Stereology in the extraction of information from images

Some important aspects for information extraction by stereology from images in surgical and experimental pathology are discussed. The relationship between stereology and morphometry is briefly discussed, with the most important conditions for stereologic analysis in pathology pointed out. The possibilities, limits and problems inherent in stereologic and morphometric analysis in pathology are explained in two examples: so-called "small airways disease" and tight junctions of hepatocytes during physiologic choleresis.     

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and K_ATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.     

Candida morphogenesis and host-pathogen interactions

The human fungal pathogen Candida albicans has many morphological forms. Recent advances in genomics and cell biology are providing an improved understanding of the molecular regulation of cell shape, and providing insights into the relationships between morphogenesis and virulence. This understanding may improve our ability to develop strategies to combat Candida infections.     

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.     

The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.In eukaryotes, the ubiquitous second messenger cyclic AMP (cAMP) is generated from ATP by membrane-integral or by cytoplasmic, CO₂-regulated cyclases (35, 44). The cAMP signal is processed by a small group of receiver proteins, including the regulatory subunit of protein kinase A (28), cAMP-gated ion channels (4), and the guanine-nucleotide-exchange proteins EPAC1 and EPAC2 (39). The cAMP signal is terminated by the action of a family of cyclic nucleotide-specific phosphodiesterases (PDEs) (9). This paradigm is rather straightforward, involves a limited number of players, and is generally well understood, at least in mammalian cells. However, much less is known about how individual cAMP signals are temporally and spatially controlled. Since most eukaryotic adenylyl cyclases are integral membrane proteins, often restricted to specific membrane subdomains (10), cAMP signaling is usually initiated at the cell membrane (40). However, diffusion of cAMP away from its site of generation is rapid, with diffusion coefficients being about 400 μm²/s (8, 15, 29), translating into diffusion velocities of 30 to 40 μm/s. As a consequence, the signal would reach the center of the cell with a diameter of 3 μm within less than 50 ms and would rapidly saturate the entire cell. While regulation through fluctuating cellular levels of cAMP represents a valid paradigm of cAMP signaling, it has become clear that other, more localized modes of cAMP signaling must also exist. Several groups have shown that the cAMP response of a given cell can differ depending on what set of receptors activates the cyclase response (14, 30, 41, 42). Similarly, the cAMP response of endothelial cells depends on the subcellular site where the cAMP is produced. They tighten their barrier function when cAMP is produced by membrane-bound adenylyl cyclases but become more permeable when cAMP is produced in the cytoplasm (17, 45). The distinct subcellular localization of cAMP signals was experimentally demonstrated using an array of techniques (29, 40, 55, 56).Physically tethered PDEs might serve to confine newly synthesized cAMP to defined microdomains. Only cAMP-binding proteins that are localized within or extend into such microdomains would be able to receive the cAMP signal (17, 49). cAMP concentrations within such domains might rise and fall rapidly, reaching peak concentrations much more rapidly and locally far beyond the steady-state cAMP levels measured in whole-cell extracts. Such spatially organized, tethered PDEs can generate local sinks into which cAMP disappears (1, 23). This paradigm would allow the simultaneous presence of numerous local cAMP concentration gradients within a single cell, allowing great flexibility in signal generation and intracellular signal transmission. This concept is based on the distinct subcellular localization and physical association of PDEs with subcellular structures and on the existence of localized subcellular cAMP pools, for which there is extensive experimental support (3, 5, 13, 50, 52). Interestingly, PDEs localized in different subcellular regions may still be able to compensate for each other. Ablation of the cilium-specific PDE1C from the olfactory neurons in the mouse did not prolong response termination, as long as the cytoplasmic PDE4 in the cell body was still present (11).The unicellular eukaryote Trypanosoma brucei is the causative agent of human sleeping sickness in sub-Saharan Africa. It belongs to the large order of the kinetoplastida, which includes many medically and economically important pathogens of humans, their livestock, and their crops worldwide (27). Trypanosomes are very small cells (about 15 by 3 μm in diameter) that carry a single flagellum (10 by 0.5 μm). The volume of a procyclic trypanosome of strain 427 is (9.6 ± 0.8) × 10⁻¹⁴ liter (Markus Engstler, personal communication), with the flagellum representing about 15% of this. A signaling threshold concentration of 1 μM cAMP corresponds to just about 30,000 molecules of cAMP per cell. Given a diffusion coefficient of 400 μm²/s (29), unrestricted diffusion of cAMP would swamp the cell within 50 ms. Obviously, temporal and spatial control of cAMP signaling is crucial for T. brucei. Strategically located, physically tethered PDEs might thus play an important role in the architecture of the cAMP signaling pathways in T. brucei.The genomes of T. brucei and of other kinetoplastids, such as T. vivax, T. cruzi, Leishmania major, L. infantum, and L. braziliensis, all code for the same set of five cyclic nucleotide-specific PDEs (25, 53). In T. brucei, the genes for T. brucei PDEB1 (TbrPDEB1; subsequently termed PDEB1) and TbrPDEB2 (PDEB2) are tandemly arranged on chromosome 9 and code for two very similar cAMP-specific PDEs, each with two GAF (mammalian cyclic GMP-dependent PDEs, Anabaena adenylyl cyclases, Escherichia coli FhlA) domains (21) in their N-terminal regions (38, 57). These two PDEs were also studied experimentally in T. cruzi (12) and L. major (24, 52), and orthologues are present in all kinetoplastid genomes available so far. Despite their high overall sequence similarity, PDEB1 and PDEB2 exhibit distinct subcellular localizations (31). PDEB1 is predominantly found in the flagellum, where it is stably associated with cytoskeletal components that are resistant to detergent extraction. In contrast, PDEB2 is mostly localized in the cell body, from where it is fully extractable by nonionic detergents. However, a minor fraction of PDEB2 also associates with the flagellar skeleton in a Triton-resistant manner, most likely through interaction with PDEB1. Earlier work has shown that both PDEB1and PDEB2 are essential enzymes in bloodstream-form T. brucei (31), while TbPDEA, TbPDEC, and TbPDED play minor roles (20; S. Kunz, unpublished data).     

Adenoviral transduction is more efficient in alginate-derived chondrocytes than in monolayer chondrocytes

Gene transfer into cultured chondrocytes by using adenoviral vectors has potential applications in treating cartilage disorders. The present study was undertaken to compare and optimize two chondrocyte culture conditions for adenoviral transduction efficacy by using primary human articular chondrocytes cultivated either directly in a monolayer condition or as outgrowths from alginate-stored chondrocyte cultures. Isolated primary chondrocytes from human articular cartilage were either immediately transduced with an EGFP (enhanced green fluorescent protein)-gene-bearing adenoviral vector (1,000 and 3,000 virus particles/cell) or cultured in alginate before transduction. Immunohistochemistry and flow cytometric analysis were employed to determine the expression of extracellular matrix proteins and of the αvβ5 integrin receptor involved in adenoviral cell entry. Monolayer chondrocytes exhibited moderate transduction rates (mean 22.2% and 46.9% EGFP-positive cells at 1,000 and 3,000 virus particles/cell by 72 h post-transduction), whereas alginate-derived chondrocytes revealed significantly higher transduction efficacies (95.7% and 99%). Both monolayer and alginate-derived chondrocytes expressed αvβ5 integrin, type II collagen and cartilage proteoglycans. The mean fluorescence intensity of type II collagen was significantly higher in the alginate-derived chondrocytes, whereas that of αvβ5 integrin was higher in the monolayer chondrocytes. Our results indicate that transduction efficacy is independent of αvβ5 integrin expression levels in chondrocytes. Moreover, adenoviral transduction of alginate-derived chondrocytes is more efficient than that for monolayer chondrocytes and may be a suitable tool to achieve sufficient numbers of transduced and differentiated chondrocytes for experimental applications and cartilage repair. Dr. Gundula Schulze-Tanzil is supported by a grant awarded by the Rahel Hirsh Foundation from the Charité Medical Schools Berlin. The study was supported by a grant from the Deutsche Arthrosehilfe e.V.     

Trypanosomes and mammalian sperm: one of a kind?

Flagellar-mediated motility is an indispensable function for cell types as evolutionarily distant as mammalian sperm and kinetoplastid parasites, a large group of flagellated protozoa that includes several important human pathogens. Despite the obvious importance of flagellar motility, little is known about the signalling processes that direct the frequency and wave shape of the flagellar beat, or those that provide the motile cell with the necessary environmental cues that enable it to aim its movement. Similarly, the energetics of the flagellar beat and the problem of a sufficient ATP supply along the entire length of the beating flagellum remain to be explored. Recent proteome projects studying the flagella of mammalian sperm and kinetoplastid parasites have provided important information and have indicated a surprising degree of similarities between the flagella of these two cell types.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.