The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

RAS-mediated suppression of PAR3 and its effects on SCC initiation and tissue architecture occur independently of hyperplasia.

Proper epithelial development and homeostasis depends on strict control of oriented cell division. Current evidence shows that this process is regulated by intrinsic polarity factors and external spatial cues. Owing to the lack of an appropriate model system that can recapitulate the architecture of the skin, deregulation of spindle orientation in human epithelial carcinoma has never been investigated. Here, using an inducible model of human squamous cell carcinoma (SCC), we demonstrate that RAS-dependent suppression of PAR3 (encoded by PARD3) accelerates epithelial disorganization during early tumorigenesis. Diminished PAR3 led to loss of E-cadherin-mediated cell adhesion, which in turn contributed to misoriented cell division. Pharmacological inhibition of the MAPK pathway downstream of RAS activation reversed the defects in PAR3 expression, E-cadherin-mediated cell adhesion and mitotic spindle orientation. Thus, temporal analysis of human neoplasia provides a powerful approach to study cellular and molecular transformations during early oncogenesis, which allowed identification of PAR3 as a critical regulator of tissue architecture during initial human SCC development.

Symptomatic bipartite patella: three subtypes, three representative cases.

Bipartite patella can be classified into three unique subtypes; type I, II, and III. The following case series describes three representative cases of each subtype and a spectrum of location-specific treatment options for surgical care of the symptomatic bipartite patella.

KLF3 Mediates Epidermal Differentiation through the Epigenomic Writer CBP.

Impairments in the differentiation process can lead to skin diseases that can afflict ∼20% of the population. Thus, it is of utmost importance to understand the factors that promote the differentiation process. Here we identify the transcription factor KLF3 as a regulator of epidermal differentiation. Knockdown of KLF3 results in reduced differentiation gene expression and increased cell cycle gene expression. Over half of KLF3's genomic binding sites occur at active enhancers. KLF3 binds to active enhancers proximal to differentiation genes that are dependent upon KLF3 for expression. KLF3's genomic binding sites also highly overlaps with CBP, a histone acetyltransferase necessary for activating enhancers. Depletion of KLF3 causes reduced CBP localization at enhancers proximal to differentiation gene clusters, which leads to loss of enhancer activation but not priming. Our results suggest that KLF3 is necessary to recruit CBP to activate enhancers and drive epidermal differentiation gene expression.

Flexion contracture due to cyclops lesion after bicruciate-retaining total knee arthroplasty.

Flexion contracture may develop after total knee arthroplasty (TKA) and is usually associated with soft tissue contracture in the posterior compartment or hamstrings. A cyclops lesion is a soft tissue mass which can form in the anterior compartment usually after anterior cruciate ligament reconstruction and has been observed after bicruciate-retaining TKA. We have treated a patient who developed progressive loss of full extension from 0° to 20° after bicruciate-retaining TKA. A large fibrous tissue mass (cyclops lesion) was identified in the anterior compartment during arthrotomy 1 year after TKA. Excision of the mass resulted in complete resolution of the flexion contracture.

HNRNPK maintains epidermal progenitor function through transcription of proliferation genes and degrading differentiation promoting mRNAs.

Maintenance of high-turnover tissues such as the epidermis requires a balance between stem cell proliferation and differentiation. The molecular mechanisms governing this process are an area of investigation. Here we show that HNRNPK, a multifunctional protein, is necessary to prevent premature differentiation and sustains the proliferative capacity of epidermal stem and progenitor cells. To prevent premature differentiation of progenitor cells, HNRNPK is necessary for DDX6 to bind a subset of mRNAs that code for transcription factors that promote differentiation. Upon binding, these mRNAs such as GRHL3, KLF4, and ZNF750 are degraded through the mRNA degradation pathway, which prevents premature differentiation. To sustain the proliferative capacity of the epidermis, HNRNPK is necessary for RNA Polymerase II binding to proliferation/self-renewal genes such as MYC, CYR61, FGFBP1, EGFR, and cyclins to promote their expression. Our study establishes a prominent role for HNRNPK in maintaining adult tissue self-renewal through both transcriptional and post-transcriptional mechanisms.

The Cohesin Complex Is Necessary for Epidermal Progenitor Cell Function through Maintenance of Self-Renewal Genes.

Adult stem and progenitor cells are critical for replenishing lost tissue due to injury or normal turnover. How these cells maintain self-renewal and sustain the tissue they populate are areas of active investigation. Here, we show that the cohesin complex, which has previously been implicated in regulating chromosome segregation and gene expression, is necessary to promote epidermal stem and progenitor cell self-renewal through cell-autonomous mechanisms. Cohesin binds to genomic sites associated with open chromatin, including DNase-I-hypersensitive sites, RNA polymerase II, and histone marks such as H3K27ac and H3K4me3. Reduced cohesin expression results in spontaneous epidermal differentiation due to loss of open chromatin structure and expression of key self-renewal genes. Our results demonstrate a prominent role for cohesin in modulating chromatin structure to allow for enforcement of a stem and progenitor cell gene expression program.

18F-fluorodeoxyglucose positron emission tomography contributes to the diagnosis and management of infections in patients with multiple myeloma: a study of 165 infectious episodes.

PURPOSE: Correctly identifying infection in cancer patients can be challenging. Limited data suggest that positron emission tomography (PET) using fluorine-18 fluorodeoxyglucose (FDG) may be useful for diagnosing infection. To determine the role of FDG-PET in the diagnosis of infection in patients with multiple myeloma (MM). PATIENTS AND METHODS: The medical records of 248 patients who had FDG-PET performed for MM staging or infection work-up revealing increased uptake at extramedullary sites and/or bones and joints that would be atypical for MM between October 2001 and May 2004 were reviewed to identify infections and evaluate FDG-PET contribution to patient outcome. RESULTS: One hundred sixty-five infections were identified in 143 adults with MM. Infections involved the respiratory tract [99; pneumonia (93), sinusitis (six)], bone, joint and soft tissues [26; discitis (10), osteomyelitis (nine), septic arthritis (one), cellulitis (six)], vascular system [18; septic thrombophlebitis (nine), infection of implantable catheter (eight), septic emboli (one)], gastrointestinal tract [12; colitis (seven), abdominal abscess (three), and diverticulitis and esophagitis (one each)], and dentition [periodontal abscess (10)]. Infections were caused by bacteria, mycobacteria, fungi, and viruses. FDG-PET detected infection even in patients with severe neutropenia and lymphopenia (30 episodes). The FDG-PET findings identified infections not detectable by other methods (46 episodes), determined extent of infection (32 episodes), and led to modification of work-up and therapy (55 episodes). Twenty silent, but clinically relevant, infections were detected among patients undergoing staging FDG-PET. CONCLUSION: In patients with MM, FDG-PET is a useful tool for diagnosing and managing infections even in the setting of severe immunosuppression.

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds.

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds' mechanisms of action--i.e., the specific molecular targets by which they kill the parasite--would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children's Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 µM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.

Safety and Efficacy of 68Ga-DOTATATE PET/CT for Diagnosis, Staging, and Treatment Management of Neuroendocrine Tumors.

UNLABELLED: Our purpose was to evaluate the safety and efficacy of (68)Ga-DOTATATE PET/CT compared with (111)In-pentetreotide imaging for diagnosis, staging, and restaging of pulmonary and gastroenteropancreatic neuroendocrine tumors. METHODS: (68)Ga-DOTATATE PET/CT and (111)In-pentetreotide scans were obtained for 78 of 97 consecutively enrolled patients with known or suspected pulmonary or gastroenteropancreatic neuroendocrine tumors. Safety and toxicity were measured by comparing vital signs, serum chemistry values, or acquisition-related medical complications before and after (68)Ga-DOTATATE injection. Added value was determined by changes in treatment plan when (68)Ga-DOTATATE PET/CT results were added to all prior imaging, including (111)In-pentetreotide. Interobserver reproducibility of (68)Ga-DOTATATE PET/CT scan interpretation was measured between blinded and nonblinded interpreters. RESULTS: (68)Ga-DOTATATE PET/CT and (111)In-pentetreotide scans were significantly different in impact on treatment (P < 0.001). (68)Ga-DOTATATE PET/CT combined with CT or liver MRI changed care in 28 of 78 (36%) patients. Interobserver agreement between blinded and nonblinded interpreters was high. No participant had a trial-related event requiring treatment. Mild, transient events were tachycardia in 1, alanine transaminase elevation in 1, and hyperglycemia in 2 participants. No clinically significant arrhythmias occurred. (68)Ga-DOTATATE PET/CT correctly identified 3 patients for peptide-receptor radiotherapy incorrectly classified by (111)In-pentetreotide. CONCLUSION: (68)Ga-DOTATATE PET/CT was equivalent or superior to (111)In-pentetreotide imaging in all 78 patients. No adverse events requiring treatment were observed. (68)Ga-DOTATATE PET/CT changed treatment in 36% of participants. Given the lack of significant toxicity, lower radiation exposure, and improved accuracy compared with (111)In-pentetreotide, (68)Ga-DOTATATE imaging should be used instead of (111)In-pentetreotide imaging where available.

Early detection of bone infection and differentiation from post-surgical inflammation using 2-deoxy-2-[18F]-fluoro-D-glucose positron emission tomography (FDG-PET) in an animal model.

Diagnosing bone infection in the context of post-surgical inflammation is problematic since many of the early signs of infection are similar to normal post-surgical changes. We used a rabbit osteomyelitis model to evaluate the use of 2-deoxy-2-[(18)F]-fluoro-d-glucose positron emission tomography (FDG-PET) as a means of detecting post-operative infection in the context of post-surgical inflammation. Comparisons were made between infected and non-infected rabbits in which infection with Staphylococcus aureus was initiated at the time of surgery. Weekly PET scans were obtained 30 and 60 min after the introduction of FDG and analyzed based on standardized uptake values (SUV) at the surgical site and visual assessment of the presence or absence of infection. Concurrent X-rays were taken immediately prior to scanning. At 4weeks post-operatively, animals were sacrificed for histologic and bacteriologic confirmation of infection. Uptake of FDG was evident in the bone of all rabbits on day 1 post-surgery, however, SUV comparisons from the surgical site could not be used to distinguish between the infected and uninfected groups until day 15. Visual analysis of FDG-PET scans revealed a significant difference (p<0.01) between the infected and uninfected groups as early as day 8. This was due in part to the ability to visualize regional lymph nodes by FDG-PET.

ErbB receptors mediate both migratory and proliferative activities in human melanocytes and melanoma cells.

Epidermal growth factor receptor (EGFR) activation by transforming growth factor alpha (TGFalpha) has been implicated in autocrine growth in melanoma, but does not alter melanocyte proliferation. This raises the possibility that different signalling pathways are activated via EGFR or ErbB receptors. Here, we demonstrate that ErbB2, ErbB3 and ErbB4 are expressed in cultured human melanocytes. Western analyses with receptor-specific antisera revealed protein bands with Mr values of 185 and 160 kDa, corresponding to ErbB2 and ErbB3, respectively. Blots probed with ErbB4 antibodies showed bands with Mr values of 180, 120 and 80 kDa, corresponding to the receptor and its reported variants. Two malignant melanoma cell lines expressed ErbB2 and ErbB3, but not the full-length ErbB4 receptor. As TGFalpha binds to EGFR and the heregulins (HRG) bind to ErbB3 and ErbB4, these growth factors were examined for effects on receptor activation and on cell growth and motility in a scratch wound closure assay. In normal melanocytes, HRGbeta1 activated the phosphorylation of tyrosine residues of proteins that immunoprecipitated with EGFR and ErbB4 antisera, and significantly enhanced cell migration but not proliferation. Neither TGFalpha nor HRGalpha1 promoted migration or growth in normal melanocytes. By contrast, TGFalpha stimulated migratory activity in the MM96L cell line, but not in the MELJG line, whereas HRGbeta1 significantly enhanced cell growth, but not migration, in both malignant cell lines. The apparent transition of HRGbeta1 from a migratory to a proliferative function after malignant transformation, and the change in TGFalpha from a non-migratory to a migratory activity in one melanoma line, suggests multiple switches in ErbB signalling pathways via EGFR/ErbB heterodimer formation.

The Bacterial Sec Pathway of Protein Export: Screening and Follow-Up.

Most noncytoplasmic bacterial proteins are exported through the SecYEG channel in the cytoplasmic membrane. This channel and its associated proteins, collectively referred to as the Sec pathway, have strong appeal as a possible antibiotic drug target, yet progress toward new drugs targeting this pathway has been slow, perhaps due partly to many researchers' focus on a single component, the SecA ATPase. Here we report on a pathway-based screen in which beta-galactosidase (ß-gal) activity is trapped in the cytoplasm of Escherichia coli cells if translocation through SecYEG is impaired. Several hit compounds passed a counterscreen distinguishing between ß-gal overexpression and impaired ß-gal export. However, the most extensively characterized hit gave limited E. coli growth inhibition (EC(50) ≥ 400 µM), and growth inhibition could not be unambiguously linked to the compound's effect on the Sec pathway. Our study and others underscore the challenges of finding potent druglike hits against this otherwise promising drug target.

Prone Versus Supine Breast FDG-PET/CT for Assessing Locoregional Disease Distribution in Locally Advanced Breast Cancer.

RATIONALE AND OBJECTIVES: Prone (18)F fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) may have advantages for breast imaging because of improved separation of deep anatomic structures. There are limited data on whether prone and supine FDG-PET/CT provide similar information regarding breast and axillary disease in the setting of locally advanced breast cancer (LABC). The purpose of this study was to compare the information on locoregional disease distribution provided by prone versus supine FDG-PET in newly diagnosed LABC. MATERIALS AND METHODS: In an Institutional Review Board-approved prospective trial, 24 patients with newly diagnosed LABC underwent both supine and prone FDG-PET/CT at the same scanning session. Three readers performed an independent review of all scans and categorized the locoregional disease distribution as breast only (BO)-unifocal, BO-multifocal, BO-multicentric, or breast + axillary involvement. For breast + axillary disease, the readers also assessed the number of involved axillary lymph nodes. Interobserver discrepancies were resolved at a consensus reading session. RESULTS: Two scanning sessions were excluded because the prone scan had omitted part of the axilla from the field of view. In the remaining 22 patients, the consensus categorization of anatomic disease distribution was concordant between prone and supine scanning in 21 patients (linear kappa 0.91, 95% confidence interval [0.79-1]). In the 16 patients with breast + axillary disease, equal numbers of involved lymph nodes were identified on prone and supine scanning in 12 patients, whereas in the remaining four patients, prone scanning resulted in a higher number of visualized lymph nodes. CONCLUSIONS: Prone and supine FDG-PET/CT provided statistically identical information on locoregional disease distribution in LABC. However, prone scanning may perform better than supine for assessing the number of involved lymph nodes. Prone FDG-PET/CT may be useful in future clinical and research efforts, including hybrid PET-magnetic resonance imaging (MRI) applications.

Introduction to PET imaging with emphasis on biomedical research.

Medical imaging is migrating from anatomic imaging to functional imaging and fused anatomic/functional imaging. The technology is being adapted for biomedical research using both clinical and small animal scanners. The ability to externally image real-time physiologic processes in both normal and deranged conditions, including various models to image gene expression, apoptosis, or drug biodistribution, has powerful impact on the exploration of biomedical and fundamental biological research. Positron emission tomography (PET) has a unique ability to not only provide such images but also to do so with high resolution (typically 1-2mm resolution for small animal scanners) and to provide both relative and absolute quantitation. This technology is revolutionizing biomedical and biological research. This article reviews the underlying principles involved in this technology, gives a brief history of its development, and then introduces the interested researcher to some of the important techniques that could be of use.

Diagnosis of infection of implantable central venous catheters by [18F]fluorodeoxyglucose positron emission tomography.

OBJECTIVE: The aim of this study was to evaluate the role of [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging in the diagnosis of infection of implantable vascular catheters. METHODS: We evaluated six patients with haematological cancer and infection of their implantable vascular catheter and who underwent FDG PET imaging around the time of their infection. RESULTS: Six patients with multiple myeloma who developed infection of their implantable device (five port pocket infections and one tunnel infection) were identified. FDG PET revealed increased uptake at the site of the implantable catheter (SUV 2.7-4.5) in all six patients, even in the absence of signs or symptoms of infection at the site of the device (three), and the presence of severe neutropenia (four). The three patients who did not have local inflammation at the site of the device were profoundly neutropenic. The FDG PET diagnosis led to removal of the device in two patients. CONCLUSION: FDG PET is a safe, rapid and accurate tool for diagnosing infection of an implantable catheter, including among those patients not exhibiting local signs and symptoms of infection, and in whom the diagnosis of infected device may be difficult. FDG PET may help prevent the unnecessary removal of implantable intravascular catheters and the unwarranted use of antibiotics.

Imaging of multiple myeloma and related plasma cell dyscrasias.

Multiple myeloma (MM) is an incurable plasma cell malignancy of the bone marrow. MM has 3 components: diffuse marrow infiltration, focal bone lesions, and soft-tissue (extramedullary) disease. The hallmark biomarker in blood or urine is a monoclonal immunoglobulin, the monoclonal protein. Waldenstrom macroglobulinemia is a similar disease with secretion of IgM. Staging is classically performed with the 1975 Durie-Salmon system, which includes conventional radiographs. Recently updated, the Durie-Salmon Plus staging system includes CT, MRI, and (18)F-FDG PET/CT. The hallmark radiographic lesion of symptomatic MM is a well-demarcated, focal osteolytic bone lesion. The number of focal bone lesions correlates inversely with outcome. Extramedullary disease is typically an aggressive, poorly differentiated form of MM that confers inferior outcome, with median survival of less than 1 y if present at diagnosis. Achievement of a complete response on (18)F-FDG PET before stem-cell transplantation correlates with a superior outcome.

New imaging tools for the diagnosis of infection.

Infection imaging became widespread in 1971 with the release of 67Ga citrate. Multiphase skeletal scintigraphy and radiolabeled white blood cells (WBCs) have since become the most widespread clinically used agents for the imaging of infection. A wide variety of other radiolabeled probes are under investigation, based on antibodies, cytokines, assorted proteins and other molecules, alone or in various combinations. However, these latter agents, with a few exceptions, are not routinely used clinically. Radiolabeled ciprofloxacin represents the first attempt to develop an infection-specific imaging agent (most infection-imaging probes localized nonspecifically to inflammation as well), but it has not proven superior to radiolabeled WBCs or 18F-fluoro-deoxy-glucose (FDG) PET. Because of the ability to combine exquisite anatomic detail with focal uptake of 18F-FDG, PET-computed tomography has achieved great success in the detection and localization of infection, including in clinically adverse conditions. Despite these advances, at this time an infection-specific imaging agent does not exist.