BAFF is a survival and maturation factor for mouse B cells.

Human B cell-activating factor (BAFF) induces mouse surface IgM+ B cells of the immature type from bone marrow and of the immature types 1 and 2 from spleen, as well as of the mature type from spleen to increased longevity in tissue culture. BAFF does so polyclonally and without inducing proliferation in any of these B cell subpopulations. BAFF induces phenotypic and functional maturation of immature to mature B cells so that all immature cells loose C1qRp (AA4.1, 493) expression and type 1 immature cells up-regulate IgD, CD21 and CD23. Immature B cells of types 1 and 2, upon pre-incubation with BAFF, change their reactiveness to Ig-specific antibodies so that they no longer enter apoptosis but now proliferate. However, BAFF does not seem to overcome negative selection of developing immature B cells in vitro.

Preparation and analysis of fetal liver extracts.

The aim of this work is to describe the techniques that have been used for preparation and analysis of whole fetal liver extracts destined for in utero transplantation. Nine fetal livers between 12 and 17 weeks of gestation were prepared: cell counts and assessment of the hematopoietic cell viability were performed on cell suspensions. Hepatocytes represented 40 to 80% of the whole cell population. The remaining cells were constituted by hematopoietic cells (mainly erythroblasts), as well as by endothelial cells. The latter expressed CD34 on their surface, interfering with the assessment of CD34+ hematopoietic cells by flow cytometry. Direct visual morphologic control using alkaline phosphatase anti-alkaline phosphatase techniques was needed to differentiate hematopoietic from extra-hematopoietic CD34+ cells. Between 3.0 and 34.6 x 10(6) CD34+ viable hematopoietic cells were collected per fetal liver. Adequate differentiation of these cells into burst-forming units erythroid (BFU-E), colony-forming units granulocyte-macrophage (CFU-GM), and colony-forming units granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) has been shown for each sample in clonogeneic cultures. In conclusion, fetal liver is a potential source of hematopoietic stem cells. Their numeration, based on the presence of CD34, is hampered by the expression of this antigen on other cells contained in the liver cell extract, in particular endothelial cells.

Characterization of tumor reactive CD8 T cells induced by peptide vaccination in melanoma patients

ABSTRACT¦Naturally acquired tumor-specific T-cells can be detected in most advanced cancer patients.¦Yet, they often fail to control or eliminate the disease, in contrast to many virus-specific CD8¦T lymphocytes. Therapeutic vaccines aim at inducing and boosting specific T-cells mediated¦immunity to reduce tumor burden. The properties of CD8 T-cells required for protection from¦infectious disease and cancer are only partially characterized.¦The objectives of this study were to assess effector functions, stage of differentiation and¦clonotype selection of tumor-reactive T lymphocytes following peptide vaccination in¦melanoma patients over time. Results were compared to protective viral-specific T-cell¦responses found in healthy individuals. We also characterized dominant versus low/non¦dominant T-cell clonotypes with the aim to further understand the in vivo function of each set¦of frequency-based specific T-cells.¦Here we developed and applied a novel approach for molecular and functional analysis of¦single T lymphocytes ex vivo. T-cell receptor (TCR) clonotype mapping revealed rapid¦selection and expansion of co-dominant T-cell clonotypes, which made up the majority of the¦highly differentiated "effector" T-cells, but only 25% of the less differentiated "effectormemory"¦cells, mostly composed of non-dominant clonotypes. Moreover, we show that¦advanced effector cell differentiation was indeed clonotype-dependent. Surprisingly, however,¦the acquisition of effector functions (cytokine production, killing) was clonotype-independent.¦Vaccination of melanoma patients with native peptide induced competent effector function in¦both dominant and non-dominant clonotypes, suggesting that most if not all clonotypes¦participating in a T-cell response have the potential to develop equal functional competence.¦In contrast, many T-cells remained poorly functional after vaccination with analog peptide,¦despite similar clonotype-dependent differentiation. Our findings show that the type of¦peptide vaccine has a critical influence on the selection and functional activation of the¦clonotypic T-cell repertoire. They also show that systematic assessment of individual T-cells¦identifies the cellular basis of immune responses, contributing to the rational development of¦vaccines.

The high road and the low road: trafficking choices in plants.

Polar transport of the signaling molecule auxin is critical for plant development and depends on both the polar distribution of auxin efflux carriers, which pump auxin out of the cell and the alignment of these polarized cells. Two papers in this issue of Cell (Michniewicz et al., 2007; Jaillais et al., 2007) address how polar transport of these carriers occurs and describe the endosomal pathways involved.

Increased neuroendocrine cells in resected metastases compared to primary colorectal adenocarcinomas.

Neuroendocrine differentiation has been described in rectal adenocarcinomas receiving neoadjuvant therapy prior to radical surgery, but its clinical relevance is controversial and no data are currently available in colorectal carcinoma metastases as compared to primary tumors. The presence of chromogranin A positive tumor cells was investigated by means of immunohistochemistry on surgical specimens from 54 primary colorectal carcinomas and their corresponding metastases, resected at diagnosis or during tumor progression. In 47 patients, tumor metastases were resected 1 month to 12 years after chemotherapy and/or radiotherapy, while in the remaining seven patients no additional therapy after primary surgery was performed. In primary tumors, neuroendocrine differentiation was found in 12/54 cases (22.2%) as compared to 25/54 metastatic lesions (46.3%; p?=?0.01). The presence of neuroendocrine phenotype was not correlated with any clinical pathological parameter nor with a different proliferation index. However, patients having neuroendocrine cells in the primary tumor had a significantly shorter survival from the time of metastatic spread than those having not (33.3 vs. 55.5 months; p?=?0.04). In summary, our data show that colorectal carcinoma metastases contain a higher percentage of neuroendocrine differentiated cells as compared to their corresponding primaries, a finding possibly related to the influence of chemotherapy in neuroendocrine differentiation during colorectal carcinoma progression.

Be fit or be sick: peroxisome proliferator-activated receptors are down the road.

Investigating metabolism by unveiling the functions of the nuclear receptors peroxisome proliferator-activated receptors (PPARs) in the numerous intricate pathways ensuring energy homeostasis and fitness has been extremely rewarding. Major lines of research were initially determined by the first-characterized crucial roles of PPARalpha in fatty oxidation and of PPARgamma in adipocyte differentiation and lipid storage. Today, the molecular bases of the functional links between glucose, lipid, and protein metabolism, under the important but nonexclusive control of PPARalpha and PPARgamma, are starting to be uncovered. In addition, in the last couple of years evidence has been provided for an important role of PPARbeta (delta) in lipid metabolism. Inevitably, such actors of metabolic homeostasis are implicated in the physiopathology of complex metabolic disorders, such as those constituting the metabolic syndrome, resulting in atherosclerosis and cardiovascular diseases. This review presents a summary of the recent findings on their dual involvement in health and disease.

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Decreased STAMP2 expression in association with visceral adipose tissue dysfunction.

CONTEXT Six-transmembrane protein of prostate 2 (STAMP2) is a counter-regulator of inflammation and insulin resistance according to findings in mice. However, there have been contradictory reports in humans. OBJECTIVE We aimed to explore STAMP2 in association with inflammatory and metabolic status of human obesity. DESIGN, PATIENTS, AND METHODS STAMP2 gene expression was analyzed in adipose tissue samples (171 visceral and 67 sc depots) and during human preadipocyte differentiation. Human adipocytes were treated with macrophage-conditioned medium, TNF-α, and rosiglitazone. RESULTS In visceral adipose tissue, STAMP2 gene expression was significantly decreased in obese subjects, mainly in obese subjects with type 2 diabetes. STAMP2 gene expression and protein were significantly and inversely associated with obesity phenotype measures (body mass index, waist, hip, and fat mass) and obesity-associated metabolic disturbances (systolic blood pressure and fasting glucose). In addition, STAMP2 gene expression was positively associated with lipogenic (FASN, ACC1, SREBP1, THRSP14, TRα, and TRα1), CAV1, IRS1, GLUT4, and CD206 gene expression. In sc adipose tissue, STAMP2 gene expression was not associated with metabolic parameters. In both fat depots, STAMP2 gene expression in stromovascular cells was significantly higher than in mature adipocytes. STAMP2 gene expression was significantly increased during the differentiation process in parallel to adipogenic genes, being increased in preadipocytes derived from lean subjects. Macrophage-conditioned medium (25%) and TNF-α (100 ng/ml) administration increased whereas rosiglitazone (2 μM) decreased significantly STAMP2 gene expression in human differentiated adipocytes. CONCLUSIONS Decreased STAMP2 expression (mRNA and protein) might reflect visceral adipose dysfunction in subjects with obesity and type 2 diabetes.

Notch tumor suppressor function

Cancer development results from deregulated control of stem cell populations and alterations in their surrounding environment. Notch signaling is an important form of direct cell-cell communication involved in cell fate determination, stem cell potential and lineage commitment. The biological function of this pathway is critically context dependent. Here we review the pro-differentiation role and tumor suppressing function of this pathway, as revealed by loss-of-function in keratinocytes and skin, downstream of p53 and in cross-connection with other determinants of stem cell potential and/or tumor formation, such as p63 and Rho/CDC42 effectors. The possibility that Notch signaling elicits a duality of signals, involved in growth/differentiation control and cell survival will be discussed, in the context of novel approaches for cancer therapy

Differential phosphorylation of some proteins of the neuronal cytoskeleton during brain development.

The cytoskeleton is important for neuronal morphogenesis. During the postnatal development of cat brain, the molecular composition of the neuronal cytoskeleton changes with maturation. Several of its proteins change in their rate of expression, in their degree of phosphorylation, in their subcellular distribution, or in their biochemical properties. It is proposed that phosphorylation is an essential mechanism to regulate the plasticity of the early, juvenile-type cytoskeleton. Among such proteins are several microtubule-associated proteins (MAPs), such as MAP5a, MAP2c or the juvenile tau proteins. Phosphorylation may also act on neurofilaments, postulated to be involved in the adult-type stabilization of axons. These observations imply that phosphorylation may affect cytoskeleton function in axons and dendrites at various developmental stages. Yet, the mechanisms of phosphorylation and its regulation cascades are largely unknown. In view of the topic of this issue on CD15, the potential role of matrix molecules being involved in the modulation of phosphorylation activity and of cytoskeletal properties is addressed.

Aggravation of chronic stress effects on hippocampal neurogenesis and spatial memory in LPA₁ receptor knockout mice.

BACKGROUND The lysophosphatidic acid LPA₁ receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA₁ receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory. METHODOLOGY/PRINCIPAL FINDINGS Male LPA₁-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice. CONCLUSIONS/SIGNIFICANCE These results reveal that the absence of the LPA₁ receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA₁ receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology.

Breast cancer 1 (BrCa1) may be behind decreased lipogenesis in adipose tissue from obese subjects.

CONTEXT Expression and activity of the main lipogenic enzymes is paradoxically decreased in obesity, but the mechanisms behind these findings are poorly known. Breast Cancer 1 (BrCa1) interacts with acetyl-CoA carboxylase (ACC) reducing the rate of fatty acid biosynthesis. In this study, we aimed to evaluate BrCa1 in human adipose tissue according to obesity and insulin resistance, and in vitro cultured adipocytes. RESEARCH DESIGN AND METHODS BrCa1 gene expression, total and phosphorylated (P-) BrCa1, and ACC were analyzed in adipose tissue samples obtained from a total sample of 133 subjects. BrCa1 expression was also evaluated during in vitro differentiation of human adipocytes and 3T3-L1 cells. RESULTS BrCa1 gene expression was significantly up-regulated in both omental (OM; 1.36-fold, p = 0.002) and subcutaneous (SC; 1.49-fold, p = 0.001) adipose tissue from obese subjects. In parallel with increased BrCa1 mRNA, P-ACC was also up-regulated in SC (p = 0.007) as well as in OM (p = 0.010) fat from obese subjects. Consistent with its role limiting fatty acid biosynthesis, both BrCa1 mRNA (3.5-fold, p<0.0001) and protein (1.2-fold, p = 0.001) were increased in pre-adipocytes, and decreased during in vitro adipogenesis, while P-ACC decreased during differentiation of human adipocytes (p = 0.005) allowing lipid biosynthesis. Interestingly, BrCa1 gene expression in mature adipocytes was restored by inflammatory stimuli (macrophage conditioned medium), whereas lipogenic genes significantly decreased. CONCLUSIONS The specular findings of BrCa1 and lipogenic enzymes in adipose tissue and adipocytes reported here suggest that BrCa1 might help to control fatty acid biosynthesis in adipocytes and adipose tissue from obese subjects.

Opioids and the skin--where do we stand?

The common ectodermal origin of the skin and nervous systems can be expected to predict likely interactions in the adult. Over the last couple of decades much progress has been made to elucidate the nature of these interactions, which provide multidirectional controls between the centrally located brain and the peripherally located skin and immune system. The opioid system is an excellent example of such an interaction and there is growing evidence that opioid receptors (OR) and their endogenous opioid agonists are functional in different skin structures, including peripheral nerve fibres, keratinocytes, melanocytes, hair follicles and immune cells. Greater knowledge of these skin-associated opioid interactions will be important for the treatment of chronic and acute pain and pruritus. Topical treatment of the skin with opioid ligands is particularly attractive as they are active with few side effects, especially if they cannot cross the blood-brain barrier. Moreover, cutaneous activation of the opioid system (e.g. by peripheral nerves, cutaneous and immune cells, especially in inflamed and damaged skin) can influence cell differentiation and apoptosis, and thus may be important for the repair of damaged skin. While many of the pieces of this intriguing puzzle remain to be found, we attempt in this review to weave a thread around available data to discuss how the peripheral opioid system may impact on different key players in skin physiology and pathology.

Cancer genes hypermethylated in human embryonic stem cells.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology

Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis. Central nervous system infection is the most common clinical presentation followed by pulmonary, skin and eye manifestations. Cryptococcosis is primarily treated with amphotericin B (AMB), fluconazole (FLC) and itraconazole (ITC). In the present work, we evaluated the in vitro effect of terbinafine (TRB), an antifungal not commonly used to treat cryptococcosis. We specifically examined the effects of TRB, either alone or in conjunction with AMB, FLC and ITC, on clinical C. neoformans isolates, including some isolates resistant to AMB and ITC. Broth microdilution assays showed that TRB was the most effective drug in vitro. Antifungal combinations demonstrated synergism of TRB with AMB, FLC and ITC. The drug concentrations used for the combination formulations were as much as 32 and 16-fold lower than the minimum inhibitory concentration (MIC) values of FLC and AMB alone, respectively. In addition, calcofluor white staining revealed the presence of true septa in hyphae structures that were generated after drug treatment. Ultrastructural analyses demonstrated several alterations in response to drug treatment, such as cell wall alterations, plasma membrane detachment, presence of several cytoplasmic vacuoles and mitochondrial swelling. Therefore, we believe that the use of TRB alone or in combination with AMB and azoles should be explored as an alternative treatment for cryptococcosis patients who do not respond to standard therapies.