An experimental study on oil-dispersion baths generated by the Jungebad apparatus.

BACKGROUND: Since the very early documentation of medical treatments, bathing is an essential part of almost all traditional medical systems. In this context the oil-dispersion bath, developed in the 1930s by Werner Junge has been developed from anthroposophic medicine. We aimed at analyzing the apparatus, which churns water and essential oils into an oil-water dispersion, by means of an experimental study. MATERIAL AND METHODS: Using three different oils (rheumatic oil, citrus oil and rosemary oil) oil volumetric flow rate and oil droplet size distribution were examined at three different water volumetric flow rates of 5, 10, and 15 l/min at a constant temperature of 40 °C. Additionally, for the rheumatic oil measurements are taken at three different temperatures, 35, 40, and 45 °C at a constant volumetric flow rate of 10 l/min. Finally results were compared with a manual oil dispersion process. RESULTS: Oil volumetric flow rate increases with increasing water volumetric flow rates. Oil flow rate increases with increasing water temperature. Droplet-size distribution shows an optimal fit with a log-normal distribution for a volumetric flow rate of 5 l/min in all oils applied with citrus and rosemary oil showing a larger mean diameter compared to the rheumatic oil. Comparing the oil droplet size distribution for a traditional oil bath, distributions behaved completely different in comparison to our distributions. Moreover it seemed not possible to create an oil-dispersion with repeatable droplet size distributions whereas the Jungebad apparatus created similar oil dispersions with predictable results, independent of the user. DISCUSSION: This is the first study to explore the mechanisms of creation of the oil-dispersion bath by means of an experimental set up. Based on these experimental results, a more fundamental theoretical approach should be carried out to complement our findings and to gain deeper insights in the hydrodynamic and droplets forming processes in the Jungebad apparatus.

Brain Region-Specific Degeneration with Disease Progression in Late Infantile Neuronal Ceroid Lipofuscinosis (CLN2 Disease).

BACKGROUND AND PURPOSE: Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene. Our hypothesis was that regional analysis of cortical brain degeneration may identify brain regions that are affected earliest and most severely by the disease. MATERIALS AND METHODS: Fifty-two high-resolution 3T MR imaging datasets were prospectively acquired on 38 subjects with CLN2. A retrospective cohort of 52 disease-free children served as a control population. The FreeSurfer software suite was used for calculation of cortical thickness. RESULTS: An increased rate of global cortical thinning in CLN2 versus control subjects was the primary finding in this study. Three distinct patterns were observed across brain regions. In the first, subjects with CLN2 exhibited differing rates of cortical thinning versus age. This was true in 22 and 26 of 34 regions in the left and right hemispheres, respectively, and was also clearly discernable when considering brain lobes as a whole and Brodmann regions. The second pattern exhibited a difference in thickness from healthy controls but with no discernable change with age (9 left hemispheres, 5 right hemispheres). In the third pattern, there was no difference in either the rate of cortical thinning or the mean cortical thickness between groups (3 left hemispheres, 3 right hemispheres). CONCLUSIONS: This study demonstrates that CLN2 causes differential rates of degeneration across the brain. Anatomic and functional regions that degenerate sooner and more severely than others compared with those in healthy controls may offer targets for directed therapies. The information gained may also provide neurobiologic insights regarding the mechanisms underlying disease progression.

Assessment of disease severity in late infantile neuronal ceroid lipofuscinosis using multiparametric MR imaging.

BACKGROUND AND PURPOSE: LINCL is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene that encodes for tripeptidyl peptidase 1, a lysosomal enzyme necessary for the degradation of products of cellular metabolism. With the goal of developing quantitative noninvasive imaging biomarkers sensitive to disease progression, we evaluated a 5-component MR imaging metric and tested its correlation with a clinically derived disease-severity score. MATERIALS AND METHODS: MR imaging parameters were measured across the brain, including quantitative measures of the ADC, FA, nuclear spin-spin relaxation times (T2), volume percentage of CSF (%CSF), and NAA/Cr ratios. Thirty MR imaging datasets were prospectively acquired from 23 subjects with LINCL (2.5-8.4 years of age; 8 male/15 female). Whole-brain histograms were created, and the mode and mean values of the histograms were used to characterize disease severity. RESULTS: Correlation of single MR imaging parameters against the clinical disease-severity scale yielded linear regressions with R2 ranging from 0.25 to 0.70. Combinations of the 5 biomarkers were evaluated by using PCA. The best combination included ADC, %CSF, and NAA/Cr (R2=0.76, P<.001). CONCLUSIONS: The multiparametric disease-severity score obtained from the combination of ADC, %CSF, and NAA/Cr whole-brain MR imaging techniques provided a robust measure of disease severity, which may be useful in clinical therapeutic trials of LINCL in which an objective assessment of therapeutic response is desired.

Therapeutic implications of Src independent calcium mobilization in diffuse large B-cell lymphoma.

We report that 38% of primary large B-cell lymphoma (DLBCL) tested expressed active Src family kinases, which are targeted by dasatinib. The expression of active Src family of kinases (SFK) in primary DLBCL tumors correlated with unfavorable prognostic markers such as Ki67 and Mum1. Using four DLBCL cell lines we found that: (1) sensitivity to dasatinib (but not imatinib) varied 400-fold; (2) dasatinib resistance was associated with distinct signaling profiles downstream of BCR activation. In particular, although Src family kinase phosphorylation was inhibited by 100-150 nM dasatinib in all cell lines, this failed to inhibit BCR-mediated Blnk phosphorylation, calcium signaling and proliferation in a dasatinib resistant cell line.

Effect of ligand density on the spectral, physical, and biological characteristics of CdSe/ZnS quantum dots.

Chemical modification of the surface of CdSe/ZnS quantum dots (QDs) with small molecules or functional ligands often alters the characteristics of these particles. For instance, dopamine conjugation quenches the fluorescence of the QDs, which is a property that can be exploited for sensing applications if the conjugates are taken up into living cells. However, different sizes and/or preparations of mercaptocarboxylic acid solubilized QDs show very different properties when incubated with cells. It is unknown what physical parameters determine a QDs ability to interact with a cell surface, be endocytosed, escape from endosomes, and/or enter the nucleus. In this study, we examine the surface chemistry of QD-dopamine conjugates and present an optimized method for tracking the attachment of small biomolecules to the surface. It is found that the fluorescence intensity, surface charge, colloidal stability, and biological interactions of the QDs vary as a function of the density of dopamine on the surface. Successful targeting of QD-dopamine to dopamine receptor positive PC12 cells correlates with greater homogeneity of particle thiol layer, and a minimum number of ligands required for specific association can be estimated. These results will enable users to develop methods for screening QD conjugates for biological activity before proceeding to experiments with cell lines and animals.

Neurological deterioration in late infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Late infantile neuronal ceroid lipofuscinosis (LINCL) is associated with progressive degeneration of the brain and retina starting in early childhood. METHODS: Thirty-two individual neurologic, ophthalmologic, and CNS imaging (MRI and MRS) assessments of 18 children with LINCL were analyzed. Disease severity was followed by two rating scales, one previously established but modified to solely assess the brain and exclude the retinal disease (modified Hamburg LINCL scale), and a newly developed scale, with expanded evaluation of the CNS impairment (Weill Cornell LINCL scale). RESULTS: For the 18 children, the Weill Cornell scale yielded a closer correlation with both age and time since initial clinical manifestation of the disease than did the modified Hamburg scale. There were no significant differences as a function of age or time since initial manifestation of the disease in the rating scales among the most frequent CLN2 mutations (G3556C, 56% of all alleles or C3670T, 22% of all alleles). Measurements of cortical MRS N-acetyl-aspartate content, MRI ventricular, gray matter and white matter volume, and cortical apparent diffusion coefficient correlated to a variable degree with the age of the children and the time since initial clinical manifestation of the disease. All imaging measurements correlated better with the Weill Cornell CNS scale compared to the modified Hamburg LINCL scale. CONCLUSION: The data suggest that the Weill Cornell late infantile neuronal ceroid lipofuscinosis (LINCL) scale, together with several of the MRI measurements, may be useful in the assessment of severity and progression of LINCL and for the evaluation of novel therapeutic strategies.

Photophysics of dopamine-modified quantum dots and effects on biological systems.

Semiconductor quantum dots (QDs) have been widely used for fluorescent labelling. However, their ability to transfer electrons and holes to biomolecules leads to spectral changes and effects on living systems that have yet to be exploited. Here we report the first cell-based biosensor based on electron transfer between a small molecule (the neurotransmitter dopamine) and CdSe/ZnS QDs. QD-dopamine conjugates label living cells in a redox-sensitive pattern: under reducing conditions, fluorescence is only seen in the cell periphery and lysosomes. As the cell becomes more oxidizing, QD labelling appears in the perinuclear region, including in or on mitochondria. With the most-oxidizing cellular conditions, QD labelling throughout the cell is seen. Phototoxicity results from the creation of singlet oxygen, and can be reduced with antioxidants. This work suggests methods for the creation of phototoxic drugs and for redox-specific fluorescent labelling that are generalizable to any QD conjugated to an electron donor.

Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death.

CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models. Although this makes CD40 an attractive target for antitumor therapies, the response of malignant B cells to CD40 signaling is variable, and CD40 stimulation can enhance proliferation and can increase chemoresistance in some cell lines. It would therefore be useful to identify markers that predict whether a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines to identify signaling pathways that are involved in CD40-mediated apoptosis. CD40-resistant lines expressed pre-B-cell markers, including RAG and VPREB, whereas CD40-sensitive cells resembled mature B cells and expressed higher levels of transcripts encoding several members of the CD40 signaling pathway, including LCK and VAV. In addition, CD40-sensitive DLBCL cell lines also displayed constitutive activation of extracellular signal-regulated kinase (ERK) and failed to undergo apoptosis when ERK phosphorylation was inhibited. In contrast, CD40-resistant lines showed no constitutive activation of ERK and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40.

Quantum dot-FRET systems for imaging of neuronal action potentials.

Fluorescent semiconductor quantum dots (QDs) can act as energy donors or acceptors with a wide variety of environmentally-sensitive molecules. Conjugation of a single QD to a select number of the selected molecule can optimize the range of sensitivity for a given application, and the relatively large size of the QDs allows them to be tracked individually in cells. Using QDs as FRET acceptors, we have created first-generation sensors for membrane potential which shows good signal to noise and time resolution, but prohibitive toxicity. The challenges of delivery, calibration, and toxicity and plans for improvement of the sensors are presented, in the context of the eventual aim of monitoring membrane potential in a cultured motor neuron model of amyotrophic lateral sclerosis.

Detection of disseminated tumor cells in peripheral blood.

Metastases are the major cause of cancer-related deaths in patients with solid epithelial malignancies, such as breast, colorectal and prostate carcinomas. Hematogenous spreading of tumor cells from a primary tumor can be considered as a crucial step in the metastasis cascade leading eventually to the formation of clinically manifest metastases. Consequently, as shown in recent studies, the detection of disseminated tumor cells in peripheral blood might be of clinical relevance with respect to individual patient prognosis and staging or monitoring of therapy. However, the rarity of disseminated tumor cells in peripheral blood renders the application of sensitive techniques mandatory for their detection. The emergence of highly sophisticated reverse transciptase-polymerase chain reaction (RT-PCR) assays, combining a preanalytical enrichment step with the assessment of multiple molecular tumor markers expressed in disseminated tumor cells, provides a powerful tool in detecting disseminated tumor cells with high sensitivity and specificity. This review will discuss currently used tumor markers as well as experimental means to enhance the sensitivity and specificity of RT-PCR assays to detect disseminated tumor cells in the peripheral blood of patients with breast, colorectal, and prostate cancers, and their clinical relevance assessed in recent studies.

Quantum dots as phototoxic drugs and sensors of specific metabolic processes in living cells.

When conjugated to CdSe/ZnS nanocrystals (quantum dots), the nucleobase adenine and the neurotransmitter dopamine quench fluorescence emission from in a manner strongly dependent upon the size of the quantum dot. The degree of quenching serves to predict the efficiency with which the conjugates are able to enter living cells. Along with quenching, the presence of specific receptors on the cells is necessary for QD binding, entry, and phototoxicity. Toxicity is manifested by opening of large membrane pores and by oxidative DNA damage, and does not require the release of Cd+2. In bacterial cells, light exposure is necessary for uptake, and procedures to reduce toxicity eliminate labeling. In mammalian cells, antioxidants prevent toxicity but not QD uptake, leading to QD-loaded cells that are nonfluorescent before light exposure. These findings provide a general procedure for rational design of nanoparticle-based photosensitizing drugs and for "off-on" fluorescent labels.

Wnt-1 and int-2 mammary oncogene effects on the beta-catenin pathway in immortalized mouse mammary epithelial cells are not sufficient for tumorigenesis.

Development of strategies for prevention of breast cancer development requires an understanding of the effects of mammary oncogenes on mammary cells at early stages in neoplastic transformation. As mammary oncogenes wnt-1 and int-2 affect different signal transduction pathways, we investigated their effects on established mouse mammary epithelial cell lines (MMECLs) reflecting early stages in tumorigenesis. Normal interactions between beta-catenin and E-cadherin were abrogated in all three immortalized MMECLs and the cells lacked beta-catenin-mediated transactivation activity, detectable using a reporter assay, suggesting that alterations in cell adhesion may be very early events in mammary tumorigenesis. Immortalized FSK4 and EL12 cells and hyperplastic TM3 cells were stably transfected with expression vectors encoding wnt-1 or int-2 or the control vector, and drug-selected pooled cells from each line were confirmed by reverse transcription-polymerase chain reaction to express the transfected oncogene; this expression persisted in the cells analysed in vitro and in vivo. Resultant phenotypic changes depended both on the oncogene and the target mammary cell line. In FSK4 cells, expression of wnt-1 or int-2 resulted in proliferative changes in vitro, including reduced contact inhibition, increased beta-catenin expression, and decreased p53 transcriptional activity, but neither oncogene conferred upon those cells the ability to produce tumors in vivo. EL12 cells were highly refractory to the effects of both oncogenes, with the only measurable changes being increased E-cadherin levels induced by both oncogenes and increased proliferation of the int-2-transfected cells in the absence of serum. Parental TM3 cells were phenotypically similar to wnt-1- or int-2-transfected FSK4 cells and displayed an increased rate of proliferation in vitro and markedly increased tumorigenicity in vivo following transfection with int-2 but not with wnt-1. These results suggest that wnt-1 signaling is redundant in the hyperplastic TM3 cells and indicate that wnt-1-induced effects in the immortalized FSK4 and EL12 cells were not sufficient to mediate a tumorigenic phenotype. This study showed that the wnt-1 and int-2 oncogenes have similar but distinguishable effects on immortalized MMECLs and that the genetic background of the mammary cells greatly influences the consequences of oncogene expression at early stages of cell transformation.

The distinct roles of T cell-derived cytokines and a novel follicular dendritic cell-signaling molecule 8D6 in germinal center-B cell differentiation.

Germinal center-B (GC-B) cells differentiate into memory B cells and plasma cells (PC) through interaction with T cells and follicular dendritic cells (FDC). Activated T cell and FDC play distinct roles in this process. The detailed kinetic experiments revealed that cytokines secreted by activated T cells determined the pathway of GC-B cell differentiation. IL-4 directs GC-B cells to differentiate into memory B cells, whereas IL-10 steers them into PC. FDC/HK cells do not direct either pathway, but provide signals for proliferation of GC-B cells. A novel FDC-signaling molecule 8D6 (FDC-SM-8D6) produced by FDC augments PC generation in the GC. FDC-SM-8D6-specific mAb blocked PC generation and IgG secretion but not memory B cell proliferation. COS cells expressing FDC-SM-8D6 enhanced GC-B cell proliferation and Ab secretion, which was blocked by mAb 8D6. In the cultures with B cell subsets, PC generation was inhibited by mAb 8D6 in the cultures with CD27(+) B cells but not in the culture with CD27(-) B cells, suggesting that CD27(+) PC precursor is the specific target of FDC-SM-8D6 stimulation.

Specific blockade by CD54 and MHC II of CD40-mediated signaling for B cell proliferation and survival.

Regulation of B lymphocyte proliferation is critical to maintenance of self-tolerance, and intercellular interactions are likely to signal such regulation. Here, we show that coligation of either the adhesion molecule ICAM-1/CD54 or MHC II with CD40 inhibited cell cycle progression and promoted apoptosis of mouse splenic B cells. This resulted from specific blockade of NF-kappa B induction, which normally inhibits apoptosis. LPS- or B cell receptor (BCR)-induced proliferation was not inhibited by these treatments, and mAb-induced association of CD40 with other B cell surface molecules did not have these effects. Addition of BCR or IL-4 signals did not overcome the effect of ICAM-1 or MHC II on CD40-induced proliferation. FasL expression was not detected in B cell populations. These results show that MHC II and ICAM-1 specifically modulate CD40-mediated signaling, so inhibiting proliferation and preventing inhibition of apoptosis.

Proliferation and cytokine profile of T. annulata-infected ovine, caprine, and bovine lymphoblastoid cells.

T. annulata, the causative agent of tropical theileriosis in cattle, can also infect ovine and caprine leukocytes in vitro. In vivo studies showed that this parasite causes a mild infection in both these animal species, and in sheep merozoite stage development seems to be inhibited. Since the nature of T. annulata infected caprine and ovine cells is not known, all three cell lines were karyotyped and phenotypically characterized by flow cytometry. They all express mRNA of cytokines IL-1 alpha, IL-1 beta, IL-8, and TNF-alpha, but not of IFN-gamma, IL-2, and IL-4. In contrast, IL-6 mRNA was expressed in the cattle cell line only, while mRNA of IL-10 was exclusively produced by the sheep cell line. The observed differences in cytokine mRNA expression may be responsible for the different pathogenesis of T. annulata infection in cattle and sheep.

(1R*,11R*)-Bicyclo[9.4.1]hexadecane-12,16-dione.

The title compound, C(16)H(26)O(2), (I), prepared by oxidation of (1R*,11R*)-12-hydroxybicyclo[9.4.1]hexadecan-16-one using pyridinium dichromate, has a trans configuration of the two fused rings and represents an interesting precursor for the synthesis of macrocyclic structures.

Follicular dendritic cells and T cells: nurses and executioners in the germinal centre reaction.

The humoral immune response constitutes an efficient system to protect the organism against diseases caused by invading pathogens. To guarantee a highly efficient defence, the humoral immune system has to be tightly regulated. Two cell subsets in particular, T cells and follicular dendritic cells (FDCs), contribute to the success of these regulation processes. Whereas the particular role of T cells is the elimination of autoreactive clones, the main role of FDCs is to present unprocessed antigen and check B-cell clones for higher affinity. B-cell clones unsuited for improved humoral immune response will be specifically killed. Involvement of Fas-mediated apoptosis might be an additional tool not only in T-cell-mediated regulation, but also in FDC-B cell interaction in the germinal centre.