SummaryThe programmed cell death 1 (PD‐1) receptor plays a major role in regulating T cell activation. Our aim was to determine how inflammation influences PD‐1‐mediated T cell suppression. Flow cytometry analysis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) synovial fluid (SF) mononuclear cells showed an increase in the percentage of PD‐1+ cells within the CD4+ and CD8+ T cell compartment compared to paired peripheral blood (PB). Upon in‐vitro T cell receptor (TCR) stimulation of healthy control (HC) CD4+ T cells in the presence of plate‐bound PD‐L1fc chimera, significantly decreased proliferation and interferon (IFN)‐γ secretion was observed. In contrast, CD4+ T cells from RA and PsA PB and SF appeared resistant to such PD‐1‐mediated inhibition. Addition of the proinflammatory cytokines tumour necrosis factor (TNF)α, interleukin (IL)‐6 and IL‐1β, which were increased in RA and PsA SF compared to osteoarthritis (OA) SF, consistently abrogated PD‐1‐mediated suppression in HC CD4+ T cell cultures. This effect was reversed by inhibitors of these cytokines. Soluble PD‐1 (sPD‐1) levels were increased in cell culture supernatants from TNFα and IL‐6‐stimulated cultures compared to untreated controls, and also in RA and PsA, but not in OA, serum and SF. Functionally, addition of sPD‐1fc counteracted PD‐1‐mediated suppression of HC CD4+ T cells, and increased T cell proliferation in HC CD4+ T cell/monocyte co‐cultures. These in‐vitro findings indicate that CD4+ T cells from patients with RA and PsA show increased resistance to PD‐1‐mediated suppression, which may be explained in part by the presence of soluble PD‐1 in the inflammatory environment.

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

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ISRCTN registry, http://isrctn.com, ISRCTN22288225 and EudraCT, https://eudract.ema.europa.eu, 2011-005831-19.

Rat tissues were surveyed for proteins which bind cGMP. Binding activity was high in extracts of lung, cerebellum, and small intestine, but was low in those of liver, adipose tissue, and skeletal muscle. DEAE-cellulose chromatography resolved two peaks of cGMP-binding activity in most tissues. The binding protein in peak 1 was eluted in the flowthrough volume and was most abundant in extracts of intestine.It had a sedimentation coefficient of 6 S and was highly specific for cGMP at pH 7.0 (dissociation constant KD = 0.05 ,1M We report here the separation and distribution in rat tissues of proteins which bind cGMP with high specificity. The relationship of binding activity to protein kinase activity has been examined. MATERIALS AND METHODSMaterials. Cyclic GMP was purchased from Nutritional Biochemicals and was found to be pure as judged by thin-layer chromatography. Cyclic AMP and calf-thymus histone type II-A and IV were from Sigma.[3H]cGMP (21 Ci/mmol) was from Amersham-Searle and [3H]cAMP (37 Ci/mmol) was purchased from New England Nuclear. All chemicals were reagent grade.Preparation of the cGMP-Binding Proteins. Male Sprague-Dawley rats (200-300 g) were decapitated and the tissues were quickly excised, rinsed in 20 mM sodium phosphate-2 mM EDTA, pH 7.0, and homogenized in 5 volumes (wt/vol) of the same buffer at 40. The homogenate was centrifuged at 12,000 X g for 20 min. For purification of the lung binding proteins approximately 35 ml of the supernatant were applied to a DEAE-cellulose column (1.6 X 8 cm) equilibrated with the same buffer. The column was rinsed with 10 volumes of buffer and eluted with a linear gradient of NaCl from 0 to 0.4 M. Fractions (about 8 ml) were collected and those with cGMP-binding activity were pooled and dialyzed against 20 mM sodium phosphate-2 mM EDTA, pH 7.0. In some cases, the peak 2 binding protein was concentrated by precipitation with 50% saturated ammonium sulfate followed by extensive dialysis against the same buffer. Other purification methods are described in the figure legends.Cyclic Nucleotide Binding Assays. The binding assay was performed in a total volume of 0.1 ml of sodium phosphate buffer at pH 7.0 containing 0.5 MM 3H-labeled (0.1 ,uCi) cyclic nucleotide (either cGMP or cAMP) and, in some cases, 10,uM of the other nonradioactive nucleotide (either cAMP or cGMP). The binding was initiated by the addition of 90 ,ul of protein and tubes were incubated at 4°for 30 min. Free and bound 3H-labeled cyclic nucleotides were separated by the Gilman technique (15) on Millipore filters which were dried at 150°and counted in a toluene-based scintillant. In order to assess the retention of the nucleotide protein complex on the filters, parallel assays were performed in which bound [3H]cGMP was separated on a Sephadex G-25 column (0.9 X 10 cm) and counted. Estimates of bound cGMP by both methods agreed closely. Protein was determined by the method of Lowry et al. (16).

This study investigated the validity of the Health Belief Model when applied to adolescents' responses to the threat of AIDS. A sample of 195 sexually active 18-year-old university students was questioned about their sexual behaviour. their perceptions of the seriousness of AIDS, their own susceptibility to the disease, the costs and benefits of using condoms, their general health motivation, their sources of information, and their knowledge about AIDS. There were gender differences in health beliefs, with young women perceiving AIDS as more serious than males and perceiving more benefits of condom use. For young men, the Health Belief Model failed to predict sexual risk-taking either with casual partners or with regular (steady) partners. For young women, the model significantly predicted sexual risk with casual but not regular partners. Of the health beliefs, only perceived susceptibility contributed significantly to the variance in these young women's sexual risk with casual partners. In no case was knowledge about AIDS a significant predictor. Gender and partner differences are explained in tern of the multiple determinants of adolescent sexual behaviour, including the differing constructions placed on relationships by young men and women.

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17Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic 18 flatworms known as schistosomes. Humans become infected by free-swimming, water-borne 19 larvae, which penetrate the skin. The earliest intra-mammalian stage, called the 20 schistosomulum, undergoes a series of developmental transitions. These changes are critical 21 for the parasite to adapt to its new environment as it navigates through host tissues to reach its 22 niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive 23 intra-mammalian development requires knowledge of the spatial organisation and 24 transcriptional dynamics of different cell types that comprise the schistomulum body. To fill 25 these important knowledge gaps, we performed single-cell RNA sequencing on two-day old 26 schistosomula of Schistosoma mansoni. We identified likely gene expression profiles for 27 muscle, nervous system, tegument, parenchymal/primordial gut cells, and stem cells. In 28 addition, we validated cell markers for all these clusters by in situ hybridisation in 29 schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-30 type atlas for the early intra-mammalian stage of this devastating metazoan parasite. 31 32 the parasite to survive for decades 5,6 . The schistosomula make their way into blood or 51 lymphatic vessels and, one week after infection, reach the lung capillaries 7 . The migration 52 through the lung requires coordinated neuromuscular activities, including cycles of muscle 53 elongation and contraction 8 , to squeeze through capillaries and reach the general circulation 7 . 54Over the following weeks, the parasites mature further into sexually reproducing adults. 55Dramatic changes to the parasite are required that include posterior growth, remodelling of the 56 musculature 9 and nervous system 10,11 as well as the development of the gonads 12 and gut 13 . 57This extensive tissue development starts in the schistosomula, with stem cells driving these 58 transitions 6,14,15 . However, to decipher cellular and molecular mechanisms underlying 59 schistosomula development, a detailed understanding of the spatial organisation and 60 transcriptional programs of individual cells are needed. 61 Important insights into major processes that underlie the transformations across the life cycle 62 have been gained from bulk transcriptomic studies 5,6,14-24 . However, these studies are not able 63 to quantify the relative abundance of different cell types from the absolute expression per cell, 64 and the signal from highly expressed genes in a minority of cells can often be masked by a 65 population averaging effect. Single-cell RNA sequencing has previously been used 66 successfully to characterise cell types [25][26][27][28][29][30][31][32] and understand how the cell expression profile 67 changes during differentiation 31-38 . Notable examples include recent studies in the free-living 68 planarian flatworm Schmidtea mediterranea 31,32,39 , a well-establishe...