Biomedical subjects

W C Hall

Publications and source records attributed to W C Hall.

At least 19 recordsLinked to original sources

PubMed2001

Excitatory and inhibitory circuitry in the superficial gray layer of the superior colliculus.

Stratum griseum superficiale (SGS) of the superior colliculus receives a dense cholinergic input from the parabigeminal nucleus. In this study, we examined in vitro the modulatory influence of acetylcholine (ACh) on the responses of SGS neurons that project to the visual thalamus in the rat. We used whole-cell patch-clamp recording to measure the responses of these projection neurons to electrical stimulation of their afferents in the stratum opticum (SO) before and during local pressure injections of ACh. These colliculothalamic projection neurons (CTNs) were identified during the in vitro experiments by prelabeling them from the thalamus with the retrograde axonal tracer wheat germ agglutinin-apo-HRP-gold. In a group of cells that included the prelabeled neurons, EPSCs evoked by SO stimulation were significantly reduced by the application of ACh, whereas IPSC amplitudes were significantly enhanced. Similar effects were observed when the nicotinic ACh receptor agonist lobeline was used. Application of the selective GABA(B) receptor antagonist 3-[[(3,4-dichlorophenyl)-methyl]amino]propyl](diethoxymethyl)phosphinic acid blocked ACh-induced reduction in the evoked response. In contrast, the ACh-induced reduction was insensitive to application of the GABA(A) receptor antagonist bicuculline. The ACh-induced reduction was also diminished by bath application of muscimol at the low concentrations that selectively activate GABA(C) receptors. Because GABA(C) receptors may be specifically expressed by GABAergic SGS interneurons (Schmidt et al., 2001), our results support the hypothesis that ACh reduces CTN activity by nicotinic receptor-mediated excitation of local GABAergic interneurons. These interneurons in turn use GABA(B) receptors to inhibit the CTNs.

Acetylcholine↗

PubMed2001

Humanization and characterization of the anti-HLA-DR antibody 1D10.

1D10 is a previously described antibody that binds to cells from a majority of B-cell malignancies. The current studies were designed to further evaluate the antigen specificity of 1D10 and its potential as an immunotherapeutic agent. Studies with transfectants and immunoprecipitation demonstrated that 1D10 recognizes some, but not all, of the human HLA-DR beta chains. Both normal and malignant B cells can express the 1D10 antigen. A humanized version of 1D10 was produced using CDR grafting. The resulting antibody has an affinity that is similar to that of the parental murine antibody. In addition, the humanized antibody is capable of inducing complement-mediated cytotoxicity, antibody-dependent cell cytotoxicity, and direct apoptosis of 1D10-expressing B cells. Based on these in vitro anti-tumor activities, we conclude humanized 1D10 deserves further evaluation as an immunotherapeutic agent.

Amino Acid Sequence↗

PubMed2001

Disinhibition in rat superior colliculus mediated by GABAc receptors.

The stratum griseum superficiale (SGS) of the superior colliculus contains a high concentration of the recently described GABA(C) receptor. In a previous study, it was postulated that activation of these receptors on inhibitory interneurons functions to disinhibit projection cells that relay visual information to the thalamus and brainstem. To test this model, we used in vitro whole-cell patch-clamp methods to measure effects of GABA and muscimol on EPSCs and IPSCs evoked in rat SGS by electrical optic layer stimulation. The neurons were filled with biocytin for later morphological characterization. As expected, bath applications of GABA and muscimol always strongly depressed evoked PSCs at concentrations of >100 and >1 micrometer, respectively. However, at lower agonist concentrations, which most likely activate GABA(C) but not GABA(A) receptors, effects were not uniform. Evoked responses were suppressed by both agonists in 48% of the neurons, whereas the remaining cells exhibited enhanced responses with increased evoked EPSCs, decreased evoked IPSCs, or both types of change. Most morphologically identified cells with suppressed responses (14 of 17 cells) had morphological characteristics of putative GABAergic interneurons, whereas almost all cells with enhanced responses (8 of 10 cells) had morphological characteristics of projection cells. Finally, all effects of GABA and muscimol at low concentrations were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid, a specific GABA(C) receptor antagonist, but not by the specific GABA(A) receptor antagonist bicuculline. Taken together, these results indicate that in SGS, GABA(C) receptors are predominantly expressed by GABAergic neurons and that activation of these receptors leads to disinhibition of SGS projection cells.

Animals↗

PubMed2001

Pathogenesis of experimental neonatal woodchuck hepatitis virus infection: chronicity as an outcome of infection is associated with a diminished acute hepatitis that is temporally deficient for the expression of interferon gamma and tumor necrosis factor-alpha messenger RNAs.

Surgical biopsies of the liver were obtained from woodchuck hepatitis virus (WHV)-infected neonatal woodchucks at 2 time points before the self-limited or chronic outcomes became obvious by serologic criteria. Following segregation of outcomes, livers were analyzed for intrahepatic type 1 cytokine messenger RNAs (mRNAs) (interleukin 2 [IL-2], interferon gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha]) and leukocyte inflammatory phenotype (IgG+ plasma cells, lysozyme+ macrophages, CD3+ T cells). Baselines were assessed using age-matched uninfected control livers. At week 8 (early acute phase), intrahepatic type 1 cytokine mRNAs were similarly low in both outcome settings and no different from age-matched uninfected controls. This was consistent with the minimal initial viral loads and lack of histologic inflammation at this time. At week 14 (mid-acute phase), changes in viral load between outcome groups related inversely to the intrahepatic inflammatory responses. Animals that eventually became resolved had increased intrahepatic expression of IFN-gamma and TNF-alpha mRNAs and robust inflammation by CD3+ T cells, plasma cells, and macrophages. At the same time point of infection, animals that eventually became chronic carriers had an acute hepatitis involving the same cell types, but at diminished levels, and markedly deficient intrahepatic expression of IFN-gamma and TNF-alpha mRNAs. IL-2 mRNA remained at baseline control levels in both outcome groups. These cotemporal comparisons map a critical deviation in host response to the acute stage of an evolving chronic infection. They strongly suggest that increasing viral load and chronicity as an outcome of neonatal WHV infection result from a temporal deficiency in the acute intrahepatic effector mechanisms mediated by IFN-gamma and TNF-alpha.

Actins↗

PubMed2000

Contribution of superficial layer neurons to premotor bursts in the superior colliculus.

In vitro whole-cell patch-clamp methods were used to examine the contribution of one component of intracollicular circuitry, the superficial gray layer, to the generation of bursts of action potentials that occur in the intermediate layer and that command head and eye movements in vivo. Applying a single brief (0.5 ms) pulse of current to the superficial layer of rat collicular slices evoked prolonged bursts of excitatory postsynaptic currents (EPSCs) in the cells of the intermediate layer. The EPSCs were sufficient to elicit bursts of action potentials that lasted as long as 300 ms and resembled presaccadic command bursts. To examine the contribution of neurons within the superficial layer to the production of these bursts, we determined how superficial neurons respond to the same current pulses that evoke bursts in the intermediate layer. Recordings from 61 superficial layer cells revealed 19 neurons that produced multiple action potentials following stimulation. Nine of these 19 neurons were wide- and narrow-field vertical cells, which are known to project to the intermediate layer and could contribute to producing the EPSC bursts. The remaining cells (n = 42) did not generate trains of action potentials and 21 of these showed only subthreshold potential changes in response to the stimulus. Our results indicate that most superficial cells do not directly contribute to production of the EPSC bursts, but a small number do have the properties necessary to provide a prolonged excitatory drive to the premotor neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1999

Cholinergic projections to the visual thalamus and superior colliculus.

The parabrachial region of the brainstem reticular formation projects to the dorsal lateral geniculate nucleus of the thalamus and to the intermediate gray layer of the superior colliculus. We used the retrograde axonal transport of two fluorescent labels to demonstrate that individual parabrachial cells project to both structures. The results suggest that cholinergic cells of the parabrachial region may coordinate the relay of visuosensory information to the cortex with the onset of orienting movements.

Acetylcholine↗

PubMed1999

Local excitatory circuits in the intermediate gray layer of the superior colliculus.

We have used photostimulation and whole cell patch-clamp recording techniques to examine local synaptic interactions in slices from the superior colliculus of the tree shrew. Uncaging glutamate 10-75 microm from the somata of neurons in the intermediate gray layer elicited a long-lasting inward current, due to direct activation of glutamate receptors on these neurons, and brief inward currents caused by activation of presynaptic neurons. The synaptic responses occurred as individual currents or as clusters that lasted up to several hundred milliseconds. Excitatory synaptic responses, which reversed at membrane potentials near 0 mV, could be evoked by uncaging glutamate anywhere within 75 microm of an intermediate layer neuron. Our results indicate the presence of extensive local excitatory circuits in the intermediate layer of the superior colliculus and support the hypothesis that such intrinsic circuitry contributes to the development of presaccadic command bursts.

Animals↗

PubMed1997

Role of intrinsic synaptic circuitry in collicular sensorimotor integration.

The superficial gray layer of the superior colliculus contains a map that represents the visual field, whereas the underlying intermediate gray layer contains a vector map of the saccades that shift the direction of gaze. These two maps are aligned so that a particular region of the visual field is represented directly above the neurons that orient the highest acuity area of the retina toward that region. Although it has been proposed that the transmission of information from the visuosensory to the motor map plays an important role in the generation of visually guided saccades, experiments have failed to demonstrate any functional linkage between the two layers. We examined synaptic transmission between these layers in vitro by stimulating the superficial layer while using whole-cell patch-clamp methods to measure the responses of intermediate layer neurons. Stimulation of superficial layer neurons evoked excitatory postsynaptic currents in premotor cells. This synaptic input was columnar in organization, indicating that the connections between the layers link corresponding regions of the visuosensory and motor maps. Excitatory postsynaptic currents were large enough to evoke action potentials and often occurred in clusters similar in duration to the bursts of action potentials that premotor cells use to command saccades. Our results indicate the presence of functional connections between the superficial and intermediate layers and show that such connections could play a significant role in the generation of visually guided saccades.

Action Potentials↗

PubMed1997

Reciprocal connections between the zona incerta and the pretectum and superior colliculus of the cat.

The goal of the present experiments was to examine the relationships of the zona incerta with two structures associated with visuomotor behavior, the superior colliculus and pretectum. The experiments were carried out in the cat, a species commonly used in studies of visuomotor integration, and utilized wheat germ agglutinin horseradish peroxidase and biocytin as retrograde and anterograde neuronal tracers. Retrograde axonal transport demonstrated that most cells in the ventral subdivision of the zona incerta project to the superior colliculus. Anterograde tracers demonstrated that the incertotectal terminal field is most dense in the intermediate gray layer, which is the primary source of the descending pathway from the superior colliculus to brainstem gaze centers. Further experiments showed that scattered cells within the intermediate gray layer give rise to a reciprocal pathway that terminates in both the dorsal and ventral subdivisions of the zona incerta. The distribution of both labeled incertotectal cells and tectoincertal terminals extends dorsolateral to the zona incerta proper, between the reticular thalamic nucleus and the external medullary lamina. Electron microscopic examination of labeled tectoincertal terminals demonstrated that they contain mainly spherical vesicles and have slightly asymmetric to symmetric synaptic densities. Labeled terminals were observed contacting labeled cells in the zona incerta, suggesting that the reciprocal pathway may be monosynaptic. The zona incerta is also reciprocally interconnected with the pretectum. The anterior pretectal nucleus provides a dense projection to the ventral part of the zona incerta and receives a sparse reciprocal projection. The posterior pretectal nucleus and nucleus of the optic tract may also project to the zona incerta. The pretectoincertal fibers form terminals that contain primarily spherical vesicles and make distinctly asymmetric synaptic contacts. In summary, these results indicate that the deep layers of the superior colliculus, which are important for controlling saccades, are the target of a projection from the ventral subdivision of the zona incerta. Like the substantia nigra, the zona incerta may play a permissive role in the tectal initiation of saccadic eye movements. The incertotectal terminal field in the cat is less dense than that observed previously in the rat, suggesting species differences in the development of this pathway. An additional finding of this study is that one of the main sources of input to these incertotectal cells is the anterior pretectal nucleus. This pretectal incertal tectal pathway is likely to play a role in the guidance of tectally initiated saccades by somatosensory stimuli.

Animals↗

PubMed1997

Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus.

Neutralizing polyclonal antibody to respiratory syncytial virus (RSV) has been shown to be an effective prophylactic agent when administered intravenously in high-risk infants. This study describes the generation of a humanized monoclonal antibody, MEDI-493, that recognizes a conserved neutralizing epitope on the F glycoprotein of RSV. The affinity of MEDI-493 was found to be equal to or slightly better than an isotype-matched chimeric derivative of the parent antibody. In plaque reduction, microneutralization, and fusion-inhibition assays, MEDI-493 was significantly more potent than the polyclonal preparation. Broad neutralization of a panel of 57 clinical isolates of the RSV A and B subtypes was demonstrated. Pretreatment of cotton rats with MEDI-493 resulted in 99% reduction of lung RSV titers at a dose of 2.5 mg/kg, corresponding to a serum concentration of 25-30 microg/mL. Further, MEDI-493 did not induce increased RSV infection or pathology in either a primary or a secondary challenge.

Amino Acid Sequence↗

PubMed1997

Toxicological properties of several novel oligonucleotide analogs in mice.

The toxicological properties of ISIS 3082, a phosphorothioate oligonucleotide, and five structurally related analogs of ISIS 3082, were examined in Balb/c mice. Comparisons were made between the uniform phosphorothioate oligonucleotide (ISIS 3082), and a 2' propoxy modified phosphodiester (ISIS 9044), a 2' propoxy phosphorothioate (ISIS 9045), a chimeric oligonucleotide comprised of 2' propoxy diester wings and phosphorothioate deoxy center (ISIS 9046), a 5' C18 amine phosphorothioate (ISIS 9047), or a 5' cholesterol modified phosphorothioate (ISIS 8005) oligonucleotide. Oligonucleotides were administered at 50 mg/kg by i.v. bolus injection (tail vein) every other day for 14 days. In general, the spectrum of alterations observed for ISIS 3082 and all of the analogs were relatively similar. Balb/c mice treated with ISIS 3082 were observed to have increases in liver transaminases and a decrease in triglycerides consistent with results from previous studies performed in CD-1 mice. Spleen weights were also increased in ISIS 3082-treated mice, but no histopathological alterations were noted. ISIS 9046 resulted in a toxicity profile that was very similar to that described for ISIS 3082 with the exception of a slightly lower cholesterol level. Alterations induced by ISIS 9045, ISIS 9047 and ISIS 8005 were qualitatively similar to ISIS 3082, but in general more pronounced, with greater reductions in cholesterol and platelet counts, or increases in blood urea nitrogen relative to ISIS 3082. Red blood cell (RBC) counts and hematocrit were also reduced in mice treated with ISIS 9046, ISIS 9047 and ISIS 8005 relative to the ISIS 3082 treatment group. Kupffer cell hypertrophy and basophilic inclusions in Kupffer cells were observed in mice treated with ISIS 9045, ISIS 9047 and ISIS 8005, but not in ISIS 3082-treated mice. A unique renal lesions was noted in mice treated with ISIS 9044 only that was characterized as mild atrophy of proximal convoluted tubules associated with interstitial fibrosis. With the exception of the renal lesions observed in ISIS 9044 treated mice, the toxicity profiles of various oligonucleotide analogs examined in this study were similar to that observed for ISIS 3082.

Alanine Transaminase↗

PubMed1996

Experimental infection of guinea pigs with Venezuelan hemorrhagic fever virus (Guanarito): a model of human disease.

Venezuelan hemorrhagic fever (VHF), a newly described disease caused by an arenavirus (Guanarito), has resulted in multiple human deaths in Venezuela. To develop an animal model of this disease, strain 13 and Hartley strain guinea pigs were inoculated subcutaneously with Guananto strain 95551 of arenavirus in a pilot study to determine susceptibility of the species to the virus. All animals were killed when moribund 12-14 days following inoculation. Animals were necropsied and tissues were fixed and examined by both light and electron microscopy. Viral antigen was demonstrated in the tissues by immunohistochemistry at both the light and electron microscopic levels. Lesions were characterized by single cell necrosis of epithelium of the gastrointestinal tract, interstitial pneumonia, lymphoid and hematopoietic cell necrosis, and the presence of platelet thrombi in occasional blood vessels associated with hemorrhage. Viral antigen was demonstrated in lymphoid tissues and macrophages, endothelial cells of multiple organs, pulmonary epithelium, epithelium of the gastrointestinal tract, and in miscellaneous other tissues and cells. Intact virions and typical arenavirus inclusions were demonstrated by immunoelectron microscopy in these tissues. Based on these findings, the guinea pig appears to be a valid animal model of the human disease.

Animals↗

PubMed1993

Interlaminar connections of the superior colliculus in the tree shrew. I. The superficial gray layer.

One of the most persistent problems in the study of the superior colliculus is the relationship between its superficial and deep layers. The superficial tier of layers is considered to be visuosensory in function, whereas the deep tier is multisensory and has premotor functions. This fundamental distinction is the primary basis for the view that a visually triggered shift in the direction of gaze depends on the transfer of information from sensory cells in the superficial tier to premotor cells in the deep tier. The goal of the present experiments was to examine the interlaminar projections of the superficial gray layer in the tree shrew Tupaia belangeri. We used biocytin as the marker for tracing the pathways. The tree shrew was chosen because its large and distinctly laminated superior colliculus facilitates the task of examining connections between the layers. Biocytin was used because of its sensitivity and because it allowed us to place very small injections restricted entirely to the superficial gray layer. The results demonstrated that a prominent pathway originates in the superficial gray layer and terminates in stratum opticum. In comparison, the projection from the superficial gray layer to the layers beneath stratum opticum is extremely sparse. The pathway from the superficial gray layer to stratum opticum has a columnar distribution, extending about 100 microns rostrally and caudally from the center of the injection site. There were no signs of more remote intracollicular connections, nor of patches or bands of terminals. The biocytin injection sites also labeled pathways to nuclei as distant from the superior colliculus as the diencephalon, including the dorsal and ventral lateral geniculate bodies, and the pulvinar. The results suggest that stratum opticum may serve as a link between the superficial gray layer and the deeper layers.

Animals↗

PubMed1993

Localization of Hantaan viral envelope glycoproteins by monoclonal antibodies in renal tissues from patients with Korean hemorrhagic fever H.

The role of viruses in several renal diseases is not documented clearly. The authors attempted to localize envelope glycoproteins of Hantaan virus in biopsy specimens from patients with Korean hemorrhagic fever (KHF) as evidence of direct viral invasion of renal tissues. The authors studied sequential sections of kidney biopsy specimens from 23 of 35 patients with serologically confirmed KHF diagnosed between June 1985 and December 1989. The sections were stained with the avidin-biotin-peroxidase complex method with monoclonal antibodies to G1 and G2 envelope glycoproteins. Control antibodies of the same isotype were used to rule out nonspecific staining, and hyperimmune rabbit sera or convalescent sera of patients with KHF were used for blocking tests. Normal renal tissues and kidney biopsy tissues from minimal-change nephrotic syndrome were used as negative control sections. The kidney biopsies were performed between the fifth and thirtieth days after onset of fever. The authors detected viral glycoproteins in renal tissues from 22 of the 23 patients. The viral glycoproteins were localized in the cytoplasm of the tubular epithelial cells, and the distribution of viral glycoproteins in the tubules was focal. Glycoproteins also were localized in the cytoplasm of the sloughed renal tubular epithelial cells, where tubular degenerative changes were prominent. These findings suggest the direct invasion of renal tubules by the virus and may partly explain the pathogenesis of acute renal failure in KHF.

Adolescent↗

PubMed1992

Pathway from the zona incerta to the superior colliculus in the rat.

In order to test the proposal that the zona incerta contributes to the generation of orienting movements, we examined the synaptic relationships between the incertotectal pathway and the cells of origin of the predorsal bundle. The predorsal bundle cells give rise to the major premotor pathway from the superior colliculus to the brainstem gaze centers. First, cytochrome oxidase histochemistry, gamma-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) immunocytochemistry, and the axonal transport of markers were used to define the borders of a ventral subdivision of the zona incerta. This subdivision projects topographically to the same sublamina of the intermediate grey layer of the superior colliculus that contains the vast majority of the predorsal bundle cells. Experiments in which incertotectal cells were labeled by both retrograde transport and immunocytochemistry showed that this pathway is GABAergic. Retrograde and anterograde experiments also showed that this pathway is reciprocated by a pathway from the intermediate grey layer of the superior colliculus to the same ventral subdivision of the zona incerta. Finally, experiments combining axonal transport and electron microscopic methods showed that the incertotectal pathway is the source of a monosynaptic GABAergic input to the cells of origin of the predorsal bundle. The ventral subdivision of the zona incerta is contrasted with a second source of GABAergic input to the predorsal bundle cells, the substantia nigra pars reticulata.

Afferent Pathways↗

PubMed1992

The nigral projection to predorsal bundle cells in the superior colliculus of the rat.

Predorsal bundle cells give rise to the major efferent pathway from the superior colliculus to the premotor centers of the brainstem and spinal cord responsible for initiating orienting movements. The activity of predorsal bundle cells is profoundly influenced by an inhibitory pathway from substantia nigra pars reticulata that uses gamma aminobutyric acid (GABA) as a neurotransmitter. The present study examines the morphological basis for this influence of substantia nigra on predorsal bundle cells in the rat. In the first experiments, the laminar distributions of the nigrotectal tract terminals and the predorsal bundle cells were compared. The predorsal bundle cells were labeled by the retrograde axonal transport of horseradish peroxidase from either the decussation of the predorsal bundle or the cervical spinal cord, while the terminations of the pathway from substantia nigra pars reticulata were labeled by anterograde axonal transport from the substantia nigra. Either horseradish peroxidase, wheat germ agglutinin conjugated to horseradish peroxidase, or Phaseolus vulgaris leucoagglutinin were used as anterograde tracers. The results showed that the distributions of both the predorsal bundle cells and the nigrotectal terminals are restricted almost entirely to the intermediate grey layer and that they overlap extensively. Predorsal bundle cells varied in size. Within the areas of maximum overlap, the majority, regardless of size, was closely apposed by nigrotectal terminals. In a second series of experiments, the synaptic contacts between nigrotectal terminals and the tectospinal component of the predorsal bundle were examined in tissue in which both the terminals and the tectospinal cells were labeled for electron microscopy. In the final experiments, the distribution and fine structure of the nigrotectal terminals were compared to those of terminals that had been labeled immunocytochemically with an antibody to glutamic acid decarboxylase, the synthesizing enzyme for GABA. The results showed that nigrotectal terminals contain large numbers of mitochondria and pleomorphic vesicles, and form synaptic contacts with the somas and proximal dendrites of tectospinal cells. These synapses have modest postsynaptic densities. In both their distribution and fine structure, these terminations resemble the glutamic acid decarboxylase immunoreactive terminals that contact tectospinal cells. Taken together, these results support the view that the nigrotectal tract is an important source of GABAergic input to most, if not all, predorsal bundle cells.

Animals↗

PubMed1992

Histopathologic observations in weanling B6C3F1 mice and F344/N rats and their adult parental strains.

Weanling Fischer 344/N (F344) rats and the first filial hybrid of C57BL/6 x C3H (B6C3F1) mice and retired breeders from the parental stocks of these strains were monitored over a 5-yr-period by examining the histopathology of selected organs and comparing those results to viral and mycoplasmal serology and the intestinal tract bacterial flora of each animal on an individual basis. Serology gave no evidence of viral infection, but Mycoplasma arthriditis antibodies were detected. Reactivity of serum of adult C57BL/6 female mice with control cells or media (tissue culture, TC) was seen in a significant number of mice. TC reactivity correlated positively with lymphoid perivascular infiltrates, predominantly of the lungs, suggesting an allergic response in development of the lesions. Other lesions of note consisted of Harderian gland inflammation of rats, focal necrotizing lesions of the liver of both species, and thickening of the pleura and adjacent pulmonary interstitium of weanling rats. Embolization of bacteria from the gastrointestinal tract to the liver was considered a possible cause of the liver necrosis in both species. Although lesions of the lung and Harderian gland of the rats are similar to those caused by known viral agents, the cause of the latter could not be determined as these animals were negative for viral antibodies and the former was considered to be related to incomplete pulmonary development in the young rat. Features differentiating the lesions observed in animals of this survey from those caused by viral infection are discussed.

Aging↗

PubMed1991

Demonstration of yellow fever and dengue antigens in formalin-fixed paraffin-embedded human liver by immunohistochemical analysis.

Two immunohistochemical techniques to determine the presence of yellow fever and dengue antigens in fixed tissue samples were developed for the purpose of making retrospective diagnoses of these viral diseases in humans. A horseradish peroxidase label was used for one technique and an alkaline phosphatase label for the other. In the former technique, acid hematin was removed from the tissues, iron-containing pigments were counterstained with Prussian blue, and the product of the diaminobenzidine reaction was enhanced with a dilute solution of osmium tetroxide that differentiated antigen from lipofuscin. In the latter technique, alkaline phosphatase was used as the enzyme labeling system with a red chromogen that contrasted nicely with the pigments in the tissues, as mentioned above. Thus, pigment removal or differentiation from antigen was not required. Replicate sections were cut and mouse polyclonal antibodies for yellow fever and all dengue types were applied to individual sections. On samples positive for dengue antigen, monoclonal antibodies were applied to additional replicate sections to demonstrate antigen of dengue types 1 and 4. In order to test the assay, samples of formalin-fixed liver tissue from Brazilian and Peruvian individuals who had died from a variety of causes as long as eight years earlier were received in a blinded fashion for immunohistochemical analysis. The techniques appeared to be highly reliable for yellow fever diagnosis; however, not enough cases were observed to adequately evaluate the procedures for dengue diagnosis. Both procedures appeared to have similar sensitivity.

Adolescent↗