Flow microfluorometric analysis of mouse thymus development in vivo and in vitro

The phenotypic properties of lymphoid cells in the developing embryonic thymus were characterized using monoclonal antibodies and flow microfluorometry. CBA/J-T6/T6 thymocytes stained with antibodies directed against Thy-1.2, Lyt-1, Lyt-2 or H-2K^k were simultaneously analyzed for fluorescence intensity and forward light scatter (FLS), a cell size-related parameter. Whereas Thy-1 and Lyt-1 antigens were already present on 15-day fetal thymocytes, Lyt-2 expression was first detectable on day 16 and increased rapidly thereafter to reach adult levels by day 19. Concomitant with these phenotypic changes, rapid changes in FLS occurred during this time period. The FLS distribution of Lyt-2⁺ cells was initially homogeneously high (day 16) but became biphasic at days 17–18. Thereafter, the lower FLS subpopulation predominated. FLS changes in Lyt-2^? cells could be dissociated kinetically from changes in the Lyt-2⁺ subpopulation. Thus high FLS Lyt-2^? cells were the predominant subpopulation throughout the entire fetal period and could still be detected after birth, when a population with lower FLS first appeared. The embryonic thymus developing in vivo was then compared with the 13-day embryonic thymus maintained for 14 days in an in vitro organ culture system. Based on a combination of fluorescence and FLS analysis, the organ-cultured thymus appeared to share certain phenotypic properties with the 18–19 day in vivo developing thymus.     

Both IFN-γ and IL-4 Induce MHC Class II Expression at the Surface of Mouse Pro-B Cells

Pro-B cells are early B-cell progenitors that retain macrophage potential. We have studied MHC class II molecules and invariant chain inducibility on four class II negative mouse pro- B-cell clones. We analyzed the effects of IL-4 and IFN-γ, which represent the major inducers of class II in the B-lymphoid and monocytic/macrophage lineages, respectively. After 48 h of treatment with either cytokine, three pro-B-cell clones (C2.13, A1.5, and F2.2) expressed intracellular invariant chain and cell-surface class II molecules. One clone (D2.1) remained negative. As already reported, more differentiated 70Z/3 pre-B cells were inducible by IL-4 only. These data suggest that the induction of class II and invariant-chain genes are subject to regulation throughout B-cell differentiation.     

Therapy by written communication

A case of marital and sexual therapy using behavioural techniques was concluded using written communication. It was a successful and cost-effective intervention.     

What is the best clinical test for Achilles tendinopathy?

BackgroundDifferential diagnosis of Achilles pathology is demanding. This study evaluates the diagnostic accuracy of clinical tests identified for a chronic mid body Achilles tendinopathy. Ultrasound scanning provides the reference standard.MethodsTwenty-one participants with, and without, an Achilles tendinopathy, had an ultrasound scan followed immediately by the application of ten clinical tests. The accuracy and reproducibility of each test was determined.ResultsThe most valid tests are; pain on palpation of the tendon (sensitivity 84%, specificity 73%, kappa 0.74–0.96) and the subjective reporting of pain 2–6 cm above the insertion into the calcaneum (sensitivity 78%, specificity 77%, kappa 0.75–0.81).ConclusionOnly location of pain and pain on palpation were found to be sufficiently reliable and accurate, to be recommended for use.     

Hypofibrinolysis in type 2 diabetes: the role of the inflammatory pathway and complement C3

Aims/hypothesis

Plasminogen activator inhibitor-1 (PAI-1) has been regarded as the main antifibrinolytic protein in diabetes, but recent work indicates that complement C3 (C3), an inflammatory protein, directly compromises fibrinolysis in type 1 diabetes. The aim of the current project was to investigate associations between C3 and fibrinolysis in a large cohort of individuals with type 2 diabetes.

Methods

Plasma levels of C3, C-reactive protein (CRP), PAI-1 and fibrinogen were analysed by ELISA in 837 patients enrolled in the Edinburgh Type 2 Diabetes Study. Fibrin clot lysis was analysed using a validated turbidimetric assay.

Results

Clot lysis time correlated with C3 and PAI-1 plasma levels (r?=?0.24, p?<?0.001 and r?=?0.22, p?<?0.001, respectively). In a multivariable regression model involving age, sex, BMI, C3, PAI-1, CRP and fibrinogen, and using log-transformed data as appropriate, C3 was associated with clot lysis time (regression coefficient 0.227 [95% CI 0.161, 0.292], p?<?0.001), as was PAI-1 (regression coefficient 0.033 [95% CI 0.020, 0.064], p?<?0.05) but not fibrinogen (regression coefficient 0.003 [95% CI ?0.046, 0.051], p?=?0.92) or CRP (regression coefficient 0.024 [95% CI ?0.008, 0.056], p?=?0.14). No correlation was demonstrated between plasma levels of C3 and PAI-1 (r?=??0.03, p?=?0.44), consistent with previous observations that the two proteins affect different pathways in the fibrinolytic system.

Conclusions/interpretation

Similarly to PAI-1, C3 plasma levels are independently associated with fibrin clot lysis in individuals with type 2 diabetes. Therefore, future studies should analyse C3 plasma levels as a surrogate marker of fibrinolysis potential in this population.     

Making interactive decision support for patients a reality

Interactive decision support applications might help patients to make difficult decisions about their health care. They lie in the context of traditional decision aids, which are known to have effects on a number of patient outcomes, including knowledge and decisional conflict. The problem of restricted uptake with decision aids may be addressed by interactive applications, particularly if associated with health information websites. We suggest that there may be an impact on the doctor-patient relationship and that this presents a number of opportunities. However, there are ethical challenges such as information bias and commercialisation.     

New insights into the proliferation and differentiation of early mouse thymocytes

Early thymocyte development was compared in normal, recombinase-activating gene 2-inactivated (RAG-2 KO) and pre-T cell receptor alpha-inactivated (pre-Talpha KO) mice, mutants representing either a complete (RAG-2 KO) or partial (pre-Talpha KO) block in progenitor development. Using three colour analysis with antibodies to CD117, CD44 and CD25, cell numbers in each progenitor subset were quantified, demonstrating an accumulation of cells prior to the block. Progenitor number was influenced both by the nature of the genetic block and thymus size, as shown in the enlarged thymus of a transgenic mouse line. By four colour staining for CD3, CD117, CD44 and CD25 and deliberately not gating out CD3(-) cells, a novel aspect of gamma delta T cell development in pre-Talpha KO mice was identified. 5-bromodeoxyuridine labelling and subsequent four colour staining for BrdU, CD117, CD44 and CD25 showed firstly that DN1 cells were cycling, secondly that the developmental block in pre-Talpha KO mice corresponded to a decrease in DN4 cell proliferation, and thirdly provided a novel 'snapshot' of T cell receptor beta-selected cells transiting the DN3 to DN4 compartment. Taken together, these results emphasise the need for a more detailed qualitative and quantitative analysis of the progenitor compartment in the thymus.     

Manufactured but not imported: new directions for research in shared decision making support and skills

Significant conceptual work on shared decision making has taken place but there are still significant challenges in achieving it in routine clinical practice. This paper outlines what research has identified to date that may promote shared decision making, and the further research that is required to enable continuing progress. Greater understanding of the models of decision making and instruments to identify them in practice are still required. Specifying consumer competences, developing instruments to assess these and interventions to enhance them may also be important. Clarifying all these aspects may enable those charged with training professionals to improve the content of professional development programmes. This may be particularly important in the field of cancer treatments where the stakes are high-patients usually desire much information but their desire for involvement in decision making is more variable. The consequences of getting this balance right or wrong are significant with much to be gained or lost. Continued development and evaluation of decision aids and decision explorers that use interactive technology will also be important in identifying how to progress with consumer involvement. If we can learn these lessons, then wider implementation of shared decision making or consumer involvement may become a nearer prospect.     

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4⁺ T cells toward the Th17 phenotype was examined. CD4⁺ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.