Antioxidant vitamins and lysophospholipids are critical for inducing mouse spermatogenesis under organ culture conditions.

In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.

Amplification of bacterial genomic DNA from all ascitic fluids with a highly sensitive polymerase chain reaction.

Due to varying positive rates of polymerase chain reaction (PCR) amplification, interpretation of conventional PCR results for non­infectious ascites remains problematic. The present study developed a highly sensitive PCR protocol and investigated the positive rate of PCR for the 16S ribosomal (r)RNA gene in non­infectious ascites. Following the design of a new PCR primer pair for the 16S rRNA gene (800F and 1400R), the sequences of PCR products were analyzed and the lower limit for bacterial DNA detection evaluated. The positive rate of PCR for 16S rRNA gene in non­infectious ascites was also evaluated. PCR with the primer pair amplified the genomic DNA of 16S rRNA genes of major disease­causing bacterial strains. Additionally, PCR with this primer pair provided highly sensitive detection of bacterial genomic DNA (lower limit, 0.1 pg of template DNA). When DNA samples isolated from ascites were used, the 16S rRNA gene was amplified independently of the presence of bacterial infection. PCR products contained the genomic DNA fragments of multiple bacterial species. Bacterial genomic DNA can be amplified from all ascitic fluids using a highly sensitive PCR protocol. Careful attention is required to interpret the results based on simple amplification of 16S rRNA gene with conventional PCR.

[Viewing sepsis and autoimmune disease in relation with infection and NETs-formation].

Neutrophil has been widely recognized as body's first line of defence against pathogens. NETosis was first reported in 2004 as a programmed cell death of neutrophil and distinguished from apoptosis and necrosis. This phenomenon has been already observed in both basic and clinical research. NETosis is induced by various stimulants such as PMA, IL-8, DAMPs/PAMPs, bacteria, and antigen-antibody complex including self-antibody such as ANCA. It is known that there are two types of NETosis following bacterial infections. Although both of them have the ability to capture and kill bacteria, they also damage the host tissues. The inhibition of the NETs-related enzymes prevents the NETs formation at that time. The production of O2- from respiratory burst of neutrophils triggers NETs formation. In the first type of NETosis, neutrophils are completely collapsed, while in the second type, they maintain the morphology and the ability of phagocytosis. However, bacteria can escape from NETs by degrading NETs with their secreting nucleases. Thus the animal models of infection, using these bacteria, oftentimes suffer from severe infectious diseases. Human CGD (Chronic Granulomatosis Disease) patients who do not have Nox2 are immunocompromised, and highly susceptible to infection due to the defect of NETs formation. On the other hand, SLE patients are unable to break down the NETs as their serum inhibits the DNase1 activity, which results in autoantibody generation against NETs as well as self-DNA. It is getting clear that there is a relationship between inflammatory diseases, including infectious diseases, Sepsis and autoimmune diseases, and NETs. Therefore, it is important to re-evaluate the inflammatory disorders from NETs' perspective, and to incorporate the emerging concepts for better understanding the mechanisms involved.

In vitro mouse spermatogenesis with an organ culture method in chemically defined medium.

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

Combinational approach using in situ hybridization targeting 23S ribosomal RNA genes and blood cultures for bacterial identification in patients with neutropenia and fever.

BACKGROUND: A new 23S ribosomal RNA genes-targeted in situ hybridization (ISH) probe to detect global bacterial genomic DNA (59 species from 35 genera; referred to as the GB probe) phagocytized in leukocytes was recently developed. This method provided early and direct evidence of bacterial infection with high sensitivity and specificity in spontaneous bacterial peritonitis ascites. However, the utility of this method in febrile neutropenia (FN) is unknown. METHODS: We prospectively evaluated the utility of the ISH approach using the GB probe and previously reported probes in patients with neutropenia and fever undergoing chemotherapy at our institution between June 2011 and July 2013. Blood samples for culture analysis and ISH tests were collected simultaneously at the onset of fever; the latter were performed repeatedly. RESULTS: Fifty febrile episodes were evaluated. In 24 episodes of fever of unknown origin and 15 episodes of local infection (all negative for blood cultures), ISH tests identified causal bacteria in 21% and 13% of cases, respectively, at the onset of fever. In seven sepsis cases (all positive for blood culture), positive ISH test results at fever onset were achieved in 71%; for two patients with neutrophil counts of 0/µl and 171/µl, respectively, negative results were obtained. CONCLUSIONS: This new ISH approach could prove useful for early detection of bacteria in patients with neutropenia and blood culture-negative, with fever of unknown etiology after chemotherapy. Using this method in combination with blood culture, even in cases with extremely low neutrophil counts, might contribute to better management of FN.

Single human sperm cryopreservation method using hollow-core agarose capsules.

OBJECTIVE: To develop an efficient cryopreservation method using a single sperm. DESIGN: Experimental study. SETTING: Laboratory of a private institute. PATIENT(S): A fertile donor. INTERVENTION(S): We produced hollow-core capsules with agarose walls. A single human sperm was injected into each capsule as per the conventional intracytoplasmic sperm injection (ICSI) method. The capsules that contained the spermatozoa were cryopreserved on polycarbonate or nylon mesh sheets using nitrogen vapor. Before their use, the capsules were thawed and recovered. The motile spermatozoa in the capsules were counted. MAIN OUTCOME MEASURE(S): The recovery rates of the agarose capsules and the spermatozoa in these capsules after thawing and the mortality and survival rates of the spermatozoa. RESULT(S): The recovery rates of the capsules were 91.5% (75/82) using polycarbonate sheets (PS) and 98.3% (59/60) using mesh sheets (MS) after thawing. The recovered capsules were not at all damaged. The recovery rates of the spermatozoa were 91.5% (75/82) using PS and 96.7% (58/60) using MS. Sperm motility rates were 85.3% (64/75) and 82.8% (48/58), whereas the survival rates of the immotile spermatozoa by the hypoosmotic swelling test were 81.8% (9/11) and 50.0% (5/10); furthermore, the total survival rates of the spermatozoa were 97.3% (73/75) and 91.4% (53/58) using PS and MS, respectively. There was no significant difference between the results obtained using PS and MS. CONCLUSION(S): A cryopreservation method for a single sperm using an agarose capsule has been developed. The method is expected to be useful in ICSI treatment in patients with few spermatozoa.

Diagnosis of spontaneous bacterial peritonitis and an in situ hybridization approach to detect an "unidentified" pathogen.

Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication in cirrhotic patients with ascites. Although identifying the pathogen(s) plays a major role in the management of infectious diseases, ascitic fluid cultures often show negative results in patients with clinical signs and symptoms of SBP, and ascitic fluid cell analyses are the gold standard method for diagnosing SBP. SBP is generally diagnosed based on an increased number of polymorphonuclear neutrophils in the ascitic fluid (>250/mm(3)), and the identification of the causal pathogen may not be given consideration. We newly developed an in situ hybridization (ISH) method to provide early and direct evidence of bacterial infection in ascites in patients with SBP. This paper will review the diagnosis of SBP, including our novel approach with ISH method to detect bacterial DNA in SBP ascitic fluid.

A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method.

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).

Development of a new in situ hybridization method for the detection of global bacterial DNA to provide early evidence of a bacterial infection in spontaneous bacterial peritonitis.

BACKGROUND & AIMS: Despite the importance of identifying the causative pathogen(s), ascitic fluid cultures are occasionally negative in patients with spontaneous bacterial peritonitis (SBP). A novel strategy using the in situ hybridization (ISH) method was introduced to detect the bacterial genomic DNA phagocytized in the blood of patients with sepsis. In the present study, we developed a new ISH probe to detect global bacterial DNA (named as GB probe) and evaluated its utility for detecting the phagocytized bacterial DNA in SBP ascites. METHODS: Hybridization of bacterial DNA with the GB probe was examined by dot-blot and ISH tests. In addition, the utility of the ISH method to detect the bacterial DNA in the leukocytes of SBP ascites was evaluated. RESULTS: The GB probe hybridized with the genomic DNA of all 59 bacterial strains tested (59 species of 36 genus). Eleven of 51 patients with ascites (out of total 542 cirrhotic inpatients) were categorized as SBP. The ISH tests showed positive results in 10 of 11 SBP cases. However, the ISH tests all showed negative results in the 40 non-SBP ascitic samples. Therefore, the ISH tests yielded highly sensitive and specific results for detecting the phagocytized bacterial DNA in the leukocytes of SBP ascites. Moreover, all of the ISH test results were obtained within only one day. CONCLUSIONS: Our newly established ISH method was found to provide both a rapid and sensitive detection of bacterial DNA in SBP ascites, thus suggesting its utility for providing early and direct evidence of bacterial infection in SBP ascites.

Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli.

In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.

Development of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus.

A cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10-100 CFU tube(-1) by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner.

Comparative analysis of cytolethal distending toxin (cdt) genes among Campylobacter jejuni, C. coli and C. fetus strains.

The cytolethal distending toxin (cdt) gene clusters of Campylobacter coli strain Co1-243 and C. fetus strain Col-187 were cloned and sequenced to understand the importance of Cdt as a virulence factor. The cdt genes of C. coli and C. fetus consist of three closely linked genes termed cdtA, cdtB, cdtC whose sizes are 774, 801, and 570 bp, and 702, 798, and 546 bp, respectively. The homologies of each subunit of cdt genes between C. jejuni and C. coli, C. jejuni and C. fetus, or C. coli and C. fetus are 59.6%, 40.3%, or 46.5% for cdtA, 70.2%, 62.4%, or 61.3% for cdtB, 61.3%, 52.3%, or 50.1% for cdtC, respectively. Colony hybridization assay revealed that the genes homologous to the cdtABC gene were distributed in all 27, 19, 20 strains of C. jejuni, C. coli, and C. fetus, respectively, isolated from patients and animals in species-specific manner. Furthermore, nucleotide sequence of the cdt operon, including flanking region, of 10 strains of each species indicated that though the size of the cdtB gene was conserved in each species, those of cdtA and cdtC genes varied particularly among C. coli strains. Amino acid residues demonstrated to be important for toxin activity in CdtB, corresponding to H152, D185, D222, D258, H259 in Cj-CdtB, were also conserved in Cc-CdtB and Cf-CdtB. The cdt gene cluster was located in different sites among different species but in the same site of genomes of the same species. Cdt activity produced by C. jejuni and C. fetus varied among strains, however, any C. coli strains exhibited Cdt activity on HeLa cells. These data indicate that the cdt gene may have a potential for virulence factor at least in C. jejuni and C. fetus.

Peripheral blood mononuclear cells are possible extrahepatic replication sites for hepatitis C virus.

BACKGROUND/AIMS: Hepatitis C virus is a major causative agent of chronic liver disease and hepatocellular carcinoma and is considered to be a hepatotropic virus. It remains controversial whether hepatitis C virus exists in peripheral blood mononuclear cells and replicates there. In order to resolve this issue, we performed nested RT-PCR (reverse transcription polymerase chain reaction) and RT-PCR in situ hybridization in peripheral blood mononuclear cells of patients with chronic hepatitis C. METHODOLOGY: We collected peripheral blood mononuclear cells from patients with chronic hepatitis C, extracted total RNA from the samples, and performed nested RT-PCR to detect hepatitis C virus RNA in the peripheral blood mononuclear cells lysates. We also fixed peripheral blood mononuclear cells of the patients in 4% paraformaldehyde and performed RT-PCR in situ hybridization with a digoxigenin-labeled RNA probe to detect hepatitis C virus RNA in the cells. RESULTS: Using these methods, we detected both positive- and negative-stranded hepatitis C virus RNA in peripheral blood mononuclear cells of hepatitis C patients. To determine in which cell population of peripheral blood mononuclear cells hepatitis C virus is present, we performed PCR in situ hybridization after incubation with fluorescent latex microbeads which could be phagocytozed by monocytes. We obtained positive signals of the replicative hepatitis C virus genome not only in lymphocytes but also in monocytes. CONCLUSIONS: RT-PCR in situ hybridization with a nonradioactive probe was found to be useful for in situ detection of hepatitis C virus RNA. Our findings suggest that peripheral blood mononuclear cells may be extrahepatic replication sites for hepatitis C virus.