Cutavirus Infection in Large-Plaque Parapsoriasis, a Premalignant Condition of Mycosis Fungoides.

BACKGROUND: Cutavirus (CuV) is associated with mycosis fungoides; however, the CuV status in parapsoriasis en plaques (PP), a premalignant inflammatory condition of mycosis fungoides, has not been fully delineated. METHODS: Fifty-five Japanese patients with chronic inflammatory skin diseases, including 13 patients with PP, were studied. RESULTS: CuV DNA was detected significantly more frequently in biopsies of the lesional skin from patients with PP (38%; 4 of 13) than in those from patients with other inflammatory skin diseases (2%; 1 of 42; P = .009). All CuV-positive PP cases were of the large-plaque parapsoriasis (LPP) subtype. The viral loads ranged from 83 450 to 2 164 170 copies/103 cells. We recovered near-full-length CuV sequences from the CuV-positive LPP biopsies, all of which were of the Japanese/Asian genotype. The CuV genome appeared to be present within lymphoid cells infiltrating the epidermis and dermis. CuV NS1 and VP1 gene transcripts were also detected in the affected tissues. CONCLUSIONS: The detection of high levels of CuV DNA with the expression of viral mRNA suggests a potential role for CuV in the pathogenesis of LPP, making it necessary to study further the impact of CuV, especially regarding the viral genotype, on the outcomes of patients with CuV-positive LPP.

Cutavirus Infection in Large Plaque Parapsoriasis, a Premalignant Condition of Mycosis Fungoides.

BACKGROUND: Cutavirus (CuV) is associated with mycosis fungoides; however, the CuV status in parapsoriasis en plaques (PP), a premalignant inflammatory condition of mycosis fungoides, has not been fully delineated. METHODS: Fifty-five Japanese patients with chronic inflammatory skin diseases, including 13 patients with PP, were studied. RESULTS: CuV DNA was detected significantly more frequently in biopsies of the lesional skin from patients with PP (38% [4/13]) than in those from patients with other inflammatory skin diseases (2% [1/42]; P = 0.009). All CuV-positive PP cases were of the large plaque parapsoriasis (LPP) subtype. The viral loads ranged from 83,450 to 2,164,170 copies/103 cells. We recovered near-full-length CuV sequences from the CuV-positive LPP biopsies, all of which were of the Japanese/Asian genotype. The CuV genome appeared to be present within lymphoid cells infiltrating the epidermis and dermis. CuV NS1 and VP1 gene transcripts were also detected in the affected tissues. CONCLUSIONS: The preferential detection of high levels of CuV DNA with the expression of viral mRNA suggests a potential role for CuV in the pathogenesis of LPP, making it necessary to study further the impact of CuV, especially regarding the viral genotype, on the outcomes of patients with CuV-positive LPP.

EBV-positive pyothorax-associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma associated with chronic inflammation (DLBCL-CI) develops in the setting of long-standing inflammation. This type of lymphoma may have specific expression profiles of chemokines involved in the pathogenesis of DLBCL-CI. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and represents a valuable model for the study of this disease category. Using a panel of PAL cell lines, we found that PAL cells expressed and secreted C-X-C motif chemokine ligands 9 and 10 (CXCL9 and CXCL10), the ligands of CXCR3, in contrast to EBV-negative DLBCL cell lines, which did not. Culture supernatants from PAL cell lines attracted CXCR3-expressing CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells from human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CXCR3-positive cytotoxic lymphocytes that expressed interferon-γ. The expression of CXCL9 and CXCL10 was detected in PAL tumor biopsy samples from patients, and CXCR3-positive lymphocytes were abundant in the tissue samples. Collectively, these findings suggest that CXCL9 and CXCL10 are produced by PAL cells and can elicit cytotoxic responses via CXCR3. This chemokine system is also likely to contribute to tissue necrosis, which is a signature histological feature of DLBCL-CI. Further studies are warranted to determine whether the CXCL9-CXCL10/CXCR3 axis exerts antitumor effects in DLBCL-CI.

Intracameral Bacteriophage Injection as Postoperative Prophylaxis for Enterococcus faecalis-Induced Endophthalmitis After Cataract Surgery in Rabbits.

Purpose: Post-cataract surgery bacterial endophthalmitis is a serious postoperative complication, and Enterococcus spp.-induced endophthalmitis reportedly has a particularly poor visual prognosis. This study aimed to demonstrate the prophylactic effect of postoperative intracameral phage administration in Enterococcus faecalis-induced endophthalmitis after cataract surgery in rabbits. Methods: Endophthalmitis was induced in rabbits by injecting E. faecalis into the anterior chamber just after lensectomy while simultaneously administering either phage phiEF24C-P2 or vehicle. Retinal function was evaluated using electroretinography. The number of viable bacteria and myeloperoxidase (MPO) activity in the eye and histopathologic examinations were analyzed 48 hours after infection. Results: In the vehicle-treated group, retinal function at 24 hours after infection was impaired, and the number of viable bacteria and MPO activity in the eye increased 48 hours later. In the phage-administered group, retinal function was maintained; the number of viable bacteria and MPO activity were significantly suppressed. Histopathologic examinations showed disruption of the retinal layers and the presence of numerous E. faecalis in the lens capsule and vitreous cavity in vehicle-treated eyes. In contrast, retinal structures were intact, and no E. faecalis staining was observed in phage-treated eyes. No retinal dysfunction was observed in the group that received phage only without lensectomy; almost no phage was detected in the eyes after 14 days of treatment. Conclusions: Phage administration in the anterior chamber did not cause retinal dysfunction and suppressed postoperative endophthalmitis in rabbits. Translational Relevance: In vivo results of intracameral phage administration suggest that phages are a promising prophylactic candidate for postoperative endophthalmitis.

Development of a novel cell line-derived xenograft model of primary herpesvirus 8-unrelated effusion large B-cell lymphoma and antitumor activity of birabresib in vitro and in vivo.

BACKGROUND: Primary human herpesvirus 8 (HHV8)-unrelated effusion large B-cell lymphoma is a clinical disease entity distinct from HHV8-positive primary effusion lymphoma (PEL). However, the lack of experimental HHV8-unrelated effusion large B-cell lymphoma models continues to hinder the pathophysiologic and therapeutic investigations of this disorder. METHODS: The lymphoma cells were obtained from the pleural effusion of a patient with primary HHV8-unrelated effusion large B-cell lymphoma and cultured in vitro. RESULTS: We established a novel HHV8-unrelated effusion large B-cell lymphoma cell line, designated Pell-1, carrying a c-MYC rearrangement with features distinct from those of HHV8-positive PEL. Moreover, we developed an HHV8-unrelated effusion large B-cell lymphoma cell line-derived xenograft model. Pell-1 cells induced profuse lymphomatous ascites and subsequently formed intra-abdominal tumors after intraperitoneal implantation into irradiated nonobese diabetic/severe combined immunodeficient mice. Thus, this xenograft mouse model mimicked the clinical phenomena observed in patients and recapitulated the sequential stages of aggressive HHV8-unrelated effusion large B-cell lymphoma. The bromodomain and extraterminal domain (BET) inhibitors JQ1 and birabresib (MK-8628/OTX015) reduced the proliferation of Pell-1 cells in vitro through the induction of cell cycle arrest and apoptosis. The antitumor effect of BET inhibition was also demonstrated in vivo, as birabresib significantly reduced ascites and suppressed tumor progression without apparent adverse effects in the xenografted mice. CONCLUSION: These preclinical findings suggest the therapeutic potential of targeting c-MYC through BET inhibition in HHV8-unrelated effusion large B-cell lymphoma.

Complete Genome Sequence of Helicobacter pylori Strain 3401, a Suitable Host for Bacteriophages KHP30 and KHP40.

Helicobacter pylori 3401, isolated from a patient with duodenal ulcers in Japan, is susceptible to the bacteriophages KHP30 and KHP40. In this study, we report the complete genome sequence of H. pylori 3401. This study may lead to the establishment of phage therapy against H. pylori infection.

In Vitro and In Vivo Evaluation of Three Newly Isolated Bacteriophage Candidates, phiEF7H, phiEF14H1, phiEF19G, for Treatment of Enterococcus faecalis Endophthalmitis.

Post-operative endophthalmitis caused by Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Therefore, novel alternative treatments that are effective against enterococcal endophthalmitis are required. Bacteriophage therapy has the potential to be an optional therapy for infectious diseases. Therefore, we investigated the therapeutic potential of three newly isolated enterococcal phages, phiEF7H, phiEF14H1, and phiEF19G, in E. faecalis-induced endophthalmitis. These phages could lyse the broad-range E. faecalis, including strains derived from endophthalmitis and vancomycin-resistant E. faecalis in vitro, as determined by the streak test. Morphological and genomic analyses revealed that these phages were classified into the Herelleviridae genus Kochikohdavirus. The whole genomes of these phages contained 143,399, 143,280, and 143,400 bp, respectively. Endophthalmitis was induced in mice by injection of three strains of E. faecalis derived from post-operative endophthalmitis or vancomycin-resistant strains into the vitreous body. The number of viable bacteria and infiltration of neutrophils in the eye were both decreased by intravitreous injection of phiEF7H, phiEF14H1, and phiEF19G 6 h after injection of all E. faecalis strains. Thus, these results suggest that these newly isolated phages may serve as promising candidates for phage therapy against endophthalmitis.

Human Polyomavirus 6 Detected in Cases of Eosinophilic Pustular Folliculitis.

BACKGROUND: Human polyomaviruses (HPyVs) have been associated with several cutaneous inflammatory conditions. More investigation is needed to identify further presentations of cutaneous pathology associated with HPyVs. Our aim was to investigate the possible association of skin-tropic HPyVs with folliculitis, particularly eosinophilic pustular folliculitis (EPF). METHODS: This study included 55 Japanese patients, comprising 13 patients with EPF and 42 patients with suppurative folliculitis. HPyV DNAs were detected by quantitative polymerase chain reaction. Expression of viral antigen and geographically related viral genotypes were also assessed. RESULTS: Human polyomavirus 6 (HPyV6) DNA was found in 9 of 13 (69%) patients with EPF, a rate significantly higher than that found in suppurative folliculitis (1/42; 2%). Of the 7 HPyV6 DNA-positive EPF specimens analyzed, 4 were positive for HPyV6 small tumor antigen. All the HPyV6 strains detected in this study were of the Asian/Japanese genotype. CONCLUSIONS: The predominant detection of HPyV6 DNA and the expression of viral antigen suggest a possible association between HPyV6 infection and EPF in a subset of patients. Worldwide studies are warranted to determine whether Asian/Japanese genotype HPyV6 is associated preferentially with the incidence and pathogenesis of this eosinophil-related skin disease that has an ethnic predilection for the East Asian population.

Epstein-Barr virus-positive pyothorax-associated lymphoma expresses CCL17 and CCL22 chemokines that attract CCR4-expressing regulatory T cells.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas associated with chronic inflammation (DLBCL-CI) develop in patients with chronic inflammation but without any predisposing immunodeficiency. Given the expression of the EBV latent genes, DLBCL-CI should have mechanisms for evasion of host antitumor immunity. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and may provide a valuable model for the study of immune evasion by DLBCL-CI. This study demonstrates that PAL cell lines express and secrete CCL17 and/or CCL22 chemokines, the ligands of C-C motif chemokine receptor 4 (CCR4), in contrast to EBV-negative DLBCL cell lines. Accordingly, culture supernatants of PAL cell lines efficiently attracted CCR4-positive regulatory T (Treg) cells in human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CCR4-expressing Treg cells. Furthermore, this study confirmed that CCR4-expressing Treg cells were abundantly present in primary PAL tissues. Collectively, these findings provide new insight into the mechanisms of immune evasion by PAL, and further studies are warranted on whether such mechanisms eventually lead to the development of DLBCL-CI.

Piperacillin and ceftazidime produce the strongest synergistic phage-antibiotic effect in Pseudomonas aeruginosa.

The combined use of phage and antibiotics can show synergistic antimicrobial effects, so-called phage-antibiotic synergy (PAS). Here, we screened and examined PAS against Pseudomonas aeruginosa in vitro. Testing four different phages infecting P. aeruginosa, phage KPP22 classified within the family Myoviridae genus Pbunavirus showed PAS with the widest range of antibiotics, and showed PAS with anti-Pseudomonas drugs such as piperacillin and ceftazidime. Thus, evidence suggests that the combined use of phage and antibiotics is a promising therapeutic strategy against P. aeruginosa infections, with consideration needed regarding the optimal selection and adequate application timing of these phages and antibiotics.

Recovery of mycobacteriophages from archival stocks stored for approximately 50 years in Japan.

Mycobacteriophage archival stocks have been kept for ca. 20-50 years in Japan. In this study, we attempted to recover mycobacteriophages from 50 archival stocks and briefly analyzed the recovered phages. The phages were recovered from 72.2% (13/18) of the lyophilized stocks that had been stored for 47-56 years. Moreover, the analysis of 12 representative recovered phages led to their classification as belonging to the family Siphoviridae, and seven of them were typed by polymerase chain reaction (PCR) targeting the gene that encodes the tape measure protein. Considering these results, lyophilization seems to be suitable for phage archival storage.

Isolation of Bacteriophages for Fastidious Bacteria.

One of the most important factors for successful bacteriophage therapy is, undoubtedly, the isolation of excellent therapeutic candidate bacteriophages. There are only a few reports about active bacteriophages in the fastidious bacteria Helicobacter pylori. In this chapter, we describe a method for isolating and purifying KHP30-like bacteriophages in H. pylori, which have lytic and pseudolysogenic life cycles.

Virus purification by CsCl density gradient using general centrifugation.

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Adsorption of Staphylococcus viruses S13' and S24-1 on Staphylococcus aureus strains with different glycosidic linkage patterns of wall teichoic acids.

The group of phages belonging to the family Podoviridae, genus P68virus, including Staphylococcus viruses S13' and S24-1, are important because of their benefits in phage therapy against Staphylococcus aureus infections. The O-glycosidic linkage patterns of wall teichoic acids (WTAs) in S. aureus cell walls seem to be important for adsorption of this phage group. In this study, the adsorption of Staphylococcus viruses S13' and S24-1 to S. aureus was examined using strains with modified WTA glycosidic linkage patterns. We found that the ß-O-N-acetylglucosamine of WTAs was essential for S13' adsorption, while N-acetylglucosamine, regardless of the α- and ß-O-glycosidic linkages of the WTAs, was essential for S24-1 adsorption. Next, examining the binding activities of their receptor-binding proteins (RBPs) to cell walls with different WTA glycosidic patterns, the ß-O-N-acetylglucosamine of the WTAs was essential for S13' RBP binding, while N-acetylglucosamine, regardless of the α- and ß-O-glycosidic linkages of the WTAs, was essential for S24-1 RBP binding. Therefore, the results of the RBP binding assays were consistent with those of the phage adsorption assays. Bioinformatic analysis suggested that the RBPs of Staphylococcus viruses S13' and S24-1 were structurally similar to the RBPs of phage phi11 of thefamily Siphoviridae. Phylogenetic analysis of the RBPs indicated that two phylogenetic subclusters in the family Podoviridae were related to the glycosidic linkage patterns required for phage adsorption, possibly mediated by RBPs. We hope that this study will encourage the future development of therapeutic phages.

Screening of KHP30-like prophages among Japanese Helicobacter pylori strains, and genetic analysis of a defective KHP30-like prophage sequence integrated in the genome of the H. pylori strain NY40.

We have recently reported the active Helicobacter pylori bacteriophages (phages), KHP30 and KHP40, the genomic DNAs of which exist as episomes in host bacterial strains isolated in Japan (i.e. pseudolysogeny). In this study, we examined the possibility of the lysogeny of active KHP30-like phages in Japanese H. pylori strains, because their genomes contain a putative integrase gene. Only the NY40 strain yielded partial detection of a KHP30-like prophage sequence in PCR among 174 Japanese H. pylori isolates, except for strains producing the above active phages. Next, according to the genomic analysis of the NY40 strain, the KHP30-like prophage sequence was found to be located from ca. 524 to 549 kb in the host chromosome. The attachment sites, attL and attR, in the NY40 genome showed almost the same genomic location and sequence as those detected in a French isolate B38, suggesting that an active parental KHP30-like phage had integrated into the ancestral NY40 genome in a site-specific manner. The prophage found in the NY40 genome was assumed to have been genetically modified, after site-specific integration. These, together with the data in the KHP30-like prophages of other H. pylori genomes, suggest that the lysogenic state of the KHP30-like phages is generally unstable.

Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus).

UNLABELLED: Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. IMPORTANCE: Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4-like viruses isolated in Japan.

Bacteriophages (phages) belonging to the family Podoviridae genus N4-like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4-like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4-like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.

Genome Analysis of Pseudomonas aeruginosa Bacteriophage KPP23, Belonging to the Family Siphoviridae.

Bacteriophage (phage) therapy is expected to become an alternative therapy for Pseudomonas aeruginosa infections. P. aeruginosa phage KPP23 is a newly isolated phage belonging to the family Siphoviridae and may be a therapeutic phage candidate. We report its complete genome, which comprises 62,774 bp of double-stranded DNA containing 95 open reading frames.

Characterization of a novel Pseudomonas aeruginosa bacteriophage, KPP25, of the family Podoviridae.

Pseudomonas aeruginosa phages belonging to the family Podoviridae are one of the well-characterized phage groups. In this study, a novel P. aeruginosa phage, KPP25, was isolated and characterized. Phage KPP25's morphology was indicative of the family Podoviridae; however, analyses of the whole genome and the virion proteins suggested that it did not belong to any of the known podophage genera. Based on these analyses, phage KPP25 appears to be a novel podophage infecting P. aeruginosa.