Indirect Fluorescent Technique Research Articles

Infection rate by TOXOPLASMA GONDII in free-range and broiler chickens in the states of Rio Grande do Norte and Paraíba, Brazil

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, a protozoan that has a cosmopolitan geographic distribution and low specificity for intermediate hosts. Domestic chickens are among the most important hosts in toxoplasmosis epidemiology, since they are potential sources of infection for humans, in addition to indicating environmental contamination by protozoa. In this work, we studied the prevalence of T. gondii infection in chickens in different breeding systems from distinct mesoregions of Rio Grande do Norte and Paraiba States: broiler chickens from commercial farms and free-range chickens from small farms. Indirect fluorescent antibody test (IFAT) and ELISA techniques were used for detecting specific antibodies in blood samples from the birds. There were no seropositive samples among the broilers tested, indicating that intensive management limited the chances of infection for these animals. Amongthe free-range chickens, the frequency of IgG anti-T. gondii detected by IFAT and ELISA were 37.9% and 40.4% respectively. Among the seropositive samples by IFAT, 33 (27.1%) were positive at a dilution of 1:16; in 1:32, 31 (25.4%); in 1:64, 24 (19.7%); 15 (12.3%) in 1:128, and 19 presented titers greater than or equal to 1:256 (15.6%). The substantial concordance observed between IFAT and ELISA signifies these methods are effective methodologies for the diagnosis of avian toxoplasmosis. The high prevalence of specific antibodies among poultry in the studied region highlights the potential risk of T. gondii transmission to humans through consumption of infected meat.KEY WORDS: Toxoplasma gondii; chicken infection; toxoplasmosis transmission; seroprevalence.

Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates.

The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum . PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples.

Structural and Mechanical Effects of Calcium on the Lipid Bilayer

Calcium is a ubiquitous divalent cation involved in fundamental biochemical and biophysical processes. Its integral roles in cell signaling, protein-mediated fusion, and action potential propagation have been well studied with a variety of techniques. Calcium is also commonly maintained at strong asymmetric concentration gradients, which often span several orders of magnitude, across mammalian cell membranes in proper physiological conditions. Yet, there has been a comparative lack of investigation of the effect of divalent cations on lipid bilayer structure and mechanical properties. Of the few studies done, experimental methods are limited to undefined resolutions and indirect spectroscopic or fluorescent techniques. This is in part due to the inherent difficulty in preparing well aligned samples with ions for high resolution experiments; common multilayer preparation techniques are subject to miscibility limits and sample segregation. Computationally, molecular dynamics simulations have predicted a calcium “condensing” effect that is associated with bilayer thickening and increased hydrocarbon chain order. However, these methods are limited by still-developing approaches to modeling divalent cation structure and interactions. Here, we report lamellar x-ray diffraction measurements of lipid bilayer thickness in partially charged multilayers containing calcium. Additionally, we report an apparent calcium-induced mechanical softening effect in giant unilamellar vesicles (GUVs) as measured by micropipette aspiration. This effect is marked by a measurable reduction in the elastic stretching coefficient of GUVs in calcium-rich environments. Both the thickening and mechanical softening effects may be important for proteins and molecules that couple directly to the lipid bilayer in physiological calcium concentrations. Future investigations involving PG-containing membranes and bacterial protoplasts may help lead to a better understanding of how invading bacteria thrive in calcium-rich environments, or if calcium-rich membrane domains have some non-trivial influence on the mechanisms of antibiotics and/or host defense antimicrobial peptides.

Some Studies on Prevalence and Effect of Thieleria Infection on Erythrocytes Profile in Camel in Some Localities at New-Valley, Governorate, Egypt

Theileria spp is protozoan parasites infecting wild and domestic animals throughout the world and affected the healthy state of the infected animals for that this study was carried to evaluate the effects of natural infection of camels with Theileria spp on blood picture and efficacy of indirect fluorescent antibody technique in diagnosis this infection, where blood samples were obtained from125 apparently healthy dromedary camels aged 1-9 years ,held in some localities in New-Valley governorate and classified into two groups, the first group (100 camels) as infected group and the rest number 25 as a control group where all of them examined by direct smear and indirect fluorescent antibody technique. Direct smear revealed 9 out 100 camel are positive for Theileria spp in ratio of 9% while indirect fluorescent antibody technique revealed 11out of 100 camels are positive (11%), with one sample as false negative and 3 samples as false positive so we can consider the indirect fluorescent technique remain the most convenient test for theileria spp diagnosis in camels. Hematological analysis revealed significant decrease in PCV, HB, RBCs count with significant increase in MCV, MCH and MCHC in infected group than control one. The frequency of theileriosis in camels is considerable and Theileria spp do not seem to induce significant alteration in clinical signs in naturally-infected dromedary camels but laboratory seem to induce significant decrease in hematological parameters which translated to anemia.

Some studies on prevalence and effect of Thieleria infection on erythrocytes profile in camel in some localities at New-Valley, Governorate, Egypt

Theileria spp is a protozoan parasite infecting wild and domestic animals throughout the world and affect the healthy state of the infected animals. Therefore this study was carried to evaluate the effects of natural infection of camels with Theileria spp on blood picture and efficacy of indirect fluorescent antibody technique in the diagnosis of this infection. The blood samples were collected from125 apparently healthy dromedary camels aged 1-9 years, held in some localities in New-Valley Governorate and classified into two groups according to infection, the first group is suspected clinically infected and considered as infected group (100 camels) and the remaining number (25) was clinically and laboratory healthy and considered as a control group where all of them were examined by thin blood smear, fecal examination and indirect fluorescent antibody technique. Thin blood smears revealed that 9 out of 100 camels were positive for Theileria spp in ratio of 9% while indirect fluorescent antibody technique revealed that 11out of 100 camelswere positive (11%), with one sample as false negative and 3 samples as false positive. Therefore the indirect fluorescent technique remains the most convenient test for theileria spp diagnosis in camels. Hematological analysis revealed a significant decrease in PCV, HB, RBCs count with a significant increase in MCV, MCH and MCHC in infected group when compared with the control one. The frequency of theileriosis in camels is low and Theileria spp do not seem to induce a significant alteration in clinical signs of naturally-infected dromedary camels but by laboratory means a significant decrease in hematological parameters which translated to anemia was resulted.

CHARACTERISTICS OF THE СOURSE OF MAXILLARY SINUSITIS IN CHILDREN WITH VERIFIED CHLAMYDIA INFECTION

To explore the detection rate of chlamydia infection and immunological features of children with acute maxillary sinusitis. Clinical epidemiologic characteristics of the course of acute maxillary sinusitis were studied in the way of medical examination provided for 58 children in the ages from 3 to 15 years, patients of ENT department. In order to identify Chlamydia structures and specific antibodies we used direct (polymerase-chain reaction, direct immune fluorescence) and indirect (immune enzyme analysis) methods. The research for cellular link of immunity was carried out by indirect immune fluorescence technique, using monoclonal antibodies. The authors report on high level of Chlamydia infection in epithelium of nasal mucosa, and also type structure of verified Chlamydia, clinical signs and peculiarities of immune response in children with acute maxillary sinusitis associated with this infection. Chlamydia was detected in 48% of children with acute maxillary sinusitis infection with prevalence of Chlamydophila pneumoniae. Optimal for detection of chlamydia infection is a disease duration not exceeding 2 weeks. The special features of clinical and immunological manifestations in children with acute sinusitis, paired with Chlamydia infection are the more frequent complaints of pain in the affected sinuses, expressed symptoms of intoxication, and the presence of an imbalance in cellular immunity, manifested a relative lymphopenia and activation of T-lymphocytes CD8+.

Adult stem / progenitor cells of the rat thyroid: side population distribution, intermediate filament expression, and long-term in vitro expansion

We recently identified adult stem / progenitor cells in the male rat thyroid, based on expression of the multipotency maker, ATP-binding cassette subfamily G member 2 (ABCG2) (1). To characterize these cells, we have now determined their distribution as a side population of the adult thyroid gland, identified their epithelial vs mesenchymal commitment by the presence of cytoplasmic intermediate filaments, and enriched their number using long-term, in vitro expansion of adherent elements. Sprague-Dawley male rats (50-75 gr) were used as thyroid donors. Following penthobarbital anesthesia rats were thyroidectomised, and primary cells prepared using enzymatic breaking of the gland. After 72 hs in standard monolayer culture, adherent cells were trypsinized, and either incubated for 90 min with the vital dye, Hoechst 33342 (Hch) + the ABCG2 inhibitor, verapamil (VE, 150 mM) followed by cytospin (1300 RPM x 8 min) for single, double, and triple light microscopic immunocytochemistry (IC), or re-seeded (20 x103 / cm2) in monolayer and grown up to 4 months, using a starvation protocol based on a single weekly change of culture medium (low glucose DMEM / 15 % FBS-FHS serum). Co-localization of nuclear Hoch with immunoreactive (IR) ABCG2 (rabbit anti-human polyclonal antiserum, 1:300), IR-cytokeratin (CTK, mAb 1:200), and IR-vimentin (VIM, mAb 1:100) was assessed by the ABC and indirect fluorescence techniques, using DAB, FITC and TRITC as chromogens. Rat kidney, human keratinocyte cell line, NTCT 2544 (courtesy of C. Pellegrini), and primary mouse and human fibroblasts (courtesy of D. Mattioli) were used as positive controls for IC. A consistent increase in Hch-positive nuclei was observed in VE-treated cultures, as opposed to VE-untreaded monolayers. In addition, an inverse staining relationship occurred between nuclear Hch and IR-CTK, as opposed to a direct relationship between nuclear Hch and IR-VIM. Co-localization of IR-ABCG2 with IR-CTK was seen in some cells, whereas that of IR-ABCG2 with IR-VIM was only occasionally detected. Finally, longterm expansion of primary thyrocytes resulted in 30% increase in IR-ABCG2 cells, as opposed to less than 1% IR-ABCG2 elements in standard culture. We conclude that ABCG2-positive cells of the rat thyroid are a side population of stem / progenitor elements, they are primarily committed to the epithelial phenotype, and can be enriched in vitro as adherent cells, suggesting clonal expansion.

Evaluation of Antioxidant Response Mechanism in Fattening Cattle Calves Suffering from Babesiosis in New-Valley- Governorate. Egypt

Invasion of animal\'s erythrocytes by Babesia parasites causes alteration in antioxidant potential of the red cells so this study was conducted to assess the effect of Babesiosis infection on some oxidative stress and antioxidant agent in calf naturally infected with babesi where the study was carried on 70 fattening calves aged in 2-3 years, from different localities in New-Valley Governorate, Egypt. The animal under the study classified into three groups, group (3) include 40 fattening calves naturally infected by babesia and ticks, group (2) include 15 fattening calves infected with ticks only and group (1) include 15 fattening calves clinically and laboratory healthy (control group).The parasitological diagnosis was confirmed by indirect fluorescent technique (IFT) and the diseased calves divided into 4 subgroups according to parasitiaemia rates (0.5% ,1% ,1.5% ,2% ). The calves under the study were free from internal parasite by fecal analysis. The study revealed a significant increase in oxidative marker (NO and MDA) in the diseased group with Babesiosis than tick infected group and control group while the biochemical serum analysis for antioxidant markers revealed an highly significant decrease in all antioxidant parameters under study, SOD, GSH-R, Catalos, Ascorbic acid and TAA, as the fallowing values, 290.13±31.21, 2.01±0.92, 0.93±0.04, 99.75±2.47 and 0.83±0.32 respectively. The study indicated that the Babesiosis calves are at greater risk of oxidative damage than other groups studied, hence early diagnosis ,treatment and addition of dietary antioxidants are highly encouraged and essential requests for improve of productivity of fattening calves.

Study on the inclusion interactions of β-cyclodextrin and its derivative with dyes by spectrofluorimetry and its analytical application

The inclusion interactions of β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) with dyes were developed by spectrofluorimetry, and the inclusion constants of inclusion complexes were determined by direct fluorescence technique. The main factors (the host molecule, the guest molecule, and the pH) for the inclusion interaction were discussed in detail. At the same time, the inclusion interaction of HP-β-CD and vitamin B 6 (VB 6) was investigated with the competitive fluorescence inclusion method and the inclusion constant of HP-β-CD and vitamin B 6 (VB 6) was obtained by indirect fluorescence technique. On the basis of the linear relationship between the change of fluorescence intensity (Δ F) and the concentration of VB 6, a competitive fluorescence inclusion method was used to the determination of VB 6. The method has been successfully applied to the analysis of VB 6 in synthetic samples, tablets and injections with satisfactory results.

Counterstaining Improves Visualization of the Myenteric Plexus in Immunolabelled Whole-Mount Preparations

Immunocytochemistry has emerged as a powerful research tool in neurobiology. One of the widely used methods is an indirect fluorescence technique that uses FITC- conjugated IgG to visualise protein expression within tissues, but a major drawback of this technique is the high background fluorescence due to non-specific antibody binding. Gut innervation is complex and best visualized in three-dimensions in whole mount preparations. We describe a simple and easy to use counterstaining procedure in conjunction with an indirect immunofluorescence technique in gut whole mount preparations that largely eliminates background fluorescence and creates a contrasting background against the bright antigen-antibody complexes. Furthermore, this technique allows the detailed qualitative and quantitative study of myenteric plexuses in whole-mount preparations.

The role of myocardin in hypoxia-induced transdifferentiation of pulmonary artery endothelial cells into smooth muscle-like cells

To investigate the transdifferentiation of pulmonary artery endothelial cells (PAECs) into smooth muscle-like cells under hypoxia and the role of myocardin therein. Recombinant plasmid psimyocardin (pSi), capable of silencing the expression of myocardin gene, was constructed by RNAi to be used to transfect the PAECs. Endothelial cells of adult pig pulmonary small arteries were purified by immunomagnetic purification technique, and divided into 4 groups: normoxia group (to be cultured in normoxic cell culture box containing 21% O(2), 5% CO2, and 74% N(2)), normoxia + pSi group, hypoxia group (to be cultured in cell box containing 1% O(2), 5% CO2, and 74% N(2)), and hypoxia + pSi group to be cultured for 1, 4, and 7 days respectively. Indirect fluorescence technique and morphological examination were used to identify the smooth muscle (SM) -like cells and the alpha-SM-actin positive cell ratio. RT-PCR was used to detect the mRNA expression of myocardin. Alpha-SM-actin positive cell could not be seen in the normoxia group and hypoxia 1 d group. The alpha-SM-actin positive cell rate of the hypoxia 7 days group was 2.07% +/- 0.06%, significantly higher than that of the hypoxia 4 d group (0.96% +/- 0.08%, P < 0.01). mRNA expression of myocardin gene could not be seen in the normoxia, normoxia + pSi, hypoxia 1 days, and hypoxia + pSi 1 days groups. The mRNA expression of myocardin gene of the hypoxia 7 days group was 0.23 +/- 0.03, significantly higher than that of the hypoxia 4 days group (0.14 +/- 0.01, P < 0.01). The mRNA expression levels of myocardin gene of the hypoxia + pSi 4 days and 7 days groups 0.03 +/- 0.02 and 0.05 +/- 0.01, both significantly lower than those of the hypoxia 4 days and 7 days groups respectively (0.14 +/- 0.01 and 0.23 +/- 0.03, both P < 0.01). The SM-like cell transdifferentiation rates of the hypoxia + pSi 4 days and 7 days groups were 0.19% +/- 0.07% and 0.21% +/- 0.04% respectively, both significantly lower than those of the corresponding hypoxia groups (0.96% +/- 0.08 and 2.07% +/- 0.06% respectively, both P < 0.01). Some PAECs have the potential to transdifferentiate into SM-like cells and may be one of the resources of muscularization of peripheral small vessels. Hypoxia remarkably promotes this transdifferentiation and myocardin may play an important role in this process.

Prevalencia de anticuerpos contra Bartonella henselae en niños, en adolescentes y en una población de riesgo ocupacional en Chile

Bartonella henselae is the causative agent of cat-scratch disease. To study the seroepidemiology of Bartonella henselae in healthy Chilean children and in a population with occupational risk. Serum IgG antibodies were determined by indirect fluorescence technique in 181 children and adolescents and in 107 technical and professional workers involved in the care of cats. Samples with titers equal to or greater than 64 were considered positive. Twenty four (13.3%) children and 11 (10.3%) occupational risk subjects were seropositive. No significant differences by age and gender were observed. Assuming that seroprevalence indicates level of exposure to Bartonella henselae, these results suggest that this infection is endemic in Chile and, for this reason, the best antibody titer to diagnose acute cat-scratch disease should be higher than the figure recommended by the Centers for Disease Control in the in United States.

Localisation of GYIRFamide immunoreactivity inMacrostomum hystricinum marinum (Plathelminthes, Macrostomida)

The distribution of GYIRFamide immunoreactivity in the nervous system ofMacrostomum hystricinum marinum has been demonstrated by an indirect fluorescence technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was extensive in both the central (CNS) and peripheral (PNS) nervous systems, revealing detailed information on the microanatomy of the peptidergic nervous system of this free-living plathelminth. In the CNS, immunoreactive nerve cell bodies and nerve fibres occurred in the brain and along two pairs of longitudinal nerve cords: the main nerve cords and the ventral nerve cords. In the PNS, immunostaining was prevalent in nerve cells and fibres innervating the pharynx and the gut. The employed antibody is directed against a recently characterised FMRF-amide-related peptide (FaRP), GYIRFamide, isolated from two species of the Tricladida,Dugesia tigrina andBdelloura candida. Phylogenetically, GYIRFamide represents the most ancient neuropeptide thus far identified within the Bilateria

The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase.

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

レーザーサイトメーターを用いたheat shock protein 72の加温および抗癌剤添加による動態の検討

Heat shock proteins (HSPs) are regarded as the proteins most related to the development of thermotolerance. Recently, not only their role in thermotolerance, but also their role in resistance to anticancer agents is gathering concern. In this study, the kinetics of hsp 72 in HeLa cells treated with heating and/or anticancer agents were studied. Hsp 72 was immunostained by the indirect fluorescent technique using a monoclonal anti-hsp 72 antibody (Amersham). The staining pattern was observed and analyzed using a laser cytometer, ACAS 570 (Meridian). Hsp 72 was normally found in the cytoplasm at 37°C and moved rapidly into the nucleus with heating at 43°C for 2 hours. It then returned to the cytoplasm 4 to 6 hours after heating. The hsp 72 content reached a peak at 8 hours after heating. Hsp 72 was induced in all cells treated with cisplatin, adriamycin, peplomycin, or etoposide for 48 hours. In the cells treated with both heating at 43°C for 2 hours and these anticancer agents, hsp 72 induction was most suppressed by adriamycin. However, translocation of hsp 72 to the nucleus was specific for heating and was not affected by the anticancer agents. By laser cytometry the intracellular localization of hsp 72 and the changes of its content were simultaneously detected. Moreover, the change pattern of hsp 72 content measured by laser cytometry coincided with that measured by the Western blotting procedure.

Changes in the cytoskeletal structure of cultured smooth muscle cells induced by calyculin-A.

Changes in the cytoskeletal structure of cultured A10 smooth muscle cells induced by calyculin-A (CL-A), a potent inhibitor of types 1 and 2A protein phosphatases, were analyzed using indirect fluorescence techniques. In the presence of 1 x 10(-7) M CL-A the cells became round and subsequently detached from the substratum. The effect of CL-A was inhibited by a non-selective kinase inhibitor, K-252a, but not by EGTA. In rounded cells stress fibers were absent and staining for F-actin appeared in patches. Vinculin, one of the components of focal contacts, was localized at the periphery of control cells. CL-A treatment moved the focal contacts towards the inside of the cell along the stress fibers, and this was followed by the rounding up of the cell. In addition, rapid and marked changes in microtubule structure were observed in CL-A-treated cells. Many 'nicks' or 'gaps' were observed along the microtubules in the attached, spread cells. A filamentous network of microtubules was not observed in the detached cells, i.e. after longer exposure to CL-A. These results suggest that CL-A may change the structure of focal contacts, resulting in the rounding up of the cell, and inducing a microtubule-severing activity. These effects were independent of the external Ca2+ concentration. The changes in cytoskeletal structure may be caused by disturbing the balance of phosphorylation and dephosphorylation in the cell.

Immunotherapy of tularemia: characterization of a monoclonal antibody reactive with Francisella tularensis

An IgM monoclonal antibody (mAb) recognized surface antigens specific to Francisella tularensis wild-type (Schu4) and live vaccine strain (LVS), and reacted with both in ELISA and slide agglutination tests. This mAb also reacted with LVS microorganisms in tissues of infected mice as assessed by an indirect fluorescence technique. Western blot analysis showed the mAb to react with antigens associated with F. tularensis LPS.

Prenatal development of glucocorticoid receptor gene expression and immunoreactivity in the rat brain and pituitary gland: a combined in situ hybridization and immunocytochemical analysis.

By means of in situ hybridization and immunocytochemical techniques it has been possible to follow the prenatal development of glucocorticoid receptor (GR) messenger RNA (mRNA) expression and GR immunoreactivity (IR) in the rat brain from embryonic day (E) 15 to 22. A 700-base-pair GR cDNA fragment was used for RNA probe generation. In the immunocytochemical analysis a mouse monoclonal antibody (IgG2a) against the rat liver GR was used in combination with the indirect fluorescence technique or the avidin-biotin immunoperoxidase method. At E15 till E22 a moderate to strong GR mRNA signal was observed within the neuro-epithelium from the medulla oblongata to the telencephalon. A moderate to strong labelling was also present within the paraventricular hypothalamic nucleus, the arcuate nucleus, the nucleus raphe magnus, the nucleus raphe obscurus and the locus coeruleus. In these areas a weak to moderate nuclear GR IR developed in nerve cells 1 or 2 days after the appearance of the GR mRNA signal. From E15 the adenohypophysis showed the strongest expression of GR mRNA. At E17 a strong GR IR was especially demonstrated in the nuclei of many pituitary cells, some exhibiting adrenocorticotropin IR. The results open up the possibility that there exist active GR in embryonic life capable of regulating proliferation events within the adenohypophysis and the neuro-epithelia of the brain. This embryonic GR may modulate the development of inter alia neuro-endocrine areas such as the paraventricular and arcuate nuclei and arousal-related areas such as the central 5-hydroxytryptamine and noradrenaline neuronal systems. Provided that this embryonic GR is capable of becoming activated by glucocorticoids in fetal life, it may mediate several neurochemical and behavioural impairments caused by prenatal stress.

Immunohistochemical distribution of keratin proteins in human gingival heterotransplants in nude mice

Clinically healthy human gingivae from deciduous molar regions were transplanted to subcutaneous sites of nude mice (nu/nu NC). Transplants were harvested after posttransplantation periods of 5, 6, 7, 8.5, 10.5 and 12 weeks and examined histologically after staining with hematoxylin-eosin (H.E.), bisbenzimide, and a panel of mouse monoclonal anti-keratin antibodies in an indirect fluorescence technique. Central parts of transplants contained human connective tissue covered by human stratified squamous epithelium which were unkeratinized in 5- to 7-wk-old transplants and most frequently (75%) parakeratinized in 8.5-wk to 12-wk transplants. Comparison of keratin expression before and after transplantation revealed a progressive keratin reconstitution, i.e., keratin markers of basal/suprabasal cells preceded those of suprabasal/spinous cell layers and immunohistochemical markers of keratinization preceded routine histologically observed parakeratinization. Original keratin staining and essential features of histodifferentiation were reconstituted and maintained after 8.5 wk but graft recovery rate decreased drastically 12 wk after transplantation. This study shows that the human gingiva/nude mouse model is useful in experimental studies of the gingival keratin profile in the period 8.5 to 10.5 wk after transplantation.

Immunohistochemical distribution of keratin proteins in clinically healthy human gingival epithelia

In clinically healthy/subclinically inflamed biopsies of marginal gingiva, the immunohistochemical distribution of keratin proteins was studied in junctional (JE), sulcular (SE), oral gingival (OGE) and in a few samples of alveolar mucosal epithelium (AE) by means of various mouse monoclonal anti-keratin antibodies in an indirect fluorescence technique. All regions stained in a nearly similar way with AE3 (keratins 1-8, all cells) and BE14 (keratin 5, basal and supra/parabasal cells). AE8-staining (keratin 13, supra/parabasal and spinous cells) was primarily confined to the stratified, nonkeratinized epithelia SE and AE, but also a variable part of JE and less frequently OGE were positive. The parakeratinized OGE was distinct in showing a homogeneous staining with AE2 (keratins 1/2, 10) and AE5 (keratin 3) throughout spinous cell layers. These antibodies did not stain JE and AE whereas SE stained in a scattered way with AE5 and sometimes also with AE2. The latter finding might indicate initial keratinization at molecular level. The JE was distinct in retaining basal characteristics throughout the epithelium with PKK2 (keratin 7, 16, 17, 19) and BE14 (keratin 5) although some initial suprabasal maturation, as observed with AE8, cannot be excluded. Differences in keratin staining of gingival epithelia and the AE was found with respect to AE1-reactivity (keratins 10, 14-16, 19) which was suprabasal in JE, SE and OGE but basal in AE.