Methodical problems for evidence of the hepatitis-B-surface and hepatitis-B-core antigens in tissue (author's transl)]

Liver biopsy material of 22 in the serum HBsAg positive patients was tested with the fluorescent antibody technique for the localization of HBcAg and HBsAg in the liver tissue. Comparative studies were done with the following tissue preparation techniques: Cryostat technique, freeze drying, freeze substitution, cold ethanol paraffin embedding technique (SAINTE MARIE) and isolated liver cells. The investigations revealed the following results: 1. No HB-components could be detected with the cold ethanol paraffin embedding technique and freeze substitution. 2. Using the cryostat technique HBsAg could be demonstrated in 16/22 (cytoplasmatic localization) and HBcAg in 8/22 (nuclear localization). 3. With freeze drying HBsAg and HBcAg could be found in the same cases. The excellent tissue preparation allowed a correct localization of the HB-components to the cell structure. 4. In comparison to cryostat sections in isolated liver cells HBcAg could be demonstrated in 11/16 and HBcAg in 8/8 cases.     

The N-terminal flanking region of the invariant chain peptide augments the immunogenicity of a cryptic "self" epitope from a tumor-associated antigen

The N-terminal flanking region of the invariant chain peptide termed CLIP appears to have superagonistic properties interacting with the T cell receptor and the MHC class II molecule at or near the binding site for the bacterial superantigen Staphylococcal enterotoxin B (SEB). The present studies explored the hypothesis that the N-terminal segment of CLIP can augment the immunogenicity of cryptic "self" tumor-associated antigens. A chimeric construct of an MHC class II binding peptide from the c-erb oncogene (Her-2/neu) containing the N-terminal flanking region of CLIP elicited potent antitumor activity against a Her-2/neu-positive tumor in a rat model system. Comparatively, the unmodified parent peptide was ineffective. The induction of effective antitumor immunity, however, required presentation of the chimeric peptide construct on irradiated tumor cells or the peptide construct in concert with a Her-2/neu MHC class I-restricted peptide from Her-2/neu. As revealed by adoptive transfer studies, effective protective antitumor immunity in this setting required the CD4 T helper subset. Additionally, in vitro analysis revealed that immunization with the parent peptide resulted in a weak immune response to the unmodified peptide consisting of both type 1 (IL-2, IFN-gamma) and type 2 (IL-4, IL-10) cytokine-producing cells analyzed by RT-PCR (qualitative and quantitative) and by limiting dilution assay. Comparatively, immunization with the chimeric construct elicited a potent immune response to the parent peptide with predominantly type 1 cytokine-producing cells. Taken together, the results suggest that immunization with the chimeric Her-2/neu peptide induced protective antitumor immunity. Associated with this immunization strategy was the enhancement of a type 1 cytokine response.     

Correlation between transmission and structure in avian ciliary ganglion synapses

1. Extracellular responses from post-ganglionic axons of pigeon and chick isolated ciliary ganglia were elicited by stimulation of the presynaptic nerve. Intracellular recordings were also obtained from newly hatched pigeon and chick ganglion cells. The fine structure of ganglia from pigeons of various ages was examined with the electron microscope.2. In ganglia from chick embryos and pigeons up to 10 days old, the extracellular response was unimodal with a long latency and could be blocked by the addition of D-tubocurarine (D-TC) or hexamethonium to the bathing solution. A bimodal extracellular response appeared in pigeons about 10 days after hatching. Only the second peak of the response could be blocked by D-TC or hexamethonium. The response recorded from 22 to 26-day-old pigeons was similar to that seen in the adult.3. The intracellular recordings from ganglion cells of 2-week-old pigeons exhibit two post-synaptic potentials elicited by presynaptic stimulation. The first post-synaptic potential appears to be due to current flow through the ganglion cell during the presynaptic action potential. The second is chemically mediated. In pigeons from 1 to 6 days old, only the second post-synaptic potential is observed.4. The presynaptic terminals in the 4-day-old birds were in the form of calyces. In pigeons 7 days old or older, boutons appeared. The boutons were presumably formed as a result of cleavage of calyciform nerve terminals. Myelin was seen first in the 7-day-old pigeon, was well developed in the 16-day-old bird, and persisted in the adults.5. In adult ganglia, the first component of the extracellular response decreased and was finally abolished after 10-12 hr of superfusion with Tyrode solution. The second component of the response increased concomitantly. The only anatomical change noted in the ganglia after soaking was the disruption and separation of the myelin lamellae from each other and from around the ganglion and presynaptic terminals.6. It is concluded that the myelin is necessary for electrical transmission in the pigeon ciliary ganglion.     

Involvement of nitric oxide synthase in the physiology and pathophysiology of facial nerve function and dysfunction

To date few reports have discussed the presence and function of nitric oxide (NO) in structures of the facial nerve. We performed nicotinamide adenine dinucleotide phosphate (NADPH-d)-diaphorase-histochemistry and immunohistochemistry on the intratemporal portion of the facial nerve, including the geniculate ganglion, of guinea pigs using specific antibodies to the three known isoforms of NO synthase and soluble guanylyl-cyclase (sGC). Normal facial nerves were compared to those treated intratympanically with bacterial lipopolysaccharides (LPS) and tumor necrosis factor-α (TNF-α). Both constitutive NOS isoforms and sGC could be detected in the bipolar ganglion cells of normal animals, while the inducible isoform (iNOS or NOS II) was not found. Endothelial NOS (NOS III) and sGC were present in blood vessels and were predominantly found in the perineurial sheath and less in the endoneurium. sGC could be detected in all fibers in a cross section of the facial nerve. LPS and TNF treatment led to the detection of iNOS in the perikaryia of the geniculate ganglion and the perineural sheath. These findings imply that NO may be involved in neurotransmission at least in the visceroafferent system. NO regulates vascular tone of nutrient blood vessels in the perineural sheath and endoneurium. The presence of sGC indicates that NO acts via its second messenger cGMP. NOS II expression may be a contributing factor to facial nerve palsy via two different mechanisms: NOS II-generated NO may lead to an overstimulation of the visceroefferent nerve fibers and motor fibers of the facial nerve. Dysregulation in facial nerve blood vessels could lead to edema and elevated pressure on the nerve within its osseous canal. Received: 13 April 1999 / Accepted: 12 August 1999     

The delivery of aerosolized steroids from MDIs with nozzle extensions: Quantitative laboratory evaluation of a method to improve aerosol delivery to intubated patients

Objective Pulmonary deposition of aerosolized drug from a metered dose inhaler (MDI) is low with intubated patients. In the laboratory, extension of the MDI nozzle to the endotracheal tube tip has been shown to increase the delivered dose of albuterol. The objectives of this study were to determine the dose of aerosolized steroid (beclomethasone and triamcinolone) delivered through a MDI nozzle extension, the effect of nozzle extension length and number of actuations on the delivered dose, and particle size delivered through the nozzle extension.Design A 19-G catheter was used as the MDI nozzle extension. The nozzle extension was attached to a 60-ml syringe via the Luer-Lok connection, and the distal end was directed through a hole drilled into a 15-ml capped tube. The MDI was placed into the syringe and actuated by pressing the syringe plunger. Drug delivered through the nozzle extension into the tube was dissolved in methanol (beclomethasone) or ethanol (triamcinolone). Nozzle extension lengths of 10 cm, 20 cm and 30 cm were studied. For each nozzle extension length, delivery was assessed using one, two, three and five actuations of each drug. Drug remaining in the nozzle extension was recovered by rinsing with the appropriate solvent. Aerosol particle size leaving the nozzle extension was determined using a seven-stage cascade impactor. Beclomethasone and triamcinolone concentrations were determined by spectrophotometry at 239 nm.Setting Respiratory care laboratory of a university teaching hospital.Results For the pooled results, 70.2±14.1% of the dose was delivered through the nozzle extension, with no difference between beclomethasone and triamcinolone (p=0.838). The proportion of drug delivered through the 10-cm extension (76.7±8.4%) was greater than that from the 20-cm (66.1±16.5%) and 30-cm (67.7±13.9%) extensions (p=0.001). Less drug was delivered through the extension with one actuation (54.1±17.7%) than with two (71.2±7.7%), three (77.2±5.5%), or five actuations (78.2±4.3%) (p<0.001). There was a decrease in MMAD with increasing nozzle extension length (3.14±0.61 m for 10 cm, 2.97±0.28 m for 20 cm, 2.37±0.27 m for 30 cm;p=0.005).Conclusions A high proportion of aerosolized steroid was delivered with a MDI actuated through a nozzle extension. The proportion delivered through the nozzle extension was significantly less with longer nozzle extensions and with fewer actuations, but this may not be clinically important. Although particle sizes were smaller from longer nozzle extensions, all were within the respirable range. These results suggest that steroids can be delivered efficiently using a MDI nozzle extension.Presented in part at the 1994 International Conference, ALA/ATS, in Boston, MA, May 23, 1994Work completed in the Respiratory Care Laboratory and Henry K. Beecher Anesthesia Laboratory, Massachusetts General Hospital, Boston, MA. Supported in part by the Puritan-Bennett Corporation and the American Respiratory Care Foundation