Histomorphological structure of the Bursa cloacalis (Bursa of Fabricius) in young Leiothrix lutea: a comparative study using light microscopy and transmission electron microscopy / Estructura histomorfológica de la Bursa cloacalis (Bursa de Fabricius) en jóvenes Leiothrix lutea: un estudio comparativo utilizando microscopía óptica y microscopía electrónica de transmisión

SUMMARY: This study aimed to investigate the microstructure and ultrastructure of the Bursa cloacalis (Bursa of Fabricius) (BC) in young Leiothrix lutea at various days of age (a few days after hatching) using light microscopy and transmission electron microscopy. The bird BC was sampled at 1, 5, 7, and 9 days of age. Immediately after dissection, the structure and integrity of the BC (not degenerative) were retained and the specific temporal features could be visualized precisely. After hematoxylin-eosin staining and uranyl acetate/lead citrate staining, the microstructure and ultrastructure of the BC, respectively, could be observed clearly. The microscopic observations revealed the following: in addition to change in the size of BC or lymphoid follicles, many cavities were found in the BC; the distribution of the lymphoid follicles in Leiothrix lutea was different from that in other birds; and the segregating line between the bursal cortex and medulla became increasingly clear as the age increased. In conclusion, the structural data obtained in this study provides a better understanding of the specific immunological function of the BC in Leiothrix lutea.

RESUMEN: Este estudio tuvo como objetivo investigar la microestructura y ultraestructura de la Bursa cloacalis (BC) en Leiothrix lutea joven unos días después de la eclosión, utilizando microscopía óptica y microscopía electrónica de transmisión. La BC se muestreó a los 1, 5, 7 y 9 días de edad del Leiothrix lutea. Inmediatamente después de la disección, se observó la estructura y la integridad de la CB (no degenerativa) y se pudo visualizar con precisión las características temporales específicas. Después de la tinción con hematoxilina-eosina y con acetato de uranilo /citrato de plomo, pudimos observar claramente la microestructura y ultraestructura de la BC. Las observaciones microscópicas revelaron el cambio en el tamaño de la CB o de los folículos linfoides y además, se encontraron numerosas cavidades en la CB; la distribución de los folículos linfoides en Leiothrix lutea era diferente a la de otras aves; y la línea de segregación entre la corteza bursal y la médula se hizo cada vez más clara a medida que aumentaba la edad. En conclusión, los datos estructurales obtenidos en este estudio proporcionan una mejor comprensión de la función inmunológica específica de la Bursa cloacalis en Leiothrix lutea.

Immune Profiling of Human Gut-Associated Lymphoid Tissue Identifies a Role for Isolated Lymphoid Follicles in Priming of Region-Specific Immunity.

The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1+ high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites. IgA sequencing analysis indicated that human ILFs are sites where intestinal adaptive immune responses are initiated in an anatomically restricted manner. Our findings position ILFs as key inductive hubs for regional immunity in the human intestine, and the methods presented will allow future assessment of these compartments in health and disease.

Histological and immunohistochemical characterization of the porcine ocular surface.

The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 µm) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6-8 epithelial cell layers, stroma, Descemet's membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 µm (mean±standard error of the mean) in the center and increased to 1496.9±138.2 µm at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Imaging lymphoid tissues in nonhuman primates to understand SIV pathogenesis and persistence.

CD4+ T cells are the primary HIV-1 target cell, with the vast majority of these cells residing within lymphoid tissue compartments throughout the body. Predictably, HIV-1 infection, replication, localization, reservoir establishment and persistence, as well as associated host immune and inflammatory responses and disease pathology principally take place within the tissues of the immune system. By virture of the fact that the virus-host struggle is played out within lymphoid and additional tissues compartments in HIV-1 infected individuals it is critical to understand HIV-1 infection and disease within these relevant tissue sites; however, there are obvious limitations to studying these dynamic processes in humans. Nonhuman primate (NHP) research has provided a vital bridge between basic and preclinical research and clinical studies, with experimental SIV infection of NHP models offering unique opportunities to understand key processes of HIV-1 infection and disease that are either not practically feasible or ethical in HIV-1 infected humans. In this review we will discuss current approaches to studying the tissue based immunopathogenesis of AIDS virus infection in NHPs, including both analyses of tissues obtained at biopsy or necropsy and complementary non-invasive imaging approaches that may have practical utility in monitoring HIV-1 disease in the clinical setting.

Morphology of mucosa-associated lymphoid tissue in odontocetes.

This study describes the mucosa-associated lymphoid tissue (MALT) in odontocetes from the Brazilian coast and freshwater systems. Seven species were evaluated and tissue samples were analyzed by light, scanning and transmission electron microscopy, and immunohistochemistry. Laryngeal tonsil was a palpable oval mass located in the larynx, composed of a lymphoepithelial complex. Dense collections of lymphocytes were found in the skin of male fetus and calf. Clusters of lymphoid tissue were found in the uterine cervix of a reproductively active juvenile female and along the pulmonary artery of an adult female. Lymphoid tissues associated with the gastrointestinal tract were characterized by diffusely arranged or organized lymphocytes. The anal tonsil was composed of an aggregate of lymphoid tissue occurring exclusively in the anal canal, being composed of squamous epithelium branches. MALT was present in different tissues and organic systems of cetaceans, providing constant protection against mucosal pathogens present in their environment.

Light and electron microscopic study of the eyelids, conjunctiva-associated lymphoid tissue and lacrimal gland in Bilgorajska Goose (Anser anser).

Normal structure of the accessory organs of the eye is essential for normal eye physiology. Among the most important accessory organs of the eye are the eyelids, the conjunctiva-associated lymphoid tissue (CALT) and the lacrimal gland (LG). The aim of this study was to demonstrate the histological structure of the eyelids and LG by histochemical and ultrastructural analysis. The study was performed on 13 adult female Bilgorajska geese. Eyelid samples were stained with the Alcian blue (AB pH 2.5) and periodic acid-Schiff (PAS) methods. Staining methods used for LG were AB pH 2.5, aldehyde fuchsin (AF), PAS and Hale's dialysed iron (HDI). Within the connective tissue of the eyelids, well-developed, diffuse, CALT follicles were observed, mostly under the conjunctival epithelium. Numerous lymphocytes were present within loose connective tissue. Staining of the eyelids with the PAS method demonstrated the presence of goblet cells of a mucous nature, and AB pH 2.5 staining indicated the presence of sulfated acid mucopolysaccharides. PAS staining of LG revealed the presence of secretory cells containing weakly PAS-positive granules. All epithelial cells of the corpus glandulae and the duct systems reacted positively to AB pH 2.5. HDI staining detected the presence of carboxylated acid mucopolysaccharides. Transmission electron microscopy investigations revealed two types of secretory epithelial cells in LG. Both types of LG cells contained drop-like secretory vesicles of different sizes with low or high electron density in cytoplasm, as well as small and large lipid vacuoles, and numerous small primary lysosomes.

Ultrastructure of the human palatine tonsil and its functional significance.

The human palatine tonsils represent a mucosa-associated lymphoid tissue with a significant function in mucosal protection against alimentary and airborne pathogens. The ultrastructure of different morphological compartments in the human palatine tonsil was studied in eighteen tonsils obtained from the patients who had undergone elective tonsillectomy due to chronic tonsillitis. The tonsillar specimens were analyzed by scanning and transmission electron microscopy. The results showed the presence of tight junctions between superficial epithelial cells of the oropharyngeal tonsillar surface. The crypt epithelium is a sponge-like structure infiltrated by non-epithelial cells, mostly lymphocytes, and is characterized by the presence of small pores - microcrypts occupied by large microvillus cells and/or lymphocytes. Antigen-presenting Langerhans cells with typical intracytoplasmic Birbeck granules were also found in the crypt epithelium. The lymphoid follicles are composed of lymphocytes and two types of non-lymphoid follicular cells: small fibroblast-like cells and large cells, morphologically consistent with antigen-bearing follicular dendritic cells or macrophages. The interfollicular areas consisted of a dense network of reticular cells and reticular fibers; many lymphocytes were interspersed between the reticular fibers. In addition to arterioles and high endothelial venules in the interfollicular lymphoid tissue, some fenestrated capillaries were seen intraepithelially and subepithelially. The complex ultrastructure of the human palatine tonsil provides a microenvironment necessary for antigen uptake, antigen processing and immune response.

Spatiotemporal basis of innate and adaptive immunity in secondary lymphoid tissue.

Secondary lymphoid tissues are the sites of both innate and adaptive host defense. Aside from the relatively static nonhematopoietic stromal elements and some macrophages and dendritic cells, most of the cells in these tissues are in constant movement, but the organs maintain a defined microanatomy with preferred locations for the bulk of T cells, B cells, and other lymphocytes and subsets of myeloid cells. Here we describe both the cell dynamics and spatial organization of lymph nodes and review how both physical features and molecular cues guide cell movement to optimize host defense. We emphasize the role of locality in improving the efficiency of a system requiring rare cells to find each other and interact productively through membrane-bound or short-range secreted mediators and highlight how changes in steady-state cell positioning during an infectious challenge contribute to rapid generation of productive responses.

Ultrastructural and histochemical study on the Paneth cells in the rat ascending colon.

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, ß-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon.

PAS-positive extracellular deposits within germinal centers of hyperplastic follicles during SIV infection in a rhesus macaque.

BACKGROUND: Lymphoid tissue remodeling is characteristic of chronic simian immunodeficiency virus infection. METHODS: A rhesus macaque infected with SIVmac239 was necropsied and its lymphoid tissues subjected to histopathology characterization. RESULTS: Germinal centers in spleen and lymph nodes contained PAS-positive, non-amyloid extracellular deposits, decreased T follicular helper cells, and normal density of Ki67(+) B cells. CONCLUSIONS: A possible mechanism for PAS-positive deposits includes exaggerated involution of SIV-induced follicular hyperplasia secondary to virus-associated immune reaction.

Development, alteration and real time dynamics of conjunctiva-associated lymphoid tissue.

PURPOSE: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. METHODS: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. RESULTS: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min. CONCLUSIONS: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.

Morphological features of the lymphoepithelial structures of the jejunum after the stress in rats.

We studied the effect of acute emotional stress on functional status of lymphoid epithelial structures of the jejunum in rats with different behavioral activity. Morphological and functional characteristics of lymphoid tissue were assessed using morphometric, histological and electron microscopic methods. In behaviorally active and passive rats, reduction in villus height, area of the epithelium and lymphoid tissue of the jejunum was recorded on the third day after acute emotional impact. At that, the largest number of destructively modified lymphoid cells was identified by comparison with the other time points. Moreover, destruction of the apical part of the jejunal villi was observed on days 3 and 7 after stress exposure. Recovery of the lymphoepithelial structures of the jejunum after acute stress exposure was detected on day 14.

Renal subcapsular tertiary lymphoid aggregates in chronic kidney diseases.

In the last decade, it has been accepted the formation of tertiary lymphoid organs in the renal parenchyma during inflammatory conditions. These organized cellular aggregates contain B- and T-lymphocytes, dendritic cells, surrounded by neo-lymphatic vessels. They have been described in renal allografts, acute and chronic interstitial nephritis, IgA and membranous nephropathies. The functional characteristics of these lymphoid nodules remained still under consideration. After investigating the renal biopsies of 268 patients with primary and secondary nephropathies, we have selected 20 cases showing lymphoid-like cellular aggregates located just beneath the renal capsule and having close contacts with this kidney envelope. All of these cases also showed an associated medium sized lymphatic vessel. The ultrastructure of these nodules proved to contain more or less the same cellular composition: lymphocytes, dendritic cells, seldom plasma cells and macrophages. We consider these particular subcapsular lymphoid-like nodules to be tertiary lymphatic structures in close association with the perirenal lymphatics, and the first to develop in any type of inflammatory and autoimmune renal condition.

Characterization of membranous (M) cells in normal feline conjunctiva-associated lymphoid tissue (CALT).

OBJECTIVE: To characterize conjunctival lymphoid nodules obtained from the nictitans of healthy cats to determine if the follicle-associated epithelium (FAE) of conjunctiva-associated lymphoid tissue (CALT) in this species contains membranous (M)-cells analogous to those described in other regions of mucosa-associated lymphoid tissue (MALT). METHODS: Lymphoid follicles from nictitan bulbar surfaces of 10 healthy cats (20 eyes total) were examined. Nictitans from five cats were harvested immediately post-mortem and a minimum of 12 lymphoid nodules from each third eyelid were isolated using a Zeiss operating microscope. At least three lymphoid follicles from each eye were examined using light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) using standard fixation and embedding protocols. Nictitan-lymphoid follicles from another five healthy cats were processed for immunohistochemistry to characterize the distribution of T- and B-lymphocytes present beneath the FAE. RESULTS: The FAE overlying CALT from 10 healthy cats demonstrated morphology characteristic of M-cells including attenuated apical cell surface with blunted microvilli and microfolds, invaginated basolateral membrane forming a cytoplasmic pocket, and diminished distance between the apical and pocket membrane. Immunohistochemistry of lymphoid tissue subtending the FAE demonstrated B-cell dependent regions in the germinal centers surrounded by T-cell dependent interfollicular zones. CONCLUSIONS: Healthy feline CALT contains morphologic features analogous to those described in other regions of MALT. Documentation of feline conjunctival M-cells is of clinical relevance in the study of primary infectious, allergic, and autoimmune ocular diseases, as well as a potential means of vaccination or drug delivery.

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens.

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

Histopathology, immunohistochemistry, in situ apoptosis, and ultrastructure characterization of the digestive and lymphoid organs of new type gosling viral enteritis virus experimentally infected gosling.

Here, for the first time, to colocalize new type gosling viral enteritis virus (NGVEV) with histological lesions and in situ apoptosis in the digestive organs (esophagus, proventriculus, gizzard, small intestine, cecum, rectum, liver, and pancreas) and the lymphoid organs (bursa of Fabricius, thymus, Harderian gland, and spleen) of experimentally infected goslings, portions of tissues were collected at sequential infection time points and examined by histopathology for histological lesions, immunohistochemical staining for viral antigens, ultrastructural observation by transmission electron microscope (TEM) for virus particles and apoptotic cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay for in situ apoptosis. The hyperemia, hemorrhage, infiltration of lymphocytes, progressive lymphoid depletion, apoptosis, and necrosis were readily observed in the lymphoid organs and intestine tract by histopathological examination. The NGVEV particles and viral antigens widely appeared in the small intestine and bursa of Fabricius as early as 2 d postinfection (PI) by TEM and immunohistochemical staining, and the presence and quantity of it reached a maximum during 6 to 12 d PI. The principal sites for NGVEV were endothelial cells, epithelia, mucosal cells, glandular cells, fibrocytes, macrophages, and lymphocytes. A series of apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, and formation of apoptotic body were observed by TEM, and the number of apoptotic cells was largely increased from 4 d PI and peaked at 9 d PI by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling analysis. The histological organ lesions and apoptosis in vivo were generally associated with sites of NGVEV localization, which can be regarded as the cause of death. This work may shed light on the pathogenesis of new type gosling viral enteritis and put new insight into the pathogenesis of goose adenovirus.

Ultramicroscopic examination of the ovine tonsillar epithelia.

As solid morphological knowledge of ovine tonsillar epithelia might contribute to a better understanding of the pathogenesis of several diseases including prion diseases, the epithelia of all tonsils of 7 one-year-old Texel sheep were examined using scanning and transmission electron microscopy. Major parts of the pharyngeal and tubal tonsils were covered by pseudostratified columnar ciliated epithelia that were interrupted by patches of epithelium containing cells with densely packed microfolds or microvilli, and cells with both microvilli and cilia. Smaller parts were covered by either flattened polygonal cells with densely packed microvilli or microfolds, squamous epithelial cells, or patches of reticular epithelium. The palatine and paraepiglottic tonsils were mainly lined by squamous epithelial cells with apical microplicae or short knobs. Additionally, regions of reticular epithelium containing epithelial cells with apical microvilli were seen. The lingual tonsil was uniformly covered by a keratinized squamous epithelium and devoid of microvillous cells and patches of reticular epithelium. The rostral half of the tonsil of the soft palate was lined by a pseudostratified columnar ciliated epithelium with characteristics of the pharyngeal and tubal tonsils. The epithelium of the caudal part resembled the epithelia of the palatine and paraepiglottic tonsils. Putative M cells, mainly characterized by apical microvilli or microfolds and a close association with lymphoid cells, seem manifestly present on the nasopharyngeal tonsils. The reticular epithelium of the palatine and paraepiglottic tonsils also harbor cells with small apical microvilli. The exact nature of these presumptive M cells should, however, be elucidated in functional studies.

Imaging circulating cells and lymphoid tissues with iron oxide nanoparticles.

The use of nanometer-sized iron oxide nanoparticles and micron-sized iron oxide particles as magnetic resonance (MR) contrast agents has garnered a high degree of interest in diverse areas of biology and medicine. Applications such as cell tracking, molecular imaging, gene detection, and lymphography are being explored to provide insight into disease mechanisms, monitor therapeutic efficacy, and facilitate diagnostic imaging. What makes iron oxide so appealing is a number of favorable properties including high detectability by MR, biodegradability and low toxicity. Here we describe the recent progress on the use of magnetic nanoparticles in imaging circulating cells and lymphoid tissues. The study of the lymph system and the biodistribution of various circulating immune cells is important in the diagnosis, prognosis, and treatment of a wide range of diseases and is expected to have a profound effect on patient outcome.

Id2-, RORgammat-, and LTbetaR-independent initiation of lymphoid organogenesis in ocular immunity.

The eye is protected by the ocular immunosurveillance system. We show that tear duct-associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid-related orphan receptor gammat, lymphotoxin (LT) alpha1beta2-LTbetaR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity.

A novel in vivo siRNA delivery system specifically targeting dendritic cells and silencing CD40 genes for immunomodulation.

Translation of small interfering RNA (siRNA)-based approaches into practical therapeutics is limited because of lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to dendritic cells (DCs) in vivo using CD40 siRNA-containing immunoliposomes (siILs) that were decorated with DC-specific DEC-205 mAb. Administration of CD40 siILs resulted in DC-specific cell targeting in vitro and in vivo. On treatment with CD40 siILs, the expression of CD40 in DCs, as well allostimulatory activity was inhibited. In vivo administration resulted in selective siRNA uptake into immune organs and functional immune modulation as assessed using a model antigen. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.