Host cell sensing and restoration of mitochondrial function and metabolism within Helicobacter pylori VacA intoxicated cells.

IMPORTANCE: Persistent human gastric infection with Helicobacter pylori is the single most important risk factor for development of gastric malignancy, which is one of the leading causes of cancer-related deaths worldwide. An important virulence factor for Hp colonization and severity of gastric disease is the protein exotoxin VacA, which is secreted by the bacterium and modulates functional properties of gastric cells. VacA acts by damaging mitochondria, which impairs host cell metabolism through impairment of energy production. Here, we demonstrate that intoxicated cells have the capacity to detect VacA-mediated damage, and orchestrate the repair of mitochondrial function, thereby restoring cellular health and vitality. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies.

Restoration of mitochondrial structure and function within Helicobacter pylori VacA intoxicated cells.

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

Structural Features of Microvascular Networks Trigger Blood Flow Oscillations.

We analyse mathematical models in order to understand how microstructural features of vascular networks may affect blood flow dynamics, and to identify particular characteristics that promote the onset of self-sustained oscillations. By focusing on a simple three-node motif, we predict that network "redundancy", in the form of a redundant vessel connecting two main flow-branches, together with differences in haemodynamic resistance in the branches, can promote the emergence of oscillatory dynamics. We use existing mathematical descriptions for blood rheology and haematocrit splitting at vessel branch-points to construct our flow model; we combine numerical simulations and stability analysis to study the dynamics of the three-node network and its relation to the system's multiple steady-state solutions. While, for the case of equal inlet-pressure conditions, a "trivial" equilibrium solution with no flow in the redundant vessel always exists, we find that it is not stable when other, stable, steady-state attractors exist. In turn, these "nontrivial" steady-state solutions may undergo a Hopf bifurcation into an oscillatory state. We use the branch diameter ratio, together with the inlet haematocrit rate, to construct a two-parameter stability diagram that delineates regimes in which such oscillatory dynamics exist. We show that flow oscillations in this network geometry are only possible when the branch diameters are sufficiently different to allow for a sufficiently large flow in the redundant vessel, which acts as the driving force of the oscillations. These microstructural properties, which were found to promote oscillatory dynamics, could be used to explore sources of flow instability in biological microvascular networks.

Methylergometrine-Induced Myocardial Infarction in the Setting of a Cesarean Delivery.

A 30-year-old female with no significant past medical history presented to our labor and delivery ward for induction of labor. Due to failure to progress, she was proceeded to cesarean delivery. Intraoperatively, it was noted that her uterus was hypotonic; she required supplemental methylergometrine to control the bleeding from the uterine atony. However, within three minutes of intramuscular (IM) administration, she complained of chest pain. She then subsequently developed pulmonary edema in the postoperative care unit, which required supplemental oxygen. She was found to have elevated troponin and brain natriuretic peptide (BNP), along with radiologic features of fluid overload suggestive of congestive cardiac failure, which all lead to the diagnosis of non-ST myocardial infarction. The patient had a normal computed tomography (CT) pulmonary angiogram, echocardiogram, and serial electrocardiograms (ECGs). She was successfully discharged from the hospital on postoperative day 4 with resolution of her symptoms and improving cardiac enzymes. Cardiology outpatient follow-up was arranged.

Protein interactions within and between two F-type type IV secretion systems.

Bacterial type IV secretion systems (T4SSs) can mediate conjugation. The T4SS from Neisseria gonorrhoeae possesses the unique ability to mediate DNA secretion into the extracellular environment. The N. gonorrhoeae T4SS can be grouped with F-type conjugative T4SSs based on homology. We tested 17 proteins important for DNA secretion by N. gonorrhoeae for protein interactions. The BACTH-TM bacterial two-hybrid system was successfully used to study periplasmic interactions. By determining if the same interactions were observed for F-plasmid T4SS proteins and when one interaction partner was replaced by the corresponding protein from the other T4SS, we aimed to identify features associated with the unique function of the N. gonorrhoeae T4SS as well as generic features of F-type T4SSs. For both systems, we observed already described interactions shared by homologs from other T4SSs as well as new and described interactions between F-type T4SS-specific proteins. Furthermore, we demonstrate, for the first-time, interactions between proteins with homology to the conserved T4SS outer membrane core proteins and F-type-specific proteins and we confirmed two of them by co-purification. The F-type-specific protein TraHN was found to localize to the outer membrane and the presence of significant amounts of TraHN in the outer membrane requires TraGN .

Risk factors associated with gastric malignancy during chronic Helicobacter pylori Infection.

Chronic Helicobacter pylori (Hp) infection is considered to be the single most important risk factor for the development of gastric adenocarcinoma in humans, which is a leading cause of cancer-related death worldwide. Nonetheless, Hp infection does not always progress to malignancy, and, gastric adenocarcinoma can occur in the absence of detectable Hp carriage, highlighting the complex and multifactorial nature of gastric cancer. Here we review known contributors to gastric malignancy, including Hp virulence factors, host genetic variation, and multiple environmental variables. In addition, we assess emerging evidence that resident gastric microflora in humans might impact disease progression in Hp-infected individuals. Molecular approaches for microbe identification have revealed differences in the gastric microbiota composition between cancer and non-cancerous patients, as well as infected and uninfected individuals. Although the reasons underlying differences in microbial community structures are not entirely understood, gastric atrophy and hypochlorhydria that accompany chronic Hp infection may be a critical driver of gastric dysbiosis that promote colonization of microbes that contribute to increased risk of malignancy. Defining the importance and role of the gastric microbiota as a potential risk factor for Hp-associated gastric cancer is a vital and exciting area of current research.

Cooperative Effects between Hydrophilic Pores and Solvents: Catalytic Consequences of Hydrogen Bonding on Alkene Epoxidation in Zeolites.

Hydrophobic voids within titanium silicates have long been considered necessary to achieve high rates and selectivities for alkene epoxidations with H2O2. The catalytic consequences of silanol groups and their stabilization of hydrogen-bonded networks of water (H2O), however, have not been demonstrated in ways that lead to a clear understanding of their importance. We compare turnover rates for 1-octene epoxidation and H2O2 decomposition over a series of Ti-substituted zeolite *BEA (Ti-BEA) that encompasses a wide range of densities of silanol nests ((SiOH)4). The most hydrophilic Ti-BEA gives epoxidation turnover rates that are 100 times larger than those in defect-free Ti-BEA, yet rates of H2O2 decomposition are similar for all (SiOH)4 densities. These differences cause the most hydrophilic Ti-BEA to also give the highest selectivities, which defies conventional wisdom. Spectroscopic, thermodynamic, and kinetic evidence indicate that these catalytic differences are not due to changes in the electronic affinity of the active site, the electronic structure of Ti-OOH intermediates, or the mechanism for epoxidation. Comparisons of apparent activation enthalpies and entropies show that differences in epoxidation rates and selectivities reflect favorable entropy gains produced when epoxidation transition states disrupt hydrogen-bonded H2O clusters anchored to (SiOH)4 near active sites. Transition states for H2O2 decomposition hydrogen bond with H2O in ways similar to Ti-OOH reactive species, such that decomposition becomes insensitive to the presence of (SiOH)4. Collectively, these findings clarify how molecular interactions between reactive species, hydrogen-bonded solvent networks, and polar surfaces can influence rates and selectivities for epoxidation (and other reactions) in zeolite catalysts.

Intradermal delivery of recombinant vaccinia virus vector DIs induces gut-mucosal immunity.

Antigen-specific mucosal immunity is generally induced by the stimulation of inductive mucosal sites. In this study, we found that the replication-deficient vaccinia virus vector, DIs, generates antigen-specific mucosal immunity and systemic responses. Following intradermal injection of recombinant DIs expressing simian immunodeficiency virus gag (rDIsSIVgag), we observed increased levels of SIV p27-specific IgA and IgG antibodies in faecal extracts and plasma samples, and antibody-forming cells in the intestinal mucosa and spleen of C57BL/6 mice. Antibodies against p27 were not detected in nasal washes, saliva, and vaginal washes. The enhanced mucosal and systemic immunity persisted for 1 year of observation. Induction of Gag-specific IFN-gamma spot-forming CD8(+) T cells in the spleen, small intestinal intraepithelial lymphocytes, and submandibular lymph nodes was observed in the intradermally injected mice. Heat-inactivated rDIsSIVgag rarely induced antigen-specific humoral and T-helper immunity. Moreover, rDIsSIVgag was detected in MHC class II IA antigen-positive (IA(+)) cells at the injection site. Consequently, intradermal delivery of rDIs effectively induces antigen-specific humoral and cellular immunity in gut-mucosal tissues of mice. Our data suggest that intradermal injection of an rDIs vaccine may be useful against mucosally transmitted pathogens.

Mucosal administration of completely non-replicative vaccinia virus recombinant Dairen I strain elicits effective mucosal and systemic immunity.

We studied the immunogenicity of completely replication-deficient vaccinia virus Dairen I strain recombinant encoding simian immunodeficiency virus (SIV) gag/pol (rDIs) in both mucosal and systemic compartments. When administered either intranasally or intragastrically, rDIs elicited enhanced levels of both SIV Gag p27-specific IgA antibodies and specific plasma antibodies, and the enhanced immunity persisted for the 1-year of observation by intranasal immunization. Increases were observed in antigen-specific IgA antibody-forming cells (AFC) in intestinal mucosal tissues and in IgG AFC in spleens. Furthermore, induction of type 1 and 2 helper cytokines in CD4+ spleen T cells and of CD8+ IFN-gamma spot-forming cells in mucosal tissues was observed in the intranasally immunized mice. Moreover, not even high-dose rDIs generated an SIV gene signal in the brain tissues of immunized mice. These findings suggest that mucosal immunization with the DIs recombinant hold promise as a safe mucosal vector.

Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in diabetic foot infections.

AIMS: Diabetic foot lesions are a major medical, social, and economic problem and are the leading cause of hospitalization for patients with diabetes, worldwide. ESBL-producing bacteria may not be detectable by routine disc diffusion susceptibility test, leading to inappropriate use of antibiotics and treatment failure. There is not much information on ESBL-producing organisms causing diabetic foot infection. An attempt was therefore made to study the ESBL-producing Escherichia coli and Klebsiella pneumoniae in diabetic foot patients with type 2 diabetes mellitus. MATERIALS AND METHODS: A total of 134 isolates of E. coli and K. pneumoniae were obtained from tissue, pus swab, and wound swab samples from diabetic foot ulcers submitted for routine microbiological analysis during the period January to December 2005 from patients with diabetic foot infections who had type 2 diabetes mellitus, attending S. L. Raheja Hospital. The above isolates were tested for antimicrobial susceptibility by disc diffusion technique according to clinical and laboratory standards institute (CLSI) guidelines. The screening for ESBL production was done by phenotypic confirmatory test using ceftazidime disc in the presence and absence of clavulanic acid as recommended by CLSI. RESULTS: Among the 134 isolates, 54 (40.29%) were E. coli and 80 (59.70%) were K. pneumoniae; among which, ESBL production was detected in 31 (23.13%) isolates. Of these 31, 15 (48.38%) were E. coli and 16 (51.61%) were K. pneumoniae. All the ESBL-producing isolates were found to be 100% sensitive to carbapenem (imipenem and meropenem). Mortality was found to be 3.22%, the cause of death being septicemia leading to multiple organ failure. CONCLUSIONS: The prevalence of ESBLs among members of Enterobacteriaceae constitutes a serious threat to the current beta-lactam therapy, leading to treatment failure and consequent escalation of costs. There is an urgent need to emphasize rational use of drugs to minimize the misuse of available antimicrobials.

Molecular dynamics simulations of ligand-induced backbone conformational changes in the binding site of the periplasmic lysine-, arginine-, ornithine-binding protein.

The periplasmic lysine-, arginine-, ornithine-binding protein (LAOBP) traps its ligands by a large hinge bending movement between two globular domains. The overall geometry of the binding site remains largely unchanged between the open (unliganded) and closed (liganded) forms, with only a small number of residues exhibiting limited movement of their side chains. However, in the case of the ornithine-bound structure, the backbone peptide bond between Asp11 and Thr12 undergoes a large rotation. Molecular dynamics simulations have been used to investigate the origin and mechanism of this backbone movement. Simulations allowing flexibility of a limited region and of the whole binding site, with and without bound ligands, suggest that this conformational change is induced by the binding of ornithine, leading to the stabilisation of an energetically favourable alternative conformation.

Infection of macaques with an R5-tropic SHIV bearing a chimeric envelope carrying subtype E V3 loop among subtype B framework.

To establish simian/human immunodeficiency virus (SHIV) clones bearing a chimeric envelope carrying subtype E V3 loop among subtype B envelope, four subtype E V3 sequences were substituted into SHIV(MD14), a SHIV clone bearing an envelope derived from a CXCR4 (X4)/CCR5 (R5)-dual tropic subtype B HIV-1 strain. SHIV-TH09V3, an only V3-chimera clone capable of replicating in human and macaque peripheral blood mononuclear cells (PBMCs), was propagated in pig-tailed macaque PBMCs and in cynomolgus macaque splenic mononuclear cells. The propagated virus stocks were intravenously inoculated into respective macaque species. SHIV-TH09V3 infected both macaque species as shown by plasma RNA viremia, isolated viruses from PBMCs and plasma, and antibody production against viral proteins. To assess how the substituted V3 sequence affected coreceptor usage, SHIV-TH09V3 stocks propagated in vitro and after isolation from macaques were verified for their corecepor usage by GHOST cells assay. SHIV-TH09V3 maintained R5-tropic phenotype both in vitro and after isolation from macaques, in contrast to the X4/R5-dual tropic SHIV(MD14). This indicates the substituted V3 sequence among the backbone of SHIV(MD14) governs coreceptor usage. Future study of infecting macaques with SHIV-TH09V3 and SHIV(MD14) will focus on differences of the outcome caused by the different V3 sequences in connection with coreceptor usage.

Comparison of neuropathogenicity of poliovirus type 3 in transgenic mice bearing the poliovirus receptor gene and cynomolgus monkeys.

To clarify the similarities of poliovirus infection in cynomolgus monkeys and transgenic mice bearing the poliovirus receptor, TgPVR21, we compared the pathological changes of these animals following intraspinal inoculation of two strains of poliovirus type 3 using immunohistochemical detection of the capsid antigen. All of the monkeys inoculated with 10(6) TCID(50) viruses showed flaccid paralysis 2 or 3 days post-inoculation (p.i.). TgPVR21 mice showed paralysis starting from 2 to 3 days p.i. Histologically, neurons having pyknotic nuclei and eosinophilic cytoplasm and neuronophagia were characteristically observed in both animals, but central chromatolysis was not observed in infected TgPVR21. The median lesion scores in the monkeys and TgPVR21 were well correlated, though the distribution of poliovirus-infected lesions in the central nervous system was different. In both animals the motor neurons and the brainstem nuclei responsible for flaccid paralysis were infected by the virus, while the cerebral cortex and thalamus were infected in the monkeys but not in TgPVR21. These results confirmed the reliability of neurovirulence tests using TgPVR21 as a substitute for monkeys, in respect to the spinal and brainstem lesions of poliovirus type 3.

Induction of CD95 ligand expression on T lymphocytes and B lymphocytes and its contribution to apoptosis of CD95-up-regulated CD4+ T lymphocytes in macaques by infection with a pathogenic simian/human immunodeficiency virus.

Using an established SIV/HIV-C2/1-infected cynomolgus monkey model displaying stable CD4+ T cell depletion, the kinetics of apoptosis and the levels of expression of CD95 membrane-associated CD95L on lymphocytes were investigated to test the involvement of the CD95/CD95L system in CD4+ T lymphocyte loss in vivo. Rapid depletion of CD4+ T cells occurred up to 2 weeks after infection, with chronic CD4+ T lymphopenia thereafter. During the initial CD4+ T cell loss, which was accompanied by viraemia, about 90% of the peripheral CD4+ T cell subset underwent spontaneous apoptotic cell death during 24 h of culture. Increased expression of CD95 was observed on both CD4+ and CD8+ T cell subsets, with CD95 expression on CD8+ cells declining rapidly, but high CD95 expression being maintained on CD4+ cells. Since CD95L was expressed on CD8+ T cells, B cells and to a lesser extent on CD4+ T cells, this suggests that CD95-mediated apoptosis might be controlled in an autocrine/paracrine fashion.

Anti-tetanus toxoid antibody production and protection against lethal doses of tetanus toxin in hu-PBL-SCID mice.

BACKGROUND: In this study, severe combined immunodeficiency (SCID) mice, which permit the survival of lymphoid cells of human origin, were used to study the human anti-tetanus immune response. METHODS: Human peripheral blood lymphocytes (hu-PBL) obtained from 88 healthy donors (aged from 18 to 62) were transplanted into SCID mice, and anti-tetanus toxoid (Ttd) antibody production and protection against lethal doses of tetanus toxin (Ttx) were investigated in the hu-PBL-SCID mice. RESULTS: The transfer of human PBL evoked significant human anti-Ttd IgG antibody production for 37.5% of the donors. After in vivo immunization, the percentage of donors with PBL exhibiting positive anti-TtD IgG production in the mice increased to 54.5%. Mean anti-Ttd IgG levels in the sera were also significantly elevated in response to immunization. The mean IgG titer for the mice injected with PBL from donors under the age of 40 was significantly higher than that of the mice injected with PBL from donors aged 40 or older. Four weeks after the cell transfer, the mice were challenged with Ttx. The induction of protection against Ttx challenge was observed mostly in mice with PBL transferred from donors under the age of 40. In vivo immunization in SCID mice with Ttd increased the number of cases of resistance to Ttx. CONCLUSIONS: These results suggest that hu-PBL-SCID mice might serve as a tool for predicting the protective ability against pathogens in PBL donors and also for evaluating vaccine efficacy.

Pathogenicity of Hantaan virus in newborn mice: genetic reassortant study demonstrating that a single amino acid change in glycoprotein G1 is related to virulence.

Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mu11E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-1 Ile to mu11E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mu11E10-derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.

Upgrading of flow cytometric analysis for absolute counts, cytokines and other antigenic molecules of cynomolgus monkeys (Macaca fascicularis) by using anti-human cross-reactive antibodies.

In order to effectively use cynomolgus monkeys as animal models for human diseases, more than 300 anti-human monoclonal antibodies (mAbs) were studied as to their cross-reaction with various antigens from cynomolgus monkeys (Macaca fascicularis). Two hundred twenty-nine of 339 (67.55%) anti-human mAbs that react with human antigens of CD-defined molecules, chemokine receptors, and T cell receptors were cross-reactive with the monkey antigens. Using the cross-reactive antibodies and the fluorescenced beads for calibration, the procedure for the absolute count of monkey lymphocyte subsets was developed and the mean values for CD4+ and CD8+ lymphocyte subsets in peripheral blood were 718 and 573/mm3, respectively. Moreover, intracellular cytokines, IL-2, IL-4 and IFN gamma, and intracellular apoptosis-related proteins, Bcl-2, FADD and active form of caspase-3 could be detected in peripheral blood mononuclear cells as well as various tissue cells. It is therefore practicable to detail the phenotype of leukocytes, assess the production of intracellular cytokines and enumerate T-lymphocyte subsets by using the cross-reactive human antibodies with respective antigens of cynomolgus monkeys.

Protective immune responses induced by a non-pathogenic simian/human immunodeficiency virus (SHIV) against a challenge of a pathogenic SHIV in monkeys.

A simian/human immunodeficiency virus (SHIV)-NM3n containing the human nef, but not the monkey nef, and vpr genes of SIV was inoculated into two cynomolgus monkeys, resulting in systemic infection with a minimum level of transient virus load. In order to study the nature of immune responses associated with the prevention of a pathogenic SHIV, the SHIV-NM3n-inoculated monkeys and three naive monkeys were intravenously challenged with a pathogenic SHIV containing the envelope gene of HIV-1 89.6. After the heterologous virus challenge, all of the SHIV-NM3n-inoculated animals completely avoided the loss of CD4+ T lymphocytes in PBMC as well as lymphoid tissues compared to pathogenic SHIV-injected control animals. The inhibition of CD4+ cell depletion was associated with maintaining the proliferative response of helper T-cells against SIV p27 in the previously nonpathogenic virus-inoculated animals following the pathogenic virus challenge. Furthermore, the decline of CD28+ cells, the increase in CD95+ cells, and the enhancement of in vitro apoptosis in PBMC were inhibited in the non-pathogenic virus-inoculated animals. These results suggest that nonpathogenic SHIV-NM3n infection induces the protection of monkeys from heterologous pathogenic viruses that may be associated with blocking the change in immune responses and the cell loss induced by a pathogenic virus.

Recovery of pathogenic measles virus from cloned cDNA.

Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700-705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773-5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.

Direct detection of apoptotic cells in peripheral blood from highly pathogenic SHIV-inoculated monkey.

Apoptosis in peripheral blood leukocytes (PBL) has been estimated by the enhancement of spontaneous apoptosis after in vitro culture, because apoptotic cells have not been observed directly in freshly isolated PBL in the course of HIV/AIDS. In monkeys infected with a highly pathogenic simian/human immunodeficiency virus (SHIV), which corresponds to rapid progressors of HIV infection, a high frequency of apoptotic cells was directly detected in fresh PBL by electron-microscopic studies. Peripheral blood apoptosis transiently occurred after intense plasma viremia, and peaking at 3 weeks postinfection; occurrence was not limited specifically to lymphocytes, but also occurred in other types of leukocytes. Apoptosis in peripheral lymph nodes was also detected following intense plasma viremia. However, the in vivo apoptosis was not detected in nonpathogenic SHIV-infected monkeys that showed no cell loss. Thus, we directly showed the apoptosis of PBL, which might be associated with pathogenic SHIV produced during the time of plasma viremia.