A short-term chick embryo in vivo xenograft model to study retinoblastoma cancer stem cells

Purpose:Cancer stem cells (CSCs) reported in various tumors play a crucial role in tumorigenesis and metastasis of retinoblastoma (Rb). Following the efforts to reduce, replace, and refine the use of mammalian models, we aimed to establish a short-term xenograft for Rb to evaluate the CSC properties of CD133^- Rb Y79 cells, using the well-established chick embryo chorioallantoic membrane (CE-CAM) assay.Methods:Y79 cells were cultured, labeled with two different dyes (CM-Dil Y79 and enhanced green fluorescent protein (eGFP)) and sorted for CD133^- and CD133 + subsets. Two million cells from each of the labeled groups were transplanted onto the abraded CAM on embryonic day 7 (E7). On E14, the tumor nodule formation on CAM and spontaneous metastasis to the embryos were evaluated by confocal microscopy, in vivo imaging, and histology.Results:Y79 cells formed pink–white raised perivascular nodules with feeder vessels on the CAM with both the types of labeled CD133^- cells. CD133^- cells, when compared to CD133 + cells, demonstrated significantly larger tumor volume (40.45 ± 7.744 mm³ vs 3.478 ± 0.69 mm³, P = 0.0014) and higher fluorescence intensity (CM-Dil: AUF = 6.37 × 10⁷ ± 7.7 × 10⁶ vs 1.08 × 10⁷ ± 1.6 × 10⁶; P < 0.0001; eGFP: AUF = 13.94 × 10⁴ ± 2.54 × 10⁴ vs AUF = 1.39 × 10⁴ ± 0.4 × 10⁴; P = 0.0003). The metastatic potential of CD133^- cells was also observed to be higher as noted by in vivo imaging and histopathology.Conclusion:This study highlights that CE-CAM is a feasible alternative nonmammalian model for evaluating tumorigenicity and metastatic potential of Y79 CSCs. Increased tumorigenicity and metastatic potential of CD133^- subset of tumor cells substantiate their CSC properties.     

Snake bites by the jararacucu (Bothrops jararacussu): clinicopathological studies of 29 proven cases in Sao Paulo State, Brazil

The jararacucu, one of the most dreaded snakes of Brazil, southern Bolivia, Paraguay and northeastern Argentina, is a heavily-built pit viper which may grow to a length of 2.2 m. Up to 1000 mg (dry weight) of highly-lethal venom may be milked from its venom glands on a single occasion. It has accounted for 0.8% to 10% of series of snake bites in Sao Paulo State, Brazil. We examined 29 cases of proven jararacucu bites recruited over a 20-year period in two Sao Paulo hospitals. Severe signs of local and systemic envenoming, (local necrosis, shock, spontaneous systemic bleeding, renal failure) were seen only in patients bitten by snakes longer than 50 cm; bites by shorter specimens were more likely to cause incoagulable blood. Fourteen patients developed coagulopathy, six local necrosis (requiring amputation in one) and five local abscesses. Two became shocked and four developed renal failure. Three patients, aged 3, 11 and 65 years, died 18.75, 27.75 and 83 h after being bitten, with respiratory and circulatory failure despite large doses of specific antivenom and intensive-care- unit management. In two patients, autopsies revealed acute renal tubular necrosis, cerebral oedema, haemorrhagic rhabdomyolysis at the site of the bite and disseminated intravascular coagulation. In one survivor with chronic renal failure, renal biopsy showed bilateral cortical necrosis; the patient remains dependent on haemodialysis. Effects of polyspecific Bothrops antivenom were not impressive, and it has been suggested that anti-Bothrops and anti-Crotalus antivenoms should be given in combination.      

Phenotypic markers and BCL-1 gene rearrangements in B-cell chronic lymphocytic leukemia: a Cancer and Leukemia Group B study

The markers, CD11b, CD11c, CD14, CD21, CD23, CD25, CD38, and FMC7 were correlated with morphologic and other laboratory and clinical characteristics of 127 patients with untreated CD5+ chronic lymphocytic leukemia (CLL). Only CD38 and CD21 were significantly associated with atypical CLL morphology. The integrin associated markers CD11b and CD11c were associated with lower leukocyte count (white blood cell count [WBC]) and lower Rai stage. By contrast, the activation antigen CD23 was associated with a higher WBC, higher Rai stage, younger age group, and the presence of lymphadenopathy. Therefore, we conclude that CD23 positivity may reflect a more aggressive form of CLL, and CD11b and CD11c positivity a less aggressive form. The BCL-1 gene rearrangement was present in 5 of 84 (6%) CLL cases examined and was associated with atypical morphology and surface expression of CD11b. Patients with a BCL-1 gene rearrangement may represent a CLL subset or possibly a different B-cell disease.     

Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.     

Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age

Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune- mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ- CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.     

Organization, expression and polymorphism of the human persyn gene

Persyn is a recently identified member of the synuclein family with a distinct pattern of expression during pre- and postnatal development of the mouse peripheral and central nervous systems. As with other synucleins, persyn is believed to be involved in the pathogenesis of human neurodegenerative diseases. However, in contrast to other synucleins, high levels of persyn mRNA expression were also found in advanced breast carcinomas, suggesting an involvement of the encoded protein in breast tumour progression. Here we have used an antibody specific to human persyn to demonstrate that the level of this protein is increased in ageing cerebral cortex and in breast tumours. We cloned, characterized and sequenced the human persyn genomic locus and localized it to the long arm of chromosome 10 in the q23.2-q23.3 region. Sequence information was used to search for specific mutations in the protein coding regions of persyn mRNA and the persyn gene in breast tumours and tumour cell lines. No tumour-specific mutations were found, but two linked polymorphisms in the coding region were detected, both in mRNA and exons III and IV of the gene. These results suggest that development of breast tumours correlates with overexpression of the wild-type persyn protein. Detailed characterization of the human persyn locus is important for further studies of the involvement of persyn in neurodegeneration and malignancy.      

Prebiotics in infant milk formulas: new perspectives

In recent years it has become accepted that healthy human intestinal microflora may play an important part in priming the infants' systemic and mucosal immunity. Dietary modulation of the gut microbiota is a topical area of nutritional sciences and the main focus of many current functional foods such as non-digestible oligosaccharides (NDOs). Fructo-oligosaccharides (FOS) and trans-β-galacto-oligosaccharides (TOS) have been claimed to benefit the health of the colon by selectively stimulating the growth of bifidobacteria and lactobacilli (prebiotic effect). It could be of clinical interest to manipulate colonic flora because it is supposed that specific bacteria in the gut microbial microflora could promote potentially antiallergenic processes and play a key part in atopic disease prevention. Supporting this view is the finding that analysis of the composition of the intestinal bacterial populations showed different microbial patterns between healthy and allergic individuals. Assuming that non-digestible TOS and FOS can affect the intestinal ecosystem beneficially, the opportunity for gut flora manipulation arises in bottle-fed infants. New preterm and term infant milk formulas, supplemented with a mixture of TOS and FOS as prebiotic ingredients induced a significantly higher colonization of bifidobacteria and lactobacilli. In the future, selective manipulation of the intestinal microbiota might be an approach to novel prophylactic and therapeutic intervention strategies of atopy, by redirecting allergic Th-2 responses in favour of Th-1 responses.     

High-resolution real-time ultrasonography of thyroid nodules

High-resolution real-time ultrasonography was used to evaluate 98 patients with palpable abnormalities of the thyroid and positive isotopic studies. It confirmed 37 of 73 (51%) suspected solitary nodules. Of 25 patients thought to have multinodular goiter, sonography was supportive in 21 (84%). In patients with adenoma or adenomatous nodules, characteristic features included a sonolucent "halo". Colloid nodules tended to be more sonolucent than normal thyroid tissue, whereas Hashimoto thyroiditis was characterized by an enlarged gland and decreased echogenicity.