Volume 124 ·  Issue 18 ·  2011

"We dreamt of it; they did it!" Reading the paper by WEN et al in this issue of the Chinese Medical Journal,1 we can not help but remember when our colleague Prof.Jean-Philippe Miguet, a hepatologist who was the first at the beginning of the 1970s to review the clinical cases of alveolar echinococcosis (AE)observed in our region of Eastern France and to renew our interest in that disease, used to tell Prof.Michel Gillet,his colleague hepatic surgeon who was the first in 1986 to succeed in transplanting a liver in a patient with AE:2 "You should rather resect the parasitic lesions from the diseased liver of the patient after removing it from the patient's body and re-transplant it!" We were at the beginning of the 1980s; it sounded like a joke, and for the first time, 30 years later, it has become true.In addition to a technical performance, this success in doing ex vivo resection of AE lesions and auto-transplanting the remaining liver is likely to become a demonstration of the best indication of that technique。

Abstract：Background Keratinocyte serum-free medium (K-SFM) is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells (CSCs) using K-SFM.Methods A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer.RPMI-1640 supplemented with 10％ fetal calf serum (FCS) was used as a control. CSCs were identified with flow cytometry using CD44+/CD24-as molecular markers. The expression of a variety of CSC markers (Oct-4, ABCG2, Nanog,N-cadherin, and E-cadherin) was analyzed with real-time PCR.Results Much higher percentage of CSCs was achieved with K-SFM: 17.3％ for MCF-7 cells, 17.4％ for SKBR-3, and 20.0％ for primary breast cancer culture. Less than 1％ CSC was achieved using RPMI-1640 supplemented with 10％ FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4,ABCG2, Nanog and N-cadherin, and lower level of E-cadherin.Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs。

Abstract：The terminology related to the hydatid cyst is sometimes inappropriate and wrong designations are used, based on incorrect concepts. "Hydatid cyst" is the larval form of the tapeworms of the genus Echinococcus granulosus, a parasite found in the small intestine of carnivores. The disease, called cystic echinococcosis, is among the most neglected parasitic diseases despite the development of new drugs and other treatment modalities. Although all of us know the vital cycle of the parasite and the different aspects of the disease, the designations around the parasite, its evolution and some therapeutic procedures are not uniform. It would be useful, for all of us, to use the same nomenclature and it is absolutely necessary that the nomenclature is correct and universally accepted. In this paper I remember the correct terms related to all aspects of the hydatid cyst's nomenclature, including especially the anatomical and surgical terminology, as well as the criteria to define the cyst inactivity and the way to know when the cyst range the inactive stage after therapy。

Abstract：Background For patients with end-stage hepatic alveolar echinococcosis (AE), in vivo resection of the involved parts of the liver is usually very difficult, therefore, allogenic liver transplantation is indicated. However, we hypothesize that for selected patents, ex vivo liver resection for thorough elimination of the involved tissues and liver autotransplantation may offer a chance for clinical cure.Methods We presented a 24-year-old women with a giant hepatic AE lesion who was treated with hepatectomy, ex vivo resection of the involved tissue and hepatic autotransplantation. The patient had moderate jaundice and advanced hepatic AE lesion which involved segments Ⅰ, Ⅳ, Ⅴ, Ⅵ, Ⅶ, Ⅷ and retrohepatic inferior vena cava. The lateral segments (Ⅱ and Ⅲ) of the left liver remained normal with over 1000 ml in its volume. No extrahepatic metastases (such as to the lung or brain) could be found. As the first step of treatment, X-ray guided percutaneous transhepatic cholangiodrainage (PTCD) was performed twice for bile drainage in segment Ⅲ and Ⅱ separately until her serum total bilirubin decreased gradually from 236 to 88 μmol/L. Total liver resection was then performed, followed by extended right hepatic trisegmentectomy and the entire retrohepatic vena cava was surgically removed en bloc while her hemodynamics parameters were stable. Neither veino-veinous bypass nor temporary intracorporeal cavo-caval or porto-caval shunt was used during the 5.7-hour anhepatic phase. The remained AE-free lateral segments of the left liver were re-implanted in situ. The left hepatic vein was directly anastomosed end-to-end to the suprahepatic inferior vena cava due to the lack of the retrohepatic inferior vena cava with AE total infiltration. Because compensatory retroperitoneal porto-caval collateral circulation developed, we enclosed remained infrahepatic inferior vena cava at renal vein level without any haemodynamics problems.Results During a 60-day following-up after operation, the patient had a good recovery except for a mildly elevated serum total bilirubin.Conclusions As a radical approach, ex vivo liver resection and liver autotransplantation in a case has shown a optimal potential for treatment of the end-stage hepatic AE. Strict compliance with its indications, evaluation of vessels of patients pre-operatively, and precise surgical techniques are the keys to improve the prognosis of patients。

Abstract：Background Hepatic alveolar echinococcosis (AE) is a parasitic disease in humans and caused by the Echinococcus multilocularis (Em). Orthotopic liver transplantation (OLT) may be the only effective treatment for end-stage hepatic AE. However, in some AE patients, extrahepatic Em can not be completely eliminated after OLT. We aimed to study whether the immunological changes caused by Em evasion may influence the rejective response.Methods Rat modles of AE were established by injecting the Em suspension into abdomen of Brown Norway (BN) rats.Three months later, in the experimental group, the liver was transplanted from Lewis (LEW) rats to Em-infected BN rats.In the control group, transplantation was from LEW rats to healthy BN rats. Liver tissue and peripheral blood (PB)samples were collected on days 1, 3, 5, and 7 after OLT. Liver tissue was analyzed after hematoxylin and eosin (H&E)staining; numbers of CD4, CD8, and CD28 on peripheral blood cells were detected by flow cytometry; and expression of the chemokine fractalkine (Fkn) was detected by reverse transcription PCR (RT-PCR). Interleukin-10 (IL-10) was measured in the serum by enzyme-linked immunosorbent assay (ELISA). In every group, eight BN rats were retained for observing survival time.Results The survival times of recipients in the experimental group were prolonged compared with those in the control group. The rejective response occurred later and was milder in the experimental group, percentage of CD4, CD8, CD28 T-cells and Fkn mRNA expression were lower in the experimental group. While the serum IL-10 levels were higher in the experimental group than those in the control group.Conclusions Acute rejective response after OLT was attenuated in the rats with Em infection, and the recipients survival time was prolonged. Em may play a role in this process by elevating IL-10 secretion, decreasing the effector T cells, inhibiting the expression of Fkn, which lead to reduce the inflammatory cells infiltration into the liver。

Abstract：Background Although the computer tomography (Cr) or magnetic resonance imaging (MRI) findings of alveolar echinococcosis (AE) have been well documented, the consecutive imaging changes of this disease in each PNM stage (parasite lesion, neighboring organ invasion, metastases) were not described accurately. The aim of this study was to analyze the correlation between imaging type and PNM stage and diameter of AE lesions, and to explore the development features of this disease.Methods A total of 87 patients with AE were examined using CT and MRI before medical management. Imaging features including the maximum diameter, calcification pattern, and imaging type of lesion were retrospectively assessed.The correlation of imaging type with PNM stage, diameter and calcification pattern was analyzed.Results Lesions (n=111) in 87 patients were divided into three types based on imaging characteristics; solid type (33.3％, 37/111, a solid lesion without liquid necrosis or only small patches of necrosis), mixed type (41.4％, 46/111, solid component surrounding large and/or irregular liquid necrosis area), and pseudo-cystic type (25.2％, 28/111, large cyst without visible solid component). Lesion calcification in the alveolar echinococcosis was categorized into three patterns;mild calcification (45.1％, 50/111, i.e. inconspicuous calcification or punctuate scattered calcification), moderate calcification (46.8％, 52/111, coastline calcification located at the periphery of the lesion, with or without the central dot-calcification) and abundant calcification (8.1％, 9/111, large calcified deposits). Significant differences were found between pseudo-cystic type and other two types in PNM stage, maximum diameter and calcification (P ＜0.05), but there was no significant difference between solid type and mixed type in those mentioned aspects (P ＞0.05). No correlation was observed between calcification patterns and maximum diameter (P ＞0.05).Conclusions Solid and mixed type lesions showed some similarities during the course of the disease and accounted for the major form of advanced AE. Pseudo-cystic type represented neither earlier nor advanced stage of AE, but a special presentation during AE development。

Abstract：Background Cystic echinococcosis (CE) is a zoonosis caused by the cestodes of the Echinococcus species. Its life cycle involves dogs and other canids as definitive hosts for the intestinal tapeworm, as well as domestic and wild ungulates as intermediate hosts for the tissue-invading metacestode (larval) stage. The disease has a special impact on disadvantaged pastoralist communities and is listed now among the three top priority neglected tropical disease (NTD).Therefore, CE is a neglected disease even in high endemicity regions. This study aimed at investigation of the prevalence of CE in different animals slaughtered for food consumption in Sinnar area, Blue Nile states in Sudan.Methods A survey of CE in livestock was conducted from April 2009 to March 2011 in Sinnar area, Blue Nile state in Sudan. Location, parasitological status and fertility conditions were determined. In addition, 120 hydatid cysts (30 from camels, 62 from cattle and 28 from sheep) were examined by polymerase chain reaction (PCR) and mitochondrial gene sequencing for the genetic allocation of Echinococcus strains or species Results The prevalence of CE was 29.7％ (30/101) in camels, 2.7％ (62/2310) in cattle and 0.6％ (26/4378) in sheep. It was shown that infection rates increased with age in camels, cattle and sheep. In camels, 67％ (20/30) of the infected animals were aged between 2-5 years whereas 58％ (36/62) of the infected cattle were ＞5 years. In sheep, the prevalence rate was distributed equally between animals ranging 2-5 years and ＞5 years. Even though multiple cysts were found in some animals, the average number of cysts per animal was close to 1 in all examined species. Lungs were found to be the predilection sites for the parasite in both camels and cattle, while most of the cysts found in sheep were located in the liver. About 63.4％ of cysts encountered in camels were considered as large (5-7 cm), whereas those in cattle and sheep were medium (2-4 cm) and small (＜2 cm) respectively. The highest fertility rate was found in camel cysts with 85.4％ (35/41) followed by cattle (50.0％, 32/64) and sheep (39.0％, 11/28). All examined cysts belonged to Echinococcus canadensis G6, which was confirmed to be the overwhelmingly predominant species in that area.Conclusion The epidemiological situation in Sinnar area, Blue Nile state is characterized by intense transmission of Echinococcus canadensis G6, thereby closely resembling the situation in most other regions of Sudan。

Abstract：Background The rodentia and lagomorpha animals are the intermediate hosts of Echinococcus multilocularis, their distribution and infection of this parasite may facilitate the infection of definitive hosts such as dogs. The aim of this study was to determine the distribution of the intermediate hosts of Echinococcus multilocularis in Shiqu County, Sichuan, China.Methods A systematic sampling method was used to investigate the density of burrows of rodents and lagomorphs at 97 pasture sites in winter and summer pastureland and remote sensing (RS) technology was used to correlate their densities to the distribution of these animals in different landscape types.Results Based on the densities of Ochotona curzoniae, Microtus fuscus (dependent variable) and their burrow densities (independent variable) in survey points, regression equations were fitted respectively (Ochotona curzoniae, P＜0.0001, R2=0.8705; Microtus fuscus, P ＜0.0001, R2=0.9736). Their burrow density in summer pastureland was higher than in winter pastureland (F=36.65, P ＜0.0001). The burrow densities of Ochotona curzoniae and Microtus fuscus in bareland and half-bareland are higher than in grassland (F=7.73, P ＜0.001).Conclusions The regression relationship between the densities of Ochotona curzoniae and Microtus fuscus and their burrow densities indicate that the burrow densities could reflect the animal densities and that the burrow density was greater in summer pastureland than in winter pastureland. The main distribution areas of the intermediate hosts were in bareland and half-bareland。

Abstract：Background Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs ofthe host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage.Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK)mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.Methods DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.Results We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.Conclusions We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and protoscolex. These findings will be useful in further study of the biological functions of the gene in the growth and development of Echinococcus and will contribute to research on novel anti-echinococcosis drug targets。

Abstract：Background Alveolar echinococcosis (AE) is a disease in human and animals, and the cure rate is unsatisfactory. This study aimed to investigate the curative efficacy of different doses of locally applied radiotherapy on alveolar echinococcosis in rats.Methods Rats infected with Echinococcus multilocularis were randomly divided into 4 groups of 15 rats each: low-,middle-, and high-irradiation groups and a control group. Rats in the control group underwent no treatment, while rats in the irradiation groups received 6-MeV radiotherapy at 20 Gy/8 f, 40 Gy/8 f, and 60 Gy/8 f respectively, once every 3 days for a total of 8 times. One month after radiotherapy, wet weight and AE vesicle inhibitory rate were detected in rats of each group. Histopathologic and ultrastructural observations of tissues with AE lesions were performed.Results In the treatment groups, an obvious inhibitory effect was found in AE rats; the inhibitory rates were 50％, 72％,and 82％, respectively. There were also statistical differences in pathological changes and average wet weight of the lesions compared with the control group (P ＜0.05). In the treatment groups, injuries of various degrees were found in the ultrastructure of the laminated and germinal layers in the capsular wall of AE, and injury was most severe in the high-dose group.Conclusion Radiotherapy has a dose-dependent inhibitory effect on the growth of AE。

Abstract：Background Echinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China.Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.Methods The genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.)granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program,and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.Results Fifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.Conclusion We found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis。

Abstract：Background Most hydatid cysts with calcified walls are biologically and clinically silent and inactive. Transforming growth factor-beta 1 (TGF-β1) plays a critical role in the calcification process of cells. The aim of this study was to assess the effect of modulating TGF-β1 signaling on the calcification of hydatid cysts.Methods Pericyst cells isolated from hepatic hydatid cysts were cultured with osteogenic media. These cells were assessed for alkaline phosphatase activity and mineralization capacity using Alizarin Red staining. Cells were also treated with recombinant human TGF-β1 and TGF-β inhibitor, and the expression profiles of osteoblast markers (RUNX2,osterix, and osteocalcin) were analyzed using Western blotting. The effects of inhibiting TGF-β1 signaling on calcification of pericyst walls were assessed using different doses of TGF-β inhibitor for 7 weeks in a preclinical disease model of liver cystic echinococcosis.Results Cells within the pericyst displayed high levels of alkaline phosphatase activity and mineralized nodule formation, as induced by osteogenic media. These activities, as well as expression profiles of osteoblast markers (RUNX2, osterix, and osteocalcin) could be inhibited by addition of recombinant human TGF-β1 (rhTGF-β1) and enhanced by TGF-β inhibitor. In the animal model of cystic echinococcosis, inhibition of TGF-β1 signaling increased calcification of the pericyst wall, which was associated with decreased cyst load index and lower viability of protoscoleces.Conclusions Cells within the pericysts adopt an osteoblast-like phenotype and have osteogenic potential. Inhibition of TGF-β1 signaling increases hydatid cyst calcification. Pharmacological modulation of calcification in pericysts may be a new therapeutic target in the treatment of hydatid disease。

Abstract：Background A previous study has shown that rs548234 polymorphism at PRDM1-ATG5 region is associated with rheumatoid arthritis (RA) in Caucasian populations. The aim of this study was to investigate the effect of rs548234 polymorphism at PRDM1-ATG5 region on susceptibility to RA in Chinese Han population.Methods We genotyped 848 RA patients and 1431 matched healthy controls for rs548234 single-nucleotide polymorphism (SNP) with a predesigned TaqMan SNP genotyping assay. Association analyses were performed on the whole data set and on rheumatoid factors (RF) and anti-cyclic citrullinated peptides (anti-CCP) antibody. Finally, we carried out combined analysis of rs548234 association with RA based on the published data.Results No significant difference in the genotype distribution between RA patients and healthy controls for rs548234 (C/T) polymorphism was found in Chinese Han population, neither in whole data set nor in stratified subsets, e.g. RF and anti-CCP status. Association analysis in different ethnic groups showed that rs548234 at PRDM1-ATG5 region was associated with RA in Caucasian ancestry but not in East Asian population.Conclusions Our results showed no involvement of rs548234 at PRDM1-ATG5 region in the susceptibility or clinical relevance of RA in Chinese Han population。

Abstract：Since the first 2 cases observed in southern Germany and the correct identification of a parasite at the origin of the disease by the famous scientist Rudolf Virchow in 1855, the borders of the endemic area of alveolar echinococcosis (AE)have never stopped to expand. The parasite was successively recognized in Switzerland, then in Russia, Austria and France which were long considered as the only endemic areas for the disease. Cases were disclosed in Turkey in 1939;then much attention was paid to Alaska and to Hokkaido, in Japan. The situation totally changed in 1991 after the recognition of the Chinese endemic areas by the international community of scientists. The world map was completed in the beginning of the 21st century by the identification of AE in most of the countries of central/eastern Europe and Baltic States, and by the recognition of cases in central Asia. Up to now, the disease has however never been reported in the South hemisphere and in the United Kingdom. In the mid-1950s, demonstration by Rausch and Schiller in Alaska, and by Vogel in Germany, of the distinction between 2 parasite species responsible respectively for cystic echinococcosis ("hydatid disease") and AE put an end to the long-lasting debate between the "dualists", who believed in that theory which eventually proved to be true, and the "unicists", who believed in a single species responsible for both diseases. At the end of the 20th century, molecular biology fully confirmed the "dualist" theory while adding several new species to the initially described E. granulosus; within the past decade, it also confirmed that little variation existed within Echinococcus (E.)multilocularis species, and that AE-Iooking infection in some intermediate animal hosts on the Tibetan plateau was indeed due to a new species, distinct from E. multilocularis, named E. shiquicus. Since the 1970s, the unique ecological interactions between the landscape, the hosts, and E. multilocu/aris have progressively been delineated. The important role of the rodent/lagomorph reservoir size for the maintenance of the parasite cycle has been recognized within the last 2 decades of the 20th century. And the discovery of a close relationship between high densities of small mammals and particularities in land use by agriculture/forestry has stressed the responsibility of political/economic decisions on the contamination pressure. Urbanization of foxes in Europe and Japan and the major role of dogs in China represent the new deals at the beginning of the 21 st century regarding definitive hosts and prevention measures。

Abstract：Background Echinococcosis is still endemic in many countries, including China, where it is especially prevalent in the northwest. The aim of this study was to enrich the international literature about the treatment of intracranial hydatid cysts.Methods We retrospectively reviewed the clinical features, radiological manifestations, and surgical outcome of 97 patients with intracranial hydatid cysts, who received surgical treatment at the Neurosurgical Department of First Affiliated Hospital of Xinjiang Medical University from 1985 to 2010 and followed up the patient via sending a questionnaire or telephone contact. Clinical outcome was evaluated using the Karnofsky Performance Scale Index.Results Headache and vomiting were the most common initial symptoms in our patients. Neurological deficits caused by the mass effect of the cysts were seen in 82 cases. On the X-ray, significant bone erosion was seen in only two cases with epidural hydatid cysts. Round-shaped and thin-walled homogeneous low-density cystic lesions without surrounding edema and enhancement were the main findings on computerized tomography (CT) in 95 patients with intraparenchymal hydatid cysts, while two cases with epidural hydatid cysts presented as a heterodensity lesions. On magnetic resonance imaging (MRI), hydatid cyst presented as a round-shaped low signal lesion in T1-weighted images and high signal lesion in T2-weighted images, without enhancement after contrast media injection, while the two cases with epidural cysts presented as mixed signal masses. Surgical removal of cyst was performed in all cases. Total removal was achieved in 93 cases without rupturing the cyst wall. Only two cysts ruptured during the dissection, resulting in two surgery-related mortalities. There was no other additional neurological deficit caused directly by surgery. In 97.2％ of the patients, the Karnofsky Performance Scale score was 80 to 90 at the last follow-up.Conclusions Intracranial hydatid cyst is still a main cause of increased intracranial pressure among the patients in endemic areas for echinococcosis. CT and MRI are the best diagnostic methods and surgery is the treatment of choice for intracranial hydatid cysts。

QIAN Yi-jian, M.D., chief editor emeritus of the Chinese Medical Journal, passed away on July 6, 2011.Yi-jian was my closest classmate from St.John's University School of Medicine, Shanghai (Figure 1).Over the years,Yi-jian was always close by my side in all the important meetings in China (Figure 2) and the United States(Figure 3)。

Dr.QIAN Yi-jian, the noted cardiologist and geriatrician in China died of liver cancer at the Beijing Hospital on July 6,2011, where he had served for 52 years.He was 86.We grieved at his death not only for his life-long service to patients but also for his deep involvement in editing and publication of medical journals of the Chinese Medical Association (CMA)。