Methyl Green-pyronin Staining Research Articles

Secretary IgA Concentrations and plasma Cell Count Changes Associated with the Estrous Cycle in Ewes

Problems statement: The level of uterine Secretory-IgA (S-IgA) and num bers of plasma cells was measured to observe the differences betwe en two stages of estrous cycle (follicular and luteal phase) in the healthy cycling non pregnant e wes. Approach: Twelve ewes were used in this study and they were divided into two groups of 6 an imals each according to the stages of estrous cycle. All ewes were subjected to estrous synchroni zation and allowed to undergo one natural estrous cycle after the removal of the sponge. All animals were then slaughtered at the end of the experiment. The uterine mucus was collected by flushing with a mixture of protease inhibitor cocktail in distilled water. For both stages, the level of uterine S-IgA was quantified by using ELISA and Methyl Green Pyronine staining was used to observe the plasma ce ll in the tissues of the uterine horn and oviduct o f ewe's genital tract. Results: The results were analyzed by independent sample t-t est and presented as mean ±SEM. This study showed the relationship of the estr ous cycle stages to uterine S-IgA concentration (µg mL -1 ) and populations of plasma cell in the healthy non -pregnant cycling ewes. The concentration (µg mL -1 ) of S-IgA (0.20±0.01) in the follicular phase was highly significant (p<0.01) as compared with the luteal phase (0.17±0.002). In add ition, the populations of the plasma cells were significantly higher (p<0.01) in the uterine horn ( 4.97 ±0.32) and oviduct (3.82 ±0.33) during follicular phase compared to the luteal phase (3.87 ±0.30) and (1.90 ±0.21), respectively. Conclusion: The main reason for the immunosuppression during the luteal phase did not fully justified, especially with the presence of potential acquired infection during coi tus in the follicular phase and at the same time immune system should decrease accordingly to prevent newly attached fetus rejection by the mother immune system.

Secretary IgA Concentrations and plasma Cell Count Changes Associated with the Estrous Cycle in Ewes

Problems statement: The level of uterine Secretory-IgA (S-IgA) and num bers of plasma cells was measured to observe the differences betwe en two stages of estrous cycle (follicular and luteal phase) in the healthy cycling non pregnant e wes. Approach: Twelve ewes were used in this study and they were divided into two groups of 6 an imals each according to the stages of estrous cycle. All ewes were subjected to estrous synchroni zation and allowed to undergo one natural estrous cycle after the removal of the sponge. All animals were then slaughtered at the end of the experiment. The uterine mucus was collected by flushing with a mixture of protease inhibitor cocktail in distilled water. For both stages, the level of uterine S-IgA was quantified by using ELISA and Methyl Green Pyronine staining was used to observe the plasma ce ll in the tissues of the uterine horn and oviduct o f ewe's genital tract. Results: The results were analyzed by independent sample t-t est and presented as mean ±SEM. This study showed the relationship of the estr ous cycle stages to uterine S-IgA concentration (µg mL -1 ) and populations of plasma cell in the healthy non -pregnant cycling ewes. The concentration (µg mL -1 ) of S-IgA (0.20±0.01) in the follicular phase was highly significant (p<0.01) as compared with the luteal phase (0.17±0.002). In add ition, the populations of the plasma cells were significantly higher (p<0.01) in the uterine horn ( 4.97 ±0.32) and oviduct (3.82 ±0.33) during follicular phase compared to the luteal phase (3.87 ±0.30) and (1.90 ±0.21), respectively. Conclusion: The main reason for the immunosuppression during the luteal phase did not fully justified, especially with the presence of potential acquired infection during coi tus in the follicular phase and at the same time immune system should decrease accordingly to prevent newly attached fetus rejection by the mother immune system.

Immunohistochemical identification of messenger RNA-related proteins in basophilic inclusions of adult-onset atypical motor neuron disease

This report concerns an immunohistochemical investigation on RNA-related proteins in the basophilic inclusions (BIs) from patients with adult-onset atypical motor neuron disease. Formalin-fixed, paraffin-embedded sections of the motor cortex and the lumbar spinal cord were examined. The BIs appeared blue in color with H&E and Nissl stain, and pink with methylgreen-pyronin stain. Ribonuclease pretreatment abolished the methylgreen-pyronin staining, suggesting that the BIs contained RNA. Immunohistochemically, the BIs were distinctly labeled with the antibodies against poly(A)-binding protein 1, T cell intracellular antigen 1, and ribosomal protein S6. These proteins are essential constituents of stress granules. In contrast, the BIs were not immunoreactive for ribosomal protein L28 and decapping enzyme 1, which are core components of transport ribonucleoprotein particles and processing bodies, respectively. Moreover, the BIs were not immunopositive for TDP-43. Our results imply that translation attenuation could be involved in the processes of BI formation in this disorder.

Appearance of Apoptotic Cells and Granular Cells in the Silkworm Midgut Lumen During Larval-Pupal Ecdysis

To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.

Trichodinids (Ciliophora: Peritrichida) parasitic on gills of freshwater fishes, Carassius auratus and Aristichthys nobilis from China, with the description of Trichodina subtilihamata sp. nov.

Five trichodinid ectoparasites (Ciliophora: Peritrichida) were isolated from two freshwater fishes, Carassius auratus and Aristichthys nobilis collected from Chongqing, China. Trichodina subtilihamata sp. nov. is described from the gills of Carassius auratus and characterized by a slender hooked blade. The other four species of trichodinids are: Trichodina mutabilis Kazubski &amp; Migala, 1968, Trichodina uniforma Van As &amp; Basson, 1989, Trichodina nigra Lom, 1960 and Trichodina kazubski Van As &amp; Basson, 1989. Comparative descriptions, with photomicrographs and morphometric data, are presented of these trichodinids to establish locality records. Descriptions presented here were obtained by examinations of specimens prepared using the dry silver nitrate impregation and the methyl green-pyronin staining. Comparisons with closely related species are provided for the new species.

Polyamines in spermatocytes and residual bodies of rat testis

Immunocytochemistry for polyamines in the rat testis revealed intense staining of small bodies close to the lumen of seminiferous tubules of spermatogenic stage VII and VIII as well as of spermatocytes. Methyl green-pyronin and propidium iodide staining combined with DNase or RNase predigestion showed that the small bodies contained RNA, but not DNA and double fluorescence staining showed that polyamines (PAs) colocalized with RNA in the bodies. Electron microscopy confirmed the absence of nuclei in the bodies and revealed that PA immunoreactivity was associated with ribosomes. These results strongly suggest that the small bodies correspond to the residual bodies, and agree with previous results showing localization of PAs to ribosomes in neurons and gastrointestinal epithelial cells. The accumulation of PAs in residual bodies may reflect a termination of their role in spermiogenesis with respect to protein synthesis.

Diagnosis of chronic endometritis in biopsies with stromal breakdown

Plasma cells are the hallmark of chronic endometritis but are not specific for upper tract infection. Plasma cells have also been noted in hormonally mediated endometrial disorders in association with gland architectural changes ("disordered proliferative" and "anovulatory" patterns), and stromal breakdown. We reviewed benign endometrial biopsies diagnosed at Beth Israel Deaconess Medical Center over a 2-year period described as disordered/anovulatory patterns +/- stromal breakdown. Cases were excluded if tissue was not available; women were younger than 50 years where most diagnoses were atrophic or cancer; or diagnoses were secretory, menstrual endometrium, or polyps. The remaining 61 cases were compared to 33 samples of unremarkable proliferative endometrium. Plasma cells were quantified on hematoxylin and eosin-stained sections and using a histochemical stain methyl green pyronin. The indication for biopsy was an abnormal pattern of bleeding in 34 cases, infertility workup in 7, incidental part of workup for pain, or other findings in 5. The majority of disordered proliferative endometrium had plasma cells (61% grade 1, 17% grade 2) all seen on methyl green pyronin staining only. Two thirds of proliferative endometrium with breakdown showed plasma cells (19% grade 1, 39% grade 2, 10 % grade 3). Plasma cells were rare in inactive endometrium and noted in only 18% of unremarkable proliferative endometrium, all grade 1. Plasma cells are commonly present in the endometrium of women with dysfunctional uterine bleeding and focal stromal breakdown. Given the lack of clinical evidence for infection, the inflammation likely represents a physiologic process.

Infectivity rate and transmission potential of Hyalomma anatolicum anatolicum ticks for Babesia equi infection

The infectivity rate of Babesia equi in the salivary glands of Hyalomma anatolicum anatolicum was assessed. The hungry nymphs were fed on a donkey experimentally infected with B. equi. The engorged dropped-off nymphs were collected at different levels of parasitaemia and kept in BOD incubator. After ecdysis, the hungry adults were prefed on rabbits for different time intervals, thereafter the salivary glands were dissected out and acini were examined after methyl green pyronin (MGP) staining. A total of 134 male and 139 female ticks were dissected out. Average infected acini per tick were found to be significantly higher ( p < 0.05) in male as compared to the female ticks. Further, maximum infected acini in both male and female ticks were found at 24 h of prefeeding on rabbits and overall infected acini per tick increased with rise in parasitaemia. The release of infected ticks on susceptible donkeys resulted in development of clinical babesiosis.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

BackgroundGene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays.ResultsThe MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases.ConclusionRNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

The calyx fluid of Microplitis rufiventris parasitoid and growth of its host Spodoptera littoralis larvae

The morphology of the female reproductive system of pupal and adult stages of Microplitis rufiventris and the ultrastructure of the ovaries are described and illustrated. Two morphologically distinct types of particles of nuclear origin, i.e., polydnavirus (PDV) and a virus-like filamentous particle (VLFP) were detected in the ovarian calyx fluid of the female wasp. It is likely that these particles are injected into the host during oviposition. PDV initiated replication in the calyx of mid-aged pupae and in pharate adults and were present throughout adult life. VLFP were only seen in the calyx fluid of newly emerged adults, and therefore observed after the PDV. Feulgen and methyl-green pyronin staining revealed the presence of DNA in both types of particles. The effects of injection of Spodoptera littoralis larvae with a combination of parasitoid viruses and venom of M. rufiventris females (CxFV) were investigated and the results were compared with two control groups, i.e., larvae injected with Pringle's saline (PS) and naturally parasitized larvae. CxFV-larvae showed significant declines ( P < 0.05 ) in food consumption, weight of ejected faeces and weight gain when compared with PS-larvae. However, naturally parasitized larvae (parasitoid egg+CxFV+ovarian protein) displayed a high significance score ( P < 0.01 ) in comparison with those of PS-larvae. The approximate digestibility (AD) values of S. littoralis larvae were positively affected as early as day 2 post-treatment by either injection of CxFV or parasitization. However, a reduction in AD was observed in both PS- and CxFV-larvae on day 3–7 in comparison with naturally parasitized larvae. Other indices of food utilization were unchanged in CxFV-larvae when compared to saline treated or parasitized controls.

Tissue Tropism and Bursal Transformation Ability of Subgroup J Avian Leukosis Virus in White Leghorn Chickens

In Experiment 1, a monoclonal antibody against the envelope glycoprotein (gp85) of subgroup J avian leukosis virus (ALV-J) was used to study the distribution of ALV-J in various tissues of White Leghorn chickens inoculated as embryos with the strain ADOL-Hcl of ALV-J. At 2 and 6 wk of age, various tissues from infected and control uninfected chickens were tested for the presence of ALV-J gp85 by immunohistochemistry. In Experiment 2, using the methyl green-pyronine (MGP) stain, sections of bursa of Fabricius (BF) from chickens of line 15I5 x 7(1), inoculated with ALV-J or Rous-associated virus-1 (RAV-1), a subgroup A ALV, at hatch were examined for transformation of bursal follicles at 4 and 10 wk of age. In Experiment 1, specific staining indicative of the presence of ALV-J gp85 was noted at both 2 and 6 wk of age in the adrenal gland, bursa, gonads, heart, kidney, liver, bone marrow, nerve, pancreas, proventriculus, spleen, and thymus. In Experiment 2, by 10 wk of age, transformed bursal follicles were detected in MGP-stained sections of BF in only one of five (20%) chickens inoculated with ALV-J at hatch, compared with five of five (100%) chickens inoculated with RAV-1. The data demonstrate distribution of ALV-J gp85 in various tissues of White Leghorn chickens experimentally inoculated as embryos with the virus. The data also confirm our previous observation that ALV-J is capable of inducing transformation of bursal follicles, albeit the incidence is less frequent than that induced by subgroup A ALV.

Assessment of contact allergens by dissociation of irritant and sensitizing properties

The human organotypic skin explant culture (hOSEC) model is a promising alternative in vitro model for screening contact allergens. In this model, the chemical-induced migration of Langerhans cells (LCs) out of the epidermis, evaluated after a 24-h exposure period, is used as a measure of sensitizer potential. As skin irritants can also induce LC migration it is essential that concentrations of test chemicals are used that are not even weakly irritant. Using the hOSEC irritation model chemicals are classified as weak irritants if they are toxic after a 48-h exposure period. Toxicity is determined by methyl green-pyronine (MGP) staining of hOSEC. We studied three frequently used non-sensitizing skin irritants and six potent or frequent human sensitizers in a dose–response. A complete discrimination between non-sensitizers and contact sensitizers was obtained for the chemicals tested when the concentrations used were lower than the weak irritant concentrations. Frequency of positive allergen reactions in patch test of human populations correlated with the difference between weak irritant concentrations and the lowest concentration inducing significant LC migration. Sensitizer potency correlated with chemical irritancy as determined by keratinocyte death. For the compounds tested, the hOSEC model predicted allergenicity in humans better than the guinea pig maximization test and the mouse local lymph node assay.

Response of White Leghorn Chickens of Various Genetic Lines to Infection with Avian Leukosis Virus Subgroup J

In Experiment 1, chickens from various white leghorn experimental lines were inoculated with strain ADOL-Hcl of subgroup J avian leukosis virus (ALV-J) either as embryos or at 1 day of age. At various ages, chickens were tested for ALV-J induced viremia, antibody, and packed cell volume (PCV). Also, at 4 and 10 wk of age, bursal tissues were examined for avian leukosis virus (ALV)-induced preneoplastic lesions with the methyl green-pyronine (MGP) stain. In Experiment 2, chickens harboring or lacking endogenous virus 21 (EV21) were inoculated with strain ADOL-Hcl of ALV-J at hatch. All embryo-inoculated chickens in Experiment 1 tested positive for ALV-J and lacked antibody throughout the experimental period of 30 wk and were considered viremic tolerant, regardless of line of chickens. By 10 wk of age, the incidence of ALV-J viremia in chickens inoculated with virus at hatch varied from 0 (line 0 chickens) to 97% (line 1515); no influence of ALV-J infection was noted on PCV. Results from microscopic examination of MGP-stained bursal tissues indicate that ALV-J can induce typical ALV-induced transformation in bursal follicles of white leghorn chickens. Lymphoid leukosis and hemangiomas were the most common ALV-J-induced tumors noted in chickens in Experiment 1. At termination of Experiment 2 (31 wk of age), 54% of chickens harboring EV21 were viremic tolerant compared with 5% of chickens lacking EV21 after inoculation with ALV-J at hatch. The data indicate that genetic differences among lines of white leghorn chickens, including the presence or absence of EV21, can influence response of chickens to infection with ALV-J.

An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures

We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen sections using a modified methyl-green pyronine (MGP) staining procedure, was used as a marker of irritancy. A decrease in epidermal RNA after a 4-, 24- or 48-h exposure to a certain concentration of a test chemical equated to a MGP score of 3, 2 or 1, respectively. The MGP score was 0 if there was no keratinocyte cytotoxicity after a 48-h exposure. A minimum of three donors were used per chemical and the average MGP score was used to classify the chemical as irritant or not. Chemicals with an average MGP score ⩾1.5 were classified as irritants (R38), at that concentration. Chemicals with a MGP score <1.5 were not classified (NC), at that concentration. The results obtained using human skin in vitro were compared with published data obtained using cultured porcine skin, the cutaneous Draize test (from this point referred to as the “rabbit skin irritation test”) and volunteer studies. There was an excellent correlation between the classification of a chemical, as R38 or NC, based on hOSEC and results of volunteer studies. The hOSEC model predicted perfectly the irritation hazard of the 22 chemicals for which volunteer data were available. The porcine OSEC correctly predicted the classification of 21 of 22 (95%) chemicals and the rabbit skin irritation test correctly predicted the classification of 14 of 15 chemicals (93%) for which data were available. In conclusion, MGP staining of human skin explant cultures can be used to predicted human skin irritancy in vivo. In addition, the data validate the use of porcine skin as an alternative to human skin for screening for dermal irritants in vitro.

Pathological evaluation of rat endometriosis model

Objective: To clarify a pathogenesis and evaluate a therapeutic effect in a disease, it is important that experimental animal model is established and that the lesions in the model are examined pathologically. Experimental rat endometriosis has been developed by uterine autotransplantation already, and has been used in various investigations of endometriosis. However, almost all previous reports are only focused on implanted uterine tissues, therefore, little is known about morphology of the lesions in detail, specifically in peritoneum attached uterine transplants. Design: In this experiment, we induced rat endometriosis by surgical autotransplantation of uterine tissue and examined morphological alterations of uterus-attached peritoneum with light and electron microscope following the time lapse after the implantation. Materials/Methods: SLC-Sprague-Dawley Rats (8-week old) were maintained on a 12-hour (light) to 12-hour (dark) for 2 weeks, and these rats were used for induction of endometriosis models by the surgical autotransplantation technique of uterine square (5 × 5 mm). The mesenteries were autotransplanted as a comparative control. The implanted areas of peritoneum including abdominal muscle were obtained from anesthetized rats at 4, 7, 14 days after uterine autotransplantation and were examined under a light and electron microscope. To detect DNA fragmentation, these samples were analyzed by TUNEL method. In order to examine localization of mast cells and plasma cells in each specimens, these were performed with Toluidine blue stain (for mast cells) and Methylgreen pyronin stain (for plasma cells). Results: On our light and electron microscopic observations in rat endometriosis models, stromal tissues of uterus-attached peritoneum showed proliferation and infiltration of mast cells, eosinophil, plasma cells, lymphocytes and macrophages. These lesions increased following time lapse after implantation, however, these infiltrated cells disappeared and the proliferation declined finally. Our findings suggest that uterine autotransplantation induced infiltration of allergic inflammatory-related cells and proliferative lesion in peritoneal stroma attached endometrial epithelium, these lesions in rat models are similar to those in endometriosis patients. Conclusions: Our findings suggest that uterine autotransplantation induced immune response, which may be hypersensitivity reaction, in peritoneal stroma attached endometrial epithelium. We believe that these morphological data of rat endometriosis are very important to clarify pathogenesis and progress of human endometriosis. Supported By: No support.

Immunohistochemical Study of Interstitial Keratitis in an Animal Model

Purpose: We performed an immunohistochemical study on the development of interstitial keratitis in a rabbit model sensitized by ovalbumin (OA). Methods: A mixture of OA (5 μg/mL) and Freund's complete adjuvant was initially injected subcutaneously (0.4 mL) into the rabbit's back and footpad (0.2 mL). After a week, the rabbits were sensitized by an injection of the same solution into the back (0.5 mL) again. After 4 weeks the rabbits underwent the same treatment as a booster shot. Then a week later OA solution (0.05 mL) was injected into the corneal stroma. We observed the cornea on days 1–3, 7, and 10 after the corneal treatment. Corneas on days 3 and 10 were examined by immunohistological methods using hematoxylin-eosin stain, methylgreen pyronin stain, and by immunohistochemical methods with anti-CD 4, anti-CD 8, and anti-OA antibodies. The localization of specific antibodies was identified by fluorescent-antigen method. Electron-microscopic observation was also done. Results: Rabbits developed corneal opacity and edema on day 1, corneas had on immune ring with a peak intensity on day 3, and vascularization on day 7 after corneal treatment. Microscopic examination revealed infiltration of neutrophils into the area of the immune ring on day 3 and many plasma cells at the limbus and stroma on day 10. CD 4 positive cells were found at the limbus and stroma on days 3 and 10. CD 8 positive cells were found at the limbus and stroma only on day 10. OA positive findings were observed at the immune ring and its inner area. In the fluorescent-antigen method, specific antibodies were found in plasma cells at the limbus on days 3 and 10. Conclusions: In our experimental model of interstitial keratitis with immune ring and vascularization, the Arthus reaction was dominant. Infiltration of antigen specific plasma cells in the stroma with vascularization caused a modification of pathologic reaction in keratitis.

Methyl green-pyronine staining of porcine organotypic skin explant cultures: an alternative model for screening for skin irritants.

We describe a new alternative method for screening for skin irritants by using fresh intact porcine skin biopsies. Test chemicals were applied to the epidermis of the biopsies, which were then incubated for different times in tissue culture medium at 37°C and with 5% carbon dioxide. A decrease in epidermal keratinocyte RNA staining, visualised in frozen sections by using a modified methyl-green pyronine (MGP) staining procedure, was employed as a marker of irritancy. If a decrease in epidermal RNA was observed after incubation for 4 hours (strong irritant), the chemical had an MGP score of 3; if after incubation for 24 hours (moderate irritant), the MGP score was 2; and if after incubation for 48 hours (weak irritant), the MGP score was 1. If no keratinocyte cytotoxicity was observed after incubation for 48 hours, the chemical was classified as non-irritant (MGP score = 0). At least three ears were used per chemical. The average MGP score was used to classify the chemical. Based on the MGP score for 20% sodium dodecyl sulphate, chemicals classified as strong or moderate irritants by using the MGP test were grouped together as category R38 chemicals. Weak irritants or non-irritants were not classified (NC). The MGP staining correctly identified 23 of 25 skin irritants for which reference data were available.

Focal Necrotizing Endometritis

From routine sign-out of endometrial biopsy specimens, a group of 15 endometria were identified that have a characteristic histologic pattern of inflammation that is not included in present classifications of endometritis. All but one of the women were premenopausal, and all presented with abnormal vaginal bleeding. The lesion is characterized by a patchy, focal inflammation, usually composed of lymphocytes with a variable number of neutrophils and rare macrophages centered around endometrial glands and extending into the glandular lumen with disruption and partial or subtotal necrosis of the endometrial glandular epithelium. These foci were widely dispersed, never confluent, and could be overlooked easily. Plasma cells were not found in any of the endometrial specimens despite methyl green pyronine staining of the samples. Based on the histologic characteristics of this process we have designated it focal necrotizing endometritis. The clinical significance, if any, of focal necrotizing endometritis is currently unknown.

A reappraisal on the modes of cell death in pilomatricoma.

The modes of cell death in pilomatricoma were analysed using 27 nodules. The shadow cells with lost nuclei and preserved cytoplasms were morphologically considered to be derived from basaloid cells via transitional cells, which showed gradual loss of nucleic acid by Feulgen reaction and methylgreen-pyronin stain and no nuclear fragmentation. This process was regarded as cell death associated with terminal differentiation into hair. Amorphous debris among the shadow cells contained many fragmented nuclei (apoptotic bodies), which were directly derived from basaloid cell layer focally undergoing apoptosis and did not have any transition to transitional cells. Although in situ 3'-tailing reaction for a detection of dying cells labelled both transitional cells and apoptotic bodies in the amorphous debris, these two components were considered to be on different histogenetic pathways. Immunostainings for the Bcl-2 family revealed no expression of Bax, Bad or Bak protein in any components, whereas Bcl-2 protein was detected in the periphery of basaloid cell layer, but not in the transitional or shadow cells. These results indicate that there are two different modes of cell death in pilomatricoma: i) terminal differentiation into shadow cells; and ii) conventional apoptosis observed as amorphous debris.

In situ localization of heat-shock and histone proteins in honey-bee (apis mellifera l.) larvae infected with paenibacillus larvae

The immunohistochemical localization of the heat shock proteins (Hsp70 and Hsp90) and histone protein in healthy and Paenibacillus larvae infected honeybee (Apis mellifera L.) larvae has been studied. Hsp70 was found in the nuclei and the cytoplasm of infected midgut, salivary gland cells and haemocytes, but not in uninfected larvae. Hsp90 was localized in both infected and uninfected cells. Exposed histone proteins were localized in the nuclei of dying uninfected cells undergoing programmed cell death. The distribution of histone protein in uninfected cells of midgut, salivary gland, and other tissues was nuclear and indicative of normal programmed cell death at levels between 1 and 5%.After applying histone protein antibodies to P. larvae infected honeybee larvae, the DAB based reaction product was located in the nuclei or immediate surroundings of all larval cells. The Hsp70, Hsp90 and histone protein distribution patterns are discussed in relation to the morphological, cytochemical and immunocytochemical characteristics of programmed cell death and pathological necrosis. Results produced by methyl green-pyronin staining confirm an elevation of RNA levels in normal programmed cell death and a reduced staining for RNA in necrotic infected cells.