Defining mechanisms of action of interleukin-11 on the progression of radiation-induced oral mucositis in hamsters.

Oral ulcerative mucositis is a common toxicity associated with drug and radiation therapy for cancer. It impacts on quality of life and economic outcomes, as well as morbidity and mortality. Mucositis is often associated with dose limitations for chemotherapy or is a cause for dose interruption for radiation. The complexity of mucositis as a biological process has only been recently appreciated. It has been suggested that the condition represents a sequential interaction of oral mucosal cells and tissues, pro-inflammatory cytokines and local factors such as saliva and the oral microbiota. The recognition that the pathophysiology of mucositis is a multifactorial process was partially suggested by the observation that interleukin-11 (IL-11), a pleotropic cytokine, favorably altered the course of chemotherapy-induced mucositis in an animal model. In the current study, we evaluated a series of biologic and morphologic outcomes to determine their roles and sequence in the development of experimental radiation-induced mucositis and to evaluate the effects of IL-11 in attenuating them. Our results suggest that IL-11 favorably modulates acute radiation-induced mucositis by attenuating pro-inflammatory cytokine expression. Data are also presented which help define the pathobiological sequence of mucositis.

Age-related alterations in IL-1beta, TNF-alpha, and IL-6 concentrations in parotid acinar cells from BALB/c and non-obese diabetic mice.

IL-1beta, TNF-alpha, and IL-6 have been implicated in the destruction of parotid gland acinar cells (but not duct cells) in autoimmune sialoadenitis. Here we report the temporal alterations of these cytokines in parotid acinar cells that may lead to this specificity in cell death in the non-obese diabetic (NOD) mouse model for Sjögren's syndrome. Immunohistochemistry on paraffin sections of parotid gland from 5- and 10-week-old BALB/c and NOD mice confirmed the presence of many peri-acinar lymphoid nodules but few T-cells and macrophages between acinar cells. RT-PCR on enzymatically dispersed mouse parotid acinar cells (MPACs) showed no bands for CD3varepsilon, CD20, or F4/80 regardless of mouse strain or age. By ELISA, MPACs from 10-week-old NODs showed a small but highly significant (p<0.003) increase in IL-1beta and a large significant decrease (p<0.008) in IL-6 compared to 5-week-old NODs. Norepinephrine-stimulated amylase release from MPACs was not different regardless of mouse strain or age. These data show that alterations in acinar cell production of IL-1beta and IL-6 in aging NODs precede periductal lymphoid aggregates and acinar cell secretory dysfunction. (J Histochem Cytochem 48:1033-1041,2000)

Calibration and standardization of microwave ovens for fixation of brain and peripheral nerve tissue.

Rapid and reproducible fixation of brain and peripheral nerve tissue for light and electron microscopy studies can be done in a microwave oven. In this review we report a standardized nomenclature for diverse fixation techniques that use microwave heating: (1) microwave stabilization, (2) fast and ultrafast primary microwave-chemical fixation, (3) microwave irradiation followed by chemical fixation, (4) primary chemical fixation followed by microwave irradiation, and (5) microwave fixation used in various combinations with freeze fixation. All of these methods are well suited to fix brain tissue for light microscopy. Fast primary microwave-chemical fixation is best for immunoelectron microscopy studies. We also review how the physical characteristics of the microwave frequency and the dimensions of microwave oven cavities can compromise microwave fixation results. A microwave oven can be calibrated for fixation when the following parameters are standardized: irradiation time; water load volume, initial temperature, and placement within the oven; fixative composition, volume, and initial temperature; and specimen container shape and placement within the oven. Using two recently developed calibration tools, the neon bulb array and the agar-saline-Giemsa tissue phantom, we report a simple calibration protocol that identifies regions within a microwave oven for uniform microwave fixation.

IL-1 beta and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release.

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1 beta and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10(-5) M norepinephrine at 37 degrees C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1 beta and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1 beta (P < 0.03) and IL-6 (P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1 beta and IL-6 were significantly higher (P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1 beta and IL-6 and release these cytokines from their granules after alpha- and beta-adrenergic stimulation.

Immunocytochemical localization of chymase to cytoplasmic vesicles after rat peritoneal mast cell stimulation by compound 48/80.

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 micrograms/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p < 0.01) increases in the area and number of chymase-immunoreactive vesicles per microns2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.

Assessment of preclinical problem-based learning versus lecture-based learning.

Academic performance on a standardized oral comprehensive exam (OCE) was compared for students taught basic science in a problem-based learning (PBL) curriculum and a lecture-based learning (LBL) curriculum. The OCE was administered to the graduating classes of 1991-1994 (n approximately 20/class) six months after completion of their basic science courses. The OCE contained six components including: Organization and Thoroughness, Diagnosis, Primary Treatment Plan, Alternate Treatment Plan, Science and Medical Knowledge, and Dental Knowledge. Six to eight examiners graded each of the students by using a standardized scoring system and by subjective comments. The class of 1991 was taught by LBL, classes of 1993 and 1994 by PBL, and the class of 1992 by an incomplete PBL teaching method. Mean OCE scores were not significantly different between classes; however, the Science and Medical Knowledge component score was significantly better for the class of 1994 than for 1991 (p < 0.05). There was a non-significant 40 percent increase (p = 0.07) in honors and a 269 percent (p < 0.001) increase in cumulative positive examiner comments between 1991 and 1994.

Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

Tumor necrosis factor alpha immunoreactivity of rat peritoneal mast cell granules decreases during early secretion induced by compound 48/80: an ultrastructural immunogold morphometric analysis.

We used fast (seconds) and ultrafast (milliseconds) microwave energy-assisted chemical fixation protocols, postembedding immunogold staining, and a morphometric analysis to investigate the early morphological changes and the TNF-alpha immunoreactivity in the cytoplasmic granules of rat peritoneal mast cells that had been stimulated to secrete by exposure to compound 48/80. Exposure to compound 48/80 induced the development of increased numbers of cytoplasmic granules that exhibited decreased electron density; these granules often also appeared swollen. These granule alterations were accompanied by a significantly decreased proportion of granules that were positive for TNF-alpha immunoreactivity. We also calculated the density of TNF-alpha labeling/mu 2 in both dense (unaltered) and altered granules in specimens. TNF-alpha immunoreactivity was present in dense granules (regardless of whether or not the specimens had been stimulated with compound 48/80) and in cells that were fixed with either fast or ultrafast microwave energy. However, altered granules exhibited a decreased density of TNF-alpha label. These findings show that changes in the immunolocalization and/or density of TNF-alpha immunoreactivity occur very rapidly upon stimulation of rat peritoneal mast cells with compound 48/80.

Rapid primary microwave-aldehyde and microwave-osmium fixation: improved detection of rat parotid acinar cell secretory granule alpha-amylase using a post-embedding immunogold ultrastructural morphometric analysis.

Studies of methods for improved fixation are becoming increasingly important in the field of quantitative immunocytochemistry. We used microwave (MW)-assisted chemical fixation to show improved retention of salivary gland acinar cell secretory granule alpha-amylase detected by a quantitative immunogold method. Blocks (4-mm3) of rat parotid gland were fixed by the following methods: (a) MW irradiation in an aldehyde fixative (AF) for 6 sec; (b) immersion in AF for 1.5 hr; (c) MW irradiation in osmium tetroxide (OT) for 9 sec; (d) immersion in OT for 1.5 hr; or (e) Sequential MW AF, 10 sec, MW OT rapid treatment (SMAORT), 10 sec. Specimens were processed routinely for transmission electron microscopy. Thin sections of Epon-embedded tissues were exposed first to rabbit IgG anti-human salivary alpha-amylase and second to gold-conjugated goat anti-rabbit IgG. Granule area was obtained by a point counting method. Labeling density was calculated as the number of gold particles/micron 2 +/- SD. Specimens fixed in seconds by MW-AF, MW-OT, or SMAORT showed ultrastructural preservation similar to immersion fixation in AF or OT for 1.5 hr. Immunogold labeling density of granule alpha-amylase was highest for SMAORT (874 microns 2) compared to MW-AF (295 microns 2), MW-OT (248 microns 2), routine sequential immersion in AF and OT (229 microns 2), or immersion in OT (no aldehyde) (190 microns 2). This study establishes the improved retention of salivary gland acinar cell secretory granule alpha-amylase and markedly enhanced fixation speed for ultrastructural studies made possible by MW-chemical fixation protocols that use aldehydes and osmium.

Application of microwave fixation techniques in pathology to neuroscience studies: a review.

The introduction of microwave energy into the scientist's repertoire of fixation modalities offers for the first time in relatively large specimens the potential for 'instantaneous' preservation of cellular structure for light and electron microscopy with minimal alteration of cellular biochemistry and antigenicity. Because of the rapid evolution of this new technology, we provide a classification system of newly generated microwave methods as applied to specimen preservation for microscopic analysis. With emphasis on neuronal tissue, we review qualitative and quantitative microscopy data of specimens fixed by two microwave methods in common use: (1) microwave stabilization and (2) fast and ultrafast, primary microwave-chemical fixation. In addition, we provide a table of neuropeptides or proteins in neuronal tissues that are preserved by various microwave fixation methods for histochemistry, immunohistochemistry, and immuno-electron microscopy studies. Commercial microwave ovens have limitations which can result in irreproducible fixation results. Therefore, we present a calibration protocol that is used to identify the best locations for fixation within large cavity (i.e., household) microwave ovens. We also provide a standardization protocol to improve the reproducibility of microwave fixation in calibrated, large-cavity microwave ovens.

Ultrastructural immunogold localization of tumor necrosis factor-alpha to the cytoplasmic granules of rat peritoneal mast cells with rapid microwave fixation.

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional, proinflammatory cytokine, which can be produced by mast cells and several other cell types. We used a newly developed microwave energy-assisted aldehyde fixation method to prepare purified rat peritoneal mast cells for the postembedding immunogold ultrastructural localization of TNF-alpha. These fixation methods were superior to chemical fixation alone in preserving both the ultrastructural morphology and immunoreactive TNF-alpha in rat mast cells. The percent of TNF-alpha-positive mast cells in samples prepared with microwave-assisted fixation in low (84%) and standard (81%) glutaraldehyde concentrations exceeded that for low (56%) and standard (15%) glutaraldehyde concentrations without the assistance of microwave energy. TNF-alpha was identified in the large storage granules of rat mast cells. The percent of positive granules in microwave-assisted standard (44%) and low (40%) glutaraldehyde samples was considerably higher than the percent of positive granules in standard (5%) and low (10%) glutaraldehyde-fixed samples without microwave assistance. This location of TNF-alpha in rat peritoneal mast cells suggests that this cytokine can use the regulated secretory route(s) for release from appropriately stimulated rat mast cells into the microenvironment.

Methods of microwave fixation for microscopy. A review of research and clinical applications: 1970-1992.

Microwave fixation methods are important because excellent preservation of both cell structure and antigenicity can be attained several orders of magnitude faster than by routine chemical fixation methods. Fast and ultrafast microwave fixation have yielded significant logistic advantages over another fast fixation approach-rapid freezing at liquid helium temperatures. For example, specimens used for microwave fixation can be as large as 1 cm3 and cells can remain in suspension. We review in detail both qualitative and quantitative morphologic results obtained by using microwave fixation in sample preparation. We provide tables of biological molecules that are preserved in a variety of human and animal tissues by various microwave fixation methods for histochemistry, immunohistochemistry, cytochemistry, immunocytochemistry, and affinity labelling studies. Limitations of large cavity (e.g., household) microwave ovens often result in irreproducible fixation results. We present calibration and standardization protocols for microwave fixation in large cavity microwave ovens that emphasize a) localization of oven hot spots (i.e., high power) using a neon bulb array, b) magnetron warm-up, c) the use of a water load, d) the use of an agar-saline-Giemsa model to predict the uniformity of irradiation in small samples, e) the use of specimen containers with one dimension less than 1.5 cm, and f) fast specimen handling to prevent conductive heating artifacts after irradiation. Although microwave ovens are commonplace their unique applications in the laboratory environment require special safety considerations, which are reviewed. Advances in microwave technology are providing new means to study the structure-function relationships of cellular and biochemical activities.

A review of rapid microwave fixation technology: its expanding niche in morphologic studies.

Microwave (MW) fixation methods are important because excellent preservation of both cell structure and antigenicity can be attained several orders of magnitude faster than by routine chemical fixation methods. However, because of the limitations of commercial MW ovens, fixation results are often irreproducible. We present a standardization protocol for MW fixation in household MW ovens that emphasizes magnetron warm-up; the use of a water load during sample irradiation, of an agar/saline/Giemsa model to evaluate uniformity of irradiation within the MW cavity, and of specimen containers with one dimension less than 1.5 cm; and fast specimen handling to prevent conductive heating artifacts after irradiation. We describe a prototypic MW device that improves the precision of sample irradiation and fixes blocks of tissue and cells in suspension in milliseconds. The solutions used to immerse the specimen during irradiation influence the specimen morphology. Aldehyde- or osmium-containing solutions used simultaneously with MW irradiation resulted in the best morphologic preservation of specimens up to 1 cm3. Using MW fixation methods and a postembedding, ultrastructural immunogold-labeling approach, we have localized granule chymase and histamine in rat mast cells and amylase in rat parotid acinar cells.

Immunocytochemical localization of histamine in secretory granules of rat peritoneal mast cells with conventional or rapid microwave fixation and an ultrastructural post-embedding immunogold technique.

We used a post-embedding immunogold labeling approach to define the fine-structural localization of histamine in rat peritoneal mast cells that were fixed using either standard aldehyde fixation or a fast microwave-aldehyde fixation method. Specimens were processed routinely for electron microscopy. Thin sections were exposed first to guinea pig antihistamine antiserum and then to gold-conjugated goat IgG directed against guinea pig IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with non-immune sera did not show labeling of mast cells. Adsorption of antihistamine antiserum with purified histamine or histamine bound to agarose showed a significant reduction (p less than 0.005) in granule staining. We also confirmed that our isolation procedures yielded functionally competent mast cells which released histamine when stimulated with sheep anti-rat IgE antiserum or with compound 48/80. These studies define the conditions of fixation for electron microscopy that are appropriate for the localization of histamine in the granule matrix of rat peritoneal mast cells.

Microwave fixation provides rapid, primary fixation for light and electron microscopy and for immunohistochemistry and immunocytochemistry.

A relatively new approach to specimen preservation for morphologic studies uses microwave energy and chemicals. Microwave fixation can produce fixation results equal in quality to chemical fixation methods and equal in speed to freeze fixation methods. The importance of this microwave fixation technology lies in its potential to provide a standardized fixation approach in histopathology, immunohistochemistry, and immunocytochemistry.

Rapid primary microwave-osmium fixation. I. Preservation of structure for electron microscopy in seconds.

Microwave irradiation (MWIr) of tissues immersed in aldehydes has been used to preserve fine structure in seconds. The purpose of this study was to extend these findings to include rapid primary osmium fixation in a microwave (MW) device with a high volume exhaust. Blocks of rat heart and liver were trimmed to approximately 4 mm3 and exposed to 0.2 M symcollidine-buffered 2% osmium tetroxide for a period of 6-7 sec during MWIr (final solution temperature approximately 45 degrees C). We also evaluated rapid fixation of tissues exposed to MWIr simultaneously with immersion in dilute Karnovsky's fixative (6-7 sec to approximately 50 degrees C) followed by MWIr of specimens immersed in osmium (7 sec to approximately 45 degrees C). Tissues were stored in 0.1 M sodium cacodylate buffer (pH 7.3, 4 degrees C) up to 2 weeks and were stained en bloc in uranyl acetate, dehydrated in a graded series of alcohols, and embedded in propylene oxide-Epon sequence. Thin sections were stained with lead citrate and examined by transmission electron microscopy. We demonstrate that fine structural preservation of tissue blocks can be achieved by MWIr in aldehyde and/or osmium in seconds.

Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils.

The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils. This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein. The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome. Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods. Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques. Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum. CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules. These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules. The crystalloid-containing, large, specific granules did not stain for CLC protein. Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein. The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections. The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct "primary" granule population that serves as a major intracytoplasmic repository for this enzyme.

Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy.

It was demonstrated that microwave energy used simultaneously in combination with low concentrations of glutaraldehyde (0.05%) and formaldehyde (2.0%) rapidly preserved light microscopic histology and excellent fine structural details, as well as a variety of cytoplasmic and membrane-bound antigens. Specimen blocks up to 1 cm3 can be fixed in as brief a time as 26 ms using a specially designed microwave device (ultrafast microwave fixation method). The fast microwave fixation method, using a commercially available device, was successfully used to preserve granule-bound rat mast cell chymase which was subsequently detected by a postembedding immunogold procedure. Control of the following parameters is important to the microwave fixation method: (1) specimens with one dimension less than 1 cm; (2) irradiation temperatures lower than 50 degrees C; (3) irradiation times less than 50 s; (4) immediate replacement of the postirradiation solution with cold storage buffer; (5) fixing the specimen within 15 min after it is removed from its blood supply.

Rapid microwave fixation of rat mast cells. I. Localization of granule chymase with an ultrastructural postembedding immunogold technique.

We defined the ultrastructural localization of chymase in rat peritoneal mast cells using standard aldehyde fixation and a newly described microwave fixation method (Login GR, Dvorak AM: Microwave energy fixation for electron microscopy. Am J Pathol 120: 230, 1985; Login GR, Stavinoha WB, Dvorak AM: Ultrafast microwave energy fixation for electron microscopy. J Histochem Cytochem 34:381, 1986) and postembedding immunogold labeling. Thin sections were exposed first to goat IgG anti-rat chymase and second to gold-conjugated rabbit Ig directed against goat IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with nonimmune sera did not exhibit labeling of mast cells. Thin sections treated simultaneously with purified rat mast cell chymase and anti-chymase antibody in competition studies, showed a marked reduction in granule staining. These findings demonstrate that a microwave fixation method can be used to rapidly fix cell suspensions for postembedding immunocytochemical studies.