Cell-specific Antibodies Research Articles

SAT0212 Circulating Endothelial Microparticles Reflect Microvascular Impairment in Patients with Systemic Sclerosis

BackgroundEndothelial injury plays a key role in the pathogenesis of systemic sclerosis (SSc), but its quantitative assessment has yet to be established clinically. Circulating endothelial microparticles (EMPs) are considered markers...

Neonatal SVZ EGFP-labeled cells produce neurons in the olfactory bulb and astrocytes in the cerebral cortex by in-vivo electroporation

Neural progenitors/stem cells (NSCs) exist in neonatal mouse subventricular zone (SVZ). To explore the differentiation of the NSCs in neonatal mouse SVZ and the distribution of the progeny cells derived from these NSCs in early adulthood, the enhanced green fluorescent protein (EGFP) plasmid was transferred into the NSCs in the lateral ventricle of newborn mice (P0) by in-vivo electroporation to trace these cells and their progeny cells. Thirty days after electroporation, histological sections of mouse brain were prepared for immunofluorescence with cell-specific antibodies to identify the type(s) of cells that were marked by EGFP. The results showed that EGFP-positive cells were distributed mainly in the olfactory bulb (OB), cortex, and SVZ, and double labeled with NeuN (neuron marker) in OB, glial fibrillary acidic protein (GFAP) (astrocyte marker) in the cortex, and Blbp and GFAP (astrocyte marker) in SVZ. However, there was no-EGFP-positive cell in the hippocampus. The present results indicate that the NSCs in SVZ of the neonatal mouse can give rise to neurons in the OB and astrocytes in the cortex in early adulthood, but not generate progeny cells residing in the hippocampus. In addition, there are still neural progenitors in SVZ until early adulthood.

Mo1909 Patient Predictors of Adherence to Therapy for Barrett's Esophagus-Associated Neoplasia

Introduction: Barrett's esophagus (BE) is a precancerous condition caused by gastroesophageal reflux disease. The principle of molecular endoscopy is to visualize disease specific fluorescence labeled probes using confocal laser endomicroscopy (CLE). Aims: Main study objective was to assess the potential of molecular imaging for diagnosis of BE using a novel fluorescence labeled probe highly specific for BE. Material & Methods: Patients with long segment BE were included. Random biopsies were taken for histologic and subsequent molecular imaging. The goblet cell specific Muc2 antibody was labeled with Fluorescein isothiocyanate (FITC) and then topically applied to the esophageal biopsies. Subsequently, CLE was performed and confocal images were digitally stored. Confocal images were than scored by two investigators according to BE specific fluorescence signals. Histopathology served as the reference standard. Results: Overall, 300 confocal images were recorded. Molecular imaging achieved an overall sensitivity, specificity and accuracy of 98.9%, 88.2%, and 97.5%, for the detection of BEspecific signals. Positive and negative predictive values were 98.3% and 91.8%. Interobserver and intraobserver agreement for prediction of BE were excellent with kappa values of 0.8597 and 0.9521, respectively. Conclusion: Molecular imaging with the novel highly-specific fluorescent Muc2 antibody enabled excellent results for prediction of BE. The technique should now be transferred to an in vivo setting in order to improve diagnosis and surveillance of BE-patients.

Mo1908 Molecular Imaging With Fluorescent MUC2 Antibodies for Highly Specific Diagnosis of Barrett's Esophagus

Introduction: Barrett's esophagus (BE) is a precancerous condition caused by gastroesophageal reflux disease. The principle of molecular endoscopy is to visualize disease specific fluorescence labeled probes using confocal laser endomicroscopy (CLE). Aims: Main study objective was to assess the potential of molecular imaging for diagnosis of BE using a novel fluorescence labeled probe highly specific for BE. Material & Methods: Patients with long segment BE were included. Random biopsies were taken for histologic and subsequent molecular imaging. The goblet cell specific Muc2 antibody was labeled with Fluorescein isothiocyanate (FITC) and then topically applied to the esophageal biopsies. Subsequently, CLE was performed and confocal images were digitally stored. Confocal images were than scored by two investigators according to BE specific fluorescence signals. Histopathology served as the reference standard. Results: Overall, 300 confocal images were recorded. Molecular imaging achieved an overall sensitivity, specificity and accuracy of 98.9%, 88.2%, and 97.5%, for the detection of BEspecific signals. Positive and negative predictive values were 98.3% and 91.8%. Interobserver and intraobserver agreement for prediction of BE were excellent with kappa values of 0.8597 and 0.9521, respectively. Conclusion: Molecular imaging with the novel highly-specific fluorescent Muc2 antibody enabled excellent results for prediction of BE. The technique should now be transferred to an in vivo setting in order to improve diagnosis and surveillance of BE-patients.

Whitening Effects of Adipose-Derived Stem Cells: A Preliminary In Vivo Study

Many studies have reported various growth factors secreted from adipose-derived stem cells (ADSCs). In particular, regenerative effects in skin have received much attention in the clinical fields. The in vitro whitening effects of ADSCs have been reported. A previous study demonstrated that ADSCs secrete growth factors that inhibit both melanin synthesis and tyrosinase activity. This study aimed to investigate the in vivo whitening effect of ADSCs using mouse models. In the study, ADSCs were isolated from the adipose tissue of C57BL/6 mice and cultured. The ADSCs (1×10(6) cells in 30μl of Hanks' balanced salt solution [HBSS]) then were injected intradermally in the dorsal area of the right ear, and 30μl of HBSS was injected on the left ear as a control. After 7days, both ears were irradiated with ultraviolet B (UVB) (150mJ/cm(2)) three times at 2-day intervals. The sections of each ear were stained with hematoxylin-eosin, Fontana-Masson, and HMB-45 (a melanocytic cell-specific monoclonal antibody). The histologic parameters evaluated included inflammation (+/-), erosion (+/-), and melanin formation (graded on a scale of 1 to 3). No significant differences in inflammation or erosion were observed by hematoxylin and eosin staining (inflammation: p=0.388; erosion: p=0.355). However, significantly more melanin formation was observed in the control group than in the ADSC injection group by Fontana-Masson and HMB-45 staining (Fontana-Masson: p=0.025; HMB-45: p=0.015). The study findings suggest that ADSCs inhibit melanin formation induced by UV exposure. Potentially, ADSCs may be used as anti-aging agents, including skin whitening. Before human clinical studies can be conducted, further study is required to determine the mechanisms underlying the whitening effects of ADSCs and the safety of ADSC use. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

In vivo depletion of leukocytes and platelets following injection of T cell-specific antibodies into mice

In vivo T cell depletion experiments are widely used to establish the role of these cells in a variety of immunological processes. Different clones of monoclonal antibody targeting the CD3 molecular complex (mainly 145-2C11 and 17A2) have been successfully used for T cell depletion. In the present work, we assessed the specificity of monoclonal antibody-mediated CD3 T cell depletion in mouse peripheral blood. We showed that treatment of BALB/C mice with monoclonal antibodies (clones 145-2C11 and 17A2) not only efficiently depletes T cells in vivo, but also leads to a substantial reduction in B cell, granulocyte and platelet counts. In contrast, T cell depletion using a combination of anti-CD4 and anti-CD8 antibodies was efficient and produced less deleterious effects on other blood cell populations. Therefore, the results obtained from T cell depletion experiments using anti-CD3 antibodies must be interpreted with caution prior to draw definitive conclusions on the role of T cells in a given immunological process.

Rat Embryonic Mast Cells Originate in the AGM

Mast cells originate from pluripotent hematopoietic stem cells. Two mast cell specific antibodies, mAbsAA4 and BGD6, have previously been used to identify and study committed mast cell precursors (MCcps) in the bone marrow of adult mice and rats. However, the embryonic origin of MCcps is still not known. In the present study, we identified MCcps in rat embryos using these previously characterized mast cell specific antibodies. The MCcps were found in the AGM (aorta-gonad-mesonephros) region of rat embryos at E11.5. These cells were BGD6+, CD34+, c-kit+, CD13+, FcεRI−, AA4− CD40−, and Thy-1−. By PCR the cells contained message for the α and β subunits of FcεRI and mast cell specific proteases. In vitro, the MCcps differentiated into metachromatic mast cells. With age of gestation the percent of MCcps diminished while the percent of mast cell progenitors increased. An increased knowledge of the biology and embryonic origin of mast cells may contribute to a greater understanding of allergy, asthma, and other mast cell related diseases.

Cerebellar Purkinje Cells Exhibit Increased Expression of HMGB-1 and Apoptosis in Congenital Hydrocephalic H-Tx Rats

Highly integrated anatomic and functional interactions between the cerebrum and the cerebellum during development have been reported. In our previous study, we conducted a proteome analysis to identify the proteins present in the congenital noncommunicating hydrocephalus in the cerebellum. We found higher expression of high-mobility group box-1 protein (HMGB-1) in hydrocephalic H-Tx rats. We studied the expression pattern of HMGB-1 in the cerebellum. We studied congenital hydrocephalic H-Tx rats aged 1 day and 7 days along with age-matched nonhydrocephalic H-Tx and Sprague-Dawley rats as controls. Gene and protein expressions of HMGB-1 in the cerebellum were assayed by real-time polymerase chain reaction and Western blotting, respectively; furthermore, immunohistochemical analyses were performed by using HMGB-1 (indicator of apoptosis), single-stranded DNA; adhesion factor related to cell migration, HNK-1; and the Purkinje cell-specific antibody, calbindin. Cytoplasmic HMGB-1 expression observed in Purkinje cells in the 1-day-old hydrocephalic group was stronger than that in the nonhydrocephalic and Sprague-Dawley groups. Double fluorescent staining with single-stranded DNA confirmed that Purkinje cells were undergoing apoptosis. HNK-1 expression was lower in the Purkinje cell layer in the 7-day-old rats in the hydrocephalic group, and Purkinje cells were disrupted in comparison with the control groups. Morphological changes in the cerebellum were observed in the 7-day-old rats in the hydrocephalic group in comparison with the control groups. Our results suggest that cerebellar neuronal cell damage in the early postnatal period may be related to the higher expression of HMGB-1 in the Purkinje cells.

Evaluation of a Novel Non-Destructive Catch and Release Technology for Harvesting Autologous Adult Stem Cells

BackgroundCell based therapies are required now to meet the critical care needs of paediatrics and healthy ageing in an increasingly long-lived human population. Repair of compromised tissue by supporting autologous regeneration is a life changing objective uniting the fields of medical science and engineering. Adipose stem cells (adSCs) are a compelling candidate for use in cell based medicine due to their plasticity and residence in numerous tissues. Adipose found in all animals contains a relatively high concentration of stem cells and is easily isolated by a minimally invasive clinical intervention; such as liposuction.MethodsThis study utilised primary rat adipose to validate a novel strategy for selecting adult stem cells. Experiments explored the use of large, very dense cell-specific antibody loaded isolation beads (diameter 5x–10x greater than target cells) which overcome the problem of endocytosis and have proved to be very effective in cell isolation from minimally processed primary tissue. The technique also benefited from pH mediated release, which enabled elution of captured cells using a simple pH shift.ResultsLarge beads successfully captured and released adSCs from rat adipose, which were characterised using a combination of microscopy, flow cytometry and PCR. The resultant purified cell population retains minimal capture artefact facilitating autologous reperfusion or application in in vitro models.ConclusionAlthough evidenced here for adSCs, this approach provides a technological advance at a platform level; whereby it can be applied to isolate any cell population for which there is a characterised surface antigen.

Selective blood-DNA extraction from mixed stain using ABO antibody for short tandem repeat typing

Short tandem repeat (STR) typing of DNA extracted from various kinds of mixed samples can be difficult because the origin of the blood donor cannot be easily discriminated. We present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody captured cell separation. This method has proven highly successful in the recovery of DNA profiles corresponding to ABO blood type. The methodological steps presented here are simple: leukocyte agglutination by ABO antibody followed by centrifugal separation of agglutinated leukocytes. Using this method, selective DNA can be extracted from mixtures of blood and/or different cells, using cell specific antibodies. The method presented here is applicable to DNA-based identification of various kinds of contaminated samples.

Immunologic Effect of Attenuated Salmonella enteric Serovar Choleraesuis C501 Expressing Recombinant Mycoplasma hyopneumoniae P97R1 Adhesin and NrdF Antigens in Mice

The immunogenicity of the P97 adhesin repeat region R1 (P97R1) and the ribonucleotide reductase R2 subunit (NrdF) antigens of Mycoplasma hyopneumoniae were evaluated as vaccines in mice. Mice were immunized orally or injected intramuscularly (IM) with attenuated Salmonella enterica serovar Choleraesuis strain C501 harboring a prokaryotic expression vector encoding P97R1 and NrdF (pYA97R1N). Local and systemic immune responses were analyzed in vaccinated mice. The results showed that P97R1N-specific serum IgG and IgA antibodies were detected in serum and lung samples. P97R1-specific serum IgG and IgA antibodies were also detected in serum, but in lung sample only P97R1-specific IgG response was induced. ELISA assays demonstrated that IFN-U and IL-4 production was induced in cultural supernatants from splenocytes stimulated with specific antigens in vitro. M. hyopneumoniae whole cell-specific antibody was induced in vaccinated mice and a good immunogenic effect by this vaccine was indicated. Importantly, animals vaccinated by IM had higher levels of P97R1N-specific IgG and IgA than those vaccinated by oral route. Furthermore, mice immunized with the C501 vaccine elicited significantly higher levels of P97R1N-specific IgA and IgG in lungs and serum (P<0.05), and IFN-U and IL-4, than those who received the live M.hyopneumoniae vaccine. Taken together, these results suggest that IM vaccination with the C501 vaccine might provide an effective method of inducing an immune response to M. hyopneumoniae.

Lipid-lowering therapy with statins reduces microparticle shedding from endothelium, platelets and inflammatory cells

Hyperlipidaemia is a causal factor in the ethiopathogenesis of atherosclerosis. Statins are the cornerstone drug therapy for LDL-cholesterol (LDL-c) lowering, that exert beneficial effects beyond lipid lowering. Circulating microparticles (cMPs), microvesicles released by activated cells into the bloodstream, are markers of vascular and inflammatory cell activation with tentative role in disease progression. However, the role of statins on cMPs seems controversial. We aimed at the evaluation of the effects of lipid-lowering treatment (LLT) on cMP generation in patients in primary prevention of atherosclerosis. A case-control study was conducted in hypercholesterolaemic patients receiving LLT with statins and normocholesterolaemic controls (LLT+ and LLT-, respectively, n=37/group), matched by age, gender and LDL-c levels. cMPs were characterised by flow cytometry using annexin-V and cell-specific antibodies. In LLT+-patients overall numbers of cMPs (p<0.005) were lower than in controls. Levels of cMPs carrying parental cell markers from vascular and circulating cell origin (platelet, endothelial cell, pan-leukocyte and specific-leukocyte subsets) were significantly lower in blood of LLT+ compared to LLT--patients. Moreover, MPs from LLT+-patients had reduced markers of activated platelets (αIIbβ3-integrin), activated inflammatory cells (αM-integrin) and tissue factor. The effect of LLT on cMP shedding was found to be accumulative in years. cMP shedding associated to cardiovascular risk in LLT+-patients. In summary, at similar plasma cholesterol levels patients on statin treatment had a significant lower number of cMPs carrying markers of activated cells. These findings indicate that statins protect against vascular cell activation.

Cancer cell spheroids as a model to evaluate chemotherapy protocols

To determine whether the spheroid culture can be used to evaluate drug efficacy, we have evaluated the toxicity of free or carrier-associated doxorubicin as a single drug or in combination with other antineoplastic agents using the spheroid cultures of drug-resistant cancer cells. Paclitaxel, cisplatin, dexamethasone, mitoxantrone, sclareol or methotrexate were used in combination with doxorubicin. The effect of the treatment protocols on free, micellar and liposomal doxorubicin accumulation in spheroids and on resulting toxicity was evaluated by fluorescence and lactate dehydrogenase release, respectively. Enhanced doxorubicin accumulation and toxicity were observed after spheroid pretreatment with mitoxantrone or paclitaxel. Effects of the drug combination with doxorubicin were sequence dependent, use of doxorubicin as the first drug being the least inducer of toxicity. Finally, spheroids were recognized by a cancer cell-specific antibody. Our results suggest the usefulness of spheroids to evaluate chemotherapy combinations.

Contribution of Cells Derived from the Area Pellucida to Extraembryonic Mesodermal Cell Lineages in Heterospecific Quail Chick Blastodermal Chimeras

The current study has two main objectives: first, to determine if cells derived from the area pellucida are able to populate extraembryonic membranes, and second, to determine if donor cells have the potential to differentiate to endothelial (EC) and hematopoietic cells (HC) in the yolk sac and allantois, the two extraembryonic membranes functioning as hematopoietic organs in the avian embryo. To this end, quail chick chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in the allantois, yolk sac, amnion, and chorion of resulting putative chimeras was examined using quail cell-specific antibody against a perinuclear antigen (QCPN) after 6 days of incubation. The presence of EC, HC, and smooth muscle cells among the QCPN⁺ donor cells was examined using QH-1, a quail-specific marker identifying HC and EC and an anti-α-smooth muscle actin antibody. Evidence gathered in the present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes of resulting heterospecific quail chick chimeras and, most importantly, give rise to HC, EC, and smooth muscle cells, all of the three main mesodermal lineages derived from the posterior mesoderm both in the yolk sac and allantois.

Flow cytometry assay for intracellular detection of Infectious Pancreatic Necrosis virus (IPNV) in Atlantic salmon (Salmo salar L.) leucocytes

Infectious Pancreatic Necrosis virus (IPNV) is traditionally detected in adherent leucocytes using immunofluorescence labelled specific antibodies, PCR or by further cultivation of infected cell material in cell lines.We present a flow cytometry (FCM) assay for detection of intracellular IPNV in salmon leucocytes, where each single cell is analysed for presence of virus. The method is established using in vitro challenge of salmon leucocytes and CHSE-214 cells. For detection of intracellular virus antigen the Cytofix/Cytoperm kit from BD is optimal compared with paraformaldehyde or acetone/methanol for cell permeabilisation. This is combined with labelling procedures allowing both internal virus antigen labelling and external antibody labelling of cell markers to identify B-cells and neutrophils. The secondary antibodies were Alexa Fluor 647 for the internal labelling and RPE for the external labelling of bound cell subtype specific antibodies. The presences of virus within cells are also demonstrated by confocal and light microscopy of infected cells.IPNV is successfully detected in blood and head kidney leucocyte samples. IPNV is found both in B-cells and neutrophils as well as in other types of leucocytes that could not be identified due to lack of cell-specific antibodies. Serial samples from cultivation of in vitro infected leucocytes and CHSE-214 cells analysed by flow cytometry showed that number of infected cells increased with increasing number of days.The flow cytometry protocol for detection of intracellular IPNV is verified using CHSE-214 cells persistently infected with IPNV. These analyses are compared with virus titre and virus infected naive CHSE-214 cells. The detection of IPNV in persistently infected cells indicates that carrier fish can be analysed, as such cells are considered to have virus titres similar to carriers.

Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG–PE micelles in ovarian cancer cell spheroid model

We describe the evaluation of doxorubicin-loaded PEG–PE micelles targeting using an ovarian cancer cell spheroid model. Most ovarian cancer patients present at an advanced clinical stage and develop resistance to standard of care platinum/taxane therapy. Doxorubicin is also approved for ovarian cancer but had limited benefits in refractory patients. In this study, we used drug-resistant spheroid cultures of ovarian carcinoma to evaluate the uptake and cytotoxicity of an antibody-targeted doxorubicin formulation. Doxorubicin was encapsulated in polyethylene glycol–phosphatidyl ethanolamine (PEG–PE) conjugated micelles. The doxorubicin-loaded PEG–PE micelles (MDOX) were further decorated with a cancer cell-specific monoclonal 2C5 antibody to obtain doxorubicin-loaded immunomicelles (2C5-MDOX). Targeting and resulting toxicity of doxorubicin-loaded PEG–PE micelles were evaluated in three dimensional cancer cell spheroids. Superior accumulation of 2C5-MDOX compared to free doxorubicin or untargeted MDOX in spheroids was evidenced both by flow cytometry, fluorescence and confocal microscopy. Interestingly, even higher toxicity was measured by lactate dehydrogenase release and terminal deoxynucleotidyl transferase dUTP nick end labeling of targeted doxorubicin micelles in Bcl-2 overexpressing adriamycin-resistant spheroids. Overall, these results support use of spheroids to evaluate tumor targeted drug delivery.

Tonsil-derived mesenchymal stromal cells: evaluation of biologic, immunologic and genetic factors for successful banking

Although mesenchymal stromal cells (MSC) from human palatine tonsils (tonsillar MSC, T-MSC) have been isolated, whether T-MSC isolated from multiple donors are feasible for cell banking has not been studied. T-MSC before and after a standard protocol of cryopreservation and thawing were assessed regarding several basic characteristics, including colony-forming unit-fibroblast features, MSC-specific surface antigen profiles, and inhibition of alloreactive T-cell proliferation. In vitro mesodermal differentiation potentials to adipocytes, osteocytes and chondrocytes were detected by staining with either cell-specific dyes or antibody after incubation with each appropriate differentiation medium. Expression of mesoderm-specific genes was also quantified by real-time polymerase chain reaction (PCR) assay. Expression profiles of endoderm-specific genes were identified by reverse transcription PCR assay. The feasibility of T-MSC in future engraftment was tested by short tandem repeat (STR) analysis using genomic DNA isolated randomly from three independent subjects. Both fresh and cryopreserved-thawed T-MSC showed a similar high proliferation capacity and expressed primitive cell-surface markers. Hematopoietic cell markers, HLA-DR, co-stimulatory molecules and follicular dendritic cell markers were not detected. In addition to mesodermal differentiation, fresh and cryopreserved-thawed cells also underwent endodermal differentiation, as evidenced by the expression of endoderm-specific genes including forkhead box A2 (FoxA2), SIX homeobox 1 (Six1) and chemokine (C-C motif) ligand 21 (CCL21). Both cells significantly decreased phorbol 12- myristate 13-acetate (PMA)-induced T-cell proliferation. T-MSC from three independent donors formed chimerism in STR analysis. Our results demonstrate for the first time that T-MSC are a potentially good source for MSC banking.

Follicle-Stimulating Hormone and Luteinizing Hormone Mediate the Androgenic Pathway in Leydig Cells of an Evolutionary Advanced Teleost1

The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC(50) 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.

Chicken dendritic cells and type II pneumocytes express a common intracellular epitope

1. CVI-ChNL 74·3, a dendritic cell-specific monoclonal antibody (mAb) also identifies chicken lung granular pneumocytes (type II pneumocytes), which produce surfactant. 2. The 74·3 mAb does not cross-react with any other avian or mammalian granular pneumocyte, and provides a convenient tool for monitoring the status of type II pneumocytes in the chicken lung.

T Cell-Specific siRNA Delivery Using Antibody-Conjugated Chitosan Nanoparticles

The intracellular delivery of small interfering RNA (siRNA) plays a key role in RNA interference (RNAi) and provides an emerging technique to treat various diseases, including infectious diseases. Chitosan has frequently been used in gene delivery applications, including siRNA delivery. However, studies regarding the modification of chitosan with antibodies specifically targeting T cells are lacking. We hypothesized that chitosan nanoparticles modified with T cell-specific antibodies would be useful for delivering siRNA to T cells. CD7-specific single-chain antibody (scFvCD7) was chemically conjugated to chitosan by carbodiimide chemistry, and nanoparticles were prepared by a complex coacervation method in the presence of siRNA. The mean diameter and zeta potential of the scFvCD7-chitosan/siRNA nanoparticles were approximately 320 nm and +17 mV, respectively, and were not significantly influenced by the coupling of antibody to chitosan. The cellular association of antibody-conjugated nanoparticles to CD4+ T cell lines as well as gene silencing efficiency in the cells was significantly improved compared to nonmodified chitosan nanoparticles. This approach to introducing T cell-specific antibody to chitosan nanoparticles may find useful applications for the treatment of various infectious diseases.