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.

Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation.

Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83(+) cells by CD16(+) NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44(+)CD83(bright) activated DC but spared CD83(dim/-) DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4(+) FoxP3(+) CD25(+) Treg cells and also viral-specific CD8(+) T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

Long-term survival in multiple myeloma is associated with a distinct immunological profile, which includes proliferative cytotoxic T-cell clones and a favourable Treg/Th17 balance.

Despite improved outcomes in multiple myeloma (MM), a cure remains elusive. However, even before the current therapeutic era, 5% of patients survived >10 years and we propose that immune factors contribute to this longer survival. We identified patients attending our clinic, who had survived >10 years (n=20) and analysed their blood for the presence of T-cell clones, T-regulatory cells (Tregs) and T helper 17 (Th17) cells. These results were compared with MM patients with shorter follow-up and age-matched healthy control donors. The frequency of cytotoxic T-cell clonal expansions in patients with <10 years follow-up (MM patients) was 54% (n=144), whereas it was 100% (n=19/19) in the long-survivors (LTS-MM). T-cell clones from MM patients proliferated poorly in vitro, whereas those from LTS-MM patients proliferated readily (median proliferations 6.1% and 61.5%, respectively (P<0.0001)). In addition, we found significantly higher Th17 cells and lower Tregs in the LTS-MM group when compared with the MM group. These results indicate that long-term survival in MM is associated with a distinct immunological profile, which is consistent with decreased immune suppression.

A robust flow-cytometric protocol for assessing growth rate of hatchery-reared barramundi Lates calcarifer larvae.

In this study, a flow-cytometric cell cycle analysis method to assess instantaneous growth rate of whole larvae of the Australian barramundi Lates calcarifer was developed and validated. High-resolution DNA measurements of either fresh, frozen or RNAlater-preserved larvae (gap0-gap1, G(0) -G(1), coefficient of variation (c.v.) < 3, 4 and 5%, respectively) enabled the deconvolution of the DNA histogram and assignment of the proportion of nuclei into cell cycle compartments G(0) -G(1), S (DNA synthesis) and G(2) -M (Gap2-Mitosis). This technique can be also used for individual fish tissues such as brain, liver, fin and muscle. For the first time, the combined proportion of replicating nuclei (into S and G(2) -M phases) of whole fish larvae and absolute growth rate in length (mm day(-1)) has been correlated in commercial aquaculture conditions. Fast growing L. calcarifer larvae had an overall hyperplasia advantage as indicated by a greater proportion of cells in the S+G(2) -M phase compared with slow growing larvae, which might explain the increasing differences in size during culture. In a fasting trial, larvae ceased growth while maintaining the constant initial rates of cell division throughout a 6 day period. For a highly fed fast growing control group, cell division rates significantly increased after day 4. Flow-cytometric cell cycle analysis of whole fish larvae may provide fish biologists and aquaculturists with a better understanding of how cell division rates influence early growth in natural and artificial environments.

In vitro mitogenesis of peripheral blood lymphocytes from rainbow trout (Salmo gairdneri).

1. In vitro mitogenesis of rainbow trout peripheral blood lymphocytes (RBT PBL) was investigated to assess the applicability of this procedure in assessment of fish health. The assay variables of media, mitogen type and concentration, serum supplementation, lymphocyte isolation procedure, and duration of incubation were assessed. 2. Concanavalin A (Con A) stimulated greater proliferation of RBT PBL than did lipopolysaccharide (LPS), phytohemagglutinin (PHA), or pokeweed mitogen (PWM). 3. RBT PBL, cultured with 10 micrograms Con A/ml and incubated for four or five days, exhibited greater proliferation than with other treatment combinations. 4. The degree of Con A-induced PBL proliferation varied significantly (P less than 0.05) among fish. The mean was positively correlated with the relative standard deviation and thus exhibited significant heteroscedasticity. 5. Human serum, as an alternative to FBS supplementation of the culture medium, did not enhance RBT PBL proliferation or reduce variation in mean proliferation. 6. Power analysis with variance estimates from this study reveal that sample size requirements of further studies under the given conditions could severely limit the applicability of this procedure for RBT health assessment. Further work in this area should center around standardization of culture conditions pertaining to the source of protein supplementation.

Oxygen diffusion in the trout retina.

Oxygen tensions were measured in vivo within the different layers of the rainbow trout retina. Oxygen microelectrodes were advanced in 10 micron increments through the retinas and the PO2 measured at each location. Mean retinal PO2 ranged from 124 mmHg at the retinal-vitreal interface to 381 mmHg at the choriocapillaris. These data reaffirm that the cellular layers of the normal trout retina are continually exposed to supra-arterial oxygen tensions that are known to cause toxicity in other species. The intraretinal PO2 gradient was mathematically characterized and results indicate that the in vivo oxygen profile can be described by a two-component exponential function. In order to gain a better understanding of oxygen delivery to the retina, the data were also subjected to an analysis based upon the classical equation for planar diffusion. The trout retina is an excellent model for studying oxygen diffusion in vivo since this tissue is supplied with oxygen from a single source, thereby simplifying the mathematical analysis. Calculations yielded values of 1.86 X 10(-5) and 0.58 X 10(-5) ml O2 min-1 cm-1 atm-1 (at 9 degrees C) for the Krogh permeation coefficient (DS) for the photoreceptor region and the remaining neural retina, respectively.

Action of organophosphates on the electroretinogram of rainbow trout.

Electroretinograms (ERGs) were recorded in vitro from eyes of rainbow trout that had received intraperitoneal injections of either TOCP (triorthocresyl phosphate) or DEF (S, S, S-tributyl phosphorotrithioate). Data obtained after 24 h indicated that these organophosphates caused alterations in four of five ERG parameters in the case of TOCP and all five parameters in the DEF treated specimens. These data were compared with data obtained from experiments with eserine and carbachol and led to the conclusion that the effects of the organophosphates on the retina were independent of any cholinesterase inhibitor activity of the compounds. These organophosphates affect (a) the non-cholinergic photoreceptor layer of the retina which produces the a-wave ERG component, and (b) the other neural layers of the retina known to be responsible for generation of the b-wave component. Based on data obtained 15 days after exposure there was no evidence that TOCP or DEF has any delayed neurotoxic effect on the retina of rainbow trout.

Factors affecting 3H2O transfer capacity of isolated perfused trout gills.

Tritiated water (3H2O) transfer capacity (PdA) and vascular impedance (Zg) of isolated trout (Salmo gairdneri) gills perfused with or without epinephrine were studied under conditions of altered osmotic gradients, fluid stirring, perfusion rate (Qa), and efferent pressure (Pe). Transfer capacity was unaffected by osmotic gradients, indicating that 3H2O moves across gills by diffusion and that PdA is independent of hydraulic water movement. Fluid stirring increased PdA asymptotically, suggesting that boundary layers significantly affect diffusion across isolated gills. Transfer capacity was directly related to Qa; Zg was inversely related to Qa. The PdA results can be explained in terms of lamellar recruitment and the distribution of flow between secondary lamellae. Increased Qa reduced Zg due to recruitment and distension of gill vessels. Elevated Pe decreased Zg and PdA. The effects of Pe on Zg resulted from distension of the gill vasculature and increased venous drainage, whereas the effects of Pe on PdA can be explained by changes in the distribution of perfusion between secondary lamellae.

Short circuiting the ocular oxygen concentrating mechanism in the teleost Salmo gairdneri using carbonic anhydrase inhibitors.

Ocular oxygen concentration by the process of counter current multiplication in rainbow trout (Salmo gairdneri) was rapidly suppressed after intraperitoneal injections of the carbonic anhydrase inhibitor CL-11,366. The rapidity with which this drug acted suggested a short circuiting of the choroidal rete mirabile. A comparison was made between the time after injection of inhibitor at which oxygen concentrating ability was lost to the time after injection of inhibitor at which its presence in red blood cells, choroidal rete, pseudobranch, and retinal tissue was first noted. A scheme for the possible role of carbonic anhydrase from each of these tissues in the process of ocular oxygen concentration is given.