MEK/ERK-mediated phosphorylation of Bim is required to ensure survival of T and B lymphocytes during mitogenic stimulation

Survival and death of lymphocytes are regulated by the balance between pro- and antiapoptotic members of the Bcl-2 family; this is coordinated with the control of cell cycling and differentiation. Bim, a proapoptotic BH3-only member of the Bcl-2 family, can be regulated by MEK/ERK-mediated phosphorylation, which affects its binding to pro-survival Bcl-2 family members and its turnover. We investigated Bim modifications in mouse B and T lymphoid cells after exposure to apoptotic stimuli and during mitogenic activation. Treatment with ionomycin or cytokine withdrawal caused an elevation in Bim(EL), the most abundant Bim isoform. In contrast, in mitogenically stimulated T and B cells, Bim(EL) was rapidly phosphorylated, and its levels declined. Pharmacological inhibitors of MEK/ERK signaling prevented both of these changes in Bim, reduced proliferation, and triggered apoptosis of mitogen-stimulated T and B cells. Loss of Bim prevented this cell killing but did not restore cell cycling. These results show that during mitogenic stimulation of T and B lymphocytes MEK/ERK signaling is critical for two distinct processes, cell survival, mediated (at least in part) through phosphorylation and consequent inhibition of Bim, and cell cycling, which proceeds independently of Bim inactivation.

Does quantification of USPIO uptake-related signal loss allow differentiation of benign and malignant normal-sized pelvic lymph nodes?

Ultrasmall superparamagnetic iron oxide (USPIO) particles are promising contrast media, especially for molecular and cellular imaging besides lymph node staging owing to their superior NMR efficacy, macrophage uptake and lymphotropic properties. The goal of the present prospective clinical work was to validate quantification of signal decrease on high-resolution T(2)-weighted MR sequences before and 24-36 h after USPIO administration for accurate differentiation between benign and malignant normal-sized pelvic lymph nodes. Fifty-eight patients with bladder or prostate cancer were examined on a 3 T MR unit and their respective lymph node signal intensities (SI), signal-to-noise (SNR) and contrast-to-noise (CNR) were determined on pre- and post-contrast 3D T(2)-weighted turbo spin echo (TSE) images. Based on histology and/or localization, USPIO-uptake-related SI/SNR decrease of benign vs malignant and pelvic vs inguinal lymph nodes was compared. Out of 2182 resected lymph nodes 366 were selected for MRI post-processing. Benign pelvic lymph nodes showed a significantly higher SI/SNR decrease compared with malignant nodes (p < 0.0001). Inguinal lymph nodes in comparison to pelvic lymph nodes presented a reduced SI/SNR decrease (p < 0.0001). CNR did not differ significantly between benign and malignant lymph nodes. The receiver operating curve analysis yielded an area under the curve of 0.96, and the point with optimal accuracy was found at a threshold value of 13.5% SNR decrease. Overlap of SI and SNR changes between benign and malignant lymph nodes were attributed to partial voluming, lipomatosis, histiocytosis or focal lymphoreticular hyperplasia. USPIO-enhanced MRI improves the diagnostic ability of lymph node staging in normal-sized lymph nodes, although some overlap of SI/SNR-changes remained. Quantification of USPIO-dependent SNR decrease will enable the validation of this promising technique with the final goal of improving and individualizing patient care.

Goal-directed colloid administration improves the microcirculation of healthy and perianastomotic colon

BACKGROUND: The aim of this study was to compare the effects of goal-directed colloid fluid therapy with goal-directed crystalloid and restricted crystalloid fluid therapy on healthy and perianastomotic colon tissue in a pig model of colon anastomosis surgery. METHODS: Pigs (n = 27, 9 per group) were anesthetized and mechanically ventilated. A hand-sewn colon anastomosis was performed. The animals were subsequently randomized to one of the following treatments: R-RL group, 3 ml x kg(-1) x h(-1) Ringer lactate (RL); GD-RL group, 3 ml x kg(-1) x h(-1) RL + bolus 250 ml of RL; GD-C group, 3 ml x kg(-1) x h(-1) RL + bolus 250 ml of hydroxyethyl starch (HES 6%, 130/0.4). A fluid bolus was administered when mixed venous oxygen saturation dropped below 60%. Intestinal tissue oxygen tension and microcirculatory blood flow were measured continuously. RESULTS: After 4 h of treatment, tissue oxygen tension in healthy colon increased to 150 +/- 31% in group GD-C versus 123 +/- 40% in group GD-RL versus 94 +/- 23% in group R-RL (percent of postoperative baseline values, mean +/- SD; P < 0.01). Similarly perianastomotic tissue oxygen tension increased to 245 +/- 93% in the GD-C group versus 147 +/- 58% in the GD-RL group and 116 +/- 22% in the R-RL group (P < 0.01). Microcirculatory flow was higher in group GD-C in healthy colon. CONCLUSIONS: Goal-directed colloid fluid therapy significantly increased microcirculatory blood flow and tissue oxygen tension in healthy and injured colon compared to goal-directed or restricted crystalloid fluid therapy.

Event-relating to potential dementia. Semantic processing in the course of healthy and pathological aging

While most healthy elderly are able to manage their everyday activities, studies showed that there are both stable and declining abilities during healthy aging. For example, there is evidence that semantic memory processes which involve controlled retrieval mechanism decrease, whereas the automatic functioning of the semantic network remains intact. In contrast, patients with Alzheimer’s disease (AD) suffer from episodic and semantic memory impairments aggravating their daily functioning. In AD, severe episodic as well as semantic memory deficits are observable. While the hallmark symptom of episodic memory decline in AD is well investigated, the underlying mechanisms of semantic memory deterioration remain unclear. By disentangling the semantic memory impairments in AD, the present thesis aimed to improve early diagnosis and to find a biomarker for dementia. To this end, a study on healthy aging and a study with dementia patients were conducted investigating automatic and controlled semantic word retrieval. Besides the inclusion of AD patients, a group of participants diagnosed with semantic dementia (SD) – showing isolated semantic memory loss – was assessed. Automatic and controlled semantic word retrieval was measured with standard neuropsychological tests and by means of event-related potentials (ERP) recorded during the performance of a semantic priming (SP) paradigm. Special focus was directed to the N400 or N400-LPC (late positive component) complex, an ERP that is sensitive to the semantic word retrieval. In both studies, data driven topographical analyses were applied. Furthermore, in the patient study, the combination of the individual baseline cerebral blood flow (CBF) with the N400 topography of each participant was employed in order to relate altered functional electrophysiology to the pathophysiology of dementia. Results of the aging study revealed that the automatic semantic word retrieval remains stable during healthy aging, the N400-LPC complex showed a comparable topography in contrast to the young participants. Both patient groups showed automatic SP to some extent, but strikingly the ERP topographies were altered compared to healthy controls. Most importantly, the N400 was identified as a putative marker for dementia. In particular, the degree of the topographical N400 similarity was demonstrated to separate healthy elderly from demented patients. Furthermore, the marker was significantly related to baseline CBF reduction in brain areas relevant for semantic word retrieval. Summing up, the first major finding of the present thesis was that all groups showed semantic priming, but that the N400 topography differed significantly between healthy and demented elderly. The second major contribution was the identification of the N400 similarity as a putative marker for dementia. To conclude, the present thesis added evidence of preserved automatic processing during healthy aging. Moreover, a possible marker which might contribute to an improved diagnosis and lead consequently to a more effective treatment of dementia was presented and has to be further developed.

Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.

FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P3 and phosphoinositide 3-kinase

Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation.

Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium-formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.

Chemical inhibitors of the calcium entry channel TRPV6

Calcium entry channels in the plasma membrane are thought to play a major role in maintaining cellular Ca(2+) levels, crucial for growth and survival of normal and cancer cells. The calcium-selective channel TRPV6 is expressed in prostate, breast, and other cancer cells. Its expression coincides with cancer progression, suggesting that it drives cancer cell growth. However, no specific inhibitors for TRPV6 have been identified thus far.

Distinct molecular composition of blood and lymphatic vascular endothelial cell junctions establishes specific functional barriers within the peripheral lymph node

Lymph nodes are strategically localized at the interfaces between the blood and lymphatic vascular system, delivering immune cells and antigens to the lymph node. As cellular junctions of endothelial cells actively regulate vascular permeability and cell traffic, we have investigated their molecular composition by performing an extensive immunofluorescence study for adherens and tight junction molecules, including vascular endothelium (VE)-cadherin, the vascular claudins 1, 3, 5 and 12, occludin, members of the junctional adhesion molecule family plus endothelial cell-selective adhesion molecule (ESAM)-1, platelet endothelial cell adhesion molecule-1, ZO-1 and ZO-2. We found that junctions of high endothelial venules (HEV), which serve as entry site for naive lymphocytes, are unique due to their lack of the endothelial cell-specific claudin-5. LYVE-1(+) sinus-lining endothelial cells form a diffusion barrier for soluble molecules that arrive at the afferent lymph and use claudin-5 and ESAM-1 to establish characteristic tight junctions. Analysis of the spatial relationship between the different vascular compartments revealed that HEV extend beyond the paracortex into the medullary sinuses, where they are protected from direct contact with the lymph by sinus-lining endothelial cells. The specific molecular architecture of cellular junctions present in blood and lymphatic vessel endothelium in peripheral lymph nodes establishes distinct barriers controlling the distribution of antigens and immune cells within this tissue.

Cathepsin W expressed exclusively in CD8+ T cells and NK cells, is secreted during target cell killing but is not essential for cytotoxicity in human CTLs

OBJECTIVE: Cathepsin W (CatW, lymphopain) is a putative cysteine protease with restricted expression to natural killer (NK) cells and CD8(+) T cells and so far unknown function and properties. Here, we characterize in detail, the regulation of human CatW during T-cell development in response to different stimuli and its functional involvement in cytotoxic lymphocyte effector function. MATERIALS AND METHODS: Western blots and real time polymerase chain reaction of sorted, unstimulated, and stimulated cell subsets (thymocytes, T cells, NK cells) and their culture supernatants were used to study regulation and expression of CatW. Primary CD8(+) T cells and short-term T-cell lines were transfected with small interfering RNA to study the involvement of CatW in effector function such as target cell killing and interferon-gamma production. RESULTS: Levels of CatW expression correlate closely with cytotoxic capacity both during development and in response to factors influencing cytotoxicity. Furthermore, CatW is secreted during specific target cell killing. However, knockdown of CatW expression by small interfering RNA neither influences target cell killing nor interferon-gamma production. CONCLUSION: Despite being expressed in the effector subset of CD8(+) and NK cells and of being released during target cell killing, our functional inhibition studies exclude an essential role of CatW in the process of cytotoxicity.

Humans and Scavengers: The Evolution of Interactions and Ecosystem Services

Since the origin of early Homo species during the Late Pliocene, interactions of humans with scavenging birds and mammals have changed in form through shifting ecological scenarios. How humans procured meat during the Quaternary Period changed from confrontational scavenging to hunting; shepherding of wild animals; and, eventually, intensive husbandry of domesticated animals. As humans evolved from carcass consumers to carcass providers, the overall relationship between humans and scavengers shifted from competition to facilitation. These changing interactions have translated into shifting provisioning (by signaling carcass location), regulating (e.g., by removing animal debris and controlling infectious diseases), and cultural ecosystem services (e.g., by favoring human language and social cooperation skills or, more recently, by enhancing ecotourism) provided by scavenging vertebrates. The continued survival of vultures and large mammalian scavengers alongside humans is now severely in jeopardy, threatening the loss of the numerous ecosystem services from which contemporary and future humans could benefit.

PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells

The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, (2) repressed by PML-RARA, and (3) functionally involved in neutrophil differentiation and cell viability of APL cells.