Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV), a betaherpesvirus, has developed several ways to evade the immune system, notably downregulation of cell surface expression of major histocompatibility complex class I heavy chains. Here we report that HCMV has devised another means to compromise immune surveillance mechanisms. Extracellular accumulation of both constitutively produced monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor-superinduced RANTES (regulated on activation, normal T cell expressed and secreted) was downregulated in HCMV-infected fibroblasts in the absence of transcriptional repression or the expression of polyadenylated RNA for the cellular chemokine receptors CCR-1, CCR-3, and CCR-5. Competitive binding experiments demonstrated that HCMV-infected cells bind RANTES, MCP-1, macrophage inflammatory protein (MIP)-1beta, and MCP-3, but not MCP-2, to the same receptor as does MIP-1alpha, which is not expressed in uninfected cells. HCMV encodes three proteins with homology to CC chemokine receptors: US27, US28, and UL33. Cells infected with HCMV mutants deleted of US28, or both US27 and US28 genes, failed to downregulate extracellular accumulation of either RANTES or MCP-1. In contrast, cells infected with a mutant deleted of US27 continues to bind and downregulate those chemokines. Depletion of chemokines from the culture medium was at least partially due to continuous internalization of extracellular chemokine, since exogenously added, biotinylated RANTES accumulated in HCMV-infected cells. Thus, HCMV can modify the chemokine environment of infected cells through intense sequestering of CC chemokines, mediated principally by expression of the US28-encoded chemokine receptor.

Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae.

Diaphragmed fenestrae (DF) are sites of increased vascular permeability. The anionic charge distribution at the luminal aspect of the DF of the endothelium of the bone marrow vessels has been studied after aldehyde fixation by means of colloidal iron (CI), native ferritin (NF), and polycationic ferritin (PCF). At pH 1.8, these cationic agents are bound by the nonmodified luminal endothelial cell surface but not at the sites of the DF. PCF was used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels, whereas NF which has a pI of 4.5 is anionic above this point). PCF shows increased binding at the DF from pH 3.5 upwards. PCF binding at pH 1.8 at the nonmodified luminal cell surface is significantly diminished by neuraminidase treatment which, however, does not perceptibly reduce PCF binding at the higher pH levels. It is concluded that there are exposed sialic acid groups at the lunimal cell surface which are absent or significantly fewer at the sites of the DF, whereas other anionic materials possibly with a pKa higher than that of sialic acid (pKa 2.6) are present both at the DF and at the nonmodified endothelial cell surface.

Nonrandom distribution of sialic acid over the cell surface of bristle-coated endocytic vesicles of the sinusoidal endothelium cells.

Previous studies with protein tracers have shown that the luminal surface of the vascular endothelium of the bone marrow is endocytic. The endocytosis occurs through the formation of large bristle-coated vesicles (LCV). The anionic charge distribution in this process was examined at the luminal surface of the endothelial cell, At pH 1.8, colloidal iron (CI), native ferritin, and polycationic ferritin (PCF) are bound by the luminal surface of the endothelial cell, but not at the sites of LCV formation. PCF used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels) revealed LCV binding of this agent in increasing manner from pH 3.5 upwards. PCF binding at low pH (1.8) at the endothelial cell surface was markedly reduced by neuraminidase. Neuraminidase did not reduce PCF binding by the endothelial cell surface nor by the LCV at higher pH levels. It is concluded that the luminal surface of the endothelial cell has exposed sialic acid groups which are absent or significantly diminished at endocytic sites. The free surface of the endothelial cells as well as the sites of endocytosis have, in addition, anionic material with a pKa higher than that of sialic acid (pKa 2.6). These anionic materials may be different at the sites of endocytosis as compared to those present at the free cell surface.

Implication of gammadelta T cells in the human immune response to cytomegalovirus.

In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.

Diagnosis and treatment of cytomegalovirus iridocyclitis without retinal necrosis.

AIM: To describe the diagnostic and therapeutic management of cytomegalovirus (CMV) anterior uveitis unassociated with retinal necrosis in immunocompetent patients. METHODS: Patients referred between 2001 and 2003 for management of unilateral, chronic, recurrent uveitis associated with secondary glaucoma underwent extensive investigation including laboratory tests for herpes virus infections. Specific antiviral treatment was initiated in all cases and the level of ocular inflammation was evaluated during the follow up. RESULTS: Five patients, three men and two women, were included. Median age was 50 years old (range 30-80 years). Anterior unilateral uveitis without iris atrophy was observed in all cases. Uveitis was chronic in three cases and recurrent in two cases. Glaucoma was observed in all patients with a median intraocular pressure of 30 mm Hg (range 22-43 mm Hg). Five patients responded initially to specific anti-CMV therapy. Even though glaucoma surgery was necessary in two cases, both ocular inflammation and glaucoma were controlled in all cases. Relapses occurred in three cases after cessation of therapy, requiring prolonged maintenance therapy with valganciclovir. CONCLUSIONS: CMV infection and specific antiviral therapy should be considered in all cases of relapsing or chronic iridocyclitis and secondary glaucoma. Maintenance regimens of valganciclovir may be necessary to prevent further relapses.

Drug resistance-reversal strategies: comparison of experimental data with model predictions.

We previously developed a mathematical model to describe the emergence and dynamic growth of a drug-resistant subpopulation in a tumor. In the present study, our objective was to test the model's ability to mimic two strategies for reversal of drug resistance. We present data from one in vitro cell proliferation assay with drug-resistant LS174T human colon carcinoma variants and one in vivo assay of survival after treatment of female (C57BL/6 x DBA/2)F1 mice inoculated with doxorubicin-resistant P388/ADR leukemia cells. The in vitro assay examined the effects of inhibiting the biosynthesis of glutathione in cells resistant to alkylating agents or cisplatin. The in vivo assay compared the effects on cell survival of low-level continuous infusion versus high-intensity bolus dosing, with or without coadministration of the drug efflux pump blocker verapamil. Results in vitro and in vivo were comparable for qualitative accuracy and predictability to results with the model. Both the in vitro study and the model showed that, for resistant cells with high levels of glutathione, short-term cell survival was dose dependent and that even high doses of drug did not eliminate all of these cells. Addition of an inhibitor of glutathione biosynthesis did, however, augment elimination of the resistant cells. Resistant cells with low levels of glutathione could be eliminated with high drug doses or coadministration of drug and a glutathione synthesis inhibitor. In vivo, coadministration of doxorubicin with verapamil increased animal survival when either continuous infusion or bolus dosing regimens were used. The effectiveness of the blocker is crucial; when a partially (50%) effective blocker is used, continuous infusion achieves better elimination of resistant cells, but a completely (100%) effective blocker is efficacious in both dosing scenarios. Careful interpretation of these findings is necessary because the pharmacokinetics of drug in the small populations of cells in the model are not easily extrapolated to those in large tumors. This model may be useful in determining resistance mechanisms, their levels of effectiveness, and concentrations of compounds required at target sites to overcome them.

Emergence of the drug-resistant phenotype in tumor subpopulations: a hybrid model.

A mathematical model is proposed that describes the emergence of drug resistance in a tumor cell population. The model is termed a hybrid in the sense that the population-wide dynamics are described by a stochastic birth-death-migration model with transition probabilities dependent on the deterministic distribution of drug within the average cell. In the model, the probability that a cell dies is proportional to the concentration of drug within the target site in the cell. The micropharmacology describing the distribution of drug within the average cell is described by a standard well-mixed compartment model. Possible mechanisms that can confer drug resistance on a cell are described: decreased drug uptake, increased drug efflux, intracellular metabolism or inactivation, or both, of a drug, and a change in the level or sensitivity of a target. The biologic mechanisms underlying resistance and potential strategies for overcoming it are discussed within the context of our model. Results from a numerical simulation are presented as verification of the initial theory.

Hypoxic fractions in xenografted human colon tumors.

We investigated the percentage of radiobiologically hypoxic cells within 11 different xenografted human colon tumors using an in vivo-in vitro excision assay technique. Tumors were excised at average volumes of 750 mm3, and it was found that hypoxic fractions varied from less than 1% (clone D) to over 80% (HCT-8). The geometric mean hypoxic percentage was 10.4% (95% confidence interval, 4.9 to 22.1%). Comparison of the percentage of hypoxia results from the xenografted human colon tumors to published data from xenografted melanomas suggests that transplanted colorectal tumors as a class contain significantly less hypoxia than do the melanomas.

Effect of preirradiation of transplantation site on growth characteristics and hypoxic fractions in human colon tumor xenografts.

The volumetric growth curves and hypoxic fractions of seven different human colon tumor lines (clone A, clone D, WiDR, SW480, SW620, DLD-2, and HCT-8) xenografted into the flank regions of either unirradiated nude mice or mice that had received 17.5 Gy of 250-kVp X-rays 1 day prior to implantation were biomathematically analyzed using the Verhulstian equation. Significant variation was found among tumors with respect to both initial growth rates (r, days-1) and theoretical final volumes (carrying capacities, K, mm3). In radiation-damaged normal tissue, tumors grew relatively well for about the first 2 wk postimplantation, attaining volumes of about 70 to 155 mm3. Then, tumor growth rates altered. This effect varied from relatively minor effects on growth rate (tumors of clones A and D) to inhibition of growth, with actual decreases in tumor volume (e.g., WiDr, SW480, SW620, HCT-8, and DLD-2). After this short-term transience in growth kinetics, neoplasms began to steadily regrow at about 3 wk postimplantation, albeit at a slower rate than that seen in controls. Tumor bed effect values were calculated using the ratio of times at which control tumors and tumors growing in the radiation-injured tissue reached a volume of 7.5% of the K values derived from the respective control growth curves. Values for clone D, clone A, and WiDR, SW480, SW620, DLD-2, and HCT-8 tumors were, respectively, 1.89, 2.41, 3.48, 3.62, 2.82, 3.66, and 3.65, indicating that tumor bed effect responses varied by almost 100%, even for cancers of the same neoplastic class. Also, the hypoxic fractions of all tumors growing in radiation-damaged sites were increased as compared with levels in controls.

Modification of the growth rates and hypoxic fractions of xenografted A431 tumors by sialoadenectomy or exogenously supplied epidermal growth factor.

We studied A431 epidermoid carcinomas xenografted into male nude mice either in the unperturbed state or after either surgical removal of the salivary glands or i.p. injection of exogenous epidermal growth factor (0.2 mg/kg daily for 7 days). The percentage of hypoxic cells in unperturbed tumors was 10.5% (95% confidence limits, 6.6-16.8%). In mice that received epidermal growth factor injections, hypoxic percentages decreased to 3.7% (1.7-7.8%), and tumor growth rates increased. In sialoadenectomized mice, hypoxic percentages increased to 35.2% (27.1-45.6%), and tumor growth rates decreased. These data indicate that the biology of solid tumors can be significantly modified by the host status.

Growth properties of artificial heterogeneous human colon tumors.

Two clonal cell lines (designated as clones A and D), originally isolated from the heterogeneous DLD-1 human colon adenocarcinoma, were used to produce xenograft tumors in nude mice. Neoplasms produced from either A or D cells alone were compared to those produced from a range of percentage admixtures of the two subpopulations. Then, Gompertzian growth parameters (initial growth rates, retardation rates) were determined, along with estimation of the final asymptotic volumes. It was found that the growth kinetics of the various artificial heterogeneous tumors could not be predicted from knowledge of the growth parameters of the pure clonal xenograft tumors. Additionally, both pure clonal and artificial heterogeneous tumors were enzymatically disaggregated as a function of time postinjection, and it was found that the admixed tumors became more zonal in composition as time progressed. Further, admixtures of extreme composition (i.e., 9% A plus 91% D or 88% A plus 12% D) remained stable with time, while those of intermediate initial composition (i.e., 50% A plus 50% D) did not. All of these data (growth kinetics, zonality, compositional stability) indicate that the growth properties of heterogeneous tumors are very complex.

Compositional stability of artificial heterogeneous tumors in vivo: use of mitomycin C as a cytotoxic probe.

A major part of the overall response of solid cancers to cytotoxic treatments will be due to the differential sensitivities of the neoplastic cell subpopulations present. To quantitatively investigate this, artificial heterogeneous human colon xenograft tumors were constructed using two clonally related cell lines (A and D) which were mixed to create compositions of approximately 9:1 or 1:9 A:D cells. Then, the pure A, D, or admixed tumors were challenged with mitomycin C which kills A cells more efficiently than D cells by a factor of about 2.3 as determined by in vitro survival curve inactivation slopes. This difference was also exhibited in vivo by the post-mitomycin C cell survival responses determined by excision assay and by the shapes of the regrowth curves of the pure A and D or admixed tumors. By approximately 30 days after treatment, artificial heterogeneous tumors had reached a new stable cellular composition which could be quantitatively predicted based on the individual survival of A and D cells from pure clonal tumors measured 24 h after treatment. Thus, in this model system, cytotoxic treatment of heterogeneous neoplasms produced predictable and stable, albeit altered, percentage admixtures of subpopulations. This result is consistent with the observed decreased clinical responsivity of primary tumors to sequential courses of therapy due to selection of preexisting resistant subpopulations.

Viral chemokine receptors and chemokines in human cytomegalovirus trafficking and interaction with the immune system. CMV chemokine receptors.

The ubiquitous, opportunistic pathogen human cytomegalovirus (CMV) encodes several proteins homologous to those of the host organism. Four different CMV genes encode chemokine receptor-like peptides. These genes, UL33, UL78, US27, and US28, are expressed at various stages of infection in vitro. Their functions remain largely unknown. To date, chemokine binding and signalling has only been demonstrated for the US28 gene product. Putative ligands for the other CMV-encoded chemokine receptors are discussed on basis of phylogenetic analysis. The potential roles of these receptors in virus trafficking, persistence, and immune evasion are summarized. Similarly, modulation of expression of the host chemokines IL-8, MCP-1a and RANTES in relation to viral dissemination and persistence is reviewed.

Polymerase chain reaction detection of cytomegalovirus DNA in peripheral blood leukocytes as a predictor of cytomegalovirus disease in HIV-infected patients.

OBJECTIVE: To describe and evaluate a polymerase chain reaction (PCR) method for early diagnosis and prompt management of cytomegalovirus (CMV) retinitis in HIV-infected patients. METHODS: A total of 110 HIV-infected patients (Centers for Disease Control and Prevention stages II to IV) were sampled sequentially for isolation of CMV from peripheral blood leukocytes (PBL; n = 560) and for amplification of CMV DNA in PBL. Semiquantitative analysis of the PCR product was performed and each PCR-positive specimen was assigned a score between 1+ and 4+ (corresponding to four points on a standard curve of dilutions: 80, 800, 8000 and 80,000 CMV genome copies). RESULTS: Levels of CMV DNA in blood increased with HIV infection stage. We focused on eight patients who developed one or more episodes of retinitis during longitudinal follow-up, in whom we found a strong correlation between viraemia, high PCR signal (3+ or 4+) (P < 0.0001) and clinical symptoms. Relapse was preceded by an increase in CMV DNA and resolution correlated with clearance of CMV DNA from blood. CONCLUSIONS: Persistent high PCR levels always preceded virus isolation and may be the first indication of organ involvement and thus early treatment. PCR scores were consistently useful as indicators of drug efficacy and for monitoring of treatment.

TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2.

OBJECTIVE: The HIV Tat protein is a transcriptional transactivator of the HIV-1 long terminal repeat (LTR) promoter element. Its activity depends on its direct interaction with the trans-activation response (TAR) element, although TAR-independent activation by Tat has been demonstrated in different cells. Herpesviruses in general and human cytomegalovirus (HCMV) in particular are often isolated from HIV-1-infected patients and could play a role in the activation of latent HIV and in a subsequent increase in HIV replication. HCMV immediate early gene products (IE1 and IE2) are nuclear phosphoproteins that play a pivotal role in HCMV replication and have been shown to transregulate both viral and cellular gene expression. It has repeatedly been shown that HCMV IE1/IE2 can independently transactivate HIV-1 LTR. The aim of this study was to investigate IE1/IE2 transactivation of HIV-1 LTR in a CD4+ T-cell line in the absence and presence of HIV-1 Tat to establish whether IE1/IE2 can synergize with Tat. METHODS: HIV-1 LTR transactivation by HCMV IE1/IE2 in the presence and absence of HIV-1 Tat was determined by transient transfection experiments of J-Jhan lymphoblastoid cells with a series of different expression vectors. RESULTS: We found a strong synergistic transactivation between HIV Tat and the IE1-IE2 complex on HIV LTR activity using vectors driven either by wild-type LTR or by the nuclear factor NF-kappa(B) response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated for TAR-deleted LTR, which cannot be activated by Tat alone. This cooperation is abolished when the region in IE2 that binds the TATA box binding protein is deleted. CONCLUSIONS: The results obtained indicate that Sp1-binding and TAR sequences are not strictly required for Tat responsiveness when Tat is directed to the HIV promoter by HCMV IE1-IE2. This synergistic effect is mediated by the IE2 and TATA-binding region, and could play a major role in HIV activation when cells are infected by both viruses, a feature often observed in AIDS patients.

Levels of selected growth factors in viable and necrotic regions of xenografted HCT-8 human colon tumours.

Xenografted tumours were produced in nude mice by injection of HCT-8 human colon tumour cells. At average volumes of about 750 mm3, animals were injected with fast green vital dye, and 20 min later, tumours were excised and dissected into viable (stained) and necrotic portions (unstained). Viable and necrotic regions were then examined for cell yields, colony forming efficiencies, and levels of basic fibroblast growth factor (FGF-2), transforming growth factors-beta 1 and -alpha (TGF-beta 1, TGF-alpha), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) using enzyme-linked immunoassay (ELISA) procedures. Levels in the viable and necrotic regions were compared to levels in unseparated tumours. The average extent of necrosis in HCT-8 tumours of this size was 64%. The data for cell yields, colony forming efficiencies FGF-2, VEGF, TGF-beta 1 and TGF-alpha indicated that values determined in the unseparated tumours could be understood on the basis of the weighted average between viable and necrotic tissue, with the higher values occurring in the viable tissue. Low levels of FGF-2 and VEGF were found in the necrotic portions of the tumour while no measurable levels of TGF-beta 1 and TGF-alpha could be determined. PDGF levels were, however, equivalent in both the viable and necrotic regions indicating that necrotic tissue could be an important reservoir for this growth factor.

Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration.

Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up-regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT-8 human colon tumour cells in vitro (22 h at 37 degrees C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O2) or the 0.01% O2 mixture respectively increased from about 73 to 1270, and 1.5 to 1180 pg/10(6) cells (about 17- and 80-fold increases). The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT-8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O2 were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/10(6) cells/h) in 21% O2 was 62.5. This rate increased to 428.8 pg/10(6) cells/h at 0.01% O2, a 7-fold increase. For HCT-8 cells, levels in the medium at 21% O2 were too low to be measured by ELISA. However, between 10% and 0.01% O2, secretion rates increased from 5.0 to 376.0 pg/10(6) cells/h, a 75-fold increase. Therefore, at very low O2 levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT-8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm3.

Verhulstian analysis of the growth of transplantable mammary tumours in sialoadenectomized mice.

In this report we have analysed data published in 1989 by Inui et al. (Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice. J. Natl Cancer Inst. 81, 1660) involving the effects of perturbation of the epidermal growth factor (EGF) status of mammary tumour-bearing mice on subsequent volumetric responses. Removal of an endogenous EGF stimulus by surgical ablation of the submaxillary glands, the major EGF-producing organ in mice, produced significantly slower growth of rodent mammary neoplasms, decreased success rate of transplantation, and an increase in the latent period before growth occurred. Administration of i.p. EGF (5 micrograms/mouse/day) to sialadectomized tumour-bearing mice would however, increase tumour growth rate. Data were analysed using the Verhulst equation which indicated that the observed effects on tumour volumetrics by either sialoadenectomy or EGF administration could be interpreted as being produced through paracrine pathways. The use of the Verhulstian analysis indicates that it is possible to analyse neoplastic responses and infer whether paracrine or autocrine pathways are involved.

Simple and fast purification of Escherichia coli adenylate kinase.

Adenylate kinase from E. coli (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. The enzyme from crude extracts of both mutant and parent strain was bound to blue-Sepharose at pH 7.5, thereafter specifically eluted with 0.05 mM P1,P5-di(adenosine-5')pentaphosphate. A second chromatography on Sephadex G-100 yielded pure enzyme. E. coli adenylate kinase was strongly inhibited by P1,P5-di(adenosine-5')pentaphosphate (Ki 0.6 microM for adenylate kinase of strain CR341 and 2.1 microM in the case of mutant enzyme). After denaturation in 6 M guanidinium hydrochloride both mutant and parent adenylate kinase returned rapidly to the native, active state by dilution of guanidinium hydrochloride.

Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission.

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.