Growth transformation of antigen-specific T cell lines from rhesus monkeys by herpesvirus saimiri

This study aims in establishing the in vitro basis for a primate model to evaluate potential applications of H. saimiri-transformed T cells. T cell lines specific for myelin basic protein and streptolysin O were derived from rhesus monkeys and transformed to stable antigen-independent growth with strain C488 of H. saimiri. The transformed T cells from rhesus monkeys did not produce infectious virus and harbored the H. saimiri genome exclusively in an episomal form, whereas transformed T cells from the New World monkey Calltithrix jacchus released infectious virus. Transformed T cells from rhesus monkeys showed an unaltered surface expression of CD2 and CD3, of the activation markers CD25 and CD69, and of the costimulatory molecule CD80 (B7.1). Remarkably, both transformed and nontransformed T cell lines were largely double-positive for CD4 and CD8. In contrast to the parental cell lines, the transformed cells constitutively expressed major histocompatibility complex-DR antigens and were able to present antigen to each other. The transformed T cells from rhesus monkeys continued to express a functionally intact T cell receptor and responded to recognition of their antigen with enhanced proliferation and production of Th1-type cytokines. In conclusion, H. saimiri-transformed rhesus monkey T cells may open a way to primate models for adoptive immunotherapy and studies on the pathogenesis of autoaggressive T cells.     

STP and Tip are essential for herpesvirus saimiri oncogenicity

Mutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS deltaSTP-C488 and HVS deltaTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.     

Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation

Oncogenic potential of human mycoplasmas was studied using cultured mouse embryo cells, C3H/10T1/2 (C3H). Mycoplasma fermentans and Mycoplasma penetrans, mycoplasmas found in unusually high frequencies among patients with AIDS, were examined. Instead of acute transformation, a multistage process in promotion and progression of malignant cell transformation with long latency was noted; after 6 passages (1 wk per passage) of persistent infection with M. fermentans, C3H cells exhibited phenotypic changes with malignant characteristics that became progressively more prominent with further prolonged infection. Up to at least the 11th passage, all malignant changes were reversible if mycoplasmas were eradicated by antibiotic treatment. Further persistent infection with the mycoplasmas until 18 passages resulted in an irreversible form of transformation that included the ability to form tumors in animals and high soft agar cloning efficiency. Whereas chromosomal loss and translocational changes in C3H cells infected by either mycoplasma during the reversible stage were not prominent, the onset of the irreversible phase of transformation coincided with such karyotypic alteration. Genetic instability--i.e., prominent chromosomal alteration of permanently transformed cells--was most likely caused by mutation of a gene(s) responsible for fidelity of DNA replication or repair. Once induced, chromosomal alterations continued to accumulate both in cultured cells and in animals without the continued presence of the transforming microbes. Mycoplasma-mediated multistage oncogenesis exhibited here shares many characteristics found in the development of human cancer.     

Genetic variation of herpesvirus saimiri subgroup A transforming protein and its association with cellular src

Herpesvirus saimiri strain 11 of subgroup A contains a gene called the saimiri transformation-associated protein, STP, which is not required for viral replication but is required for in vitro immortalization and for the lymphoma-inducing capacity of the virus. To assess the effects of sequence variation on STP function, STP genes from six subgroup A isolates were cloned and sequenced. Sequence comparisons revealed extensive amino acid substitutions within the central region, but the acidic amino terminus and the hydrophobic carboxyl terminus were well conserved. Amino acid identities varied from 73 to 99% among all two-way comparisons. The highly conserved YAEV/I motif at amino acid residues 115 to 118 was preceded by negatively charged glutamic acid residues and thus matched very well the consensus sequence for binding to SH2 domains of src family kinases. The STPs of these subgroup A strains were shown to associate with cellular src and to be an in vitro substrate for src kinase. Mutational analysis of STP-A11 showed that binding to src kinase required the tyrosine residue at 115, showing that YAEV/I is a likely binding motif for src. Also, tyrosine phosphorylation of STP-A11 by src led to subsequent binding to lck and fyn in vitro. Thus, the association of STP with src is likely to be important for T-cell transformation by subgroup A strains of herpesvirus saimiri.     

T-cell lymphoma caused by herpesvirus saimiri C488 independently of ie14/vsag, a viral gene with superantigen homology

The immediate-early gene ie14/vsag of herpesvirus saimiri has homology with murine superantigens. We compared the pathogenesis of infection with either ie14/vsag deletion mutants or wild-type virus C488 in cottontop tamarin monkeys (Saguinus oedipus). Two weeks after infection, all animals developed acute T-cell lymphomas independently of the presence of the viral ie14/vsag gene.     

Advances in the treatment of herpesvirus infection: the role of famciclovir

Shingles (herpes zoster) is the result of reactivation of varicella-zoster virus after years of latency. The acute phase is self-limiting but is often associated with moderate-to-severe pain; postherpetic neuralgia is the most frequent and debilitating complication of shingles, occurring in 3.4 per 1000 individuals per year. In the case of genital herpes, herpes simplex virus can reactivate to cause recurrent episodes as often as several times a year, sometimes for the remainder of a person's life. Antiviral agents such as famciclovir, valacyclovir, and acyclovir can be used to shorten the course and decrease the severity of these diseases and may suppress the virus itself, thereby preventing future outbreaks of genital herpes. This article presents a brief synopsis of the etiology of herpes zoster and genital herpes and reviews 12 key studies that demonstrate the efficacy of famciclovir in the management of these two conditions.     

Primary human herpesvirus 7 infection: a comparison of human herpesvirus 7 and human herpesvirus 6 infections in children

Atrial fibrillation is an important and independent risk factor for cerebrovascular disease and vascular dementia. There is increasing evidence that atrial fibrillation is associated with an increased risk of asymptomatic or silent cerebral infarction and as a result may confer an increased risk of progressive cognitive impairment on a person. In this study we sought to determine whether this hypothesis could be explored in a prospective case controlled design. Twenty seven patients with non-valvular atrial fibrillation (NVAF) and no history of stroke, transient ischaemic attack, dementia, and thyrotoxicosis were compared with 54 age and sex matched controls in sinus rhythm. All cases underwent clinical examination, ECG, and psychological assessment using a battery of nine neuropsychological tests. Between group analysis and a comparison of mean test scores of paired controls with cases were undertaken. The presence of atrial fibrillation was consistently associated with poorer performances on all the subtests of the neuropsychological battery. There was no association between duration of atrial fibrillation and performance. These results provide evidence to justify further examination of the hypothesis in a larger prospective study to determine whether antithrombotic therapy may protect against cognitive decline in patients at maximal risk of silent cerebral ischaemia and associated cognitive decline.     

Mechanisms of persistent airway inflammation in asthma. A role for T cells and T-cell products

The role of T cells in human allergic inflammation is just beginning to be understood. However, the data presented indicate how the T cell may be a pivotal cell to direct features of allergic inflammation in asthma, how the T cell may be able to transfer hyperresponsiveness, which is a feature of bronchial asthma, what some of the genetic factors are that may determine this process, and how an important precipitant of asthma, viral respiratory infections, may participate in this process. Its cells are isolated from patients with asthma and studied for their ability to generate proinflammatory failure. An expanded understanding of the chronic, persistent nature of asthma will become apparent.     

Immortalization with herpesvirus saimiri modulates the cytokine secretion profile of established Th1 and Th2 human T cell clones

T blasts of six established human CD4+ T cell clones with defined Ag specificity and cytokine secretion profile (3 Th1 and 3 Th2) were immortalized with Herpesvirus saimiri (HVS) and compared with their uninfected counterparts for their ability to proliferate, produce cytokines, and express cytolytic activity. HVS-transformed Th1 and Th2 clones neither substantially changed their original surface markers nor lose their ability to proliferate in response to their specific Ag but did acquire the ability to proliferate in response to contact signals delivered by SRBC or autologous APC alone. In addition, transformation by HVS substantially enhanced the lectin-dependent cytolytic activity of Th1 clones and enabled noncytolytic Th2 clones to exert cytolytic activity. HVS-transformed Th1 clones but not their uninfected counterparts spontaneously transcribed and secreted Th1-type cytokines (IL-2, IFN-gamma, and TNF-beta) and such a production was further enhanced by stimulation with either SRBC or PMA plus anti-CD3 mAb. HVS transformed but not uninfected Th2 clones constitutively expressed both IL-4 and IL-2 mRNA and secreted IFN-gamma. Stimulation with PMA plus anti-CD3 mAb induced uninfected Th2 clones to secrete high amounts of IL-4 and IL-5 but not Th1-type cytokines, whereas the same HVS-transformed Th2 showed minimal IL-4 and IL-5 secretion with concomitant high production of IL-2, IFN-gamma, and TNF-beta. Transformation by HVS also resulted in up-regulation of TNF-alpha and IL-3 production by both Th1 and Th2 clones. The ongoing proliferation of HVS-transformed clones was partially inhibited by either anti-IL-2 or anti-IL-3 antibodies and virtually abolished by the combined addition of the two anticytokine antibodies, suggesting that both IL-2 and IL-3 can function as autocrine growth factors for HVS-transformed Th1 and Th2 clones.     

Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma

To investigate the effects of adenosine A1 receptor activation on energy metabolism and RNA and protein biosynthesis in central neurons, cultured neurons from the rat forebrain were exposed for 1 hr to 72 hr to various concentrations (10 nM-100 microM) of the selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) or the A1 receptor antagonist 8-cyclopentyltheophylline (CPT). At all concentrations tested, the adenosinergic compounds did not affect cell viability within 72 hr of treatment, except for CPT, which reduced viability by 19.7% when used at the concentration of 100 microM. Energy metabolism was analysed by studying the specific uptake of 2-D-[3H]deoxyglucose ([3H]2DG). Rates of RNA and protein biosynthesis were assessed by the measurement of [3H]uridine and [3H]leucine incorporation, respectively. Neuronal [3H]2DG uptake was increased by 16% (P < 0.01) after 8 hr in the presence of 100 microM CCPA, whereas 100 microM CPT for 24 hr also increased [3H]2DG uptake (8%, P < 0.01). At these concentrations, both ligands inhibited [3H]uridine incorporation after a 3-hr treatment by 92% and 30%, respectively. CCPA never altered [3H]leucine incorporation when compared to controls, and CPT significantly inhibited protein synthesis only at 10-100 microM. Additional experiments to analyse the influence of A1 ligands on the transport of [3H]2DG, [3H]leucine and [3H]uridine suggested that CCPA and CPT, which interact functionally with adenosine receptors by regulating cyclic AMP production in this model, are able to alter energy metabolism and RNA synthesis in central neurons in a nonspecific manner by interacting with glucose and uridine transporters.     

A case of congenital mumps infection complicated with persistent pulmonary hypertension

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.     

A role for perforin in downregulating T-cell responses during chronic viral infection

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf -/-) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf -/- mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral "exhaustion" of activated CD8 T cells occurred less efficiently in perf -/- mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf -/- mice; approximately 50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.     

Human herpesvirus 6 reactivation is associated with cytomegalovirus infection and syndromes in kidney transplant recipients at risk for primary cytomegalovirus infection

A potential association between human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) following kidney transplantation was explored by retrospectively testing serial serum specimens for HHV-6 IgG and IgM antibody. HHV-6 reactivation occurred in 35 (66%) of 53 transplant recipients. Fungal or parasitic opportunistic infections, graft rejection or loss, and mortality were not associated with HHV-6 reactivation. HHV-6 reactivation was associated with primary CMV infection (P=.001) and CMV syndrome (P=.003) and with trends for CMV-related hepatitis (P=.095), CMV-related neutropenia (P=.104), and serious CMV disease (P=.085). After controlling for CMV immune globulin (CMVIG) prophylaxis, the association between HHV-6 reactivation and primary CMV infection and syndrome remained significant (P=.002 and 0.006, respectively). The reduction in CMV syndrome among those receiving CMVIG prophylaxis remained significant (P=.007) after controlling for HHV-6 reactivation. HHV-6 reactivation in kidney transplant recipients at risk for primary CMV infection is associated with CMV infection and CMV-related disease, and these effects are independent of CMVIG prophylaxis.     

Sexual transmission and the natural history of human herpesvirus 8 infection

BACKGROUND: Although human herpesvirus 8 (HHV-8) has been suspected to be the etiologic agent of Kaposi's sarcoma, little is known about its seroprevalence in the population, its modes of transmission, and its natural history. METHODS: The San Francisco Men's Health Study, begun in 1984, is a study of a population-based sample of men in an area with a high incidence of human immunodeficiency virus (HIV) infection. We studied all 400 men infected at base line with HIV and a sample of 400 uninfected men. Base-line serum samples were assayed for antibodies to HHV-8 latency-associated nuclear antigen (anti-LANA). In addition to the seroprevalence and risk factors for anti-LANA seropositivity, we analyzed the time to the development of Kaposi's sarcoma. RESULTS: Anti-LANA antibodies were found in 223 of 593 men (37.6 percent) who reported any homosexual activity in the previous five years and in none of 195 exclusively heterosexual men. Anti-LANA seropositivity correlated with a history of sexually transmitted diseases and had a linear association with the number of male sexual-intercourse partners. Among the men who were infected with both HIV and HHV-8 at base line, the 10-year probability of Kaposi's sarcoma was 49.6 percent. Base-line anti-LANA seropositivity preceded and was independently associated with subsequent Kaposi's sarcoma, even after adjustment for CD4 cell counts and the number of homosexual partners. CONCLUSIONS: The prevalence of HHV-8 infection is high among homosexual men, correlates with the number of homosexual partners, and is temporally and independently associated with Kaposi's sarcoma. These observations are further evidence that HHV-8 has an etiologic role in Kaposi's sarcoma and is sexually transmitted among men.     

Identification of persistent infection in experimental syphilis by PCR

The studies described herein were designed to evaluate the usefulness of the PCR in detecting persistent syphilitic infection. Three groups of animals were used: a nonimmune group infected with Treponema pallidum (NI/TP), a nonimmune group injected with heat-killed treponemes (NI/HKTP), and an immune and reinfected group (I/TP). All animals were inoculated with similar numbers of organisms distributed at 10 sites on the clipped back and in both testes. The persistence of the treponemes was examined by PCR and the rabbit infectivity test (RIT). The kinetic studies and statistical analysis of their results demonstrated that the rate of bacterial clearance from the NI/TP group was very low and incomplete at 4 months after infection. It was significantly different from those of both the NI/HKTP (P < 0.001) and I/TP (P < 0.05) groups. No statistically significant differences in treponemal elimination were found between the NI/HKTP and I/TP groups. PCR can detect the DNA of dead organisms, but the latter are eliminated by the host relatively quickly (15 to 30 days) as compared to elimination of live treponemes (>120 days). PCR results correlated well with RIT results. These data suggest that PCR-positive specimens obtained from an untreated patient(s) or collected weeks after treatment indicate persistent infection. They also show that the process of elimination of T. pallidum from primary sites of infection is prolonged and incomplete.     

Human herpesvirus 8 infection occurs following adolescence in the United States

Most recent evidence suggests that human herpesvirus 8 (HHV-8) infection is restricted to persons with Kaposi's sarcoma (KS) or to persons who may subsequently develop KS. To accurately determine the prevalence of infection in the United States, children and adults with AIDS were examined for evidence of HHV-8 infection to see whether HHV-8 (like other herpesviruses) would be readily detected in immunosuppressed persons. By use of nested polymerase chain reaction, DNA specific for HHV-8, Epstein-Barr virus, and cytomegalovirus was detected in blood leukocytes from 0, 26 (51%), and 9 (18%), respectively, of 51 children. Similarly, HHV-8-specific antibodies were not detected in analyses of sera from the children. By contrast, HHV-8 DNA was detected in 9 (27%) of 33 adult AIDS patients without KS. These findings suggest that the pattern of transmission of HHV-8 in the United States differs from that of other herpesviruses in that primary infection occurs predominantly in adults.     

Interferon-gamma mediates long-term persistent Chlamydia psittaci infection in vitro

The long-term anti-chlamydial effects of recombinant ovine interferon gamma (rOvIFN-gamma) were studied in ovine ST-6 fibroblasts infected with the S26/3 strain of Chlamydia psittaci. Chlamydial multiplication was assessed by enzyme-linked immunosorbent assay analysis of supernate lipopolysaccharide, titration of inclusion-forming units in culture supernates, and enumeration of inclusion bodies in cultured cells at 7-day intervals. Concentrations of 250 and 1000 U/ml of rOvIFN-gamma resulted in a microbistatic inhibition of C. psittaci growth, which appeared to become microbicidal when rOvIFN-gamma was maintained in the cultures for 14 days or more. There were no signs of C. psittaci multiplication when cultures were maintained in 25 or 100 U/ml of rOvIFN-gamma. However, subsequent removal of rOvIFN-gamma from these cultures resulted in a re-emergence of viable, infectious chlamydiae, which eventually killed all the fibroblasts. This re-emergence was more rapid in cultures initially treated with 25 U/ml of rOvIFN-gamma than in those treated with 100 U/ml.