Optimization of 100 µm alginate-poly-L-lysine-alginate capsules for intravitreous administration.

The field of cell microencapsulation is advancing rapidly. Particle size plays a critical role in terms of biocompatibility and limits decisively its applicability. Producing reduced size microcapsules involves broadening the possibilities to employ this technology in the treatment of many disorders. Nervous system diseases (NSD) represent a clear example of that. This work describes the feasibility of reducing the size of alginate-poly-L-lysine-alginate (APA) microcapsules up to 100 µm in a highly monodisperse way using the novel Flow Focusing technique. C(2)C(12) myoblasts genetically engineered to express the triple reporter gene thymidine kinase-green fluorescent protein-luciferase (TGL) and secrete vascular endothelial growth factor soluble receptor 2 (VEGFR2, also known as KDR) were encapsulated for further characterization. Resulting new particles were assayed in vitro to explore whether their functionality might be affected due to the physicochemical changes arising from such dramatic size reduction. Not only were negative effects at this level not noticed in terms of cell viability, cell proliferation and KDR secretion, but once again the suitability of APA microcapsules was also reinforced against other microcapsule designs. Furthermore, the fully viable and functional biosystems were successfully administered in the intravitreous space of rats, where the activity of encapsulated cells was monitoring over 3 weeks.

Overexpression of TMPRSS4 in non-small cell lung cancer is associated with poor prognosis in patients with squamous histology.

BACKGROUND: Mortality rates in lung cancer patients have not decreased significantly in recent years, even with the implementation of new therapeutic regimens. One of the main problems is that a large proportion of patients present local or distant metastasis at the time of diagnosis. The need for identification of novel biomarkers and therapeutic targets for a more effective management of lung cancer led us to investigate TMPRSS4, a protease reported to promote tumour growth and metastasis. MATERIAL AND METHODS: In all, 34 lung cancer cell lines were used to evaluate the TMPRSS4 expression. Cell migration and clonogenic assays, and an in-vivo lung metastasis model were used for functional analysis of the TMPRSS4 downregulation in H358, H441 and H2170 cell lines. The TMPRSS4 expression analysis in normal and malignant lung tissue samples was performed by qPCR. Five different microarray-based publicly available expression databases were used to validate our results and to study prognosis. RESULTS: The TMPRSS4 knock down in H358, H441 and H2170 cells resulted in a significant reduction in proliferation, clonogenic capacity and invasion. A significant (P<0.05) decrease in the lung colonisation and growth was found when mice were injected with TMPRSS4-depleated H358-derived clones, as compared with controls. Expression of TMPRSS4 showed a >30-fold increase (P<0.001) in tumours in comparison with non-malignant samples. Levels in tumours with squamous cell carcinoma (SCC) histology were found to be significantly higher (P<0.001) than those with adenocarcinoma (AC) histology, which was confirmed in data retrieved from the microarrays. Kaplan-Meier curves demonstrated that high levels of TMPRSS4 were significantly associated (P=0.017) with reduced overall survival in the patients with SCC histology, whereas no correlation was found for the AC histology. CONCLUSION: Our results demonstrate that TMPRSS4 has a role in the lung cancer development. The potential use of TMPRSS4 as a biomarker for lung cancer detection or as a predictor of patient's outcome warrants further investigation.

The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer.

Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 microM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg(-1) P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer.

Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis.

Metastasis is the primary cause of death in patients with breast cancer. Overexpression of c-myc in humans correlates with metastases, but transgenic mice only show low rates of micrometastases. We have generated transgenic mice that overexpress both c-myc and vascular endothelial growth factor (VEGF) (Myc/VEGF) in the mammary gland, which develop high rates of pulmonary macrometastases. Gene expression profiling revealed a set of deregulated genes in Myc/VEGF tumors compared to Myc tumors associated with the increased metastatic phenotype. Cross-comparisons between this set of genes with a human breast cancer lung metastasis gene signature identified five common targets: tenascin-C(TNC), matrix metalloprotease-2, collagen-6-A1, mannosidase-alpha-1A and HLA-DPA1. Signaling blockade or knockdown of TNC in MDA-MB-435 cells resulted in a significant impairment of cell migration and anchorage-independent cell proliferation. Mice injected with clonal MDA-MB-435 cells with reduced expression of TNC demonstrated a significant decrease (P<0.05) in (1) primary tumor growth; (2) tumor relapse after surgical removal of the primary tumor and (3) incidence of lung metastasis. Our results demonstrate that VEGF induces complex alterations in tissue architecture and gene expression. The TNC signaling pathway plays an important role in mammary tumor growth and metastases, suggesting that TNC may be a relevant target for therapy against metastatic breast cancer.

Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells.

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (-5.7 to -3.3 kb) which can not only drive expression of a (beta)-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2(beta)-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.

The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha.

1. Myocardial dysfunction during septic shock is associated with enhanced production of cytokines such as interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha). These cytokines depress cardiac mechanical function by a mechanism which is not well defined. 2. Bacterial endotoxin or cytokines cause the expression of Ca(2+)-independent nitric oxide (NO) synthase in cardiac myocytes, vascular endothelial cells and endocardial endothelial cells, causing enhanced production of NO. As NO has negative inotropic actions on cardiac muscle, we tested the sum effects of IL-1 beta plus TNF-alpha in the intact heart to determine whether enhanced expression of NO synthase activity in the cells that comprise the heart is involved in cardiac depression associated with cytokine stimulation. 3. Rat isolated working hearts perfused with IL-1 beta plus TNF-alpha showed a markedly greater depression in contractile function, measured as cardiac work, after 2 h of perfusion compared with time-matched control hearts. The depressant action of IL-1 beta plus TNF-alpha was first apparent after 1 h of perfusion; no early (15 min) cardiac depressant actions were seen. 4. The competitive inhibitor of Ca(2+)-dependent and Ca(2+)-independent NO synthases, NG-nitro-L-arginine methyl ester (L-NAME, 3 microM) when given concurrently with IL-1 beta plus TNF-alpha prevented the loss in contractile function such that these hearts after 2 h of perfusion had similar function to time-matched controls. L-NAME did not acutely reverse the loss of contractile function in hearts exposed for 2 h to IL-1 beta plus TNF-alpha. The protective action of L-NAME in the presence of cytokines was concentration-dependent and was not seen at a higher concentration (10 micro M) due to the significant reduction in coronary flow observed at this concentration.5. In contrast, when L-NAME (3 micro M) was given in the absence of IL-l beta plus TNF-alpha it depressed contractile function over the 2 h perfusion period by significantly reducing coronary flow.6. Inhibition of protein synthesis with cycloheximide (Cx) abolished the loss in function that occurred over 2h in both control and IL-1 beta plus TNF-a-treated hearts.7. Inducible, Ca2+-independent NO synthase activity was not observed in freshly isolated hearts but was observed in control hearts perfused for 2 h in vitro and was doubled in hearts perfused with IL-1 beta plus TNF-a. Cx prevented the expression of Ca2+-independent NO synthase in both control and cytokine-treated hearts.8. In summary, these results suggest that the depression of myocardial function by IL-l beta plus TNF-alpha is mediated, at least in part, by induction of Ca2+-independent NO synthase activity in the heart.

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.

[3H]iloprost and prostaglandin E2 compete for the same receptor site on cardiac sarcolemmal membranes.

We have previously demonstrated that high-affinity PGE receptors are present on purified cardiac sarcolemmal (SL) membrane from bovine heart (Lopaschuk et al. (1989) Circ. Res. 65, 538-545). In this study we determined whether PGI2 receptors are also present on the cardiac SL membrane. Due to the extreme lability of prostacyclin (PGI2) under physiological conditions, the PGI2 analogue, Iloprost was substituted for PGI2. 3H-Iloprost specifically bound to two sites on the SL membrane; one of high affinity (Kd = 0.3 nM, Bmax = 97.0 fmol/mg SL), and one of lower affinity (Kd = 20.6 nM, Bmax = 1589 fmol/mg SL). Competition studies demonstrated that the concentrations of PGE2 and PGE1 necessary to displace 50% of the specific binding of 20 nM [3H]Iloprost on cardiac SL were 15-fold lower than the concentrations of unlabelled Iloprost necessary to displace 50% of binding. In contrast, a 15-fold higher concentration of unlabelled Iloprost was needed to displaced 50% of specific binding of 2 nM [3H]PGE2 compared to the concentrations of PGE1 or PGE2 required to displace 50% of [3H]PGE2 binding. In summary, our results indicate that a prostacyclin receptor is present on the cardiac sarcolemmal membrane, and that PGI2 competes for the same receptor site as PGE2.

Phospholipids in inflammatory synovial effusions.

The concentration of phospholipids and proteins was determined in 23 inflammatory synovial fluids obtained from human knee joints. The synovial fluid to plasma phospholipid ratio (0.48 and 0.37 at high and low inflammatory state) was lower than the value found for the total protein content (0.68 and 0.53, respectively) indicating that phospholipids were more discriminated than proteins in their transfer from plasma to the synovial space. Constant amounts of phosphatidylinositol were found in all synovial fluids, whereas trace amounts of lysophosphatidylethanolamine and phosphatidylserine were more frequent in the active inflammatory state. A decrease in the relative amounts of phosphatidylcholine and phosphatidylinositol with respect to plasma suggested the possibility of phospholipid hydrolysis in the synovial compartment. In agreement, determinations of phospholipase activity disclosed the presence of a phospholipase A2 in the fluid phase of synovial effusions. Phospholipid derivatives formed in the synovial space may thus contribute to the amplification of the inflammatory response.

Freeze-fracture characterization of the outermost Golgi cisterna (OGC) in rat pancreatic acinar cells.

The outermost Golgi cisterna (OGC) frequently exhibits 55 nm diameter protuberances in P fracture faces and corresponding depressions, or pits, in E faces. When these protuberances (pits) appear regularly disposed, OGC faces are recognizable at relatively low electron microscopic magnifications. The mean particle width is less in OGC (6.4 nm) than in rough ER (7.9 nm) P faces, while particle number per unit area is respectively 70% and 100% greater in OGC P and E faces than in corresponding ER faces. The rather small OGC P face particles are better resolved at a relatively low shadowing angle. These differences in particle size (for P faces) and density between OGC and rough ER faces are detectable by inspection and may be used in the recognition of OGC faces devoid of protuberances (in P faces) and pits (in E faces). Fusion of presumably rough ER-derived microvesicles form short double-bossed tubular elements which constitute the OGC initial structure. These tubules enlarge by addition of microvesicle membrane and contents forming a characteristic sheet-like bossed structure.

[Chronic lymphatic leukemia and chromosomic anomalies (author's transl)]. / Leucemia linfatica cronica ed anomalie cromosomiche.

A cytogenetic study carried out by a blood culture from aspirated medulla of a patient with chronic lymphatic leukemia is here reported. It was possible to observe an anomaly consisting in a small supernumerary acrocentric chromosome of G group. The anomalies associated with CLL related in the literature are shortly reviewed, and the variability of the chromosomopathy in this blood disease are emphazised.

[Agnogenic myeloid metaplasia (author's transl)]. / Metaplasia mieloide agnogenica

The AA., present a case of "agnogenic myeloid metaplasia" (MMA). They describe the principal findings that have led to this diagnosis: Fe59-erythrocynesis, radiologic study of skeleton and cytogenetic study. Fe59-erythrocynesis have shown an hypergetting of spleen and liver and an hypogetting of skeleton. The radiography of skeleton and particularly of humerus, femur and pelvis have shown a diffuse osteosclerosis with rare osteolytic areas. The cytogenetic study have shown an alteration with C-group trisomy.

[Waldenström's syndrome and chromosome aberrations]. / Sindrome di Waldenström ed anomalie cromosomiche

The AA. present a case of Waldenström syndrome with chromosomic alterations. The cytogenetic study demonstrated that 3% of metaphases are represented by maps with 47 chromosomes for a supernumerary sub-metacentric chromosome like the A-3 group. On 12% of euploid maps they found similary the sub-metacentric chromosome a-3 group like (chromosome MG) and, at the same time, the lack of an element of the 18-group.