT demonstrated morphology characteristic of M cells, including attenuated apical cell surface with blunted microvilli and microfolds, invaginated basolateral membrane forming a cytoplasmic pocket containing lymphocytes and macrophages, and diminished distance between the apical and pocket membrane. Heat-killed bacteria were bound to the surface and transcytosed to the cytoplasmic pocket of CALT M cells. Immunohistochemistry of organized lymphoid tissue subtending the FAE demonstrated B-cell germinal centers with T-cell predominant apical caps. CONCLUSIONS: In canine CALT, the FAE overlying lymphoid follicles, as well as the distribution of T and B lymphocytes subtending this region, contain morphologic and functional features analogous to MALT described in other regions. Documentation of canine conjunctival M cells is of clinical relevance in the study of primary ocular diseases, as well as a potential means of vaccination or drug delivery.

Cooperation in the care of the institutionalized elderly in a rural community.

OBJECTIVE: To develop a method of addressing and minimizing the institutional, cultural, and regulatory barriers to the care of nursing facility residents in our community. METHODS: Nurses, administrators, and medical directors from all the nursing facilities in our community plus representatives from the community hospital participated in a monthly meeting where difficult issues in the care of nursing facility residents were discussed. The committee developed responses to these issues that were implemented throughout the community. RESULTS: This committee has provided an opportunity for the whole community to address problems in the care of the institutionalized elderly. Systems have been developed which have improved communication between nurses and physicians and between nursing facilities and the hospital. Community standards for the care of common problems in nursing facility residents have also been developed. Other unexpected benefits have included community discussion of regulatory concerns, nurse assistant education, and care at the end of life, as well as coordination of laboratory services in the nursing facilities. Other rural communities may find a similar approach useful.