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PubMedDate not supplied

Age-related changes in the epithelium of the monkey larynx.

An experiment was designed to measure the area of subglottic squamous epithelium in a group of 14 adult monkeys. The animals were much older than any so far available for medical research. The larynges were strained using pyronin Y, which stains respiratory epithelium magenta and squamous epithelium pale pink. The areas of squamous epithelium inferior to the free margin of the vocal cord were measured. The results showed that the subglottic epithelium increased with age (r = +0.61, p = less than 0.05). This finding may have importance in the staging of laryngeal carcinoma.

Aging↗
PubMedDate not supplied

Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2.

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.

Animal Testing Alternatives↗
PubMed1992

Characterization of morphologic changes and lymphocyte subset distribution in lymph nodes from cats with naturally acquired feline immunodeficiency virus infection.

Lymph nodes were collected at biopsy or necropsy from 18 cats with naturally acquired symptomatic feline immunodeficiency virus (FIV) infection and from 18 seronegative cats. Thirty-five of the cats were domestic shorthairs and one was a Persian cross. The cats ranged from 7 months to 16 years of age and were mainly obtained from California veterinary practitioners, a California cattery, and a Veterinary Teaching Hospital. Based on clinical signs present at tissue collection, ten FIV-infected cats fell into the acquired immunodeficiency syndrome (AIDS)-related complex (ARC) clinical stage and eight in the terminal (AIDS) stage of FIV disease. All cats were FeLV negative by antigen ELISA. Histologic sections of lymph nodes from each cat were examined blindly and were categorized as hyperplastic, involuting, mixed hyperplastic and involuting, depleted, or normal based upon subjective evaluation of follicles and paracortex. The relative abundance of plasma cells was evaluated in methyl green pyronin (MGP) and hematoxylin and eosin-stained sections. Similar numbers of FIV-seropositive and -seronegative cats fell into each lymph node category. The only difference evident between FIV-infected cats and control cats was in the degree of plasmacytosis present; moderate to marked plasmacytosis was present in 13/18 FIV-infected cats but in only 3/18 control cats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Cytochemical reactions of human hematopoietic cells in liquid culture.

Growth of human bone marrow in liquid suspension cultures has been used to study normal hematopoietic cell differentiation and abnormalities in blood diseases. A variety of cytochemical stains were applied to human marrow cells cultured in vitro for up to 14 days. AS-D- CHLOROACETATE ESTERASE AND ALPHA-NAPHYHYL BUTYrate esterase were most useful in distinguishing different cell lines in culture. Peroxidase activity disappeared with mononuclear phagocyte morphogenesis and diminished with culture in intermediate and mature granulocytes. Acid phosphatase activity and methyl greed pyronin staining intensity increased with macrophage maturation.

Adult↗
PubMed2001

Replicative stress after allogeneic bone marrow transplantation: changes in cycling of CD34+CD90+ and CD34+CD90- hematopoietic progenitors.

To further characterize hematopoietic "replicative stress" induced by bone marrow transplantation (BMT), the cell-cycle status of CD90+/- subsets of marrow CD34+ cells obtained 2 to 6 months after transplantation from 11 fully chimeric recipients was examined. Cycling profiles, derived by flow cytometry after staining with Hoechst 33342 and pyronin Y, were compared with those of 14 healthy marrow donors. Primitive CD34+CD90+ cells represented a smaller proportion of CD34+ cells in recipients (10% +/- 4% versus 19.6% +/- 5.3% in donors; P <.0001) and were more mitotically active, with the proportion of cells in S/G2/M nearly 4-fold higher than in donors (15.6% +/- 3% and 4.4% +/- 1.6%, respectively; P <.0001). By comparison, there was a modest increase in the proportion of CD34+CD90- progenitors in S/G2/M after BMT (10.9% +/- 1% vs 9.6% +/- 2% in donors; P =.04). Replicative stress after BMT is borne predominantly by cells in a diminished CD34+CD90+ population.

Adult↗
PubMed2002

Early hematopoietic reconstitution after clinical stem cell transplantation: evidence for stochastic stem cell behavior and limited acceleration in telomere loss.

Our inability to purify hematopoietic stem cells (HSCs) precludes direct study of many aspects of their behavior in the clinical hematopoietic stem cell transplantation (HSCT) setting. We indirectly assessed stem/progenitor cell behavior in the first year after HSCT by examining changes in neutrophil telomere length, X-inactivation ratios, and cycling of marrow progenitors in 25 fully engrafted allogeneic HSCT recipients. Donors were sampled once and recipients at engraftment and 2 to 6 months and 12 months after HSCT. Telomere length was measured by an in-gel hybridization technique, X-inactivation ratios were measured by the human androgen receptor assay, and cell cycle status was determined by flow cytometric analysis of pyronin Y- and Hoechst 33342-stained CD34(+)CD90(+) and CD34(+)CD90(-) marrow cells. Compared with their donors, recipients' telomeres were shortened at engraftment (-424 base pairs [bp]; P <.0001), 6 months (-495 bp; P =.0001) after HSCT, and 12 months after HSCT (-565 bp; P <.0001). There was no consistent pattern of change in telomere length from 1 to 12 months after HSCT; marked, seemingly random, fluctuations were common. In 11 of 11 informative recipients, donor X-inactivation ratios were faithfully reproduced and maintained. The proportion of CD34(+)CD90(+) progenitors in S/G(2)/M was 4.3% in donors, 15.7% at 2 to 6 months (P <.0001) after HSCT, and 11.5% at 12 months after HSCT (P <.0001, versus donors; P =.04, versus 2-6 months). Cycling of CD34(+) CD90(-) progenitors was largely unchanged. We infer that (1) HSCT-induced accelerated telomere loss is temporary and unlikely to promote graft failure or clonal hematopoietic disorders and (2) the striking fluctuations in telomere length and variation in pattern of telomere loss reflect stochastic determination of HSC fate after HSCT.

Adult↗
PubMed2001

Segregation of nucleolar components coincides with caspase-3 activation in cisplatin-treated HeLa cells.

We studied morphological changes of the nucleoli in HeLa cells treated with cisplatin and compared them with induction of markers of programmed cell death and TUNEL staining. We used different light microscopic nucleolar staining methods allowing us to visualize not only nucleolar proteins but also nucleolar RNA. Our results show predominantly compact, centrally localized nucleoli in intact control HeLa cells. In cisplatin-treated HeLa cells, we found an early onset of nucleolar segregation of proteins detected by argyrophilic nucleolar organizer regions and anti-nucleolar monoclonal antibody as well as an increased immunoreactivity for activated caspase-3 after 6 hours. Staining with Toluidine Blue and Methyl-green Pyronine revealed segregated nucleoli 12 hours after the treatment with cisplatin. TUNEL positivity in cisplatin-treated HeLa cells was accompanied by the aggregation of the argyrophilic proteins in the central portion of nucleus, disappearance of nucleolar RNA and shrinkage of the nucleus after 24 hours. Monitoring of the biochemical changes by immunoblotting revealed that activation of distinct caspases and degradation of their downstream protein substrates is executed in two phases. During an early apoptotic stage beginning 4.5 hours post treatment an activation of caspase-9 and caspase-3 was observed. This was accompanied by proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The caspase-9 activation seems to be mediated by recruitment by the activating factor Apaf-1 because the increased accumulation of Apaf-1 and cytochrome C in cytosol preceded the generation of mature caspase-9 form. A second phase of apoptosis occurring between 10 and 15 hours post treatment was characterized by degradation of other nucleolar and nuclear proteins such as nuclear lamins, topoisomerase I and B23. In conclusion, remarkable segregation of nucleolar argyrophilic proteins, nucleolar RNA and a simultaneous activation of the cascade of caspases markedly preceded the TUNEL positivity in cisplatin-treated HeLa cells thereby substantiating the hypothesis that the nucleolus is a preferred target for caspase-3-dependent proteolysis in cisplatin-treated HeLa cells.

Apoptosis↗
PubMed1990

Infection rates of Theileria sergenti in Haemaphysalis longicornis ticks collected from the field in Japan.

For a period of one year Haemaphysalis longicornis ticks were collected in a pasture from vegetation by dragging flannel cloth and from soil samples at monthly intervals. Nymphal ticks were assessed for Theileria infection. Salivary glands were stained with methylgreen-pyronin and examined for parasite masses. Nymphal H. longicornis infected with Theileria sergenti were found in all samples during 12 months including the winter. After shortening the prefed period on rabbits to 24 hours, the parasite masses could be detected in the salivary glands of nymphs collected in the grazing season, from May to October, while no parasite masses were detected in other season. It was suggested that the environmental factors in the grazing season might influence on the maturation of parasites in the salivary glands of ticks.

Animals↗
PubMed1990

Maturation of Theileria sergenti in salivary glands of Haemaphysalis longicornis induced by temperature stimulation.

The parthenogenetic Haemaphysalis longicornis larvae engorged on cattle naturally infected with Theileria sergenti were reared at 24 degrees C. The resultant nymphal ticks were incubated at 37 degrees C to clear the effect of incubation on the development and maturation of sporozoites. The sporozoites in the salivary glands of the nymphal ticks exposed to 37 degrees C for 16 days were observed by the methyl green pyronin staining method. The ticks exposed to 37 degrees C were ground up in a mortar and the supernatant of the tick suspension in PBS was inoculated into cattle. The cattle showed parasitemia and specific antibody response 18 days after inoculation. Consequently, the parasites in the tick salivary glands became infective to cattle by incubating infected. H. longicornis nymphs at 37 degrees C.

Animals↗
PubMed2004

Phenotypic and functional analyses of CD34NEG hematopoietic precursors from mobilized peripheral blood.

Methods are described for the characterization of CD34 antigen modulation and its relationship to cell proliferation in early human hematopoietic cells. Toward that end, quiescent primitive CD34+ and CD34NEG cells are purified from mobilized peripheral blood (MoPB). Unlike CD34NEG cells from other sources, those from MoPB grow readily in stroma-free culture, facilitating their analysis. Using a lineage-depleted, low-density mononuclear cell fraction, CD34NEGCD38NEGLINNEG and CD34+CD38NEGLINNEG cells are purified by cell sorting. Cells are cultured in serum-free medium supplemented with early acting cytokines. Up- and downmodulation of CD34 antigen can be observed within 40 h of incubation. Samples are removed for analysis of expression of CD34, CD38 and lineage-commitment antigens as well as for cell proliferation as determined by expression of Ki67 antigen and uptake of pyronin Y. This approach permits an assessment of changes in CD34 and CD38 antigen expression by primitive LINNEG cells as they are activated for growth or remain in a quiescent state.

ADP-ribosyl Cyclase↗
PubMed2000

Histochemical characterization of the lead intranuclear inclusion bodies.

A battery of histological and histochemical techniques was applied on the lead intranuclear bodies that have resuted in the kidneys of adult Wistar male rats receiving lead acetate in their diet to determine their nature. The intranuclear inclusion bodies have stained strongly with xanthene, anthraquinone, and trisulfonated basophilic dyes and weakly with dyes containing both positive and negative radicals, and they have responded negatively to acidophilic cationic dyes. They have also reacted positively to proteins and lead stains, but weakly to lipid stains, and negatively to Feulgen and methyl green pyronin techniques. The intranuclear bodies proved to be lead lipoprotein complexes containing sulfyhydryl groups and are basic in nature with orthochromatic, eosinophilic, argyrophilic, osmophilic, fuchsinophilic, and sudanophilic characteristics.

Animals↗
PubMed1980

Cytological examination of cerebrospinal fluid in eight patients with neuro-Behçet's disease.

In eight patients with Neuro-Behçet's disease cerebrospinal fluid (CSF) cells were studied in relation to the clinical symptoms, clinical course and treatment. The results obtained were as follows: (1) The mean value of CSF cell counts was 126 +/- 177/3 mm3 in 88 specimens from the patients, and 2.9 +/- 2.4/3 mm3 in 30 specimens from healthy control subjects, the former being significantly higher than the latter (p less than 0.01). (2) In the patients' CSF, the average percentages of mononuclear and polymorphonuclear cells were 67 +/- 23% and 32 +/- 23%, respectively. (3) In CSF cytogram from 37 specimens from the patients, the average percentage of small lymphocytes was 59 +/- 19%, which was significantly lower (p less than 0.01) than control (71 +/- 8%). The average percentage of activated lymphocytes, activated monocytes and neutrophils which were not observed in control were 0.5 +/- 1.2%. 1.9 +/- 2.2% and 17 +/- 16%, respectively. (4) The ratio of nucleic acids (RNA/DNA) measured by microspectrophotometry (Zeiss UMSP-1) in CSF lymphocytes stained with methylgreen and pyronin was 0.58 +/- 0.12 in 17 specimens from the patients and 0.55 +/- 0.07% in 10 specimens from the control; there was no significant difference between them. These results suggested that the pleocytosis in Neuro-Behçet's disease may represent perivascular inflammatory process in the central nervous system.

Adult↗
PubMed2003

Morphological quantitative changes in the number of lymphocytes, macrophages and plasma cells in the uterus and lymph nodes of rats exposed to the systemic administration of BCG.

This study was designed to investigate the effect of systemic administration of BCG on the morphological quantitative changes in the number of lymphocytes, macrophages and plasma cells in the uterus and lymph nodes of rats. Thirty female virgin Wistar Albino rats, aging 6 months and weighing between 200-250 g, were assigned to the two experimental groups; BCG treated and controls (n = 15). BCG group received 0.1 ml BCG in tail skin and control group received 0.1 ml saline at the same place. Two weeks after injections, rats in both groups were anesthesized with a high dose of ether and decapitated. Uterus and ileocecal lymph nodes were processed to determine a napthyl acid esterase (ANAE)-positive T lymphocytes and macrophages. The plasma cells were stained with the methyl green-pyronin method. It was found that the numbers of T lymphocytes, macrophages and plasma cells in the uterus and the ileocecal lymph nodes of BCG treated group significantly increased indicating the presence of an immune response to the systemic BCG administration. It was concluded that the systemic administration of BCG increases humoral and cellular immunity in endometrium, myometrium and regional lymph nodes. The immune deficiency system plays an important role in the pathogenesis of endometriosis. Therefore, the endometriosis might be prevented by using periodical administration of BCG. However, further experimental and clinical studies associated with these issue are required.

Animals↗
PubMed2004

Analysis of primitive CD34- and CD34+ hematopoietic cells from adults: gain and loss of CD34 antigen by undifferentiated cells are closely linked to proliferative status in culture.

There is limited understanding of CD34- hematopoietic cells and the linkage between CD34 antigen expression and cell proliferation. In this study, early CD34- CD38- LIN- (CD34-) cells were purified from mobilized adult peripheral blood and carefully analyzed in vitro for growth and modulation of CD34. Mobilized CD34+CD38- LIN- (CD34+) cells were used for comparison. Expression of CD34, CD38, and LIN antigens was determined, and proliferative responses were assessed with PKH tracking dye, expression of Ki67 antigen, and uptake of pyronin Y. Suspension cultures of adult CD34- cells generated CD34+ cells and progenitors for >8 weeks. Stromal cultures demonstrated the presence of long-term culture-initiating cells within the CD34- fraction. While CD34- cells were slower to initiate growth than the CD34+ cells were, no significant difference in hematopoietic cell output was found. Upon cultivation of CD34- cells, CD34 antigen appeared within 48 hours but was restricted to those cells that had initiated growth. Surprisingly, CD34+ precursors lost CD34 expression in culture if they remained in G0 for more than 2 days. Those cells later regained expression of CD34 antigen upon initiation of growth. Comparison of cells that did or did not rapidly modulate CD34 antigen revealed no differences in long-term growth potential. In conclusion, in vitro expression of CD34 by CD34- and CD34+ populations is tightly linked to cellular proliferation. In this culture system, expression of CD34 antigen by LIN- cells constitutes an early hallmark of growth. Measurement of CD34 expression by LIN- cells in expansion culture underestimates the total content of hematopoietic cells.

Adult↗
PubMedDate not supplied

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.

Animals↗
PubMed2004

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.

Animals↗
PubMed1978

Characterization of the DNA-synthesizing cells in rheumatoid synovial tissue.

DNA-synthesizing cells from the rheumatoid arthritis synovial tissue of 7 patients and from 5 patients with traumatic and degenerative joint lesions were studied by incubating fresh synovial tissue samples with tritiated thymidine. Labelled cells were identified using autoradiography. The tissue sections were stained with methyl green pyronine. It was found that 0.66% (range 0.38-1.4) of the cells were labelled with 3H-thymidine whereas in control materials the percentage was 0.17 (range 0-0.38). Approximately half of the labelled subsynovial cells in rheumatoid patients (range 37-67%) were small lymphocytes, according to morphological criteria. About one-tenth of the labelled cells (range 0-17%) looked like medium-sized lymphocytes, while the rest had the characteristics of fibroblasts or were unidentifiable.

Adult↗
PubMed1980

Staining and histochemistry of undecalcified bone embedded in a water-miscible plastic.

Undecalcified bone fixed in a variety of fixatives and embedded in a new formulation of 2-hydroxypropyl methacrylate at 4 c has been sectioned at 1 to 5 microns. The embedding mixture contains 2-butoxyethanol as plasticizer and triethyleneglycol dimethacrylate as cross-linker. The accelerator was benzoyl peroxide and the catalyst was N,N-dimethylaniline. With proper embedding and care in sectioning it is possible to obtain sections with relatively little bone compression, excellent preservation of cellular elements, and a minimum of wrinkling. A wide variety of stains have been used for these sections and those reported here are Gill's hematoxylin-eosin, Nocht's azure-eosin, Feulgen, Hoechst 33258 (bisbenzimid H 33258), methyl green-pyronin, PAS, alizarin red, and von Kossa silver stain. There was excellent preservation of acid and alkaline phosphatase activities. A new method of prestaining immunofluorescent labeling was also applied to bone and examples of staining with anticollagen I and antifibronectin are presented.

Animals↗