The significance of low bcl-2 expression by CD45RO T cells in normal individuals and patients with acute viral infections. The role of apoptosis in T cell memory.

The bcl-2 gene product has been shown to prevent apoptotic cell death. We have now investigated the bcl-2 protein expression by resting and activated mature T cell populations. Freshly isolated CD45RO+ T cells within CD4+ and CD8+ subsets expressed significantly less bcl-2 than CD45RO- (CD45RA+) T cells (p < 0.001). When CD45RA+ T cells within both CD4+ and CD8+ subsets were activated in vitro, the transition to CD45RO phenotype was associated with a decrease in bcl-2 expression. Patients with acute viral infections such as infectious mononucleosis caused by Epstein-Barr virus infections or chickenpox, resulting from varicella zoster virus infection, had circulating populations of activated CD45RO+ T cells which also showed low bcl-2 expression. In these patients, a significant correlation was seen between low bcl-2 expression by activated T cells and their apoptosis in culture (r = 0.94, p < 0.001). These results suggest that the primary activation of T cells leads to the expansion of a population that is destined to perish unless rescued by some extrinsic event. Thus the suicide of CD45RO+ T cells could be prevented by the addition of interleukin 2 to the culture medium which resulted in a concomitant increase in the bcl-2 expression of these cells. Alternatively, apoptosis was also prevented by coculturing the activated T lymphocytes with fibroblasts, which maintained the viability of lymphoid cells in a restinglike state but with low bcl-2 expression. The paradox that the CD45RO+ population contains the primed/memory T cell pool yet expresses low bcl-2 and is susceptible to apoptosis can be reconciled by the observations that maintenance of T cell memory may be dependent on the continuous restimulation of T cells, which increases their bcl-2 expression. Furthermore, the propensity of CD45RO+ T cells to extravasate may facilitate encounter with fibroblast-like cells in tissue stroma and thus be an important additional factor which promotes the survival of selected primed/memory T cells in vivo.

The effect of cytomegalovirus infection on the host response to foreign and hapten-modified self histocompatibility antigens.

The effect of murine cytomegalovirus (MCMV) infection on the host's ability to respond to allogeneic or hapten-modified syngeneic histocompatibility antigens was characterized by an early suppressive phase followed by a phase of enhancement. Suppression of the potential to respond to allogeneic H-2 antigens, as measured by an in vitro cell-mediated lympholysis assay, was seen on days 2-4 postinfection with MCMV, and was greatest at larger doses of virus and in mice with H-2 haplotypes associated with genetic susceptibility to MCMV. In mice of the C57BL genetic background, such responses had returned to normal levels by day 6 and thereafter (days 7-13) the potential of the infected host to respond to allogeneic histocompatibility determinants was markedly enhanced compared with uninfected control mice. This enhancement of alloreactivity was maximal at lower doses of virus and was coincident with the emergence of reactivity to self antigens, as measured by autoantibody production. A similar pattern of time-dependent suppression and enhancement of the hapten modified self cytotoxic response was observed. Enhancement of alloreactivity during MCMV infection was dependent on the genetic constitution of the host with the response of BALB/c mice remaining in a suppressive phase for up to 17 days postinfection. The possibility that this in vitro finding of enhanced alloreactivity during MCMV infection underlies the clinical observations of association between episodes of kidney allograft rejection and cytomegalovirus infection is discussed.

Cytomegalovirus induced up-regulation of LFA-3 (CD58) and ICAM-1 (CD54) is a direct viral effect that is not prevented by ganciclovir or foscarnet treatment.

Cytomegalovirus (CMV) is a major pathogen in transplant recipients and AIDS patients, and the virus may also play a role in allograft rejection. Previous work from this laboratory demonstrated increased cell surface expression of the adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58) following CMV infection in vitro. We investigated whether the induction of adhesion molecules by CMV was a direct viral effect or secondary to cytokine induction. Cytokines known to up-regulate ICAM-1, such as TNFalpha or IL-1beta, were not detected in the supernatants of infected fibroblasts, and neutralizing antibodies against these cytokines did not abrogate the induction of either ICAM-1 or LFA-3 by CMV. Infected cell supernatants had increased levels of IL-6, IL-8 and IFNbeta however, the addition of recombinant forms of these cytokines did not affect adhesion molecule expression. Neither virus-free infected cell supernatants nor UV-inactivated virus up-regulated adhesion molecules, demonstrating that the induction of ICAM-1 and LFA-3 by CMV was a direct effect requiring infectious virus. Effective antiviral treatment with ganciclovir or foscarnet accentuated rather than abrogated the up-regulation of adhesion molecules, suggesting that CMV immediate early/early gene expression, which is not blocked by such treatment, was responsible for the adhesion molecule induction. Thus, despite effective antiviral therapy in the transplant recipient, CMV infected cells may continue to provide a focus of proinflammatory activity, which could contribute to immunopathology and/or accentuate graft rejection or graft-versus-host disease in vivo.

Augmentation of graft-versus-host reaction by cytomegalovirus infection resulting in interstitial pneumonitis.

The severity of the graft-versus-host (GVH) reaction, judged by splenomegaly and immunosuppression, was augmented by murine cytomegalovirus (MCMV) infection. Profound GVH-induced immunosuppression was seen in adult unirradiated MCMV-infected F1, mice even after challenge with extremely low doses of parental spleen cells. Mice receiving MCMV+GVH challenge died from days 16-21, with interstitial pneumonia being the most prominent pathological lesion. Pulmonary disease was unrelated to levels of viral replication in the lung. These results suggest that in human marrow recipients, cytomegalovirus infection may play a primary role both in provoking or accentuating GVH disease, as well as in the development of interstitial pneumonia.

Cytomegalovirus and beta 2 microglobulin in urine specimens. Reciprocal interference in their detection is responsible for artifactually high levels of urinary beta 2 microglobulin in infected transplant recipients.

We have previously shown that the binding of host beta 2 microglobulin (beta 2 m) by cytomegalovirus (CMV) in body fluids masks the viral antigenic determinants preventing its detection by CMV-specific monoclonal antibodies. We now report that the recognition of CMV-bound beta 2m by anti-beta 2m-specific antibodies in assays for beta 2m, results in erroneously high readings for beta 2m levels in urine specimens containing CMV. Urinary beta 2m levels have previously been reported to be elevated in patients with CMV infection--however, when virion bound beta 2m was removed by ultracentrifugation of urine specimens, the levels of free beta 2m were not found to be elevated in these patients. Since CMV is frequently excreted by transplant recipients and acquired immunodeficiency syndrome patients, our data suggest that measurements of urinary beta 2m levels in such patients are unreliable unless the urine specimens are confirmed to be free of CMV before analysis.

IL2 activated killer cells may contribute to cytomegalovirus induced marrow hypoplasia after bone marrow transplantation.

Cytomegalovirus (CMV) infection remains the commonest cause of infective death following allogeneic bone marrow transplantation (BMT). CMV disease post-BMT occurs in the context of compromised cellular defence mechanisms and is associated with marrow hypoplasia and pneumonitis. However, CMV infection induces release of interleukin 2 (IL2) which in turn generates MHC unrestricted lymphokine activated killer (LAK) cells. We have investigated whether recruitment of IL2 activated MHC unrestricted defence mechanisms post-transplant can be implicated in the marrow hypoplasia that frequently accompanies CMV infection. The results show that IL2 activated peripheral blood mononuclear cells (PBMC) have substantial cytotoxicity against MHC matched and MHC mismatched marrow fibroblasts but that this activity is not specific for CMV infected fibroblasts; uninfected target cells are also equally killed. Furthermore, post-BMT PBMC show greater responsiveness to IL2 than normal PBMC in killing of marrow fibroblasts. We provide a hypothesis from these observations which may explain some of the consequences of CMV infection post-BMT. Local production of IL2 activated cytotoxic cells which would be generated during CMV infection would damage uninfected as well as infected marrow fibroblasts and thereby could compromise haemopoietic growth factor production by marrow fibroblasts. Similarly generated cytotoxicity in the lung may accompany CMV pneumonitis. Our results suggest that administration of anti-IL2 receptor antibody may have a therapeutic role in CMV disease post-BMT as has recently been shown in graft-versus-host disease.

An improved radioimmunoassay method for the detection of IgG antibodies against cytomegalovirus.

The non-specific binding seen with human sera in a radioimmunoassay for the detection of IgG antibodies specific for CMV can be reduced greatly by using a murine monoclonal antibody as a radiolabelled detecting antibody. Such non-specific binding formerly obtained with a polyclonal detecting antibody was due to the binding of the polyclonal reagent to factors on the solid phase other than IgG molecules.

Biochemistry below 0 degrees C: nature's frozen vertebrates.

Although alien to man, the ability to endure the freezing of extracellular body fluids during the winter has developed in several species of terrestrially hibernating frogs and turtles as well as in many species of insects and other invertebrates. Wood frogs, for example, can endure freezing for at least 2 weeks with no breathing, no heart beat or blood circulation, and with up to 65% of their total body water as ice. Our studies are providing a comprehensive view of the requirements for natural freezing survival and of the physical and metabolic protection that must be offered for effective cryopreservation of vertebrate organs. Molecular mechanisms of natural freeze tolerance in lower vertebrates include: 1) control over ice crystal growth in plasma by ice nucleating proteins, 2) the accumulation of low molecular weight cryoprotectants to minimize intracellular dehydration and stabilize macromolecular components, and 3) good ischemia tolerance by all organs that may include metabolic arrest mechanisms to reduce organ energy requirements while frozen. Cryomicroscopy of tissue slices and magnetic resonance imaging (MRI) of whole animals is revealing the natural mode of ice propagation through an organism. MRI has also revealed that thawing is non-uniform; core organs (with high cryoprotectant levels) melt first, facilitating the early resumption of heart beat and blood circulation. Studies of the production and actions of the natural cryoprotectant, glucose, in frogs have shown its importance in maintaining a critical minimum cell volume in frozen organs and new work on the metabolic effects of whole body dehydration in 3 species of frogs has indicated that adaptations supporting freeze tolerance grew out of mechanisms that deal with desiccation resistance in amphibians. Studies of the regulation of cryoprotectant glucose synthesis by wood frog liver have shown the role of protein kinases and of alpha and beta adrenergic receptors in regulating the glycemic response, and of changes in membrane glucose transporter proteins to facilitate cryoprotectant distribution.

Virologic and pathogenetic aspects of cytomegalovirus infection.

Cytomegalovirus (CMV) is a ubiquitous agent that rarely causes disease in immunocompetent humans but is an important cause of morbidity and mortality in the immunocompromised. A number of host and viral factors are associated with pathology following CMV infection. CMV can be found at many sites in the body but only causes disease at some of these and then only in certain patient populations. In some situations the mechanism underlying disease is direct viral replication, but in others, particularly CMV pneumonitis in allogeneic transplant recipients, an immunopathologic basis is strongly implicated. An important factor in the pathogenesis of infection and the expression of symptomatic disease is the source of CMV infection-whether it arises from an exogenous source or is due to reactivation of latent endogenous virus. Exogenous infection can occur in previously seronegative individuals or in those with prior exposure to the virus. In renal transplant recipients both types of exogenous infection have been associated with disease. Another factor that affects the interaction between CMV and its host is the modulating effect of the virus on the host immune response, the mechanism of which remains unknown.

Beta 2 microglobulin on the envelope of urinary cytomegalovirus is not associated with host class I human leukocyte antigen alpha chain.

Previous studies have shown that beta 2 microglobulin (beta 2m) is associated with glycoproteins present on the envelope of urinary human cytomegalovirus (CMV). beta 2m is non-covalently associated with the alpha chain of human leukocyte antigen (HLA) class I antigens and therefore it was of interest to determine whether the class I alpha chain is also associated with the beta 2m-CMV complex. Using a panel of monoclonal antibodies recognizing different conformational determinants on the HLA class I heterodimer or free beta 2m, we have shown that beta 2m but not the alpha chain of HLA class I could be immunoprecipitated from 125I-surface-labelled virions purified directly from urine. We therefore conclude that host class I HLA alpha chains are not associated with beta 2m on the envelope of urinary CMV.

Down-regulation of the class I HLA heterodimer and beta 2-microglobulin on the surface of cells infected with cytomegalovirus.

Cytotoxic T cell recognition of virus-infected cells requires the presentation of viral peptides by class I HLA molecules on the cell surface. We report here that cytomegalovirus (CMV) infection of human fibroblasts results in a progressive decrease in the cell surface expression of class I HLA and beta 2-microglobulin (beta 2m) such that in the late stages of infection the majority of infected cells have no detectable surface class I HLA. Coincident with decreased surface expression of class I HLA was an increase in his cytoplasmic expression. Confocal scanning laser microscopic analysis demonstrated that class I HLA and beta 2m accumulate in a perinuclear compartment inside the CMV-infected cell. Our data thus support the concept that CMV infection induces altered transport of class I HLA to the cell surface. We suggest that the virus has evolved this mechanism as a strategy to avoid T cell recognition of infected cells.

Human cytomegalovirus induces an Fc gamma receptor (Fc gammaR) in endothelial cells and fibroblasts that is distinct from the human cellular Fc gammaRs.

The expression of a trypsin-sensitive receptor for the Fc portion of IgG (Fc gammaR) was demonstrated by flow cytometry on the surface of human umbilical vein endothelial cells and fibroblasts infected with human cytomegalovirus (CMV). Double-labeling experiments showed strong expression of the CMV Fc gammaR in a perinuclear region of infected cells but not in bystander uninfected cells. The CMV Fc gammaR did not react with a panel of murine monoclonal antibodies directed against the known human IgG Fc receptors, Fc gammaRI, Fc gammaRII, and Fc gammaRIII. The cytoplasmic form but not the cell surface form of CMV Fc gammaR bound murine IgG3 moderately and murine IgG1 more weakly, while both forms bound rabbit IgG almost as strongly as human IgG. The function of CMV Fc gammaR is unclear, but it may allow CMV to evade host antibody responses. However, the binding of immune complexes to infected endothelium might also contribute to immunopathology.

Urea and salt effects on enzymes from estivating and non-estivating amphibians.

The effects of urea, cations (K+,NH4,Na+,Cs+,Li+), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle were analyzed in two anuran amphibians, an estivating species, the spadefoot toad Scaphiopus couchii, and a semi-aquatic species, the leopard frog Rana pipiens. Urea, which accumulates naturally to levels of 200-300 mM during estivation in toads, had only minor effects on the Vmax, kinetic constants and pH curves of PK from either species and no effects on PFK Vmax or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the Vmax of toad PFK and of PK from both species and altered the Km values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the Vmax of PK from either species was reduced by more than 80% by the addition of 200 mM NH4Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the Km values for substrates of toad lactate dehydrogenase and strongly reduced the Vmax of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid early diagnosis of cytomegalovirus infection.

Cytomegalovirus infection can now be diagnosed rapidly in the laboratory by methods which are reviewed here. Together with the clinical investigation of patients early in their infection before extensive tissue damage has occurred, these methods offer the option of considering therapeutic intervention in some patients.