Immunofluorescence Procedures Research Articles

Microdistribution of Targeted, Fluorescently Labeled Anti–Carcinoembryonic Antigen Antibody in Metastatic Colorectal Cancer: Implications for Radioimmunotherapy

Most radioimmunotherapy studies on radiolabeled antibody distribution are based on autoradiographic and radioluminographic data, which provide a lack of detailed information due to low resolution. We used fluorescently labeled anti-carcinoembryonic antigen (CEA) antibody (A5B7) to investigate quantitatively the kinetics and microdistribution of antibody in a clinically relevant orthotopic colorectal cancer model (LS174T) using high-resolution digital microscopy. Nude mice bearing LS174T liver orthotopic tumors received a single i.v. injection of fluorescently labeled A5B7 and were sacrificed at 10 minutes, 1 hour, or 24 hours postinjection. Before sacrifice, mice were injected with the perfusion marker Hoechst 33342. An anti-CD31 antibody was used to detect blood vessel distribution. Cryostat sections were processed with immunofluorescence procedures and analyzed with fluorescence microscopy and image analysis techniques. The fluorescence images were related to morphologic images of the same or adjacent tumor sections. Fluorescently labeled antibody showed rapid, selective uptake into tumor deposits, with a strong negative correlation with tumor size at 10 minutes and 1 hour (P < or = 0.01). By 24 hours, the correlation was no longer significant. The study showed movement of antibody across the tumor with time and a tendency to localize more uniformly by later time points (24 hours). The rate of antibody motility was similar in small and large tumor metastases, but small deposits showed more rapid antibody localization. Intratumoral vessels were positively related to tumor size (P < or = 0.001). The obtained data suggest that radioimmunotherapy can be highly efficient in an adjuvant or minimal residual disease setting.

Adipose triglyceride lipase (ATGL) expression in human skeletal muscle is type I (oxidative) fiber specific

Accumulation of triacylglycerol (TAG) and lipid intermediates in skeletal muscle plays an important role in the etiology of insulin resistance and type 2 diabetes mellitus. Disturbances in skeletal muscle lipid turnover and lipolysis may contribute significantly to this. So far, knowledge on the regulation of muscle lipolysis is limited. Recently the identification of a new lipase was reported: adipose triglyceride lipase (ATGL). ATGL deficient animals show significant lipid accumulation in skeletal muscle, which may indicate that ATGL plays a pivotal role in skeletal muscle lipolysis. However, until now, it is still unknown whether ATGL protein is expressed in human skeletal muscle. Therefore, the aim of the present study was to investigate whether ATGL is expressed at the protein level in human skeletal muscle, and to examine whether its expression is fiber-type specific. To accomplish this, we established an imunohistochemical and immunofluorescent staining procedure to study ATGL protein expression in relation to fiber type in human vastus lateralis muscle of eight male subjects (BMI range: 21.0–34.5 kg/m2 and age: 38–59 years). In the present paper we report for the first time that ATGL protein is indeed expressed in human skeletal muscle. Moreover, ATGL is exclusively expressed in type I (oxidative) muscle fibers, suggesting a pivotal role for ATGL in intramuscular fatty acid handling, lipid storage and breakdown.

Phenotypic characterisation of intestinal dendritic cells in sheep

The present study was undertaken to identify dendritic cells (DCs) in the ileum and rectum of lambs and adult sheep. The distribution of these cells in four different intestinal compartments, i.e. lamina propria, lymphoid follicles, domes and interfollicular areas was assessed, and the presence of these cells in lambs and adult sheep was compared. Specimens were examined by using a number of potential DC markers (CD11c, CD205, MHC class II (MHCII), CD1b and CD209) in immunohistochemical and multicolour immunofluorescent procedures. The ovine ileal and rectal mucosa contain many CD11c+/CD205+ cells with a dendritic morphology, and the majority of these cells co-expressed MHCII. These double-positive cells were also labelled with the CD209 antibody in the lamina propria and interfollicular regions. Only very few cells expressed CD1b. In conclusion, a major DC population in ileum and rectum of sheep co-expressed the CD11c, CD205 and MHCII molecules. The CD209 antibody appeared to be a novel marker for a subpopulation of ovine intestinal DCs.

Autoimmune liver serology: Current diagnostic and clinical challenges

Liver-related autoantibodies are crucial for the correct diagnosis and classification of autoimmune liver diseases (AiLD), namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis variants in adults and children. AIH-1 is specified by anti-nuclear antibody (ANA) and smooth muscle antibody (SMA). AIH-2 is specified by antibody to liver kidney microsomal antigen type-1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1). SMA, ANA and anti-LKM antibodies can be present in de-novo AIH following liver transplantation. PBC is specified by antimitochondrial antibodies (AMA) reacting with enzymes of the 2-oxo-acid dehydrogenase complexes (chiefly pyruvate dehydrogenase complex E2 subunit) and disease-specific ANA mainly reacting with nuclear pore gp210 and nuclear body sp100. Sclerosing cholangitis presents as at least two variants, first the classical primary sclerosing cholangitis (PSC) mostly affecting adult men wherein the only (and non-specific) reactivity is an atypical perinuclear antineutrophil cytoplasmic antibody (p-ANCA), also termed perinuclear anti-neutrophil nuclear antibodies (p-ANNA) and second the childhood disease called autoimmune sclerosing cholangitis (ASC) with serological features resembling those of type 1 AIH. Liver diagnostic serology is a fast-expanding area of investigation as new purified and recombinant autoantigens, and automated technologies such as ELISAs and bead assays, become available to complement (or even compete with) traditional immunofluorescence procedures. We survey for the first time global trends in quality assurance impacting as it does on (1) manufacturers/purveyors of kits and reagents, (2) diagnostic service laboratories that fulfill clinicians' requirements, and (3) the end-user, the physician providing patient care, who must properly interpret test results in the overall clinical context.

Immunocytochemistry and Laser Capture Microdissection for Real-time Quantitative PCR Identify Hindbrain Neurons Activated by Interaction Between Leptin and Cholecystokinin

Current evidence suggests that leptin reduces food intake in part by enhancing the hindbrain neuronal response to meal-related gastrointestinal signals, including cholecystokinin (CCK), but the phenotypes of the relevant cells are not known. To identify neurons that participate in this interaction in the rat nucleus of the solitary tract (NTS), we induced c-Fos gene expression in NTS neurons with leptin and CCK. We focused on NTS catecholamine neurons because these cells have been implicated in the feeding response to CCK. Hindbrain sections from rats that received CCK with or without leptin pretreatment were immunostained for c-Fos and tyrosine hydroxylase (TH) by a double immunofluorescence procedure. Leptin pretreatment increased the number of NTS cells expressing c-Fos-like immunoreactivity (cFLI) 3-fold relative to CCK alone, but the number of TH-positive cells with cFLI was increased 6-fold. Next, cells detected by immunofluorescence for TH were collected by laser capture microdissection and pooled for real-time quantitative PCR of c-Fos mRNA. Here, neither le0ptin nor CCK alone affected the relative amount of mRNA in the TH cell-enriched samples, but leptin plus CCK substantially increased c-Fos mRNA content. These histochemical findings identify hindbrain catecholamine cells as potential mediators of the interaction between leptin and CCK.

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster ( Crassostrea virginica ) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.

Blind trials evaluating in vitro infectivity ofCryptosporidiumoocysts using cell culture immunofluorescence

An optimized cell culture immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in blind trials to determine the sensitivity and reproducibility of measuring the infectivity of flow-cytometry-prepared inocula of Cryptosporidium parvum oocysts. In separate trials, suspensions consisting of between 0% and 100% viable oocysts were prepared at the US Environmental Protection Agency, shipped to the American Water Laboratory, and analyzed blindly by cell culture IFA. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to cell culture dose-response curve data developed previously at the American Water Laboratory. For test samples containing oocyst suspensions of unknown infectivity, cell culture IFA analyses indicated a high degree of correlation (r2 = 0.89; n = 26) with the values expected by the US Environmental Protection Agency. Cell culture infectivity correlates well with neonatal mouse infectivity assays, and these blind validation trials provide credibility for the cell culture IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.

Detection of β-1-4-galactan in compression wood of Sitka spruce [Picea sitchensis (Bong.) Carrière] by immunofluorescence

Abstract β-1-4-Galactan has been detected in Sitka spruce [Picea sitchensis (Bong.) Carrière] by an immunofluorescence procedure. The anti-β-1-4-galactan monoclonal antibody LM5 was found to bind to compression wood (CW) tissue as identified by cell morphology features. In cross-sections, year rings with very fine bands of galactan-containing tissue were found. Cells in these bands expressed no morphological CW features. This finding was interpreted to imply that β-1-4-galactan synthesis is one of the first physiological reactions of CW formation. In cases where the CW character was pronounced, galactan was exclusively located in the outer cell-wall layers and was absent from the majority of the inner S2 layer.

Responses of reactive astrocytes containing S100β protein and fibroblast growth factor‐2 in the border and in the adjacent preserved tissue after a contusion injury of the spinal cord in rats: implications for wound repair and neuroregeneration

This paper demonstrates glial reaction and changes in the S100beta protein and basic fibroblast growth factor (bFGF, FGF-2) in the border and in the adjacent preserved tissue of the rat spinal cord after a contusion. In view of the expression of FGF-2 and S100beta in reactive glial cells and their ability to promote gliogenesis and neuronal trophism, the molecules have been considered to participate in the wound repair and regenerative events after nervous tissue injury. Adult rats were submitted to a moderate spinal cord (10th thoracic level) contusion induced by a New York University Impactor by dropping a 10 g rod from a distance of 25 mm onto the dorsal surface of the exposed dura spinal cord. Impactor curves and parameters were used to monitor the severity of the trauma. Control rats were submitted to sham operation. The motor behavioral spontaneous recovery was demonstrated by means of a BBB test and the combining behavior score up to 3 weeks after injury. Animals were killed 72 hours, 2, and 3 weeks after surgery and spinal cords were processed for immunohistochemistry to show glial fibrillary acidic protein positive astrocytes and OX-42-positive microglia/macrophages as well as changes in the S100beta and FGF-2 in the border and in the adjacent preserved tissue of the lesioned cords. The changes in the immunoreaction products were quantified by means of morphometric/microdensitometric image analysis, and the cell type expressing S100beta and FGF-2 was analyzed by means of two-color immunofluorescence procedures. Massive increases of S100beta and FGF-2 were found in reactive astrocytes, not in reactive microglia, in the border and in the white and gray matters of adjacent preserved tissue of the contused spinal cord in the periods studied. The results are discussed in view of possible paracrine trophic actions of the reactive astrocytes, mediated by S100beta and FGF-2, triggering wound repair events in the border of the trauma, and also leading to neurotrophism and neuronal plasticity in the adjacent regions. These cellular and molecular responses may interfere with the pattern of behavioral recovery after a contusion injury of the spinal cord.

Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells

This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca(2+) depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in approximately 50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.

The expression profile of PKC isoforms during MC3T3-E1 differentiation

Protein kinase C (PKC) is a family of kinases whose isoforms show subtle differences in physiological and biochemical responses, with their expression being cell- specific. We hypothesize that there may be a specific profile of expression of PKC isoforms in differentiating osteoblastic cells (OBC) with individual isoforms having specific functions. Herein, the MC3T3-E1 cell line was used as a differentiating model, which was induced from the pre-osteoblast stage to mature osteoblast and characterized with several phenotypic markers, including alkaline phosphatase activity, osteocalcin and bone sialoprotein. The expression of PKC isoforms was monitored using Western blot analysis. Upon induction of osteogenesis, the intracellular localization of PKC eta and theta was determined using immunofluorescence. Lastly, the effect of P38 MAP kinase inhibition was determined using SB203580. Results show 1) PKC alpha, delta, lambda were all highly expressed in MC3T3-E1 osteoblastic cells, 2) the expression of PKC theta was significantly down-regulated upon induction of osteoblastic differentiation; 3) PKC eta was non-detectable at certain cell culture days; however, was up-regulated as the cells transit from each differentiation phase. The increased expression of PKC eta correlated with increases in OC, BSP levels and alkaline phosphatase activity. Immunofluorescence procedure confirmed the Western blot results with an increase in PKC eta and a decrease in PKC theta upon osteogenic stimulation. The inhibition of p38 resulted in a marked down-regulation of PKC eta. The data demonstrate that there is a specific profile of expression of PKC isoforms in differentiating osteoblasts; the different expression pattern of individual isoforms may be either a consequence of the differentiation itself or plays a role in the regulatory mechanism of osteoblastic differentiation. This study has provided primary information on the temporal pattern of expression of PKC isoforms in the differentiating osteoblast and further insight into their possible role in osteoblastic cell maturation.

Leptin Increases Axonal Growth Cone Size in Developing Mouse Cortical Neurons by Convergent Signals Inactivating Glycogen Synthase Kinase-3β

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3beta (GSK3beta), an event inactivating this kinase. Leptin-mediated GSK3beta phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Lepr(db/db) mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3beta phosphorylation and mimicked by the GSK3beta inhibitor SB216763. At concentrations preventing GSK3beta phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3beta activity.

Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum

Detection of antinuclear (ANA) and antineutrophil cytoplasmic (ANCA) antibodies is extensively used for establishing a diagnosis in patients with clinical features suggestive of autoimmune disorders. The most common methods for the identification of positive patients' sera for ANA or ANCA are indirect immunofluorescence (IIF) and ELISA-based procedures. Considerable effort has been made in developing simpler automated assays for routine laboratory use. Recently a commercially available microsphere-based fluorescent assay has been introduced for the detection of ANA and ANCA. The aim of this study was to compare this technology with routinely used IIF and ELISA procedures, in patients with a suggested autoimmune disorder. A highly significant correlation between ELISA procedures for specific antibodies and the microsphere-based assays were obtained for both ANA and ANCA as well as for extractable nuclear antigens ELISA screening, indicating that multiplex technology could replace individual ELISA tests for the measurement of specific autoantibodies. However, a low sensitivity for identifying IIF-positive cases was obtained for both ANA (58.0%) and ANCA (59.1%), although there was a significant correlation between the assays. In conclusion, our data show that a microsphere-based fluorescent assay may be a valid platform for the simultaneous determination of circulating individual ANA and ANCA autoantibodies. Furthermore, multiplexing technology offers several advantages that will probably make it an attractive tool in the future. Nevertheless, until further studies are conducted that determine the clinical performance of the multiplex technology, the initial screening of patients for autoantibodies with IIF is still considered necessary.

Comparison of HER2 status between disseminated tumor cells (DTC) and corresponding tumors in breast cancer patients

Aims: Breast cancer patients with tumor cell persistence in the bone marrow may benefit from a secondary adjuvant immunotherapy. HER2 protein is suggested as one of those promising targets. However, the antigen pattern of disseminated cells (DTC), to which immunotherapy might be targeted, could differ from the antigen profile of the primary tumor. Therefore, the aims of this study were (a) to determine the HER2 status of DTC and (b) to compare the HER2 status between DTC and corresponding primary tumors. Methods: 135 primary breast cancer patients were included in this study. The isolation of DTC was performed by density centrifugation through Bicoll. To assess the HER2 status of DTC, an immunofluorescent double staining procedure was performed. The DTC were detected by the pan anti-cytokeratin antibody C11 conjugated to FITC and double labeled with polyclonal rabbit antibody CB 11 against the HER2 antigen. CB11 was detected by a secondary antibody conjugated to Texas Red. The HER2 status of the primary tumor was assessed by the HERCEP-test (Dako Co., CA). Results: 58 of 135 patients had DTC in the bone marrow. In 24 of these patients, HER2 positive DTC could be detected. The HER2 expression was heterogeneous in 11 of 23 patients with two or more tumor cells. HER2 positivity (score +2/+3) could be demonstrated in 12 of 55 primary tumors. The concordance rate between primary tumor and DTC was only 67%. HER2 positive DTC were detected in 15 patients with HER2 negative (0/+1) tumors. In contrast, three patients with HER2 positive primary tumors had HER2 negative DTC. Conclusion: (1) The HER2 status of DTC and corresponding primary tumor can differ. Therefore, the decision for antibody based strategies should also be based on the HER2 status of DTC. (2) The HER2 expression of DTC is heterogeneous in individual patients which may reduce efficacy of an immunotherapy based strategy only directed against HER2.

A possible role for humoral immunity in the pathogenesis of Parkinson's disease

The pathogenesis of idiopathic Parkinson's disease is unknown, but nigral degeneration and depigmentation are associated with microglial inflammation and anti-inflammatory medications appear to protect against the disease. The possibility that humoral immunity may play a role in initiating or regulating the inflammation has been suggested by experimental studies triggering dopamine cell death using a variety of transfer strategies and the observation of CD8+ T lymphocytes and complement in the nigra in Parkinson's disease. We analysed the association between degeneration and humoral immune markers in brain tissue of patients with idiopathic (n = 13) or genetic (n = 2 with alpha-synuclein and n = 1 with parkin mutations) Parkinson's disease and controls without neurological disease (n = 12) to determine the humoral immune involvement in Parkinson's disease. Formalin-fixed tissue samples from the substantia nigra and primary visual cortex for comparison were stained for alpha-synuclein, major histocompatibility complex II (HLA), immunoglobulin M (IgM), immunoglobulin G (IgG), IgG subclasses 1-4 and IgG receptors FcgammaR I-III. Antigen retrieval and both single immunoperoxidase and double immunofluorescence procedures were employed to determine the cell types involved and their pattern and semiquantitative densities. Significant dopamine neuron loss occurred in all patients with Parkinson's disease, negatively correlating with disease duration (r = -0.76, P = 0.002). Although all patients had increased inflammatory HLA immunopositive microglia, the degree of inflammation was similar throughout the disease (r = 0.08, P = 0.82). All patients with Parkinson's disease had IgG binding on dopamine neurons but not IgM binding. Lewy bodies were strongly immunolabelled with IgG. A mean 30 +/- 12% of dopamine nigral neurons were immunoreactive for IgG in Parkinson's disease with the proportion of IgG immunopositive neurons negatively correlating with the degree of cell loss in the substantia nigra (r = -0.67, P < 0.0001) and positively correlating with the number of HLA immunopositive microglia (r = 0.51, P = 0.01). Most neuronal IgG was the IgG1 subclass with some IgG3 and less IgG2 also found in the damaged substantia nigra. The high affinity activating IgG receptor, FcgammaRI, was expressed on nearby activated microglia. The low affinity activating IgG receptor, FcgammaRIII was expressed on cells morphologically resembling lymphocytes, whereas immunoreactivity for the inhibitory IgG receptor FcgammaRII was absent in all cases. This pattern of humoral immune reactivity is consistent with an immune activation of microglia leading to the targeting of dopamine nigral neurons for destruction in both idiopathic and genetic cases of Parkinson's disease.

Partial Characterization of an Adenovirus-Like Virus Isolated from Broiler Chickens with Transmissible Viral Proventriculitis

Transmissible viral proventriculitis (TVP) was experimentally reproduced in specific-pathogen-free chickens using a homogenate of proventricular tissue obtained from TVP-affected commercial broiler chickens. Thin-section electron microscopy revealed intranuclear, approximately 70-nanometer (nm), adenovirus-like viruses (AdLV) within proventricular lesions. The AdLV, designated AdLV (R11/3), could not be propagated using various avian and mammalian cell cultures or by inoculation of embryonated chicken eggs by yolk, allantoic, or chorioallantoic membrane routes. However, AdLV (R11/3) was successfully propagated by amniotic inoculation of embryonated chicken eggs, with detection of the virus in proventriculi and intestinal contents of hatched 2-day-old chicks (8 days postinoculation). Virus propagation was evident in in ovo-inoculated chicks by (1) gross and microscopic lesions in proventriculi consistent with TVP, (2) immunohistochemical localization of AdLV (R11/3) antigens in proventricular epithelium, (3) thin-section electron microscopic detection of intranuclear, approximately 70-nm AdLVs within proventricular epithelium, and (4) negative-stain electron microscopic detection of extracellular, approximately 70-nm AdLVs in intestinal contents. Indirect immunofluorescence and polymerase chain reaction procedures that specifically recognize groups I, II, and III avian adenoviruses failed to recognize AdLV (R11/3). The findings suggest an etiologic role for AdLV (R11/3) in TVP and indicate that this virus is distinct from known avian adenoviruses.

A Novel Immunocompetent Rat Model of HCV Infection and Hepatitis

Hepatitis C virus (HCV) infects millions of people worldwide. Therapy is limited, and treatment does not produce a sustained response in the majority of patients. Development of new agents has been hampered by the lack of a convenient animal model. The aim of this study was to determine whether an immunocompetent rat, tolerized and transplanted with a human hepatoma cell line (Huh 7 cells), could be used to sustain an HCV infection. Fetal rats were tolerized in utero with 10(5) Huh 7 cells. One day after birth, rats were transplanted with 5 x 10(6) Huh 7 cells and, a week later, inoculated with HCV, genotype 1. In tolerized, transplanted, and HCV-infected rats, Huh 7 cells were found in the liver, and HCV viral replication was detected by the presence of negative strand HCV RNA. HCV levels in serum were measured at 11,000 copies/mL at week 4, peaked at 22,500 copies/mL by week 12. In tolerized, transplanted, inoculated rats, but not controls, serum alanine aminotransferase (ALT) values increased to 60 IU/L by week 4 and reached a peak of approximately 120 IU/L by week 13. Histology showed foci of mononuclear infiltrates in portal and central regions. HCV-inoculated immunocompetent rats tolerized and transplanted with Huh 7 cells support HCV gene expression, viral replication, and develop biochemical and histologic evidence of hepatitis.

Proprioception and Palisade Endings in Extraocular Eye Muscles

Palisade endings occur only in extraocular muscles, and their function is unknown. They form a cuff of nerve terminals around the tips of muscle fibers. We describe here the advantages of using antibodies to a synaptosomal-associated protein (SNAP-25) to study properties of palisade endings in man, monkey, and rat. The stain can be combined readily with other immunofluorescence procedures, and results suggest that the synapses of palisade endings do not bind alpha-bungarotoxin (i.e., are not motor), nor do they contain substance P. These double-labeling data support the hypothesis that palisade endings are non-nociceptive sensory receptors, and could serve a proprioceptive function. With SNAP-25 immunolabeling, palisade endings were identified in the rat for the first time. Thus, palisade endings appear to be present in all vertebrate extraocular muscles studied to date. Their apparent universality, which contrasts with the more variable manifestation of extraocular muscle spindles and Golgi tendon organs, would be expected if proprioceptive feedback is necessary to the function of the ocular motor system, and if palisade endings are the critical proprioceptive structure.

Palisade endings in extraocular eye muscles revealed by SNAP-25 immunoreactivity.

Palisade endings form a cuff of nerve terminals around the tip of muscle fibres. They are found only in extraocular muscles, but no definite evidence for their role in eye movements has been established. Palisade endings have been reported in all species so far investigated except the rat. In this study we demonstrate that antibodies against SNAP-25, the synaptosomal associated protein of 25 kDa, reliably visualize the complete motor, sensory and autonomic innervation of the extraocular muscles in human, monkey and rat. The SNAP-25 antibody can be combined with other immunofluorescence procedures, and is used here to study properties of palisade endings. With SNAP-25 immunolabelling putative palisade endings are identified in the rat for the first time. They are not well branched, but fulfil several criteria of palisade endings, being associated with non-twitch fibres as shown by double labelling with 'myosin heavy chain slow-twitch' antibodies. The putative palisade endings of the rat lack alpha-bungarotoxin binding, which implies that these synapses are sensory. If palisade endings are sensory then they could function as an eye muscle proprioceptor. They seem to be a general feature of all vertebrate eye muscles, unlike the other two extraocular proprioceptors, muscle spindles and Golgi tendon organs, the presence of which varies widely between species.