Genetic evidence for a new type of major histocompatibility complex class II combined immunodeficiency characterized by a dyscoordinate regulation of HLA-D alpha and beta chains.

Major histocompatibility complex (MHC) class II combined immunodeficiency (CID), also known as type II bare lymphocyte syndrome, is an autosomal recessive genetic disorder characterized by the complete lack of expression of MHC class II antigens. The defect results from a coordinated lack of transcription of all class II genes. Cell fusion studies using many patient- and experimentally derived class II-negative cell lines have identified four distinct genetic complementation groups. In this report, we present genetic evidence that cell lines derived from two newly described MHC class II-deficient patients, KER and KEN, represent a fifth complementation group. In addition, the KER and KEN cell lines display a unique pattern of dyscoordinate regulation of their MHC class II genes, which is reflected in a new phenotype of in vivo promoter occupancy as revealed by in vivo genomic footprinting. These data point to a new defect that can result in the MHC class II-deficient phenotype.

Molecular characterization of major histocompatibility complex class II gene expression and demonstration of antigen-specific T cell response indicate a new phenotype in class II-deficient patients.

Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency. The disease is known as bare lymphocyte syndrome (BLS). BLS is characterized by a lack of constitutive MHC class II expression on macrophages and B cells as well as a lack of induced MHC class II expression on cells other than professional antigen-presenting cells (APCs) due to the absence of mRNA and protein of the human leukocyte antigen (HLA) class II molecules, designated HLA-DR, -DQ, and -DP. The defect in gene expression is located at the transcriptional level and affects all class II genes simultaneously. Here we have analyzed transcription and protein expression of class II antigens in Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines and mononuclear cells (MNCs) of twin brothers. Whereas flow cytometric analysis failed to detect class II antigens on the cell surface of the patients' EBV-B cells and MNCs, examination of the genes coding for HLA-DR, -DQ, -DP, and the invariant chain (Ii) by reverse transcriptase-polymerase chain reaction amplification resulted in an unusual mRNA pattern in the B cell lines of the patients (HLA-DR alpha +, -DR beta, -DQ alpha +, -DQ beta -, -DP alpha -; -DP beta +, Ii+). In accordance with these findings no HLA-DR beta-specific protein was detected by immunoblotting, whereas low levels of HLA-DR alpha and normal levels of Ii were present. In contrast to EBV-B cells, the MNCs of both patients displayed a residual HLA-DR beta, -DQ beta, and -DP alpha mRNA signal. Furthermore, HLA-DR beta-specific protein was found in addition to HLA-DR alpha by immunoblotting of cell lysates, even though it was clearly decreased as compared with controls. Our results indicate that the defect in class II antigen expression is not necessarily present to the same extent in B cells and cells of other lineages. mRNA levels of HLA-DR beta were found to be enriched in adherent cells within the MNC fraction. Further investigations indicated that the MHC class II expressed is functional in antigen presentation, as the two boys' CD4+ T cells became activated and expressed interleukin-2R after stimulation of peripheral blood mononuclear cell cultures with recall antigen (tetanus toxoid). Furthermore, T cells tested in one of the two patients responded to both MHC class I and II allostimulation, and this response was inhibited by monoclonal antibodies of the respective specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiological anatomy of the ambient cistern in children.

Ambient cistern (AC) is a thin extension of the subarachnoid space surrounding the brainstem at the level of the mesencephalon and pons. Despite various definitions, it constitutes an important landmark in clinical assessment of intracranial volume reserve. Although it is indisputably useful, there exists no defined standard for radiological examination for the dimensions and ranges in specific age groups. This paper aims to describe the ambient cistern anatomically and give the ranges of dimensions for proper radiological interpretation. The study was performed on 160 axial computed tomography (CT) examinations of Polish children of both sexes, aged 1-18 years, admitted to the hospital because of mild brain concussion. Pictures were made using a Siemens 8-row CT scanner, without contrast administration. We estimated distances at the level of the pons and midbrain, based on axial cross-sections, according to standard radiological protocol. The parameters included the width of the AC in its anterior and posterior part, the width of the tentorial notch, and the distance from the pons and sella. All measurements were analyzed statistically with StatSoft Statistica 8.0 software. The average width of the AC differs between age groups. It is greatest at 1-3 years (2.8 +/- 0.6 mm) and lowest at 4-10 years (2.4 +/- 0.6 mm). AC is more likely to be greater in its anterior part in boys. The distance from the sella to the pons is greatest in 1-3-year-old girls (6.9 +/- 1.3 mm), and the tentorial notch is widest in the 15-18-year-old group (24.6 +/- 2.4 mm). Dimensions of the AC correlate with intracranial reserve volume. This is particularly visible in the youngest children. Thin and narrow AC is not always a sign of raised intracranial pressure. It may be specific for the child's age.

Transient immunoglobulin and antibody production. Occurrence in two patients with common varied immunodeficiency.

Two patients with common varied immunodeficiency both had increased immunoglobulin responses for a limited period of time on acquiring a bacterial infection. In one patient a relatively short-lived high-antibody response was produced on vaccination with tetanus toxoid. Although the patients varied in their course of immunoglobulin and/or antibody production, our results demonstrate that some patients with common varied immunodeficiency are able to produce both under certain circumstances.

Recombinant gp160 as a therapeutic vaccine for HIV-infection: results of a large randomized, controlled trial. European Multinational IMMUNO AIDS Vaccine Study Group.

OBJECTIVES: The primary objective of this study was to expand the safety and immunogenicity database of recombinant gp160 as a therapeutic vaccine in the treatment of HIV-infection. Preliminary efficacy data was also sought. DESIGN: This trial was a randomized, double-blind, placebo-controlled study. Two-hundred and eight volunteers, 96 therapy-naive with CD4 cell count >500x10(6)/l (group A) and 112 with CD4 cell count of 200-500x10(6)/l (group B, 51 out of 112 on treatment with one or two nucleoside analogues), received monthly injections of rgp160 IIIB vaccine or placebo for the first 6 months of the study; booster immunizations with rgp160 MN or placebo were given at times 15, 18, and 21 months. METHODS: Safety and immunogenicity data were obtained and measurements of CD4 cell count, plasma viral RNA, and proviral DNA were performed. Clinical outcome was recorded for the 24 months of study. RESULTS: The vaccine was safe and well tolerated. Despite the induction of new rgp160-specific lymphoproliferative responses and the presence of positive delayed type hypersensitivity skin tests to rgp160 at the end of the 24 month study, no effect on the natural history of HIV infection was detected. Within 24 months, AIDS-defining illnesses had occurred in 19 of the vaccinated volunteers and in 18 of the placebo recipients. Persons with higher plasma viral RNA levels and higher proviral DNA had a more rapid decline in CD4 cell count when compared to persons with lower values. Vaccine did not alter viral RNA or proviral DNA levels. CONCLUSION: There was no clinical benefit to therapeutic immunizations with rgp160, despite the induction of new lymphoproliferative responses.

Defective TCR surface expression associated with impaired TCR beta-chain assembly in a patient with cutaneous T-cell lymphoma.

We report on a patient with cutaneous T-cell lymphoma (CTCL) of long-standing duration. Phenotypic analysis of his peripheral blood mononuclear cells revealed an increased CD4+ T-helper subset and a decreased CD8+ cytotoxic T-cell population. Eighty-three to ninety-three percent of the patient's CD4+ T cells in the peripheral blood and 70% of the CD4+ T cells in the lesional skin lacked surface expression of the TCR/CD3 complex and showed a clonal rearrangement pattern of the TCR gamma-chain gene (V11-J1/J2). The lack in TCR surface expression correlated with defective assembly of the TCR beta-chain. Although mRNA for the TCR constant region beta 1 was found in the patient's purified CD4+ TCR-CD3- T cells, no intracytoplasmic TCR beta protein was detectable. In contrast, the patient's purified CD4+ TCR-CD3- T cells not only expressed mRNA specific for the TCR alpha-chain and for all CD3 chains, but intracytoplasmic TCR alpha and CD3 epsilon proteins could also be found. The lack of TCR beta protein clearly explains the defective surface expression of the TCR/CD3 complex in the patient's malignant T cells.

Nitrocellulose particles adsorbed to immunoglobulins are a new and effective approach to induce cell activation dependent on receptor aggregation.

Nitrocellulose (NC) has proved to be a versatile tool for the isolation and characterization of various biomolecules. In this report we extend its scope by using antibody-coated NC particles to cross-link molecules on the surface of living cells. Ligation of receptors in Jurkat cells with NC-bound specific antibodies induced protein tyrosine phosphorylation patterns of cellular proteins comparable to conventional antibody cross-linking. In addition, the present study shows that application of NC particles coated with human IgA significantly activated monocytic cells via the Fc alpha receptor (Fc alphaR), whereas cross-linking of receptor-ligand complexes with isotype-specific antibody was less efficient. Subsequent immunoprecipitation and immunoblot analysis of aggregated Fc receptors (FcRs) complexed to Ig-adsorbed particles permits fast identification of molecules involved in the transmission of signals. Therefore, ligand-coated NC particles can be used to examine receptor-mediated cell activation events dependent upon extensive receptor aggregation.

Detection of phosphatidylinositol glycan class A gene transcripts by RT in situ PCR hybridization. A comparative study using fluorescein, Texas Red, and digoxigenin-11 dUTP for color detection.

Gene-specific probes labeled with fluorescein, Texas Red, and digoxigenin-11 dUTP (DIG) were used for RT in situ PCR hybridization to detect PIG-A gene (phosphatidylinositol glycan class A) transcripts. The PIG-A gene is responsible for biosynthesis of the glycosylphosphatidyl-inositol (GPI) anchor. Lack of GPI anchor expression due to mutations can cause an acquired clonal hematologic disorder called paroxysmal nocturnal hemoglobinuria (PNH). In this RT in situ PCR study, two types of labeling methods, a direct method (using fluorescein and Texas Red) and an indirect method (using DIG-11 dUTP) were compared. Both were successfully applied to detect and localize the PIG-A gene transcripts within single cells of the cell lines AA2, H9, and JY. Furthermore, similar results for sensitivity and reproducibility were obtained. Advantages and disadvantages of the different labeling techniques are discussed. In addition, peripheral blood mononuclear cells from PNH patients were also included in this study.

Down regulation of Fc receptors by IVIgG.

IVIgG preparations are now widely applied for immune modulatory treatment in various forms of autoimmune and immune complex diseases. Several controlled studies clearly demonstrated the clinical efficacy of this type of treatment; the underlying pathophysiological mechanisms, however, have yet to be elucidated. Among the mechanisms suggested to play a role in this context is the interaction of gamma globulin with Fc gamma receptors (Fc gamma R) expressed in the membrane of immunocompetent cells. Our studies concentrated on these aspects and focused on possible functional consequences of IgG-Fc gamma R interaction. By using the peripheral blood monocyte as a model system for an Fc gamma R-bearing cell, we confirmed previous reports by showing differences in Fc gamma R binding and Fc gamma R modulation induced by IgG in its various forms (monomeric IgG, Polymeric IgG, immune complexes). As biological consequences of Fc gamma R modulation, changes in effector and accessory function of these cells were observed. The results presented in this brief review emphasize especially the difference between ligand-oriented Fc gamma R diffusion (induced by surface-bound IgG) and true long-term down-modulation of Fc gamma R (mediated by fluid-phase IgG polymers) and show that only the down-modulation of Fc gamma R correlated with impaired functions of the affected cell.

CD3, CD8 double-positive cells from HIV-1-infected chimpanzees show group-specific inhibition of HIV-1 replication.

The CD8-positive (CD8+) lymphocyte population in the chimpanzee is composed of two major subsets, a CD3-positive, CD8-positive (CD3+CD8+) and a CD3-negative, CD8-positive (CD3-CD8+) population. The aim of this study was to delineate the function of CD3+CD8+ T cells with respect to inhibition of HIV-1 replication. It could be shown that this cell population had the capacity to control the growth of HIV-1 in exogenously infected CD4-positive (CD4+) lymphocytes. This effect was observed only with cells from HIV-1-infected chimpanzees, was operative only in an autologous and not in a homologous situation, and was not due to cytotoxicity. CD3+CD8+ lymphocyte-mediated inhibition of HIV-1 replication was group-specific in that CD3+CD8+ cells of HIV-1 (IIIB)-infected chimpanzees were capable of inhibiting replication of HIV-1 strains IIIB, MN, and RF. The effect observed was operational during the early stages of HIV-1 replication only; the effector cells had to be added to CD4+ cells within 3 days after HIV-1 infection in order to suppress growth of the virus. The presence of CD3+CD8+ lymphocytes with anti-HIV-1 activity in the circulation of HIV-1-infected chimpanzees might contribute to the lack of progression toward AIDS observed in this species.

An experimental prime-boost regimen leading to HIV type 1-specific mucosal and systemic immunity in BALB/c mice.

Induction of mucosal as well as systemic immunity to HIV-1 is considered to have high priority in current concepts of future AIDS vaccines. Here we show that the desired immune responses can be elicited by an experimental prime-boost regimen consisting of mucosal (intragastric) application of a recombinant vaccinia virus carrying the HIV-1 env gene (vSC25), followed by parenteral (intradermal) immunization with the recombinant HIV-1 glycoprotein 160 (rgp160). Following intragastric immunization of mice with vSC25 in combination with the mucosal adjuvant cholera toxin (CT), HIV-1 env-specific IgA was secreted by B cells of Peyer's patches and the lamina propria. Moreover, mucosal (intragastric and intranasal) application of vSC25 (both in presence or absence of CT) induced a long-lasting, HIV-1 env-specific systemic cytotoxic T cell response. Subsequent intradermal boosters with rgp160 led to HIV-1-specific T cell memory and serum antibodies.

Dual tropism of HIV-1 IIIB for chimpanzee lymphocytes and monocytes.

In humans, macrophages serve as a major reservoir of human immunodeficiency virus (HIV-1) in the infected host and may play a role in the pathogenesis of the disease. In HIV-1-infected chimpanzees, however, virus could not be recovered from cells of the monocyte/macrophage lineage, leaving the question of macrophage tropism of HIV-1 in this species unresolved. The data reported that HIV-1 IIIB shows dual tropism and is infectious for both chimpanzee monocytes and lymphocytes in vitro. Viral replication in chimpanzee monocytes was clearly demonstrated by infection of allogeneic phytohemagglutinin (PHA) blasts in vitro and by electron microscopy (EM). EM revealed HIV particles associated with 10-15% of the HIV-1 IIIB-infected chimpanzee monocytes. Viral particles budding from the monocyte surface in the typical crescent form were noted as well. This is in contrast to the human situation, where monocytotropic HIV strains preferentially bud into and accumulate in cytoplasmic vacuoles. These results indicate that both lymphocytes and cells of the monocyte/macrophage lineage replicate virus in the chimpanzee; the cell tropism of viral strains, however, is different in chimpanzees and humans.

Role of preimmunization virus sequences in cellular immunity in HIV-infected patients during HIV type 1 MN recombinant gp160 immunization.

The effect of patient preimmunization virus sequences on CTL responses during gp160 immunization were studied. Ten HLA-A2+, HIV+ asymptomatic patients with CD4+ T cells >500/mm3 were given two courses of HIV-1 MN rgp160 vaccine over a 2-year period. Envelope epitope-specific CTL responses, using PBMCs, were measured against peptide-coated autologous B lymphoblastoid cell lines. Optimum CTL epitopes were determined by HLA-A2-binding affinity of 9- to 10-mer peptides containing the HLA-A2.1-binding motif. Ten of the high- or intermediate-binding peptides were conserved among >50% of reported clade B HIV strains. These peptide-specific CTL activities and the patient virus sequences in peptide-coding regions were monitored. Six patients showed envelope peptide-specific CTL responses, which correlated with the presence of whole envelope antigen-specific CTL responses. Five of these patients, who showed responses to epitopes in the gp41 region (aa 814-824), had preimmunization virus similar to the vaccine sequence in this region. Three patients who did not show these epitope-specific responses had initially different sequences in the HIV gene encoding that region. The epitope-specific CTL responses appear to reflect recall responses, as only patients infected with virus containing the vaccine sequence developed them and they could be recalled with a second set of vaccine injections. This appears to be reminiscent of the concept of T cell "original antigenic sin." This vaccine was also immunogenic as measured by gp160-specific lymphocyte-proliferative responses. However, increased immune responses did not impact the HIV load or CTL epitope sequences during therapy.

Allostimulated lymphocytes inhibit replication of HIV type 1.

Previous reports demonstrated that alloantigen- or xenoantigen-specific antibodies displayed neutralizing activity toward human or simian immunodeficiency viruses. In the present article we have addressed the question of alloantigen-induced cell-mediated anti-HIV activity. We show that allostimulation resulted in a lymphocyte population (largely of the CD8-positive phenotype) with the capacity to inhibit HIV-1 replication in PHA blasts of homologous and, unexpectedly, also autologous origin. The allostimulated effector cells exerted their activity via a noncytolytic mechanism. Experiments in which direct cell-to-cell contact between allostimulated effectors and HIV-1-infected PHA blasts was prevented by a semipermeable membrane indicated that soluble mediators were involved in inhibition of HIV-1 replication. As such allostimulated effectors not only would have the capacity to prevent viral replication in allogeneic HIV-1-infected cells (known to play an important role in HIV-1 transmission in vivo), but also might inhibit HIV-1 growth in autologous lymphocytes, the concept of an AIDS vaccine containing both HIV-1 antigens and alloantigens warrants further consideration.

Effects of cyclosporin A upon humoral and cellular immune parameters in insulin-dependent diabetes mellitus type I: a long-term follow-up study.

Patients who had been included in a randomized double-blind placebo-controlled trial on the efficacy of cyclosporin A (CyA) in producing remissions in insulin-dependent diabetes mellitus (IDDM) type I were investigated for humoral and cellular immunologic parameters. Whereas metabolic derangement before the initiation of insulin treatment led to small but significant decreases in the percentage of CD4-positive lymphocytes as well as of the activity of natural killer (NK) cells and antibody-dependent cellular cytotoxicity (ADCC), the administration of CyA did not influence any of the immunologic parameters tested, which included proliferative lymphocyte responses to mitogens and alloantigens and serum concentrations of immunoglobulins G, A and M. Thus NK cell activity, ADCC as well as the percentage of CD4-positive lymphocytes returned to normal levels in parallel with the normalization of glycosylated haemoglobin (HbAlc), but were not further influenced in their course by the administration of CyA, as compared with patients receiving placebo. Interferon-induced augmentation of NK cell activity did not differ between patients with IDDM on placebo and those under CyA therapy. All other investigated parameters also remained unchanged during the time of CyA therapy. We conclude that metabolic derangement leads to a reversible disturbance of certain cellular immune functions, but their normalization achieved by insulin treatment and their further course remains uninfluenced by the administration of CyA.

Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity.

The human immunodeficiency virus (HIV-1) envelope gene was expressed in large-scale microcarrier cultures of Vero cells using a system involving coinfection with two recombinant vaccinia viruses. One recombinant contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second contained the HIV-1 gp160 gene flanked by T7 promoter and termination sequences. The protein was expressed on the surface of infected cells, and it was shown to have a molecular weight of 160 kD and to react with gp41 and gp120 specific monoclonal antibodies. After purification by successive affinity and ion-exchange chromatography, the protein was demonstrated to be present in a particulate form with a diameter in the range of 15-30 nm. When injected into goats a high-titer gp160 specific antibody response was elicited and group-specific neutralizing activity could be demonstrated in vitro. The immunogenicity of the protein was also studied in conjunction with a number of adjuvant formulations, and the highest potency in mice was obtained using a preparation with 0.2% Al(OH)3 and 0.25% deoxycholate.

Immunization of chimpanzees with the HIV-1 glycoprotein gp160 induces long-lasting T-cell memory.

The goal of the present study was to investigate the antigen-specific T-cell response to the recombinant HIV envelope glycoprotein (gp160) and to test the effect of various adjuvant formulations on the efficiency of T-cell priming as well as on magnitude and longevity of the gp160-specific T-cell response. Our studies revealed that, in combination with an appropriate adjuvant (lipid-based adjuvant or mineral carrier complex), immunization with recombinant gp160 led to the appearance of gp160-primed T cells. The T-cell response obtained was substantial (proliferative response of greater than 100,000 delta dpm after one primary and two booster immunizations), gp160-specific (proliferation only in response to gp160, no proliferation after addition of a mock gp160 preparation), and long-lasting (T cell responses of greater than 50,000 delta dpm were observed more than one year after the last booster). The results presented here differ from those of previous studies in that they show the presence of substantial and long-lasting T-cell memory toward the immunogen gp160. Therefore further investigations on the use of these preparations as HIV candidate vaccines appear to be justified.

A pilot clinical trial of HIV antigen-pulsed allogeneic and autologous dendritic cell therapy in HIV-infected patients.

A pilot study was carried out to assess the safety and antigen-presenting properties of allogeneic or autologous dendritic cells (DCs) in six HLA-A2+, HIV-infected patients. Allogeneic DCs obtained from the peripheral blood of HLA-identical, HIV-seronegative siblings were pulsed with recombinant HIV-1 MN gp160 or synthetic peptides corresponding to HLA-A2-restricted cytotoxic epitopes of envelope, Gag, and Pol proteins. The antigen-pulsed cells were infused intravenously six to nine times at monthly intervals and HIV-specific immune responses were monitored. One allogeneic DC recipient with a CD4+ T cell count of 460/mm3 showed increases in envelope-specific CTL- and lymphocyte-proliferative responses, as well as in IFN-gamma and IL-2 production. Another allogeneic DC recipient with a CD4+ T cell count of 434/mm3 also showed an increase in HIV envelope-specific lymphocyte-proliferative responses. A recipient of autologous DCs with a CD4+ T cell count of 730/mm3 showed an increase in peptide-specific lymphocyte-proliferative responses after three infusions. Three other allogeneic DC recipients with CD4+ T cell counts <410/mm3 did not show increases in their HIV-specific immune responses. No clinically significant adverse effects were noted in this study and CD4+ T cell numbers and plasma HIV-1 RNA detected by RT-PCR of all six patients were stable during the study period. Thus, both allogeneic and autologous DC infusions were well tolerated and in patients with normal or near normal CD4+ T cell counts administration of these antigen-pulsed cells enhanced the immune response to HIV. However, since no effect on viral load was observed there was no evidence that this approach provided clinical benefit.

HIV-1MN recombinant glycoprotein 160 vaccine-induced cellular and humoral immunity boosted by HIV-1MN recombinant glycoprotein 120 vaccine. National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

We evaluated prime-boost immunization with two recombinant envelope glycoprotein subunit vaccines (HIV-1MN recombinant gp160 vaccine in alum adjuvant [MN rgp160] and HIV-1MN recombinant gp120 vaccine in alum adjuvant [MN rgp120]) for safety and immunogenicity in healthy, HIV-1-uninfected adults. The rationale was to combine the helper T cell memory and binding antibody responses typically induced by rgp160 vaccines with the superior neutralizing antibody responses induced by rgp120 vaccines. In a double-blinded, controlled trial, volunteers were randomly assigned to receive MN rgp160 or adjuvant placebo, and a subset later received MN rgp120. The two vaccines were safe, but reactions to MN rgp160 and its adjuvant placebo exceeded those to MN rgp120. MN rgp160 induced IgG binding antibodies, including all IgG subclasses, to MN rgp160 in all vaccine recipients. HIV-1MN-neutralizing and anti-V3 MN peptide-binding antibodies were observed in a majority of volunteers after the fourth MN rgp160 immunization, but at lower levels compared with immunization with MN rgp120 in historical controls. HIV-1-binding, neutralizing, and fusion inhibition antibodies were boosted to the highest levels among MN rgp160 recipients after MN rgp120 booster injections. MN rgp120 boosting appeared to alter the distribution of MN rgp160 vaccine-induced, anti-MN rgp160 IgG subclass antibodies. MN rgp160 induced helper T cell memory, measured by lymphocyte proliferation, Thl and Th2 cytokine production, and skin testing. Strategies including both subunit vaccines may help maximize antibody and helper T cell memory responses to HIV-1 envelope glycoprotein.

Cytomegalovirus diagnosis in renal and bone marrow transplant recipients: the impact of molecular assays.

BACKGROUND: Cytomegalovirus (CMV) infections are a major threat in transplant recipients. In recent years, new assays for routine CMV diagnosis, based on molecular techniques, have become available. OBJECTIVE: The impact of molecular assays for CMV diagnosis in transplant recipient was evaluated. STUDY DESIGN: A total of 51 transplant recipients were screened for CMV infection. Serological (AxSYM CMV IgG and recombinant CMV IgM assays), antigenemia, CMV DNA (qualitative in house PCR and the quantitative COBAS AMPLICOR CMV MONITOR Test), and CMV mRNA (NucliSens CMV pp67 Test) tests were compared. RESULTS: In 11/20 bone marrow transplant (BMT) recipients and 10/31 renal transplant (RTX) recipients there was no evidence of active CMV infection. Ten RTX recipients and one BMT recipient were antigenemia positive, 21 RTX and seven BMT recipients were PCR positive (qualitative CMV PCR). There were more BMT recipients CMV DNA positive in serum (7/21) than antigenemia positive (1/21). CMV mRNA was found positive in two BMT recipients (one case with no other evidence of CMV infection, the other one CMV DNA positive and antigenemia negative). The only antigenemia positive BMT recipient was found negative for CMV mRNA, but positive in all other tests. Eight RTX recipients were found positive for CMV mRNA. Six of them were also antigenemia positive and five of those were also found positive for CMV IgM. One CMV mRNA positive RTX recipient was CMV IgM positive but antigenemia negative and the other one CMV mRNA positive RTX recipient was found negative in all other tests. Two antigenemia positive RTX recipients were found negative for mRNA and CMV IgM. CONCLUSION: Antigenemia was found to be a good screening test for CMV infection in RTX recipients. In BMT recipients, tests based on molecular techniques appeared to be superior compared to antigenemia.