Demonstration of Borna disease virus RNA in peripheral blood mononuclear cells derived from Japanese patients with chronic fatigue syndrome.

CFS, a recently named heterogeneous disorder, is an illness of unknown etiology. The association of CFS with viral infections has been suggested. A common association between CFS and several viruses examined has not been confirmed. Here, we centered on the possible link between CFS and BDV infection. By nested RT-PCR followed by hybridization, BDV RNA was demonstrated as a clear signal in PBMCs in 3 out of 25 CFS patients. The amplified cDNA fragments were cloned and sequenced. A total of 16 clones were studied. Intra-patients divergencies of the p24 were 2-9%, 3-20%, and 3-11% in the deduced amino acids. Inter-patient divergencies among the 16 clones were 3-24%. Antibodies to recombinant BDV p24 protein were detected in 6 CFS patients including one carrying BDV RNA. Overall, these gave the prevalence of 32% (8/25) in Japanese CFS patients, suggesting that Japanese CFS is highly associated with active infection of BDV, or a related agent.

Intracellular glutathione as a possible direct blocker of HIV type 1 reverse transcription.

In AIDS patients, chronic inflammation and elevated levels of cytokines seem to be associated with reduced levels of glutathione (GSH). GSH has been proposed to inhibit the activation of NF-kB, which results in the inhibition of HIV-1 replication. Here, we show the evidence that GSH and N-acetylcysteine, but not L-cysteine or dithiothreitol, could inhibit the reverse transcriptase (RT) process of HIV-1. Such inhibition was not observed with the RT of murine leukemia virus.

Infectious DNA clone of HIV type 1 A/G recombinant (CRF02_AG) replicable in peripheral blood mononuclear cells.

We constructed an infectious DNA clone of the HIV-1 A/G recombinant 97GH-AG2, which was isolated in Ghana in 1997 and was classified originally as subtype A. By phylogenetic and recombination breakpoint analyses, p97GH-AG2 was grouped in the circulating form of A/G recombinants (CRF02_AG) and was found to contain the least amount of subtype G-derived region among the known CRF02_AG HIV-1 DNAs. This result suggests that CRF02_AG may be a predominant form in Ghana. Virions produced by transfection of p97GH-AG2 into 293T cells grew in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs). 97GH-AG2 also replicated efficiently in CCR5-expressing HeLa cells, MAGIC5, but only weakly in the parent MAGI cells, indicating that 97GH-AG2 uses mostly CCR5 as a coreceptor. Isolation of the first HIV-1 (CRF02_AG) DNA clone that replicates in PBMCs will accelerate the molecular analysis of this subtype.

Establishment of a MAGI-derived indicator cell line that detects the Nef enhancement of HIV-1 infectivity with high sensitivity.

The Nef protein of the simian and human immunodeficiency viruses (SIV and HIV) is regarded as one of the critical determinants of the pathogenicity of HIV-1 in vivo. The positive effect of Nef on viral replication is examined most easily in vitro by the use of indicator cells such as HeLa-CD4-LTR-beta-gal cells (MAGI) or MAGIC5 cells, which are MAGI-derived, CCR5-expressing cells. However, Nef increases the infectivity of many HIV-1 strains no more than 10-fold in these indicator cells. It was noted that MAGI cells expressing a lower level of CD4 enabled us to discriminate more clearly between wild-type and Nef-defective virions. A MAGIC5-derived cell line, MAGNEF, which stably expressed a low level of CD4, was established. The infectivity of the Nef-defective HIV-1 NL4-3 strain was consistently less than one-twentieth of that of the wild type in MAGNEF cells. By using MAGNEF cells, it was shown that Nef enhanced the infectivity of a subtype C HIV-1, Indie-C1 strain, although the effect of Nef on Indie-C1 was significantly less than that on the subtype B strains NL4-3 and SF2. These results validate the versatility of MAGNEF cells for use in the simple and sensitive assay for the level of Nef dependence of various HIV-1 isolates.

Retroperitoneal approach for laparoscopic nephroureterectomy with stripping technique: extracorporeal ligation of ureter and ureteral catheter.

INTRODUCTION: The pluck and stripping techniques are used for lower ureter management in renal pelvic cancer patients. Herein, we report our experience of extracorporeal ligation of the ureter and the ureteral catheter through the trocar port, which differs from conventional laparoscopic ligation in the retroperitoneal space. This technique was selected to reduce the time needed for ureter management using the stripping technique and to provide secure ligation. MATERIALS AND SURGICAL TECHNIQUE: We performed this stripping technique in patients with T1 and T2 stage renal pelvic cancer without imaging-evident lymph node metastasis. After transurethrally placing a ureteral catheter, we resected the circumference of the ureteral orifice. After laparoscopic nephrectomy via a retroperitoneal approach, the ureteral catheter and distal ureter were ligated extracorporeally. The catheter was pulled to invaginate the ureter so it could then be pulled through the external urethral orifice. DISCUSSION: This technique of extracorporeal ligation ensures more a secure ligation of the ureter and ureteral catheter. This modified stripping technique does not require lower ureter management with laparotomy, and it is also useful in shortening the operative time. This method is effective for relatively early stage renal pelvic cancer.

A chain section containing epitopes for cytotoxic T, B and helper T cells within a highly conserved region found in the human immunodeficiency virus type 1 Gag protein.

Cell-mediated immune responses constitute a major defense against the spread of human immunodeficiency virus type 1 (HIV-1). However, multiple alterations within a particular epitope may accumulate during disease progression, allowing the virus to escape cytotoxic T lymphocytes (CTLs). Therefore, the best candidate for a peptide vaccine that would prevent the onset of the disease might be a chain section containing epitopes for the generation of CTLs in regions of conserved sequences among different HIV-1 isolates. We previously showed that immunizing mice with synthetic peptides consisting of 23-amino acids (Gag-23mer; 287-309 amino acid residues) in a highly conserved region derived from the major core protein p24 of HIV-1 generates specific CTLs as well as antibodies. Here, we identified one CTL (T-1; 291-300) and two B-cell (B-1; 290-299 and B-2; 300-309) epitopes, all of which consisted of 10 amino acids within the region. In addition, helper T cells primed by the Gag-23mer peptide were proliferated by in vitro stimulation with a 21mer (H-1; 289-309) or a 19mer (H-2; 291-309) peptide, but not with a 17mer peptide (293-309) or 19mer peptide (287-305). Immunization with the H-1 peptide generated an antibody reactive to B-1, but not B-2, whereas that with H-2 generated an antibody reactive to B-2, but not B-1. CTLs were not generated by immunization with these peptides, indicating that the entire sequence of Gag-23mer is the helper epitope for CTLs. Thus, the Gag-23mer is a chain section containing epitopes for cytotoxic T, B and helper T-cells within a highly conserved region of HIV-1 Gag protein.

Dependence on host cell cycle for activation of human immunodeficiency virus type 1 gene expression from latency.

Human immunodeficiency virus type 1 (HIV-1) establishes latent infection of a certain population of CD4+ host cells which could be long-term reservoirs for HIV-1. The expression of viral genes in such long-term infected cells is strongly regulated by cellular status, such as the phase of the cell cycle or stage of cell differentiation. Here, viral gene expression in synchronized U1 cells, a monocytic cell clone latently infected with HIV-1, was characterized. The expression of HIV-1 antigens was detected exclusively at G2/M phase in U1 cells, irrespective of phorbol myristate acetate (PMA) treatment. The induction of HIV-1 gene expression in PMA-treated cells was due to the recruitment of NF-kappaB with DNA-binding activity at G2/M phase. Activated NF-kappaB was induced only by PMA treatment during the late G1 to S, but not after entering G2 phase, indicating that the transcriptional factor(s) involved in viral gene expression is also largely regulated by the host cell cycle. In contrast, the enhancement of antigen expression by treatment with tumour necrosis factor-alpha (TNF-alpha) was cell cycle-independent. In fact, NF-kappaB was activated 2 h after TNF-alpha treatment at all stages of the cell cycle. Thus, the mechanisms of HIV-1 activation from latency in U1 cells by PMA and TNF-alpha treatment are different. The model system using U1 cells shown here may provide insight into the mechanisms responsible for HIV-1 gene expression from latency.

Requirement of nef for HIV-1 infectivity is biased by the expression levels of Env in the virus-producing cells and CD4 in the target cells.

The nef gene of human immunodeficiency virus type 1 (HIV-1) encodes a 27 to 34 kDa myristoylated protein, which enhances viral infectivity in a single-round infection assay. The level of Nef enhancement of HIV-1 infectivity depends on the viral strains, on the target cells, and on the cells used for propagating the viruses. In this study, we aimed at clarifying the molecular basis of these differences in the requirement for Nef. We found that the requirement for Nef was increased when we decreased the quantity of Env protein in the virus-producing cells or the quantity of CD4 in the target cells. Both the wild-type and Nef-defective HIV-1 viruses were propagated in 293T cells, which did not express any CD4; therefore, Nef-induced CD4 down-regulation did not explain this phenomenon. Moreover, we did not observe any increase in the viral entry or fusion activity of gp120env in the wild-type HIV-1 compared to that in the Nef-defective HIV-1. Thus, we propose that Env on the virion and CD4 on the target cells have inhibitory effects on the post-entry step of the HIV-1 replication cycle, and that Nef functions to counteract this negative effect.

CalDAG-GEFIII activation of Ras, R-ras, and Rap1.

We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Ras and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Ras family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in situ hybridization. CalDAG-GEFIII activated ERK/MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CalDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, co-activation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Ras family G proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.

Exposure of normal monocyte-derived dendritic cells to human immunodeficiency virus type-1 particles leads to the induction of apoptosis in co-cultured CD4+ as well as CD8+ T cells.

The depletion of immune T cells by human immunodeficiency virus type-1 (HIV-1) infection is a major mechanism involved in the pathogenesis of AIDS. Here, we examined a possible effector function of blood monocyte-derived dendritic cells (DCs) to induce apoptosis in bystander CD4+ and CD8+ T cells. The DCs were generated by culturing monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs exposed to HIV-1 particles were co-cultured with healthy donor-derived blood T cells at a ratio of 1:20. Analyses by percent cell mortality, staining with propidium iodide and reactivity with Annexin V revealed the induction of apoptosis in both CD4+ and CD8+ target T cells. Further, this apoptosis occurred without stimulation with mitogens when the cell cycle of target T cells shifted from G0 to G1, probably due to the mitogenic effect of the DCs. Thus, induction of apoptosis in both CD4+ and CD8+ T cells occurred via interaction with DCs adsorbed with HIV-1 particles.

Possible correlation between Borna disease virus infection and Japanese patients with chronic fatigue syndrome.

Borna disease virus (BDV) is a neurotropic, as yet unclassified, non-segmented, negative-sense, single-strand RNA virus. Natural infection with this virus has been reported to occur in horses and sheep. In addition, antibodies to BDV in plasma or BDV RNA in peripheral blood mononuclear cells (PBMCs) were also found in patients with neuropsychiatric diseases. We describe here the possible link between the patients with chronic fatigue syndrome (CFS) and infection with BDV.