Biomedical subjects

Dirk P Dittmer

Publications and source records attributed to Dirk P Dittmer.

At least 19 recordsLinked to original sources

PubMed2023

Intra-Host Evolution Provides for the Continuous Emergence of SARS-CoV-2 Variants.

Variants of concern (VOC) in SARS-CoV-2 refer to viruses whose viral genomes differ from the ancestor virus by ≥3 single-nucleotide variants (SNVs) and that show the potential for higher transmissibility and/or worse clinical progression. VOC have the potential to disrupt ongoing public health measures and vaccine efforts. Still, too little is known regarding how frequently new viral variants emerge and under what circumstances. We report a study to determine the degree of SARS-CoV-2 sequence evolution in 94 patients and to estimate the frequency at which highly diverse variants emerge. Two cases accumulated ≥9 SNVs over a 2-week period and one case accumulated 23 SNVs over 3 weeks, including three nonsynonymous mutations in the spike protein (D138H, E554D, D614G). The remainder of the infected patients did not show signs of intra-host evolution. We estimate that in as much as 2% of hospitalized COVID-19 cases, variants with multiple mutations in the spike glycoprotein emerge in as little as 1 month of persistent intra-host virus replication. This suggests the continued local emergence of variants with multiple nonsynonymous SNVs, even in patients without overt immune deficiency. Surveillance by sequencing for (i) viremic COVID-19 patients, (ii) patients suspected of reinfection, and (iii) patients with diminished immune function may offer broad public health benefits. IMPORTANCE New SARS-CoV-2 variants can potentially disrupt ongoing public health measures and vaccine efforts. Still, little is known regarding how frequently new viral variants emerge and under what circumstances. Based on this study, we estimate that in hospitalized COVID-19 cases, variants with multiple mutations may emerge locally in as little as 1 month, even in patients without overt immune deficiency. Surveillance by sequencing for continuously shedding patients, patients suspected of reinfection, and patients with diminished immune function may offer broad public health benefits.

Humans↗

PubMed2007

Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions.

Kaposi's sarcoma (KS) and its causative agent, Kaposi's sarcoma associated herpesvirus (KSHV/HHV-8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the "oncoweed" hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV-associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the "oncoweed" hypothesis by demonstrating basic biological plausibility.

Biological Assay↗

PubMed2006

Functional p53 signaling in Kaposi's sarcoma-associated herpesvirus lymphomas: implications for therapy.

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is associated with Kaposi's sarcoma (KS) as well as primary effusion lymphomas (PEL). The expression of viral proteins capable of inactivating the p53 tumor suppressor protein has been implicated in KSHV oncogenesis. However, DNA-damaging drugs such as doxorubicin are clinically efficacious against PEL and KS, suggesting that p53 signaling remains intact despite the presence of KSHV. To investigate the functionality of p53 in PEL, we examined the response of a large number of PEL cell lines to doxorubicin. Two out of seven (29%) PEL cell lines harbored a mutant p53 allele (BCBL-1 and BCP-1) which led to doxorubicin resistance. In contrast, all other PEL containing wild-type p53 showed DNA damage-induced cell cycle arrest, p53 phosphorylation, and p53 target gene activation. These data imply that p53-mediated DNA damage signaling was intact. Supporting this finding, chemical inhibition of p53 signaling in PEL led to doxorubicin resistance, and chemical activation of p53 by the Hdm2 antagonist Nutlin-3 led to unimpaired induction of p53 target genes as well as growth inhibition and apoptosis.

Animals↗

PubMed2006

Rapamycin is efficacious against primary effusion lymphoma (PEL) cell lines in vivo by inhibiting autocrine signaling.

The antitumor potency of the mTOR inhibitor rapamycin (sirolimus) is the subject of intense investigations. Primary effusion lymphoma (PEL) appears as an AIDS-defining lymphoma and like Kaposi sarcoma has been linked to Kaposi sarcoma-associated herpesvirus (KSHV). We find that (1) rapamycin is efficacious against PEL in culture and in a murine xenograft model; (2) mTOR, its activator Akt, and its target p70S6 kinase are phosphorylated in PEL; (3) rapamycin inhibits mTOR signaling as determined by S6 phosphorylation; (4) KSHV transcription is unaffected; (5) inhibition of IL-10 signaling correlates with drug sensitivity; and (6) addition of exogenous IL-10 or IL-6 can reverse the rapamycin growth arrest. This validates sirolimus as a new treatment option for PEL.

Animals↗

PubMed2006

Baboon model for West Nile virus infection and vaccine evaluation.

Animal models that closely mimic the human condition are of paramount significance to study pathogenic mechanisms, vaccine and therapy scenarios. This is particularly true for investigations that involve emerging infectious diseases. Nonhuman primate species represent an alternative to the more intensively investigated rodent animal models and in a number of instances have been shown to represent a more reliable predictor of the human response to infection. West Nile virus (WNV) has emerged as a new pathogen in the Americas. It has a 5% fatality rate, predominantly in the elderly and immune compromised. Typically, infections are cleared by neutralizing antibodies, which suggests that a vaccine would be efficacious. Previously, only macaques had been evaluated as a primate model for WNV vaccine design. The macaques did not develop WNV disease nor express the full complement of IgG subclasses that is found in humans. We therefore explored baboons, which exhibit the similar four IgG subclasses observed in humans as a new model for WNV infection and vaccine evaluation. In this present report, we describe the experimental infection of baboons with WNV and test the efficacy of an inactivated WNV vaccination strategy. All experimentally infected animals developed transient viremia and subsequent neutralizing antibodies. Anti-WNV IgM antibodies peaked at 20 days post-infection. Anti-WNV IgG antibodies appeared later and persisted past 60 days. Prior vaccination with chemically inactivated virus induced neutralizing titers and a fast, high titer IgG recall response, which resulted in lower viremia upon challenge. This report is the first to describe the development of the baboon model for WNV experimental infection and the utility of this model to characterize the immunologic response against WNV and a candidate WNV vaccine.

Animals↗

PubMed2006

Promoter switching allows simultaneous transcription of LANA and K14/vGPCR of Kaposi's sarcoma-associated herpesvirus.

Latent transcription of the latency-associated nuclear antigen (LANA/ORF73) of Kaposi's sarcoma-associated herpesvirus is driven by the LANAp-c. Complexity arises during lytic reactivation, however, as the bicistronic K14/vGPCR transcript initiates 32 bp downstream of LANAp-c in the opposite orientation. We identify an Rta/ORF50-inducible LANA promoter (LANAp-i) that is distinct from the LANAp-c. LANAp-c is unaffected by Rta/ORF50. Utilization of the second, downstream LANAp-i explains how LANA and K14/vGPCR are simultaneously transcribed during de novo infection or lytic reactivation. Transactivation of LANAp-i and K14/vGPCRp requires the C-terminal activation domain of Rta/ORF50 and is mediated by DNA-binding-dependent and -independent Rta/ORF50 mechanisms. Transcriptional profiling following viral reactivation support promoter reporter phenotypes. In sum, cis-elements within the LANAp were selected to ensure faithful expression of LANA and other genes regulated by LANAp during all stages of the KSHV lifecycle despite potential interference from K14/vGPCRp activity.

Antigens, Viral↗

PubMed2006

Immortalization of primary endothelial cells by the K1 protein of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to three different human cancers: Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The Kaposi's sarcoma lesion expresses high levels of angiogenic factors and is comprised of a mixed cell population, including endothelial cells that are infected with KSHV. We find that the KSHV K1 protein is expressed in Kaposi's sarcoma lesions and can immortalize and extend the life span of primary human umbilical vein endothelial cells in culture. Vascular endothelial growth factor (VEGF) is critical for the survival of endothelial cells, and we show that expression of K1 in endothelial cells resulted in increased levels of secreted VEGF and the activation of key signaling pathways, including the VEGF/VEGF receptor and the phosphatidylinositol-3'-OH-kinase (PI3K) pathway. The SH2 binding motifs present in the cytoplasmic tail of K1 were critical for K1's ability to activate these pathways. Activation of PI3K by K1 results in activation of Akt kinase and mammalian target of rapamycin and inactivation of the proapoptotic proteins FKHR, glycogen synthase kinase-3, and Bad, which are events indicative of cell survival. Because activation of the PI3K pathway is critical for transformation of many human cells, we suggest that PI3K activation by K1 is involved in endothelial cell immortalization and contributes to KSHV-associated tumorigenesis. We also report that K1 enhances angiogenesis in vivo and increases tumor vasculature and tumor size.

Animals↗

PubMed2006

The latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus induces B cell hyperplasia and lymphoma.

Kaposi sarcoma-associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA's contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+ IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis.

Animals↗

PubMed2006

Long-term-infected telomerase-immortalized endothelial cells: a model for Kaposi's sarcoma-associated herpesvirus latency in vitro and in vivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.

Animals↗

PubMed2006

A naturally occurring fatal case of Herpesvirus papio 2 pneumonia in an infant baboon (Papio hamadryas anubis).

Here we describe the unusual finding of herpesvirus pneumonia in a 7-d-old infant baboon (Papio hamadryas anubis). This animal had been separated from its dam the morning of its birth and was being hand-reared for inclusion in a specific pathogen-free colony. The baboon was presented for anorexia and depression of 2 d duration. Physical examination revealed a slightly decreased body temperature, lethargy, and dyspnea. The baboon was placed on a warm-water blanket and was given amoxicillin-clavulanate orally and fluids subcutaneously. The animal's clinical condition continued to deteriorate despite tube feeding, subcutaneous fluid administration, and antibiotic therapy, and it died 2 d later. Gross necropsy revealed a thin carcass and severe bilateral diffuse pulmonary consolidation. Histopathology of the lung revealed severe diffuse necrotizing pneumonia. Numerous epithelial and endothelial cells contained prominent intranuclear herpetic inclusion bodies. Virus isolated from lung tissue in cell culture was suspected to be Herpesvirus papio 2 (HVP2) in light of the viral cytopathic effect. Real-time polymerase chain reaction (PCR) analysis and DNA sequencing of PCR products both confirmed that the virus was HVP2. This case is interesting because the age at onset suggests perinatal transmission at or immediately after birth, and the disease course suggests inoculation of the virus into the respiratory tract.

Animals↗

PubMed2005

Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.

Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein-Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.

DNA Primers↗

PubMed2005

Methylene blue photoinactivation abolishes West Nile virus infectivity in vivo.

The prevalence of West Nile virus (WNV) infections and associated morbidity has accelerated in recent years. Of particular concern is the recent demonstration that this virus can be transmitted by blood products and can cause severe illness and mortality in transfusion recipients. We have evaluated methylene blue (MB)+light as a safe and cost-effective means to inactivate WNV in vitro. This regimen inactivated WNV with an IC50 of 0.10 microM. Up to 10(7)pfu/ml of WNV could be inactivated by MB+light with no residual infectivity. MB+light inactivated three primary WNV isolates from the years 1999, 2002 and 2003 and prevented mortality in a murine model for WNV infection. Since MB is already approved for human use at a dose of 100mg/kg/day, we conjecture that MB+light treatment of blood products for high-risk patients will be efficacious and suitable for use in resource-limited settings.

Animals↗

PubMed2005

Azidothymidine inhibits NF-kappaB and induces Epstein-Barr virus gene expression in Burkitt lymphoma.

The antiviral compound azidothymidine (AZT), alone or in combination with other agents, induces apoptosis in early-passage, Epstein-Barr virus-positive Burkitt lymphoma (EBV+ BL) lines and has clinical activity in EBV+ BL. We report here a mechanism of AZT's antitumor activity. The nuclei of these cells contain activated nuclear factor-kappaB (NF-kappaB) subunits p50, c-Rel, RelB, and p52, but not p65. Treatment of primary EBV+ BL lines with AZT inhibited NF-kappaB within 1 to 2 hours. This was followed by up-regulation of EBV gene expression including viral thymidine kinase (vTK) and apoptosis. Subclones of EBV+ BL cells that demonstrated activated p65 were resistant to AZT. In EBV+ BLs, AZT but not ganciclovir (GCV) was highly phosphorylated to its monophosphate form (AZT-MP). Phosphorylation, as well as apoptosis, was markedly enhanced in the presence of hydroxyurea. AZT inhibits NF-kappaB and up-regulates EBV gene expression in primary EBV+ BLs. AZT with hydroxyurea may represent an inexpensive, targeted regimen for endemic BL.

Apoptosis↗

PubMed2005

Whole-genome transcription profiling of rhesus monkey rhadinovirus.

Rhesus monkey rhadinovirus (RRV) and Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) belong to the gamma-2 grouping of herpesviruses. RRV and KSHV share a high degree of sequence similarity, and their genomes are organized in a similar fashion. RRV serves as an excellent animal model system to study the gamma herpesvirus life cycle both in vitro and in vivo. We have developed a high-sensitivity, high-throughput, high-specificity real-time quantitative reverse transcriptase-based PCR assay for RRV and have used this assay to profile transcription from the whole RRV genome during de novo productive infection of rhesus fibroblasts. Using this assay, we demonstrate that the genome-wide transcription profile for RRV closely parallels the genome-wide transcription profile for KSHV.

Animals↗

PubMed2005

Role of Notch signal transduction in Kaposi's sarcoma-associated herpesvirus gene expression.

Kaposi's sarcoma-associated herpesvirus (KSHV) RTA transcription factor is recruited to its responsive elements through interaction with a Notch-mediated transcription factor, RBP-Jkappa, indicating that RTA mimics cellular Notch signal transduction to activate viral lytic gene expression. To test whether cellular Notch signal transduction and RTA are functionally exchangeable for viral gene expression, human Notch intracellular (hNIC) domain that constitutively activates RBP-Jkappa transcription factor activity was expressed in KSHV-infected primary effusion lymphoma BCBL1 cells (TRExBCBL1-hNIC) in a tetracycline-inducible manner. Gene expression profiling showed that like RTA, hNIC robustly induced expression of a number of viral genes, including viral interleukin 6 (vIL-6), K3, and K5. Unlike RTA, however, hNIC was not capable of evoking the full repertoire of lytic viral gene expression and thereby lytic replication. To further understand the role of Notch signal transduction in KSHV gene expression, vIL-6 growth factor and K5 immune modulator genes were selected for detailed analysis. Despite the presence of multiple RBP-Jkappa binding sites, hNIC targeted the specific RBP-Jkappa binding sites of vIL-6 and K5 promoter regions to regulate their gene expression. These results indicate that cellular Notch signal transduction not only is partially exchangeable with RTA in regard to activation of viral lytic gene expression but also provides a novel expression profile of KSHV growth and immune deregulatory genes that is likely different from that of RTA-independent standard latency program as well as RTA-dependent lytic reproduction program.

Cell Line↗

PubMed2005

Enhanced cytotoxicity without internuclear spread of adenovirus upon cell fusion by measles virus glycoproteins.

The efficiency of viruses in cancer therapy is enhanced by proteins that mediate the fusion of infected cells with their neighbors. It was reported that replication-competent adenovirus particles can spread between nuclei within fusion-generated syncytia. To assess this conjecture, we generated fusogenic adenoviruses that express a balanced ratio of the F and H glycoproteins of measles virus. The viruses displayed enhanced cytotoxicity but largely unchanged replication efficiencies compared to a nonfusogenic virus. Most notably, the virus genomes did not spread through fusion-generated multinuclear cells. Hence, adenovirus replication in syncytia remains largely restricted to initially transduced nuclei.

Adenoviridae↗

PubMed2005

Real-time quantitative PCR analysis of viral transcription.

Whole-genome profiling using DNA arrays has led to tremendous advances in our understanding of cell biology. It has had similar success when applied to large viral genomes, such as the herpesviruses. Unfortunately, most DNA arrays still require specialized and expensive resources and, generally, large amounts of input RNA. An alternative approach is to query entire viral genomes using real-time quantitative PCR. We have designed such PCR-based arrays for every open reading frame of human herpesvirus 8 and describe here the general design criteria, validation procedures, and detailed application to quantify viral mRNAs. This should provide a useful resource either for whole-genome arrays or just to measure transcription of any one particular mRNA of interest. Because these arrays are RT-PCR-based, they are inherently more sensitive and robust than current hybridization-based approaches and are ideally suited to query viral gene expression in models of pathogenesis.

Base Sequence↗

PubMed2004

De novo generation of CD4 T cells against viruses present in the host during immune reconstitution.

T cells recognizing self-peptides are typically deleted in the thymus by negative selection. It is not known whether T cells against persistent viruses (eg, herpesviruses) are generated by the thymus (de novo) after the onset of the infection. Peptides from such viruses might be considered by the thymus as self-peptides, and T cells specific for these peptides might be deleted (negatively selected). Here we demonstrate in baboons infected with baboon cytomegalovirus and baboon lymphocryptovirus (Epstein-Barr virus-like virus) that after autologous transplantation of yellow fluorescent protein (YFP)-marked hematopoietic cells, YFP+ CD4 T cells against these viruses were generated de novo. Thus the thymus generates CD4 T cells against not only pathogens absent from the host but also pathogens present in the host. This finding provides a strong rationale to improve thymopoiesis in recipients of hematopoietic cell transplants and, perhaps, in other persons lacking de novo-generated CD4 T cells, such as AIDS patients and elderly persons.

Acquired Immunodeficiency Syndrome↗