Global genetic diversity of Spirometra tapeworms

@#Spirometra larvae are etiological agents of human sparganosis. However, the systematics of spirometrid cestodes has long been controversial. In order to determine the current knowledge on the evolution and genetic structure of Spirometra, an exhaustive population diversity analysis of spirometrid cestodes using the mitochondrial gene: cytochrome c oxidase subunit 1 (cox1) was performed. All publicly available cox1 sequences available in the GenBank and 127 new sequencing genes from China were used as the dataset. The haplotype identify, network, genetic differentiation and phylogenetic analysis were conducted successively. A total of 488 sequences from 20 host species, representing four spirometrid tapeworms (S. decipiens, S. ranarum, S. erinaceieuropaei and Sparganum proliferum) and several unclassified American and African isolates from 113 geographical locations in 17 countries, identified 45 haplotypes. The genetic analysis revealed that there are four clades of spirometrid cestodes: Clade 1 (Brazil + USA) and Clade 2 (Argentina + Venezuela) included isolates from America, Clade 3 contained African isolates and one Korean sample, and the remainders from Asia and Australia belonged to Clade 4; unclassified Spirometra from America and Africa should be considered the separate species within the genus; and the taxonomy of two Korea isolates (S. erinaceieuropaei KJ599680 and S. decipiens KJ599679) was still ambiguous and needs to be further identified. In addition, the demographical analyses supported population expansion for the total spirometrid population. In summary, four lineages were found in the spirometrid tapeworm, and further investigation with deeper sampling is needed to elucidate the population structure.

Effects of vacuum sealing drainage on the treatment of cranial bone-exposed wounds in rabbits

This study was designed to assess the efficacy of vacuum sealing drainage (VSD) on skull exposure wounds in rabbits and to investigate the underlying mechanism of the process. Full-thickness excisional circular wounds 2×2 cm with or without periosteum involvement were created in 88 New Zealand white rabbits (mean body weight: 3.0±0.65 kg). Animals were randomly divided into 4 groups: periosteum-intact wounds treated with traditional dressing (p+control), periosteum-intact wounds treated with VSD (p+VSD), periosteum-lacking wounds treated with traditional dressing (p-control) and periosteum-lacking wounds treated with VSD (p-VSD). The wounds treated with traditional dressing were covered with Vaseline gauze, while VSD treatment was accompanied with continuous -120 mmHg pressure. Finally, wound tissues were harvested for analysis of hydroxyproline content and histologic detection. VSD hastened the wound healing process significantly (P<0.05) compared to the corresponding control groups. VSD alleviated the inflammation reaction, accelerated re-epithelialization and facilitated the organization of collagen fibers into neat rows. During the wound healing process, the hydroxyproline content increased overtime [i.e., postoperative days (POD) 7, POD 10 and POD 15] in all four groups, and it peaked in the p+VSD group. VSD also promoted angiogenesis via increasing number and quality of collagen. We concluded that VSD can promote healing in bone-exposed wounds via increasing hydroxyproline content and vessel density, reducing inflammatory responses and generating ordered collagen arrangement.

Clinical and prognostic significance of serum transforming growth factor-beta1 levels in patients with pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate of 5%. Biomarkers for the early detection of pancreatic cancer are urgently needed. Transforming growth factor-beta1 (TGF-β1) is elevated in the tissues and plasma of patients with PDAC. However, no studies systemically report prognostic significance of plasma TGF-β1 levels in PDAC. In the present study, we assessed the prognostic significance of serum TGF-β levels in patients with PDAC. TGF-β levels were determined in serum from 146 PDAC patients, and 58 patients with benign pancreatic conditions. Regression models were used to correlate TGF-β levels to gender, age, stage, class, and metastasis. Survival analyses were performed using multivariate Cox models. Serum levels of TGF-β1 distinguished PDAC from benign pancreatic conditions (P<0.001) and healthy control subjects (P<0.001). Serum levels of TGF-β also distinguished tumor stage (P=0.002) and lymph node metastasis (P=0.001). High serum levels of TGF-β1 were significantly correlated with reduced patient survival. Multivariate analysis revealed that TGF-β1, lymph node metastasis and tumor stage were independent factors for PDAC survival. Our results indicate that serum TGF-β1 may be used as a potential prognostic marker for PDAC.

Plasma levels and placental expression of vaspin in pregnant women with diabetes mellitus

The present study aimed to investigate visceral adipose tissue-specific serpin (vaspin) concentrations in serum and term placentas and relate these values to insulin resistance and lipid parameters in women with gestational diabetes mellitus (GDM). A total of 30 GDM subjects and 27 age-matched pregnant women with normal glucose tolerance (NGT, control) were included. Serum glucose, glycated hemoglobin (HbA1c), lipid profile, insulin, and vaspin were measured at the end of pregnancy, and homeostasis model of assessment-insulin resistance (HOMA-IR) values were calculated. Vaspin mRNA and protein levels in placentas were measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Serum vaspin levels were significantly lower in the GDM group than in controls (0.49±0.24 vs 0.83±0.27 ng/mL, respectively; P<0.01). Three days after delivery, serum vaspin levels were significantly decreased in subjects with GDM (0.36±0.13 vs 0.49±0.24 ng/mL, P<0.01). However, in the GDM group, serum vaspin levels were not correlated with the parameters evaluated. In contrast, in the control group, serum vaspin levels were positively correlated with triglycerides (TG; r=0.45, P=0.02) and very low-density lipoprotein cholesterol (VLDL-C; r=0.42, P=0.03). Placental mRNA vaspin (0.60±0.32 vs 0.68±0.32, P=0.46) and protein (0.30±0.08 vs 0.39±0.26; P=0.33) levels in the GDM group did not differ significantly from those in the control group, but were negatively correlated with neonatal birth weight in the GDM group (r=-0.48, P=0.03; r=-0.88; P<0.01). Our findings indicated that vaspin may be an important adipokine involved in carbohydrate and lipid metabolism and may also play a role in fetal development.

Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.

Rat Dlx5 is expressed in the subventricular zone and promotes neuronal differentiation

The molecular mechanisms and potential clinical applications of neural precursor cells have recently been the subject of intensive study. Dlx5, a homeobox transcription factor related to the distal-less gene in Drosophila, was shown to play an important role during forebrain development. The subventricular zone (SVZ) in the adult brain harbors the largest abundance of neural precursors. The anterior SVZ (SVZa) contains the most representative neural precursors in the SVZ. Further research is necessary to elucidate how Dlx5-related genes regulate the differentiation of SVZa neural precursors. Here, we employed immunohistochemistry and molecular biology techniques to study the expression of Dlx5 and related homeobox genes Er81 and Islet1 in neonatal rat brain and in in vitro cultured SVZa neural precursors. Our results show that Dlx5 and Er81 are also highly expressed in the SVZa, rostral migratory stream, and olfactory bulb. Islet1 is only expressed in the striatum. In cultured SVZa neural precursors, Dlx5 mRNA expression gradually decreased with subsequent cell passages and was completely lost by passage four. We also transfected a Dlx5 recombinant plasmid and found that Dlx5 overexpression promoted neuronal differentiation of in vitro cultured SVZa neural precursors. Taken together, our data suggest that Dlx5 plays an important role during neuronal differentiation.

Field assessment of recombinant Schistosoma japonicum 26 kDa glutathione S-transferase in Chinese water buffaloes.

We have shown previously that anti-fecundity immunity can be induced experimentally against recombinant 26 kDa glutathione S-transferase (reSjc26GST) in Chinese water buffaloes (Bos buffelus), important reservoir hosts for Schistosoma japonicum in China. In the field study described here, we immunized buffaloes with reSjc26GST to induce protective immunity against S. japonicum and to evaluate its effectiveness in controlling schistosomiasis japonica. We selected two villages as test and control groups in inside-embankment areas endemic for schistosomiasis japonica. The buffaloes in the test village were vaccinated with reSjc26GST, whereas those in the control village were not. The indicators of the effect of the vaccine included the generation of specific IgG antibodies in the vaccinated buffaloes, changes in the prevalence and infection intensity in buffaloes and village children, changes in the density of infected snails, and changes in the infectivity of water bodies (assessed by sentinel mice) in transmission areas adjacent to both villages. Twenty months after vaccination, the infection rate of buffaloes in the test village was decreased by 60.4% (from an initial prevalence of 13.5% to 5.4%), and 67.9% when compared with that in the control village (initial prevalence of 16.7%). However, the infection rate in village children remained unchanged. The density of infected snails decreased by 71.4%, from 0.0049/0.11 m2 to 0.0014/0.11m2 in the high transmission area outside the embankment in the test village. There was no change in the infectivity of the water body transmission areas between the test and control villages. The levels of specific antibodies to reSjc26GST showed a continuous increase after vaccination. These results indicate that protective immunity was induced and maintained in buffaloes after vaccination with reSjc26GST. The vaccine could thus play a significant role in reducing S. japonicum transmission caused by water buffaloes in the Lake region of China.

Analysis of the 1991-2000 influenza epidemic in Guangdong Province, China.

Influenza surveillance networks in Guangdong were established to investigate the epidemiological characteristics of influenza and influenza epidemics. Influenza activity peaked annually from March to July in Guangdong in 1991-2000; influenza H3N2 predominated in the epidemic (7 years of 10); the outbreak of influenza in 1996 was the remarkable result of antigenic drift of H3N2 strain. Ten isolates of H9N2 strains were discovered from human subjects in 1998 and 1999: chicken strains isolated after the Hong Kong fowl influenza outbreak. It was found that there was just one influenza activity season per annum in Guangdong and that the influenza H3N2 subtype still predominates in Guangdong. Further research into the pathogenicity of influenza H9N2 in humans warranted.

Hydroxychloroquine sulphate inhibits in vitro apoptosis of circulating lymphocytes in patients with systemic lupus erythematosus.

The serological hallmark of systemic lupus erythematosus (SLE) is the presence of antibodies against double-stranded DNA. However, several studies have suggested that it is not DNA itself, but nucleosomes that are the immunogenic particles involved both in the induction of anti-DNA antibodies, and in the pathophysiology of SLE. Meanwhile, It has been demonstrated that there is an accelerated in vitro apoptosis of lymphocytes from patients with SLE. Therefore, one can postulate that the process of apoptosis may provide a source of nuclear antigens to drive the autoantibody response seen in SLE. Our study has demonstrated that hydroxychloroquine exhibits an anti-apoptotic action and this anti-apoptotic effect is dependent on monocyte coexistence. We used both morphology assessment and fluorescent antibody cell sorter (FACS) analysis to measure the apoptotic percentage of lymphocytes from 25 SLE patients in medium alone (control) or with the addition of different concentrations of hydroxychloroquine. Our results have shown that there is a significant decrease in the percentage of apoptosis at the therapeutic concentration (10(-6) M) as compared with the control (p < 0.05). It has been reported that the anti-rheumatic properties of hydroxychloroquine result from its interference with antigen processing in macrophages and other antigen-presenting cells. We propose that this results in decreased stimulation of autoreactive lymphocytes reactive with self-peptides, and consequently diminution of activation-induced cell death (apoptosis) of mature peripheral lymphocytes.

The effect of allergen immunotherapy on in vitro IL-4 and IFN-gamma production by peripheral mononuclear cells in house dust-sensitive Chinese patients with bronchial asthma.

The IFN-gamma produced by Th1 cells and IL-4 produced by Th2 cells are two most important cytokines in the regulation of IgE production. House dust immunotherapy has been tried in the treatment of house dust-sensitive Chinese asthmatic patients with good results. We examined the influence of such treatment on in vitro IL-4 and IFN-gamma production by peripheral blood mononuclear cells in house dust-sensitive asthmatic patients. Allergen immunotherapy in house-dust sensitive asthmatic patients can significantly decrease IL-4 production from peripheral mononuclear cells (p<0.05). The production levels of IL-4 in patients without treatment had higher levels than those in patients with hyposensitization (p<0.01). Such therapy also have some effect on promotion of IFN-gamma production in asthmatic patients. In conclusion, immunotherapy with house dust may have the potential ability to shift the Th1/Th2 balance of immune response to allergens and to create a favorable cytokine microenvironment to suppress the allergic reaction in the asthmatic airway.

The possibility of GST antigen from Chinese strain of Schistosoma japonicum for immunodiagnosis of schistosomiasis.

The GST antigen (called 26-28 kDa antigen) extracted and purified from Schistosoma japonicum adult worms was applied to the detection of specific antibodies in sera of infected mice and mice immunized with the above protein antigen by ELISA technique. The 26-28 kDa antigen was better than crude antigens (SEA, SWAP) when used to detect specific antibodies in sera from immunized mice. As with crude antigens (SEA and SWAP), the 26-28 kDa antigen could be used to detect specific antibodies in infected sera, with titers as high as 1:160-1:320. There were no false positive reactions and a positivity rate as high as that using SWAP occurred when the 26-28 kDa antigen was used in schistosomiasis patients and normal subjects by intradermal test. It is suggested that the 26-28 kDa antigen may be a suitable candidate for immunodiagnosis of schistosomiasis.

The temperature--dependent expression of GST of Schistosoma japonicum (Philippine strain).

Obtained from pSj5, the cDNA gene encoding GST antigen of Schistosoma japonicum (Philippine strain) was ligated with efficient temperature-dependent PBV220 vector which was constructed in CAPM, and then introduced into host bacterium-DH5 alpha (E. coli) by transformation. Transformants were selected by ampicillin and recombinant clones were identified by restriction mapping. The result showed that recombinant clone 43 was the one carrying recombinant plasmid PBV 220 with the correct insertion of the gene fragment. The GST expression ability of clone 43 was investigated by GST enzymic activity assay and SDS-PAGE. A relatively high level of GST enzymic activity was expressed by this clone under the temperature-dependent condition, that is, cultured at 30 degrees C and expressed at 42 degrees C. A more strongly stained 26 kDa protein band was identified by SDS-PAGE. The result indicated that GST of S. japonicum (Philippine strain) could be expressed not only by IPTG induction, but also by the temperature-dependent method.

The antigenicity of GST antigen extracted from Chinese strain of Schistosoma japonicum.

The GST antigen, similar to Sj26 (Philippine strain), which plays an important role in inducing protective immunity against Schistosoma japonicum, can be extracted and purified from adult worms of the Chinese strain of S. japonicum. There are two bands at 26 kDa and 28 kDa of GST antigen called the 26-28 kDa GST antigen as identified by SDS-PAGE, and these have GST activities. Mice were immunized with the 26-28 kDa antigen and the specific antibody response in serum was assayed by ELISA, IFA and western blot. The antigenicity of the 26-28 kDa GST antigen in mice was significant. For example, the antigen could stimulate mice to increase the level of serum IgM and IgGl; the antibodies in serum of immunized mice could be localized in the antigenic determinants of tegument or body of the worms; specific antibodies against the antigens increased markedly after immunization as measured by ELISA or IFA; the antibody from mice immunized with the 26-28 kDa GST antigen can recognize 26-28 kDa antigenic molecules, identified by immunoblot assay.

Comparative study on antigenicity and immunogenicity of 26-28 kDa antigen and recombinant Sj26 (RSj26) of Schistosoma japonicum.

This paper reports a comparison of the recombinant Sj26 (rSj26) antigen derived from the Philippine strain and the 26-28 kDa antigen isolated and purified from the Chinese strain of Schistosoma japonicum with respect to their antigenicity and immunogenicity. The results showed that there were obvious cross reactions between rSj26 and 26-28 kDa antigen when rSj26 antigen was tested against specific antibodies in sera of mice infected with the Chinese strain of S. japonicum or the 26-28 kDa antigen was tested against specific anti-rSj26 antibodies by ELISA, IFA and Western blotting. Both the 26-28 kDa and the rSj26 antigen had weak cross reactions with SEA antigen. The worm reduction rate after challenging with Chinese strain cercariae in mice immunized with rSj26 was 26-32%, similar to that in mice immunized with 26-28 kDa antigen. It is suggested that rSj26 antigen can induce a certain level of specific protective immunity in the host against infection by the Chinese strain of S. japonicum cercariae.

Acetylcholine receptor antibody in patients with myasthenia gravis.

The determination of acetylcholine receptor antibody (AChR Ab) titer by an enzyme-linked immunosorbent assay (ELISA) in patients with myasthenia gravis was introduced. The optimal conditions were determined by chequerboard determination. The specificity was confirmed by inhibition tests. The sensitivity is 9 p mole. The comparison of AChR Ab titers among 49 myasthenic patients, 19 non-myasthenic neurological patients and 20 healthy blood donors has shown that it is a highly sensitive, specific, reproducible, rapid, simple and inexpensive method for determining AChR Ab and that it is highly valuable for the diagnosis of myasthenia gravis.