Vaccination with short-term-cultured autologous PBMCs efficiently activated STLV-1-specific CTLs in naturally STLV-1-infected Japanese monkeys with impaired CTL responses.

A small proportion of human T-cell leukemia virus type-1 (HTLV-1)-infected individuals develop adult T-cell leukemia/lymphoma, a chemotherapy-resistant lymphoproliferative disease with a poor prognosis. HTLV-1-specific cytotoxic T lymphocytes (CTLs), potential anti-tumor/virus effectors, are impaired in adult T-cell leukemia/lymphoma patients. Here, using Japanese monkeys naturally infected with simian T-cell leukemia/T-lymphotropic virus type-1 (STLV-1) as a model, we demonstrate that short-term-cultured autologous peripheral blood mononuclear cells (PBMCs) can serve as a therapeutic vaccine to activate such CTLs. In a screening test, STLV-1-specific CTL activity was detectable in 8/10 naturally STLV-1-infected monkeys. We conducted a vaccine study in the remaining two monkeys with impaired CTL responses. The short-term-cultured PBMCs of these monkeys spontaneously expressed viral antigens, in a similar way to PBMCs from human HTLV-1 carriers. The first monkey was subcutaneously inoculated with three-day-cultured and mitomycin C (MMC)-treated autologous PBMCs, and then boosted with MMC-treated autologous STLV-1-infected cell line cells. The second monkey was inoculated with autologous PBMC-vaccine alone twice. In addition, a third monkey that originally showed a weak STLV-1-specific CTL response was inoculated with similar autologous PBMC-vaccines. In all three vaccinated monkeys, marked activation of STLV-1-specific CTLs and a mild reduction in the STLV-1 proviral load were observed. Follow-up analyses on the two monkeys vaccinated with PBMCs alone indicated that STLV-1-specific CTL responses peaked at 3-4 months after vaccination, and then diminished but remained detectable for more than one year. The significant reduction in the proviral load and the control of viral expression were associated with CTL activation but also diminished 6 and 12 months after vaccination, respectively, suggesting the requirement for a booster. The vaccine-induced CTLs in these monkeys recognized epitopes in the STLV-1 Tax and/or Envelope proteins, and efficiently killed autologous STLV-1-infected cells in vitro. These findings indicated that the autologous PBMC-based vaccine could induce functional STLV-1-specific CTLs in vivo.

Short-term cultured autologous peripheral blood mononuclear cells as a potential immunogen to activate Tax-specific CTL response in adult T-cell leukemia patients.

Activation of CD8+ Tax-specific CTL is a new therapeutic concept for adult T-cell leukemia (ATL) caused by HTLV-1. A recent clinical study of the dendritic cell vaccine pulsed with Tax peptides corresponding to CTL epitopes showed promising outcomes in ATL patients possessing limited human leukocyte antigen (HLA) alleles. In this study, we aimed to develop another immunotherapy to activate Tax-specific CTL without HLA limitation by using patients' own HTLV-1-infected cells as a vaccine. To examine the potential of HTLV-1-infected T-cells to activate CTL via antigen presenting cells, we established a unique co-culture system. We demonstrated that mitomycin C-treated HLA-A2-negative HTLV-1-infected T-cell lines or short-term cultured peripheral blood mononuclear cells (PBMC) derived from ATL patients induced cross-presentation of Tax antigen in co-cultured HLA-A2-positive antigen presenting cells, resulting in activation of HLA-A2-restricted CD8+ Tax-specific CTL. This effect was not inhibited by a reverse transcriptase inhibitor. IL-12 production and CD86 expression were also induced in antigen presenting cells co-cultured with HTLV-1-infected cells at various levels, which were improved by pre-treatment of the infected cells with histone deacetylase inhibitors. Furthermore, monocyte-derived dendritic cells induced from PBMC of a chronic ATL patient produced IL-12 and expressed enhanced levels of CD86 when co-cultured with autologous lymphocytes that had been isolated from the same PBMC and cultured for several days. These findings suggest that short-term cultured autologous PBMC from ATL patients could potentially serve as a vaccine to evoke Tax-specific CTL responses.

Involvement of EZH2 inhibition in lenalidomide and pomalidomide-mediated growth suppression in HTLV-1-infected cells.

Immunomodulatory imide drugs (IMiDs), such as lenalidomide and pomalidomide, exert pleiotropic effects, e.g., antitumor effects in multiple myeloma, by binding the protein Cereblon and altering its substrate specificity. Lenalidomide is approved for the treatment of adult T-cell leukemia/lymphoma (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1), although the precise mechanisms responsible for its effectiveness have not been fully elucidated. Here, we used HTLV-1-infected cell lines to investigate how IMiDs exert anti-ATL effects. In three of four tested HTLV-1-infected cell lines, the cells treated with lenalidomide or pomalidomide exhibited mild growth suppression without apoptosis, which was associated with decreased IRF4, c-Myc, and phosphorylated STAT3 levels as well as enhanced SOCS3 expression. Additionally, the levels of enhancer of zeste homolog 2 (EZH2) and trimethyl histone 3 Lys27 (H3K27me3) were decreased following IMiD treatment in all three susceptible cell lines. An IMiD-mediated reduction of EZH2 and H3K27me3 levels was also observed in a multiple myeloma cell line. Furthermore, treatment with an EZH2-inhibitor reproduced the IMiD-mediated effects in HTLV-1-infected cells and multiple myeloma cells. These findings strongly suggest that a reduction of EZH2 expression is involved in the mechanism underlying the antitumor effects of IMiD.

Maintenance of long remission in adult T-cell leukemia by Tax-targeted vaccine: A hope for disease-preventive therapy.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive lymphoproliferative disease caused by human T-cell leukemia virus type 1 (HTLV-1). Multi-agent chemotherapy can reduce ATL cells but frequently allows relapses within a short period of time. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) following chemotherapy is now a standard therapy for ATL in Japan as it can achieve long-term remission in approximately one-third of recipient ATL patients; however, it also has a risk of treatment-related mortality. Allo-HSCT often induces HTLV-1 Tax-specific cytotoxic T cells (CTL) as well as graft-versus-host (GVH) response in ATL patients. This observation led to development of a new therapeutic vaccine to activate Tax-specific CTL, anticipating anti-ATL effects without GVH response. The newly developed Tax-DC vaccine consists of autologous dendritic cells pulsed with Tax peptides corresponding to CTL epitopes that have been identified in post-allo-HSCT ATL patients. In a pilot study of Tax-DC therapy in three ATL patients after various initial therapies, two patients survived for more than 4 years after vaccination without severe adverse effects (UMIN000011423). The Tax-DC vaccine is currently under phase I trial, showing a promising clinical outcome so far. These findings indicate the importance of patients' own HTLV-1-specific T-cell responses in maintaining remission and provide a new approach to anti-ATL immunotherapy targeting Tax. Although Tax-targeted vaccination is ineffective against Tax-negative ATL cells, it can be a safe alternative maintenance therapy for Tax-positive ATL and may be further applicable for treatment of indolent ATL or even prophylaxis of ATL development among HTLV-1-carriers.

Impact of host immunity on HTLV-1 pathogenesis: potential of Tax-targeted immunotherapy against ATL.

Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. There is no disease-specific difference in viral strains, and it is unclear how HTLV-1 causes such different diseases manifesting as lymphoproliferation or inflammation. Although some progress has been made in therapies for these diseases, the prognosis for ATL is still dismal and HAM/TSP remains an intractable disease. So far, two regulatory proteins of HTLV-1, Tax and HBZ, have been well studied and shown to have pleiotropic functions implicated in viral pathogenesis. Tax in particular can strongly activate NFκB, which is constitutively activated in HTLV-1-infected cells and considered to contribute to both oncogenesis and inflammation. However, the expression level of Tax is very low in vivo, leading to confusion in understanding its role in viral pathogenesis. A series of studies using IL-2-dependent HTLV-1-infected cells indicated that IL-10, an anti-inflammatory/immune suppressive cytokine, could induce a proliferative phenotype in HTLV-1-infected cells. In addition, type I interferon (IFN) suppresses HTLV-1 expression in a reversible manner. These findings suggest involvement of host innate immunity in the switch between lymphoproliferative and inflammatory diseases as well as the regulation of HTLV-1 expression. Innate immune responses also affect another important host determinant, Tax-specific cytotoxic T lymphocytes (CTLs), which are impaired in ATL patients, while activated in HAM/TSP patients. Activation of Tax-specific CTLs in ATL patients after hematopoietic stem cell transplantation indicates Tax expression and its fluctuation in vivo. A recently developed anti-ATL therapeutic vaccine, consisting of Tax peptide-pulsed dendritic cells, induced Tax-specific CTL responses in ATL patients and exhibited favorable clinical outcomes, unless Tax-defective ATL clones emerged. These findings support the significance of Tax in HTLV-1 pathogenesis, at least in part, and encourage Tax-targeted immunotherapy in ATL. Host innate and acquired immune responses induce host microenvironments that modify HTLV-1-encoded pathogenesis and establish a complicated network for development of diseases in HTLV-1 infection. Both host and viral factors should be taken into consideration in development of therapeutic and prophylactic strategies in HTLV-1 infection.

Impact of 5'-end nucleotide modifications of HIV-1 genomic RNA on reverse transcription.

Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5'-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (7mG), a hallmark of transcripts generated by RNA polymerase II, at the 5'-end G nucleotide (5'-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3' terminal region of viral RNA (3'-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5'-G with cap or triphosphate form demonstrated that the 5'-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5'-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3'-GGG tract. Lack of apparent contribution of the 3'-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3'-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that 7mG of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3'-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5'-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5'-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5'-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5'-G with 3'-end region of viral RNA.

IL-10-mediated signals act as a switch for lymphoproliferation in Human T-cell leukemia virus type-1 infection by activating the STAT3 and IRF4 pathways.

Human T-cell leukemia virus type-1 (HTLV-1) causes two distinct diseases, adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since there are no disease-specific differences among HTLV-1 strains, the etiological mechanisms separating these respective lymphoproliferative and inflammatory diseases are not well understood. In this study, by using IL-2-dependent HTLV-1-infected T-cell lines (ILTs) established from patients with ATL and HAM/TSP, we demonstrate that the anti-inflammatory cytokine IL-10 and its downstream signals potentially act as a switch for proliferation in HTLV-1-infected cells. Among six ILTs used, ILTs derived from all three ATL patients grew much faster than those from three HAM/TSP patients. Although most of the ILTs tested produced IFN-γ and IL-6, the production of IL-10 was preferentially observed in the rapid-growing ILTs. Interestingly, treatment with exogenous IL-10 markedly enhanced proliferation of the slow-growing HAM/TSP-derived ILTs. The IL-10-mediated proliferation of these ILTs was associated with phosphorylation of STAT3 and induction of survivin and IRF4, all of which are characteristics of ATL cells. Knockdown of STAT3 reduced expression of IL-10, implying a positive-feedback regulation between STAT3 and IL-10. STAT3 knockdown also reduced survivin and IRF4 in the IL-10- producing or IL-10- treated ILTs. IRF4 knockdown further suppressed survivin expression and the cell growth in these ILTs. These findings indicate that the IL-10-mediated signals promote cell proliferation in HTLV-1-infected cells through the STAT3 and IRF4 pathways. Our results imply that, although HTLV-1 infection alone may not be sufficient for cell proliferation, IL-10 and its signaling pathways within the infected cell itself and/or its surrounding microenvironment may play a critical role in pushing HTLV-1-infected cells towards proliferation at the early stages of HTLV-1 leukemogenesis. This study provides useful information for understanding of disease mechanisms and disease-prophylactic strategies in HTLV-1 infection.

HTLV-1 Tax-Specific CTL Epitope-Pulsed Dendritic Cell Therapy Reduces Proviral Load in Infected Rats with Immune Tolerance against Tax.

Adult T cell leukemia/lymphoma (ATL), a CD4+ T cell malignancy with a poor prognosis, is caused by human T cell leukemia virus type 1 (HTLV-1) infection. High proviral load (PVL) is a risk factor for the progression to ATL. We previously reported that some asymptomatic carriers had severely reduced functions of CTLs against HTLV-1 Tax, the major target Ag. Furthermore, the CTL responses tended to be inversely correlated with PVL, suggesting that weak HTLV-1-specific CTL responses may be involved in the elevation of PVL. Our previous animal studies indicated that oral HTLV-1 infection, the major route of infection, caused persistent infection with higher PVL in rats compared with other routes. In this study, we found that Tax-specific CD8+ T cells were present, but not functional, in orally infected rats as observed in some human asymptomatic carriers. Even in the infected rats with immune unresponsiveness against Tax, Tax-specific CTL epitope-pulsed dendritic cell (DC) therapy reduced the PVL and induced Tax-specific CD8+ T cells capable of proliferating and producing IFN-γ. Furthermore, we found that monocyte-derived DCs from most infected individuals still had the capacity to stimulate CMV-specific autologous CTLs in vitro, indicating that DC therapy may be applicable to most infected individuals. These data suggest that peptide-pulsed DC immunotherapy will be useful to induce functional HTLV-1-specific CTLs and decrease PVL in infected individuals with high PVL and impaired HTLV-1-specific CTL responses, thereby reducing the risk of the development of ATL.

Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.

Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. IMPORTANCE: Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.

Critical Role for Monocytes/Macrophages in Rapid Progression to AIDS in Pediatric Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Infant humans and rhesus macaques infected with the human or simian immunodeficiency virus (HIV or SIV), respectively, express higher viral loads and progress more rapidly to AIDS than infected adults. Activated memory CD4+ T cells in intestinal tissues are major primary target cells for SIV/HIV infection, and massive depletion of these cells is considered a major cause of immunodeficiency. Monocytes and macrophages are important cells of innate immunity and also are targets of HIV/SIV infection. We reported previously that a high peripheral blood monocyte turnover rate was predictive for the onset of disease progression to AIDS in SIV-infected adult macaques. The purpose of this study was to determine if earlier or higher infection of monocytes/macrophages contributes to the more rapid progression to AIDS in infants. We observed that uninfected infant rhesus macaques exhibited higher physiologic baseline monocyte turnover than adults. Early after SIV infection, the monocyte turnover further increased, and it remained high during progression to AIDS. A high percentage of terminal deoxynucleotidyltransferase dUTP nick end label (TUNEL)-positive macrophages in the lymph nodes (LNs) and intestine corresponded with an increasing number of macrophages derived from circulating monocytes (bromodeoxyuridine positive [BrdU+] CD163+), suggesting that the increased blood monocyte turnover was required to rapidly replenish destroyed tissue macrophages. Immunofluorescence analysis further demonstrated that macrophages were a significant portion of the virus-producing cells found in LNs, intestinal tissues, and lungs. The higher baseline monocyte turnover in infant macaques and subsequent macrophage damage by SIV infection may help explain the basis of more rapid disease progression to AIDS in infants.IMPORTANCE HIV infection progresses much more rapidly in pediatric cases than in adults; however, the mechanism for this difference is unclear. Using the rhesus macaque model, this work was performed to address why infants infected with SIV progress more quickly to AIDS than do adults. Earlier we reported that in adult rhesus macaques, increasing monocyte turnover reflected tissue macrophage damage by SIV and was predictive of terminal disease progression to AIDS. Here we report that uninfected infant rhesus macaques exhibited a higher physiological baseline monocyte turnover rate than adults. Furthermore, once infected with SIV, infants displayed further increased monocyte turnover that may have facilitated the accelerated progression to AIDS. These results support a role for monocytes and macrophages in the pathogenesis of SIV/HIV and begin to explain why infants are more prone to rapid disease progression.

Correction to: Resistance Mechanism in a Terbinafine-Resistant Strain of Microsporum canis.

In the initial online publication, the name of author Hock Siew Han was given incorrectly as Han Hock Siew. The original article has been corrected.

Resistance Mechanism in a Terbinafine-Resistant Strain of Microsporum canis.

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2-4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.

A novel mother-to-child human T-cell leukaemia virus type 1 (HTLV-1) transmission model for investigating the role of maternal anti-HTLV-1 antibodies using orally infected mother rats.

Human T-cell leukaemia virus type 1 (HTLV-1) is a human retrovirus that is a causative agent of adult T-cell leukaemia/lymphoma (ATL) and is mainly transmitted from an infected mother to her child via breastfeeding. Such an HTLV-1 infection during childhood is believed to be a risk factor for ATL development. Although it has been suggested that an increased proviral load (PVL), a higher titre of antibody (Ab) in the infected mother and prolonged breastfeeding are associated with an increased risk of mother-to-child transmission (MTCT), the mechanisms underlying MTCT of HTLV-1 remain largely unknown. In this study, we developed an MTCT model using orally HTLV-1-infected rats that have no Ab responses against viral antigens, such as Gag and Env. In this model, HTLV-1 could be transmitted from the infected mother rats to their offspring at a high rate (50-100 %), and the rate of MTCT tended to be correlated with the PVL of the infected mother rats. Furthermore, passive immunization of uninfected adult rats and an infected mother rat with a rat anti-HTLV-1 Env gp46-neutralizing mAb was unable to suppress primary oral HTLV-1 infection to the adult rats and vertical HTLV-1 transmission to the offspring, respectively. Our findings indicate that this MTCT model would be useful to investigate not only the mechanisms of MTCT but also the role of anti-HTLV-1 Ab in MTCT of HTLV-1. They also provide some information on the role of maternal Abs in MTCT, which should be considered when designing a strategy for prevention of MTCT of HTLV-1.

Multi-azole-resistant strains of Cryptococcus neoformans var. grubii isolated from a FLZ-resistant strain by culturing in medium containing voriconazole.

A Cryptococcus neoformans var. grubii strain, NUBS14020, was the first fluconazole (FLZ)-resistant strain isolated from a feline cryptococcosis. Subsequent work demonstrated that multi-azole-resistant strains are readily isolated from FLZ-resistant strains by culturing in medium containing voriconazole (VRZ). The resulting clones were assessed for mutation and expression of known target genes, including the loci encoding lanosterol 14-α demethylase (ERG11), an ATP-binding cassette (ABC) transporter (AFR1), or a multidrug efflux pump (MEP); mutation and/or overexpression of these genes is known to be associated with azole resistance. We also examined the interaction between an efflux blocker (FK506, calcineurin inhibitor) and VRZ in the multi-azole-resistant strains. The ERG11 genes from multi-azole-resistant strains encoded a protein with a G344S substitution. Expression levels of AFR1, ERG11, and MEP in the multi-azole-resistant strains were not higher than those in the VRZ-susceptible parent strain (NUBS14020) when cultured in Sabourad's dextrose broth containing VRZ. Synergistic effects between FK506 and VRC were observed in all of the multi-azole-resistant strains. The minimal inhibitory concentrations (MICs) of the combination of VRZ and FK506 in multi-azole-resistant strains were 4 to 8 times lower that the MICs of VRZ alone. To the best of our knowledge, this work represents the first report that multi-azole-resistant strains of C. neoformans encode a G344S substitution in Erg11p. Further investigation will be needed to determine the mechanism of multi-azole resistance in C. neoformans, given that feline cryptococcosis due to multi- azole-resistant strains is readily transmitted from cats to humans.

Differentiation Kinetics of Blood Monocytes and Dendritic Cells in Macaques: Insights to Understanding Human Myeloid Cell Development.

Monocyte and dendritic cell (DC) development was evaluated using in vivo BrdU pulse-chase analyses in rhesus macaques, and phenotype analyses of these cells in blood also were assessed by immunostaining and flow cytometry for comparisons among rhesus, cynomolgus, and pigtail macaques, as well as African green monkeys and humans. The nonhuman primate species and humans have three subsets of monocytes, CD14(+)CD16(-), CD14(+)CD16(+), and CD14(-)CD16(+) cells, which correspond to classical, intermediate, and nonclassical monocytes, respectively. In addition, there exist presently two subsets of DC, BDCA-1(+) myeloid DC and CD123(+) plasmacytoid DC, that were first confirmed in rhesus macaque blood. Following BrdU inoculation, labeled cells first appeared in CD14(+)CD16(-) monocytes, then in CD14(+)CD16(+) cells, and finally in CD14(-)CD16(+) cells, thus defining different stages of monocyte maturation. A fraction of the classical CD14(+)CD16(-) monocytes gradually expressed CD16(+) to become CD16(+)CD14(+) cells and subsequently matured into the nonclassical CD14(-)CD16(+) cell subset. The differentiation kinetics of BDCA-1(+) myeloid DC and CD123(+) plasmacytoid DC were distinct from the monocyte subsets, indicating differences in their myeloid cell origins. Results from studies utilizing nonhuman primates provide valuable information about the turnover, kinetics, and maturation of the different subsets of monocytes and DC using approaches that cannot readily be performed in humans and support further analyses to continue examining the unique myeloid cell origins that may be applied to address disease pathogenesis mechanisms and intervention strategies in humans.

In vitro resistance of Aspergillus fumigatus to azole farm fungicide.

Azole resistance in Aspergillus fumigatus is mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which encodes the target of azole fungicides. Moreover, overexpression of CYP51B or multidrug resistance (MDR) gene is supposedly related to the mechanism of azole resistance in A. fumigatus. In this study, we tried to induce resistance to tetraconazole, an azole fungicide, in strains of A. fumigatus from a farm and then investigated mutation and expression of their CYP51A, CYP51B, and multidrug resistance (MDR) genes. Three tetraconazole resistant strains were induced and their minimum inhibitory concentration (MIC) for tetraconazole was 145 mg/L. However, the MICs of itraconazole (ITZ), posaconazole (POS), and voriconazole (VRZ) obtained by an E-test of the three tetraconazole resistant strains were 0.064-0.19 mg/L for ITZ, 0.023-0.32 mg/L for POS, and 0.047-0.064 mg/L for VRZ. No gene mutations were detected in the CYP 51A sequence amplified in these strains. RT-PCR of cyp51A and cyp51B indicated that the tetraconazole resistant strains more highly expressed these genes than the susceptible strain in tetraconazole containing medium.

Short communication: ELISA system for screening of bovine mastitis caused by Prototheca zopfii.

Prototheca zopfii is an achlorophyllic alga that causes bovine mastitis, resulting in a reduction in milk production and the secretion of thin, watery milk with white flakes. This study evaluated the use of an ELISA system for distinguishing cows with mastitis due to P. zopfii genotype 2 from healthy cows and cows with chronic candidal mastitis. We also investigated the transitional changes of specific antibody titers in healthy cows injected with inactivated P. zopfii genotype 2 cells. The ELISA system exhibited the highest sensitivity (94%) and specificity (100%) for chronic protothecal mastitis when the positive cutoff value was set at 43.4 ELISA units. Anti-protothecal IgG titers were positive in all cows after they were inoculated with inactivated P. zopfii genotype 2 cells. These results indicated that ELISA detection of anti-protothecal IgG in serum provided specificity and sensitivity sufficient for diagnosing protothecal mastitis. Thus, an ELISA system incorporating this specific antiserum is expected to be valuable for definitive field-based diagnosis of bovine mastitis due to P. zopfii genotype 2.

Mating Type Gene (MAT) and Itraconazole Susceptibility of Trichophyton tonsurans Strains Isolated in Japan.

Infection by Trichophyton tonsurans is an emerging fungal epidemic in Japan. Itraconazole (ITZ) and terbinafine have been used for the treatment of this infection for 15 years. However, patients with T. tonsurans infections have been shown to remain uncured or to become reinfected, suggesting that subclinical infection or polyphyletic strains and/or antifungal drug-resistant strains might be occurring in Japan. In this study, PCR analysis was performed to confirm the presence of the mating type locus MAT in genomic DNA from 60 Japanese clinical isolates of T. tonsurans, and to assess the previously postulated clonal origin of clinical isolates of this species. Antifungal susceptibility testing on isolates also was performed to confirm the absence of strains resistant to ITZ. PCR analysis proved that all 60 strains contained the MAT1-1 allele, while none contained the MAT1-2 allele. As determined by E-test, the mean MIC of ITZ in the 60 strains was 0.023 mg/L (range 0.002-0.125 mg/L). All strains of T. tonsurans isolated in Japan were clonal and were not resistant to ITZ. Therefore, dermatophytosis due to T. tonsurans is expected to respond to ITZ, since clinical isolates of T. tonsurans tested to date have been susceptible to this antifungal. This infection is proliferating as a subclinical infection in Japan.

Clinical outcomes of a novel therapeutic vaccine with Tax peptide-pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study.

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type-I (HTLV-I)-infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti-ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate- to high-risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax-specific CTL responses were observed with peaks at 16-20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide-pulsed DC vaccine is a safe and promising immunotherapy for ATL.