Use of tumor dynamics to clarify the observed variability among biochemical recurrence nomograms for prostate cancer.

BACKGROUND: Nomograms for biochemical recurrence (BCR) of prostate cancer (PC) after radical prostatectomy can yield very different prognoses for individual patients. Since the nomograms are optimized on different cohorts, the variations may be due to differences in patient risk-factor distributions. In addition, the nomograms assign different relative scores to the same PC risk factors and rarely stratify for tumor growth rate. METHODS: We compared BCR-free probabilities from the GPSM model with a cell kinetics (CK) model that uses the individual's tumor state and growth rate. We first created a cohort of 143 patients that reproduced the GPSM patient distribution in Gleason score, Prostate specific antigen (PSA), Seminal vesicle involvement and Margin status since they form the GPSM score. We then performed 143 CK calculations to determine BCR-free probabilities for comparison with the GPSM results for all scores and with four other prominent nomograms for a high-risk patient. RESULTS: The BCR-free probabilities from the CK model agree within 10% with those from the GPSM study for all scores once the CK model parameters are stratified in terms of the GPSM risk factors and the PSA doubling time (PSADT). However, the probabilities from widely used nomograms vary significantly. CONCLUSIONS: The CK model reproduces the observed GPSM BCR-free probabilities with a broad stratification of model parameters for PC risk factors and can thus be used to describe PC progression for individual patients. The analysis suggests that nomograms should stratify for PSADT to be predictive.

Secular trends in hip fracture incidence and recurrence.

UNLABELLED: The decline in hip fracture incidence is now accompanied by a further reduction in the likelihood of a recurrent hip fracture among survivors of the first fracture. INTRODUCTION: Hip fracture incidence is declining in North America, but trends in hip fracture recurrence have not been described. METHODS: All hip fracture events among Olmsted County, Minnesota residents in 1980-2006 were identified. Secular trends were assessed using Poisson regression, and predictors of recurrence were evaluated with Andersen-Gill time-to-fracture regression models. RESULTS: Altogether, 2,752 hip fractures (median age, 83 years; 76% female) were observed, including 311 recurrences. Between 1980 and 2006, the incidence of a first-ever hip fracture declined by 1.37%/year for women (p < 0.001) and 0.06%/year for men (p = 0.917). Among 2,434 residents with a first-ever hip fracture, the cumulative incidence of a second hip fracture after 10 years was 11% in women and 6% in men with death treated as a competing risk. Age and calendar year of fracture were independently associated with hip fracture recurrence. Accounting for the reduction in first-ever hip fracture rates over time, hip fracture recurrence appeared to decline after 1997. CONCLUSION: A recent reduction in hip fracture recurrence is somewhat greater than expected from the declining incidence of hip fractures generally. Additional research is needed to determine the extent to which this can be attributed to improved patient management.

Intragenic codon bias in a set of mouse and human genes.

To better conceptualize the mechanism underlying the evolution of synonymous codons, we have analysed intragenic codon usage in chosen "regions" of some mouse and human genes. We divided a given gene into two regions: one consisting of a trinucleotide repeat (TNR) and the other consisting of the "rest of the coding region" (RCR). Usually, a TNR is composed of a repetitive single codon, which may reflect its frequency in a gene. In contrast, a non-random frequency of a codon in the RCR versus TNR (or vice versa) of a gene should indicate a bias for that codon within the TNR. We examined this scenario by comparing codon frequency between the RCR and the cognate TNR(s) for a set of human and mouse genes. A TNR length of six amino acids or more was used to identify genes from the Genbank database. Twenty nine human and twenty one mouse genes containing TNRs coding for nine different amino acid runs were identified. The ratio of codon frequency in a TNR versus the corresponding RCR was expressed as "fold change" which was also regarded as a measure of codon bias (defined as preferential use either in TNR or in RCR). Chi-square values were then determined from the distribution of codon frequency in a TNR vs. the cognate RCR. At p<0.001, 22% and 27%, respectively, of human and mouse TNRs showed codon bias. Greater than 40% of the TNRs (29 out of 69 in human, and 18 of 42 in mouse) showed codon bias at p<0.05. In addition, we identify eight single-codon TNRs in mouse and ten in human genes. Thus, our results show intragenic codon bias in both mouse and human genes expressed in diverse tissue types. Since our results are independent of the Codon Adaptation Index (CAI) and starvation CAI, and since the tRNA repertoire in a cell or in a tissue is constant, our data suggest that other constraints besides tRNA abundance played a role in creating intragenic codon bias in these genes.

Development of a cellular model to study alternative splicing of type II collagen gene.

To study regulation of alternative splicing of type II collagen (COL2) pre-mRNA, we constructed a mouse COL2 "minigene" containing genomic sequences spanning exon 1 to exon 4 of COL2 downstream of a cytomegalovirus (CMV) promoter. This minigene was introduced into ATDC5 cells, which undergo chondrocytic differentiation when treated with insulin. Alternative splicing of the COL2 minigene was evaluated by comparing the expression of the two mRNAs transcribed from the minigene to the expression of alternatively spliced transcripts from the endogenous COL2 gene. This analysis suggested that regulation of alternative splicing of pre-mRNAs from the minigene and the endogenous COL2 gene are accomplished by similar mechanisms. We conclude that the cloned genomic fragment contains key sequences necessary for alternative splicing of COL2 pre-mRNA. This system provides a useful model to begin the process of identifying cis- and trans-acting factors that carry out alternative splicing of COL2 pre-mRNA during chondrocyte differentiation.

Identification of twenty-two candidate markers for human osteogenic sarcoma.

Since osteogenic sarcoma (OGS) predominantly affects children, its etiology and progression may be determined more by genetic than environmental factors. A few genes have been associated with OGS, however, their value in the diagnosis and/or prognosis of the disease remains poor. Evidently, more markers need to be identified for improving management of patients with OGS. To identify potential genetic markers for OGS, we have extended preferential amplification of coding sequences (PACS) to screen multiple samples simultaneously. The extended method is termed multi-PACS. Multi-PACS was applied between a normal osteoblast and four OGS-derived cell lines to identify differentially expressed coding sequence tags (dCST) that identified 145 dCSTs. Subsequently, differential mRNA expression was validated for a chosen subset of 22 dCSTs. These chosen dCSTs include among others cyclins D and E, two cyclin dependent kinases, two other kinases, transcription factors E2F4, E2F5, and p130, a DNA repair gene, a gene for the signalosome subunit, and potential guanine nucleotide binding factors. We infer that these genes could be so easily identified because PACS preferentially identifies coding instead of non-coding sequences. We also infer that these genes identify signaling pathways pertinent to OGS. mRNA expression profile of these 22 genes/dCSTs generated distinct expression signature of the OGS-derived cell lines suggesting that further work on clinical samples with these dCSTs will yield valuable information for OGS. We conclude that these 22 genes/dCSTs are candidate markers for OGS.

A method for accurate detection of translocation junctions in Ewing family of tumors.

The Ewing family of tumors (ET) generally contain translocations involving the EWS gene and the FLI or ERG genes. Identification of the translocation confirms the diagnosis of ET. Currently, diagnosis of the translocation is made by several methods. In general, these methods require different primer sets for amplifying different translocations and subsequent efforts to identify the amplified product. The need to employ different sets of primers to amplify different translocation junctions presents some limitations. We have developed a method based on PCR with consensus primers followed by direct automated sequencing of the amplified product. With this method we have correctly determined known as well as unknown ET-associated EWS-FLI and EWS-ERG translocations in appropriate specimens. Use of our consensus primers eliminates the need for separate PCRs to amplify EWS-FLI and EWS-ERG translocation junctions, and because direct sequencing is used for confirming the identity of the amplification product, the accuracy of detection becomes 100%. The method might also accurately diagnose ET-associated translocations other than EWS-FLI and EWS-ERG translocations.

An integrated approach to assess structure-to-function relationships in the skeleton.

In general, the disciplines of biomechanics, morphology, densitometry, biochemistry, cell biology and molecular biology have advanced independently of one another. In spite of this fragmentation, there have been incremental increases in our understanding of the organization, mechanical properties, growth, remodeling and repair of the tissues comprising the skeleton. As a practical application, this increased knowledge has greatly improved our capabilities for early diagnosis of bone loss and has proven similarly useful in determining the efficacy of interventions to prevent osteoporosis. This approach, however, has been much less successful in countering several other important musculoskeletal disorders, including arthritis. In the immediate future, a major emphasis will be placed on tissue regeneration (engineering) to restore lost mechanical function to a compromised skeleton. To accomplish this goal, it will be necessary to employ much more sophisticated approaches toward evaluating the structure-to-function relationships, ones which will include integration of the respective contributions of gene expression, cell number and activity, matrix composition and architecture to achieve adequate tissue function.

Differential mRNA fingerprinting by preferential amplification of coding sequences.

The need for rapid identification of differentially expressed genes will persist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquires expressed sequence tags (ESTs) from the extreme 3' non-coding end of mRNAs. Such ESTs have limitations for downstream applications. We have developed a method, termed preferential amplification of coding sequences (PACS), that was applied to identify differentially expressed coding sequence tags (dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequences in mRNAs and another set of primers to anchor at a PCR-amplifiable distance from AUG sequences. An initial screen identified 103 candidate dCSTs after screening approximately 15% of the expressed genes between the two cell types. Of these sequences, 27 represent CSTs of known genes and two are from 3'-ESTs of known mRNAs. Thus, PACS identified CSTs approximately 13.5 times more often than it identified 3' ESTs, attesting to the objective of the method. Since many of the dCSTs represent known genes, their identity and potential relevance to osteosarcoma could be immediately hypothesized. Differential expression of many of the dCSTs was further demonstrated by northern blotting or RT-PCR. Since PACS is not dependent on the existence of a poly A tail on an mRNA, it should have application to identify dCSTs for both prokaryotic and eukaryotic organisms. Additionally, PACS should aid in the identification of cell-specific or tissue-specific genes and bidirectional acquisition of cDNA sequence enabling rapid retrieval of full-length cDNA sequence of novel genes.

Identification of differentially expressed genes by mutually subtracted RNA fingerprinting.

A mutually subtracted RNA fingerprinting (SuRF) method has been developed that allows efficient identification of differentially expressed sequence tags between two samples. Mutual subtractions of two RNA samples are achieved by first synthesizing cDNAs using oligo(dT) coupled with magnetic beads which are then reciprocally hybridized to starting RNA samples to remove common mRNAs between them. The second step involves differential fingerprinting of the subtracted RNA samples by polymerase chain reaction with specially designed degenerate primers. SuRF was applied to identify alteration in gene expression pertinent to osteogenic sarcoma which was achieved by employing the method between FOB (an immortalized fetal osteoblast) and MG63 (an osteosarcoma) cell lines. An estimated 10% of the total expressed genes in these two cell types were screened by the method. This analysis identified 96 differentially expressed sequences, none of which was identified repeatedly. A subset of these sequences was subsequently confirmed to have differential expression between the two cell types. Removal of common mRNAs prior to differential display should diminish redundant identification of abundant genes and increase the chance of identifying rare differentially expressed genes.

Ciprofloxacin inhibition of experimental fracture healing.

BACKGROUND: Fluoroquinolones, such as ciprofloxacin, have an adverse effect on growing cartilage and endochondral ossification in children. This study was carried out to determine whether ciprofloxacin also has an adverse effect on the healing of experimental fractures. METHODS: Sixty male 300-gram Wistar rats were divided equally into three groups, which received ciprofloxacin, cefazolin, or no treatment for three weeks, beginning seven days after production of a closed, nondisplaced, bilateral femoral fracture. The serum concentrations of the ciprofloxacin and the cefazolin were 2.4 and 146 micrograms per milliliter, respectively. Radiographic, histological, and biomechanical studies were used to evaluate fracture-healing. RESULTS: Radiographs revealed significantly more advanced healing of the control fractures compared with the fractures in the ciprofloxacin-treated group (average stage, 2.1 compared with 1.5, p = 0.01). The cefazolin-treated group was not different from the controls with respect to radiographic healing (average stage, 1.8 compared with 2.1, p = 0.18). Torsional strength-testing of fracture callus exposed to ciprofloxacin revealed a 16 percent decrease in strength compared with the controls (284 compared with 338 newton-millimeters, p = 0.04) and a 49 percent decrease in stiffness (twenty compared with thirty-nine newton-millimeters per degree, p = 0.001). The biomechanical strength in the cefazolin-treated group was not different from that of the controls. Fracture calluses in the animals treated with ciprofloxacin showed abnormalities in cartilage morphology and endochondral bone formation and a significant decrease in the number of chondrocytes compared with the controls (0.77 x 10(4) compared with 1.3 x 10(4) cells per square millimeter, p = 0.004). CONCLUSIONS: These data suggest that experimental fractures exposed to therapeutic concentrations of ciprofloxacin in serum demonstrate diminished healing during the early stages of fracture repair. The administration of ciprofloxacin during early fracture repair may compromise the clinical course of fracture-healing.

Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression.

We evaluated the effect of low intensity-pulsed ultrasound stimulation on rat chondrocytes in vitro using two different 1.0-MHz ultrasound signals with spatial and temporal average intensities of 50 or 120 mW/cm2. The pulses had a duration of 200 microseconds and were repeated every millisecond, with corresponding average peak-pressure amplitudes of 230 or 360 kPa, respectively. Cells were stimulated one, three, or five times for 10 minutes each day starting the third day after plating. One group of cells was exposed to sham ultrasound as a control. The cultures were evaluated for cell proliferation (by [3H]thymidine incorporation and DNA measurement), steady-state mRNA levels of alpha1(I) and alpha1(II) procollagens and aggrecan (by Northern blotting), and proteoglycan synthesis (by [35S]sulfate incorporation). The results revealed that ultrasound causes increases in the level of aggrecan mRNA (p < 0.05) and in proteoglycan synthesis (p < 0.03) after three and five treatments. Expression of mRNA for alpha1(II) procollagen increased over time, but ultrasound had no stimulatory effect. Expression of mRNA for alpha1(I) procollagen was initially low and remained unchanged with time. Although cell proliferation increased with time in both groups, there was no statistically significant difference between the cultures treated with ultrasound and the controls (p = 0.1). The in vitro results support our previous in vivo findings that low-intensity ultrasound stimulates aggrecan mRNA expression and proteoglycan synthesis by chondrocytes, which may explain the role of ultrasound in advancing endochondral ossification, increasing the mechanical strength of fractures, and facilitating fracture repair.

Inhibition of endochondral ossification during fracture repair in experimental hypothyroid rats.

Using a rat fracture model, we investigated the effects of a decrease in serum levels of thyroid hormone on the fracture-repair process. Rats were divided into the following groups: (a) controls, (b) those treated with methimazole for the duration of the experiment, and (c) those treated with methimazole and L-thyroxine, receiving both for the same duration. Three weeks after the initiation of pharmacologic treatment, closed femoral fractures were produced. The formation of cartilage tissue in the fracture callus in all rats was not obviously different on day 7 after fracture. In the rats treated with methimazole, differentiation from proliferating to hypertrophic chondrocytes in the fracture callus was less advanced and vascular invasion was clearly inhibited on day 12. Gene expression of alkaline phosphatase and osteocalcin in the callus was significantly lower in these rats than in the controls on days 10, 12, and 14. The mechanical properties of the fracture callus were also significantly weaker in these animals than in the controls on day 21, resulting in impaired fracture repair. These results demonstrate that hypothyroidism inhibits endochondral ossification, resulting in an impaired fracture-repair process. L-thyroxine replacement in the rats treated with methimazole caused the impaired repair process to revert to normal. These results indicate that thyroid hormone is one of the critical systemic factors for fracture repair.

Initial evidence for the involvement of bone morphogenetic protein-2 early during periosteal chondrogenesis.

The potential of periosteum to form cartilage makes periosteal transplantation a viable approach to repairing defects in articular cartilage, which has a limited potential for repair. However, cartilage repair, including that by periosteal chondrogenesis, is poorly understood. Consequently, a thorough understanding of its molecular mechanisms will help to achieve the quality of neocartilage required for its clinical application in damaged joints. An in vitro model was used to study the early molecular events of periosteal chondrogenesis. During the search for the expression of transforming growth factor-beta-related mRNAs in this model system, bone morphogenetic protein-2 mRNA expression was found to be upregulated 20-fold within the first 12 hours of culture. This stimulation was dependent on the explants being suspended in agarose and did not occur with explants cultured in liquid medium. The upregulation of bone morphogenetic protein-2 mRNA expression was also enhanced by exogenously added transforming growth factor-beta1 in the presence of fetal calf serum. The upregulation, however, was not transient; rather, it persisted over a prolonged period in both transforming growth factor-beta1-treated and untreated explants. Further data indicate that this stimulation of bone morphogenetic protein-2 mRNA expression was regulated at the transcriptional level and that no new protein synthesis was required for this. Bone morphogenetic protein-2 is known to influence developmental chondrogenesis; therefore, these observations direct our attention toward an important potential role of it as a regulator of the early events in cartilage repair. Furthermore, because periosteum produces fracture (cartilage) callus, these findings may be important in defining the molecular mechanisms of fracture healing.

Repeat cDNA synthesis and RT-PCR with the same source of RNA.

A simple method has been developed that enables reextraction of RNA from an RNA-cDNA mixture. The reextracted RNA was converted to cDNA followed by polymerase chain reaction (PCR). Thus, cDNA synthesis (followed by PCR) was carried out two times on the same source of RNA. The method has been applied to 40 RNA samples of diverse tissue origin with a success rate of 100%. Thus, the method offers more versatile use of small but valuable RNA sources than currently possible.

High temperature cDNA synthesis by AMV reverse transcriptase improves the specificity of PCR.

The enzyme avian myeloblastosis virus reverse transcriptase (AMV-RT) is routinely used for cDNA synthesis, which is generally carried out at temperatures between 37 degrees C and 42 degrees C. We show that this enzyme can support cDNA synthesis, at temperatures as high as 70 degrees C. We have utilized this property of the AMV-RT to improve the specificity of polymerase chain reaction (PCR). Furthermore, this apparently thermophilic property of the enzyme, which is an important constituent of a mesophilic organism, raises intriguing questions regarding evolution of the enzyme structure.

Selective differential fingerprinting. A method for identifying differentially expressed genes in a family between two samples.

A method termed selective differential fingerprinting (SDF) has been developed that enables one to investigate the level of expression for a family of genes between two samples. SDF produces a fingerprint (on a sequencing gel) on reverse transcription polymerase chain reaction (RT-PCR) of a sample with degenerate primers designed from conserved regions of a family of genes. By comparing fingerprints obtained after SDF with primers representing the transforming growth factor-beta (TGF beta) family of growth factors between a low-grade and a high-grade tumor from the same patient, a TGF beta family member known as osteogenic protein 1 (OP-1) or bone morphogenic protein 7 (BMP-7) was found to be greatly overexpressed in the high-grade tumor compared to the low-grade one. SDF also has the potential to identify novel genes. SDF offers a general way to identify differentially expressed genes for a family between two given samples.

Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR.

The determination of unknown DNA sequences around a known locus has important applications in molecular genetics, specifically in genomic walking and genome mapping. Several PCR-based methods have been reported to address this issue, but they often involve multiple, time-consuming steps. We have previously described a technique known as restriction site PCR (RS-PCR) that allows sequence acquisition faster than the existing methods. The method involves PCR using four separate universal primers that are representative of given restriction enzyme sites (RS primers), and a specific primer from one end of the known sequence. We have now significantly improved the technique by mixing the four universal primers into one PCR tube with the first specific primer. This is followed by a nested PCR with the mixed RS primers and an internal specific primer, after which the product is sequenced by direct automated sequencing. The technique, called multiplex RS-PCR (mRS-PCR), is reproducible and can be used to obtain unknown sequence adjacent to known sequences in both the upstream and downstream directions. We illustrate the application of mRS-PCR in the acquisition of approximately 780 bp of genomic sequence starting from a known sequence of approximately 120 bp. Multiplex RS-PCR appears to be the fastest of all methods that address the issue of unknown sequence retrieval adjacent to a known region.

Artificial cavitation nuclei significantly enhance acoustically induced cell transfection.

The efficiency of ultrasound-mediated gene transfection was enhanced three- to fourfold, compared to previous results, through the use of green fluorescent protein reporter gene, cultured immortalized human chondrocytes and artificial cavitation nuclei in the form of Albunex. Cells were exposed to 1.0-MHz ultrasound transmitted through the bottom of six-well culture plates containing immortalized chondrocytes, media, DNA at a concentration of 40 micrograms/mL and Albunex at 50 x 10(6) bubbles/mL. Transfection efficiency increased linearly with ultrasound exposure pressure with a transfection threshold observed at a spatial average peak positive pressure (SAPP) of 0.12 MPa and reaching about 50% of the living cells when exposed to 0.41 MPa SAPP for 20 s. Adding fresh Albunex at 50 x 10(6) bubbles/mL prior to sequential 1-s, 0.32- or 0.41-MPa exposures increased transfection with each exposure, reaching 43% transfection after four exposures. Efficient in vitro and in vivo transfection now appear possible with these enhancements.

Oligoclonal T-cell proliferation and interferon-gamma production in periprosthetic inflammation.

Total joint arthroplasty has dramatically changed the treatment options for patients with destructive joint disease. The materials used to manufacture implants are regarded as biologically inert; accordingly, arthroplasty is a very successful intervention for most patients. However, a subset of patients develops an inflammatory reaction around the prosthesis, causing implant loosening and irreversible bone destruction. To identify mechanisms leading to periprosthetic inflammation, the function and composition of macrophages and T cells accumulated in the pseudosynovia were examined. Tissue-infiltrating macrophages synthesized a spectrum of proinflammatory cytokines including IL-1beta, IL-6, and TGF-beta. T cells recruited to the periprosthetic inflammatory lesions were characterized by restricted diversity of T-cell receptors and the emergence of dominant clonal populations. T cells with identical T-cell receptor sequences, and thus with identical antigen specificity, were isolated from anatomically distinct and independent regions of the tissue. Transcription of IL-2, IFN-gamma, and, in some patients, IL-4 genes in the periprosthetic membrane indicated functional activation of infiltrating T cells. Correlation of periprosthetic osteolysis with the tissue cytokine pattern demonstrated a relationship between IFN-gamma transcription and bone loss. We propose that antigen-recognition events are critically involved in the development of periprosthetic inflammation and that the functional commitment of T cells recruited to the periprosthetic region influences whether periprosthetic inflammation is complicated by bone destruction.

PIG-B: a homemade monophasic cocktail for the extraction of RNA.

An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent are Phenol, Isoamyl alcohol, Guanidinium isothiocyanate, and Beta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.