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Biomedical subjects

Jan Cerny

Publications and source records attributed to Jan Cerny.

At least 19 recordsLinked to original sources
PubMed2007

Murine allogeneic in vivo stem cell homing(,).

Stem cell homing has been studied in syngeneic models and appears to be rapid (<1 h) and dependent on cellular adhesion and migration factors. We utilized a full H2-mismatched transplantation model to determine the basics of allogeneic homing. C57BL/6J Lin-Sca-1+ cells were labeled with CFSE and injected in non-myeloablated BALB/c mice. Fluorescent cell detection was via high-speed FACS analysis. Alternatively, B6.SJL whole bone marrow cells were injected in lethally irradiated BALB/c mice (10 Gy). One, 3, 6, and 24 h after transplant, marrow was harvested and cells were either plated for high proliferative potential colony-forming cell (HPP-CFC) assay or secondarily injected into myeloablated (8 Gy) C57BL/6J mice using 10% competing C57BL/6J marrow. Chimerism was evaluated at 8 weeks. CFSE+ cells were detected in the bone marrow 1, 3, and 6 h after injection. The numbers were moderately lower when compared to syngeneic homing possibly due to strain effect. Conversely, utilizing a surrogate or secondary assay, we observed a decline of secondary engraftment of harvested cells over time, but not of HPP-CFC. Combining experiments and normalizing the 1-h time point to 100% (to allow comparison), we observed a mean relative engraftment of 87 +/- 29%, 72 +/- 21%, 84 +/- 35% of the 1 h level at 3, 6, and 24 h respectively. HPP-CFC assay showed no significant variation as a homing surrogate over 1-6 h. These data indicate a rapid homing into allogeneic recipients with a plateau at 1 h. The decline of secondary engraftability over time may indicate a phenotype alteration of homed cells.

Animals↗
PubMed2006

Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis.

Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1(l)) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1(l)) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.

Animals↗
PubMed2006

Clinical response of myelodysplastic syndromes patients to treatment with coenzyme Q10.

The myelodysplastic syndromes (MDS) are a collection of hematopoietic disorders with varying degrees of mono- to trilineage cytopenias and bone marrow dysplasia. In recent years much progress has been made in the treatment of MDS and there are now several therapeutic compounds used with varying levels of success. These compounds typically cause side effects that make them unattractive for treatment of patients in the early stages of MDS. Naturally occurring compounds that are not toxic may provide a means to treat patients in the initial stages of disease. We conducted a pilot study to test the efficacy of coenzyme Q10 (coQ10) in MDS patients with low to intermediate-2 risk disease. A variety of responses were observed in 7 of 29 patients including two trilineage and two cytogenetic responses. Sequencing mitochondrial DNA (mtDNA) from pretreatment bone marrows showed multiple mutations, some resulting in amino acid changes, in 3/5 nonresponders, 1/4 responders and in two control samples. We conclude that coQ10 may be of clinical benefit in a subset of MDS patients, but responders cannot be easily pre-selected on the basis of either the conventional clinical and pathologic characteristics or mtDNA mutations.

Aged↗
PubMed2006

Molecular characterization of chronic lymphocytic leukemia with two distinct cell populations: Evidence for separate clonal origins.

We report a case of an elderly woman with persistent lymphocytosis in whom flow cytometric immunophenotyping revealed 2 distinct clonal B-cell populations with different light chain restrictions. One clone was CD19+ (dim), CD5+ (bright), CD23+ (moderate), and kappa+; the other clone was CD19+ (moderate), CD5+ (bright), CD23+ (dim), and lambda+. We separated the 2 clones by flow cytometry from peripheral blood lymphocytes for further molecular and cytogenetic analysis. Polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain (IgH) genes revealed that the clones had bands of slightly different size. The PCR products of the framework 3 (FR3) region were cloned, and sequence analysis confirmed that each population had distinct, clone-specific IgH gene rearrangements. Fluorescence in situ hybridization (FISH) analysis revealed trisomy 12 in the lambda-restricted B-cell clone, whereas the k-restricted population had normal FISH patterns. Our results demonstrated that the immunophenotypically different cell populations originated from 2 separate clones.

Aged, 80 and over↗
PubMed2006

Chronic myelomonocytic leukemia evolving from preexisting myelodysplasia shares many features with de novo disease.

The majority of chronic myelomonocytic leukemia (CMML) cases arise de novo; cases evolving from preexisting myelodysplasia (MDS) or myeloproliferative diseases have not been well-studied. We conducted the present study to determine the clinicopathologic features and to study possible underlying molecular and cytogenetic mechanisms involved in this evolution. Between April 1995 and November 2005, we identified 120 CMML cases, of which 20 (16.7%) had a previous diagnosis of MDS. Of the 20 patients with MDS, 6 had relative monocytosis at diagnosis. At the time of MDS to CMML evolution, mutations in JAK2 (V617F), FLT3 (ITD), K-ras-2, or N-ras were not acquired, and only 1 (6%) of 17 evaluable cases showed cytogenetic progression. The median time to evolution from MDS to CMML was 29 months, and the median survival following CMML development was 13 months. Three cases (17%) transformed to acute myeloid leukemia. These findings indicate that in some cases of otherwise typical MDS, the progenitor cells may have some capacity for monocytic proliferation at diagnosis and manifest rapid disease progression once a monocytic proliferation supervenes.

Aged↗
PubMed2005

Insertion of the dibasic motif in the flanking region of a cryptic self-determinant leads to activation of the epitope-specific T cells.

Efficient induction of self tolerance is critical for avoiding autoimmunity. The T cells specific for the well-processed and -presented (dominant) determinants of a native self protein are generally tolerized in the thymus, whereas those potentially directed against the inefficiently processed and presented (cryptic) self epitopes escape tolerance induction. We examined whether the crypticity of certain determinants of mouse lysozyme-M (ML-M) could be attributed to the nonavailability of a proteolytic site, and whether it could be reversed to immunodominance by engraftment of a novel cleavage site in the flanking region of the epitope. Using site-directed mutagenesis, we created the dibasic motif (RR or RK; R = arginine, K = lysine), a target of intracellular proteases, in the region adjoining one of the three cryptic epitopes (46-61, 66-79, or 105-119) of ML-M. Interestingly, the mutated lysozyme proteins, but not unmutated ML-M, were immunogenic in mice. The T cell response to the altered lysozyme was attributable to the efficient processing and presentation of the previously cryptic epitope, and this response was both epitope and MHC haplotype specific. In addition, the anti-self T cell response was associated with the generation of autoantibodies against self lysozyme. However, the results using one of three mutated lysozymes suggested that the naturally processed, dibasic motif-marked epitope may not always correspond precisely to the cryptic determinant within a synthetic peptide. This is the first report describing the circumvention of self tolerance owing to the targeted reversal of crypticity to dominance in vivo of a specific epitope within a native self Ag.

Amino Acid Motifs↗
PubMed2005

Identification of a ubiquitously active promoter of the murine activation-induced cytidine deaminase (AICDA) gene.

Somatic hypermutation and class switch recombination of immunoglobulin genes are dependent on the presence of the activation-induced cytidine deaminase (AICDA) enzyme. AICDA expression is restricted to activated B-lymphocytes in the germinal centers. It has been suggested that inappropriate expression of AICDA may lead to genome instability and aberrant affinity maturation of putative autoreactive antibodies. To better understand the molecular control of its tightly regulated expression we have identified the transcription initiation site and an upstream, conserved promoter region of the murine AICDA gene. The promoter lacks a consensus TATA box but contains an initiator (Inr) element and is active in several murine and human cell lines irrespective of endogenous AICDA expression. Mutagenesis analysis identified a functionally important Sp-binding site which binds both Sp1 and Sp3 in vitro in all cell types. Contrary to a recent report, no evidence was found for direct Pax5-binding at this DNA site. We discuss the role of ubiquitous and lymphoid-specific factors in the control of AICDA gene transcription.

Animals↗
PubMed2005

Daclizumab in the induction phase of immunosuppression in heart transplant recipients.

OBJECTIVES: Acute rejection episodes negatively effect both short-term survival and development of graft vasculopathy. METHODS: We assessed the incidence and severity of acute rejection episodes under a new protocol using a monoclonal antibody daclizumab in the induction phase of immunosuppressive therapy. The study group B (26 patients) was given daclizumab (1 mg/kg), the control group A (25 patients) was treated without daclizumab. There was no significant difference between the two groups in the important data. A total of first 10 EMB in every patient was classified according to the ISHLT recommendation. We also analysed the incidence of infections. RESULTS: During the first two months there was a significant lower incidence and/or severity of rejection episodes in the daclizumab group with p < or = 0.0016, 0.001 and 0.005 respectively. CONCLUSIONS: Induction therapy with daclizumab significantly reduces the incidence of acute rejection episodes during the first two posttransplant months in heart transplant recipients. It does not increase the risk of infection.

Adult↗
PubMed2004

Echo-guided endomyocardial biopsy in heart transplant recipients.

After heart transplantation the effect of immunosuppression is monitored by histopathology of endomyocardial biopsy (EMB). EMB is usually carried out under X-ray guidance. Between January 1998 and March 2003, 1,262 biopsies were collected under echo-guidance in 156 patients. The biopsy access was gained through the internal jugular vein, by the standard catheterization technique. The average time of the procedure was 17 min. Four or five specimens were obtained from each patient, with a success rate of 96%. Complications involved two episodes of partial pneumothorax in one patient, atrial flutter in another and ventricular fibrillation in three patients. Conversion from echo to X-ray guidance was indicated in 11 patients. No case of significant tricuspid regurgitation related to the EMB procedure was recorded. The echo-guided endomyocardial biopsy appears to be a prospective alternative to the conventional approach under X-ray guidance. Its duration is comparable, it eliminates X-ray exposure, enables continuous echocardiographic monitoring and can be performed at the bedside.

Adolescent↗
PubMed2004

Intrinsic hematopoietic stem cell/progenitor plasticity: Inversions.

Traditional concepts indicate that stem cells give rise to progenitor cells in a hierarchical system. We studied murine engraftable stem cells (ESCs) and progenitors in in vitro and found that ESC and progenitors exist in a reversible continuum, rather then a hierarchy. B6.SJL and BALB/c marrow cells were serially cultured with thrombopoietin (TPO), FLT-3 ligand (FLT-3L), and steel factor through cell cycle. Progenitors (high-proliferative potential colony-forming cells (HPP-CFC) and colony-forming unit culture (CFU-c)) and ESC capacity was determined. The cell cycle status of purified lineage(negative)rhodamine(low)Hoechst(low) stem cells was determined under the same conditions using tritiated thymidine incorporation and cell counts. We found an inverse relationship between progenitors and ESC, which occurred during the first cell cycle transit and was reversible. We have termed these progenitor/stem cell inversions and found that these inversions were consistently seen at 28-32 h of culture, representing early S-phase. We observed 13 major reversible increases in progenitor numbers from one time-point to another during the first cell cycle transit; this was coupled with 11 major ESC decreases and in 2 instances ESC were at baseline. These studies indicate that primitive marrow cells reversibly shift from ESC to progenitors without differentiation occurring. They exist as a fluctuating continuum.

Animals↗
PubMed2004

Biallelic IgH rearrangements in patients with indolent lymphoproliferative disorders: molecular and practical implications.

We report a group of patients (pts) with indolent lymphoproliferative disorder who had both alleles for the immunoglobulin heavy chain genes rearranged (biIgH). This group of 17 pts consisted of 9 small lymphocytic lymphomas (SLL) and 8 chronic lymphocytic leukemia (CLL). The polymerase chain reaction (PCR) amplification of clonal immunoglobulin heavy (IgH) rearrangement using the complementarity determining region III (CDRIII) constantly retrieved two distinct bands in all PCR informative samples of those pts. To rule out biclonality, we evaluated samples by fluorescein activated cell sorting (FACS) analysis and sequenced the PCR products. We were able to obtain both IgH sequences from 12 patients. FACS suggested biclonality in one case, which also correlated with sequencing results as both IgH rearrangements were in-frame. Recently, we reported a patient who sustained transformation into an aggressive disease after biIgH was detected in the setting of monoclonal disease (Cerny et al., 2003b, Haematologica 88(05):ECR15 B.). We decided to compare clinical characteristics and prognosis of 17 pts with biIgH and 37 pts with monoIgH rearrangements. Although we found some minor differences in disease characteristics between both groups, these did not translate into a significantly different overall survival. Our findings suggest that true biclonal cases of CLL are rare.

Adult↗
PubMed2004

Chromatin remodeling and stem cell theory of relativity.

The field of stem cell biology is currently being redefined. Stem cell (hematopoietic and non-hematopoietic) differentiation has been considered hierarchical in nature, but recent data suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. The stem cell (hematopoietic and non-hematopoietic) phenotype, the total differentiation capacity (hematopoietic and non-hematopoietic), gene expression as well as other stem cell functional characteristics (homing, receptor and adhesion molecule expression) vary throughout a cell-cycle transit widely. This seems to be dependent on shifting chromatin and gene expression with cell-cycle transit. The published data on DNA methylation, histone acetylation, and also RNAi, the major regulators of gene expression, conjoins very well and provides an explanation for the major issues of stem cell biology. Those features of stem cells mentioned above can be rather difficult to apprehend when a classical hierarchy biology view is applied, but they become clear and easier to understand once they are correlated with the underlining epigenetic changes. We are entering a new era of stem cell biology the era of "chromatinomics." We are one step closer to the practical use of cellular therapy for degenerative diseases.

Animals↗
PubMed2004

The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing.

Resealing after wounding, the process of repair following plasma membrane damage, requires exocytosis. Vacuolins are molecules that induce rapid formation of large, swollen structures derived from endosomes and lysosomes by homotypic fusion combined with uncontrolled fusion of the inner and limiting membranes of these organelles. Vacuolin-1, the most potent compound, blocks the Ca(2+)-dependent exocytosis of lysosomes induced by ionomycin or plasma membrane wounding, without affecting the process of resealing. In contrast, other cell structures and membrane trafficking functions including exocytosis of enlargeosomes are unaffected. Because cells heal normally in the presence of vacuolin-1, we suggest that lysosomes are dispensable for resealing.

Animals↗
PubMed2003

Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen.

Marginal zone (MZ) B cells play a major role in the first-line responses against blood-born T-independent bacterial antigens (TI), but the full scope of their immune functions is not known. Here we compare the responses of MZ and follicular (FO) B cells to a T-dependent antigen (TD), hapten-(4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gamma-globulin, in a cell transfer system. Consistent with the conventional paradigm, MZ B cells but not FO B cells rapidly generated the early burst of NP-specific antibody-forming cells (AFC), high levels of IgM Ab, and early IgG with relatively high affinity to NP. However, MZ B cells were also capable of forming germinal centers (GCs) albeit with a delay, compared with FO B cells. The early AFCs and the GCs originated from different MZ precursors, but the MZ- and FO-derived GCs were similar in VH gene repertoire, somatic mutation, and production of late AFC and IgG Ab. Surprisingly, the MZ but not the FO memory response included IgM Ab. We conclude that MZ B cells are heterogeneous, comprising cells for both early AFC response and GC/memory pathway against TD antigens.

Animals↗
PubMed2003

Requirements for T cell-polarized tubulation of class II+ compartments in dendritic cells.

Activation of naive CD4 T cells by dendritic cells requires the sequential interaction of many TCR molecules with peptide-class II complexes of the appropriate specificity. Such interaction results in morphological transformation of class II MHC-containing endosomal compartments. In this study, we analyze the requirements for long tubular endosomal structures that polarize toward T cell contact sites using dendritic cells from I-A(b) class II -enhanced green fluorescent protein knock-in mice and I-A(b)-restricted CD4 T cells specific for OVA. Clustering of membrane proteins and ligation of T cell adhesion molecules LFA-1 and CD2 are involved in induction of endosomal tubulation. Activation of T cells increases their ability to induce class II-enhanced green fluorescent protein-positive tubules in dendritic cells, in part through up-regulation of CD40 ligand. Remarkably, and in stark contrast with the result obtained with dendritic cells loaded with intact OVA, OVA peptide added to dendritic cells failed to evoke T cell-polarized endosomal tubulation even though both conditions allowed T cell stimulation. These results suggest the existence of microdomains on the membrane of dendritic cells that allow Ag-specific T cells to evoke tubulation in the dendritic cell.

Amino Acid Sequence↗
PubMed2003

T cells induce extended class II MHC compartments in dendritic cells in a Toll-like receptor-dependent manner.

Interaction of Ag-loaded dendritic cells with Ag-specific CD4 T cells induces the formation of long tubular class II MHC-positive compartments that polarize toward the T cell. We show involvement of a Toll-like receptor-mediated signal in this unusual form of intracellular class II MHC trafficking. First, wild-type dendritic cells loaded with LPS-free Ag failed to show formation of class II-positive tubules upon Ag-specific T cell engagement, but did so upon supplementation of the Ag with low concentrations of LPS. Second, Ag-loaded myeloid differentiation factor 88 -deficient dendritic cells failed to form these tubules upon interaction with T cells, regardless of the presence of LPS. Finally, inclusion of a cell-permeable peptide that blocks TNFR-associated factor 6 function, downstream of myeloid differentiation factor 88, blocked T cell-dependent tubulation. A Toll-like receptor-dependent signal is thus required to allow Ag-loaded dendritic cells to respond to T cell contact by formation of extended endosomal compartments. This activation does not result in massive translocation of class II MHC molecules to the cell surface.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

Antiviral Agents↗