Circulating and Endometrial Regulatory T Cell and Related Populations in Endometriosis and Infertility: Endometriosis Is Associated with Blunting of Endometrial Cyclical Effects and Reduced Proportions in Moderate-Severe Disease.

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.

Comprehensive analysis utilizing flow cytometry and immunohistochemistry reveals inflammatory changes in local endometrial and systemic dendritic cell populations in endometriosis.

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.

Foetal haemoglobin, blood transfusion, and retinopathy of prematurity in very preterm infants: a pilot prospective cohort study.

PurposeTo identify if there is an association between foetal haemoglobin (HbF) concentration and retinopathy of prematurity (ROP) in very preterm infants.Patients and methodsProspective cohort study. Infants born <32 weeks' gestational age or <1501 g in two tertiary neonatal units between January 2012 and May 2013 (n=42) were enrolled. HbF and adult haemoglobin (HbA) concentrations were measured using high-pressure liquid chromatography from blood samples sent as part of routine neonatal care once routinely requested laboratory tests had been performed. Clinical data were obtained from case notes. We calculated odds ratios (ORs) (95% confidence intervals (CIs)) to quantify the relationship between initial and mean %HbF with ROP severity (none, stages 1-3).ResultsA total of 42 infants were recruited: mean gestation 28.0 weeks (SD 1.91); mean birth weight 1042 g (SD 264). Six infants died before ROP screening; 14/36 developed ROP (39%); and 22/36 (61%) did not. Infants who developed ROP had similar initial %HbF (83.3 vs 92.3%, P=0.06), but significantly lower mean %HbF (61.75 vs 91.9%, P=0.0001) during their inpatient stay than those who did not develop ROP. In ordinal logistic regression models adjusted for birth weight, gestation and transfusion volume, mean post-natal %HbF was negatively associated with ROP severity: adjusted OR 0.94 (0.90-0.99), while initial %HbF at birth was not: adjusted OR 1.05 (0.97-1.16).ConclusionReplacing HbF by HbA during transfusion may promote ROP development by rapidly increasing oxygen availability to the retina. Conversely, maintaining a higher %HbF may be a protective factor against ROP.

Pathogenesis of Frog Virus 3 ( Ranavirus, Iridoviridae) Infection in Wood Frogs ( Rana sylvatica).

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.

Coronary computed tomography angiography and its increasing application in day to day cardiology practice.

Coronary artery disease (CAD) is the leading single cause of death in Australia affecting around 1.4 million people. Coronary computed tomography angiography has an established role in the assessment of patients with low to intermediate pretest probability for CAD who have chest pain and is typically used with the aim to rule out significant coronary artery stenosis. Use was initially limited because of concerns over radiation exposure, a Medicare rebate restricted to specialist referrals and an absence of data supporting its use as an alternative to functional testing in patients with chest pain. Recent advances in scanner technology and image sequencing, along with data from randomised control trials, have addressed these issues and indicate that coronary computed tomography angiography will play a greater role in the assessment of CAD in the coming years.

Dysregulation of vascular endothelial growth factors and their neuropilin receptors in the eutopic endometrium of women with endometriosis.

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.

Endometrial stromal cells and immune cell populations within lymph nodes in a nonhuman primate model of endometriosis.

Mounting evidence suggests that immunological responses may be altered in endometriosis. The baboon (Papio anubis) is generally considered the best model of endometriosis pathogenesis. The objective of the current study was to investigate for the first time immunological changes within uterine and peritoneal draining lymph nodes in a nonhuman primate baboon model of endometriosis. Paraffin-embedded femoral lymph nodes were obtained from 22 normally cycling female baboons (induced endometriosis n = 11; control n = 11). Immunohistochemical staining was performed with antibodies for endometrial stromal cells, T cells, immature and mature dendritic cells, and B cells. Lymph nodes were evaluated using an automated cellular imaging system. Endometrial stromal cells were significantly increased in lymph nodes from animals with induced endometriosis, compared to control animals (P = .033). In animals with induced endometriosis, some lymph node immune cell populations including T cells, dendritic cells and B cells were increased, suggesting an efficient early response or peritoneal drainage.

Fish microsporidia: immune response, immunomodulation and vaccination.

Immune response to fish microsporidia is still unknown and there are current research trying to elucidate the events involved in the immune response to this parasite. There is evidence suggesting the role of innate immune response and it is clear that adaptive immunity plays an essential part for eliminating and then mounting a solid resistance against subsequent microsporidian infections. This review article discusses the main mechanisms of resistance to fish microsporidia, which are considered under four main headings. 1) Innate immunity: the inflammatory tissue reaction associated with fish microsporidiosis has been studied at the ultrastructural level, providing identification of many of the inflammatory cells and molecules that are actively participating in the spore elimination, such as macrophages, neutrophils, eosinophilic granular cells, soluble factors and MHC molecules. 2) Adaptive immunity: the study of the humoral response is relatively new and controversial. In some cases, the antibody response is well established and it has a protective role, while in other situations, the immune response is not protective or it is depressed. Study of the cellular response against fish microsporidia is still in its infancy. Although the nature of the microsporidian infection suggests participation of cellular mechanisms, few studies have focused on the cellular immune response of infected fish. 3) Immunomodulation: glucans are compounds that can modulate the immune system and potentiate resistance to microorganisms. These compounds have been proposed that can interact with receptors on the surface of leukocytes that result in the stimulation on non-specific immune responses. 4) Vaccination: little is known about a biological product that could be used as a vaccine for preventing this infection in fish. In the Loma salmonae experience, one of the arguments that favor the production of a vaccine is the development in fish of resistance, associated to a cellular immune response. A recently proved spore-based vaccine to prevent microsporidial gill disease in salmon has recently shown its efficacy by considerably reducing the incidence of infection. This recent discovery would be first anti-microsporidian vaccine that is effective against this elusive parasite.

The effect of age on the immune response of horses to vaccination.

Few studies have investigated immunosenescence in the horse, but it is accepted that the primary and secondary (anamnestic) immune responses may differ between aged and younger horses. The aim of the present study was to determine whether aged horses have a protective immune response post-vaccination. Thirty-four aged healthy horses (> or =20 years) and 29 younger adult horses (4-12 years) of various breeds were vaccinated with commercially produced killed rabies and influenza vaccines. Rabies serum neutralizing antibody titres and equine influenza virus specific antibody subclasses (immunoglobulin IgGa and IgGb) and single radial haemolysis titres were determined. Healthy aged horses mounted a primary immune response to rabies vaccine that was similar to that of younger adult horses. However, aged horses had a significantly reduced anamnestic response to influenza vaccination in comparison with the younger adult horses, even though the pre-vaccination antibody titres of aged horses were higher. Rabies antibody titres in both groups declined significantly by 6 months post-vaccination. Serum concentrations of selenium (Se) and vitamin E were measured to test for potential confounding effects. Significant numbers of horses had suboptimal serum Se concentrations, but Se status had no significant impact on antibody production after vaccination.

Diagnosis of endometriosis by detection of nerve fibres in an endometrial biopsy: a double blind study.

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.

The effect of age on serum antibody titers after rabies and influenza vaccination in healthy horses.

BACKGROUND: The proportion of geriatric horses within the equine population has increased in the past decade, but there is limited information on the immune function of these animals. HYPOTHESIS: Aged horses will have a lesser increase in serum antibody response to vaccination. ANIMALS: Thirty-four aged healthy horses (> or = 20 years) and 29 younger adult horses (4-12 years) of various breeds. METHODS: All horses were vaccinated with vaccines of killed rabies and influenza virus. Horses in each age group were allocated to receive either rabies or influenza booster vaccine 4 weeks after the initial vaccination. Serum samples were taken at 0, 4, 8, and 24 weeks. Rabies serum neutralization titers and equine influenza virus specific antibody sub-isotypes (IgGa, IgGb, IgG(T), and IgA) as well as single radial hemolysis (SRH) titers were determined. RESULTS: Rabies antibody titers were similar in the 2 age groups at all sampling times. Aged horses had higher IgGa and IgGb influenza antibody titers before vaccination than younger horses but similar titers after vaccination (P= .004 and P= .0027, respectively). Younger horses had significantly greater increases in titer than aged horses at all sampling times for IgGa (P= .001) and at 8 and 24 weeks for IgGb (P= .041 and .01, respectively). There was no detectable serum IgG(T) at any time point. A significant booster vaccine effect was seen for both antirabies and anti-influenza titers. Anti-influenza titer before vaccination also had a significant effect on subsequent antibody response. CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy aged horses generated a primary immune response to a killed rabies vaccine similar to that of younger adult horses. Aged horses had a significantly reduced anamnestic response to influenza vaccine.

Vitreoretinal surgery for retinal detachment in retinochoroidal colobomata.

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.

Endometrial dendritic cell populations during the normal menstrual cycle.

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.

Serologic detection of Angiostrongylus vasorum infection in dogs.

Angiostrongylus vasorum, French Heartworm, is a metastrongylid nematode infecting the pulmonary arteries and right heart of wild and domestic canids in various regions of the world. Infection in dogs can result in fatal cardiopulmonary disease. A single endemic focus of A. vasorum in North America occurs in the southeastern portion of Newfoundland, Canada. Dogs are currently diagnosed by detection of first-stage larvae shed in feces using the Baermann technique or fecal flotation. However, these procedures may lack sensitivity due to intermittent fecal larval shedding. The potential for using detection of circulating worm antigen for diagnosis was investigated by developing a sandwich-ELISA using rabbit anti-whole adult worm antiserum. This test detected circulating antigen in sera from 22/24 Baermann positive dogs naturally infected with A. vasorum. Negative results (0/52) were obtained from sera collected from Baermann negative dogs from outside of the endemic region, and from sera (0/30) from dogs from non-endemic regions that were infected with Crenosoma vulpis, the fox lung worm. Receiver operating curve analysis gave a specificity of 100% and a sensitivity of 92% for the sandwich-ELISA at an optical density cut-off of 0.19. Subsequently, 239 dogs from Newfoundland displaying clinical signs of cardiopulmonary disease, were examined using both the Baermann fecal examination and the sandwich-ELISA. Larvae were detected in 10% (24/239) of these dogs by fecal examination, whereas the sandwich-ELISA detected circulating antigen of A. vasorum in serum from 18.8% (45/239) of the dogs. This suggests that fecal diagnostics may have missed approximately half of the A. vasorum infected dogs, and that the sandwich-ELISA may be a useful tool in the diagnosis of this parasite.

The aetiology of paediatric rhegmatogenous retinal detachment: 15 years experience.

PURPOSE: To provide contemporary data on the aetiology of paediatric rhegmatogenous retinal detachment (RRD) in the UK population. METHODS: Retrospective case series. RESULTS: Eighty-eight eyes in 82 patients (aged 0-16 years) were treated for RRD at Bristol Eye Hospital between 1 January 1990 and 31 December 2004. Seventy-three per cent of patients were male and the main predisposing factors were trauma (53%), associated conditions (27%), and high myopia (17%). Nineteen per cent of RRDs were idiopathic, and the majority of these were due to infero-temporal dialyses. The macula was detached on presentation in 66% of eyes. CONCLUSIONS: The principal causes of paediatric RRDs have not changed over the past 40 years. Those due to congenital cataracts, retinopathy of prematurity, uveitis, and glaucoma are now less prevalent, presumably reflecting advances in their management. Differences with other contemporary series may arise from geographical variation in the prevalence of myopia and other associated conditions, as well as institutional referral patterns. Full examination of the retinal periphery is advised for children with eye injuries (to exclude dialyses).

Development of an effective whole-spore vaccine to protect against microsporidial gill disease in rainbow trout (Oncorhynchus mykiss) by using a low-virulence strain of Loma salmonae.

In determining the effective vaccine spore dose of a low-virulence strain of Loma salmonae to limit microsporidial gill disease in trout, we found that fish receiving 10(3) to 10(5) killed spores had the best protection against experimental infection, with 85% fewer xenomas in their gills than in the controls. Intraperitoneal delivery of the vaccine was effective, and the addition of adjuvant did not improve vaccine performance against this disease-causing microsporidian.

Cellular immunity in salmonids infected with the microsporidial parasite Loma salmonae or exposed to non-viable spores.

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.