Tumor dormancy and cell signaling. II. Antibody as an agonist in inducing dormancy of a B cell lymphoma in SCID mice.

Tumor dormancy can be induced in a murine B cell lymphoma (BCL1) by immunizing BALB/c mice with the tumor immunoglobulin (Ig) before tumor cell challenge. In this report, we have investigated the immunological and cellular mechanisms underlying the induction of dormancy. BCL1 tumor cells were injected into SCID mice passively immunized with antibody against different epitopes on IgM or IgD with or without idiotype (Id)-immune T lymphocytes. Results indicate that antibody to IgM is sufficient to induce a state of dormancy. Antibodies against other cell surface molecules including IgD and CD44 (Pgp1) had no effect on tumor growth. Id-immune T cells by themselves also had no effect on tumor growth in SCID mice. However, simultaneous transfer of anti-Id and Id-immune T cells enhanced both the induction and duration of the dormant state. In vitro studies indicated that antibody to IgM induced apoptosis within several hours and cell cycle arrest by 24 h. Hyper cross-linking increased apoptosis. The Fc gamma RII receptor played little or no role in the negative signaling. Antibodies that did not negatively signal in vitro did not induce dormancy in vivo. The results suggest that anti-IgM plays a decisive role in inducing tumor dormancy to BCL1 by acting as an agonist of IgM-mediated signal transduction pathways.

Induction of B cell tumor dormancy by anti-idiotypic antibodies.

Long-term dormancy of murine B-cell lymphomas can be experimentally induced by immunizing the host with the idiotype expressed on the tumor. Interaction of the cells with anti-idiotype antibodies is sufficient to induce and maintain the dormant state. The growth of lymphoma cells interacting with anti-idiotype antibodies is arrested and they undergo dramatic changes in their morphology, cell-cycle status and oncogene expression. Regrowth of a tumor after long-term dormancy results from the emergence of a tumor cell variant that no longer responds to the antibodies with growth inhibition. These data demonstrate the feasibility of reversing a malignant phenotype of cells by specific growth arrest signals and suggest new approaches for therapeutic intervention in cancer.

Cux/CDP homeoprotein is a component of NF-muNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the bright transcription activator.

Nuclear matrix attachment regions (MARs) flanking the immunoglobulin heavy chain intronic enhancer (Emu) are the targets of the negative regulator, NF-muNR, found in non-B and early pre-B cells. Expression library screening with NF-muNR binding sites yielded a cDNA clone encoding an alternatively spliced form of the Cux/CDP homeodomain protein. Cux/CDP fulfills criteria required for NF-muNR identity. It is expressed in non-B and early pre-B cells but not mature B cells. It binds to NF-muNR binding sites within Emu with appropriate differential affinities. Antiserum specific for Cux/CDP recognizes a polypeptide of the predicted size in affinity-purified NF-muNR preparations and binds NF-muNR complexed with DNA. Cotransfection with Cux/CDP represses the activity of Emu via the MAR sequences in both B and non-B cells. Cux/CDP antagonizes the effects of the Bright transcription activator at both the DNA binding and functional levels. We propose that Cux/CDP regulates cell-type-restricted, differentiation stage-specific Emu enhancer activity by interfering with the function of nuclear matrix-bound transcription activators.

Tyrosine kinase activation in the decision between growth, differentiation, and death responses initiated from the B cell antigen receptor.

Immunoglobulin-containing receptors expressed on B lineage lymphocytes play critical roles in the development and function of the humoral arm of the immune system. The preB cell antigen receptor (preBCR) contains the immunoglobulin mu heavy chain (Ig mu) and signals to the preB cell that heavy chain rearrangement has been successful, a process termed heavy chain selection. The B cell antigen receptor (BCR) contains both Ig heavy and light chains and is expressed on immature and mature B cells before and after antigen encounter. Both receptor types from a complex with the Ig alpha and Ig beta proteins that link the predominantly extracellular Ig with intracellular signal transduction pathways. Signaling through the BCR induces different cellular responses depending on the nature of the signaling agent and the development stage of the target cell. These responses include clonal anergy and apoptotic deletion in immature B cells and survival, proliferation, and differentiation in mature B and preB cells. Several protein tyrosine kinases are activated rapidly following engagement of the BCR/preBCR complexes, including members of the Src family (Lyn and Blk), the Syk/ZAP70 family (Syk), and the Tec family (Btk). In this review, we discuss possible mechanisms by which engagement of these similar receptor complexes can give rise to different cellular responses and the role that these kinases play in this process.

Cancer dormancy: role of cyclin-dependent kinase inhibitors in induction of cell cycle arrest mediated via membrane IgM.

Anti-idiotype (anti-Id) antibody can induce tumor dormancy in a murine B lymphoma, BCL1, by its ability to induce cell cycle arrest and apoptosis (negative signaling). In human B lymphoma, there is accumulating evidence that the antitumor effect of anti-Id or several other B cell-reactive antibodies relates to their ability to act as agonists rather than conventional effector antibodies. In this study, we sought to elucidate the role of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors in anti-IgM-induced cell cycle arrest to better understand the mechanisms underlying cancer dormancy. To accomplish this, we have performed in vitro studies with a human lymphoma cell line (Daudi) because its response to anti-Id (or anti-IgM) is similar to that of a BCL1 cell line, more reagents are available, and the results would be particularly pertinent to therapy of human B cell lymphomas. Our results show that cross-linking of membrane IgM on Daudi cells induces an arrest late in G1 and prevents pRb from becoming phosphorylated. The G1 arrest is correlated with an induction of the CDK inhibitor p21 and reduced CDK2 activity, although the level of CDK2 protein was not changed. Coprecipitation of CDK2 with p21 in anti-IgM-treated cells and the unchanged level of cyclin E suggest that p21 is responsible for the reduction of CDK2 activity and therefore blockade of the cell cycle. The induction of p21 was not accompanied by changes in p53 levels. As a result of the G1 block, cyclin A levels sharply declined by 24 h after anti-IgM treatment. There was no evidence for involvement of CDK4 or CDK6 in the blockade. These results provide evidence that membrane IgM cross-linking on Daudi cells induces expression of p21 and a subsequent inhibition of the cyclin E-CDK2 kinase complex resulting in a block to pRb phosphorylation and cell cycle arrest late in G1.

Loss of p53 expression in Myc-induced B lineage tumors.

Tumors are formed following the accumulation of several genetic changes in genes which normally function to regulate cell growth. As yet it is unclear why multiple mutations are required, which type of alterations can collaborate with each other, and if collaboration is cell-type specific. In our myc transgenic mouse model system both point mutations and loss of mRNA expression for the p53 tumor suppressor gene have been found in the myc-induced B-lineage tumors arising spontaneously in these mice. This demonstrates the collaboration between these two growth control genes in cellular transformation. The observation that alterations in the expression of p53 is a common phenomenon in tumors formed in myc transgenic mice as well as a variety of different types of human tumors suggests that inactivation of the p53 growth control pathway may be required for transformation, and that alterations in p53 itself might be the most efficient way to achieve this inactivation. An analysis of the molecular mechanism for p53 alterations has implications for what kind of factors, both environmental and physiological, can influence tumor formation. The identification of collaboration groups has implications for the process of tumor formation, growth regulation, and will some day be important for the diagnosis of cancer, the prognosis of the individual and the design of specific therapeutic agents for treatment.

Kinetics of early therapeutic response as measured by quantitative PCR predicts survival in a murine xenograft model of human T cell acute lymphoblastic leukemia.

The identification of prognostic parameters and surrogate markers for defining patient risk has been beneficial in effectively guiding therapy and increasing the survival of leukemia patients. It has been hypothesized that the therapeutic response, as measured by a change in tumor burden during therapy, might serve as a new surrogate marker of survival. Here we describe the development of a murine SCID xenograft model of human T cell acute lymphoblastic leukemia (T-ALL), and the use of a sensitive, quantitative PCR assay for the measurement of tumor levels to investigate the relationships between tumor burden quantification, therapeutic response and survival. Animals engrafted with the CCRF-CEM (CEM) human T-ALL cell line develop leukemia that closely resembles the human disease. Quantitative PCR detects the expanding tumor mass in the peripheral blood of the animals several weeks before death. In response to induction therapy with chemotherapeutic agents, both the level of minimal residual disease (MRD) in peripheral blood at the end of therapy and the rate of tumor reduction in peripheral blood during therapy strongly correlated with animal survival. Thus, these surrogate markers, which can be measured during the early stages of therapy, may help improve patient survival through dynamic risk stratification.

Molecular Profile of Mitochondrial Dysfunction in Kidney Transplant Biopsies Is Associated With Poor Allograft Outcome.

BACKGROUND: In kidney transplantation (KT), progression of chronic histological damage with subclinical inflammation is associated with poor long-term allograft survival. The role of nonimmunological pathways in chronic allograft injury has not been fully assessed. METHODS: We analyzed a public microarray dataset that used 1-year protocol kidney transplant biopsy specimens to investigate whether nonimmunological genes and pathways might influence long-term allograft outcome. The selected microarray dataset included 3 patient/sample groups based on their histological findings: normal histology (n = 25), interstitial fibrosis alone (IF alone, n = 24), and interstitial fibrosis with inflammation (IF+i, n = 16). The IF+i group had lower death-censored graft survival and renal function in patients with a mean follow-up of 4 years. We performed statistical analysis comparing gene expression patterns in the 3 group samples. RESULTS: Gene cluster enrichment and group-specific expression patterns demonstrated a divergent pattern between mitochondrial and immune response genes, with downregulation of mitochondrial genes in the IF+i group. Gene ontological analysis of the downregulated mitochondrial genes identified generation of precursor metabolite and energy, and response to oxidative stress as the most significant biological processes. The transcription regulation pathway analysis of downregulated gene cluster demonstrated transcription factors involved in mitochondrial biogenesis. CONCLUSIONS: The molecular signature of mitochondrial dysfunction reflects mitochondrial energetic insufficiency, and inadequate antioxidant response involved in mitochondria biogenesis pathways is associated with IF+i and worse long-term allograft survival. Thus, mitochondria function impairment appears to be an important nonimmune factor involved in chronic allograft injury.

MARs of antigen receptor and co-receptor genes.

MARs are cis-acting DNA sequences that function both negatively and positively in conjunction with transcriptional enhancers to regulate antigen receptor and co-receptor genes. Evidence exists that certain tissue-specific nuclear proteins are involved in this regulation, including SATB1, Bright, and Cux/CDP, possibly by modulating intranuclear gene location, histone acetylation, DNA methylation, and/or nucleosome positioning.

Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients.

Human herpesviruses can cause significant morbidity and mortality in pediatric solid organ transplant recipients. It was hypothesized that viral burden quantification by polymerase chain reaction using an internal calibration standard could aid in distinguishing between viral disease and latency. Here we report the results of a 2-year prospective study of 27 pediatric solid organ (liver, kidney, or heart) transplant recipients in which multiple samples were analyzed for levels of all eight human herpesviruses by internal calibration standard-polymerase chain reaction. Herpes simplex viruses 1 and 2, varicella-zoster virus, and Kaposi's sarcoma-associated herpesvirus were not detected in any of these samples. Human herpesvirus types 6 and 7 were detected in half of the patients, but were present at low levels, similar to those found in reference populations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were detected in 89% and 56% of the patients, respectively. Viral burden analysis suggested distinct patient populations for CMV, with a natural cutoff of 10,000 viral targets/ml blood strongly associated with disease. In some cases, a dramatic increase in CMV levels preceded clinical evidence of disease by several weeks. EBV viral burden was relatively high in the only patient presenting with an EBV syndrome. However, two other patients without evidence of EBV disease had single samples with high EBV burden. Rapid reduction in both EBV and CMV burden occurred with antiviral treatment. These data suggest that viral burden analysis using internal calibration standard-polymerase chain reaction for CMV, and possibly other herpesviruses, is an effective method for monitoring pediatric transplant patients for significant herpesvirus infection and response to therapy.

Polymerase chain reaction amplification of archival material for Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, and parvovirus B19 in children with bone marrow hemophagocytosis.

Bone marrow hemophagocytosis may occur as an incidental finding, or it may be a manifestation of a systemic and potentially lethal disorder. When systemic, the proliferation is termed hemophagocytic lymphohistiocytosis (HLH), a clinicopathologic entity characterized by a widespread proliferation of benign hemophagocytic histiocytes, fever, pancytopenia, deranged liver function, and frequently coagulopathy and hepatosplenomegaly. A variety of infectious agents, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), and parvovirus B19 (PVB19), have been associated with HLH, but the relative frequency of each using one technique has not been evaluated. In addition, infectious causes of incidental bone marrow hemophagocytosis, not occurring in the setting of HLH, have not been evaluated. Review of bone marrow reports from bone marrow examinations done between December 1986 and June 1997 showed that 20 children aged 2 months to 15 years had bone marrow examinations that indicated hemophagocytosis. Archival materials from 19 patients were successfully retrieved, and DNA was extracted from archived unstained coverslips with subsequent polymerase chain reaction for EBV, CMV, HHV6, and PVB19 genomic DNA. DNA extracted from 16 bone marrow specimens of age-matched children was used as negative controls. Eleven of the 19 patients fulfilled the clinical and pathological criteria for HLH; the remaining eight patients had isolated hemophagocytosis without a systemic presentation. Viral DNA was detected in 8 of 11 patients with HLH but in none of eight patients with isolated hemophagocytosis. EBV was present in five of the bone marrows, followed in frequency by HHV6, CMV, and PVB19. Infection with more than one agent was present in three patients. Only one control patient was positive for HHV6 DNA; the remaining control patients were negative for all viruses. Viral infection, detected by PCR analysis of bone marrow, is a common finding in patients with HLH but not in patients with isolated bone marrow hemophagocytosis. This technique may provide another marker to aid in the diagnosis of HLH and suggests a different cause of hemophagocytosis occurring in patients with and without HLH.

Quantitation of 8 human herpesviruses in peripheral blood of human immunodeficiency virus-infected patients and healthy blood donors by polymerase chain reaction.

Human herpesviruses are associated with morbidity and mortality in persons with compromised immune systems, including patients infected with human immunodeficiency virus (HIV). To investigate the basis for this association, the levels of all 8 human herpesviruses (herpes simplex virus, types 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6, human herpesvirus 7, and human herpesvirus 8) were measured with the quantitative polymerase chain reaction (PCR). Viral DNA was measured in the whole blood of 20 HIV-infected patients and compared with levels in 20 healthy blood donors. There was no significant difference in the frequency of virus detection of the 8 human herpesviruses between HIV-infected patients and healthy adults. These results indicate that HIV infection is not associated with a general increase in the circulating levels of human herpesviruses, and suggest that quantitative PCR analysis is superior to qualitative PCR analysis for detection of clinically relevant disease in HIV-infected patients.

Prevalence and cellular reservoir of latent human herpesvirus 6 in tonsillar lymphoid tissue.

There are few studies that examine prevalence, quantity, and cellular proclivity of latent human herpesvirus 6 (HHV-6) in healthy populations. We examined 69 tonsils with paired blood specimens from children without evidence of acute infection. By polymerase chain reaction (PCR), HHV-6 was detected at low levels in 100% of tonsils and 39% of blood samples (n = 27), suggesting that prevalence of latent HHV-6 infection is high in children and may be underestimated by PCR analysis of blood. Although HHV-6A and HHV-6B were detected, HHV-6B predominated, being found in 97% of samples (n = 67). Tonsil sections from 7 cases were examined by in situ hybridization using 2 HHV-6 probes and immunohistochemical analysis. Using both in situ hybridization and immunohistochemical analysis, all tissues revealed marked HHV-6-specific staining in the squamous epithelium of the tonsillar crypts and rare positive lymphocytes. We conclude that HHV-6 is present universally in tonsils of children, and tonsillar epithelium may be an important viral reservoir in latent infection.

CD19 antigen in leukemia and lymphoma diagnosis and immunotherapy.

The CD19 antigen plays an important role in clinical oncology. In normal cells, it is the most ubiquitously expressed protein in the B lymphocyte lineage. CD19 expression is induced at the point of B lineage commitment during the differentiation of the hematopoietic stem cell, and its expression continues through preB and mature B cell differentiation until it is finally down-regulated during terminal differentiation into plasma cells. CD19 expression is maintained in B-lineage cells that have undergone neoplastic transformation, and therefore CD19 is useful in diagnosis of leukemias and lymphomas using monoclonal antibodies (mAbs) and flow cytometry. Interestingly, CD19 is also expressed in a subset of acute myelogenous leukemias (AMLs) indicating the close relationship between the lymphoid and myeloid lineages. Because B lineage leukemias and lymphomas rarely lose CD19 expression, and because it is not expressed in the pluripotent stem cell, it has become the target for a variety of immunotherapeutic agents, including immunotoxins. Treatment of non-Hodgkin's lymphoma (NHL) and acute lymphocytic leukemia (ALL) with anti-CD19 mAbs coupled to biological toxins has proven to be effective in vitro and in animal models, and has shown some promising results in Phase I clinical trials. Recently, the analysis of anti-CD19 effects on lymphoma cell growth has highlighted a novel mechanism of immunotherapy. Engagement of cell surface receptors like CD19 by mAbs can have anti-tumor effects by the activation of signal transduction pathways which control cell cycle progression and programmed cell death (apoptosis).

Diagnostic applications of quantitative polymerase chain reaction for cytomegalovirus.

Eight viruses in the herpes family have been identified that infect humans: herpes simplex viruses 1 and 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus (CMV), human herpesviruses 6 and 7 and the Kaposi sarcomaassociated herpesvirus (1). In immunocompetent individuals, primary infections are usually handled effectively by the host immune system without therapeutic intervention. However, these viruses are never completely eradicated by the immune response, probably because these viruses have the capacity to enter a latent state in a subset of infected cells. However, this does not normally pose a problem since the host immune system has been primed to handle any subsequent reactivation. Thus, human herpesviruses rarely cause serious problems in immunocompetent individuals.

Human herpesvirus-6 and sudden death in infancy: report of a case and review of the literature.

Investigation of sudden death in infancy is a vital function of the medical examiner's office. Surveillance of these cases may lead to recognition of new diseases or new manifestations of previously described diseases. Human herpesvirus-6 (HHV-6) is a relatively newly described virus that has been recognized as a cause of acute febrile illness in early childhood. While most cases are apparently self-limited, seven fatal cases have been reported. We present a case of a seven-month-old Latin American male with recent otitis media and vomiting who was found dead in bed. Autopsy revealed interstitial pneumonitis with an atypical polymorphous lymphocytic infiltrate in the liver, kidney, heart, spleen, lymph nodes, and bone marrow, associated with erythrophagocytosis. Polymerase chain reaction (PCR) analysis of formalin-fixed paraffin-embedded tissue was positive for HHV-6 and negative for Epstein-Barr virus (EBV) and cytomegalovirus (CMV). HHV-6 was also detected in the atypical lymphoid infiltrate by in-situ hybridization.

Metadata-driven comparative analysis tool for sequences (meta-CATS): an automated process for identifying significant sequence variations that correlate with virus attributes.

The Virus Pathogen Resource (ViPR; www.viprbrc.org) and Influenza Research Database (IRD; www.fludb.org) have developed a metadata-driven Comparative Analysis Tool for Sequences (meta-CATS), which performs statistical comparative analyses of nucleotide and amino acid sequence data to identify correlations between sequence variations and virus attributes (metadata). Meta-CATS guides users through: selecting a set of nucleotide or protein sequences; dividing them into multiple groups based on any associated metadata attribute (e.g. isolation location, host species); performing a statistical test at each aligned position; and identifying all residues that significantly differ between the groups. As proofs of concept, we have used meta-CATS to identify sequence biomarkers associated with dengue viruses isolated from different hemispheres, and to identify variations in the NS1 protein that are unique to each of the 4 dengue serotypes. Meta-CATS is made freely available to virology researchers to identify genotype-phenotype correlations for development of improved vaccines, diagnostics, and therapeutics.