Proton Pump Inhibitors and Cyclin-Dependent Kinase 4/6 Inhibitors in Patients With Breast Cancer.

BACKGROUND: Proton pump inhibitors (PPIs) reduce the bioavailability of several anticancer drugs. The impact of PPIs co-administered with cyclin-dependent kinase 4 and 6 inhibitors is controversial. We aimed to clarify whether the concomitant use of PPIs impacts palbociclib and abemaciclib effectiveness in breast cancer treatment. PATIENTS AND METHODS: This multicenter, retrospective, observational study, conducted across 4 medical institutions in Japan, consecutively included patients with endocrine-resistant metastatic breast cancer, receiving palbociclib or abemaciclib between December 2017 and August 2022. Propensity score-matched analyses were performed. Treatment efficacy and safety with and without PPIs were compared. Progression-free survival and overall survival were estimated using the Kaplan-Meier method and compared using a log-rank test. A Cox proportional hazards model was used to estimate the hazard ratio. RESULTS: The study included 240 patients. After 1:1 matching, 112 patients were treated with and without PPIs. The median progression-free survival period was 1.2 years in the PPI group and 1.3 years in the non-PPI group (hazard ratio, 1.19; 95% CI, 0.70-2.02). The median overall survival period was 3.6 years in the PPI group, whereas it was not reached in the non-PPI group (hazard ratio, 1.23; 95% CI, 0.61-2.47). Consistent results were obtained for subgroups receiving palbociclib (n = 177) and abemaciclib (n = 63) without propensity score matching. Adverse event incidence and severity were similar in both groups. CONCLUSION: The effectiveness of cyclin-dependent kinase 4/6 inhibitors is unlikely to be affected by concomitant PPI use. Future prospective pharmacokinetic studies are warranted.

Periodontal ligaments enhance neurite outgrowth in trigeminal ganglion neurons through Wnt5a production induced by mechanical stimulation.

The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

Mode-selective inhibitory effects of eugenol on the mouse TRPV1 channel.

The transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal receptor in sensory nerves and involved in pain sensation. TRPV1 has at least three distinct activation modes that are selectively induced by different stimuli capsaicin, noxious heat, and protons. Although many mode-selective TRPV1 antagonists have been developed for their anticipated analgesic effects, there have been few successful reports because of adverse effects due to burn injuries and hyperthermia. Eugenol is a vanilloid that has been used as an analgesic in the dental treatment, and its TRPV1 activation ability has been reported. However, our knowledge about the underlying mechanisms of the antagonistic effects of eugenol on TRPV1 activation induced by three different modes is limited. Here, we show that eugenol dose-dependently inhibited the capsaicin-activated inward currents of mouse TRPV1 expressed in human embryonic kidney 293 (HEK293) cells. Under low pH conditions, low concentrations of eugenol only enhanced the proton-induced TRPV1 currents, whereas high eugenol concentrations initially potentiated but then immediately abrogated TRPV1 currents. Finally, eugenol had no modulatory effects on heat-activated TRPV1 in electrophysiological and Fura-2-based Ca2+ imaging experiments. Our results demonstrate that eugenol is a mode-selective antagonist of TRPV1 and can be evaluated as a lead compound of analgesics targeting TRPV1 without serious side effects.

Two cases of bacterial meningitis due to meropenem-resistant Streptococcus pneumoniae: A threat of serotype 35B, ST 558 lineage.

Although the pneumococcal conjugate vaccine (PCV) has decreased the incidence of invasive pneumococcal disease (IPD) in children, cases of IPD caused by non-PCV serotypes have been increasing. Here, we report two cases of bacterial meningitis caused by meropenem-resistant Streptococcus pneumoniae; in both the cases, 13-valent PCV (PCV13) had been administered. The isolated S. pneumoniae strains were non-PCV13 serotype 35B and resistant to penicillin G, cefotaxime, and meropenem. In addition, multilocus sequence typing (MLST) revealed the sequence type (ST) to be 558. In case 1, a 6-month-old girl recovered without sequelae after antibiotic therapy comprising cefotaxime and vancomycin, whereas in case 2, a 9-month-old boy was treated with an empirical treatment comprising ceftriaxone and vancomycin administration. However, maintaining the blood concentration of vancomycin within the effective range was difficult, due to which the antibiotics were changed to panipenem/betamipron. During the treatment, he presented with seizures, which were effectively controlled with antiepileptic drugs. The rate of incidence of penicillin-susceptible IPD has been substantially increasing after the introduction of PCV. However, an upsurge in IPD cases due to multidrug-resistant (MDR) serotype 35B has been reported in countries where PCV13 was introduced before introducing in Japan. Moreover, an increase in the proportion of MDR serotype 35B and decrease in the susceptibility to broad-spectrum antimicrobials, including meropenem, have been reported. Hence, the number of meningitis cases caused by MDR serotype 35B/ST558 may increase in the future.

Stable Accumulation of Photosystem II Requires ONE-HELIX PROTEIN1 (OHP1) of the Light Harvesting-Like Family.

The cellular functions of two Arabidopsis (Arabidopsis thaliana) one-helix proteins, OHP1 and OHP2 (also named LIGHT-HARVESTING-LIKE2 [LIL2] and LIL6, respectively, because they have sequence similarity to light-harvesting chlorophyll a/b-binding proteins), remain unclear. Tagged null mutants of OHP1 and OHP2 (ohp1 and ohp2) showed stunted growth with pale-green leaves on agar plates, and these mutants were unable to grow on soil. Leaf chlorophyll fluorescence and the composition of thylakoid membrane proteins revealed that ohp1 deletion substantially affected photosystem II (PSII) core protein function and led to reduced levels of photosystem I core proteins; however, it did not affect LHC accumulation. Transgenic ohp1 plants rescued with OHP1-HA or OHP1-Myc proteins developed a normal phenotype. Using these tagged OHP1 proteins in transgenic plants, we localized OHP1 to thylakoid membranes, where it formed protein complexes with both OHP2 and High Chlorophyll Fluorescence244 (HCF244). We also found PSII core proteins D1/D2, HCF136, and HCF173 and a few other plant-specific proteins associated with the OHP1/OHP2-HCF244 complex, suggesting that these complexes are early intermediates in PSII assembly. OHP1 interacted directly with HCF244 in the complex. Therefore, OHP1 and HCF244 play important roles in the stable accumulation of PSII.

Genome-wide association study identifies polymorphisms associated with the analgesic effect of fentanyl in the preoperative cold pressor-induced pain test.

Opioid analgesics are widely used for the treatment of moderate to severe pain. The analgesic effects of opioids are well known to vary among individuals. The present study focused on the genetic factors that are associated with interindividual differences in pain and opioid sensitivity. We conducted a multistage genome-wide association study in subjects who were scheduled to undergo mandibular sagittal split ramus osteotomy and were not medicated until they received fentanyl for the induction of anesthesia. We preoperatively conducted the cold pressor-induced pain test before and after fentanyl administration. The rs13093031 and rs12633508 single-nucleotide polymorphisms (SNPs) near the LOC728432 gene region and rs6961071 SNP in the tcag7.1213 gene region were significantly associated with the analgesic effect of fentanyl, based on differences in pain perception latency before and after fentanyl administration. The associations of these three SNPs that were identified in our exploratory study have not been previously reported. The two polymorphic loci (rs13093031 and rs12633508) were shown to be in strong linkage disequilibrium. Subjects with the G/G genotype of the rs13093031 and rs6961071 SNPs presented lower fentanyl-induced analgesia. Our findings provide a basis for investigating genetics-based analgesic sensitivity and personalized pain control.

PCoM-DB Update: A Protein Co-Migration Database for Photosynthetic Organisms.

The identification of protein complexes is important for the understanding of protein structure and function and the regulation of cellular processes. We used blue-native PAGE and tandem mass spectrometry to identify protein complexes systematically, and built a web database, the protein co-migration database (PCoM-DB, http://pcomdb.lowtem.hokudai.ac.jp/proteins/top), to provide prediction tools for protein complexes. PCoM-DB provides migration profiles for any given protein of interest, and allows users to compare them with migration profiles of other proteins, showing the oligomeric states of proteins and thus identifying potential interaction partners. The initial version of PCoM-DB (launched in January 2013) included protein complex data for Synechocystis whole cells and Arabidopsis thaliana thylakoid membranes. Here we report PCoM-DB version 2.0, which includes new data sets and analytical tools. Additional data are included from whole cells of the pelagic marine picocyanobacterium Prochlorococcus marinus, the thermophilic cyanobacterium Thermosynechococcus elongatus, the unicellular green alga Chlamydomonas reinhardtii and the bryophyte Physcomitrella patens. The Arabidopsis protein data now include data for intact mitochondria, intact chloroplasts, chloroplast stroma and chloroplast envelopes. The new tools comprise a multiple-protein search form and a heat map viewer for protein migration profiles. Users can compare migration profiles of a protein of interest among different organelles or compare migration profiles among different proteins within the same sample. For Arabidopsis proteins, users can compare migration profiles of a protein of interest with putative homologous proteins from non-Arabidopsis organisms. The updated PCoM-DB will help researchers find novel protein complexes and estimate their evolutionary changes in the green lineage.

[Value of Fractional Exhaled Nitric Oxide after Using a Beta-2 Bronchodilator in the Differential Diagnosis of Bronchial Asthma and Chronic Obstructive Pulmonary Disease].

It has been established that an increase in fractional exhaled nitric oxide (FeNO) is one of the indicators of bronchial asthma (BA) in clinical settings. However, the differential diagnosis of BA and chronic obstructive pulmonary disease (COPD) is difficult due to pathological similarities. Therefore, to determine if FeNO may be utilized in the differential diagnosis of BA and COPD, we compared FeNO values before and after inhalation of a short-acting beta-2 agonist (SABA). There were 3 groups of subjects recruited to this study: (1) 23 normal healthy controls, (2) 36 patients with BA, and (3) 13 patients with COPD. We measured FeNO, forced vital capacity, forced expiratory volume in 1 second (FEV1), and FEV1%, calculated using spirometry. Then, after the subjects inhaled the SABA, we measured these data after 10 and 30 minutes. Here we found that after inhalation of a SABA, 8 cases in the BA group who showed reversibility of airway obstruction demonstrated significantly increased FeNO values compared to the BA patients with non-reversible airway obstruction, those with COPD, and healthy subjects. This finding may be because the obstructed pulmonary peripheral airway was expanded by inhaling a SABA, and nitric oxide, which had been produced in the peripheral airway, was then exhaled. These results suggest the possibility that FeNO may be utilized in the differential diagnosis of BA and COPD.

Functional analysis of light-harvesting-like protein 3 (LIL3) and its light-harvesting chlorophyll-binding motif in Arabidopsis.

The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. LHC contains a characteristic sequence motif, termed LHC motif, consisting of 25-30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs that are termed light-harvesting-like (LIL) proteins. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein has been shown previously to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes, even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the transmembrane domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (Glu-171, Asn-174, and Asp-189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol-biosynthesizing activity. However, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction.

Consultation-Liaison Psychiatry in the General Hospital: the Experience of UK, Italy, and Japan.

Consultation-liaison psychiatry (CLP) is a psychiatric subspecialty addressing the psychological care of medically ill patients as well as the relationship between medical and psychiatric disorders. As a specialty, it has developed over the last 30 years. Yet several cultural and organizational problems in different countries have emerged, including differences regarding implementing CLP services and in the application of CLP guidelines. This paper examines the progress and challenges of CLP in three culturally diverse countries: the UK, Italy, and Japan. In all three countries, the provision of CLP service remains insufficient, and the application of officially acknowledged standards of care is necessary.

Role of Abscisic Acid, Reactive Oxygen Species, and Ca2+ Signaling in Hydrotropism-Drought Avoidance-Associated Response of Roots.

Plant roots exert hydrotropism in response to moisture gradients to avoid drought stress. The regulatory mechanism underlying hydrotropism involves novel regulators such as MIZ1 and GNOM/MIZ2 as well as abscisic acid (ABA), reactive oxygen species (ROS), and Ca2+ signaling. ABA, ROS, and Ca2+ signaling are also involved in plant responses to drought stress. Although the mechanism of moisture gradient perception remains largely unknown, the sensory apparatus has been reported to reside in the root elongation zone rather than in the root cap. In Arabidopsis roots, hydrotropism is mediated by the action of MIZ1 and ABA in the cortex of the elongation zone, the accumulation of ROS at the root curvature, and the variation in the cytosolic Ca2+ concentration in the entire root tip including the root cap and stele of the elongation zone. Moreover, root exposure to moisture gradients has been proposed to cause asymmetric ABA distribution or Ca2+ signaling, leading to the induction of the hydrotropic response. A comprehensive and detailed analysis of hydrotropism regulators and their signaling network in relation to the tissues required for their function is apparently crucial for understanding the mechanisms unique to root hydrotropism. Here, referring to studies on plant responses to drought stress, we summarize the recent findings relating to the role of ABA, ROS, and Ca2+ signaling in hydrotropism, discuss their functional sites and plausible networks, and raise some questions that need to be answered in future studies.

Spatial distribution and size of small canopy gaps created by Japanese black bears: estimating gap size using dropped branch measurements.

BACKGROUND: Japanese black bears, a large-bodied omnivore, frequently create small gaps in the tree crown during fruit foraging. However, there are no previous reports of black bear-created canopy gaps. To characterize physical canopy disturbance by black bears, we examined a number of parameters, including the species of trees in which canopy gaps were created, gap size, the horizontal and vertical distribution of gaps, and the size of branches broken to create gaps. The size of black bear-created canopy gaps was estimated using data from branches that had been broken and dropped on the ground. RESULTS: The disturbance regime was characterized by a highly biased distribution of small canopy gaps on ridges, a large total overall gap area, a wide range in gap height relative to canopy height, and diversity in gap size. Surprisingly, the annual rate of bear-created canopy gap formation reached 141.3 m2 ha-1 yr-1 on ridges, which were hot spots in terms of black bear activity. This rate was approximately 6.6 times that of tree-fall gap formation on ridges at this study site. Furthermore, this rate was approximately two to three times that of common tree-fall gap formation in Japanese forests, as reported in other studies. CONCLUSIONS: Our findings suggest that the ecological interaction between black bears and fruit-bearing trees may create a unique light regime, distinct from that created by tree falls, which increases the availability of light resources to plants below the canopy.

LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis.

The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in α-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase.

Embryo developmental capability and pregnancy outcome are related to the mitochondrial DNA copy number and ooplasmic volume.

PURPOSE: To investigate the correlation between the ooplasmic volume and the number of mitochondrial DNA (mtDNA) copies in embryos and how they may affect fecundity. METHOD: Using real-time PCR, mtDNA quantification was analyzed in unfertilized oocytes and uncleaved embryos. The size of the ovum was also assessed by calculating the ooplasmic volume at the time of granulosa cell removal for IVF or ICSI. Quantification analysis of the mtDNA in blastomeres was performed by real-time PCR at the 7-8 cell stage of the cleaved embryos at 72 h after oocyte retrieval. We calculated the cytoplasmic volume of the blastomeres. RESULT: Our studies showed a significantly lower mtDNA copy number in unfertilized oocytes and uncleaved embryos in women who were older than 40 years of age (p < 0.05). The larger ooplasmic volume was also associated with earlier and more rapid cleavage (p < 0.05). The ooplasmic volume was also significantly larger in the group achieving pregnancy. We found a significant positive correlation between blastomere volume and the number of mtDNA copies (r = 0.76, p < 0.01, from Pearson product-moment correlation coefficient). CONCLUSIONS: We have shown that blastomere volume is directly proportional to the number of mtDNA copies. Therefore, larger cytoplasmic volume, with earlier cleavage speed, implies more mtDNA copies. Evaluation of mtDNA quantification and the measurement of ooplasmic and blastomere volume may be useful for selection of high quality embryo and pregnancy outcome.

Improvement in persistent idiopathic facial pain with comorbid ADHD using the combination of a dopamine system stabilizer and psychostimulant: A case report.

Key Clinical Message: Persistent idiopathic facial pain (PIFP) and attention-deficit/hyperactivity disorder (ADHD) may coexist and can be improved with ADHD medications. Thus, clinicians should screen for ADHD by a multidisciplinary approach when treating PIFP and differentiate between other odontogenic disorders. Abstract: We report a case of a woman with persistent idiopathic facial pain (PIFP) and attention-deficit/hyperactivity disorder (ADHD) that markedly improved with the administration of a combination of aripiprazole (APZ) and methylphenidate (MP) treatment. Screening for ADHD and administration of APZ and/or MP may be considered in treating PIFP.

Diagnosis and treatment of intractable idiopathic orofacial pain with attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) has been reported to be associated with primary chronic pain syndromes, such as fibromyalgia, migraine, and chronic low back pain. Although idiopathic orofacial pain (IOP) is classified as burning mouth syndrome or persistent idiopathic facial or dentoalveolar pain and as a primary chronic pain, the association between IOP and ADHD has not been investigated. This retrospective cohort study investigated the severity of ADHD symptoms measured using the ADHD scale and the effects of treatment using ADHD drugs and the dopamine system stabilizer aripiprazole. The participants were 25 consecutive patients with refractory IOP referred to a psychiatrist and diagnosed with coexisting ADHD according to the Diagnostic and Statistical Manual of Mental Disorders-5. The ADHD scale scores were higher in patients with intractable IOP than those in the general population. Pharmacotherapy used in this study led to clinically significant improvements in pain, anxiety/depression, and pain catastrophizing. Intractable IOP and ADHD were shown to be associated. In the future, screening and pharmacotherapy for ADHD should be considered in the treatment of intractable IOP.

Therapeutic effect of aged garlic extract on gingivitis in dogs.

Periodontal disease is one of the most common dental health problems in dogs. Clinical studies in humans have shown that aged garlic extract (AGE), which contains stable and water-soluble sulfur-containing bioactive compounds, improves the symptoms of periodontal diseases. Our previous study demonstrated that oral administration of AGE in healthy Beagle dogs at 90 mg/kg/day for 12 weeks had no adverse effects such as hemolytic anemia, which is well known to occur as a result of ingestion of Allium species, including onions and garlic, in dogs. However, the therapeutic potential of AGE in canine periodontal disease remains unclear. Accordingly, we investigated the therapeutic effects of AGE in Beagle dogs with mild gingivitis. Feeding 18 mg/kg/day of AGE for 8 weeks resulted in the improvement of gingival index score, level of volatile sulfur compounds in exhaled air, and enzyme activity of periodontal pathogens without any adverse effects on clinical signs and hematological and serum biochemical parameters. Moreover, AGE increased the concentration of salivary cathelicidin, an antimicrobial peptide that contributes to the oral innate immune response. These results suggest that AGE could be a potential therapeutic agent for canine gingivitis.

A domino-like chlamydial attachment process: concurrent Parachlamydia acanthamoebae attachment to amoebae is required for several amoebal released molecules and serine protease activity.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that infects free-living amoebae (Acanthamoeba), and is a potential human pathogen associated with hospital-acquired pneumonia. The attachment mechanism of this bacteria to host cells is crucial in bacterial pathogenesis, yet remains undetermined. Hence, we obtained monoclonal antibodies (mAbs) specific to either P. acanthamoebae or amoebae in an attempt to elucidate the attachment mechanism involved. Hybridomas of 954 clones were assessed, and we found that four mAbs (mAb38, mAb300, mAb311, mAb562) that were reactive to the amoebae significantly inhibited bacterial attachment. All mAbs recognized amoebal released molecules, and mAb311 also recognized the amoebal surface. mAbs reacted with the bacteria not only within amoebae, but also when they were released from amoebae (except mAb311). Furthermore, a serine protease inhibitor had an inhibitory effect on the bacterial attachment to amoebae, although none of the mAbs had any synergistic effect on the inhibition of attachment by the protease inhibitor. Taken together, we conclude that concurrent P. acanthoamebae attachment to amoebae is required for several amoebal released molecules and serine protease activity, implying the existence of a complicated host-parasite relationship.

Ciliates expel environmental Legionella-laden pellets to stockpile food.

When Tetrahymena ciliates are cultured with Legionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental L. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.

Chlamydia trachomatis serovar L2 infection model using human lymphoid Jurkat cells.

Chlamydia trachomatis L2 invasively attacks lymphatic and subepithelial tissues of the genital tract during the formation of primary lesions. This subsequently results in lymphadenopathy, and suggests a greater propensity for systemic dissemination. However, whether lymphocytes are a potential vehicle cell for the dissemination of this infection remains unknown. We therefore assessed the growth properties of C. trachomatis L2 in lymphoid Jurkat cells compared with those observed in epithelial HeLa cells. Both cells supported the growth of C. trachomatis with a similar increase in infective progenies. Enriched human-blood lymphocytes also supported the C. trachomatis growth as well as Jurkat cells. Bacteria infecting the Jurkat cells were more susceptible to antibiotics (doxycycline, azithromycin, ofloxacin) than those in HeLa cells. Of the sphingomyelin biosynthesis inhibitors tested, both myriocin and fumonisin B1 significantly inhibited bacterial growth in both cells types. A Jurkat cell mutant that impaired bacterial growth was established using ethylmethanesulfonate treatment. DNA microarray analysis with real-time reverse transcription-polymerase chain reaction revealed that the mutant cells over-expressed granzyme K gene. Immunofluorescence staining also indicated that granzyme K irregularly over-expressed among the mutant cells as compared with that of the wild cells, suggesting a possible mechanism refractory to C. trachomatis infection. Thus, we concluded that C. trachomatis L2 could infect Jurkat cells with lymphoid properties, providing a new tool for studying C. trachomatis dissemination to tissues via lymphocyte movement.