bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity of Sibship Determination Using the ABI Identifiler Multiplex System
Authors: Reid TM , Wolf CA , Kraemer CM , Lee SC , Baird ML , Lee RF
Published: August 2004

Fifty known siblings and fifty unrelated pairs were genotyped using the ABI Identifiler STR system and sibship indices computed for each pair. Combined sibship indices (CSIs) for the known siblings ranged from less than 10 to greater than 1 billion. CSIs for the unrelated pairs ranged from 4.5 x 10-8 to 0.12. In the known sibling group the percentage of loci where both alleles matched was approximately 40%, while the percentage of loci where neither matched was approximately 10%. In the non-sibling group, the percentage of loci where both alleles matched was approximately 6%, while the percentage of loci where neither matched was approximately 45%. Interestingly, the percentage of loci where a single allele matched was the same in both the known siblings and unrelated pairs, approximately 50%.
A Mobile System for Postmortem Genital Examinations with Colposcopy: SART-TO-GO
Authors: Crowley SR
Published: August 2004

The interpretation of genital findings in the deceased is a vital and timely issue. A paucity of information exists on the nature and appearance of the anogenital tissues in the postmortem interval. The traditional postmortem genital examination consists of gross visualization, which may preclude detection of the subtler trauma that usually constitutes injury in sexual assault. The mobile system grew out of a need to bring the examiner to the patient, e.g., in jurisdictions that lack a centralized morgue. The theoretical framework of the methodology lies in the sexual activity that culminates in the death of the sexual homicide victim. This sequential methodology was based on the Sexual Assault Response Team (SART) model, with adaptation to the autopsy milieu. Colposcopy is well-established for the medical-legal investigation of living sexual assault victims. During the author's initial rape-homicide examinations, only gross visualization was available. It soon became apparent that a system that facilitated detailed scrutiny of the anogenital tissues at various postmortem intervals was needed. Colposcopy was selected as the examination method because of its magnification, photodocumentation, and peer review potential. The sequential protocol was developed and refined during an ongoing accumulation of baseline cases. The role of the forensic nurse examiner is described within a defined scope of expertise and as a collaborative member of the homicide investigative team.
Identification of Firearms Holders by the [Fe(PDT)3]+2 complex. Quantitative Determination of Iron Transfer to the Hand and Its Dependence on Palmar Moisture Levels
Authors: Avissar YY , Sagiv AE , Mandler D , Almog J
Published: August 2004

Quantitative data are reported for the first time on the amounts of iron, which are transferred to the hand upon holding a firearm. Iron levels between 21–315 ng/cm2 were found on volunteers' palms after a single holding of a handgun. Determination of the iron traces was accomplished spectrophotometrically using 3-(2-pyridyl)-5, 6-diphenyl-1,2,4-triazine (PDT) as a complexing agent. The transfer of iron from firearms to the palm was found to be, by and large a chemical (dissolution) rather than mechanical dislodgement. The prime factor that determines the amount of iron transferred from the firearm to the hand is the moisture level on the palm. These findings are likely to be of importance in courts of law, as well as in the war against terrorism and serious crime.
Understanding Rib Fracture Patterns: Incomplete and Buckle Fractures
Authors: Love JC , Symes SA
Published: August 2004

Reconstructing traumatic thoracic events, especially when soft tissues are absent, requires an advanced understanding of ribcage fracture patterns. The morphology and orientation of ribs complicate the fracture pattern, as a single blow often causes multiple fractures at various locations. Furthermore, fracture types observed in ribs are not explained easily by current bone biomechanic literature. Using evidential skeletal material archived at the Regional Forensic Center, Memphis, the ribs of 43 blunt force trauma cases were analyzed. A total of 195 incomplete fractures and 63 buckle fractures were noted. Incomplete fractures, previously thought to be common in children but rare in adults, were found among individuals ranging in age from 21–76 years. A buckle fracture, failure resulting from compressive instability, has been undefined previously in bone trauma literature but was repeatedly observed in this sample. This study elucidates recognizable rib fracture patterns while emphasizing gross bone examination for force and mechanical factors.
Population Genetics for Three STR Loci in the Population from Southern China
Authors: Ye Q S , Tang J P
Published: August 2004

A total of 111 unrelated individuals from the Han ethnic group of Southern China (Nanning City, Guangxi, China).
Review of: Color Atlas of the Autopsy
Authors: Davis G G
Published: August 2004

Dr. Wagner wrote his Color Atlas of the Autopsy for crime scene investigators, law enforcement officials, and other professionals who interact with forensic pathologists during the course of an autopsy. Individuals who lack formal medical training have many questions upon seeing their first autopsy, and Color Atlas of the Autopsy attempts to answer these questions by capturing the process of autopsy examination as it unfolds.
Thrombopoietin and Chemokine mRNA Expression in Patient Post-Chemotherapy and In Vitro Cytokine-Treated Marrow Stromal Cell Layers
Authors: Schwartz Gretchen N. , Kammula Udai , Warren M. Kim , Park Matthew K. , Yan Xiao-Yi , Marincola Francesco M. , Gress Ronald E.
Published: August 2004

CD34+ cells and megakaryocyte progenitors were lower in marrow from patients after hematological recovery from the first cycle of 5-fluorouracil, leucovorin, adriamycin, cyclophosphamide (FLAC) chemotherapy plus PIXY321 (GM-CSF/interleukin 3; IL-3 hybrid) than in FLAC + GM-CSF or pre-FLAC marrows. Marrow stromal layers, an in vitro model of the marrow microenvironment, express a combination of stimulatory and inhibitory factors that modulate hematopoietic progenitor cell proliferation and differentiation. The TaqMan assay and quantitative reverse transcriptase-polymerase chain reaction were used to measure monocyte chemoattractant protein-1 (MCP-1), melanoma stimulatory growth activity, and monokine inducible by interferon-γ (Mig) (inhibitory chemokines for primitive or megakaryocyte progenitors) mRNA levels in in vitro PIXY and GM-CSF-treated and patient post-FLAC marrow stromal layers. Chemokine mRNA was increased after in vitro GM-CSF and to a lesser extent after PIXY treatment. MCP-1 mRNA levels were fivefold higher in FLAC + PIXY than in FLAC + GM-CSF layers, and Mig mRNA was elevated in FLAC + GM-CSF layers. Thrombopoietin (TPO), insulin-like growth factor I (IGF-I), and IGF-II (stimulatory factors for primitive and megakaryocyte progenitors) mRNA were also measured. TPO mRNA levels were 30% lower in GM-CSF and PIXY-pretreated than in control layers with no decrease in IGF mRNA. TPO mRNA in stromal layers of patients who developed grade 3 thrombocytopenia (platelets < 20 × 109/l) during the third cycle of FLAC was only 24% of levels in stromal layers of marrow from other post-FLAC patients. Results demonstrate that patient and in vitro treatment had modulatory effects on TPO and chemokine mRNA expression in marrow stromal layers.
In Vivo Demethylation of a MoMuLV Retroviral Vector Expressing the Herpes Simplex Thymidine Kinase Suicide Gene by 5′ Azacytidine
Authors: Di Ianni Mauro , Terenzi Adelmo , Di Florio Sabrina , Venditti Gigliola , Benedetti Roberta , Santucci Antonella , Bartoli Andrea , Fettucciari Katia , Marconi Pierfrancesco , Rossi Ruggero , Martelli Massimo F. , Tabilio Antonio
Published: August 2004

We constructed a functional MoMuLV-based bicistronic retroviral vector encoding the herpes simplex virus type I thymidine kinase gene, which induces sensitivity to the prodrug ganciclovir (gcv), and the reporter β-galactosidase gene (MFG-tk-IRES-lacZ). The U937 histiocytic cell line was transduced with this vector, and a clone (VB71) with high-level transgene expression was selected. Severe combined immunodeficient (SCID) mice were injected with VB71 cells to evaluate the role of long terminal repeat methylation in transgene silencing in vivo and to see whether 5-azacytidine (5′ aza-C) demethylating agent prevented it.

We found 5′ aza-C maintained gene expression at high level in vitro. In vivo, time to tumor onset was significantly longer in SCID mice receiving the VB71 cells, 5′ aza-C, and gcv compared with animals treated with either 5′ aza-C or gcv alone. The number of injected tumor cells influences tumor onset time and the efficacy of 5′ aza-C and gcv treatment. The standard gcv treatment schedule (10 mg/kg from d + 1 until the onset of tumor) controlled tumor onset better than short-term treatment with high doses. In conclusion, the results extend our previous findings that transgene methylation in vivo may be prevented with an appropriate schedule of 5′ aza-C and gcv.
Plasma Levels of SDF-1 and Expression of SDF-1 Receptor on CD34+ Cells in Mobilized Peripheral Blood of Non-Hodgkin's Lymphoma Patients
Authors: Gazitt Yair , Liu Qun
Published: August 2004

CXCR4 is the receptor for the chemokine stromal derived factor-1 (SDF-1), is expressed on CD34+ cells, and has been implicated in the process of CD34+ cell migration and homing. We studied the mobilization of CD34/CXCR4 cells and the plasma levels of SDF-1 and flt3-ligand (flt3-L) in 36 non-Hodgkin's lymphoma patients receiving cyclophosphamide (Cy) plus G-CSF (arm A), Cy plus GM-CSF (arm B), or Cy plus GM-CSF followed by G-CSF (arm C) for peripheral blood stem cell (PBSC) mobilization and autotransplantation.

We observed lower plasma levels of SDF-1 in PBSCs compared to premobilization bone marrow samples. The mean plasma SDF-1 levels were similar in PBSC collections in the three arms of the study. In contrast, SDF-1 levels in the apheresis collections of the “good mobilizers” (patients who collected a minimum of 2 × 106 CD34+ cells/kg in one to four PBSC collections) were significantly lower than the apheresis collections of the “poor mobilizers” (≥0.4 × 106 CD34+ cells/kg in the first two PBSC collections; 288 ± 82 pg/ml versus 583 ± 217 pg/ml; p = 0.0009). The mean percentage of CD34+ cells expressing CXCR4 in the apheresis collections was decreased in the PBSC collections compared with premobilization values from 28% to 19.4%. Furthermore, the percentage of CD34+ cells expressing CXCR4 in the good mobilizers was significantly lower compared with the poor mobilizers (14.7 ± 2.1% versus 33.6 ± 2.1%; p = 0.002). The good mobilizers had also significantly lower levels of flt3-L compared with the poor mobilizers (34 ± 4 pg/ml versus 106 ± 11 pg/ml; p = 0.006), Finally, the levels of flt3-L strongly correlated with SDF-1 levels (r = 0.8; p < 0.0001). We conclude: A) low plasma levels of SDF-1 and low expression of CXCR4 characterize patients with good mobilization outcome, and B) the levels of SDF-1 correlate with flt3-L, suggesting an association of these cytokines in mobilization of CD34+ cells.
Successful Allogeneic Bone Marrow Transplantation (BMT) by Injection of Bone Marrow Cells via Portal Vein: Stromal Cells as BMT-Facilitating Cells
Authors: Fan Tian-Xue , Hisha Hiroko , Jin Tie-Nan , Yu Cheng-Ze , Lian Zhe-Xiong , Guo Shu-Bin , Cui Yun-Ze , Feng Biao , Yang Guo-Xiang , Li Qing , Ikehara Susumu
Published: August 2004

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day-14 colony-forming unit-spleen (CFU-S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU-S formation nor the hemopoietic colonies in the recipient liver were observed.

These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.
Stem Cell Dose and Tumorbiologic Parameters as Prognostic Markers for Patients with Metastatic Breast Cancer Undergoing High-Dose Chemotherapy with Autologous Blood Stem Cell Support
Authors: Hensel Manfred , Schneeweiss Andreas , Sinn Hans-Peter , Egerer Gerlinde , Kornacker Martin , Solomayer Erich , Haas Rainer , Bastert Gunther , Ho Anthony D.
Published: August 2004

We report on the prognostic significance of tumorbiologic parameters and CD34+ cell dose in 120 patients with metastatic breast cancer (MBC) who received high-dose chemotherapy (HDCT) with autologous blood stem cell transplantation as first-line treatment. Her2/neu, p53, Ki67, and bcl-2 protein expression were studied using immunohistochemical staining on formalin-fixed, paraffin-embedded primary tumor sections. DNA content of tumor cells (DNA-index) and tumor cell proliferation (S-phase fraction) were measured by DNA flow cytometry. The relationship between these parameters and the CD34+ cell dose and progression free (PFS) and overall survival (OS) was analyzed.

With a median follow-up period of 40 months (range, 7-89 months), no more than two metastatic sites (relative risk [RR] = 3.84 [95% confidence interval (CI) 1.49-10]; p =.005) and hyperploidy (RR = 2.58 [95% CI 1.26-5.26]; p =.009) were independent predictors of longer PFS according to multivariate analysis. Independent prognostic factors of longer OS included one or two metastatic sites (RR = 4.16 [95% CI 1.96-4.16]; p <.001), a positive combined hormone receptor status (RR = 2.45 [95% CI 1.45-4.14]; p =.001) and a high number of infused stem cells (>7.8 × 106 CD34+ cells per kg body weight) (RR = 2.0 [95% CI 1.17-3.42]; p =.01).

In conclusion, positive hormone receptors, ≤2 metastatic sites, high DNA-index and high CD34+ cell dose (>7.8 × 106 CD34+ cells per kg) are predictors for a favorable outcome after autotransplantation for MBC. Our observation might indicate a favorable effect of HDCT in MBC patients with overexpression of Her2/neu who might have a worse prognosis when treated with conventional chemotherapy.
In Vitro Functional Gut-Like Organ Formation from Mouse Embryonic Stem Cells
Authors: Yamada Takatsugu , Yoshikawa Masahide , Takaki Miyako , Torihashi Shigeko , Kato Yoko , Nakajima Yoshiyuki , Ishizaka Shigeaki , Tsunoda Yukio
Published: August 2004

Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found that ES cells can give rise to a functional gut-like unit, which forms a three-dimensional dome-like structure with lumen and exhibits mechanical activity, such as spontaneous contraction and peristalsis. The aim of the present study was to investigate the electrophysiological and morphological properties of ES cell-derived contracting clusters.

Methods. Electrical activity was examined by an extracellular recording. Morphology and cellular components were investigated by immunohistochemistry and electron microscopy.

Results. Clusters with rhythmic contractions displayed electrical slow waves at a regular rhythm, and clusters with highly coordinated peristalsis showed regular slow waves and spontaneous spike action potentials. Immunoreactivity for c-Kit, a marker of interstitial cells of Cajal (ICC), was observed in dense network structures. Neuronal marker PGP9.5 immunoreactivity was observed only in clusters with peristalsis. The topographical structure of the wall was organized by an inner epithelial layer and outer smooth muscle layer. The smooth muscle layer was provided with an ICC network and innervated with enteric neurons.

Conclusions. ES cells can differentiate into a functional gut-like organ in vitro that exhibits physiological and morphological properties characteristic of the gastrointestinal (GI) tract. This ES cell-derived gut provides a powerful tool for studying GI motility and gut development in vitro, and has potential for elucidating and treating a variety of motility disorders.
Gene Expression Profile of Megakaryocytes from Human Cord Blood CD34+ Cells Ex Vivo Expanded by Thrombopoietin
Authors: Kim Jeong-Ah , Jung Yu-Jin , Seoh Ju-Young , Woo So-Youn , Seo Jeong-Sun , Kim Hyung-Lae
Published: August 2004

Previously, we investigated the process of megakaryocytopoiesis during ex vivo expansion of human cord blood (CB) CD34+ cells using thrombopoietin (TPO) and found that megakaryocytopoiesis was closely associated with apoptosis. To understand megakaryocytopoiesis at the molecular level, we performed a microserial analysis of gene expression (microSAGE) in megakaryocytes (MKs) and nonmegakaryocytes (non-MKs) derived from human CB CD34+ cells by ex vivo expansion using TPO, and a total of 38,909 tags, representing 8,976 unique genes, were identified. In MKs, many of the known genes, including coagulation factor VII, P-selectin (CD62P), pim-1, azurocidin, defensin, and CD48 were highly expressed; meanwhile, those genes encoding some small G proteins of the Ras family (Rab 7 and Rab 11A) and glutathione S transferase family (1, 4, A2, omega, and pi) showed lower expression levels in MKs. These gene expression profiles will be useful to understand megakaryocytopoiesis at the molecular level, including apoptosis and related signal transduction pathways.
A Subset of ES-Cell-Derived Neural Cells Marked by Gene Targeting
Authors: Xian Hai-Qing , McNichols Elizabeth , St. Clair Andrew , Gottlieb David I.
Published: August 2004

Embryonic stem cells differentiate efficiently in culture into neural progenitors, neurons, oligodendrocytes, and astrocytes. An embryonic stem (ES) cell line with green fluorescent protein (GFP) inserted into the gene for Olig2, a lineage-specific transcription factor, permits visualization and physical separation of a subset of living ES-cell-derived neural cells. GFP-expressing cells have morphological and antigenic properties of the oligodendrocyte lineage. The differentiation of living GFP-expressing cells can be followed in cultures, and they can be separated by fluorescence-activated cell sorting and cultured as pure populations. This system will allow detailed biochemical and molecular analysis of a neural differentiation pathway at a level not previously feasible. The strategy may have general applicability, since other neural lineages can be marked in an analogous manner.
Colocalization Analysis of Sialomucins CD34 and CD164
Authors: McGuckin Colin P. , Forraz Nicolas , Baradez Marc-Olivier , Lojo-Rial Carlos , Wertheim David , Whiting Karen , Watt Suzanne M. , Pettengell Ruth
Published: August 2004

Flow cytometric protocols are employed to identify and characterize hemopoietic stem/progenitor populations before transplantation. Cell surface antigens, including CD34, are employed in this process and widely used in harvest protocols, which largely ignores the potential functional role of such antigens. Transmembrane glycoprotein sialomucins, including CD34 and CD164, have been implicated in cell-to-cell interactions and activation. CD164, also expressed on early hemopoietic populations, was reported to have a possible function facilitating CD34+ cells to adhere to bone marrow stroma. In this study, we employed high-definition laser-scanning confocal microscopy to investigate CD34 and CD164 surface co-localization patterns on bone marrow and cord blood cells and to compare the expression patterns using a three-dimensional computer-generated method developed in house. Differential interference microscopy analysis revealed bone marrow membrane activity was higher than the corresponding cord blood counterpart, perhaps indicating the marrow microenvironmental nature. Fluorescence analysis of CD34 and CD164 antigens showed both were expressed first in a halo-like pattern and second in antigen-dense pockets. Three-dimensional computer analyses further revealed that this pocketing corresponded to dense crest-like surface structures appearing to rise from the point of adherence on the slide. Further, it was found that CD34 and CD164 display strong colocalization patterns on cells expressing both antigens. The dual nature of the CD34 and CD164 antigens discovered here lends further evidence to the previous literature implicating a strong functional link between these two sialomucins, which should be considered in the transplantation arena and in the function of such sialomucins as negative regulators of cell proliferation.
Potential Use of Embryonic Stem Cells for the Treatment of Mouse Parkinsonian Models: Improved Behavior by Transplantation of In Vitro Differentiated Dopaminergic Neurons from Embryonic Stem Cells
Authors: Nishimura Fumihiko , Yoshikawa Masahide , Kanda Seiji , Nonaka Masahiro , Yokota Hiroshi , Shiroi Akira , Nakase Hiroyuki , Hirabayashi Hidehiro , Ouji Yukiteru , Birumachi Jun-Ichi , Ishizaka Shigeaki , Sakaki Toshisuke
Published: August 2004

Background and Aims. The purpose of the present study was to examine the efficacy of transplantation of mouse embryonic-stem-(ES)-cell-derived tyrosine hydroxylase-positive (TH+) cells into Parkinsonian mice using behavioral tests and immunohistochemical evaluation.

Methods. Undifferentiated ES cells carrying the enhanced green fluorescent protein (EGFP) gene were differentiated into a cell population containing TH+ neurons using a five-step in vitro differentiation method. These ES-cell-derived cells were used as allografts in Parkinsonian mice, made by administering injections of 6-hydroxydopamine (6-OHDA). Fifteen hemiparkinsonian mice were divided into three groups. Four weeks after 6-OHDA injection, mice in groups 1, 2, and 3 received phosphate-buffered saline, 1 × 104 graft cells, and 1 × 105 graft cells, respectively, into their dopamine-denervated striata.

Results. Improved rotational behavior was observed in the graft-transplanted groups (groups 2 and 3) 2 weeks after transplantation. Mice in group 2 displayed a continuous maintenance of reduced rotational behavior, while those in group 3 showed ipsilateral rotation toward the lesioned side at 4, 6, and 8 weeks after transplantation. Tumor formation was observed in one mouse in group 3. TH+ cells were found at the grafted sites 8 weeks after transplantation in mice in groups 2 and 3, some of which were immunopositive to GFP, demonstrating the presence of dopaminergic neurons derived from the ES cells.

Conclusion. Transplantation of in vitro differentiated ES cells changed rotational behavior in Parkinsonian mice. Our results suggest the potential availability of ES cells for Parkinson's disease.
Engraftment Capacity of Umbilical Cord Blood Cells Processed by Either Whole Blood Preparation or Filtration
Authors: Eichler Hermann , Kern Susanne , Beck Christian , Zieger Wolfgang , Klüter Harald
Published: August 2004

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction, as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuick™E) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6), or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml, respectively, and nucleated cell (NC) content of 9.7 and 23.8 × 108 resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%-77.5%), CD34+ cells (76.3%-79.0%), and colony-forming cells (64.1%-86.3%). Moreover, we detected a relevant depletion of the transplants for RBCs (89.2%-90.0%) and platelets ([PLTs] 77.5%-86.1%). In contrast, the mean depletion rate using BC processing proved to be significantly different for PLTs (10%, p = 0.03) and RBCs (39.6%, p < 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p < 0.01), compared with a less significant MNC increase in the BC group (p < 0.05). For mice transplanted with WB-derived progenitors, we observed a mean of 15.3% ± 15.5% of human CD45+ cells within the BM compared with 19.9% ± 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34+ cells was 4.2% ± 3.1% for WB samples and 4.5% ± 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration, no negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced cells from WB transplants was found. The StemQuick™E filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume-reduced UCB transplant, including a partial depletion of granulocytes, RBCs, and PLTs without the need for centrifugation. Therefore, it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans.
Expression of Functional CXCR4 by Muscle Satellite Cells and Secretion of SDF-1 by Muscle-Derived Fibroblasts is Associated with the Presence of Both Muscle Progenitors in Bone Marrow and Hematopoietic Stem/Progenitor Cells in Muscles
Authors: Ratajczak Mariusz Z. , Majka Marcin , Kucia Magda , Drukala Justyna , Pietrzkowski Zbigniew , Peiper Stephen , Janowska-Wieczorek Anna
Published: August 2004

We found that the murine cell lines C2C12 and G7 derived from muscle satellite cells, which are essential for muscle regeneration, express the functional CXCR4 receptor on their surface and that the specific ligand for this receptor, α-chemokine stromal-derived factor 1 (SDF-1), is secreted in muscle tissue. These cell lines responded to SDF-1 stimulation by chemotaxis, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT serine-threonine kinase, and calcium flux, confirming the functionality of the CXCR4 receptor. Moreover, supernatants derived from muscle fibroblasts chemoattracted both satellite cells and human CD34+ hematopoietic stem/progenitor cells. In a similar set of experiments, supernatants from bone marrow fibroblasts were found to chemoattract CXCR4+ satellite cells just as they chemoattract CD34+ cells. Moreover, preincubation of both muscle satellite cells and hematopoietic stem/progenitor CD34+ cells before chemotaxis with T140, a specific CXCR4 inhibitor, resulted in a significantly lower chemotaxis to media conditioned by either muscle- or bone marrow-derived fibroblasts. Based on these observations, we postulate that the SDF-1-CXCR4 axis is involved in chemoattracting circulating CXCR4+ muscle stem/progenitor and circulating CXCR4+ hematopoietic CD34+ cells to both muscle and bone marrow tissues. Thus, it appears that tissue-specific stem cells circulating in peripheral blood could compete for SDF-1+ niches, and this would explain, without invoking the concept of stem cell plasticity, why hematopoietic colonies can be cultured from muscles and early muscle progenitors can be cultured from bone marrow.
Designing, Testing, and Validating a Focused Stem Cell Microarray for Characterization of Neural Stem Cells and Progenitor Cells
Authors: Luo Yongquan , Cai Jingli , Ginis Irene , Sun Yanyang , Lee Siulan , Yu Sean X. , Hoke Ahmet , Rao Mahendra
Published: August 2004

Fetal neural stem cells (NSCs) have received great attention not only for their roles in normal development but also for their potential use in the treatment of neurodegenerative disorders. To develop a robust method of assessing the state of stem cells, we have designed, tested, and validated a rodent NSC array. This array consists of 260 genes that include cell type-specific markers for embryonic stem (ES) cells and neural progenitor cells as well as growth factors, cell cycle-related genes, and extracellular matrix molecules known to regulate NSC biology. The 500-bp polymerase chain reaction products amplified and validated by using gene-specific primers were arrayed along with positive controls. Blanks were included for quality control, and some genes were arrayed in duplicate. No cross-hybridization was detected. The quality of the arrays and their sensitivity were also examined by using probes prepared by conventional reverse transcriptase or by using amplified probes prepared by linear polymerase replication (LPR). Both methods showed good reproducibility, and probes prepared by LPR labeling appeared to detect expression of a larger proportion of expressed genes. Expression detected by either method could be verified by RT-PCR with high reproducibility. Using these stem cell chips, we have profiled liver, ES, and neural cells. The cell types could be readily distinguished from each other. Nine markers specific to mouse ES cells and 17 markers found in neural cells were verified as robust markers of the stem cell state. Thus, this focused neural stem array provides a convenient and useful tool for detection and assessment of NSCs and progenitor cells and can reliably distinguish them from other cell populations.
Oct-4 Knockdown Induces Similar Patterns of Endoderm and Trophoblast Differentiation Markers in Human and Mouse Embryonic Stem Cells
Authors: Hay David C. , Sutherland Linda , Clark John , Burdon Tom
Published: August 2004

The transcription factor Oct-4 is a marker of pluripotency in mouse and human embryonic stem (ES) cells. Previous studies using a tetracycline-regulated Oct-4 transgene in the ZHBTc4 cell line demonstrated that downregulation of Oct-4 expression induced dedifferentiation into trophoblast, a lineage mouse ES cells do not normally generate. We found that transfection of Oct-4-specific short interfering RNA significantly reduced expression and functional activity of Oct-4 in mouse and human ES cells, enabling its role to be compared in both cell types. In mouse ES cells, Oct-4 knockdown produced a pattern of morphological differentiation and increase in expression of the trophoblast-associated transcription factor Cdx2, similar to that triggered by suppressing the Oct-4 transgene in the ZHBTc4 cell line. In addition, downregulation of Oct-4 was accompanied by increased expression of the endoderm-associated genes Gata6 and α-fetoprotein, and a gene trap associated with primitive liver/yolk sac differentiation. In human ES cells, Oct-4 knockdown also induced morphological differentiation coincident with the upregulation of Gata6. The induction of Cdx2 and other trophoblast-associated genes, however, was dependent on the culture conditions. These results establish the general requirement for Oct-4 in maintaining pluripotency in ES cells. Moreover, the upregulation of endoderm-associated markers in both mouse and human ES cells points to overlap between development of trophoblast and endoderm differentiation.
Behavioral Changes in Unilaterally 6-Hydroxy-Dopamine Lesioned Rats After Transplantation of Differentiated Mouse Embryonic Stem Cells Without Morphological Integration
Authors: Baier Paul Christian , Schindehütte Jan , Thinyane Keneuoe , Flügge Gabriele , Fuchs Eberhard , Mansouri Ahmed , Paulus Walter , Gruss Peter , Trenkwalder Claudia
Published: August 2004

Objective. Transplantation of fetal mesencephalic cells into the striatum has been performed in about 350 patients with Parkinson's disease and has been intensively studied in rat models of Parkinson's disease. Limited access to this material has shifted the focus toward embryonic stem (ES) cells. The grafting of undifferentiated ES cells to 6-hydroxy-dopamine (6-OHDA)-lesioned rats leads to behavioral improvements but may induce teratoma-like structures. This risk might be avoided by using more differentiated ES cells. In this study, we aimed to investigate differentiated mouse ES cells regarding their in vivo development and fate after transplantation in the striatum in the 6-OHDA rat model and the behavioral changes induced after transplantation.

Methods. Mouse ES cells were differentiated on PA6 feeder cells for 14 days before grafting. Twenty to twenty-five percent of the neurons obtained were positive for tyrosine-hydroxylase (TH). PKH26-labeled cells were transplanted in the striata of unilaterally 6-OHDA-lesioned rats.

Results. Direct PKH26 fluorescence visualization and TH staining proved the existence of cell deposits in the striata of all grafted animals, indicating cell survival for at least 5 weeks posttransplantation. There was no evidence of tumor formation. Immunocytochemical staining showed glial immunoreactivity surrounding the grafted cell deposits, probably inhibiting axonal outgrowth into the surrounding host tissue. There was a significant reduction in amphetamine-induced rotational behavior seen in grafted animals, which was not observed in sham-operated animals.

Conclusions. The findings of this study suggest that the amphetamine-induced rotational behavioral test without histological confirmation is not proof of morphological integration with axonal outgrowth within the first 4 weeks posttransplantation.
Changes in Gene Expression at the Precursor → Stem Cell Transition in Leech
Authors: Hohenstein Kristi A. , Shain Daniel H.
Published: August 2004

The glossiphoniid leech, Theromyzon trizonare, displays particularly large and accessible embryonic precursor/stem cells during its early embryonic cleavages. We dissected populations of both cell types from staged embryos and examined gene expression profiles by differential display polymerase chain reaction methodology. Among the ∼10,000 displayed cDNA fragments, 56 (∼0.5%) were differentially expressed at the precursor → stem cell transition; 29 were turned off (degraded, precursor-specific); and 27 were turned on (transcribed, stem cell-specific). Several putative differentially expressed cDNAs from each category were confirmed by Northern blot analysis on staged embryos. DNA sequencing revealed that 19 of the cDNAs were related to a spectrum of genes including the CCR4 antiproliferation gene, Rad family members, and several transcriptional regulators, while the remainder encoded hypothetical (10) or novel (27) sequences. Collectively, these results identify dynamic changes in gene expression during stem cell formation in leech and provide a platform for examining the molecular aspects of stem cell genesis in a simple invertebrate organism.
Critical Parameters for the Isolation of Mesenchymal Stem Cells from Umbilical Cord Blood
Authors: Bieback Karen , Kern Susanne , Klüter Harald , Eichler Hermann
Published: August 2004

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However, attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria.

Using optimized isolation and culture conditions, in up to 63% of 59 low-volume UCB units, cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 × 108 mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition, osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However, in contrast to MSCs, MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation.

Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours, a net volume of more than 33 ml, and an MNC count of more than 1 × 108 MNCs.

Because MSC-like cells can be isolated at high efficacy from full-term UCB donations, we regard UCB as an additional stem cell source for experimental and potentially clinical purposes.
High-Dose Chemotherapy and Peripheral Blood Progenitor Cell Transplantation in the Treatment of Breast Cancer
Authors: Peters William P. , Dansey Roger D. , Klein Jared L. , Baynes Roy D.
Published: August 2004

Each year in the USA, 180,000 new cases of breast cancer are diagnosed and about 44,000 women die of the disease. Current primary treatment consists of adjuvant chemotherapy and hormone therapy, and statistics show that combination chemotherapy favorably influences the outcomes in both node-negative and node-positive primary disease. However, a significant number of breast cancer patients succumb to the disease, and nearly every patient diagnosed with metastatic breast cancer will be dead within five years.

High-dose chemotherapy (HDC) and peripheral blood progenitor cell transplantation (PBPCT) are based upon laboratory and clinical observations of the ability to modify growth properties of quiescent and replicating cancer cells. A large number of HDC and PBPCT regimens have been evaluated for treatment of metastatic breast cancer, and recent autologous bone marrow transplantation data indicate that three HDC regimens (CPB, CTCb and cytoxan and thiotepa) predominate. Unfortunately, negative media coverage surrounding and subsequent to the presentation of preliminary findings reported at the May 1999 American Society of Clinical Oncologists, that were not allowed adequate follow-up time for full analysis of treatment results, has had a detrimental effect on the ability to conduct trials in this area.

Several randomized trials have been conducted in both the metastatic and high risk primary disease settings. Thorough analysis of these studies indicates an evaluable improvement in favor of HDC and PBPCT in three of the four randomized studies performed in metastatic breast cancer and two of the four high risk primary studies. Also, initial evaluations found that quality of life appeared comparable in patients receiving either HDC or not. Each randomized trial studied asks a different question and, depending on the intensity of HDC regimen, the intensity and duration of the standard dose chemotherapy control and the schedule of events in relation to induction chemotherapy, the outcomes may be quite variable. Still, certain general trends are indentifiable. HDC alone will not completely cure breast cancer and should be considered as part of an overall therapeutic plan. In some of these studies, significantly longer follow-up is required before definitive analysis can be completed.
Second-Line Treatment of Ovarian Cancer
Authors: Markman Maurie , Bookman Michael A.
Published: August 2004

Patients with epithelial ovarian cancer are often diagnosed with advanced-stage disease. Although clinical complete remissions are obtained in the majority of patients through a combination of cytoreductive surgery and chemotherapy, relapse is common. A number of agents with diverse biologic mechanisms have been identified with activity in the setting of recurrent disease. Strategies for management of patients with recurrent disease, including classification, treatment goals, and therapeutic options will be reviewed.
Intermittent Androgen Deprivation in Prostate Cancer Patients: Factors Predictive of Prolonged Time Off Therapy
Authors: Strum Stephen B. , Scholz Mark C. , McDermed Jonathan E.
Published: August 2004

Objectives. We hypothesize that prostate cancer (PC) patients who achieve and maintain an undetectable prostate-specific antigen (UD-PSA) on androgen deprivation therapy (ADT) have a predominantly androgen-dependent cancer cell population sensitive to apoptosis that allows for a prolonged time off ADT. This study summarizes patient- and treatment-related factors associated with a prolonged time off ADT in patients electing intermittent androgen deprivation (IAD).

Methods. Hormone-naïve patients with PC were treated with ADT using an antiandrogen and a luteinizing-hormone-releasing hormone-agonist. Of 255 consecutive patients, 216 (85%) achieved a UD-PSA (<0.05 ng/ml). Ninety-three (43%) of 216 elected to stop ADT after maintaining a UD-PSA for a median of one year. Patients were followed off therapy and advised to restart ADT if the PSA level reached ≥5.0 ng/ml. Forty-one patients received finasteride as part of IAD induction and as maintenance off therapy; these patients are excluded from the current study and are the focus of another publication. The remaining 52 patients are assessable for response being either in the off-phase of IAD ≥1 year or having restarted IAD.

Results. In the first IAD cycle, the median duration of the on-phase of IAD was 16 months (mean 19.0 months, range 3.6-71 months), and the median off-phase duration was 15.5 months (mean 24.1 months, range 3.2-87+ months). In 28 patients who maintained a UD-PSA for ≥1 year, their median off-phase duration was 29 months (mean 35.8 months, range 7.8-87+ months), with nine (32%) still off IAD after a median follow-up of 62 months. Significant (p < 0.05) independent factors associated with prolonged off-phase duration by multivariate analysis included UD-PSA on ADT ≥1 year (p = 0.010), PSA-only recurrence after local therapy (p = 0.039), and reaching a testosterone level ≥150 ng/dl in ≥4 months off ADT (p = 0.041). After a median of 66 months of follow-up, only one (2%) patient developed androgen-independent PC.

Conclusions. Hormone-naïve patients who achieve and maintain a UD-PSA for at least one year during ADT may initiate IAD and anticipate a prolonged off-phase duration. Attainment of a UD-PSA on ADT may serve as an in vivo sensitivity test of a patient's tumor cell population, and allow for better selection of those best suited for IAD.
Sedation for Intractable Distress of a Dying Patient: Acute Palliative Care and the Principle of Double Effect
Authors: Krakauer Eric L. , Penson Richard T. , Truog Robert D. , King Linda A. , Chabner Bruce A. , Lynch Thomas J.
Published: August 2004

Shortly before his death in 1995, Kenneth B. Schwartz, a cancer patient at Massachusetts General Hospital (MGH), founded The Kenneth B. Schwartz Center at MGH. The Schwartz Center is a nonprofit organization dedicated to supporting and advancing compassionate health care delivery, which provides hope to the patient, support to caregivers, and encourages the healing process. The Center sponsors the Schwartz Center Rounds, a monthly multidisciplinary forum where caregivers reflect on important psychosocial issues faced by patients, their families, and their caregivers, and gain insight and support from fellow staff members.

The case presented is of a young man dying of recurrent epithelioid hemangioendothelioma, distressed with stridor and severe pain, whose poorly controlled symptoms were successfully treated with an infusion of propofol, titrated to provide effective comfort in the last few hours of the patient's life. The tenet of double effect, which allows aggressive treatment of suffering in spite of foreseeable but unintended consequences, is reviewed. The patient's parents were invited and contributed to the Rounds, providing compelling testimony to the power of the presence of clinicians at the time of death and the importance of open communication about difficult ethical issues.
Waldenström's Macroglobulinemia
Authors: Gertz Morie A. , Fonseca Rafael , Rajkumar S. Vincent
Published: August 2004

Waldenström's macroglobulinemia is a low-grade lymphoplasmacytic lymphoma. It has an overall incidence of 2.5/million/year. The median age at diagnosis is 63 years. The clinical manifestations are hepatomegaly (20%), splenomegaly (15%), and lymphadenopathy (15%). The most common symptom is fatigue related to a normochromic, normocytic anemia, and the median hemoglobin value at diagnosis is 10 gm/dl. All patients with Waldenström's macroglobulinemia have a circulating tumor marker, the monoclonal IgM protein. Occasionally high levels of the IgM monoclonal protein can produce a hyperviscosity syndrome manifested by oronasal bleeding. Occasionally retinal hemorrhage or serious neurologic complications, such as somnolence or coma, may occur. The most important prognostic factors are hemoglobin, age, weight loss, and a cryoglobulin. Therapy has included alkylating agents, particularly chlorambucil, purine nucleoside analogs such as fludarabine or cladribine, and most recently the use of rituximab. The median survival of symptomatic patients is 65 months. Patients without symptoms should not be treated.
Cellular Suicide Therapy of Malignant Disease
Authors: Link Charles J. , Seregina Tatinia , Traynor Ann , Burt Richard K.
Published: August 2004

Adoptive cellular therapy is developing as a supplement or alternative to chemotherapy and/or radiation for malignant disease. Our focus is two ongoing clinical studies with transgeneic (genetically altered) cellular therapy; one uses allogeneic (from another person) lymphocytes to treat leukemia, and the second uses xenogeneic (from another species) fibroblast cells genetically altered to contain a toxin-producing suicide gene to treat ovarian cancer.

Allogeneic donor lymphocyte infusions (DLI) are known to induce remission of hematologic malignancies. However, the toxicity associated with DLI is related to graft-versus-host-disease, which is due to donor lymphocytes attacking normal tissue in the recipient. Therefore, we have taken the approach of infusing DLI that have been modified to contain a latent suicide gene to treat leukemia.

To treat ovarian cancer, we used xenogeneic nonimmune fibroblast-derived cells to deliver a tumor-directed cytotoxic gene to carcinoma cells. These cells release HStk transgene retroviruses that in turn transduce replicating tumor cells but not quiescent epithelium, rendering the tumor selectively susceptible to ganciclovir-mediated killing.

These initial trials summarize the early stage of allogeneic/xenogeneic adoptive cellular therapy for cancer, and although the data are limited, it is encouraging to see some patients with evidence of antitumor responses. Advances in our understanding of the basic science of these treatments, together with improvements in the technology of vector design, will be required to streamline these methodologies into broader application.