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Frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone for two reasons: some skin basement membrane zone proteins are labile to routine chemical fixation, and skin is not amenable to vibratome sectioning. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slide-mounting, usually limit immunohistochemical study to the light microscopy level. In the present study, we introduce use of unfixed, free-floating cryostat sections for characterization of immunolocalizations of selected skin basement membrane proteins at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy. In this regard, the method renders itself useful when results of immunolabeling experiments need to be elucidated quickly at histological and ultrastructural levels as required for diagnostic and accelerated investigative strategies.

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Cutaneous exposure to sulfur mustard (bis(2-chloroethyl) sulfide, HD), a chemical warfare agent, produces a delayed inflammatory skin response and severe tissue injury. Despite defined roles of inflammatory cytokines produced or released in response to skin-damaging chemicals, in vivo cytokine responses associated with HD-induced skin pathogenesis are not well understood. Additionally, there is little information on the in vivo temporal sequence of gene expression of cytokines postexposure to HD. The goal of these studies was to identify in vivo molecular biomarkers of HD skin injury within 24 hours after HD challenge. Gene expression of interleukin 1beta (IL-1beta), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), and interleukin 1alpha (IL-1alpha) in the mouse ear vesicant model was examined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). An increase in IL-1beta mRNA levels was first observed at 3 hours. IL-1beta, GM-CSF, and IL-6 mRNA levels were dramatically increased at 6-24 hours postexposure. IL-1alpha mRNA levels were not increased following HD exposure. Immunohistochemical studies demonstrated that IL-1beta and IL-6 protein was produced at multiple sites within the ear, including epithelial cells, inflammatory cells, hair follicles, sebaceous glands, the dermal microvasculature, smooth muscle, and the dermal connective tissue. An increase in the intensity of staining for IL-1beta, and IL-6 was observed in localized areas at 6 hours and was evident in multiple areas at 24 hours. Positive staining for GM-CSF immunoreactive protein was localized to the inflammatory cells within the dermis. The number of immunostaining cells was increased as early as 1 hour following HD exposure. These studies document an early increase in the in vivo expression of inflammatory cytokines following cutaneous HD exposure. An understanding of the in vivo cytokine patterns following HD skin exposure may lead to defining the pathogenic mechanisms of HD injury and the development of pharmacological countermeasures.

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Metabolically active and proliferating basal cells in the skin are most sensitive to the potent skin blistering chemical warfare compound HD (bis-(2-chloroethyl) sulfide). We previously described a Ca2+-dependent mechanism of HD (0.3-1 mM) toxicity that was inhibited by the cell-permeant Ca2+ chelator BAPTA AM (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester). We describe some cellular effects of BAPTA AM that suggest a mechanism for its protective action. Monolayer log-phase normal human epidermal keratinocytes were incubated (37 degrees C) first in keratinocyte growth medium (KGM) containing BAPTA AM (10-40 microM) for 30 min and then in KGM alone overnight prior to evaluation. The BAPTA AM inhibited cell growth in a concentration-dependent manner with some cellular degeneration above 30 microM (light microscopy). At 20-30 microM, BAPTA AM also inhibited cellular metabolic processes, as evidenced by a lower incorporation of [3H]-thymidine (DNA synthesis, 54 +/- 5%), [3H]-uridine (RNA synthesis, 29 +/- 6%) and [14C]-valine (protein synthesis, 12 +/- 2%) as well as a lower protein content per culture (30 +/- 3%) compared with corresponding untreated controls. However, 20-30 microM BAPTA AM did not cause any demonstrable cytopathology based on morphological (electron microscopy) as well as biochemical (lactate dehydrogenase release, an indicator of cell viability loss) criteria, indicating a lack of acute toxicity. These results suggest that a mechanism of protection by BAPTA AM against HD may be via decreasing some metabolic, and therefore proliferative, rates.

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Whole-body exposure to sulfur mustard (HD) produces cutaneous, respiratory and ocular impairment. Of these, ocular damage causes the most immediate incapacitation. Heretofore, characterization of HD ocular toxicity has been largely limited to gross and histological observations. In the present study we explore histological, ultrastructural and immunopathological acute effects of HD ocular exposure and establish correlations with HD toxicity data already documented for dermal exposure. Anesthetized rabbits were exposed to 0.4 microl of liquid HD placed directly on the cornea. Animals were euthanized at 6, 9 and 24 h post-exposure and the eyes were enucleated and processed for histopathology, ultrastructural and immunoperoxidase study. At 6 and 9 h, the most prominent histological feature was nuclear pyknosis, necrosis and loss of polarity of corneal epithelial basal cells to the exclusion of other epithelial cells. At 24 h, all corneal epithelial cells presented degenerative changes, with the epithelium eventually detaching from the underlying basement membrane at the level of the lamina lucida. Microblisters, a characteristic HD-induced skin pathology of the basement membrane zone of animals, were absent in this corneal study. Edema, degenerating fibroblasts and inflammatory cellular infiltrates were persistent stromal responses. Immunopathological effects included changes in antigenicity of bullous pemphigoid protein, laminin, desmosonal protein, Ki67 and p53. These morphological and immunopathological effects of corneal exposure to HD appear to be largely consistent with that previously reported for dermal exposures, perhaps providing shared anatomical considerations for the development of specific HD prophylaxis and therapy.

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The vesicular neuroexocytosis process consists of two important steps: fusion of transmitter-loaded vesicles at release sites on the presynaptic nerve terminal membrane; followed by the release of transmitter molecules into the synaptic cleft. We previously reported that in nerve growth factor (NGF)-differentiated PC12 cells, arachidonic acid (AA) release is associated with acetylcholine (ACh) release, botulinum neurotoxin A (BoNT/A) inhibits both processes and AA itself or a phospholipase A(2) (PLA(2)) activator can cause ACh release in BoNT/A-poisoned cells in which SNAP-25 has supposedly been hydrolyzed. In the present study, we examined the roles of two endogenous intraterminal components in neuroexocytosis: the membrane fusogenic agent AA; and the vesicle fusion protein SNAP-25. A PLA(2) activator, mastoparan, was used to induce the release of AA and ACh from NGF-differentiated PC12 cells. Release depended upon the mastoparan concentration, as well as Ca(2+) influx via the neuronal-type voltage-sensitive Ca(2+) channels. Release of ACh followed a rise in intracellular free Ca(2+) concentration; the increased Ca(2+) activated PLA(2) and, thereby, increased the AA level. Scanning and transmission electron microscopy confirmed that mastoparan-induced ACh and AA release were not due to simple diffusion through damaged plasma membranes. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K(+) induced ACh exocytosis. The results support the conclusion that PLA(2) and AA have important roles in neuroexocytosis that are independent of SNAP-25. Both PLA(2) and AA have been shown to be involved in actin cytoskeletal organization related to vesicle fusion and exocytosis. This mechanism may be an alternative target of BoNT/A other than SNAP-25.

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Effects of the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were investigated on cytotoxicity in clonal NG108-15 neuroblastoma-glioma hybrid cells. Three min after addition of 100 microM TPEN, cells began to retract their neurites and lose their characteristic multipolar shape; by 3-4 hr of exposure, most cells detached from the substrate, either singly or as variable-sized aggregates. Viability was assessed by monitoring uptake of calcein AM and propidium iodide, fluorescent dyes that served as markers for live and dead cells, respectively. Incubation of cultures in 100 microM TPEN led to a gradual decrease in the population exhibiting calcein fluorescence (viable cells) and a corresponding increase in the population displaying propidium iodide fluorescence (nonviable cells). Loss of cell viability reached 12% at 8 hr, 61% at 24 hr and 83% by 48 hr. Ultrastructural examination of TPEN-treated cells revealed condensed chromatin and fragmented nuclei, characteristic of apoptosis, as well as plasma membrane defects and organelle swelling, generally associated with necrosis. Addition of an equimolar concentration of Zn2+ or Cu2+ but not Fe2+ or Mn2+ prevented morphological abnormalities and cell death.

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Sulfur mustard (2,2-dichlorodiethyl sulfide, HD) is a chemical warfare agent that is a threat to both troops and civilians. The focus of HD research has been on intracellular adduct formation leading to apoptosis and/or necrosis in cutaneous lesions. However, there is work which suggests that HD may have a more direct effect on the basement membrane zone. Immunohistochemical staining to desmosomal proteins, cellular fibronectin, laminin 1, laminin 5, collagen IV, collagen VII, p53, Bcl-2, and PCNA was performed on weanling pig skin exposed to vesicating doses of HD, GB3, an antibody to laminin 5, showed a progressive decrease with loss of expression during the time period of clinical vesiculation. The other basement membrane proteins showed no change or inconsistent changes. PCNA, and p53 staining increased in the overlying epidermis in areas of vesiculation without significant necrosis. Bcl-2 positive cells were decreased or absent after exposure. This study implicates laminin 5 as the main basement membrane protein affected acutely by HD exposure. The patterns of staining of PCNA, Bcl-2, and p53 within the epidermis suggest that apoptosis and cellular necrosis both may play a role in cell death secondary to HD.

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Stimulus-induced acetylcholine (ACh) exocytosis from presynaptic nerve terminals involves two important steps: fusion of ACh loaded vesicles at presynaptic release sites, followed by release into the synaptic cleft. We studied the role of the putative vesicle fusion protein SNAP-25 in this process. The nerve growth factor-differentiated PC12 cell line was used as an experimental model. A bee venom tetradecapeptide (INLKALAALAKKIL-NH2) phospholipase A2 (PLA2) activator, mastoparan, was used to induce ACh release. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K+ induced ACh exocytosis. The results indicate that in PC12 cells, ACh exocytosis due to mastoparan plus K+ can occur in the absence of SNAP-25.

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Although the exact pathogenesis of mustard gas-induced dermal toxicity remains elusive, morphopathological data gathered in controlled animal and in vitro investigations is providing important clues as to approximate mechanisms. Our laboratory has been studying dermal effects of the chemical warfare agent, sulfur mustard, in a variety of animal models, cultured isolated human cells, and in vitro organotypic skin models. Published anatomical, pathological, and ultrastructural results of these studies have documented consistent cellular and basement membrane zone effects irrespective of the model. Cellular effects include the early targeting of basal cells of the stratum basale to the exclusion of other epidermal cells, with nuclear and cytoplasmic indications of cell injury and cell death. Effects on the basement membrane zone include the formation of characteristic microvesicles in the lamina lucida of those models which possessed structural components of a true basement membrane. We are now investigating effects on proteins of the basement membrane microenvironment and correlate in the present paper the morphopathology of sulfur mustard dermal lesions with immunohistochemical study of bullous pemphigoid antigen, laminin, type IV collagen, and type VII collagen.

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The mechanism of action of the alkylating agent bis-(2-chloroethyl)sulfide (sulfur mustard, SM) was studied using the in vitro mouse neuroblastoma-rat glioma hybrid NG108-15 clonal cell line model. Following 0.3 mM SM exposure, cell viability remained high (> 80% of untreated control) up to 9 hr and then declined steadily to about 40% of control after 20-24 hr. During the early period of SM exposure, when there was no significant cell viability loss, the following effects were observed. The cellular glutathione level decreased 20% after 1 hr and 34% after 6 hr. Between 2 and 6 hr, there was a time-dependent increase (about 10 to 30%) in intracellular free calcium (Ca2+), which was localized to the limiting membrane of swollen endoplasmic reticula and mitochondria, to euchromatin areas of the nucleus, and to areas of the cytosol and plasma membrane. Moreover, there was also a time-dependent increase in the release of isotopically labeled arachidonic acid ([3H]AA) from cellular membranes. Increase in [3H]AA release was 28% at 3 hr and about 60-80% between 6 and 9 hr. This increase in [3H]AA release was inhibited by quinacrine (20 microM), which is a phospholipase (PLA2) inhibitor. At 16 hr after SM exposure, there was a large increase (about 200% of control) in [3H]AA release, which was coincident with a 50% loss of cell viability. These results suggest a Ca(2+)-mediated toxic mechanism of SM via PLA2 activation and arachidonate release.

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A commercially available human skin equivalent (HSE) was used as an in vitro organotypic skin model to study temporal morphological effects of sulfur mustard gas (HD). Light and electron microscopic analyses of the HD-human skin equivalent model (HD-HSE) were compared to the HD-hairless guinea pig model (HD-HGP). HSE samples were exposed to 10 microliters HD vapor for 8 min and harvested at selected times up to 24 h. Skin sites of HGP were exposed to the same vapor dose or to 2.0 microliters liquid HD for 30 min and collected at 12 and 24 h. In both models, basal cells of the stratum germinativum were selectively affected. The HD-HSE study revealed that basal cell changes began 3 to 6 h following exposure. These early cellular changes included an acantholysis of some basal cells with widening of intercellular spaces, disruption of desmosomal attachments, nuclear pyknosis, perinuclear blebbing and repositioning of cytoplasmic tonofilaments to a perinuclear position. At 12 to 24 h, basal cell pathology progressed to diffuse swelling of endoplasmic reticula, cytoplasmic vacuolations and necrosis which now extended to supra basal cell layers. Comparing basement membrane zone effects, HD-HGP consistently developed characteristic microblisters at the dermal-epidermal junction; however, HD-HSE with its absence of a morphologically distinguishable basement membrane did not. Instead, cellular fragments, granules and debris accumulated early in this area to thicken regions usually assigned to the lamina lucida and lamina densa of a true basement membrane leading to complete separation of dermis from epidermis at later time periods.

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In the presence of paraoxon, the amplitudes of chloride currents activated by acetylcholine (ACh) or gamma-aminobutyric acid (GABA) were reduced in cell R2 of Aplysia californica. IC50 values were 12 and 9.7 microM for ACh and GABA responses, respectively. Paraoxon did not affect resting membrane potential, input resistance, or chloride reversal potential. Both the slopes and maxima of ACh and GABA concentration-response curves were reduced by paraoxon, suggesting that paraoxon antagonism of these responses is not competitive. The antagonism of ACh and GABA responses by paraoxon was not related to inhibition of acetylcholinesterase.

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The total cholinesterase activity in canine tracheal smooth muscle was found to consist of butyrylcholinesterase and acetylcholinesterase in a ratio of 3:1. Most of the acetyl- and butyrylcholinesterase sites were distributed on the muscle surface; the remaining hydrolytic sites were associated with internal structures. Intracellular acetylcholinesterase staining was associated with the perinuclear envelope, sarcoplasmic reticulum and Golgi apparatus. Intracellular butyrylcholinesterase was associated with the perinuclear envelope, sarcoplasmic reticulum and the contractile filaments. Inhibition of acetylcholinesterase by the selective agent 1,5,bis(allyl-dimethylammoniumphenyl)-pentane-3-one dibromide (BW 284C51) led to a parallel leftward shift in the concentration-response curve for bath-applied acetylcholine. A similar shift was observed in the frequency-response curve for neurally released acetylcholine. Inhibition of butyrylcholinesterase by the selective agent tetraisopropyl-pyrophosphoramide potentiated the response to bath-applied and neurally released acetylcholine; the potentiation was limited to acetylcholine concentrations greater than or equal to 1 microM and frequencies greater than or equal to 10 Hz. It is concluded that both acetyl- and butyrylcholinesterase participate in the hydrolysis of acetylcholine in canine tracheal smooth muscle. The role of acetylcholinesterase is evident over the entire range of concentrations (1 nM to 100 microM) and frequencies (1 to 90 Hz) examined, whereas the role of butyrylcholinesterase is confined to the higher end of the concentration and frequency ranges used.

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Soman, an organophosphorous irreversible inhibitor of acetylcholinesterase, was studied for its effect on the rat blood-brain barrier (BBB) during the first 24 h of intoxication. Young adult male Sprague-Dawley rats, injected with Evans blue-dye and surviving a subsequent single convulsive dose of soman (114 micrograms/kg, 0.9LD50), presented focal and diffuse penetration of dye in areas of brain normally considered protected by the BBB. Invasion was widest during the first hour when signs of excitation, respiratory distress and convulsions peaked and was absent at 24 h. During this time period, cholinesterase inhibition, as measured by enzyme assay, persisted in brain and blood at 10% and 6% of control values respectively. Brains of nonconvulsing animals and animals pretreated with nembutal (45 mg/kg, I.P.) or with diazepam (10 mg/kg, I.P.) were free of extravasated dye. A ranking of dye-breached brain areas suggested that cerebellar and cerebral cortex were most frequently involved while brain stem was rarely stained. Ultrastructural analysis of breached areas with horseradish peroxidase as a tracer molecule, revealed that the probable subcellular mechanism of the induced breach was enhanced vesicular transport, a mechanism similarly described for seizure. Consequences of the breach were emphasized with the detection of significantly elevated levels of an exogenously administered quaternary compound, 3H-hexamethonium. These findings present additional evidence that an anticholinesterase-induced breach of the rat blood-brain barrier is convulsive dependent, demonstrates BBB mechanisms similar to that of seizure, and can allow CNS penetration of blood-borne drugs and circulatory proteins that normally would be slowed or excluded by an intact BBB.(ABSTRACT TRUNCATED AT 250 WORDS)

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Sulfur mustard (HD; 1,1'-thiobis[2-chloroethane]) induces fluid-filled blisters in man but not in conventional laboratory animals. An animal model is needed to emulate both cytotoxic (vesicant) and vascular (irritant) responses of human skin to HD exposures. An acceptable model must permit reproducible comparisons of uniformly graded and dose-related HD control responses with reduced responses that may follow antivesicant treatments. Hairless guinea pigs were evaluated by exposing six or eight dorsal skin sites 12 mm in diameter to similar HD vapor concentrations for graded intervals (1-16 min). HD vapor was delivered under occlusive caps holding 10 microliters of HD in filter paper located 5 mm above the skin. Four-minute exposures induced moderate erythema, slight edema, and microblisters in 1 of 39 sites. Eight-minute exposures induced severe erythema, moderate edema, and microblisters in 31 of 40 sites. Gross blistering was not seen after use of vapor cups, but damage to basal cells resembled lesions of vesicant injury in man. The hairless guinea pig model, with graded HD vapor exposures, provides acceptable comparisons of responses. Exposures of both 4- and 8-min durations were used to show the feasibility of using this model to bioassay antivesicant topical protectants. These methods may be useful for measurements of irritant and cytotoxic responses of skin to other toxic vapors.

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An apparent wasting syndrome was observed in newly arriving 3 to 4 week old guinea pigs characterized by anorexia, weight loss, diarrhea, perineal staining and death. Diagnostic efforts to attribute the disease to husbandry, environmental factors or to known guinea pig pathogens were unsuccessful. Clinical signs, enteric histopathological lesions and diagnostic transmission electron microscopy identification of typical coronavirus-like virions in fecal samples were consistent with enteric coronaviral diseases seen in other species.

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Sulfur mustard (HD) has been shown to cause a concentration-dependent decrease in viability of human lymphocytes in vitro as measured by dye exclusion; this decrease is preventable by inhibitors of poly(adenosine diphosphatase ribose) polymerase such as niacinamide. The present study investigates the morphologic correlates of the protection afforded by niacinamide through scanning and transmission electron microscopic analysis of human lymphocytes incubated in the presence or absence of 10(3) M niacinamide for 24 h at 37 degrees C and exposed in vitro to 10(-3) M HD. Lymphocytes exposed to HD alone demonstrated 30% to 40% viability and loss of microvilli, large cytoplasmic vacuoles, extensive blebbing of the perinuclear envelope, loss of cytoplasmic organelles, condensation of nuclear chromatin, and multiple perforations of the plasmalemma. In the presence of niacinamide HD-treated lymphocytes had a viability of 87% and, except for blunting of the microvilli, essentially normal ultrastructure. Although the sequence of observed ultrastructural changes was not established, results of this morphologic study suggest that, in addition to the prevention of plasmalemmal defects and dye infusion, the mechanism of niacinamide protection appears to include preservation of the morphologic and functional integrity of cellular organelles.

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A biochemical hypothesis explaining the generation of pathology in human skin by mustard gas (HD) is presented which links the initiation of DNA damages to local alterations of metabolism and subsequent development of blisters. The proposed sequence involves HD alkylation of purines in DNA which are processed to form apurinic sites. Apurinic endonucleases act at these sites to produce backbone breaks in DNA which cause activation of the chromosomal enzyme poly(ADP-ribose)polymerase. This enzyme utilizes NAD+ as a substrate and, at vesicating doses of HD, would deplete the cells of their NAD+ content. The depletion in NAD+ would cause inhibition of glycolysis, and the resulting accumulation of common intermediates would stimulate the NADP+-dependent hexosemonophosphate shunt (HMS). Such stimulation of the HMS has been associated with DNA damage and enhancement of protease synthesis and release. These proteases could be responsible for development of subepidermal blisters which result from fluid accumulation in the cavity created by separation of the moribund basal cell layer from the basement membrane--a characteristic feature of HD-exposed human skin. Partial validation of this biochemical hypothesis has been achieved. DNA alkylated with either monofunctional or bifunctional sulfur mustards, followed by spontaneous or enzymatic depurination, was shown to be sensitized to degradation by apurinic endonuclease. Studies on the effect of HD on human skin grafted to athymic nude mice demonstrated dose- and time-related decreases in NAD+ levels. These decreases in NAD+ levels preceded and correlated to the predicted severity of pathology. The participation of poly(ADP-ribose)polymerase activity in the HD-induced NAD+ loss was substantiated by prevention of this loss in the presence of inhibitors of the enzyme. Additional supporting evidence for the proposed mechanism was obtained at the cellular level by studies which utilized human leukocytes. The subsequent involvement of the HMS and proteases in HD-induced vesication is discussed.

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Dermal tuberculous lesions were produced in rabbits with Bacille Calmette Guèrin and biopsied on days 4, 14, 21, 35, and 56. Frozen sections prepared from the biopsy specimens were stained by the peroxidase-antiperoxidase immunocytochemical technique with antisera against whole tubercle bacilli and antisera against the protein B, polysaccharide I, and wax D fractions of tubercle bacilli. In the primary lesions, protein B was often undetectable at 21 days, and polysaccharide I was often undetectable at 35 days. Wax D disappeared more slowly than the other bacillary components evaluated, and some wax D was still found at 56 days (if the lesions had not completely healed). Antisera against intact tubercle bacilli produced results similar to those produced by antiserum against wax D. These studies suggest that the chronicity of tuberculous lesions is due, at least in part, to the persistence of the wax D-like component of tubercle bacilli.

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