Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.     

CT quantification of pulmonary emphysema: assessment of lung structure and function

Accurate diagnosis and quantification of pulmonary emphysema in vivo is important to understand the natural history of the disease, to assess the extent of the disease, and to evaluate and follow-up therapeutic interventions. Because pulmonary emphysema is defined by pathology, new diagnostic methods for quantification should be validated by reference to pathological and histological standards. Recent studies have addressed the capability of computed tomography (CT) to accurately quantify pulmonary emphysema. These studies that have been overviewed in this article have been based on CT scans obtained after deep inspiration or expiration, on subjective visual grading, and on objective measurements of attenuation values by using dedicated software providing numerical data on two-dimensional and on three-dimensional approaches, and compared CT data with pulmonary function tests. More recently, fractal and textural analyses were applied to CT scans to assess the presence, extent, and types of emphysema. Quantitative CT has already been used in patient selection for surgical treatment of pulmonary emphysema and in pharmacotherapeutical trials. However, despite numerous and extensive studies already available, this technique has not yet been standardized, and important questions about how to best use CT for the quantification of pulmonary emphysema remain to be addressed.     

Bullous pemphigoid,primary biliary cirrhosis and vitiligo: a multiple autoimmune syndrome?

Bullous pemphigoid is the most frequent autoimmune blistering skin disease. There have been few reports of an association with primary biliary cirrhosis and vitiligo. We report the simultaneous occurrence of bullous pemphigoid and primary biliary cirrhosis in an 86-year-old patient who also suffered from vitiligo. Multiple autoimmune syndrome, proposed by Humbert and Dupond, can be divided into three groups based on preferential associations of autoimmune disorders. The association of bullous pemphigoid, cirrhosis biliary primary and vitiligo has been reported three times in the literature. This association is probably not fortuitous and suggests a pathogenic relationship. This association is not typical of the multiple autoimmune syndrome as defined by Humbert and Dupond but the collection of such observations may contribute to revise the classification of autoimmune disease and provide a better understanding of the pathophysiological mechanisms of autoimmunity.     

Smooth muscle dysfunction in resistance arteries of the staggerer mouse, a mutant of the nuclear receptor RORalpha

Retinoic acid receptor-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor superfamily. The mouse mutant staggerer (sg/sg) carries a deletion within the RORalpha gene. RORalpha plays a major role in cellular differentiation during development and growth. In the present study, we found a lower mean arterial blood pressure in sg/sg than in +/+ mice (80.1+/-1.2 and 87.0+/-0.9 mm Hg, respectively; P<0.0002) and a smaller increase in blood pressure after in vivo injections of phenylephrine. To elucidate the mechanisms responsible for this phenotype, we investigated the vascular reactivity of large vessels (aorta and carotid arteries) and small resistance mesenteric arteries in response to mechanical forces or vasoactive agents. Arteries from sg/sg and +/+ mice were studied in vitro in arteriographs. Vascular responses of large vessels to all stimuli were similar in both groups. However, we found a markedly altered vascular function in mesenteric arteries from sg/sg mice. Flow-induced dilation, pressure-induced myogenic tone, responses to endothelium-dependent or -independent vasodilators, and responses to vasoconstrictors were significantly reduced in sg/sg compared with +/+ mice. We also determined by Western blot analysis the expression of smooth muscle (SM)-myosin, calponin, and heavy (h)-caldesmon, in large and small arteries of sg/sg and +/+ mice, and found a marked decrease in the expression of these contractile proteins in mesenteric arteries of sg/sg mice. Our findings provide the first evidence that functional RORalpha is required for normal contractile phenotype of smooth muscle cells (SMCs) in small resistance arteries and suggest that RORalpha might be involved in the differentiation of SMCs in mesenteric arteries.     

Combined detection of mannanaemia and antimannan antibodies as a strategy for the diagnosis of systemic infection caused by pathogenic Candida species

A novel strategy for the diagnosis of systemic candidosis was evaluated, based on the combination of two enzyme immunoassays that detect a candida oligomannoside repetitive epitope expressed in large amounts by Candida albicans (Platelia Candida Ag), and antibodies against C. albicans mannan, the major cell-wall immunogen in which this epitope is present (Platelia Candida Ab). Sera were selected retrospectively from intensive care and haematology patients with clinically suspected systemic candidosis, and from whom Candida spp. had been isolated from normally sterile sites. Of the 21 patients infected with C. albicans, 13 had positive antigenaemia and 14 had a positive antibody response, including eight patients who were antigenaemia negative. The sensitivity of the combined tests was 100%. In patients infected with C. glabrata (n = 12) or C. tropicalis (n = 10), the sensitivity was 83% and 80%, respectively. For the remaining patients, infected with C. parapsilosis (n = 10), C. krusei (n = 8) or C. kefyr (n = 2), the sensitivity of the combined tests was 40%, 50% and 50%, respectively. At least one of the serological tests was positive before yeast growth occurred in 60% of patients for whom a serum sample was available before blood culture sampling. An increase in serological test positivity to >80% was observed for sera obtained around the date of positive culture, irrespective of the Candida species isolated. These results suggest that regular serological monitoring for both mannanaemia and anti-mannan antibodies in at-risk patients may contribute to the early diagnosis of candidosis.     

Identification of histological features associated with metastatic potential in thin (<1.0 mm) cutaneous melanoma with metastases. A study on behalf of the EORTC Melanoma Group

Contrary to expectations, a small number of thin (<1 mm) melanomas do metastasize. This collaborative study was performed in an attempt to identify the morphological basis of such aggressive behaviour. Regression was expected to be the explanation for the lack of thickness in some cases. Whether a vertical growth phase (VGP) was present in the remainder was carefully assessed. A pilot study had identified two other patterns associated with metastasis in thin melanomas. These were termed 'junctional expansion nodules' and 'melanomatous follicular invasion'. Both were seen in the absence of other dermal invasion. These two patterns were included in the study, which comprised 54 cases and 56 controls, which were thin melanomas which had not metastasized 5 years after excision. Regression was present in 50% of test cases (30.4% in controls, p=0.036) and VGP was present in 59.3% of cases and 48.2% of controls. The thinnest metastasizing melanoma without regression was 0.27 mm. Eight (14.8%) cases, however, had metastasized but showed neither regression nor VGP; seven of these showed a junctional expansion nodule, present in only three controls (p=0.016). Five of these seven also showed melanomatous follicular invasion. One of these five showed this follicular involvement without a junctional nodule. Melanomatous follicular invasion was not seen in the control cases (p=0.012). Mitoses were seen in the VGP of both test and control cases, but high counts (>3 per mm(2)) were much more common in the metastasizing lesions (p=0.007). These findings support the idea that in most cases, regression and/or a VGP are required for metastasis to occur. However, a small number of thin melanomas without these features, as conventionally described, still metastasize. This implies that VGP may require redefinition and that junctional expansion nodules and melanomatous follicular invasion may be variants of VGP.     

Transgenic B lymphocytes expressing a human cold agglutinin escape tolerance following experimental infection of mice by Mycoplasma pulmonis

Several microbial infections, including Mycoplasma pneumoniae respiratory infection, are capable, in man, of transiently inducing the expression of anti-red blood cell autoantibody called cold agglutinins (CA). To analyze the mechanisms by which immune tolerance is broken following a mycoplasma infection, we used transgenic mice expressing a pathogenic human CA, designated CA-GAS, specific for sialylated carbohydrates. In these mice peripheral deletion of autoreactive B lymphocytes and receptor editing, prevent the development of autoimmune hemolytic anemia. Experimental infections of transgenic mice with Mycoplasma pulmonis resulted in a high anti-mycoplasma antibody response (despite a severe B cell depletion at the onset of infection), and an important induction of serum CA concentrations, reaching in some mice pathological titers. Whereas in na?ve mice, only a small percentage of CA-expressing cells could be detected, in infected mice, a majority of circulating B lymphocytes were large B220(-) cells, which expressed the transgenic immunoglobulin. Immunization of the transgenic mice with keyhole limpet hemocyanin and Freund's adjuvant, to nonspecifically stimulate the expression of the passenger transgenes, only moderately increased the CA titers. These results indicate that M. pulmonis infection is capable of breaking immune tolerance in the CA-transgenic mice, in part through specific activation of CA-expressing B lymphocytes. This experimental infection mimics the induction of CA in humans and provide an animal model for studying the genesis of the autoimmune hemolytic anemia.