Publications by authors named "Uluhan Sili"

25 Publications

  • Page 1 of 1

Frequency and associated factors for carbapenem-non-susceptible Bacteroides fragilis group bacteria colonization in hospitalized patients: Case control study in a university hospital in Turkey.

Indian J Med Microbiol 2021 Mar 31. Epub 2021 Mar 31.

Department of Microbiology, Marmara University School of Medicine, Istanbul, Turkey. Electronic address:

Purpuse: The carbapenem-resistant Bacteroides fragilis group (CR-BFG) bacteria have been reported in several countries recently with increasing global attention. The high incidence of CR-BFG isolated from our hospitalized patients has become an important problem. Therefore, we aimed to determine the frequency and associated factors for intestinal colonization by carbapenem-non-susceptible BFG (CNS-BFG) among adult patients hospitalized at intensive care units, neurosurgery and internal medicine wards in our hospital.

Methods: Rectal swabs (n = 1200), collected from 766 patients between February 2014 and March 2015, were inoculated onto kanamycin-vancomycin-leaked blood agar containing 0.125 mg/L meropenem. The isolates were identified by MALDI-TOF MS. Susceptibility testing was performed by agar dilution method. The carbapenemase gene (cfiA) was detected by PCR. Logistic regression analysis was used to evaluate the associated factors for intestinal colonization by CNS-BFG.

Results: A total 180 non-duplicate BFG isolates were obtained from 164 patients. Ten different species, including Parabacteroides distasonis (n = 46, 25.6%), and Bacteroides fragilis (n = 30; 16.6%), were identified. Twenty-five percent of the isolates were non-susceptible to meropenem (MIC >2 mg/L). The highest prevalence of meropenem resistant strains (MIC >8 mg/L) was detected among B. fragilis (n = 12), followed by Parabacteroides spp. (n = 4). All but one B. fragilis strains were cfiA gene positive. Hospital admission, increasing Charlson score, use of antibiotics; including carbapenems in past three months, colonization with other accompanying carbapenem-resistant Gram negative bacteria (Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa), and having undergone surgical operations were significantly associated with RCS- BFG colonization.

Conclusions: The high carriage rate of CNS-BFG in hospitalized patients may lead to worse clinical outcomes, such as serious infections and mortality, and deserves attention.
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http://dx.doi.org/10.1016/j.ijmmb.2021.03.018DOI Listing
March 2021

Untreated herpes simplex virus encephalitis without a fatal outcome.

J Neurovirol 2021 Jun 31;27(3):493-497. Epub 2021 Mar 31.

Department of Medical Microbiology, Medical School of Cerrahpasa, Istanbul University, Istanbul, Turkey.

Herpes simplex virus encephalitis (HSE) is the most common sporadic fatal encephalitis. Although timely administered acyclovir treatment decreases mortality, neuropsychiatric sequelae is still common among survivors. Magnetic resonance imaging is frequently utilized for the diagnosis of HSE, which typically involves temporal lobe(s) and can be mixed with brain tumors involving the same area. Here, we report a case of HSE, who received acyclovir with a delay of 90 days because of presumptive tumor diagnosis and survived with minimal sequelae.
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http://dx.doi.org/10.1007/s13365-021-00968-yDOI Listing
June 2021

Tuberculosis and COVID-19: An overlapping situation during pandemic.

J Infect Dev Ctries 2020 Jul 31;14(7):721-725. Epub 2020 Jul 31.

Marmara University School of Medicine, Department of Medical Microbiology, Pendik Training and Research Hospital, İstanbul, Turkey.

Introduction: The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19). First COVID-19 case was detected in March, 10, 2020 in Turkey and as of May, 18, 2020 148,067 cases have been identified and 4096 citizens have died. Tuberculosis (TB) is a worldwide public health concern, incidence of tuberculosis (per 100,000 people) in Turkey was reported at 14, 1 in 2018. During pandemic COVID-19 was the main concern in every clinic and as we discuss here overlapping respiratory diseases may result in delaying of the diagnosis and treatment.

Methodology: There were 4605 respiratory samples examined between March 23 and May 18 for COVID-19 and 185 samples for Mycobacterium tuberculosis in our laboratory. The Xpert Ultra assay was performed for the diagnosis of pulmonary tuberculosis; SARS-CoV-2 RNA was determined by real-time PCR (RT-PCR) analysis in combined nasopharyngeal and deep oropharyngeal swabs of suspected cases of COVID-19.

Results: Both of SARS-CoV-2 and M. tuberculosis tests were requested on the clinical and radiological grounds in 30 patients. Here we discussed 2 patients who were both COVID-19 and TB positive. One patient already diagnosed with tuberculosis become COVID-19 positive during hospitalization and another patient suspected and treated for COVID-19 received the final diagnosis of pulmonary TB and Human Immunodeficiency Virus infection.

Conclusions: We want to emphasize that while considering COVID-19 primarily during these pandemic days, we should not forget one of the "great imitators", tuberculosis within differential diagnoses.
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http://dx.doi.org/10.3855/jidc.13152DOI Listing
July 2020

Hand hygiene knowledge, perception and practice of healthcare workers in a Turkish university hospital intensive care unit.

J Infect Dev Ctries 2019 08 31;13(8):744-747. Epub 2019 Aug 31.

Department of Infectious Diseases and Clinical Microbiology, School of Medicine, Marmara University, Istanbul, Turkey.

Introduction: While improvement of hand hygiene (HH) compliance is considered as the best approach to reduce healthcare-associated infections, the instructional interventions in HH among healthcare workers of intensive care unit (ICU) of our hospital was not successful enough. The following study was conducted to evaluate HH knowledge, perception, practice and effectiveness of the trainings among healthcare workers of ICU in our hospital.

Methodology: A cross-sectional study was conducted in the ICU containing 8 medical and 16 surgical beds with 284 filled questionnaires about HH knowledge and 1187 observed opportunities for HH compliance.

Results: Overall observed HH compliance rate was 40.6%; lowest compliance was 21.7% for "before clean/aseptic procedure" indication and highest compliance was 68.6% for "after touching a patient" indication. Although > 90% healthcare workers correctly identified the World Health Organization's five indications for HH, 82 - 85% failed to recognize non-indications, i.e. when it was not necessary to perform HH. Our study showed that 15.1% of healthcare workers neither received nor felt the need for HH training.

Conclusions: Despite regular HH trainings, healthcare workers could not differentiate when HH was not required which suggested failure to understand HH rationale. This may explain poor HH compliance rates. A systematic study is needed in order to find out the reasons behind of this noncompliance and improve HH training methods for educating healthcare workers.
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http://dx.doi.org/10.3855/jidc.10916DOI Listing
August 2019

A qualitative study of hand hygiene compliance among health care workers in intensive care units.

J Infect Dev Ctries 2019 02 28;13(2):111-117. Epub 2019 Feb 28.

Department of Public Health, School of Medicine, Marmara University, Istanbul, Turkey.

Introduction: Studies indicate that adherence to hand hygiene guidelines is at suboptimal levels. We aimed to explore the reasons for poor hand hygiene compliance.

Methodology: A qualitative study based on the Theory of Planned Behavior as a framework in explaining compliance, consisting four focus group discussions and six in-depth interviews.

Results: Participants mostly practiced hand hygiene depending on the sense of "dirtiness" and "cleanliness". Some of the participants indicated that on-job training delivered by the infection control team changed their perception of "emotionally" based hand hygiene to "indication" based. Direct observations and individual feedback on one-to-one basis were the core of this training. There was low social cohesiveness and a deep polarization between the professional groups that led one group accusing the other for not being compliant.

Conclusions: The infection control team should continue delivering one-to-one trainings based on observation and immediate feedback. But there is need to base this training model on a structured behavioral modification program and test its efficacy through a quasi-experimental design. Increasing social cohesiveness and transforming the blaming culture to a collaborative safety culture is also crucial to improve compliance. High workload, problems related to work-flow and turnover should be addressed.
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http://dx.doi.org/10.3855/jidc.10926DOI Listing
February 2019

Assessment of Transmitted HIV-1 Drug Resistance Mutations Using Ultra- Deep Pyrosequencing in a Turkish Cohort.

Curr HIV Res 2018 ;16(3):216-221

Department of Infectious Diseases and Clinical Microbiology, School of Medicine, Marmara University, Istanbul, Turkey.

Background: Antiretroviral treatment (ART) reduces morbidity and mortality caused by human immunodeficiency virus (HIV) infection; however, the emergence of drug-resistant strains poses an important obstacle to treatment success. Using conventional sequencing methods to determine antiretroviral resistance, mutations present in ≥20% of quasispecies can be identified, but drug-resistant minority variants can lead to virologic failure.

Objective: We aimed to assess transmitted drug resistance mutations (TDRMs) within minority variants using ultra-deep pyrosequencing (UDPS).

Method: Treatment-naive adult patients were included in this observational study. Surveillance TDRMs were classified as ≥20% or at minority variant level (≥2% - <20%). Genotypic sensitivity score calculated by using all pre-treatment drug resistance mutations (PDRMs) was also evaluated.

Results: Thirty-six patients were analyzed. Any TDRM at ≥20% level was detected in 8.3% of the patients (n=3). This prevalence increased to 30.6% (n=11) with the inclusion of minority variants. All non-nucleoside reverse transcriptase inhibitor and protease inhibitor-related TDRMs were within minority variants. The genotypic sensitivity score of rilpivirine-based regimens was considerably diminished when minority variants were included in the PDRM analysis.

Conclusion: UDPS was used for the first time to assess TDRM in a Turkish HIV cohort and uncovered several mutations hidden within minority variants. UDPS may be preferred to detect PDRMs for avoiding virologic failure with rilpivirine-based ART regimens.
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http://dx.doi.org/10.2174/1570162X16666180910130112DOI Listing
April 2019

HCV-specific lymphocyte responses in individuals with positive anti-HCV but negative HCV-RNA.

J Clin Virol 2015 Jun 15;67:73-7. Epub 2015 Apr 15.

Department of Infectious Diseases and Clinical Microbiology, Cerrahpasa School of Medicine, Istanbul University, Istanbul, Turkey.

Background: Hepatitis C virus (HCV) status cannot be reliably predicted in anti-HCV positive/HCV-RNA negative individuals who may either have recovered spontaneously or have a false-positive test due to antibody cross-reaction. Investigating T lymphocyte responses in individuals with different HCV status may help understand the cellular immune mechanisms underlying spontaneous recovery, treatment response, and chronicity.

Objective: We aimed to determine whether anti-HCV positive, HCV-RNA negative individuals are truly spontaneous recoverers from acute HCV infection.

Study Design: We used enzyme-linked immunosorbent spot (ELISPOT) assay to compare HCV-specific lymphocyte response among anti-HCV positive/HCV-RNA negative individuals, patients with sustained virological response to interferon-γ/ribavirin treatment, and patients with chronic HCV infection.

Results: We found that 83% of anti-HCV positive/HCV-RNA negative individuals without a past medical history of acute icteric hepatitis had an HCV-specific T lymphocyte response in peripheral blood. Lymphocyte responses in these individuals were similar in magnitude to treatment responders unlike patients with chronic HCV whose virus-directed immunity was significantly suppressed.

Conclusions: Detection of HCV-specific T lymphocyte responses using ELISPOT is a feasible method to ascertain past asymptomatic acute HCV infection.
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http://dx.doi.org/10.1016/j.jcv.2015.04.014DOI Listing
June 2015

Successful treatment of an invasive fungal infection caused by Talaromyces sp. with voriconazole.

Med Mycol Case Rep 2015 Jun 24;8:21-3. Epub 2015 Feb 24.

Department of Infectious Diseases and Clinical Microbiology, School of Medicine, Marmara University, Istanbul, Turkey.

Invasive fungal infections (IFI) are on the rise due to increasing numbers of immunosuppressed and critically ill patients. A malignant-looking pulmonary nodule in an immunosuppressed patient may indeed be caused by a fungal organism. We report a patient, who was eventually diagnosed with an IFI caused by an agent of hyalohyphomycosis, Talaromyces sp. determined via molecular methods and succesfully treated with voriconazole.
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http://dx.doi.org/10.1016/j.mmcr.2015.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354872PMC
June 2015

Comparison of cellular immunity in patients with chronic hepatitis B, inactive hepatitis B surface antigen carriers and spontaneously recovered individuals.

Hepatogastroenterology 2014 Nov-Dec;61(136):2326-9

Different clinical outcomes of acute HBV infection have been partially explained by individual differences in immune response. In this study we investigated interferon gamma (IFN-γ) secretion of peripheral blood mononuclear cells (PBMC) in vitro against specific (Hepatitis B core antigen; recombinant HB-cAg) and non-specific (CMV, EBV, Influenza peptide pool; CTL CEF peptide pool "plus") antigens using enzyme linked immunospot (ELISPOT) assay in 7 patients with chronic hepatitis B (CHB group), 8 inactive carriers Of HBV (carrier group) and 8 subjects who spontaneously recovered from acute HBV infection as detected by anti-HBs positivity (immune group). Phytohemaglutinin served as the positive test control. Response against recombinant HBcAg was 88±135, 50±110, 105±150 spot forming cell (SFC)/10(5) PBMC, in CHB, carrier and immune groups, respectively. HBcAg-specific T-cell response was slightly higher in the immune group; however, statistically there was no significant difference between the groups. Assessment of cellular immunity by IFN-γ ELISPOT was not sufficient to explain the various outcomes of HBV infection such as resolution, chronicity and carriership.
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April 2015

Compliance of healthcare workers with hand hygiene practices in neonatal and pediatric intensive care units: overt observation.

Interdiscip Perspect Infect Dis 2014 25;2014:306478. Epub 2014 Nov 25.

Department of Pediatrics, Division of Pediatric Infectious Diseases, Marmara University Medical Faculty, Istanbul, Turkey.

Background. The objective of this study was to assess the compliance of hand hygiene (HH) of healthcare workers (HCWs) in the neonatal and pediatric intensive care unit in a tertiary university hospital in Istanbul. Methods. An observational study was conducted on the compliance of HH for the five World Health Organization (WHO) indications. HCWs were observed during routine patient care in day shift. The authors also measured the technique of HH through hand washing or hand hygiene with alcohol-based disinfectant. Results. A total of 704 HH opportunities were identified during the observation period. Overall compliance was 37.0% (261/704). Compliance differed by role: nurses (41.4%) and doctors (31.9%) [P = 0.02, OR: 1.504, CI 95%: 1.058-2.137]. HCWs were more likely to use soap and water (63.6%) compared to waterless-alcohol-based hand hygiene (36.3%) [P < 0.05]. Conclusion. Adherence to hand hygiene practice and use of alcohol-based disinfectant was found to be very low. Effective education programs that improve adherence to hand hygiene and use of disinfectants may be helpful to increase compliance.
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http://dx.doi.org/10.1155/2014/306478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262750PMC
December 2014

Herpes simplex virus encephalitis: clinical manifestations, diagnosis and outcome in 106 adult patients.

J Clin Virol 2014 Jun 25;60(2):112-8. Epub 2014 Mar 25.

Department of Internal Medicine, Istanbul Medipol University School of Medicine, Istanbul, Turkey.

Background: Herpes simplex virus (HSV) is one of the most common causes of sporadic encephalitis worldwide.

Objective: We aimed to determine clinical characteristics and prognosis of HSV encephalitis (HSVE) cases reviewed retrospectively from several collaborating centers.

Study Design: We searched hospital archives of the last 10 years for patients with HSVE diagnosis, i.e. clinical presentation compatible with encephalitis and brain involvement on magnetic resonance imaging (MRI) and/or detection of HSV DNA in the cerebrospinal fluid by polymerase chain reaction (PCR). Clinical characteristics were noted and patients were phone-interviewed. HSVE cases were grouped and analyzed as proven and probable, based on virological confirmation by PCR. Univariate and multivariate analyses were used to determine factors associated with prognosis.

Results: A total of 106 patients (63 males and 43 females; mean age, 44 years; range, 18-83 years) were included. Most common symptoms were changes in mental status, fever, headache, and seizure. HSV PCR was positive in 69% of patients tested, while brain involvement was detected on MRI in 95%. Acyclovir was started mostly within five days of main symptom and continued for ≥14 days. Case fatality rate was 8%, while 69% of patients recovered with sequelae. Favorable prognosis was observed in 73% of patients. Multivariate analysis identified the duration of disease before hospital admission (odds ratio (OR)=1.24) and the extent of brain involvement on MRI at the time of admission (OR=37.22) as two independent risk factors associated with poor prognosis.

Conclusions: Although HSVE fatality regressed considerably with acyclovir treatment, many patients survive with sequelae. Our results emphasize the importance of early diagnosis and prompt treatment of HSVE.
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http://dx.doi.org/10.1016/j.jcv.2014.03.010DOI Listing
June 2014

Candidal psoas abscess following persistent pyuria in a renal transplant recipient.

Int Urol Nephrol 2014 Jan 8;46(1):269-73. Epub 2012 Nov 8.

Division of Nephrology, Department of Internal Medicine, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey.

Candidal infections occur commonly in renal transplant recipients especially in genitourinary system. Although the epidemiology of candiduria has not been well characterized in renal transplant population, it is the most common cause of fungal infections. However, candidal psoas abscess is very rare in the literature. We report a 42-year-old male renal transplant recipient with prolonged pyuria and candiduria followed by candidal psoas abscess formation. The treatment consisted of prolonged antifungal therapy along with percutaneous drainage. However, eventually, a surgical drainage had to be performed for the successful eradication.
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http://dx.doi.org/10.1007/s11255-012-0316-8DOI Listing
January 2014

Production of good manufacturing practice-grade cytotoxic T lymphocytes specific for Epstein-Barr virus, cytomegalovirus and adenovirus to prevent or treat viral infections post-allogeneic hematopoietic stem cell transplant.

Cytotherapy 2012 Jan;14(1):7-11

Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital, The Methodist Hospital, Houston, Texas 77030, USA.

Infections with a range of common community viruses remain a major cause of mortality and morbidity after allogeneic hematopoietic stem cell transplantation. T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenoviruses can safely prevent and infections with these three most common culprits, but the manufacture of individual T cell lines for each virus would be prohibitive in terms of time and cost. We have demonstrated that T cells specific for all three viruses can be manufactured in a single culture using monocytes and EBV-transformed B lymphoblastoid cell lines (LCLs), both transduced with an adenovirus vector expressing pp65 of CMV, as antigen-presenting cells. Trivirus-specific T cell lines produced from healthy stem cell donors could prevent and treat infections with all three viruses, not only in the designated recipient, but in unrelated, partially-HLA-matched third party recipients. We now provide the details and logistics of T cell manufacture.
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http://dx.doi.org/10.3109/14653249.2011.636963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705934PMC
January 2012

Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex).

J Immunother 2010 Apr;33(3):305-15

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA.

The clinical manufacture of antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy is limited by the complexity and time required to produce large numbers with the desired function and specificity. The culture conditions required are rigorous, and in some cases only achieved in 2-cm wells in which cell growth is limited by gas exchange, nutrients, and waste accumulation. Bioreactors developed to overcome these issues tend to be complex, expensive, and not always conducive to CTL growth. We observed that antigen-specific CTLs undergo 7 to 10 divisions poststimulation. However, the expected CTL numbers were achieved only in the first week of culture. By recreating the culture conditions present during this first week-low frequency of antigen-specific T cells and high frequency of feeder cells-we were able to increase CTL expansion to expected levels that could be sustained for several weeks without affecting phenotype or function. However, the number of 24-well plates needed was excessive and cultures required frequent media changes, increasing complexity and manufacturing costs. Therefore, we evaluated novel gas-permeable culture devices (G-Rex) with a silicone membrane at the base allowing gas exchange to occur uninhibited by the depth of the medium above. This system effectively supports the expansion of CTL and actually increases output by up to 20-fold while decreasing the required technician time. Importantly, this amplified cell expansion is not because of more cell divisions but because of reduced cell death. This bioprocess optimization increased T-cell output while decreasing the complexity and cost of CTL manufacture, making cell therapy more accessible.
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http://dx.doi.org/10.1097/CJI.0b013e3181c0c3cbDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946348PMC
April 2010

Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.

Nat Med 2006 Oct 24;12(10):1160-6. Epub 2006 Sep 24.

Center for Cell and Gene Therapy, Baylor College of Medicine, The Methodist Hospital and Texas Children's Hospital, Houston, Texas 77030, USA.

Immunocompromised individuals are at high risk for life-threatening diseases, especially those caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus. Conventional therapeutics are primarily active only against CMV, and resistance is frequent. Adoptive transfer of polyclonal cytotoxic T lymphocytes (CTLs) specific for CMV or EBV seems promising, but it is unclear whether this strategy can be extended to adenovirus, which comprises many serotypes. In addition, the preparation of a specific CTL line for each virus in every eligible individual would be impractical. Here we describe genetic modification of antigen-presenting cell lines to facilitate the production of CD4(+) and CD8(+) T lymphocytes specific for CMV, EBV and several serotypes of adenovirus from a single cell culture. When administered to immunocompromised individuals, the single T lymphocyte line expands into multiple discrete virus-specific populations that supply clinically measurable antiviral activity. Monoculture-derived multispecific CTL infusion could provide a safe and efficient means to restore virus-specific immunity in the immunocompromised host.
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http://dx.doi.org/10.1038/nm1475DOI Listing
October 2006

A comparison of gene transfer and antigen-loaded dendritic cells for the generation of CD4+ and CD8+ cytomegalovirus-specific T cells in HLA-A2+ and HLA-A2- donors.

Biol Blood Marrow Transplant 2004 Nov;10(11):761-71

Westmead Institute for Cancer Research, Westmead Millennium Institute, University of Sydney, Sydney, New South Wales, Australia.

Dendritic cells have been used effectively to select for human cytomegalovirus (CMV)-specific T cells for immunotherapy applications. The ability to process and present relevant major histocompatibility complex class I and II peptides to T cells makes them ideal for selecting CD4+ and CD8+ T cells regardless of HLA tissue type. This study compared the generation of CMV-specific T cells by using dendritic cells loaded with either CMV pp65495-503 peptide or CMV lysate or transduced with adenovirus encoding the pp65 gene (Ad5pp65GFP) for the generation of CD4+ and CD8+ CMV-specific T cells in HLA-A2+ and HLA-A2 - donors. In HLA-A2+ donors, CD8+ tetramer+ T cells increased with all antigens but were greatest in peptide- and Ad5pp65GFP-stimulated T cells. The CD4+ /CD8+ ratio in the stimulated T-cell cultures proved to be dependent on the antigen used. CMV lysate-stimulated cells were primarily CD4+, whereas peptide- and Ad5pp65GFP-stimulated cultures were mostly CD8+. Analysis of cells from lysate-stimulated or gene-transduced-stimulated cultures showed expansion of CMV-specific CD4+ T cells, indicating that major histocompatibility complex class II peptides were present in both antigens. Furthermore, CMV-specific T cells were generated from HLA-A2 - donors by using Ad5pp65GFP transduction or CMV lysate stimulation and were able to recognize a pp65 peptide restricted to the HLA-B35 allele. These data indicate that either CMV lysate or adenovirus encoding CMV antigenic genes may be useful for the generation of both CD4+ and CD8+ CMV-specific T cells in donors irrespective of HLA tissue type and may be applicable to clinical immunotherapy.
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http://dx.doi.org/10.1016/j.bbmt.2004.05.011DOI Listing
November 2004

Conserved CTL epitopes on the adenovirus hexon protein expand subgroup cross-reactive and subgroup-specific CD8+ T cells.

Blood 2004 Oct 20;104(8):2432-40. Epub 2004 Jul 20.

Center for Cell and Gene Therapy, Department of Pediatrics, 6621 Fannin St, MC 3-3320, Houston, TX, USA.

Adenoviruses often cause lethal infections in immunocompromised individuals. Adoptive transfer of immune T cells offers a therapeutic option, but this strategy has been hindered by the paucity of information on molecular targets of cellular immunity and by the immunologic heterogeneity of the 51 human adenoviruses, which are grouped from A to F on the basis of genome size, composition, homology, and organization. Clonal analysis of the adenovirus-specific cytotoxic T lymphocyte (CTL) responses of seropositive individuals identified 5 novel CD8(+) T-cell epitopes, all located in conserved regions of the capsid protein hexon. Reactive T cells were cross-reactive between 2 to 4 groups, while no T cells specific for a single subgroup were detected. Thus, by exploiting these peptide targets, it is possible to prepare a T-cell population capable of reacting with most adenoviruses that cause disease in immunocompromised patients.
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http://dx.doi.org/10.1182/blood-2004-02-0646DOI Listing
October 2004

Optimization of gene expression in nonactivated circulating lymphocytes.

Mol Ther 2003 Oct;8(4):629-36

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030, USA.

Circulating lymphocytes are important target cells for the treatment of HIV-related and autoimmune diseases and for stimulating anti-tumor immunity. To date, gene transfection of these nonactivated cells after intravenous delivery of viral or nonviral vectors remains low although these circulating cells are highly accessible. Optimized lentiviral vectors currently can transduce less than 10% of nonactivated circulating lymphocytes. Here we report transfection of up to 15% of these nonactivated cells using liposomes directed to human CCR5 displayed on the surface of helper T cells and macrophages in transgenic mice. Attachment of modified MIP-1 beta to the surface of DNA-liposome complexes increased gene delivery and expression in nonactivated circulating lymphocytes approximately sixfold. In vitro data using these complexes to transfect PM1 cells that have elevated levels of CCR5 supported our data obtained in vivo. Therefore, ligands that bind to cell surface receptors on circulating lymphocytes can be used with optimized systemic liposomes to increase transfection and gene expression in these cells without activation.
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http://dx.doi.org/10.1016/s1525-0016(03)00231-4DOI Listing
October 2003

Fiber-modified adenoviruses generate subgroup cross-reactive, adenovirus-specific cytotoxic T lymphocytes for therapeutic applications.

Blood 2004 Feb 2;103(3):1011-9. Epub 2003 Oct 2.

Center for Cell and Gene Therapy, Department of Pediatrics, 6621 Fannin S, MC 3-3320, Houston, TX 77030, USA.

Adenovirus (Ad) infections are responsible for considerable morbidity and mortality, particularly in pediatric hematopoietic stem cell transplant (HSCT) recipients. To date there is no therapy. The present study was motivated by the potential for using adoptive immunotherapy as either prophylaxis or treatment for Ad infections and associated diseases. The authors have developed a protocol to reactivate Ad-specific memory T cells from peripheral blood mononuclear cells (PBMCs) using a clinical-grade adenoviral vector. Such lines contain a specific CD4 and CD8 T-cell component and are capable of recognizing and lysing target cells infected with wild-type Ad serotypes from different Ad groups. Furthermore, the frequency of Ad-specific precursors can be determined in PBMCs ex vivo and used as a means to assess changes in Ad-specific T-cell memory responses after infusion. This is the first report of a simple and reproducible method to activate and expand Ad-specific cytotoxic T lymphocytes (CTLs), which should be protective against the range of different Ad subtypes that affect transplant recipients.
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http://dx.doi.org/10.1182/blood-2003-07-2449DOI Listing
February 2004

Induction of antigen-specific regulatory T cells following overexpression of a Notch ligand by human B lymphocytes.

J Virol 2003 Oct;77(20):10872-80

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030, USA.

In mice, activation of the Notch pathway in T cells by antigen-presenting cells overexpressing Notch ligands favors differentiation of regulatory T lymphocytes responsible for antigen-specific tolerance. To determine whether this mechanism operates in human T cells, we used Epstein-Barr virus-positive lymphoblastoid cell lines (EBV-LCL) as our (viral) antigen-presenting cells and overexpressed the Notch ligand Jagged-1 (EBV-LCL J1) by adenoviral transduction. The EBV-LCL J1s were cocultured with autologous T cells, and the proliferative and cytotoxic responses to EBV antigens were measured. Transduction had no effect on EBV-LCL expression of major histocompatibility complex (MHC) antigens or of costimulatory molecules CD80, CD86, and CD40. However, we observed a 35% inhibition of proliferation and a >65% reduction in cytotoxic-T-cell activity, and interleukin 10 production was increased ninefold. These EBV-LCL J1-stimulated T lymphocytes act as antigen-specific regulatory cells, since their addition to fresh autologous T cells cultured with autologous nontransduced EBV-LCL cells significantly inhibited both proliferation and cytotoxic effector function. Within the inhibitory population, CD4(+)CD25(+) and CD8(+)CD25(-) T cells had the greatest activity. This inhibition appears to be antigen-specific, since responses to Candida and cytomegalovirus antigens were unaffected. Hence, transgenic expression of Jagged-1 by antigen-presenting cells can induce antigen-specific regulatory T cells in humans and modify immune responses to viral antigens.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC224961PMC
http://dx.doi.org/10.1128/jvi.77.20.10872-10880.2003DOI Listing
October 2003

Large-scale expansion of dendritic cell-primed polyclonal human cytotoxic T-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy.

J Immunother 2003 May-Jun;26(3):241-56

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.

Dendritic cells (DCs) have been shown to activate cytotoxic T-lymphocytes (CTLs) for many tumor and virus-associated antigens in vitro. In this study, the authors tested the feasibility of using DCs to expand polyclonal, cytomegalovirus (CMV)-specific CTL lines for adoptive immunotherapy. Two stimulations with DCs expressing pp65, the immunodominant antigen of CMV, effectively activated and expanded MHC-class I restricted, CMV-specific CTLs from peripheral blood mononuclear cells. However, limiting monocyte-derived DC numbers precluded the authors from expanding the CTLs to the numbers required for adoptive transfer protocols. Nonspecific stimulation methods failed to expand CTL lines specifically. However, the authors found that lymphoblastoid cell lines (LCLs) expressing pp65 expanded pp65-specific CTL lines without competition from EBV-specific CTLs. An unlimited source of antigen presenting cells that could present antigen in the appropriate MHC context emerged as a critical point for expansion of polyclonal, antigen-specific CTL lines.
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http://dx.doi.org/10.1097/00002371-200305000-00008DOI Listing
January 2004

Selective depletion of donor alloreactive T cells without loss of antiviral or antileukemic responses.

Blood 2003 Sep 22;102(6):2292-9. Epub 2003 May 22.

Department of Bone Marrow Transplantation, Great Ormond Street Children's Hospital, London, United Kingdom.

Poor immune reconstitution after haploidentical stem cell transplantation results in a high mortality from viral infections and relapse. One approach to overcome this problem is to selectively deplete the graft of alloreactive cells using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient peripheral blood mononuclear cells (PBMCs), and this can result in graft versus host disease (GVHD). We have refined this approach using recipient Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as stimulators to activate donor alloreactive T cells. Our studies demonstrate that allodepletion with an anti-CD25 immunotoxin following stimulation with HLA-mismatched host LCLs more consistently depleted in vitro alloreactivity than stimulation with host PBMCs, as assessed in primary mixed lymphocyte reactions (MLRs). Allodepletion using this approach specifically abrogates cytotoxic T-cell responses against host LCLs. In interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assays, antiviral responses to adenovirus and cytomegalovirus (CMV) were preserved following allodepletion. Likewise, using HLA-A2-pp65 tetramers, we have shown that the frequency of CMV-specific T cells is unaffected by allodepletion. Moreover, the donor anti-EBV response is partially retained by recognition of EBV antigens through the nonshared haplotype. Finally, we studied whether allodepletion affects the response to candidate tumor antigens in myeloid malignancies. Using HLA-A2-PR1 tetramer analysis, we found that the frequency of T cells recognizing the PR1 epitope of proteinase 3 was not significantly different in allodepleted and unmanipulated PBMCs from patients with chronic myeloid leukemia (CML) undergoing transplantation. Based on these data, we have embarked on a phase 1 clinical trial of addback of allo-LCL-depleted donor T cells in the haplo-identical setting.
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http://dx.doi.org/10.1182/blood-2002-11-3516DOI Listing
September 2003

Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies.

Blood 2003 Mar 31;101(5):1905-12. Epub 2002 Oct 31.

Center for Cell and Gene Therapy, Texas Children's Cancer Center, Departments of Pediatrics, Medicine, and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.

The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in EBV-positive Hodgkin disease and is a potential target for cytotoxic T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is low, and so far the generation of LMP1-specific CTLs has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DCs) for CTL stimulation, and we reasoned that an inactive, nontoxic LMP1 mutant (DeltaLMP1) could be expressed in DCs and would enable the activation and expansion of polyclonal LMP1-specific CTLs. Recombinant adenoviral vectors expressing LMP1 or DeltaLMP1 were tested for their ability to transduce DCs. LMP1 expression was toxic within 48 hours whereas high levels of DeltaLMP1 expression were achieved with minimal toxicity. DeltaLMP1-expressing DCs were able to reactivate and expand LMP1-specific CTLs from 3 healthy EBV-seropositive donors. LMP1-specific T cells were detected by interferon-gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) assays using the HLA-A2-restricted LMP1 peptide, YLQQNWWTL (YLQ). YLQ-specific T cells were undetectable (less than 0.001%) in donor peripheral blood mononuclear cells (PBMCs); however, after stimulation the frequency increased to 0.5% to 3.8%. Lysis of autologous target cells by CTLs was dependent on the level of LMP1 expression. In contrast, the frequency of YLQ-specific CTLs in EBV-specific CTLs reactivated and expanded using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. Thus, DeltaLMP1 expression in DCs is nontoxic and enables the generation of LMP1-specific CTLs for future adoptive immunotherapy protocols for patients with LMP1-positive malignancies such as EBV-positive Hodgkin disease. Targeting LMP1 in these malignancies may improve the efficacy of current adoptive immunotherapy approaches.
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http://dx.doi.org/10.1182/blood-2002-05-1514DOI Listing
March 2003

Transfusion Medicine: New Clinical Applications of Cellular Immunotherapy.

Hematology Am Soc Hematol Educ Program 2000 :356-375

There is now clear clinical evidence that adoptive cellular immunotherapy can eradicate hematologic malignancy and cure otherwise lethal viral infections. With this knowledge comes the challenge of improving the effectiveness and safety of the approach and of simplifying the methodologies required whilst still meeting appropriate federal regulatory guidelines. This review provides an overview of the current status of cellular immunotherapies and addresses how they may be implemented and the future directions they are likely to take. In Section I, Dr. Brenner with Drs. Rossig and Sili reviews the clinical experience to date with adoptive transfer of viral antigen-specific T cells for the successful treatment of Epstein-Barr virus-associated malignancies as well as viral infectious diseases. Genetic modification of the T cell receptor of the infused cells to potentiate such T cells as well as modifications to improve safety of the infusions are described. In Section II, Dr. Young describes the hematopoietic lineages of human dendritic cells and some of their immunotherapeutic applications. The critical importance of dendritic cells to T cell immunity and the capacity to generate dendritic cells in large numbers has spawned enormous interest in the use of these specialized leukocytes to manipulate cellular immunity. Successful cytokine-driven differentiation of dendritic cells reveal two types, myeloid- and plasmacytoid or lymphoid-related dendritic cells. The effects of maturation on phenotype and function of the dendritic cells and their use as immune adjuvants in dendritic cell vaccines to elicit antitumor and antiviral immunity are reviewed. In Section III, Professor Goulmy illustrates some current and future approaches towards tumor-specific cellular therapy of hematopoietic malignancy. Minor histocompatibility antigen (mHag) disparities between HLA-matched bone marrow donor and recipient can induce allo-responses that may participate in post bone marrow transplantation (BMT) graft-versus-leukemia (GVL) reactivities. A lack of such allo-reactivity may result in relapse of leukemia after BMT. In these patients, adoptive immunotherapy with cytotoxic T cells (CTLs) specific for hematopoietic system-restricted mHags may be used as an extension of current efforts using immunotherapy with donor lymphocyte infusions. Adoptive immunotherapy with CTLs specific for the hematopoietic system-restricted mHags, however, offers the prospect of greater and more predictable effectiveness in the absence of graft-versus-host disease.
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http://dx.doi.org/10.1182/asheducation-2000.1.356DOI Listing
January 2000
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