Detection of HHV-8 (human herpesvirus-8) genomes in induced peripheral blood mononuclear cells (PBMCs) from US blood donors.

BACKGROUND: Human herpesvirus-8 (HHV-8) causes Kaposi's sarcoma and can be detected and induced in peripheral blood mononuclear cells (PBMCs) from infected individuals. The prevalence of viral genomes in induced/cultured PBMCs from healthy blood donors has not been systematically studied. MATERIALS AND METHODS: PBMCs from 164 donors were purified and stored as two equal aliquots in liquid nitrogen. One aliquot was used for CD19+ B-cell purification with a fraction reserved for DNA extraction. The second aliquot was cultured for 2 or 4 days in culture media containing n-butyrate and tetradecanoyl phorbol acetate. DNA was extracted from all four cell sources: PBMCs, purified B cells, induced PBMCs harvested at days 2 and 4 of culture. A sensitive real-time PCR with a DNA equivalent of 3×10(5) cells per reaction was run in duplicate for all samples along with a quantitative HHV-8 DNA standard ranging from 1.6 to 200 copies. RESULTS: For all 164 donors, HHV-8 genomes were not detected in the DNA equivalent of 3-6×10(5) of PBMCs and induced/cultured PBMCs with a real-time PCR assay (95% CI: 0-3.5/164). HHV-8 DNA was not detected from DNA equivalent of 1.5 (0.5-5.6)×10(5) CD19+ B cells from 139/164 donors. HHV-8 antibodies were detected in 7 of the 164 donors (4.3%). CONCLUSIONS: HHV-8 genomes were not detected from PBMCs, induced/cultured PBMCs and CD19+ B cells from 164 blood donors. The level of detectable HHV-8 genomes in blood donors seems to be extremely low, if they exist.

Chimpanzee bipedalism: cineradiographic analysis and implications for the evolution of gait.

Bipedal chimpanzees reorient the pelvis to achieve an upright posture but retain the same pattern of femoral flexion and extension as in quadrupedal walking. Major differences from human gait are the abducted, relatively more flexed excursion of the femur and the timing of pelvic tilt, which raises during the swing phase. The femoral head morphology in the fossil hominid Australopithecus robustus is evidence of an approximately vertical excursion of the femur in contrast to the adducted pattern of modern man and the abducted pattern of chimpanzees.

Limb movements in a monotreme (Tachyglossus aculeatus): a cineradiographic analysis.

In a walking echidna the principal movement of the humerus is long-axis rotation. The humerus remains approximately perpendicular to the sagittal plane, but the femur is directed anterolaterally at angles from 35 degrees to 50 degrees . In addition to long-axis rotation, the femur elevates and depresses in an arc which usually varies between 40 degrees and 90 degrees . The femoral angle, the femoral elevation and depression, and the plantar contact of the manus beneath the glenoid are features found also in generalized therians.

Genetic engineering of novel genomes of large DNA viruses.

Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals.

Adaptations for climbing in north american multituberculates (mammalia).

A recently discovered skeleton of Ptilodus exhibits several specializations for climbing. A survey of postcranial bones of Cretaceous and early Cenozoic multituberculates from North America reveals similar locomotor specializations. Multituberculates possessed distinctive tarsal adaptations for a range of pedal mobility characteristic of arboreal mammals that descend trees headfirst. The divergent hallux could move independently of the other digits. The long robust tail of Ptilodus possessed musculoskeletal features that, among living mammals, are associated with prehensility.

Mammalian locomotor-respiratory integration: implications for diaphragmatic and pulmonary design.

Diaphragmatic function and intrapulmonary respiratory flow in running mammals were found to differ substantially from the corresponding conditions known in resting mammals. In trotting dogs, orbital oscillations of the diaphragm were driven by inertial displacements of the viscera induced by locomotion. In turn, oscillations of the visceral mass drove pulmonary ventilation independent of diaphragmatic contractions, which primarily served to modulate visceral kinetics. Visceral displacements and loading of the anterior chest wall by the forelimbs are among the factors that contribute to an asynchronous ventilation of the lungs and interlobar gas recycling. Basic features of mammalian respiratory design, including the structure of the diaphragm and lobation of the lungs, appear to reflect the mechanical requirements of locomotor-respiratory integration.

American Jurassic symmetrodonts and Rhaetic "pantotheres".

The molar morphology of the symmetrodonts Tinodon and Eurylambda from the late Jurassic of North America is virtually identical to that of so-called "pantotheres" from the Rhaetic of Wales. Therefore a primitive symmetrodont molar pattern was probably present in the phylogeny of pantotherian and tribosphenic molars. Occlusion of Tinodon and Eurylambda produced complex wear facets unlike the simple trigon-trigonid shear surfaces of Spalacotherium and Peralestes.

Mesozoic mammals from Arizona: new evidence on Mammalian evolution.

Knowledge of early mammalian evolution has been based on Old World Late Triassic-Early Jurassic faunas. The discovery of mammalian fossils of approximately equivalent age in the Kayenta Formation of northeastern Arizona gives evidence of greater diversity than known previously. A new taxon documents the development of an angular region of the jaw as a neomorphic process, and represents an intermediate stage in the origin of mammalian jaw musculature.

A cineradiographic analysis of bird flight: the wishbone in starlings is a spring.

High-speed x-ray movies of European starlings flying in a wind tunnel provide detailed documentation of avian skeletal movements during flapping flight. The U-shaped furcula (or "wishbone," which represents the fused clavicles) bends laterally during downstroke and recoils during upstroke; these movements may facilitate inflation and deflation of the clavicular air sac. Sternal movements are also coupled with wingbeat, ascending and retracting on downstroke and descending and protracting on upstroke in an approximately elliptical pathway. The coupled actions of the sternum and furcula appear to be part of a respiratory cycling mechanism between the lungs and air sacs.

Modern turtle origins: the oldest known cryptodire.

The discovery of a turtle in the Early Jurassic(185 million years before present) Kayenta Formation of northeastern Arizona provides significant evidence about the origin of modern turtles. This new taxon possesses many of the primitive features expected in the hypothetical common ancestor of pleurodires and cryptodires, the two groups of modern turtles. It is identified as the oldest known cryptodire because of the presence of a distinctive cryptodiran jaw mechanism consisting of a trochlea over the otic chamber that redirects the line of action of the adductor muscle. Aquatic habits appear to have developed very early in turtle evolution. Kayentachelys extends the known record of cryptodires back at least 45 million years and documents a very early stage in the evolution of modern turtles.

Human granulocyte-macrophage colony-stimulating factor enhancer function is associated with cooperative interactions between AP-1 and NFATp/c.

The promoter of the human granulocyte-macrophage colony-stimulating factor gene is regulated by an inducible upstream enhancer. The enhancer encompasses three previously defined binding sites for the transcription factor NFAT (GM170, GM330, and GM550) and a novel NFAT site defined here as the GM420 element. While there was considerable redundancy within the enhancer, the GM330, GM420, and GM550 motifs each functioned efficiently in isolation as enhancer elements and bound NFATp and AP-1 in a highly cooperative fashion. These three NFAT sites closely resembled the distal interleukin-2 NFAT site, and methylation interference assays further defined GGA(N)9TCA as a minimum consensus sequence for this family of NFAT sites. By contrast, the GM170 site, which also had conserved GGA and TCA motifs but in which these motifs were separated by 15 bases, supported strong independent but no cooperative binding of AP-1 and NFATp, and this site functioned poorly as an enhancer element. While both the GM330 and GM420 elements were closely associated with the inducible DNase I-hypersensitive site within the enhancer, the GM420 element was the only NFAT site located within a 160-bp HincII-BalI fragment defined by deletion analysis as the essential core of the enhancer. The GM420 element was unusual, however, in containing a high-affinity NFATp/c-binding sequence (TGGAAAGA) immediately upstream of the sequence TGACATCA which more closely resembled a cyclic AMP response-like element than an AP-1 site. We suggest that the cooperative binding of NFATp/c and AP-1 requires a particular spacing of sites and that their cooperativity and induction via independent pathways ensure very tight regulation of the granulocyte-macrophage colony-stimulating factor enhancer.

Reduced risk of AIDS lymphoma in individuals heterozygous for the CCR5-delta32 mutation.

Non-Hodgkin's lymphoma (NHL) has been increasing in frequency in the industrialized world, but the environmental and genetic factors that contribute to susceptibility are not known. B-cell lymphomas represent a major cause of morbidity and mortality in HIV-infected individuals. The identification of a deletion in the CCR5 chemokine receptor gene that alters the risk for infection and progression to AIDS led us to examine a potential role of this gene in AIDS lymphoma. A matched case-control analysis was performed using all eligible NHL cases in the Multicenter AIDS Cohort Study. Patients were matched for age, study center, time AIDS-free, and slope of the CD4+ T-cell decline. The CCR5-delta32 allele was found to be associated with a 3-fold lower risk of NHL among individuals after controlling for time of infection and progression toward AIDS. The CCR5 gene was not associated with a difference in risk for Kaposi's sarcoma, another common malignancy in AIDS patients, or opportunistic infections. Costimulation of normal phorbol 12-myristate 13-acetate-treated B cells with the CCR5 ligand RANTES induced a proliferative response, indicating that RANTES is a mitogen for B cells. Taken together, these findings suggest that the CCR5 gene plays a role in the risk of NHL in HIV-infected patients, perhaps through a mechanism involving a decreased response of B cells to the mitogenic activity of RANTES.

Pubic osteomyelitis due to anaerobic bacteria.

Osteomyelitis of the pubic bone due to anaerobic bacteria has been reported infrequently, although an entity known as "sterile" osteitis pubis is common to the literature. We have described two cases of pubic osteomyelitis due to anaerobic bacteria, discussed two previously reported cases, and suggested that most cases of what has previously been termed sterile osteitis pubis may actually have been due to anaerobic bacteria that were not isolated because of deficiencies in collection, transport, and culture of clinical specimens. Included are the pathogenesis and an approach to the treatment of this entity.

A.E. Bennett Research Award. Developmental traumatology. Part I: Biological stress systems.

BACKGROUND: This investigation examined the relationship between trauma, psychiatric symptoms and urinary free cortisol (UFC) and catecholamine (epinephrine [EPI], norepinephrine [NE], dopamine [DA]) excretion in prepubertal children with posttraumatic stress disorder (PTSD) secondary to past child maltreatment experiences (n = 18), compared to non-traumatized children with overanxious disorder (OAD) (n = 10) and healthy controls (n = 24). METHODS: Subjects underwent comprehensive psychiatric and clinical assessments and 24 hour urine collection for measurements of UFC and urinary catecholamine excretion. Biological and clinical measures were compared using analyses of variance. RESULTS: Maltreated subjects with PTSD excreted significantly greater concentrations of urinary DA and NE over 24 hours than OAD and control subjects and greater concentrations of 24 hour UFC than control subjects. Post hoc analysis revealed that maltreated subjects with PTSD excreted significantly greater concentrations of urinary EPI than OAD subjects. Childhood PTSD was associated with greater co-morbid psychopathology including depressive and dissociative symptoms, lower global assessment of functioning, and increased incidents of lifetime suicidal ideation and attempts. Urinary catecholamine and UFC concentrations showed positive correlations with duration of the PTSD trauma and severity of PTSD symptoms. CONCLUSIONS: These data suggest that maltreatment experiences are associated with alterations of biological stress systems in maltreated children with PTSD. An improved psychobiological understanding of trauma in childhood may eventually lead to better treatments of childhood PTSD.

A case-control study of HIV-1-related dementia and co-infection with HHV-8.

This nested case-control study assessed the putative protective effect of human herpesvirus-8 (HHV-8) against HIV-1-related dementia (dementia). The HHV-8 seropositivity of 210 male age- and HIV disease stage-matched cases and controls was compared. The overall HHV-8 seropositivity of 66% was similar among demented HIV-infected cases and nondemented HIV-infected controls.

Herpes simplex virus: a tool for neuroscientists.

Herpes viruses have received a great deal of attention due to their widespread and ubiquitous prevalence in the human population and to the diverse range of diseases caused as a result of an infection. During the last 20-25 years, many research laboratories have investigated the pathogenesis and molecular biology of these viruses; particularly herpes simplex virus (HSV). As a result of this research, HSV has begun to get the attention of neuroscientists. In fact, in the last few years there has been an explosion of research involving the use of HSV and related viruses as tools or model systems for different areas of neuroscience research. This brief review will describe several of these areas including demyelinating diseases, neuronal tracings, and genetic therapy.

Deletion of the Herpes simplex 1 internal repeat sequences affects pathogenicity in the mouse.

We have isolated three different herpes simplex virus 1 (HSV-1) recombinant viruses, each frozen in either the P (prototype), IS (inversion of S component), or ILS (inversion of both components) genome arrangement. Common to all three recombinant viruses is the deletion of approximately 14 kilobases (kb) of viral DNA sequences representing greater than 95% of the internal repeat sequences and the insertion of a 9.6 kb mini-Mu genome containing a functional thymidine kinase gene. No unique DNA sequences were deleted from the viral genomes. Analyses of growth curves of the wild-type and recombinant viruses in cell culture has revealed that the recombinants grow somewhat more slowly, producing final titers within 1.5 logs of wild-type HSV-1(F). There is no discernible difference in plaque size or plaque morphology between the recombinant and wild type strains. Analysis of the recombinant viruses in mice reveals the following: I), the recombinant viruses are essentially avirulent, exhibiting drastically increased LD50 values as compared to the wild-type strain by intracerebral injection; ii), the recombinant viruses are not neuroinvasive in that they do not spread from the cornea to sensory ganglion; iii), the recombinant viruses exhibit minimal local replication both in the corneas of infected mice and in the brains of mice inoculated by intracerebral injection; and iv), the recombinant viruses do not establish a reactivable latent infection in the trigeminal ganglion following either intracerebral inoculation or inoculation of scarified corneas. These properties suggest a unique pattern of pathogenesis for HSV mutants in the mouse model.

Herpesvirus 6 variant A infection after heart transplantation with giant cell transformation in bile ductular and gastroduodenal epithelium.

Herpesvirus 6 (HHV-6) is a ubiquitous virus known to cause febrile syndromes and exanthema subitum in children. Less commonly, and particularly in organ transplant recipients, it may result in hepatitis, bone marrow suppression, interstitial pneunonitis, and meningoencephalitis. This report expands the spectrum of clinical disease associated with HHV-6 by documenting viral infection in a 44-year-old heart transplant recipient presenting with gastroduodenitis, pancreatitis, and hepatitis. On histopathologic examination, the gastric, duodenal, and bile ductular epithelium showed a multinucleate giant cell transformation similar to the cytopathic effect caused by the virus in human T-lymphocytes infected in vitro. Electron microscopy showed herpes particles with a thick tegument layer in the duodenum. Polymerase chain reaction amplified HHV-6 variant A sequences from multiple sites. Serology confirmed the presence of an acute HHV-6 infection. Thus, HHV-6 variant A can cause gastroduodenitis and pancreatitis in immunosuppressed individuals. Multinucleate giant cells and enveloped virions with a prominent tegument can be used as morphologic criteria to raise the possibility of HHV-6 infection in human biopsy tissue.

Persistence of the SV40 early region without expression in permissive simian cells.

SV40 containing recombinant vectors were introduced into permissive simian, non-permissive rodent and semi-permissive human cell lines, and assayed for transformation. All mouse and human cell clones expressed T-antigen (T-Ag) and were morphologically transformed when they contained only the wt T-Ag gene (E-SV40) or the entire wt viral genome with an interrupted late region. However, of 63 simian clones with these recombinant vectors, none became morphologically transformed and T-Ag containing cells were rare or absent. Nearly all simian cell lines made either no detectable early SV40 RNA or only small amounts of viral RNA but contained viral DNA restriction fragments similar to those in the original recombinant vectors. Functional T-Ag genes were recoverable from several cell clones and used to regenerate infectious virus. Hence, T-Ag gene expression had been suppressed. We found two conditions where T-Ag expression was activated. In a BSC-1 cell line containing E-SV40 DNA, subsequent introduction of a vector with a functional viral late coding region (L-SV40) resulted in the appearance of T-Ag and transformation. These findings suggest that L-SV40 sequences activate or enhance T-Ag expression and that this activation requires a functional Vpl gene. We found also, that vectors with E-SV40 DNA from the bipartite variant EL-SV40 consistently transformed simian CV-1 cells. Transformation was shown to be effected by the multiple alterations present in the regulatory region of this variant.