Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.

Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines.

Granulocyte colony-stimulating factor supports liver regeneration in a small-for-size liver remnant mouse model

Experimental partial hepatectomy of more than 80% of the liver weight bears an increased mortality in rodents, due to impaired hepatic regeneration in small-for-size liver remnants. Granulocyte colony-stimulating factor (G-CSF) promotes progenitor cell expansion and mobilization and also has immunomodulatory properties. The aim of this study was to determine the effect of systemically administered G-CSF on liver regeneration and animal survival in a small-for-size liver remnant mouse model. Mice were preconditioned daily for 5 days with subcutaneous injections of 5 microg G-CSF or aqua ad injectabile. Subsequently, 83% partial hepatectomy was performed by resecting the median, the left, the caudate, and the right inferior hepatic lobes in all animals. Daily sham or G-CSF injection was continued. Survival was significantly better in G-CSF-treated animals (P < 0.0001). At 36 and 48 h after microsurgical hepatic resection, markers of hepatic proliferation (Ki67, BrdU) were elevated in G-CSF-treated mice compared to sham injected control animals (P < 0.0001) and dry liver weight was increased (P < 0.05). G-CSF conditioning might prove to be useful in patients with small-for-size liver remnants after extended hepatic resections due to primary or secondary liver tumors or in the setting of split liver transplantation.

Clinical characteristics associated with isolation of small-colony variants of Staphylococcus aureus and Pseudomonas aeruginosa from respiratory secretions of patients with cystic fibrosis

During a 3-month period, small-colony variant phenotypes of both Staphylococcus aureus and Pseudomonas aeruginosa were isolated from respiratory secretions of 8.2% and 9.2%, respectively, of 98 patients with cystic fibrosis, particularly those with advanced pulmonary disease and prolonged antibiotic exposure.

Granulocyte colony stimulating factor supports liver regeneration in a surgical small for size liver remnant mouse model

ntense liver regeneration and almost 100% survival follows partial hepatectomy of up to 70% of liver mass in rodents. More extensive resections of 70 to 80% have an increased mortality and partial hepatectomies of >80% constantly lead to acute hepatic failure and death in mice. The aim of the study was to determine the effect of systemically administered granulocyte colony stimulating factor (G-CSF) on animal survival and liver regeneration in a small for size liver remnant mouse model after 83% partial hepatectomy (liver weight <0.8% of mouse body weight). Methods: Male Balb C mice (n=80, 20-24g) were preconditioned daily for five days with 5μg G-CSF subcutaneously or sham injected (aqua ad inj). Subsequently 83% hepatic resection was performed and daily sham or G-CSF injection continued. Survival was determined in both groups (G-CSF n=35; Sham: n=33). In a second series BrdU was injected (50mg/kg Body weight) two hours prior to tissue harvest and animals euthanized 36 and 48 hours after 83% liver resection (n=3 each group). To measure hepatic regeneration the BrdU labeling index and Ki67 expression were determined by immunohistochemistry by two independent observers. Harvested liver tissue was dried to constant weight at 65 deg C for 48 hours. Results: Survival was 0% in the sham group on day 3 postoperatively and significantly better (26.2% on day 7 and thereafter) in the G-CSF group (Log rank test: p<0.0001). Dry liver weight was increased in the G-CSF group (T-test: p<0.05) 36 hours after 83% partial hepatectomy. Ki67 expression was elevated in the G-CSF group at 36 hours (2.8±2.6% (Standard deviation) vs 0.03±0.2%; Rank sum test: p<0.0001) and at 48 hours (45.1±34.6% vs 0.7±1.0%; Rank sum test: p<0.0001) after 83% liver resection. BrdU labeling at 48 hours was 0.1±0.3% in the sham and 35.2±34.2% in the G-CSF group (Rank sum test: p<0.0001) Conclusions: The surgical 83% resection mouse model is suitable to test hepatic supportive regimens in the setting of small for size liver remnants. Administration of G-CSF supports hepatic regeneration after microsurgical 83% partial hepatectomy and leads to improved long-term survival in the mouse. G-CSF might prove to be a clinically valuable supportive substance in small for size liver remnants in humans after major hepatic resections due to primary or secondary liver tumors or in the setting of living related liver donation.

Granulocyte colony-stimulating factor increases hepatic sinusoidal perfusion during liver regeneration in mice

Conditioning with granulocyte colony-stimulating factor (G-CSF) promotes liver regeneration in an experimental small-for-size liver remnant mouse model. The mechanisms involved in this extraordinary G-CSF effect are unknown. The aim of this study was to investigate the influence of G-CSF on the hepatic microvasculature in the regenerating liver. The hepatic sinusoidal microvasculature and microarchitecture of the regenerating liver were evaluated by intravital microscopy in mice. Three experimental groups were compared: (1) unoperated unconditioned animals (control; n = 5), (2) animals conditioned with G-CSF 48 h after 60% partial hepatectomy (G-CSF-PH; n = 6), and (3) animals sham conditioned 48 h after 60% PH (sham-PH; n = 6). PH led to hepatocyte hypertrophy and increased hepatic sinusoidal velocity in the sham-PH and G-CSF-PH groups. Increased sinusoidal diameter and increased hepatic blood flow were observed in the G-CSF-PH group compared to the sham-PH and control groups. Furthermore, there was a strong positive correlation between spleen weight and hepatic sinusoidal diameter in the G-CSF-PH group. The increased hepatic blood flow could explain the observed benefit of G-CSF conditioning during liver regeneration. These results elucidate an unexplored aspect of pharmacological modulation of liver regeneration and motivate further experiments.

Myocardial salvage through coronary collateral growth by granulocyte colony-stimulating factor in chronic coronary artery disease: a controlled randomized trial

BACKGROUND: The efficacy of granulocyte colony-stimulating factor (G-CSF) for coronary collateral growth promotion and thus impending myocardial salvage has not been studied so far, to our best knowledge. METHODS AND RESULTS: In 52 patients with chronic stable coronary artery disease, age 62+/-11 years, the effect on a marker of myocardial infarct size (ECG ST segment elevation) and on quantitative collateral function during a 1-minute coronary balloon occlusion was tested in a randomized, placebo-controlled, double-blind fashion. The study protocol before coronary intervention consisted of occlusive surface and intracoronary lead ECG recording as well as collateral flow index (CFI, no unit) measurement in a stenotic and a > or =1 normal coronary artery before and after a 2-week period with subcutaneous G-CSF (10 microg/kg; n=26) or placebo (n=26). The CFI was determined by simultaneous measurement of mean aortic, distal coronary occlusive, and central venous pressure. The ECG ST segment elevation >0.1 mV disappeared significantly more often in response to G-CSF (11/53 vessels; 21%) than to placebo (0/55 vessels; P=0.0005), and simultaneously, CFI changed from 0.121+/-0.087 at baseline to 0.166+/-0.086 at follow-up in the G-CSF group, and from 0.152+/-0.082 to 0.131+/-0.071 in the placebo group (P<0.0001 for interaction of treatment and time). The absolute change in CFI from baseline to follow-up amounted to +0.049+/-0.062 in the G-CSF group and to -0.010+/-0.060 in the placebo group (P<0.0001). CONCLUSIONS: Subcutaneous G-CSF is efficacious during a short-term protocol in improving signs of myocardial salvage by coronary collateral growth promotion.

Staphylococcus aureus as an intracellular pathogen: the role of small colony variants

Increasing evidence indicates that Staphylococcus aureus might be a facultative intracellular pathogen. In particular, certain subpopulations, called small colony variants (SCVs), seem to be well adapted to the intracellular milieu. When compared to 'normal' staphylococcal strains, SCVs show increased uptake by host cells, resistance to intracellular defenses and reduced stimulation of host defenses. We propose that the ability to form two subpopulations with different phenotypes might allow S. aureus the option for both extra- cellular and intra-cellular survival in the host.