Immunotherapy for type 1 diabetes mellitus by adjuvant-free Schistosoma japonicum-egg tip-loaded asymmetric microneedle patch (STAMP).

BACKGROUND: Type 1 diabetes mellitus (T1DM) is an autoimmune disease mediated by autoreactive T cells and dominated by Th1 response polarization. Insulin replacement therapy faces great challenges to this autoimmune disease, requiring highly frequent daily administration. Intriguingly, the progression of T1DM has proven to be prevented or attenuated by helminth infection or worm antigens for a relatively long term. However, the inevitable problems of low safety and poor compliance arise from infection with live worms or direct injection of antigens. Microneedles would be a promising candidate for local delivery of intact antigens, thus providing an opportunity for the clinical immunotherapy of parasitic products. METHODS: We developed a Schistosoma japonicum-egg tip-loaded asymmetric microneedle patch (STAMP) system, which serves as a new strategy to combat TIDM. In order to improve retention time and reduce contamination risk, a specific imperfection was introduced on the STAMP (asymmetric structure), which allows the tip to quickly separate from the base layer, improving reaction time and patient's comfort. After loading Schistosoma japonicum-egg as the immune regulator, the effects of STAMP on blood glucose control and pancreatic pathological progression improvement were evaluated in vivo. Meanwhile, the immunoregulatory mechanism and biosafety of STAMP were confirmed by histopathology, qRT-PCR, ELISA and Flow cytometric analysis. RESULTS: Here, the newly developed STAMP was able to significantly reduce blood glucose and attenuate the pancreatic injury in T1DM mice independent of the adjuvants. The isolated Schistosoma japonicum-eggs micron slowly degraded in the skin and continuously released egg antigen for at least 2 weeks, ensuring localization and safety of antigen stimulation. This phenomenon should be attributed to the shift of Th2 immune response to reduce Th1 polarization. CONCLUSION: Our results exhibited that STAMP could significantly regulate the blood glucose level and attenuate pancreatic pathological injury in T1DM mice by balancing the Th1/Th2 immune responses, which is independent of adjuvants. This technology opens a new window for the application of parasite products in clinical immunotherapy.

Genetic Identification of Spirometra erinaceieuropaei Spargana in Liaoning and Hubei Provinces, PR China.

Spargana were collected from human and frogs in Liaoning and Hubei Provinces, China. PCR amplification and direct sequencing of A cox1 fragment was PCR-amplified from genomic DNA extracted from 7 specimens (5 from humans and 2 from frogs). The cox1 fragment (390 bp) showed 97-100% similarity to the reference sequence of S. erinaceieuropaei and 88-89% to the reference sequence of S. decipiens. There were 1-12 bases different between these worms, but no obvious genetic variation (0-3.3%) to the references. There was little difference of cox1 gene between sparganum samples of humans and frogs (1-3%). This study is the first report on S. erinaceieuropaei spargana from humans in Liaoning and Hubei Provinces.

NLRP3 inflammasome activation from Kupffer cells is involved in liver fibrosis of Schistosoma japonicum-infected mice via NF-κB.

BACKGROUND: NOD-like receptor protein 3 (NLRP3) inflammasome was reported as expressed in schistosomiasis-induced liver fibrosis (SSLF). We used an NLRP3 inflammasome inhibitor, MCC950, to investigate whether it inhibited liver fibrosis, and explored the preliminary molecular mechanism. METHODS: BALB/c mice were infected with 15 cercariae through the abdominal skin. They received intraperitoneal injections of MCC950 on the day of infection and at day 22 post-infection. We examined their SSLF phenotype and the effect on liver fibrosis, primary Kupffer cells (KCs), and HSCs. Human hepatic stellate cell lines (human LX-2 cells) were treated with soluble egg antigen (SEA) released from the eggs. We then determined the expression of NLRP3 inflammasome and liver fibrosis-associated markers, liver granuloma and ALT/AST. RESULTS: NLRP3 inflammasome expression in the liver was significantly increased, and eosinophilic granuloma and collagen deposition were found around the eggs in mice infected for 56 days. Additionally, IL-1ß, ALT/AST in plasma, and NF-κB in liver tissue and in KCs were all greatly significantly increased. The above-mentioned indicators were largely reduced in mice treated with MCC950 on the day of infection. In vitro, lipopolysaccharide (LPS)/SEA could induce LX-2 cells to express NLRP3 and fibrosis markers, and the SEA-treated group was reversed by MCC950. Furthermore, NLRP3 inflammasome and liver fibrosis-associated markers were both increased in the primary KCs and HSCs isolated from infected mice. However, this effect was not observed in the same cells from the mice treated with MCC950 on the day of infection. Contrary to the aforementioned results, MCC950 treatment at day 22 post-infection aggravated this process. Surprisingly, NLRP3 inflammasome was involved in liver fibrosis mostly from KCs. CONCLUSIONS: MCC950 acts dually on SSLF pathology and fibrosis in infected mice. Although MCC950 treatment improved SSLF on the day of infection, it exacerbated the pathological effects at day 22 post-infection. These dual effects were mediated via NF-κB. Moreover, NLRP3 inflammasome mainly came from KCs. Our results suggest that blocking NLRP3 on the day of infection may prove to be a promising direction in preventing SSLF.

Plasmodium yoelii infection inhibits murine leukaemia WEHI-3 cell proliferation in vivo by promoting immune responses.

BACKGROUND: Leukaemia is a malignant leukocyte disorder with a high fatality rate, and current treatments for this disease are unsatisfactory. Therefore, new therapeutic strategies for leukaemia must be developed. Malaria parasite infection has been shown to be effective at combating certain neoplasms in animal experiments. This study is to demonstrate the anti-leukaemia activity of malaria parasite Plasmodium yoelii (P. yoelii) infection,. METHODS: In this study, the proportion of CD3, CD19, CD11b and Mac-3 cells was analysed by flow cytometry; the levels of IFN-γ and TNF-α in individual serum samples were measured by enzyme-linked immunosorbent assay, and the phagocytic activity of macrophages and natural killer (NK) cell activity were measured by flow cytometry. RESULTS: We found that P. yoelii infection significantly attenuated the growth of WEHI-3 cells in mice. In addition, tumor cell infiltration into the murine liver and spleen was markedly reduced. We also demonstrated that malaria parasite infection elicited anti-leukaemia activity by promoting immune responses, including increasing the surface markers of T cells (CD3) and B cells (CD19); decreasing the surface markers of monocytes (CD11b) and macrophages (Mac-3); inducing the secretion of IFN-γ and TNF-α; and increasing NK cell and macrophage activity. CONCLUSIONS: Malaria parasite infection significantly decreases the number of myeloblasts and inhibits neoplasm proliferation in mice. In addition, malaria parasite infection inhibits murine leukaemia by promoting immune responses.

Thelazia callipaeda infection in a 5-month-old infant.

BACKGROUND: Thelazia callipaeda is the main causative organism in thelaziasis, commonly infecting orbital cavities and associated tissues of carnivores. Thelazia callipaeda infection is rarely reported in humans, especially in infants. CASE PRESENTATION: A 5-month-old male infant presented with 2 weeks of redness and increased secretions in the left eye. On examination, the left eye revealed the presence of one creamy thread-like mobile worm in the conjunctival sac. During surgical exploration, a total of 11 worms were extracted from the left eye. The worms were morphologically identified as seven female and four male T. callipaeda. Ocular symptoms resolved rapidly after the removal of the worms, with no recurrence after the 6-month follow-up. CONCLUSION: We present here detailed clinical and morphological information pertaining to T. callipaeda infection, which is considered to be a probably neglected parasitic disease of the eye. This case illustrates the importance of including thelaziasis into the differential diagnosis of ocular surface diseases, especially in infant patients.

Enhanced Wnt Signalling in Hepatocytes is Associated with Schistosoma japonicum Infection and Contributes to Liver Fibrosis.

Liver fibrosis is the most serious pathology caused by Schistosoma japonicum infection, which arises when schistosome eggs are deposited in the liver. Eosinophils, macrophages and hepatic stellate cells (HSCs) have been identified as major cellular contributors to the development of granulomas and fibrosis, but little is known about the effects of hepatocytes on granuloma formation. Here, we found that the levels of Wnt signalling-related molecules, transforming growth factor ß (TGF-ß) and connective tissue growth factor (CTGF) in hepatocytes were markedly elevated after S. japonicum infection. Liver fibrosis was exacerbated when exogenous Wnt3a was introduced, but was alleviated when Wnt signalling was suppressed by DKK1, accompanied by the reduced expression of TGF-ß and CTGF in hepatocytes. These results indicate that the hepatocytic expression of TGF-ß and CTGF is mediated by Wnt signalling. Additionally, the hepatocytes isolated from infected mice promoted the activation of primary HSCs in vitro, however, this effect was not observed when hepatocytes from DKK1 treated S. japonicum-infected mice was employed in the co-culture system. Our findings identify a novel pro-fibrogenic role of hepatocytes in schistosomiasis-induced liver fibrosis that is dependent on Wnt signalling, which may serve as a potential target for ameliorating hepatic fibrosis caused by helminths.

[Investigation on Lophomonas blattarum infection in Periplaneta americana in Wuhan City].

An investigation of Lophomonas blattarum infection in Periplaneta americana in Wuhan City were conducted. A total of 110 P. americana were dissected and the intestines were separated. The intestines were washed with 0.6% saline and the washing solutions were smeared on slides. The slides were stained with Giemsa stain and observed under a microscope (x1000). Out of 110 intestine washing solution samples, 44 were suspected of L. blattarum infection. The parasite was oval or pyriform in shape and 20-40 microm in size. A tuft of flagella extended down the central axis of the parasite and a trumpet-shaped calyx enveloped the flagellar area and the nucleus. An axostyle was slender and pointed posterior ends. Based on the above morphological characteristics, the parasite was identified as L. blattarum. The results showed that the infection rate of L. blattarum in P. amerivana in Wuhan City was 40.0% (44/110).

[Detecting effect comparison between MPAIA, DDIA and IHA for screening advanced schistosomiasis].

OBJECTIVE: To evaluate the effects of the magnetic particle antibody immunoassay (MPAIA), dipstick dye immunoassay (DDIA) and indirect hemagglutination assay (IHA), on detecting advanced schistosomiasis. METHODS: The sera of 224 cases of advanced schistosomiasis were detected by MPAIA, DDIA, and IHA, and the positive rates were compared. RESULTS: The positive rates of MPAIA, DDIA and IHA, were 67.14%, 14.29% and 16.52%, respectively,the positive coincidence rate of MPAIA is higher than the one of IHA and DDIA. CONCLUSION: The value of MPAIA is higher than that of DDIA or IHA in screening advanced schistosomiasis.

Delayed tail loss during the invasion of mouse skin by cercariae of Schistosoma japonicum.

A traditional assumption is that schistosome cercariae lose their tails at the onset of penetration. It has, however, recently been demonstrated that, for Schistosoma mansoni, cercarial tails were not invariably being shed as penetration took place and a high proportion of tails entered human skin under experimental conditions. This phenomenon was termed delayed tail loss (DTL). In this paper, we report that DTL also happens with S. japonicum cercariae during penetration of mouse skin. It occurred at all cercarial densities tested, from as few as 10 cercariae/2·25 cm(2) of mouse skin up to 200 cercariae. Furthermore, it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased. No such density-dependent enhancement was shown for percentage attachment over the same cercarial density range.

Enhanced protection against tuberculosis by vaccination with recombinant BCG over-expressing HspX protein.

Immunization with Mycobacterium bovis Bacille Calmette-Guerin (BCG) did not induce adequate Th1 responses to the latency antigen, HspX of M. tuberculosis. To increase the immunogenicity and protective efficacy of BCG, a recombinant BCG strain over-expressing antigen HspX (rBCG::X) was constructed. The recombinant strain rBCG::X expressed high levels of both HspX protein in the cytosol and Ag85B protein in the cytosol and supernatant. Mice vaccinated with rBCG::X produced a more consistent and enduring protective effect against infection with M. tuberculosis, showing lower bacterial load in lung and less severe lung pathology, than the control mice vaccinated with BCG strain containing the vector pMV261. The long-term protection induced by rBCG::X was associated with significant increases in antigen-specific IFN-gamma to both HspX and Ag85B proteins, while PPD-specific IFN-gamma responses declined. Our results suggest that latency antigens of M. tuberculosis may be promising targets for developing more effective recombinant BCG strains to protect against TB.

[Purification of Toxoplasma gondii Tachyzoites with cellulose membrane filtration and preparation of soluble antigen].

20 ml peritoneal lavage fluid of mice infected with Toxoplasma gondii RH strain was diluted to 250 ml with sterilized physiological saline, and filtered through cellulose membrane filters (pore size: 5 microm). The filtrate was centrifuged at 1512 x g for 15 min, and the sediment was pure T. gondii tachyzoites which were then sonicated. The soluble antigen was prepared by centrifugation at 11200 x g for 30 min. Sera of T. gondii infected SD rat and normal SD rats were collected for immunodetection of soluble antigen. The specificity and valence of soluble antigen were detected with indirect ELISA. The mean removal rates of mouse leukocytes and erythrocytes were 99.9% and 80.3%, respectively, and recovery rate of tachyzoites was 71%. The soluble antigen was extracted from purified T. gondii (1.38 mg per mouse). Indirect ELISA showed that the lowest effective antigen concentration was 5 microg/ml.

An improved whole-blood gamma interferon assay based on the CFP21-MPT64 fusion protein.

Differentiation of latent tuberculosis infection (LTBI) from a healthy, unexposed population plays a vital role in the strategy of controlling and eliminating tuberculosis (TB). Both CFP21 and MPT64, antigens encoded by the RD2 region which are restricted in the Mycobacterium tuberculosis complex, are TB-specific diagnostic candidate antigens. In this study, we designed a fusion protein by linking both CFP21 and MPT64 with a 15-amino-acid peptide, (G(4)S(1))(3), and overexpressed the fusion protein in Escherichia coli. A new whole-blood gamma interferon assay based on the recombinant fusion protein, CFP21-MPT64 (rCM-WBIA), was developed and compared with the tuberculin skin test (TST) for screening of LTBI in household contacts of patients with sputum-positive TB. rCM-WBIA had a slightly higher sensitivity (66.7%; 24/36 contacts) than that of the TST (61.1%; 22/36 contacts) for household contacts. We found that rCM-WBIA had a very high sensitivity (90.9%) and specificity (71.4%) for LTBI detection compared with TST. The overall agreement between rCM-WBIA and TST was 83.3% (k = 0.64); rCM-WBIA positivity was associated with a larger TST induration. These results suggest that rCM-WBIA, based on the recombinant fusion protein CFP21-MPT64, is a promising alternative diagnostic tool for detection of LTBI.

[Observation on mitosis of Trichomonas vaginalis cultivated in vitro using modified Giemsa staining].

The observation showed that the percentage of Trichomonas vaginalis trophozoites at the stages of interphase, binary fission and multiple fission was 66.5%, 24.1% and 9.4% respectively. Cells in binary fission could be classified as premitotic phase, prophase, metaphase, anaphase and telophase. 3 to 8 microcosms were seen in one trophozoite under multiple fission and the percentage of trophozoites with 3 and 4 microcosms occupied 69% and 24.5% respectively. Cells with abnormal morphs were also observed.

[Influence of the metabolite produced by Trichomonas vaginalis on human sperm motility in vitro].

OBJECTIVE: To investigate the effects of the metabolite produced by Trichomonas vaginalis on human sperm motility in vitro. METHODS: Trichomonas vaginalis having been cultured, the culture solution containing metabolite was obtained by removing the protozoa, then diluted into 3 kinds of concentration. Sperm was obtained from 10 healthy fertile men by masturbation and prepared by swim-up technique to produce a spermatozoon solution of high motility. Every sperm sample was divided into 4 groups (A, B, C, D). Unused culture solution was added to Group A as control, and the other 3 groups (B, C, D) were respectively incubated with the above used culture solution at 3 kinds of concentration (1.2 x 10(9)/L, 6 x 10(8)/L, 1.2 x 10(8)/L). Measurements were carried out at 30 s, 1 h, 2 h, 4 h, 6 h by CASA. RESULTS: Sperm motility decreased in both Group B and C markedly, and the effects displayed a concentration- and time-dependent manner. CONCLUSION: The metabolite of Trichomonas vaginalis can reduce human sperm motility in vitro, and may be one of the causes of infertility.