Early histological transformation of follicular lymphoma to diffuse large B-cell lymphoma indicating adverse survival: A population-based analysis and validation.

INTRODUCTION: The histological transformation (HT) of follicular lymphoma (FL) is a crucial biological event. The study aimed to evaluate the incidence, clinicial characteristics, prognosis and impact of HT time on survival of FL transforming to diffuse large B-cell lymphoma in population-based large-scale cohorts. METHODS: A retrospective cohort study of FL with HT was performed in the Surveillance, Epidemiology, and End Results database. The Hematological Malignancy Research Network FL cohort and Aristotle study FL cohort were used to assess the external validity. RESULTS: Among 44,127 FL cases from the Surveillance, Epidemiology, and End Results database, 1311 cases were pathology-proven recorded to transform to diffuse large B-cell lymphoma. The cumulative rates of HT at 5, 10, and 15 years after FL diagnosis were estimated to be 1.19%, 2.93%, and 5.01%, respectively. Significantly worse overall survival and cancer-specific survival were exhibited in patients with HT than those without HT. Early HT (transformation of FL within 48 months after FL diagnosis [TOD48]) was an independent predictor for adverse overall survival of HT patients, regardless of treatment modalities before transformation. The adverse prognostic effect of TOD48 was validated in the Hematological Malignancy Research Network cohort and Aristotle study cohort. Older age (>75 years) and B symptoms within FL at diagnosis were the independent risk factors of TOD48. Furthermore, a novel prognostic model combining TOD48 with Follicular Lymphoma International Prognostic Index (TOD48-FLIPI) was constructed and validated for risk stratification. CONCLUSION: TOD48 was a risk indicator of HT, and the novel prognostic model "TOD48-FLIPI" for HT patients was proposed.

Diffuse large B-cell lymphoma with continuously elevated immunoglobulin M following treatment: a case report with pathologic, immunophenotypic, and molecular analyses.

A rare subtype of diffuse large B-cell lymphoma (DLBCL) has been reported to be accompanied by elevated immunoglobulin M (IgM) paraprotein in the serum at diagnosis, called as IgMs-DLBCL. The monoclonal IgM paraprotein disappears soon after treatment in most of these patients. Here, we described a DLBCL patient with continuously elevated IgM following therapy. A 59-year-old male was diagnosed with DLBCL (GCB subtype per Hans algorithm, stage IA) with involvement of the right cervical lymph node. After six cycles of immuno-chemotherapy with the R-CHOP regimen, complete metabolic remission was achieved, but an elevated level of serum IgM persisted. To investigate the origin of elevated IgM, pathologic, immunophenotypic, and molecular analyses of lymph node and bone marrow (BM) samples were performed pre- and post-treatment. BM infiltration of lymphoplasmacytic cells, and a typical immunophenotypic profile by flow cytometry supported the diagnosis of Waldenström macroglobulinemia (WM). The MCD subtype of DLBCL was identified by next-generation sequencing of the lymph node at initial diagnosis characterized by co-occurring point mutations in MYD88 L265P and CD79B. Additionally, two different dominant clonotypes of the immunoglobulin heavy chain (IGH) were detected in the lymph node and BM by IGH sequencing, which was IGHV 3-11*06/IGHJ 3*02 and IGHV 3-11*06/IGHJ 6*02, respectively, speculating to be two independent clonal origins. This study will provide a panoramic understanding of the origin or biological characteristics of DLBCL co-occurring with WM.

Analysis of aptamer-target binding and molecular mechanisms by thermofluorimetric analysis and molecular dynamics simulation.

Introduction: Aptamers are valuable for bioassays, but aptamer-target binding is susceptible to reaction conditions. In this study, we combined thermofluorimetric analysis (TFA) and molecular dynamics (MD) simulations to optimize aptamer-target binding, explore underlying mechanisms and select preferred aptamer. Methods: Alpha-fetoprotein (AFP) aptamer AP273 (as the model) was incubated with AFP under various experimental conditions, and melting curves were measured in a real-time PCR system to select the optimal binding conditions. The intermolecular interactions of AP273-AFP were analysed by MD simulations with these conditions to reveal the underlying mechanisms. A comparative study between AP273 and control aptamer AP-L3-4 was performed to validate the value of combined TFA and MD simulation in selecting preferred aptamers. Results: The optimal aptamer concentration and buffer system were easily determined from the dF/dT peak characteristics and the melting temperature (Tm) values on the melting curves of related TFA experiments, respectively. A high Tm value was found in TFA experiments performed in buffer systems with low metal ion strength. The molecular docking and MD simulation analyses revealed the underlying mechanisms of the TFA results, i.e., the binding force and stability of AP273 to AFP were affected by the number of binding sites, frequency and distance of hydrogen bonds, and binding free energies; these factors varied in different buffer and metal ion conditions. The comparative study showed that AP273 was superior to the homologous aptamer AP-L3-4. Conclusion: Combining TFA and MD simulation is efficient for optimizing the reaction conditions, exploring underlying mechanisms, and selecting aptamers in aptamer-target bioassays.

Aptamer-Based Triple Serum Fluorescence Intensity Assay: A Novel and Feasible Method for the Clinical Diagnosis of Primary Hepatic Carcinoma.

Background and Aims: Aptamers are artificial ligands that bind to biological targets with high specificity and affinity. We previously selected a group of aptamers against the serum of primary hepatic carcinoma (PHC) via systematic evolution of ligands by exponential and enrichment (SELEX) method, and some of the aptamers were valuable for PHC diagnosis in polyacrylamide gel electrophoresis (PAGE) analysis. Here, we used aptamers to develop a novel method suitable for the clinical diagnosis of PHC. Methods: The intensities of serum autofluorescence, cell-free DNA (cfDNA)-related fluorescence and aptamer-related fluorescence, named the aptamer-based triple serum fluorescence intensity (ATSFI), were sequentially measured at 8 °C and 37 °C in one tube by using a real-time polymerase chain reaction (PCR) system as a fluorimeter in patients with PHC (n=346) or liver cirrhosis (n=321). The diagnostic performances of ATSFI indicators alone and in combination were evaluated by area under the receiver operator characteristic curve (AUROC), and the underlying clinical mechanisms were analyzed by bivariate correlation. Results: The measurement of ATSFI was high throughput, rapid, convenient, and low cost. The aptamer-related fluorescence indicator SEA-SE37 was the most valuable for PHC diagnosis among all fluorescence indicators and superior to alpha-fetoprotein (AFP) (AUROC 0.879 vs. 0.836). The logistic model of ATSFI indicators exhibited excellent diagnostic performance for PHC, including AFP-negative, early and small PHCs, with AUROCs of 0.935-0.950 and accuracies of 86.8-88.3%. The diagnostic performance was further improved when ATSFI indicators were combined with AFP, with AUROCs of approximately 0.95 and accuracies of approximately 90%, suggesting ATSFI was independent of but complementary to AFP in PHC diagnosis. ATSFI models were highly valuable in clinical decision-making. The aptamer-related fluorescence intensity was generally independent of the clinicopathological characteristics of PHC but correlated with laboratory characteristics of PHC serum. Conclusions: The ATSFI assay is a novel, robust and feasible method for the clinical diagnosis of PHC.

A novel four-gene signature for predicting the prognosis of hepatocellular carcinoma.

OBJECTIVE: To identify and utilize gene signatures for the prognostic evaluation of postoperative patients with hepatocellular carcinoma (HCC). METHODS: The gene mRNA expression profiles and corresponding clinicopathological data of postoperative patients with HCC were downloaded from The Cancer Genome Atlas (TCGA) database. Highly differentially expressed genes (DEGs) in tumor tissues compared to adjacent tissues were identified, and their associations with the overall survival (OS) of HCC patients were analyzed. The strongly associated genes were used to develop a prognostic score for the survival stratification of HCC, and the underlying mechanisms were analyzed using bioinformatics. RESULTS: A total of 376 DEGs were identified and four DEGs (ADH4, COL15A1, RET and KCNJ16) were independently associated with OS. A prognostic score derived from the four genes could effectively stratify HCC patients with different OS outcomes, independent of clinical parameters. Patients with high scores exhibited poorer OS than patients with low scores (HR 5.526, 95% CI: 2.451-12.461, p < .001). The four genes were involved in cancer-related biological processes and were independent of each other in bioinformatics analyses. CONCLUSION: Four genes strongly associated with the prognosis of postoperative patients with HCC were identified, and the derived prognostic score was simple and valuable for overall survival prediction.

Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli for treatment of colitis.

Colitis is a common disease of the colon that is very difficult to treat. Probiotic bacteria could be an effective treatment. The probiotic Escherichia coli Nissle 1917 (EcN) was engineered to synthesize the ketone body (R)-3-hydroxybutyrate (3HB) for sustainable production in the gut lumen of mice suffering from colitis. Components of heterologous 3HB synthesis routes were constructed, expressed, optimized, and inserted into the EcN genome, combined with deletions in competitive branch pathways. The genome-engineered EcN produced the highest 3HB level of 0.6 g/L under microaerobic conditions. The live therapeutic was found to colonize the mouse gastrointestinal tract over 14 days, elevating gut 3HB and short-chain-length fatty acid (SCFA) levels 8.7- and 3.1-fold compared to those of wild-type EcN, respectively. The sustainable presence of 3HB in mouse guts promoted the growth of probiotic bacteria, especially Akkermansia spp., to over 31% from the initial 2% of all the microbiome. As a result, the engineered EcN termed EcNL4 ameliorated colitis induced via dextran sulfate sodium (DSS) in mice. Compared to wild-type EcN or oral administration of 3HB, oral EcNL4 uptake demonstrated better effects on mouse weights, colon lengths, occult blood levels, gut tissue myeloperoxidase activity and proinflammatory cytokine concentrations. Thus, a promising live bacterium was developed to improve colonic microenvironments and further treat colitis. This proof-of-concept design can be employed to treat other diseases of the colon.

Ketone Body 3-Hydroxybutyrate Ameliorates Atherosclerosis via Receptor Gpr109a-Mediated Calcium Influx.

Atherosclerosis is a chronic inflammatory disease that can cause acute cardiovascular events. Activation of the NOD-like receptor family, pyrin domain containing protein 3 (NLRP3) inflammasome enhances atherogenesis, which links lipid metabolism to sterile inflammation. This study examines the impact of an endogenous metabolite, namely ketone body 3-hydroxybutyrate (3-HB), on a mouse model of atherosclerosis. It is found that daily oral administration of 3-HB can significantly ameliorate atherosclerosis. Mechanistically, 3-HB is found to reduce the M1 macrophage proportion and promote cholesterol efflux by acting on macrophages through its receptor G-protein-coupled receptor 109a (Gpr109a). 3-HB-Gpr109a signaling promotes extracellular calcium (Ca2+) influx. The elevation of intracellular Ca2+ level reduces the release of Ca2+ from the endothelium reticulum (ER) to mitochondria, thus inhibits ER stress triggered by ER Ca2+ store depletion. As NLRP3 inflammasome can be activated by ER stress, 3-HB can inhibit the activation of NLRP3 inflammasome, which triggers the increase of M1 macrophage proportion and the inhibition of cholesterol efflux. It is concluded that daily nutritional supplementation of 3-HB attenuates atherosclerosis in mice.

Synthesis and biological evaluation of benzofuran-based 3,4,5-trimethoxybenzamide derivatives as novel tubulin polymerization inhibitors.

A new series of derivatives characterized by the presence of the 3,4,5-trimethoxylbenzamide substituted benzofurans were synthesized and evaluated for antiproliferative activity against four cancer cell lines and one normal human cell line. Among them, derivative 6g with greatest cytotoxicity significantly inhibited the growth of MDA-MB-231, HCT-116, HT-29 and HeLa cell lines with IC50 values of 3.01, 5.20, 9.13, and 11.09 µM, respectively. Importantly, 6g possessed excellent selectivity over non-tumoral cell lines HEK-293 (IC50 > 30 µM). Moreover, mechanistic studies revealed that 6g induced HeLa cells arrested in G2/M phase in a concentration-dependent manner, and inhibited polymerization of tubulin via a consistent way with CA-4. In general, these observations suggest that 6g is a promising anti-cancer lead and is worth further investigation to generate potential antitumor agents.

Two-level percutaneous endoscopic lumbar discectomy for highly migrated upper lumbar disc herniation: A case report.

BACKGROUND: The technique of percutaneous endoscopic lumbar discectomy (PELD) as a transforaminal approach has been used to treat highly migrated lower lumbar disc herniations. However, due to the different anatomic characteristics of the upper lumbar spine, conventional transforaminal PELD may fail to remove the highly migrated upper lumbar disc nucleus pulposus. Therefore, the purpose of this study was to describe a novel surgical technique, two-level PELD, for the treatment of highly migrated upper lumbar disc herniations and to report its related clinical outcomes. CASE SUMMARY: A 60-year-old male presented with a complaint of pain at his lower back and right lower limb. The patient received 3 mo of conservative treatments but the symptoms were not alleviated. Physical examination revealed a positive femoral nerve stretch test and a negative straight leg raise test for the right leg, and preoperative visual analog scale (VAS) score for the lower back was 6 points and for the right leg was 8 points. Magnetic resonance imaging (MRI) demonstrated L2-L3 disc herniation on the right side and the herniated nucleus pulposus migrated to the upper margin of L2 vertebral body. According to physical examination and imaging findings, surgery was the primary consideration. Therefore, the patient underwent surgical treatment with two-level PELD. The pain symptom was relieved and the VAS score for back and thigh pain was one point postoperatively. The patient was asymptomatic and follow-up MRI scan 1 year after operation revealed no residual nucleus pulposus. CONCLUSION: Two-level PELD as a transforaminal approach can be a safe and effective procedure for highly migrated upper lumbar disc herniation.

[Cloning, Expression and Immungenicity Analysis of Antigen Eg-01883 Screened from Protoscoleces of Echinococcus granulosus].

Objective: To screen for the Echinococcus granulosus 01883（Eg-01883） specifically expressed at the protoscolex period, clone and express this molecule as well as analyse its immunogenicity. Methods: Eg-01883, which is highly expressed at the protoscolex period but not in oncosphere， was screened by analysing the published mRNA sequences of E. granuolosus. Total RNA of E. granuolosus was extracted, Eg-01883 was cloned by RT-PCR, and the recombinant plasmid pET28a-Eg-01883 was constructed. Expression of the recombinant protein rEg-01883 was induced by isopropyl-ß-D-thiogalactoside （IPTG）. ICR mice were randomized into 3 groups （n=12 in each group）. Mice in the immunization group received subcutaneous injections of 10 µg rEg-01883 in 100 µl PBS emulsified in Freund's adjuvant at multiple sites, followed by immune enhancement after 2 weeks. Mice in the adjuvant group were injected with PBS and adjuvant. Mice in the control group received no treatment. Blood was obtained through caudal vein before immunization, and at 1, 2, and 4 weeks after the first immunization, and through the eyeball at 6 weeks after immunization. Serum levels of IgG, IFN-γ and IL-4 were determined by ELISA. The immunogenicity of rEg-01883 was identified by Western blotting. Results: Eg-01883 was screened, cloned, expressed and purified to obtain the recombinant protein rEg-01883, which mainly existed as the inclusion body. ELISA results showed that immunization with rEg-01883 induced production of specific IgG antibody. The serum IgG level in the immunization group increased from 1 week after the first immunization, peaked at 6 weeks（2.344±0.153）, which was significantly higher than those of the adjuvant group（0.206 1±0.006） and the control group （0.241±0.01） （P<0.01）. At 6 weeks after the first immunization, the serum levels of IFN-γ （43.23 pg/ml） and IL-4（24.88 pg/ml） in the immunization group were significantly higher than those in the adjuvant group（21.77 pg/ml, 13.27 pg/ml） and the control group（17.40 pg/ml, 12.25 pg/ml）（P<0.05）. Western blot showed that the recombinant protein rEg-01883 could be recognized by His-Tag antibodies, serum of immunized mice, and serum of mice with secondary infection. Conclusion: The recombinant protein rEg-01883 shows good immunogenicity in ICR mice.

[Size distributions of aerosol particles and the impact on visibility in winter of Nanjing].

High resolution instruments were used to investigate the relationship between aerosol size distribution characteristics and meteorological factors, and its possible influence on visibility in urban Nanjing from November to December 2009. Results show that the size distribution of aerosol number concentration showed a bimodal shape with the main peak value concentrating at particle sizes of 0.04-0.1 microm. Mass concentration distribution presented a bimodal shape with the two peak values concentrating at particle sizes of 0.5-0.7 microm and 2.7 microm, and the surface area concentration distribution presented two peaks from 0.1 to 0.5 microm and from 0.5 to 0.9 microm. It is found that the diurnal and interdiurnal variations of particle concentrations are obvious. Human activities and variation of atmospheric stability had great effect on daily variation of particle concentrations, while meteorological conditions such as precipitation, wind, relative humidity and so on had strong influence on interdiurnal variation. The aerosol size distribution was significantly affected by relative humidity. When RH was lower than 54%, number concentration of aerosol particles less than 1 microm in diameter increased gradually as RH increased, and concentration of particles with diameter larger than 1 microm almost had no change. When RH was higher than 54%, number concentration of aerosol particles ranging from 0.01 to 0.2 microm and from 2.7 to 10 microm decreased with the increase of RH, in contrast, concentration of aerosol particles between 0.5 and 1.5 microm in diameter increased. In addition, the particle number size distributions were different in rainy, foggy, sunny and haze weather conditions. Compared to sunny day, concentration of particles with different sizes all decreased in rainy day. In foggy weather, The number concentration of aerosol particles ranging from 0.01 to 0.3 microm and from 2.7 to 10 microm decreased, and aerosol particles between 0.3 and 2.7 microm increased in comparison with sunny day. Scavenging action of rain and fog to particles of different sizes from high to low was that coarse particles > nuclei mode particles > accumulation mode particles. In haze day, the peak of number concentration distribution moved toward a higher value. Compared to sunny day, the number concentration of aerosol particles ranging from 0.03 to 0.1 microm decreased, and aerosol particles between 0.1 and 2.7 microm increased in haze day. Based on Mie theory, the correlation between visibility and surface area concentration with different particle sizes indicated that particles between 0.1 and 2 microm in diameter shows a good correlation with visibility, which was the major contributor to visibility degradation in Nanjing.

Characteristics of physicochemical adsorption of soluble matter by particles formed in a fluidized pellet bed reactor.

To investigate the mechanism of soluble matter removal by a fluidized pellet bed (FPB) reactor, an experimental study was conducted using a laboratory-scale FPB device for treating synthetic wastewater under a condition of no activated sludge seeding and no dissolved oxygen supply so that the physicochemical functions of the FPB reactor could be evaluated. By using polyaluminium chloride and polyacrylamide as coagulants, it was found that most of the substances in the synthetic wastewater could not be effectively removed by conventional coagulation and sedimentation. A similar condition was observed in the start-up period of the FPB operation. However, as a steady FPB was formed usually after about 10 hours' operation, the influent COD, ammonia nitrogen and total phosphorous could be quickly and effectively entrapped by the fluidized grown particle layer. Because these substances were not coagulable under normal conditions, adsorption might have performed an important role in their removal. Through an adsorption experiment using the grown pellets as adsorbent and glucose as adsorbate without dosing coagulants, it was found that the process followed well the Freundlich adsorption isotherm. The adsorption was also confirmed to be reversible by a washing experiment. A scanning electron microscopy coupled with energy dispersive spectroscopy analysis of the pellets before and after washing showed that the elements of carbon and phosphorous in the outer layer of the pellets were easily desorbed. The study results can provide an explanation, other than the coagulation mechanism, for the removal of soluble matter by the FPB.

[Microphysics of atmospheric aerosols during winter haze/fog events in Nanjing].

Intensive field observations of fog/haze events, including simultaneous measurements of aerosol particle and fog droplet size distributions, were conducted in Nanjing in November, 2007. Four weather conditions (fog, mist, wet haze and haze) were distinguished based on visibility and liquid water content firstly. Then, the microphysical characteristics of coarse and fine particles in each condition were investigated. The results showed the dominant sequence of the four weather conditions was haze<-->mist-->wet haze-->fog-->, wet haze-->mist<-->haze. The lasting time of pre-fog wet haze was longer than that of post-fog wet haze. The number, surface area and volume concentration of coarse particles with diameter larger than 2.0 micron in fog were much higher than those in the other three conditions, and the smallest concentrations were observed in haze. The size distributions of surface area and volume concentration exhibited multi-peak in fog droplets, while it showed single peak for coarse particles in haze, mist and wet haze. For the fine particles with diameter larger than 0.010 microm, the spectral shapes of surface area concentration are similar in fog (mist) and wet haze (haze) condition. The dominant size ranges of fine particle number concentration were in 0.04-0.13 microm and 0.02-0.14 microm for fog and wet haze, separately. The same dominant size ranges located in 0.02-0.06 microm for both mist and haze. During the transition processes from haze, mist and wet haze to fog, the concentration of smaller particles (less than 0.060-0.090 microm) reduced and vice versa for the corresponding larger particles. Temporal variation of aerosol number concentration correlated well with the root mean diameters negatively during the observation period. The number concentration of aerosol was the lowest and the mean diameter was the largest in fog periods.

[Study on the growth of Vibrio cholerae O139 within Acanthamoeba polyphaga and its survival in the cysts in low temperature].

OBJECTIVE: To determine whether Acanthamoeba polyphaga could affect the survival and growth of Vibrio cholerae O139 in low temperature. METHODS: V. cholerae O139 was co-cultured with the Acanthamoeba polyphaga to be examined on its intracellular growth and survival rate within cysts at low temperature, using methods as Gram-staining, electron microscope and passage culture. RESULTS: V. cholerae O139 were observed to enter into the trophozoites and grow the within the vacuoles after 8 hour incubation with Acanthamoeba polyphaga. The germs survived in the vacuole and/or endo-layer of wall and could be re-isolated from the excystment of Acanthamoeba polyphaga. At 30 degrees C, V. cholerae O139 could survive for 120 days with the amoeba while less than 45 days in PAS. At 4 degrees C, the number of viable bacteria decreased and reached undetectable levels for both study and control groups after a 30-day incubation. V. cholerae O139 could be re-isolated from the 30-, 45-, 60- and 75-day's infected cysts after excystment. However the ability of excystment for 90-day's infected cysts decreased and V. cholerae O139 within the cyst could not be isolated again because the amoebae had lysed. CONCLUSION: These findings indicated that V. cholerae O139 could grow within Acanthamoeba polyphaga and the survival time could be increased in the cysts at low temperature. It seemed that Acanthamoeba can provide an environmental reservoir for V. cholerae O139.

[Gimenez staining: a rapid method for initial identification of legionella pneumophila in Amoeba trophozoite].

OBJECTIVE: To establish a rapid staining method for facilitating initial identification of Legionella pneumophila in amoebal trophozoite. METHODS: Acanthamoeba polyphaga and Legionella pneumophila were co-cultured under laboratory condition. At consecutive time points during the culture, smears of the cultured products were made on glass slides for staining purposes. Different types of stainings including Gram's staining, Gimenez staining, Giemsa staining and immunofluorescence were used to determine the best method for the identification of amoebal pathogens. RESULTS: Gimenez staining technique is simpler and yields better results as compared with the other three stainings. Gimenez stain gives the best color and contrast for amoeba and amoebal Legionella Amoeba trophozoites and/or cysts showed a distinct purplish blue with amoebal Legionella in red. Amoebal Legionella can be distinguished clearly at an earlier time of co cul ture, providing a proper sensitivity. It takes only 10 minutes to finish the operation. The other techniques require the use of expensive reagents, are relatively time-consuming, and involve complex staining procedures. CONCLUSION: Gimenez staining is of value for the initial identification of amoebal pathogens, and it is suitable for laboratory diagnosis.

[Survival and growth of Vibrio cholerae O139 inside Acanthamoeba].

OBJECTIVE: To study the survival and growth of Vibrio cholerae inside the Acanthamoeba polyphage. METHODS: Survival and growth of Vibro cholerae O139, co-cultured with Acanthamoeba polyphaga, was observed inside the trophozoites and cysts, using Gram stain and electron microscope. RESULTS: Viable O139 was observed inside the amoebal vacuoles in 24 hours. Vacuoles were filled with more bacteria along with the longer period of co-culture. The process of O139 infection with Amoebae would include uptake, formation of O139 vacuole, multiplication, trophozoites lysed and expel under electron microscopy. Some infected trophozoites could subsequently encyst and the surviving O139 could locate in the vesicles inside the cysts. CONCLUSION: O139 might survive and multiply in the trophozoites and reside inside the cysts of Amoebae, suggesting that Acanthamoebae might serve as one of the environmental hosts of Vibro cholerae.

[A micro-culture method for continuous observation of free-living amoebae].

OBJECTIVE: To establish a method for co-culture of amoebae and endosymbionts, also for continuously observing the microphenotype of amoebae. METHODS: 24 wells culture plate with cover glass on the wells was used as containers. Amoebae and Candida albicans were co-cultured in microdrop of medium in the wells at 37 degrees C, and observed under x1000. RESULTS: Continuous observation revealed trophozoites in various shapes like letters T, K, or Y, their movement and ingestion phenomenon were observed. CONCLUSION: The micro-culture method is useful in observing the amoebal morphology and its phagocytic process to Candida albicans.

[The discovery of naked cluster particles of Parachlamydia and its developmental mechanism].

OBJECTIVE: To study the survival and developmental morphology of Parachlamydia (BN9) within Acanthamoeba. METHODS: The morphology of BN9 within Acanthamoeba was studied by inverted phase contrast microscope, electron microscope, Gimenez and AO-staining with amoebal co-culture. RESULTS: The endosomal maturation-blocked were formed after the egress of BN9. Two developmental stages-elementary and reticulate bodies, were both observed within the vacuoles. The reticulate bodies, multiplicated by binary fission, were located mainly within the vacuoles, while the elementary bodies can also be located in the plasma individually. The naked cluster particles were observed after the trophozoites cytolysis with Gimenez-staining. The light infectious trophozoites could encyst, and elementary bodies could survive within the mature cysts. CONCLUSION: The egress of BN9 could form the endosomal maturation-blocked, which was presented in two developmental stages-elementary and reticulate bodies. It exhibited the cytolysin activity that could lyse the infectious trophozoites and were expelled in the vesicles. A few light infected amoeba could encyst with survival elementary bodies in the plasma.