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Holger Hebart

Publications and source records attributed to Holger Hebart.

At least 37 records · Page 2Linked to original sources
PubMed2004

Clinical aspects of CMV infection after stem cell transplantation.

Cytomegalovirus (CMV) infection is one of the most important infectious complications after stem cell transplantation (SCT). Major improvements in the management of CMV infection have been achieved during the last decade, including the introduction of safe blood product support for CMV-seronegative patients, the development of early pre-emptive antiviral therapy based on sensitive diagnostic tests, and antiviral prophylaxis. With the better control of CMV infection during the first 100 days after allogeneic SCT an increase in the incidence of CMV infection and disease after day 100 after transplantation was observed. New methods that allow for the reconstitution of CMV-specific immune responses such as adoptive T-cell therapy are promising tools that might help to improve the management of late CMV infection and disease.

Antiviral Agents↗
PubMed2004

Preliminary evaluation of one conventional nested and two real-time PCR assays for the detection of Toxoplasma gondii in immunocompromised patients.

Toxoplasma reactivation is a serious complication in patients receiving allogenic stem cell transplantation. Real-time PCR assays allow a rapid diagnosis of toxoplasma infection; however, no comparative data are available on the performance of real-time PCR protocols under routine conditions. Therefore, the aim of this study was to amplify Toxoplasma gondii DNA from routine samples of allogenic stem cell recipients using two real-time PCR assays on a LightCycler, and using conventional nested PCR. Conventional nested PCR revealed T. gondii DNA in 16 samples. Only 12 of the 16 samples yielded a positive result in both real-time PCRs. The accuracy of the conventional PCR results was demonstrated by direct sequencing. Amplification and detection of the amplicon was completed in only 1 h using the real-time PCR assays. Thus, real-time PCR substantially accelerates the detection of T. gondii DNA in the majority of positive specimens; however, conventional nested PCR is required for detection of T. gondii DNA in some samples.

Animals↗
PubMed2004

Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4.

Invasive aspergillosis (IA) is a leading cause of mortality in haematological patients. Appropriate activation of the innate immune system is crucial for the successful clearance of IA. Therefore, we studied the Aspergillus fumigatus-mediated activation of human granulocytes and monocyte-derived immature dendritic cells (DCs), as well as murine bone marrow-derived DCs (BMDCs) from wild type, toll-like receptor (TLR)4-deficient, TLR2 knockout, and TLR2/TLR4 double deficient mice. Aspergillus fumigatus antigens induced the activation and maturation of immature DCs as characterized by CD83 expression, upregulation of major histocompatibility complex and co-stimulatory molecules. Moreover, fungal antigens enhanced the phagocytosis and production of interleukin (IL)-8 in granulocytes. The release of IL-12 by BMDCs in response to A. fumigatus antigens was dependent on the expression of TLR2, whereas the release of IL-6 was dependent on the expression of functional TLR4 molecules. The protein precipitate of A. fumigatus supernatant provided strong stimulation of DCs and granulocytes, indicating that a factor secreted by A. fumigatus might activate innate immune cells. In conclusion, A. fumigatus antigens induced the activation of DCs and granulocytes. Our results indicated that this activation was mediated via TLR2 and TLR4. Future studies are needed to assess the clinical impact of these findings in patients at high risk for IA.

Animals↗
PubMed2003

Infectious complications after allogeneic stem cell transplantation: epidemiology and interventional therapy strategies--guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO).

The risk of infection after allogeneic stem cell transplantation is determined by the underlying disease, the intensity of previous treatments and complications that may have occurred during that time, but above all, the risk of infection is determined by the selected transplantation modality (e.g. HLA-match between the stem cell donor and recipient, T cell depletion of the graft, and others). In comparison with patients treated with high-dose chemotherapy and autologous stem cell transplantation, patients undergoing allogeneic stem cell transplantation are at a much higher risk of infection even after hematopoietic reconstitution, due to the delayed recovery of T and B cell functions. The rate at which immune function recovers after hematopoietic reconstitution greatly influences the incidence and type of post-transplant infectious complications. Infection-associated mortality, for example, is significantly higher following engraftment than during the short neutropenic period that immediately follows transplantation.

Humans↗
PubMed2003

Sepsis in neutropenia--guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO).

Patients developing fever in neutropenia are at high risk of infection-related complications. Their outcome is influenced by the degree of severity (sepsis, severe sepsis and septic shock). Sepsis describes clinical syndromes resulting from systemic inflammatory response. Diagnosis of sepsis is based on simple clinical criteria. Treatment of neutropenic patients with sepsis does not differ from sepsis treatment in non-neutropenic patients. A variety of treatment options have failed (e.g. anti-cytokine strategies, anti-endotoxin antibodies), however, in recent years successful targeted treatment, the use of activated protein C or the substitution of hydrocortisol has been shown to reduce mortality rates. The outcome of neutropenic sepsis is influenced by the underlying disease as well, however survival rates of neutropenic patients treated on the intensive care unit have improved during the past decade. This paper focuses on pathophysiology, diagnosis and treatment of sepsis. Evidence based medicine (EBM) criteria are used to grade treatment recommendations [50].

Humans↗
PubMed2003

Specific infectious complications after stem cell transplantation.

Infectious complications continue to be a leading cause of morbidity and mortality after stem cell transplantation (SCT). The most important determinants for risk and outcomes are the stem cell source and the time after transplantation that the infection occurs. More recent developments in preparative regimens and graft manipulations, as well as the control of some important infectious complications after SCT by the introduction of effective prophylaxis and preemptive treatment strategies, have had a great impact on the timing and the epidemiology of infections posttransplantation. Better control of infectious complications posttransplantation has resulted in a reduced transplant-related mortality.

Cytomegalovirus Infections↗
PubMed2003

A CTL epitope from human cytomegalovirus IE1 defined by combining prediction of HLA binding and proteasomal processing is the target of dominant immune responses in patients after allogeneic stem cell transplantation.

OBJECTIVE AND METHODS: In an attempt to define HCMV IE1-derived, HLA-A(*)0201-restricted epitopes, an advanced computer-based epitope prediction combining HLA binding and proteasomal cleavages in silico was performed. RESULTS: This prediction algorithm clearly confirmed VLEETSVML to be the most likely CTL epitope. By tetramer staining, HCMV pp65 NLVPMVATV-specific CD8(+) T cells were detectable in 18/24 HCMV seropositive HLA-A(*)0201-expressing individuals (median frequency 0.58%; range 0.1%-4.7%), and IE1 VLEETSVML-specific CD8(+) T cells in 5/24 (median frequency 2.1%; range 0.1%-4.3%), respectively (p<0.01). Also in recipients of an allogeneic SCT, VLEETSVML- and NLVPMVATV-specific CD8(+) T cells were detectable in comparable frequencies, but again the number of patients with detectable pp65-specific CD8(+) T cells was higher (p=0.014). In 4/15 individuals, all demonstrating IE1 VLEETSVML-specific CD8(+) T cells prior to peptide stimulation, VLEETSVML-specific T cell lines (purity of 42.6%-98.6% of all CD3(+)/CD8(+) T cells) were successfully generated after 2-4 weeks of culture using the IFN-gamma secretion assay. CONCLUSION: In conclusion, this novel prediction strategy efficiently predicted an immunodominant viral T-cell epitope.

Adult↗
PubMed2003

Development and evaluation of the nuclisens basic kit NASBA for the detection of RNA from Candida species frequently resistant to antifungal drugs.

We describe a Nucleic Acid Sequence Based Amplification (NASBA) protocol to detect 6 different Candida species (Candida krusei, Candida glabrata, Candida inconspicua, Candida dubliniensis, Candida norvegensis, Candida lusitaniae) and compare it to a PCR assay. NASBA showed a sensitivity of 1 Colony Forming Unit and detected RNA from all 6 Candida species within 1 working day. All 5 patients with documented candidiasis showed identical results by both methods. This assay offers a sensitive, specific and fast possibility to detect yeast RNA.

Antifungal Agents↗
PubMed2003

Follow-up of patients with progressive multiple myeloma undergoing allografts after reduced-intensity conditioning.

Allogeneic stem cell transplantation (allo-SCT) after reduced-intensity conditioning was evaluated in 22 patients (median age 53, range 36-66 years) with multiple myeloma with progression after an autologous SCT. Seven patients received a transplant from a human leucocyte antigen (HLA)-identical sibling and 15 patients (68%) from an unrelated donor [including 3/22 (14%) from a HLA-mismatched unrelated donor]. Graft-versus-host disease (GVHD) prophylaxis consisted of serotherapy with antithymocyte globulin (ATG) and cyclosporine (CSA) (n = 12) or CSA plus mycophenolate mofetil (n = 10). Despite of heavy pretreatment, the transplant-related mortality (TRM) for all grafted patients was acceptable at 5/22 patients (23%). Seven of 21 patients (33%) that were evaluated developed grade II GVHD and one (5%) patient developed grade III/IV acute GVHD. Seven patients developed chronic GVHD (cGVHD), but only one was extensive. Eleven patients died of progressive disease within a median of 7 months (2-19 months) post transplant. Thirteen of all 22 patients (59%) achieved a partial or complete remission with six of these 13 patients (46%) remaining event free at a median of 24 months (range 8-36 months) post allografting. Estimated 2 year overall and event-free survival was, respectively, 25.5% and 22.0% for the whole patient group, and 62.5% and 57.1% for patients with chemosensitive disease. Chemorefractory disease prior to allogeneic stem cell transplantation (P = 0.0182) and absence of cGVHD (P = 0.069) were associated with shorter event-free survival. Thus long-term disease control can be achieved, but is restricted to patients responding to prior salvage chemotherapy.

Acute Disease↗
PubMed2002

Quantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system.

T-cell receptor excision circles (TRECs) are circular, stable extrachromosomal DNA fragments and are generated during V(D)J gene recombination, a process responsible for diversity of the T-cell receptor repertoire. Here we describe a sensitive, rapid and easy to perform real-time PCR assay based on the LightCycler technique for the quantification of TRECs among peripheral blood cells and a comparison of this assay with conventional PCR-ELISA. By LightCycler, a sensitivity of 20 copies of plasmid deltaRec-psiJalpha Signal Joint TREC DNA was achieved whereas by PCR-ELISA, a detection limit of 2 copies was demonstrated. In blood samples from healthy individuals (n=52) a median TREC count of 1.6x10(4) copies [range 2x10(1) to 2x10(5)]/2x10(5) peripheral blood mononuclear cells (PBMNCs) and in cord blood, a median TREC count of 1.45x10(5) copies [range 1.2x10(5) to 1.6x10(5)]/2x10(5) PBMNCs) could be detected. No significant difference was found in 15 individuals when unfractionated PBMNCs (median count of 1.1x10(5) copies/2x10(5) PBMNCs) or magnetic associated cell sorter (MACS)-sorted CD45RA+ T-cells (median count of 2.5x10(5) copies/2x10(5) cells) were analyzed for TREC counts. In addition, we examined the number of deltaRec-psiJalpha Signal Joint TREC in paediatric (n=6) and adult patients (n=7) after allogeneic stem cell transplantation. In children, we observed a median TREC count of 5.7x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 5.6x10(4) copies/2x10(5) PBMNCs after 2 years, and in adults, a median count of 3.6x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 1.1x10(4) copies/2x10(5) PBMNCs after 2 years. In conclusion, the LightCycler-based real-time PCR assay described offers a very sensitive and rapid tool for the quantification of TREC DNA.

Adolescent↗
PubMed2002

Analysis of T-cell responses to Aspergillus fumigatus antigens in healthy individuals and patients with hematologic malignancies.

Invasive aspergillosis has become a major cause of infection-related mortality in nonneutropenic patients after allogeneic stem cell transplantation (SCT). To assess the potential role of Aspergillus-specific T-cell responses for the successful control of invasive aspergillosis, lymphoproliferative responses to Aspergillus fumigatus antigens were studied in healthy individuals, patients with evidence of invasive aspergillosis, and patients late after allogeneic SCT. In healthy individuals, a positive lymphoproliferative response was documented to cellular extracts of A fumigatus (14 of 16), the 88-kDa dipeptidylpeptidase (4 of 16), and the 90-kDa catalase (8 of 11). A predominant release of interferon gamma (IFN-gamma) in culture supernatants on stimulation with A fumigatus antigens was demonstrated in 13 of 17 healthy individuals, indicating a T(H)1 response. In patients with clinical evidence of invasive aspergillosis, a favorable response to antifungal therapy was found to correlate with a higher IFN-gamma/interleukin 10 (IL-10) ratio in culture supernatants (n = 7; median ratio, IFN-gamma/IL-10 = 1.0; range, 0.09-24.8) compared to 10 patients with progressive or stable disease (median ratio, IFN-gamma/IL-10 = 0.1; range, 0.002-2.1; P =.04). Steroid treatment was found to suppress Aspergillus-specific lymphoproliferation (P =.037) and release of IFN-gamma in culture supernatants (P =.017). In contrast to cytomegalovirus- and tetanus toxoid-specific T-cell responses, Aspergillus-specific T-cell reconstitution late after allogeneic SCT was characterized by low stimulation indices and a low IFN-gamma/IL-10 ratio. In addition, phosphoantigen-reactive V(gamma)9/V(delta)2 T-cell clones from healthy individuals were found to produce significant amounts of tumor necrosis factor in response to A fumigatus antigens. In conclusion, these results further support the hypothesis that T cells contribute to the host defense against A fumigatus.

Adolescent↗
PubMed2002

Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.

We adoptively transferred donor-derived cytomegalovirus (CMV)-specific T-cell lines into 8 stem cell transplant recipients lacking CMV-specific T-cell proliferation. All patients, of whom one was infected by a CMV strain that was genotypically ganciclovir resistant, had received unsuccessful antiviral chemotherapy for more than 4 weeks. CMV-specific lines had been prepared by repetitive stimulation with CMV antigen, which increased the percentage of CMV-specific T cells and ablated alloreactivity completely even against patients mismatched for 1 to 3 HLA antigens. After transfer of 10(7) T cells/m(2) at a median of 120 days (range, 79-479 days) after transplantation, no side effects were noticed. Despite cessation of antiviral chemotherapy, the CMV load dropped significantly in all 7 evaluable patients, with a maximal reduction after a median of 20 days (range, 5-31 days). In 2 patients with high virus load, the antiviral effect was only transient. One of these patients received a second T-cell infusion, which cleared the virus completely. At a median of 11 days after transfer, CMV-specific T-cell proliferation was demonstrated in 6 patients, and an increase in CMV-specific CD4(+) T cells was demonstrated in 5 patients. In 6 patients, 1.12 to 41 CMV-specific CD8(+) T cells/microL blood were detected at a median of 13 days after transfer, with an increase in all patients lacking CMV-specific CD8(+) T cells prior to transfer. Hence, anti-CMV cellular therapy was successful in 5 of 7 patients, whereas in 2 of 7 patients, who received an intensified immune suppression at the time of or after T-cell therapy, only transient reductions in virus load were obtained.

Adoptive Transfer↗
PubMed2002

Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses by interferon-gamma-enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation.

Reconstitution of human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs), predominantly directed against pp65, provides protective immunity for the development of HCMV disease after allogeneic stem cell transplantation (SCT). To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-gamma release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8(+) T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide-specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen-specific CD4(+) T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35->180 days). Peak counts of peptide-specific CD8(+) T cells ranged from 0.14 to 60.6 cells/microL; those of protein-specific CD4(+) T cells ranged from 0.64 to 18.97 cells/microL. Reconstitution of HCMV-peptide-specific CD8(+) T cells and protein-specific CD4(+) T cells was associated with clearance of HCMV infection (r(2) = 0.89, P <.0001 and r(2) = 0.61, P =.0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein-specific T cells at 3 months after SCT. In conclusion, prospective monitoring of HCMV-specific CD4(+) and CD8(+) T-cell reconstitution can be performed rapidly by using flow cytometry after specific stimulation with HCMV peptides and proteins and might help to further improve clinical management of HCMV infection after allogeneic SCT.

Adult↗
PubMed2002

Polymerase chain reaction detection of aspergillus DNA in experimental models of invasive aspergillosis.

To determine the sensitivity of polymerase chain reaction (PCR) assays for the diagnosis of invasive aspergillosis, results of quantitative culture, PCR-ELISA, and a quantitative LightCycler assay (Roche Diagnostics) of blood and organ specimens of experimentally infected mice and rabbits were compared. By PCR-ELISA, 297 of 379 murine lung specimens were positive, but only 235 of 379 were culture positive. Whereas 64 culture-negative lungs were positive by PCR, Aspergillus was grown from only 2 PCR-negative samples. The PCR assay was 19.4 times more sensitive than culture. None of the 68 blood cultures from mice and rabbits were positive for Aspergillus fumigatus, whereas PCR detected Aspergillus DNA in 17 of 68 blood samples. Quantitative PCR analysis of blood samples showed a fungus load of 10(1)-10(2) cfu/mL of blood. The data confirm the superior sensitivity of PCR for the diagnosis of experimental Aspergillus infections.

Animals↗
PubMed2002

Isolation and expansion of human cytomegalovirus- specific cytotoxic T lymphocytes using interferon-gamma secretion assay.

OBJECTIVE: The aim of this study was to isolate and expand donor-derived human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs) for adoptive transfer of 10(7) cells per m(2) of body surface area to restore protective immunity after stem cell transplantation. MATERIALS AND METHODS: A new strategy to generate HCMV-specific CTLs using the interferon-gamma (IFN-gamma) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 and feeder cell stimulation, is described. RESULTS: From 1 to 5 x 10(4) HCMV peptide-specific T lymphocytes (greater than 90% CD3(+)CD8(+)) were isolated from 1 to 2 x 10(8) peripheral blood mononuclear cells comparable to 50 to 100 mL of blood from HLA-A*0201 HCMV seropositive blood donors (n = 14) and expanded ex vivo after a median of 16 days (range 8-28 days; n = 13) to greater than 10(7)/m(2) HCMV peptide-specific CTLs using autologous (n = 2) or allogeneic (n = 11) feeder cell stimulation. In three experiments, expansion to 6 weeks was performed, achieving a median of 1.6 x 10(9) cells (range 6.1 x 10(8)-3.3 x 10(9)). Characterization of these HCMV-specific CTL lines revealed an average purity of 89.2% (range 66.2-99.3%) using HCMV pp65 peptide HLA-A*0201 tetramer staining (n = 14) and 89.4% (range 64.4-99.5%) by peptide-specific IFN-gamma secretion (n = 7). A median of 82.6% (range 76.0-88.0%) showed perforin secretion (n = 3) and 57.5% (range 22.2-80.7%) specific lysis of peptide-pulsed T2 cells (n = 5). A median of 52.2% (range 35.2-7.3%) revealed specific killing of HCMV-infected autologous, but not allogeneic, fibroblasts (n = 6). CONCLUSIONS: IFN-gamma secretion assay allows development of a simple and rapid protocol with short expansion times for generation of greater than 10(7)/m(2) HCMV-specific CTLs for adoptive immunotherapy.

Adoptive Transfer↗
PubMed2002

Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cells.

The hypothesis that productive infection of monocyte-derived immature dendritic cells (DCs) by the human cytomegalovirus (HCMV) is associated with decreased immunostimulatory capacity was tested in this study. DCs were infected with 60-80% efficiency by HCMV strain TB40/E. Infected versus uninfected cells were analysed by fluorescence-activated cell sorting and by immunocytochemistry for surface expression of major histocompatibility complex (MHC) and co-stimulatory molecules as well as cytokine secretion during the 3 d after infection. The immunostimulatory capacity of these cells was measured by mixed leucocyte reaction. In spite of the fact that HCMV infection of DCs induced an increased release of tumour necrosis factor-alpha (TNF-alpha) and a decreased interleukin 10 (IL-10) production, expression of MHC class I and II, as well as CD40 and CD80 molecules, were downregulated on infected DCs. The mixed leucocyte reaction showed significantly reduced immunostimulatory capacity of infected DC cultures. Simultaneous detection of MHC antigens and virus antigens by double immunofluorescence revealed that downregulation occurred only on infected cells, but not on uninfected bystander cells. These findings demonstrate on a single cell level, together with the marked downregulation of MHC and co-stimulatory molecules in the presence of high TNF-alpha and low IL-10 levels, a direct inhibitory effect of HCMV on antigen presentation by immature DCs independent of soluble mediators.

Antigen Presentation↗