The biology of mammary transgenes: Five rules

The development of hyperplasias, dysplasias, and mammary tumors has been studied extensively in transgenic mice. It is now becoming clear that transgenes activate and participate in oncogenic pathways that govern the events surrounding neoplastic progression in transgenic mice. The oncogenic pathways control mammary growth, development, and neoplastic progression. Some of the key features of transgenic biology can be expressed in the following rules: (1) Mammary development is related to the type and amount of transgene expressed; (2) dysplasias and tumors develop from secondary mutations; (3) the transgenes determine tumor phenotype; (4) transgenes may activate dominant oncogenic pathways; and (5) the oncogenic pathway determines prognosis. The study of the comparative pathology of mammary tumorigenesis in many strains of transgenic mice provides examples of these principles.     

The Genetic Epidemiology of Breast Cancer Genes

Genetic susceptibility to breast cancer in women is conferred by a large number of genes, of which six have so far been identified. In the context of multiple-case families, BRCA1 and BRCA2 are the most important. Mutations in these genes confer high lifetime risks of breast cancer and ovarian cancer, and more moderate risks of prostate cancer and some other cancer types. Mutations in the CHEK2 and ATM genes, by contrast, cause much more modest (2-4 fold) risks of breast cancer. Genes so far identified explain approximately 20% of the familial aggregation of breast cancer. The remaining susceptibility genes have, so far, proved illusive, suggesting that they are numerous and confer moderate risks. A variety of techniques including genome-wide association studies, use of quantitative intermediate endpoints, and resequencing of genes may be required to identify them. The identification of such genes can provide a basis for targeted prevention of breast cancer.     

Proteomic dissection of dome formation in a mammary cell line

The study of the development of the mammary gland at the molecular level in animals is difficult because of the complex tissue organization. This review introduces a proteomic approach to investigate mammary gland development in a cell culture system that we have previously developed as an in vitro model for studying mammary cell differentiation. The model is based on two cell lines, one of which is able to differentiate spontaneously and produce hemispherical blisters, called domes, when confluent. Through proteomic dissection of dome-forming cells, two types of key regulatory genes have been identified: genes inducing cellular structural modifications and genes related to functional modifications. We identified several genes in the pathway leading to dome formation in vitro and showed that the functional and structural changes taking place in dome-forming cells correspond to cellular changes occurring in vivo when tubules and alveoli are developed in the mammary gland at pregnancy.     

Genetic forms of nephrotic syndrome

Mutations of NPHS1, NPHS2 , or WT1 may be responsible for severe forms of nephrotic syndrome in children, progressing to end-stage renal failure. Recent studies have shown that congenital nephrotic syndrome may be secondary to mutations of one of these three genes and that some patients have a digenic inheritance of NPHS1 and NPHS2 mutations. The clinical spectrum of NPHS2 mutations has broadened, with the demonstration that mutations in the respective gene podocin may be responsible for nephrotic syndrome occurring at birth, in childhood, or in adulthood. It is now well recognized that podocin mutations are found in 10%–30% of sporadic cases of steroid-resistant nephrotic syndrome with focal segmental glomerulosclerosis. Data from large cohorts indicate that the risk of recurrence of nephrotic syndrome after renal transplantation in patients with podocin mutations is very low.     

Nodular myofibroblastic stromal hyperplasia of the mammary gland as an accurate name for pseudoangiomatous stromal hyperplasia of the mammary gland

Pseudoangiomatous stromal hyperplasia (PASH) of the mammary gland is a well-known benign localized form of stromal overgrowth with probable hormonal etiology. We describe the histologic findings and immunohistochemistry of two cases. Two women, 16 and 58 years old, presented with a breast mass and underwent surgical excision. Grossly, they consisted of a well-circumscribed, rubbery tissue with a solid white-tan homogeneous cut surface. One of the cases showed focal cystic areas. Histologically the lesion showed a proliferation of the collagenous stroma with varying degrees of density, and hyalinization with many pseudovascular slit-like anastomosing spaces lined by spindle cells with scant cytoplasm and bland chromatin. The spindle cells lining the spaces were strongly reactive for vimentin and weakly reactive for CD34, actin, and desmin. They were negative for factor VIII, S-100, and pankeratin. In PASH, the "pseudoangiomatous" term describes a recognizable pattern but does not describe the tumor's histologic nature. We propose the name nodular myofibroblastic hyperplasia of the mammary stroma as a more accurate name.     

Effectiveness of low-dose contrimoxazole prophylaxis against Pneumocystis carinii pneumonia after renal and/or pancreas transplantation

We retrospectively examined the effectiveness of prophylaxis with cotrimoxazole in preventing Pneumocystis carnii pneumonia in recipients of kidney and combined kidney-pancreas transplants between 1985 and 1989. Cotrimoxazole prophylaxis (480 mg daily or 300 mg/m²), when used, was started within 2 months after transplantation and usually continued until 6 months after surgery. Eight (3.7%) of the 214 patients who were not given prophylaxis were infected with Pneumocystis carinii, and there were 4 fatalities (50% mortality). There were no cases among the 161 patients given prophylaxis (P 0.03). No serious adverse effects were noted in the prophylaxis group. It is concluded that prophylaxis against Pneumocystis carinii infection is well tolerated and should be given as soon as possible to all organ transplant recipients for at least 6 months.     

N-Acetylcysteine failed to improve early microcirculatory alterations of the rat liver after transplantation

The application of radical scavengers reduces reperfusion injury of liver grafts despite the natural occurrence of cellular defense mechanisms enabling the cell to tolerate moderate oxidant stress without further cell damage. The glutathione peroxidase mechanism of the liver serves to reduce hydroxyl radical-induced lipid peroxidation by releasing reduced glutathione from intracellular stores. There is evidence that the application of cysteine-providing aminoacids for glutathione synthesis could maintain or even increase liver glutathione. Therefore, the purpose of this study was to evaluate the effect of N-acetylcysteine (NAC) on oxidative stress-induced reperfusion injury after liver transplantation. This was done by applying intravital microscopy. Livers from female Sprague-Dawley rats weighing 220–260 g were stored for 20 h in University of Wisconsin (UW) solution and transplanted orthotopically using the cuff technique. Donors were given 150 mg/kg body weight NAC i. v. or placebo in a blind, random fashion 6 h prior to harvesting, followed by two injections of 50 mg/kg body weight, 4 and 2 h before explantation. In additional experimental groups, recipients were given a bolus of 83 mg/kg body weight NAC or placebo at the beginning of the recipient operations, 1 min prior to reperfusion, and 60 min after surgery. Ninety minutes after transplantation, intravital microscopy was applied and five liver lobules were recorded for 30 s after injection of acridine orange, a fluorescent leukocyte marker. Sinusoidal perfusion, sinusoidal width, and leukocyte adhesion, as well as reduced and oxidized glutathione, were determined in all livers. Neither microcirculatory disturbance nor leukocyte adhesion was less, nor was the liver glutathione in the recipient groups pretreated or treated with NAC greater than that in rats receiving the placebo. Moreover, liver glutathione was significantly decreased in livers from donors pretreated with NAC. In conclusion, the application of NAC as a pretreatment for donors and as treatment for recipients, respectively, failed to reduce early microvascular failure after liver transplantation.     

Genetic and Epigenetic Changes in Mammary Epithelial Cells May Mimic Early Events in Carcinogenesis

Studies of human mammary epithelial cells from healthy individuals are providing novel insights into how early epigenetic and genetic events affect genomic integrity and fuel carcinogenesis. Key epigenetic changes, such as the hypermethylation of the p16 (INK4a) promoter sequences, create a previously unappreciated preclonal phase of tumorigenesis in which a subpopulation of mammary epithelial cells are positioned for progression to malignancy (Romanov et al. , 2001, Nature , 409:633-637; Tlsty et al. , 2001, J. Mammary Gland Biol. Neoplasia , 6:235-243). These key changes precede the clonal outgrowth of premalignant lesions and occur frequently in healthy, disease-free women. Understanding more about these early events should provide novel molecular candidates for prevention and therapy of breast cancer that target the process instead of the consequences of genomic instability. This review will highlight some of the key alterations that have been studied in human mammary epithelial cells in culture and relate them to events observed in vivo and discussed in accompanying reviews in this volume.     

Regulation of mouse mammary gland development and tumorigenesis by the ERBB signaling network

The four ERBB receptors and their multiple polypeptide ligands are differentially expressed during development of the mouse mammary gland. Profiles suggest that ERBB1/EGF receptor (EGFR)⁴ and ERBB2/Neu are required during ductal morphogenesis, whereas the Neuregulin (NRG) receptors, ERBB3 and ERBB4, are preferentially expressed through alveolar morphogenesis and lactation. Consistent with these profiles, recent gene knockouts established that EGFR and its ligand, Amphiregulin (AR), are essential for ductal morphogenesis in the adolescent mouse and likely provide the required epithelial-stromal signal. In contrast, the phenotypes of transgenic mice expressing dominant negative ERBB2 and ERBB4 proteins suggest that these receptors differentially act to promote or maintain alveolar differentiation. This view of ERBB action provides a conceptual framework for future testing using more sophisticated conditional knockout models. New or existing transgenic mice are also being used to better understand the contributions of ERBB receptors and ligands to mammary tumorigenesis, as well as to more closely mimic the human disease. Recent studies have focused on defining molecular events in neoplastic progression, and in the case of ERBB2/Neu, the requirement for ERBB heterodimerization partners as well as the relative importance of gene amplification versus gene mutation. Collectively, these recent studies establish that normal development and homeostasis of the mammary gland is critically dependent on regulated ERBB signaling. They also illustrate the value of animal models in deciphering roles for the complex ERBB network in this dynamic tissue.     

The mammary gland: A unique organ for the study of development and tumorigenesis

The microanatomy and development of the mammary gland are unique and a reflection of its function to synthesize and deliver milk to the newborn offspring. The uniqueness of the mammary gland resides in several factors. First, the mammary parenchyma undergoes the vast majority of its growth postpubertally, thus enabling experiments on development to be performed in the juvenile or adult and presenting opportunities for experimental manipulation of the gland not available with other organs. On the basis of this characteristic, the fat pad transplantation method was developed, which resulted in the elaboration of important concepts in senescence, immortalization, and preneoplasia. Second, the accessibility of the gland and the ductal organization allows delivery and localization of specific molecules to mammary parenchyma cells, the cells which are the site of origin of neoplastic development. Third, the organ is the target of viral, chemical, and physical carcinogens, allowing development of unique and complex models for neoplastic development. Finally, the complexity of hormone and growth factor regulation of mammary gland function allows a sophisticated approach to the study of hormone action. The purpose of this review is to illustrate some unique properties of the gland which provide the basis for specialized approaches to developmental, neoplastic, and functional problems.     

Evolution of the mammary gland defense system and the ontogeny of the immune system

A decisive event in the evolution of mammals from synapsid reptiles was the modification of ventral thoracic–abdominal epidermal glands to form the mammary gland. The natural selection events that drove the process may have been the provision of certain immunological agents in dermal secretions of those nascent mammals. This is mirrored by similar innate immune factors in mammalian sebum and in protherian and eutherian milks. On the basis of studies of existing mammalian orders, it is evident that immune agents in milk such as immunoglobulins, iron-binding proteins, lysozyme, oligosaccharides, and leukocytes compensate for developmental delays in early postnatal production of antimicrobial factors. At least in human milk, anti-inflammatory and immunomodulating agents also evolved to provide different types of protection for the offspring. In addition, investigations reveal that the types or concentrations of immunological agents in milk vary depending upon the type of placenta, lactation pattern, and environment of the species.     

Opportunistic infections in children following renal transplantation

Opportunistic infections following renal transplantation in children are a major cause of severe morbidity and mortality. These infections account for the majority of early post renal-transplant deaths in children. General risk factors which affect the incidence and severity of these infections include: transmission of the infectious agent by the donor organ; history of immunity in the recipient prior to transplantation; type and amount of immunosuppression including treatment for rejection episodes; availability of specific treatment for the infection. Children are at particular risk because of the lack of exposure to certain pathogens prior to transplantation. There have been recent advances in the prevention and treatment of important infections which occur in children following transplantation, including varicella,Pneumocystis carinii pneumonia (PCP) and cytomegalovirus (CMV) disease. Varicella is treatable with acyclovir, often without decreasing immunosuppression and placing the graft at risk. Prophylaxis against PCP may be achieved by provision of alternate-day trimethoprim sulpha, but clear guidelines for determining who should be treated are lacking. Treatment of this disease with high-dose trimethoprim sulfa or pentamidine is usually successful. CMV disease is frequently severe, especially when the donor is seropositive and the recipient seronegative. In these situations, prophylactic CMV immunoglobulin reduces the morbidity and the mortality of the disease and prophylactic oral acyclovir may decrease its incidence. Treatment of severe CMV disease with gancyclovir is promising.     

Avenues for immunomodulation and graft protection by gene therapy in transplantation

Organ transplantation represents the only definitive therapy for many causes of end-organ failure. However, the universal success of this therapy is limited by chronic allograft rejection, the side effects of chronic immunosuppressive therapy, and a severe shortage of donor organs. Presently, the success of solid-organ transplantation depends on the continuous administration of toxic and nonspecific immunosuppressive agents, therapies that present risks for opportunistic infection, malignancy, and a variety of agent-specific side effects. To promote the use of transplantation with limited risk of long-term sequelae, three dominant research challenges emerge: (i) elimination of the need for exogenous immunosuppression by immunological tolerance induction; (ii) prevention of chronic rejection/graft dysfunction; and (iii) expansion of available organs for transplantation. Gene therapy may provide significant advances and solutions in each of these areas. Rejection of the graft in the immediate post-transplant period has been attacked through the transfer of immunomodulatory molecules in addition to tolerance inducing approaches. Chronic graft rejection may be similarly addressed through permanent tolerance induction or alternatively through the introduction of molecules to resist chronic graft damage. Genetic manipulation of stem cells may ultimately produce transgenic animals to serve as tissue donors to overcome the limited donor organ supply. This review will highlight ongoing developments in the translation of gene therapy approaches to the challenges inherent in transplantation.     

Renal tubule function in beta-thalassemia after hematopoietic stem cell transplantation

Advances in hematopoietic stem cell transplantation (HSCT) for beta-thalassemia major make the long-term outcome of these patients very important. Few data on long-term renal function of thalassemia patients are available. We evaluated the renal function in children after successful allogeneic HSCT for beta-thalassemia. Twenty-nine patients were included; the mean age at HSCT was 4.9 years. Mean follow-up time was 7.6 years. After HSCT, two patients developed acute renal failure and two had graft versus host disease. At last follow up, height standard deviation score (SDS) remained the same, but weight SDS had improved. Mean hemoglobin was 12.5 g/dl, and serum ferritin level was 545 ng/ml. All children had normal estimated glomerular filtration rate (GFR). One patient had hypertension and proteinuria, 10 years after HSCT. When comparing 39 children of the same age with beta-thalassemia of similar disease severity but who had not experienced HSCT, we found that the parameters of renal tubule function were better in patients that had undergone HSCT, as demonstrated by urine protein level (0.36 mg/mg creatinine vs 3.03 mg/mg creatinine, P < 0.001), osmolality (712 mosmol/kg vs 573 mosmol/kg, P = 0.006), N-acetyl-beta-D: -glucosaminidase (17.7 U/g creatinine vs 42.9 U/g creatinine, P = 0.045), and beta 2 microglobulin (0.09 microg/mg creatinine vs 0.13 microg/mg creatinine, P = 0.029). This study showed a low incidence of long-term renal impairment after HSCT and indicated that renal tubule function may be better in beta-thalassemia patients after HSCT.     

小儿活体肝移植伦理审查问题研究及探讨

目的 通过分析小儿活体肝移植伦理审查资料和审查过程,探讨小儿活体肝移植的伦理审查的要点和难点.方法 回顾性分析近5年我院小儿活体肝移植伦理审查材料937例.结果 937例中936例通过,1例未通过.结论 根据《人体器官移植条例》和《卫生部关于规范活体器官移植若干规定》要求,严格履行审查程序,充分评估和权衡手术风险,在挽...     

Indications and results of liver transplantation for<Emphasis Type="Italic"> Echinococcus</Emphasis> alveolar infection: an overview

Background Alveolar echinococcosis (AE) of the liver, caused by the larval stage of the fox tapeworm Echinococcus multilocularis, has the characteristics of a slow-growing liver cancer. It is one of the rare parasitic diseases for which a parasitolytic drug is not yet available, and AE is lethal in the absence of appropriate therapeutic management. Complete surgical resection of the parasite at an early stage of infection provides favourable prospects for cure, but, due to a long clinical latency, many cases are diagnosed at an advanced stage, so that partial liver resection can be performed in only 35% of patients. Benzimidazole (BZM) treatment is given in inoperable cases but these compounds are only parasitostatic, and lifelong therapy is required. During the past 20 years some centres have considered liver transplantation (LT) for the treatment of incurable AE.Methods Our review summarizes the results of this experience based on a series of 47 European patients who received transplants between 1985 and 2002, tries to specify the real place of LT for AE, and underlines the measures that could be undertaken in the future to improve the results.Results Five-year survival was 71%. Five-year survival without recurrence was 58%. Major technical difficulties related either to previous laparotomies or to the loco-regional involvement were observed. The nine early deaths concerned AE patients with a long past-history of symptomatic AE (iterative cholangitis, secondary biliary cirrhosis). Five late deaths were directly related to ongoing AE, located in the brain in three cases, a very rare AE location that was not investigated before LT in these patients.Conclusions In general, the pre-LT screening for distant AE metastases appeared insufficient in this series. Heavy immunosuppressive schemes, absence or delayed re-introduction of BZM after LT have clearly played a role in this unfavourable course. This unique experience indicates that, despite major technical difficulties, LT for incurable AE is feasible and could be discussed in very symptomatic cases. Before LT, interventional radiology should be preferred to repeated laparotomies. Pre-LT and post-LT BZM treatment is mandatory. A careful evaluation of possible distant metastases should be done before the decision for LT is made. After LT, the possibility of an ongoing AE must be permanently kept in mind. This could be reduced by lightening the immunosuppressants, carefully following the specific circulating antibodies, and applying a systematic radiological evaluation, not only to the graft but also to the lungs and the brain.The authors wrote this report in collaboration with a European group of clinicians, which included the following participants. France: Jean-Pierre Crumbach, Sabine Kurz, Liver Transplant Unit, CHU Besançon; Anne Minello, Patrick Hillon, CHU Dijon and Liver Transplant Unit, CHU Besançon; Didier Samuel, Henri Bismuth, Centre Hepato-biliaire, Hopital Paul-Brousse, Paris; Maxime Audet, Philippe Wolf, Daniel Jaeck, Strasbourg; Patrick Boissel, Nancy; Filomena Conti, Yves Chapuis, Hopital Cochin, Paris; Jean Chipponi, Clermont-Ferrand; Michel Pouyet, Hôpital de la Croix Rousse, Lyons; Martine Neau-Cransac, Jean-Yves Lacut, Jacques Carles, Bordeaux; Belgium: Jean Van de Stadt, Yves Carlier, Brussels; Switzerland: Philippe Morel, Gilles Mentha, Petro Majno, Geneva; Germany: Markus Golling, Gerd Otto, Heidelberg; Juergen Klempnauer, Karl Oldhafer, Hanover; Ewert Schutte-Frohlinde, Peter Vorwald, Munich; Olaf Guckelberger, Berlin; Carl Allers, Frankfurt; Johannes Scheele, Jena; Tobias Beckurts, Cologne