RESUMO
Evidence to date supports regulatory T cell (Treg) alterations in endometriosis; however, the relationship remains unclear, and Tregs have not previously been investigated with respect to infertility in endometriosis. This prospective cross-sectional cohort study details circulating and endometrial tissue-specific disturbances in Tregs and broader gated populations in women of reproductive age with and without endometriosis (n = 57 and 29, respectively) using flow cytometry and immunohistochemistry. Participants were characterised by menstrual cycle phase, r-ASRM endometriosis disease stage and fertility status.In the endometrium of women with endometriosis, endometrial Tregs and CD4+ lymphocyte proportions did not change between the proliferative and secretory phases, while in women without the disease, they significantly decreased (p = 0.045 and p = 0.039, respectively). In women with endometriosis, endometrial Tregs were lower than in women without endometriosis overall (p = 0.050 as a proportion of all CD45+ immune cells). We have shown for the first time that proportions of CD4+ lymphocytes (p = 0.021), overall lymphocytes (p = 0.034) and non-granulocytes (p = 0.027) were significantly decreased in the endometrium of women with moderate-severe (r-ASRM stages III and IV) compared to minimal-mild (r-ASRM stages I and II) endometriosis. During the secretory phase, circulating Treg proportions were significantly increased in infertile compared to fertile women (p = 0.049). This study confirms differences in endometrial Tregs in women with endometriosis, with blunting of normal menstrual cyclical variations, reduced proportions during the proliferative phase and disease stage-specific relationships.
Assuntos
Endometriose/metabolismo , Endométrio/metabolismo , Infertilidade Feminina/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Estudos Transversais , Epitélio/metabolismo , Feminino , Humanos , Estudos Prospectivos , Adulto JovemRESUMO
STUDY QUESTION: What are the detailed endometrial tissue specific and systemic dendritic cell (DC) subset disturbances in endometriosis? SUMMARY ANSWER: This study confirms myeloid DC (mDC) and plasmacytoid DC subsets are readily identified in endometrial tissue and shows both endometrial and circulating differences in DC populations in women with endometriosis, with disease stage-specific relationships evident locally in the endometrium. WHAT IS KNOWN ALREADY: Immune factors in the uterus, the peritoneal environment and systemically are implicated in the pathogenesis and progression of both endometriosis and infertility. While there is some evidence that endometrial DC populations are altered in endometriosis, DC subset involvement in both the endometrium and peripheral blood have not been comprehensively investigated so the functional consequences have been unknown. STUDY DESIGN, SIZE, DURATION: This prospective cross-sectional cohort study compares circulating and endometrial DC populations in women of reproductive age with and without endometriosis (n = 55 and 30, respectively), wherein each participant donated samples at a single time point. Study participants were surveyed for menstrual cycle phase, American Society for Reproductive Medicine (ASRM) endometriosis disease stage and fertility status (where possible). PARTICIPANTS/MATERIALS, SETTING, METHODS: Peripheral blood samples were processed into mononuclear cells for analysis by flow cytometry, and endometrial samples were analysed by immunohistochemistry and dissociated into single-cell suspension for flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: In the endometrium of women with endometriosis, IRF-8+ cells were increased during the proliferative phase (P = 0.014), total DC proportions increased in the secretory phase (P = 0.038) and normal menstrual cyclical fluctuations in CD1c+ and IRF-8+ cells blunted; indicative of a consistently inflammatory tissue environment. The inflammatory changes in CD141+ and IRF-8+ populations in the endometrium of women with endometriosis were particularly evident in more advanced ASRM stages of the disease (respective P-values 0.032 and 0.045). There was also evidence of systemic inflammation in women with endometriosis, with increased circulating CD141+ mDC proportions (overall P = 0.040, secretory phase P = 0.021). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: As is common in this type of study, one of the main limitations was small sample numbers, particularly during the menstrual phase of the cycle. WIDER IMPLICATIONS OF THE FINDINGS: Further phenotyping of local and circulating immune cell subtypes is critical to improving understanding of endometriosis pathogenesis and immune contributions to infertility associated with the disease. STUDY FUNDING/COMPETING INTEREST(S): This research was financially supported by a Sydney Medical School and Balnaves Foundation Kick Start Grant and the Department of Obstetrics, Gynaecology and Neonatology at The University of Sydney. The authors have no conflicts of interest to declare.
Assuntos
Endometriose , Estudos Transversais , Células Dendríticas , Endométrio , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Gravidez , Estudos ProspectivosRESUMO
Wood frogs ( Rana sylvatica) are highly susceptible to infection with Frog virus 3 (FV3, Ranavirus, Iridoviridae), a cause of mass mortality in wild populations. To elucidate the pathogenesis of FV3 infection in wood frogs, 40 wild-caught adults were acclimated to captivity, inoculated orally with a fatal dose of 104.43 pfu/frog, and euthanized at 0.25, 0.5, 1, 2, 4, 9, and 14 days postinfection (dpi). Mild lesions occurred sporadically in the skin (petechiae) and bone marrow (necrosis) during the first 2 dpi. Severe lesions occurred 1 to 2 weeks postinfection and consisted of necrosis of medullary and extramedullary hematopoietic tissue, lymphoid tissue in spleen and throughout the body, and epithelium of skin, mucosae, and renal tubules. Viral DNA was first detected (polymerase chain reaction) in liver at 4 dpi; by dpi 9 and 14, all viscera tested (liver, kidney, and spleen), skin, and feces were positive. Immunohistochemistry (IHC) first detected viral antigen in small areas devoid of histologic lesions in the oral mucosa, lung, and colon at 4 dpi; by 9 and 14 dpi, IHC labeling of viral antigen associated with necrosis was found in multiple tissues. Based on IHC staining intensity and lesion severity, the skin, oral, and gastrointestinal epithelium and renal tubular epithelium were important sites of viral replication and shedding, suggesting that direct contact (skin) and fecal-oral contamination are effective routes of transmission and that skin tissue, oral, and cloacal swabs may be appropriate antemortem diagnostic samples in late stages of disease (>1 week postinfection) but poor samples to detect infection in clinically healthy frogs.
Assuntos
Infecções por Vírus de DNA/veterinária , Ranavirus , Ranidae/virologia , Animais , Animais Selvagens/virologia , Infecções por Vírus de DNA/patologia , Infecções por Vírus de DNA/virologia , Masculino , Ranavirus/patogenicidade , Ranidae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
Despite the importance of neuropilins (NRPs) in a number of processes that are altered in endometriosis, such as angiogenesis and neuronal guidance, these molecules have not been previously studied in the disease. Similarly, potent lymphangiogenic factors, vascular endothelial growth factor C (VEGF-C) and VEGF-D, have not been comprehensively investigated in endometriosis. The objective of this study was to examine their expression in women with and without endometriosis. NRPs and VEGFs were quantified in 79 histologically normal uterine tissue samples (37 control and 42 endometriosis, all menstrual cycle phases) using immunohistochemistry and automated cellular imaging analysis. NRP-1 was significantly reduced in women with endometriosis (P = .004). The normal significant menstrual cyclical variations in endometrial NRP-1, NRP-2, and VEGF-C were absent in endometriosis, and VEGF-D was dysregulated. Dysregulated expression of growth factors and receptors, such as NRPs and VEGFs, likely contribute to altered angiogenesis, lymphangiogenesis, neurogenesis and immune function in endometriosis and may reflect altered hormone signals.
Assuntos
Endometriose/metabolismo , Endométrio/química , Neuropilina-1/análise , Neuropilina-2/análise , Fator C de Crescimento do Endotélio Vascular/análise , Fator D de Crescimento do Endotélio Vascular/análise , Estudos de Casos e Controles , Endometriose/fisiopatologia , Endométrio/fisiopatologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Linfangiogênese , Ciclo Menstrual/metabolismo , Neovascularização Fisiológica , NeurogêneseRESUMO
BACKGROUND: Diagnosis of endometriosis currently requires a laparoscopy and this need probably contributes to the considerable average delay in diagnosis. We have reported the presence of nerve fibres in the functional layer of endometrium in women with endometriosis, which could be used as a diagnostic test. Our aim was to assess efficacy of nerve fibre detection in endometrial biopsy for making a diagnosis of endometriosis in a double-blind comparison with expert diagnostic laparoscopy. METHODS: Endometrial biopsies, with immunohistochemical nerve fibre detection using protein gene product 9.5 as marker, taken from 99 consecutive women presenting with pelvic pain and/or infertility undergoing diagnostic laparoscopy by experienced gynaecologic laparoscopists, were compared with surgical diagnosis. RESULTS: In women with laparoscopic diagnosis of endometriosis (n = 64) the mean nerve fibre density in the functional layer of the endometrial biopsy was 2.7 nerve fibres per mm(2) (+/-3.5 SD). Only one woman with endometriosis had no detectable nerve fibres. Six women had endometrial nerve fibres but no active endometriosis seen at laparoscopy. The specificity and sensitivity were 83 and 98%, respectively, positive predictive value was 91% and negative predictive value was 96%. Nerve fibre density did not differ between different menstrual cycle phases. Women with endometriosis and pain symptoms had significantly higher nerve fibre density in comparison with women with infertility but no pain (2.3 and 0.8 nerve fibre per mm(2), respectively, P = 0.005). CONCLUSIONS: Endometrial biopsy, with detection of nerve fibres, provided a reliability of diagnosis of endometriosis which is close to the accuracy of laparoscopic assessment by experienced gynaecological laparoscopists. This study was registered with the Australian Clinical Trials Registry (ACTR) 00082242 (registered: 12/12/2007). The study was approved by the Ethics Review Committee (RPAH Zone) of the Sydney South West Area Health Service (Protocol number X05-0345) and The University of Sydney Human Research Ethics Committee (Ref. No. 10761) and all women gave their informed consent for participation.
Assuntos
Técnicas de Diagnóstico Obstétrico e Ginecológico , Endometriose/diagnóstico , Endométrio/inervação , Fibras Nervosas Amielínicas/patologia , Adulto , Biópsia , Método Duplo-Cego , Diagnóstico Precoce , Endometriose/patologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Fibras Nervosas Amielínicas/metabolismo , Sensibilidade e Especificidade , Ubiquitina Tiolesterase/metabolismo , Adulto JovemAssuntos
Doenças da Túnica Conjuntiva/complicações , Cistos/complicações , Descolamento Retiniano/complicações , Esclerite/complicações , Doenças da Túnica Conjuntiva/patologia , Doenças da Túnica Conjuntiva/cirurgia , Cistos/cirurgia , Oftalmopatias/diagnóstico , Oftalmopatias/etiologia , Oftalmopatias/cirurgia , Feminino , Humanos , Vasos Retinianos/patologia , Esclerite/patologia , Esclerite/cirurgia , Resultado do Tratamento , Acuidade Visual , VitrectomiaRESUMO
PURPOSE: To report the management and outcome of retinal reattachment surgery in retinochoroidal coloboma. METHODS: Four patients with retinochoroidal colobomata presented to the Bristol Eye Hospital (a UK tertiary referral center for vitreoretinal surgery) with retinal detachment. INTERVENTION: All were type II colobomatous detachments (three patients with type IIB, one patient with type IID). All eyes underwent vitrectomy with endolaser and/or cryotherapy and three eyes underwent scleral buckling. Two eyes had internal tamponade with gas (SF6, C3F8) while the other two had silicone oil. Endolaser was applied over healthy retinal pigment epithelium. RESULTS: At last follow-up, all (100%) remained attached, with no recurrences. Three patients achieved visual acuity of 6/120 or better and were able to perform satisfactory near work with appropriate magnifiers. The last patient began with hand movement vision and retained similar vision but subjectively felt more navigational. CONCLUSIONS: Good anatomic and functional outcomes can be achieved in this patient group with combined vitrectomy with or without scleral buckling surgery. Endolaser retinopexy is effective over healthy RPE at the margin of the coloboma combined with either gas or oil internal tamponade.
Assuntos
Corioide/anormalidades , Coloboma/cirurgia , Retina/anormalidades , Descolamento Retiniano/cirurgia , Recurvamento da Esclera , Vitrectomia , Adolescente , Adulto , Pré-Escolar , Criocirurgia , Feminino , Fluorocarbonos/administração & dosagem , Humanos , Fotocoagulação a Laser , Masculino , Pessoa de Meia-Idade , Óleos de Silicone/administração & dosagem , Hexafluoreto de Enxofre/administração & dosagem , Acuidade VisualRESUMO
BACKGROUND: Dendritic cells (DCs) are specialized antigen presenting cells that are highly involved in the stimulation and modulation of the immune response within mucosal surfaces, including the female reproductive tract. DCs have been poorly characterized in the non-pregnant endometrium. METHODS: Hysterectomy specimens were obtained from premenopausal women (n = 49) with histologically normal endometrium. Endometrial sections were stained immunohistochemically using antibodies for monoclonal mouse anti-human CD1a and CD83, two markers which are specific for populations of immature and mature DCs, respectively. RESULTS: There was a significantly higher density of endometrial CD1a+ DCs than CD83+ DCs throughout the menstrual cycle (P < 0.001). The density of CD1a+ and CD83+ DCs did not vary between the fundus and isthmus of the uterus. There was a significant increase in the density of CD1a+ DCs, but not CD83+ DCs, in the basal layer of the endometrium through the phases of the menstrual cycle. The density of CD83+ was significantly greater in the basal layer compared with the functional layer during both the proliferative (P = 0.004) and secretory phases (P = 0.001), whereas for CD1a+ DCs, the greater density in the basal layer was only observed in the secretory phase (P < 0.001). CONCLUSIONS: The highly coordinated cyclical changes in DC populations during the normal menstrual cycle reported in this study may be important for local regulatory mechanisms relevant to menstruation and implantation; alterations in this normal profile may contribute to the development of disturbances of function, fertility and even benign gynaecological disease.
Assuntos
Células Dendríticas/imunologia , Endométrio/citologia , Ciclo Menstrual/imunologia , Adulto , Antígenos CD/análise , Antígenos CD1/análise , Feminino , Humanos , Histerectomia , Imunoglobulinas/análise , Imuno-Histoquímica , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Pré-Menopausa , Antígeno CD83RESUMO
Following a per os challenge of naive rainbow trout with live spores of Loma salmonae, head kidney mononuclear cells (MNC) in culture were able to proliferate in response to crude soluble parasite extract or intact dead spores. A significant response was seen by week 2 post-exposure and a maximum response developed by week 6 or 8, respectively. During this initial challenge, spore filled cysts developed on the gills of challenged fish, and the cysts ruptured by week 12 as is typical for microsporidial gill disease of salmonids (MGDS). Two weeks following this, fish were re-challenged with live spores, and in these fish an enhanced in vitro proliferative response of MNC was immediately apparent, and spore filled cysts did not develop. In contrast, when naive trout were given dead spores by intraperitoneal injection, the most pronounced proliferative responses of MNC developed earlier (week 2 PE) and the response was greater when cells were incubated in vitro with dead spores rather than with crude soluble extract. When these fish were re-challenged per os with live spores, a heightened proliferation in MNC was observed 4 weeks after this exposure and the fish likewise resisted development of xenomas. In fish infected orally or injected intraperitoneally with spores, a marked increase in the response to the mitogen concanavalin A was seen for 22 weeks post-exposure when compared to controls not receiving any spores.
Assuntos
Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Loma/imunologia , Microsporidiose/veterinária , Oncorhynchus mykiss/imunologia , Animais , Proliferação de Células , Concanavalina A/imunologia , Brânquias/imunologia , Brânquias/microbiologia , Imunidade Celular/imunologia , Estudos Longitudinais , Microsporidiose/imunologia , Microsporidiose/microbiologia , Mitógenos/imunologia , Esporos Fúngicos/imunologia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
AIM: To document the long term outcome of congenital cataract surgery with primary posterior chamber (PC) lens implantation in the first year of life. METHOD: A retrospective review of congenital cataract surgery in the first year of life with PC lens implantation in 18 infants, eight with unilateral and 10 with bilateral cataract. The average age at surgery was 15 weeks (range 3-44 weeks). The mean follow up was 95 months (range 60-139 months). RESULTS: The best outcomes were in the bilateral group where 50% of eyes achieved 6/18 or better, with a best acuity of 6/9. Acuities were poor in the unilateral group where only 38% achieved 6/60 or better, with a best acuity of 6/24. There was a mean refractive shift between first refraction after surgery and refraction at 36 months after surgery of -3.44 dioptres with a very wide range (+2.00 to -15.50). There was a significantly greater myopic shift in the unilateral cases. Many eyes in both groups continued to show an increasing myopic shift between 36 months after surgery and their final recorded refraction. The main complications were amblyopia, especially in unilateral cataracts, and posterior capsular opacification. Amblyopia was most probably related to a combination of early onset of dense cataract in this young age group, late presentation for initial surgery, delay in capsulotomies, and imperfect compliance with a rigorous occlusion regime. CONCLUSION: Intraocular lens implantation in infants less than 1 year of age is generally a safe procedure. The spread of final refractive error was very wide. Final refraction in the unilateral group was significantly more myopic than the bilateral group. Final acuities were often disappointing especially in the unilateral group.
Assuntos
Catarata/congênito , Implante de Lente Intraocular , Ambliopia/etiologia , Catarata/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Implante de Lente Intraocular/efeitos adversos , Lentes Intraoculares , Masculino , Miopia/etiologia , Recidiva , Refração Ocular , Estudos Retrospectivos , Resultado do Tratamento , Acuidade VisualRESUMO
BACKGROUND: Endometriosis is a common gynaecological disease and is frequently associated with recurrent and serious pelvic pain such as dysmenorrhoea and dyspareunia, but the mechanisms by which these symptoms are generated are not well understood. METHODS: Histological sections of endometrial tissue were prepared from endometrial curettings and hysterectomies performed on women with endometriosis (n=25 and n=10, respectively) and without endometriosis (n=47 and n=35, respectively). These were stained immunohistochemically for the highly specific polyclonal rabbit anti-protein gene product 9.5 (PGP9.5) and monoclonal mouse anti-neurofilament protein (NF) to demonstrate both myelinated and unmyelinated nerve fibres. RESULTS: Small nerve fibres were identified throughout the basal and functional layers of the endometrium in all endometriosis patients, but were not seen in the functional layer of the endometrium in any of the women without endometriosis (P<0.001). NF-immunoreactive nerve fibres were present in the basal layer in all endometriosis patients but not in non-endometriosis patients, with one exception (P<0.001). CONCLUSIONS: Small nerve fibres detected in the functional layer in all women with endometriosis may have important implications for understanding the generation of pain in these patients. The presence of nerve fibres in an endometrial biopsy may be a novel surrogate marker of clinical endometriosis.
Assuntos
Endometriose/patologia , Endométrio/inervação , Endométrio/patologia , Fibras Nervosas/patologia , Adulto , Biópsia , Endometriose/fisiopatologia , Feminino , Humanos , Imuno-Histoquímica , Laparoscopia , Dor/fisiopatologia , Ubiquitina Tiolesterase/análiseRESUMO
The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.
Assuntos
Apoptose/imunologia , Linfócitos B/patologia , Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa/imunologia , Doenças das Aves Domésticas/virologia , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/patologia , Infecções por Birnaviridae/virologia , Linhagem Celular Tumoral , Marcação In Situ das Extremidades Cortadas/veterinária , Doenças das Aves Domésticas/patologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Transfecção/veterinária , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologiaRESUMO
Recent evidence points towards a substantial disturbance of the process of angiogenesis within the endometrium in women who are exposed continuously to low dose contraceptive progestogens. This results in the patchy appearance of abnormally small and abnormally large, thin-walled vessels in the superficial regions of exposed endometrium. Three-dimensional pictures were developed from digitised images of serial tissue sections of endometrium in which microvascular endothelial cells were labelled with antibodies to the endothelial cell surface antigen, CD34 and their basement membranes labelled with anti-Collagen IV antibodies. Microvessels from endometrium exposed to continuous low-dose levonorgestrel from a subdermal implant system (Norplant) displayed considerable variations in size and shape. No spiral arterioles were identified. Some microvessels showed considerable dilatation, distortion and variability in the presence of surrounding basement membrane components. Other endothelial structures included narrow, solid cords of endothelial cells, without basement membranes, which often connected with normal or abnormal vessels containing lumens. Some areas, especially deeper in the tissue, contained microvessels of normal size and shape surrounded by basement membrane. These images have revealed an overall picture of great variability in superficial endometrial vascular structures in some women using a low-dose levonorgestrel implant system which appears substantially different from that seen in normal endometrium.
Assuntos
Anticoncepcionais Femininos/farmacologia , Endométrio/irrigação sanguínea , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Levanogestrel/farmacologia , Microcirculação/efeitos dos fármacos , Microcirculação/ultraestrutura , Implantes de Medicamento , Endométrio/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Neovascularização PatológicaRESUMO
There has been considerable interest over many years in the precise structural relationships between microvessels and secretory glands in human endometrium. However, microcirculatory networks have rarely been studied in three-dimensions (3D) using modern computerised technologies, this has been partly due to the late arrival of suitable endothelial cell markers. This study was designed to develop a technique to visualize and to reveal the relationships between microvessels, their glandular environment and epithelial boundaries in 3D, using endometrium from human hysterectomy biopsies. Specimens were carefully selected from women with conditions unlikely to affect the microvascular networks. Monoclonal antibodies (mouse anti-human CD 34 and goat anti-mouse fluorescein (FITC)) were used to visualize the microvessels, and polyclonal antibodies (rabbit anti-human keratin and goat anti-rabbit tetramethylrhodamine (TRITC)) were used to visualize the glandular structures. The samples were studied with a Leica multiphoton system using a titanium-sapphire laser (excitation 800 nm with pulses in the 200 fs range) to obtain a stack of two-dimensional (2D) images to a minimal focus depth of 120 microm. The initial data sets acquired were volume rendered using the integrated software of the Leica system to produce 3D images. This software allowed for the acquisition of data sets from the microscope and for an observational morphological assessment to be made, but was limited in preparing the data for any quantitative analysis. The additional use of ImarisBasic 3.1 visualization software allowed for an observational morphological assessment but also included numerous tools for data manipulation.
Assuntos
Endométrio/anatomia & histologia , Endométrio/irrigação sanguínea , Técnicas Histológicas/métodos , Animais , Feminino , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Microcirculação , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microtomia/instrumentação , Microtomia/métodos , Fótons , CoelhosRESUMO
This paper describes a technique to develop high-resolution three-dimensional (3D) images of microvasculature structures in curettage, hysterectomy or endometrial resection biopsies using parallel histological serial sections. Employing a labelled streptavidin-biotin-alkaline phosphatase (LSAB(+)) method and visualising by using DAB(+) with the primary antibody, mouse anti human Q-Bend-10, the images were directly digitised from a light microscope into the KS400 Universal Image Processing and Analysis software via a CCD colour camera; binary images of the structures were created and the binary images were exported into VoxBlast 3D rendering software to view still and rotating 3D images on a computer monitor. This in turn enabled hard copies of the full sequence to be printed.
Assuntos
Endométrio/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Endométrio/irrigação sanguínea , Feminino , Humanos , Microtomia/métodosRESUMO
HIV-1 infection of human PBMC has been shown to elicit secretion of several different cytokines. TNF-alpha secretion induced by this virus has been of particular interest because it has been associated with the development of HIV-1 dementia and because TNF-alpha increases viral replication by enhancing NF-kappaB interaction with the viral promoter, the HIV-1 long terminal repeat. Thus, an autocrine pathway is potentially created in which HIV-1 stimulates its own replication. Conflicting reports exist, however, on the ability of HIV-1 to induce TNF-alpha secretion in vitro or in vivo. Using experimental protocols that controlled for potential bacterial endotoxin-induced TNF-alpha secretion, the current study demonstrates significant differences in TNF-alpha-eliciting properties among primary and laboratory obtained HIV-1. The relative TNF-alpha-inducing ability of different variants is conserved when tested using PBMC from different individuals. Elicitation of TNF-alpha secretion was not blocked by exposure of cells to zidovudine, indicating that viral integration was not required to induce secretion. Rather, the interaction between the virus and cell surface is critical for TNF-alpha induction, as Abs against CD4 or CCR5 blocked the induction of TNF-alpha synthesis by PBMC when added before virus exposure. Furthermore, the ability to induce TNF-alpha secretion mapped to a region of the HIV-1 env gene that includes the third hypervariable domain. Differences in the ability of different HIV-1 variants to elicit TNF-alpha may account for individual differences in HIV-1 disease course.
Assuntos
HIV-1/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Variação Antigênica/efeitos dos fármacos , Variação Antigênica/genética , Variação Antigênica/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Células Cultivadas , Mapeamento Cromossômico , Genes env/imunologia , Anticorpos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Receptores de HIV/imunologia , Reprodutibilidade dos Testes , Inibidores da Transcriptase Reversa/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Zidovudina/farmacologiaRESUMO
This study describes the isolation and partial characterization of a low molecular weight (approximately 14 kDa), cadmium-binding protein from rainbow trout (Onchorynchus mykiss) liver. Rainbow trout were injected intraperitoneally with 3.5 mg/kg cadmium chloride (total body dose) twice weekly for 3 wk. Livers were removed and a cadmium-binding protein was isolated. Monoclonal antibodies produced against this protein were used in the affinity purification process. Amino acid analysis showed the protein contained 3.8 mol% cysteine, 3.5 mol% phenylalanine, 2.2 mol% tyrosine and 1.9 mol% histidine. The low cysteine content suggests that it was distinct from metallothionein. The monoclonal antibodies were also used to identify the protein in liver homogenates from both cadmium-exposed and control fish and in the testes of cadmium-exposed mice lacking the gene for both metallothionein-1 and metallothionein-II. The compound identified in this study represents a non-metallothionein cadmium-binding protein that appears to be highly conserved.
Assuntos
Metalotioneína/isolamento & purificação , Oncorhynchus mykiss/fisiologia , Animais , Anticorpos Monoclonais , Cádmio/toxicidade , Fígado/química , Camundongos , Poluentes Químicos da Água/toxicidadeRESUMO
This study aimed to investigate the effects of the subdermal levonorgestrel contraceptive implant Norplant on endometrial vascular density at different durations of exposure, and the relationship between endometrial histology, vascular density, and bleeding patterns. A prospective controlled trial of Norplant implant users compared endometrial vascular density in biopsies taken before and after Norplant implant insertion. A total of 34 women with regular menstrual cycles requesting long-term contraception were recruited at the Sydney Centre for Reproductive Health Research, Australia. A significant increase in mean endometrial microvascular density was observed from as early as 3 weeks after insertion of Norplant implants. Vascular density was increased from a control secretory phase value of 189.6 (7.0 vessels/mm2 (+/- SEM) to 253.80 +/- 7 vessels/mm2 at 2-13 weeks of Norplant implant exposure (t ratio = 2.08, p = 0.01) and 212.7 +/- 12.9 vessels/mm2 at 14-42 weeks of exposure (t ratio = 2.03, p = 0.02). In those with atrophic endometrium, or in whom myometrium and basalis only were found in biopsies (20 of 66, 30%), mean endometrial vascular density was increased at 273.1 +/- 16.1 vessels/mm2 compared with 210.9 +/- 11.7 vessels/mm2 in other histological groups (F ratio = 9.74, p = 0.0028). Bleeding and spotting in the previous 30 days were less common in those with this histological appearance at a mean of 4.95 days compared with 8.22 days. This is the first study to assess endometrial vascular density in the early months of Norplant implant use. The findings suggest that the endometrial vasculature is profoundly altered in the early months of Norplant implant exposure when bleeding problems are most common.
PIP: This is a study on the effects of Norplant on endometrial microvascular density at different durations of implant exposure and on the relationship between endometrial histology, vascular density, and bleeding patterns. A total of 34 women aged 18-40 years who had regular menstrual cycles and wanted long-term contraception were recruited between May 1994 and September 1995. Results showed a marked increase in endometrial vascular density after exposure to Norplant implants. Mean endometrial vascular density in the control cycle was 189.0 + 7.0 vessels/mm, which was significantly less than that seen at 2-13 weeks of exposure (253.9 + 80.7 vessels/mm; t ratio = 2.08, p = 0.01) and at 14-42 weeks of exposure to Norplant implants (212.7 + 12.9 vessels/mm; t ratio = 2.03, p = 0.02). The increase in vascular density was observed from as early as 3 weeks after insertion of Norplant implants. Endometrial vascular density was related to histological appearance. Subjects with regressed endometrium biopsies had significantly higher mean endometrial vascular density (273.1 + 16.1 vessels/mm). This study has assessed changes in endometrial vascular density before and after Norplant-implant insertion. Bleeding and spotting were not frequently present in subjects with these histological appearances.
Assuntos
Anticoncepcionais Femininos , Endométrio/irrigação sanguínea , Endométrio/citologia , Levanogestrel , Ciclo Menstrual/fisiologia , Microcirculação/efeitos dos fármacos , Adolescente , Adulto , Implantes de Medicamento , Endométrio/efeitos dos fármacos , Estradiol/sangue , Feminino , Humanos , Estudos Longitudinais , Ciclo Menstrual/efeitos dos fármacos , Menstruação , Progesterona/sangueRESUMO
An immunofluorescent antibody test (IFAT) developed for the diagnosis for plasmacytoid leukemia was evaluated against histology under field conditions. Previously published results from a laboratory evaluation indicated that the IFAT had a much higher sensitivity than did histology. One hundred seventy-seven moribund chinook salmon from 3 farms located in British Columbia were sampled. Sensitivity, specificity and their respective quality indices were estimated for the IFAT relative to histology. The IFAT was shown to be unreliable, particularly with respect to sensitivity. Cohen's kappa was also calculated and revealed that the agreement between the 2 tests was no better than random. In contrast to previously published results the IFAT did not perform better than histology in the presence of bacterial kidney disease. The results emphasize the importance of evaluating tests in the field conditions in which they are to be used. The possible reasons for the shortcomings of the IFAT are discussed.
Assuntos
Doenças dos Peixes/diagnóstico , Leucemia Plasmocitária/veterinária , Animais , Colúmbia Britânica , Doenças dos Peixes/patologia , Técnica Indireta de Fluorescência para Anticorpo , Leucemia Plasmocitária/diagnóstico , Leucemia Plasmocitária/patologia , Oncorhynchus , Reprodutibilidade dos Testes , Água do Mar , Sensibilidade e EspecificidadeRESUMO
Progestogen-only contraception is almost invariably associated with changes in menstrual bleeding patterns. Changes in the endometrial vasculature, and in particular an increase in vascular fragility, may contribute to this bleeding. In this study, endometrial vascular density and endothelial cell basement membrane components were examined using immunohistochemistry before and after insertion of Norplant. Endometrial vascular density was increased from a mean (+/- SEM) of 189.6 +/- 7.0 vessels/mm2 during the control cycle to 253.9 +/- 80.7 vessels/mm2 at 2-13 weeks of Norplant exposure, and to 212.7 +/- 12.9 vessels/mm2 at 14-42 weeks. During the control cycle, a mean of 161.4 +/- 4.5 vessels/mm2 stained for collagen IV (85% of all vessels), while at 2-13 weeks, 144.5 +/- 13.0 vessels/mm2 stained for collagen IV (57% of all vessels) (t ratio = 2.08, P = 0.0057). By 14-42 weeks, 71% of vessels (151.0 +/- 9.8) vessels/mm2 were surrounded by collagen IV. This was not significantly different from control values (t ratio = 2.03). Endometrial vascular laminin was also reduced following Norplant insertion, from a mean of 176.0 +/- 4.2 vessels/mm2 in the control cycle (93% of vessels), to 156.3 +/- 6.7 vessels/mm2 at 2-13 weeks of exposure (57% of vessels) (t ratio = 2.08, P = 0.01). By 14-42 weeks of exposure to Norplant, 162.5 +/- 9 vessels/mm2 (76%) stained for laminin. This was not significantly different from control values (t ratio = 2.04). Endometrial vascular heparan sulphate proteoglycan (HSPG) was reduced from 58.6 +/- 3.0 vessels/mm2 during the control cycle (31% of vessels) to 43.6 +/- 5.6 vessels/mm2 (only 17% of vessels) at 2-13 weeks (t ratio = 2.08, P = 0.025). At 14-42 weeks, only 19% of vessels stained for HSPG (41.3 +/- 5.8 vessels/mm2; t ratio = 2.04, P = 0.009).