A novel xeno-organoid approach: exploring the crosstalk between human iPSC-derived PGC-like and rat testicular cells.

Specification of germ cell-like cells from induced pluripotent stem cells has become a clinically relevant tool for research. Research on initial embryonic processes is often limited by the access to foetal tissue, and in humans, the molecular events resulting in primordial germ cell (PGC) specification and sex determination remain to be elucidated. A deeper understanding of the underlying processes is crucial to describe pathomechanisms leading to impaired reproductive function. Several protocols have been established for the specification of human pluripotent stem cell towards early PGC-like cells (PGCLC), currently representing the best model to mimic early human germline developmental processes in vitro. Further sex determination towards the male lineage depends on somatic gonadal cells providing the necessary molecular cues. By establishing a culture system characterized by the re-organization of somatic cells from postnatal rat testes into cord-like structures and optimizing efficient PGCLC specification protocols, we facilitated the co-culture of human germ cell-like cells within a surrogate testicular microenvironment. Specified conditions allowed the survival of rat somatic testicular and human PGCLCs for 14 days. Human cells maintained the characteristic expression of octamer-binding transcription factor 4, SRY-box transcription factor 17, and transcription factor AP-2 gamma and were recovered from the xeno-organoids by cell sorting. This novel xeno-organoid approach will allow the in vitro exploration of early sex determination of human PGCLCs.

Derivation of germ cells from embryonic stem cells.

Embryonic stem cells (ESCs), derivatives of cells of early mammalian embryos, have proven to be one of the most powerful tools in developmental and stem cell biology. When injected into embryos, ESCs can contribute to tissues derived from all three germ layers and to the germline. Prior studies have successfully shown that ESCs can recapitulate features of embryonic development by spontaneously forming somatic lineages in culture. Amazingly, recently it has been shown that mouse ESCs can also give rise to primordial germ cells (PGCs) in culture that are capable of undergoing meiosis and forming both male and female gametes. While the full potential of these ES-derived germ cells and gametes remains to be demonstrated, these discoveries provide a new approach for studying reproductive biology and medicine.

Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences.

The Oct-4 gene encodes a transcription factor that is specifically expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that differentiate into somatic tissues lose Oct-4 expression. Regulation of Oct-4 gene transcription involves a TATA-less minimal promoter and two upstream elements: the proximal (PE) and distal enhancers (DE). We report here the nucleotide sequence of the 5' upstream regulatory regions of the human and murine Oct-4 genes. A comparative alignment analysis between these regions and those of the bovine Oct-4 ortholog reveals four conserved regions of homology (CR 1 to 4) between these species (66-94% conservation). The 1A sequence within the mouse PE is located approximately half-way between CR 2 and CR 3. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR1 are identical in all three species. A high number of CCC(A/T)CCC motifs exhibit various levels of homology in these upstream regions. We discuss the importance of these and other sequences and present candidate factors that may bind and regulate Oct-4 gene expression.

Effects of high-frequency, low-magnitude mechanical stimulus on bone healing.

Recent studies have shown osteogenic effects of high-frequency mechanical stimuli. The purpose of this study was whether externally applied, high-frequency, low-magnitude interfragmentary movements affect the process of bone healing. In 12 sheep, a transverse osteotomy with a 3 mm gap was created in the right metatarsus and externally stabilized by a rigid circular fixator. External stimulation was performed in six sheep with the use of ground-based vibration. The sheep were standing with their hind limbs on a platform that produced vertical movements resulting in interfragmentary movements of approximately 0.02 mm magnitude at 20 Hz frequency. The other six sheep remained rigidly stabilized by external fixation during the 8-week study and served as a control group. Healing was assessed postmortem by densitometric and mechanical examinations. No significant differences were found between the two groups, although callus formation was slightly enhanced (11%) in the stimulated group compared with the control group. Mechanical stimuli attributable to weightbearing in the control group were sufficient enough to initiate callus formation even under rigid, external fixation. Thus, external mechanical stimulation with the stimulation design described in the current study might not be indicated for improvement of bone healing.

Airway nitric oxide levels in cystic fibrosis patients are related to a polymorphism in the neuronal nitric oxide synthase gene.

Patients with cystic fibrosis (CF) have decreased concentrations of expired nitric oxide (FENO) as compared with healthy individuals. A number of factors, including viscous mucus as a diffusion barrier for airway NO, consumption of NO by bacterial enzymes, and decreased NO production have been hypothesized to account for these low levels of FENO. We examined the relationship between the size of an AAT repeat polymorphism in intron 20 of the NOS1 gene and FENO in 75 patients with CF. Mean FENO was significantly (p = 0.027) lower in CF patients who harbored two alleles with a high number of repeats (>/= 12) than in those who harbored alleles with fewer repeats at this locus (4.0 +/- 0.8 [mean +/- SEM] ppb versus 6.4 +/- 0.9 ppb). Colonization of the airways with Pseudomonas aeruginosa was significantly (p = 0.0358) more common in CF patients with high numbers of AAT repeats in the NOS1 gene. Significant differences between NOS1 genotypes were also observed among patients homozygous for the cystic fibrosis transmembrane regulator delta F508 mutation for FENO (2.3 +/- 0.4 ppb versus 5.3 +/- 0.7 ppb, p = 0.0006), and this was also true for colonization of the airways with P. aeruginosa (p = 0.0147) and Aspergillus fumigatus (p = 0.0221). These data provide evidence that the NOS1 gene is not only associated with the variability of FENO, but also with P. aeruginosa colonization of airways in CF patients.

The diagnostic utility of the lognormal behavior of PET standardized uptake values in tumors.

UNLABELLED: A meta-analysis of data primarily from PET oncologic investigations using FDG PET was performed. Its purpose was to establish statistical features of the distributions of standardized uptake values (SUVs) as possible aids in the diagnostic process. METHODS: We obtained 1536 values of oncologic markers from patient studies of 40 investigations in the literature. Statistical parameters were tabulated for analysis. RESULTS: A significant observation is that, unlike skewed SUV histograms, log10SUV has Gaussian behavior, which is not uncommon for biologic quantities. This was found for SUVs of FDG and 2 amino acids as well as a few other cancer markers. A possible model for explaining this is proposed. For FDG, the SD sigma of the log10SUVs for an average cancer category was 0.23. Examining data within the framework of the model points to physiologic factors as dominating SUV variability rather than PET protocols. When data for a single cancer category were available from multiple institutions, averages, mean(SUV)s, disagree beyond chance expectations. Diagnostic utility suggestions include a universal linear relationship between sensitivity and severity, defined as SUV/mean(SUV), on semilogarithmic probability paper; a generic receiver-operating-characteristic curve for all cancers; using [log10(mean(SUVmal)/mean(SUVnorm))] divided by (sigma(mal)2 + sigma(norm)2)(1/2) as a simple diagnostic effectiveness measure; and using Gaussian log10SUVs to avoid erroneous P values. CONCLUSION: Using the logarithms of markers, such as SUVs, several advantages stemming from their Gaussian nature can be achieved with benefits ensuing to the diagnostic process.

Tissue eosinophilia correlates strongly with poor prognosis in nodular sclerosing Hodgkin's disease, allowing for known prognostic factors.

Although eosinophilic granulocytes are frequently observed in lymphatic tissue of Hodgkin's patients, no substantial data reveal the prognostic role, if any, of tissue eosinophilia. Thus, eosinophilia was analyzed histologically in 1511 diagnostic biopsy specimens of patients treated under protocol therapy of the German Hodgkin's Lymphoma Study Group between 1988 and 1994. Prominent eosinophilia was seen in 38% of cases, which differed among the histologic types of Hodgkin's disease (HD): none in lymphocyte predominant, 14% in lymphocyte rich classical, 40% in nodular sclerosis grade 1 (NS-1), 55% in nodular sclerosis grade 2, 43% in mixed cellularity (MC), and 54% in lymphocyte depleted. In a multivariate analysis, tissue eosinophilia proved to be the strongest prognostic factor for freedom from treatment failure (P <. 001) and overall survival (P <.001) in a stage-stratified model. Among NS-1 patients, the effect was highly significant. In MC, no significant effect of eosinophilia on survival could be demonstrated. Eosinophils secrete CD30 ligand that is capable of binding to CD30 positive HD cells. The activation of TRAF2, followed by NF-kappaB, which occurs on CD30L/CD30 binding, may explain the neoplastic proliferation and apoptosis protection of HD cells. TRAF2 is also activated by EBV-LMP expression, which is detectable in the majority of MC but not NS cases. In addition to the possibility that eosinophils are only passive indicators for other unknown prognostic determinants, it may be concluded that the positive clinical outcome of eosinophilia-negative NS cases could be due to lower NF-kappaB activity. (Blood. 2000;95:1207-1213)

The Potential of F-18-FDG PET in Breast Cancer. Detection of Primary Lesions, Axillary Lymph Node Metastases, or Distant Metastases.

This retrospective study was done to evaluate the utility of 2-[F-18]fluoro-2-deoxy-D-glucose positron emission tomography (F-18-FDG PET) in identifying primary and recurrent breast cancer and lymph node metastases. One hundred whole-body PET scans of 87 patients were reviewed. PET results obtained with F-18-FDG and an ECAT/EXACT-921 or an ECAT-931 (Siemens/CTI) were based on visual interpretation, or standardized uptake values (SUVs), related to histology and also compared to computerized tomography (CT) and mammography results. The sensitivity for PET in detecting primary (N = 35 studies) and recurrent breast cancer (N = 65 studies) was 96% and 85% with a specificity of 91% and 73%. The sensitivity for lymph node metastases at the time of initial diagnosis was 100% with a specificity of 100%. Quantitative SUV information did not improve the accuracy of F-18-FDG PET in identifying primary breast cancers. The results suggest that whole-body PET is useful in detecting recurrence or metastases, may be useful in detecting lymph node metastases prior to initial axillary lymph node dissection, but is less sensitive in excluding axillary lymph nodes metastases later in the course of the disease.

Analysis of inter-fragmentary movement as a function of musculoskeletal loading conditions in sheep.

It is well accepted that inter-fragmentary movement influences the fracture healing process. Small axial movement can stimulate callus formation whereas larger shear movement delays the healing process. It is, therefore, essential for optimal fracture healing to minimize shear and to control axial movement. Unfortunately, the complex gap movements are mostly unknown under the large variety of clinical as well as experimental conditions of fracture fixation. To further understand the complex interactions of musculoskeletal loading and inter-fragmentary movements in bones and to reduce the need for animal experiments, a three-dimensional (3D) musculoskeletal model of the left hind limb of a sheep was developed. From 3D ground reaction forces and inverse dynamics, resultant joint loading was determined over a gait cycle. Muscle and joint contact forces were derived from an optimization routine and internal loads in the tibia and metatarsus from beam theory. Finally, inter-fragmentary movements were calculated from the bony loading condition and experimentally determined stiffness matrices of monolateral AISF external fixator constructs. Both the joint contact forces at the hip and gap movement of a mid-shaft tibial fracture agree with in vivo data reported in the literature. The bones proved to be mainly axially loaded with slightly increasing shear forces toward their ends. The results suggest that inter-fragmentary movement of metatarsal fractures is fairly independent of the fracture location whereas the movement increases in proximal tibial fractures compared to those in the distal and diaphyseal tibia. Considerable shear movement was found for all locations and external fixator mountings. However, shear movement could be minimized with a cranio-lateral rather than a cranio-medial shift from the cranial fixator plane.

Radiographic, densitometric, and biomechanical effects of recombinant canine somatotropin in an unstable ostectomy gap model of bone healing in dogs.

OBJECTIVE: To determine the effect of recombinant canine somatotropin (STH) on radiographic, densitometric, and biomechanical aspects of bone healing using an unstable ostectomy gap model. STUDY DESIGN: After an ostectomy of the midshaft radius, bone healing was evaluated over an 8-week period in control dogs (n = 4) and dogs receiving recombinant canine STH (n = 4). ANIMALS OR SAMPLE POPULATION: Eight sexually intact female Beagle dogs, 4 to 5 years old. METHODS: Bone healing was evaluated by qualitative and quantitative evaluation of serial radiographs every 2 weeks. Terminal dual-energy x-ray absorptiometry and three-point bending biomechanical testing were also performed. RESULTS: Dogs receiving STH had more advanced radiographic healing of ostectomy sites. Bone area, bone mineral content, and bone density were two to five times greater at the ostectomy sites of treated dogs. Ultimate load at failure and stiffness were three and five times greater in dogs receiving STH. CONCLUSIONS: Using the ostectomy gap model, recombinant canine STH enhanced the radiographic, densitometric, and biomechanical aspects of bone healing in dogs. CLINICAL RELEVANCE: Dogs at risk for delayed healing of fractures may benefit from treatment with recombinant canine STH.

Prospective investigation of positron emission tomography in lung nodules.

PURPOSE: Solitary pulmonary nodules (SPNs) are commonly identified by chest radiographs and computed tomography (CT). Biopsies are often performed to evaluate the nodules further. An accurate, noninvasive diagnostic test could avoid the morbidity and costs of invasive tissue sampling. We evaluated the ability of fluorine-18 deoxyglucose positron emission tomography (FDG-PET) to discriminate between benign and malignant pulmonary nodules in a prospective, multicenter trial. METHODS: Eighty-nine patients who had newly identified indeterminate SPNs on chest radiographs and CT were evaluated with FDG-PET. PET data were analyzed semiquantitatively by calculating standardized uptake values (SUVs) as an index of FDG accumulation and also by a visual scoring method. PET results were compared with pathology results. RESULTS: Sixty SPNs were malignant and 29 were benign. Using SUV data, PET had an overall sensitivity and specificity for detection of malignant nodules of 92% and 90%. Visual analysis provided a slightly higher, but not statistically significant, sensitivity of 98% and lower specificity of 69%. For SPNs < or = 1.5 cm (34 of 89), the sensitivity and specificity of SUV and visual analysis were 80% and 95% and 100% and 74%, respectively. CONCLUSION: FDG-PET can accurately characterize indeterminate SPNs. PET imaging provides a noninvasive method to evaluate indeterminate SPNs, which can reduce the need for invasive tissue biopsy.

Positron Emission Tomography (PET) with 1-Aminocyclobutane-1-[(11)C]carboxylic Acid (1-[(11)C]-ACBC) for Detecting Recurrent Brain Tumors.

This study was done to determine whether 1-[(11)C]ACBC PET has any advantages over 2-[(18)F]FDG PET, CT, or MRI in detecting recurrent brain tumors, and whether quantitative 1-[(11)C]ACBC PET information improves the accuracy of "visual" image interpretation.Twenty patients with recurrent brain tumor underwent dynamic PET. Images were analyzed by visual interpretation; in addition, standardized uptake values (SUVs) and Patlak values (k(1)*k(3)/k) were evaluated.1-[(11)C]ACBC identified 19/20 recurrent brain tumors, [18F]FDG 13/19, MRI 13/19, and CT 8/16. Based on SUVs, the average tumor-to-contralateral gray matter ratio of 1-[(11)C]ACBC was 5.0 and 0.5 for 2-[(18)F]FDG. Mean Patlak values of 1-[(11)C]ACBC were 0.044 +/- 0.047 for high and 0.034 +/- 0.026 for low grade tumors. However, visual interpretation was effective without quantitative PET data.1-[(11)C]ACBC, accurately detects recurrent tumors for selecting biopsy sites and treatment planning.