Search

Herpes simplex virus (HSV) encephalitis has a predilection for the temporal and frontal lobes but occasionally affects the brainstem. We describe a patient who developed HSV brainstem encephalitis that progressed to quadriplegia. Using MEDLINE, we conducted a comprehensive review of other published cases of HSV brainstem encephalitis. Twenty-four published cases of HSV brainstem encephalitis met our inclusion criteria. The mean age was 41.4 years (range 18-71). HSV-1 was the etiologic agent in 79% of reported HSV brainstem encephalitis cases, and HSV-2 accounted for 21% of cases. Infection was limited to the brainstem in 29% of cases and multi-focal, including the brainstem, in 71%. Common manifestations of HSV brainstem encephalitis included neuro-ophthalmologic findings (81%), cranial nerve deficits (69%), and fever (69%). Quadriplegia, as occurred in our patient, was an unusual finding (19%). The mortality rate of HSV brainstem encephalitis was 41%. Intravenous acyclovir showed a beneficial effect on mortality (75% vs. 22%, p = 0.06). HSV brainstem encephalitis is a distinct type of HSV encephalitis. With the increasing use of HSV-PCR, more cases of HSV brainstem encephalitis may be identified. A greater recognition of this syndrome will help better define its optimal treatment and prognosis.

Coagulase-negative staphylococcal species are generally considered commensal inhabitants of the skin. Often regarded as avirulent organisms, certain species such as Staphylococcus lugdunensis have been identified as the cause of severe human infections including infective endocarditis. We report on a case of a native valve, S. lugdunensis infective endocarditis relapse after initial conservative medical therapy. A literature review of the outcomes of treatment was also performed. Analyzing definitive medical versus medical-surgical treatment of native valve infective endocarditis, an odds ratio of mortality of 3.1875 (95% confidence interval [CI], 1.1855-8.5705; P < 0.03) was obtained and was significant based on 2-tailed Fisher exact probability test. This analysis reveals an increase risk of mortality with medical therapy alone versus medical-surgical therapy for native valve infective endocarditis. Our case and review of the literature emphasize the growing evidence that early medical treatment and surgical evaluation and removal of infected foreign hardware is required to prevent significant morbidity and mortality.

Background: Sexually transmissible infections (STI) have been variably associated with increased risks of prostate cancer, largely in case-control studies. Methods: In the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we examined risk of prostate cancer in relation to serum antibodies to Chlamydia trachomatis, human papillomavirus-16 and -18, herpes simplex virus-2, cytomegalovirus, and human herpesvirus-8 in 868 cases (765 Whites and 103 Blacks) and 1,283 controls matched by race, age, time since initial screening, and year of blood draw; all blood samples were collected at least 1 year before prostate cancer diagnosis, except for 43 Black cases. We also assessed risk associated with self-reported history of syphilis and gonorrhea. Results: Prevalences of the 7 STIs among controls were weakly correlated, and all were more frequent among Blacks than Whites, except for human herpesvirus-8. Among Whites, prostate cancer risk was not significantly associated with the individual infections or with their number (Ptrend = 0.1); however, men with one or more STI had slightly higher risk (odds ratio, 1.3; 95% confidence interval, 1.0-1.6). Among Blacks, excess risk was associated with IgA antibody to C. trachomatis (odds ratio, 2.1; 95% confidence interval, 1.2-3.6). Conclusion: This large prospective study of prostate cancer shows no consistent association with specific STIs and a borderline association with any versus none. Whether a shared response or correlated infection not directly measured underlies the weak association requires further study. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2374–81)

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.

Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.

Pluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo . Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications. Statement of Significance Directing stem cell differentiation and morphogenesis via biomaterials represents a novel strategy to promote cell fates and tissue formation. Our study demonstrates the ability of calcium phosphate-based mineral particles to promote osteochondrogenic differentiation of embryonic stem cell aggregates as well as modulate teratoma formation in vivo . This hybrid biomaterial–ESC aggregate approach serves as an enabling platform to evaluate the ability of biomaterials to regulate stem cell fate and regenerate functional skeletal tissues for clinical applications.

Young pregnant women are a group at particularly high risk of STDs.1,2 In a systematic literature review of sexual risk and STDs among pregnant teenagers and those who have given birth, STD incidence was 19−39% during pregnancy, and 14−39% between six and 10 months postpartum. In addition, adolescent mothers were twice as likely as their nulliparous peers to acquire an STD during a 12-month period. Furthermore, 78−88% of pregnant women engaged in sex without condoms during pregnancy, and pregnant women were only about one-fifth as likely as nonpregnant women to use condoms.3 Current guidelines from the Centers for Disease Control and Prevention (CDC) recommend STD screening for all high-risk women-usually defined as those younger than 25 and those with multiple or new partners.4 Frequently, screening for all high-risk pregnant women occurs only during initial prenatal care visits, so an infection that is acquired later in pregnancy may go undetected. Some clinics retest women who are deemed high-risk (e.g., those testing STD-positive at their initial visit) or exhibit symptoms; however, testing procedures differ from clinic to clinic, and screening behavior can vary by provider.5 In addition, because an STD diagnosis and treatment are not enough to have long-term effects on risky behavior,6 an emphasis on prevention is necessary to achieve long-term risk reduction. Prevention programs integrated with prenatal care can capitalize on women's motivation to have a healthy pregnancy and child. HIV and other STD prevention programs have been successfully integrated in psychiatric care, drug treatment and palliative medicine settings.7–9 This model for prevention could be extended to include prenatal care. For interventions to be most effective in clinical settings, they should be easy to implement and tailored to meet the needs and risks of particular patients. Thus, it may be beneficial to identify subgroups of women at varying risk of STDs during pregnancy, and implement treatment, care and prevention programs that best meet the needs of those individuals. To successfully do this, providers need to go beyond the typical characteristics obtained during prenatal care assessments. A 2002 Institute of Medicine report and more recent research syntheses suggest that ecological approaches to health, and to STD prevention, are particularly useful and needed.3,10,11 Ecological systems theory emphasizes that factors from many levels (e.g., individual, dyad, family and community) can influence health.3,10–12 The few studies on young people's sexual activity that have included factors from many levels have shown that dyad, family and community factors independently contribute to risk behavior above and beyond individual factors.13–15 In addition to using primarily individual-level predictors, most studies that have tried to estimate STD risk have used standard analytic techniques (e.g., logistic regression). However, these methods are limited because they assume primarily linear relationships between the predictors and outcomes that may not be realistic in applied settings. Furthermore, results from these techniques (e.g., odds ratios in logistic regression) are difficult to translate into clinical practice and policy.16 Classification tree analysis, also called recursive partitioning, avoids the limitations associated with logistic regression and other standard methods. The technique uses a set of predictors to create subgroups of individuals that vary in risk for the main outcome of interest, ultimately producing a user-friendly classification tree that can be employed to guide care, treatment and prevention efforts in clinical settings. For example, in a study of young adults at an STD clinic, several subgroups were classified as having a high STD risk because of particular behavioral and emotional factors (e.g., one group felt bad about themselves after having sex and had sex to relieve tension; one group fell bad about themselves after having sex and had sex to get back at someone).17 Use of classification tree analysis has been documented in the health care and managed care literature,18 and has been used in many clinical and medical settings to identify high-risk patients and to help create clinical guidelines.19–23 Clinicians often use informal decision trees when making clinical decisions. On the basis of a complex interaction of medical, demographic and behavioral factors, clinicians determine the likelihood of disease outcomes and often use these patient characteristics to guide care and treatment decisions (e.g., if a woman is having risky sex and is younger than 25, the clinician may screen for STDs because past experience suggests that the patient is at risk). The purpose of this study is to use individual, dyad, and family- and community-level characteristics to develop a clinically relevant classification tree that will help identify women who are at risk of acquiring an STD during pregnancy and will provide a formal decision-making structure for STD prevention, treatment and care.

The progressive loss of endogenous regenerative capacity that accompanies mammalian aging has been attributed at least in part to alterations in the extracellular matrix (ECM) composition of adult tissues. Thus, creation of a more regenerative microenvironment, analogous to embryonic morphogenesis, may be achieved via pluripotent embryonic stem cell (ESC) differentiation and derivation of devitalized materials as an alternative to decellularized adult tissues, such as demineralized bone matrix (DBM). Transplantation of devitalized ESC materials represents a novel approach to promote functional tissue regeneration and reduce the inherent batch-to-batch variability of allograft-derived materials. In this study, the osteoinductivity of embryoid body-derived material (EBM) was compared to DBM in a standard in vivo ectopic osteoinduction assay in nude mice. EBM derived from EBs differentiated for 10 days with osteogenic media (+β-glycerophosphate) exhibited similar osteoinductivity to active DBM (osteoinduction score = 2.50 ± 0.27 vs. 2.75 ± 0.16) based on histological scoring and exceeded inactive DBM (1.13 ± 0.13, p de novo bone formation in vivo.

Matrix metalloproteinases (MMPs) remodel the extracellular matrix (ECM) to facilitate epithelial-to-mesenchymal transitions (EMTs) and promote cell specification during embryonic development. In this study, we hypothesized that introducing degradable ECM-based biomaterials to pluripotent stem cell (PSC) aggregates would modulate endogenous proteolytic activity and consequently enhance the differentiation and morphogenesis within 3D PSC aggregates. Gelatin methacrylate (GMA) microparticles (MPs) of low (∼20%) or high (∼90%) cross-linking densities were incorporated into mouse embryonic stem cell (ESC) aggregates, and the effects on MMP activity and cell differentiation were examined with or without MMP inhibition. ESC aggregates containing GMA MPs expressed significantly higher levels of total MMP and MMP-2 than aggregates without MPs. GMA MP incorporation increased expression of EMT markers and enhanced mesenchymal morphogenesis of PSC aggregates. MMP inhibition completely abrogated these effects, and GMA MP-induced MMP activation within ESC aggregates was partially reduced by pSMAD 1/5/8 inhibition. These results suggest that GMA particles activate MMPs by protease-substrate interactions to promote EMT and mesenchymal morphogenesis of ESC aggregates in an MMP-dependent manner. We speculate that controlling protease activity via the introduction of ECM-based materials may offer a novel route to engineer the ECM microenvironment to modulate stem cell differentiation.

Background Whether community-associated methicillin-resistant Staphylococcus aureus (MRSA) genotypes (e.g., USA300) are a major cause of bloodstream infections (BSIs) and health care-associated infections has been poorly defined. Methods Consecutive MRSA isolates recovered from patients with BSIs were prospectively collected at an urban public hospital. Molecular typing studies were performed. Prevalence and risk factors for the MRSA USA300 genotype were assessed. Results One hundred thirty-two cases of MRSA BSI were documented over 7.5 months in 2004 (incidence, 6.79 per 1000 admissions); 116 isolates were available for genotyping. Characteristics of the 116 evaluable cases included: a mean age 47 years; 62% were male, 82% were African American, and 22% were HIV seropositive. The crude in-hospital mortality rate was 22%. In 107 cases (92%), there was contact with a health care facility within the year prior to infection, and a nosocomial infection (defined as positive blood culture results obtained >48 h after admission) occurred in 49 cases (42%). PFGE demonstrated that 39 (34%) of the 116 isolates were the MRSA USA300 genotype; 34 (29%) were USA100; 42 (36%) were USA500; and 1 (1%) was USA800. MRSA USA300 accounted for 28% of health care-associated BSIs and 20% of nosocomial MRSA BSIs. In multivariate analysis, isolation of the USA300 genotype was associated with injectiondrug use (OR, 3.67; 95% CI, 1.10-12.28) and skin and soft tissue infection (OR, 4.26; 95% CI, 1.08-16.84). Patients who resided in long-term care facilities (OR, 0.09; 95% CI, 0.01-0.82) and those who were treated with antimicrobials in the prior year were less likely to have MRSA USA300 genotype recovered (OR, 0.10; 95% CI, 0.02-0.49). Conclusions MRSA USA300 genotype, the predominant cause of community-associated MRSA infections in our area (Atlanta, GA), has now emerged as a significant cause of health care-associated and nosocomial BSI. MRSA USA300 as a nosocomial pathogen presents new challenges to infection control programs.

To the Editor: Multidrug-resistant tuberculosis (MDR TB), i.e., TB resistant to at least the 2 most effective first-line antituberculous drugs (isoniazid [INH] and rifampin [RIF]), is increasing globally. World Health Organization estimations of 390,000–510,000 new MDR TB cases and 150,000 related deaths in 2008 highlight the need for timely drug susceptibility testing and improved therapies (1). Although novel rapid drug susceptibility testing tools are increasingly available, their clinical applicability is unsettled. We report a patient with pulmonary TB relapse with discordant genotypic and in vitro phenotypic drug susceptibility testing results associated with a mutation outside the RIF resistance determining region (RRDR) of the rpoB gene. In August 2009, a 45-year-old homeless woman with AIDS (CD4+ T-cell count 3 cells/mm3) and a history of substance abuse sought care for fever, night sweats, weight loss, and cough (Table A1). Pulmonary TB had been diagnosed in June 2008. At that time, she received, by directly observed therapy, 6 weeks of INH, RIF, pyrazinamide (PZA), and ethambutol (EMB) through the local health department and was switched to RIF, PZA, and EMB on week 7 after the isolate was determined by liquid culture with BD BACTEC MGIT 960 Mycobacterial Testing System (BD Diagnostics, Sparks, MD, USA) to be INH resistant. During that period, she resided in American Lung Association–supported housing for TB patients and had 97% medication adherence by dose count. Her condition clinically improved, infiltrates completely resolved according to chest radiograph, 12 sputum inductions failed to yield sufficient material for analysis, and she began highly active antiretroviral therapy (HAART). In December 2008, however, because of crack cocaine use and belligerent behavior, she lost housing privileges. Caseworkers could not locate her to complete the 9-month planned directly observed therapy. The woman was hospitalized in January and again in February 2009 with dyspnea and off medications. In both instances, chest radiographs showed no new changes, sputum specimens were negative for acid-fast bacilli (AFB) by microscopy and culture, and she was treated for presumptive Pneumocystis pneumonia and showed clinical improvement. In August 2009, she was readmitted with cough and new cavitation on chest radiograph. Chest computed tomographic scan demonstrated right upper lobe infiltrates, bilateral lower lobe cavitation, and hilar and mediastinal lymphadenopathy. Sputum AFB smear was positive (graded 4+), and nucleic acid amplification (Amplified Mycobacterium Direct Test; Gen-Probe, San Diego, CA, USA) was positive for Mycobacterium tuberculosis complex. INH, RIF, PZA, and EMB, along with moxifloxacin (MXF) and amikacin (AMK), were initiated in accordance with 2003 national TB treatment guidelines for possible MDR TB (2). Shortly thereafter, a line probe assay (GenoType MTBDRplus; HAIN Lifescience, Nehren, Germany) performed by Southeastern National TB Center (Gainesville, FL, USA) on the culture of the sputum specimen obtained at admission indicated an inhA point mutation but no mutation in the RRDR region of the rpoB gene, which suggested that the isolate was INH resistant but RIF susceptible. AMK was discontinued, and the patient was discharged with RIF, PZA, EMB, and MXF. One week later, drug susceptibility testing (BD BACTEC MGIT 960 System) results from the state mycobacteriology laboratory demonstrated that the M. tuberculosis isolate was resistant to INH and RIF. The patient was readmitted to resume injectable aminoglycoside therapy. After 5 weeks, sputum culture became negative, clinical and radiographic improvement was apparent, and HAART was reinitiated. She completed 2 months’ INH/PZA/EMB/MXF/AMK inpatient therapy and was discharged to complete 6 additional months of PZA/EMB/MXF and streptomycin followed by 16 months of PZA/EMB/MFX. With HAART, her plasma HIV RNA viral load became undetectable, but her CD4 count remained low (9 cells/mm3). She died from a motor vehicle accident 10 months after recurrent TB was diagnosed. In this patient, RIF resistance was not predicted by line probe assay but was identified phenotypically by an automated system (BD BACTEC MGIT 960 System) that continuously monitors for growth and detection of mycobacteria. Through genotyping and DNA sequencing of the 2008 and 2009 M. tuberculosis isolates, the Mycobacteriology Laboratory Branch at the Centers for Disease Control and Prevention (Atlanta, GA, USA) established that the 2009 infection was a relapse, not re-infection, and confirmed the inhA mutation in both isolates. Using primers extending beyond the RRDR (the rpoB region surveyed by rapid molecular tests and responsible for >95% of RIF resistance mutations) the laboratory identified a novel rpoB gene mutation at codon 480 (ACC→AAC; Thr→Asn) and another previously described (3–5) mutation at codon 176 (GTC→TTC; Val→Phe) in the 2009 isolate, which has been implicated in RIF resistance. The role of the T480N mutation in RIF resistance is being investigated. This case demonstrates the limitations of rapid molecular drug susceptibility testing (6). Rapid molecular diagnostics are valuable adjuncts to conventional phenotypic testing because they can quickly confirm clinically suspected MDR TB and have high agreement with other genotypic and phenotypic methods (7–10). However, they should not supplant phenotypic testing, and clinicians should understand their limitations. When rapid molecular tests are negative but suspicion for MDR TB is high, MDR TB treatment should be continued until phenotypic susceptibility results are available. DNA sequencing may be best suited for evaluating suspected drug-resistant M. tuberculosis isolates with discordant results for phenotypic susceptibility and rapid molecular testing.

Introduction The Centers for Disease Control and Prevention has recommended using a nucleic acid amplification test (NAAT) for diagnosing pulmonary tuberculosis (TB) but there is a lack of data on NAAT cost-effectiveness. Methods We conducted a prospective cohort study that included all patients with an AFB smear-positive respiratory specimen at Grady Memorial Hospital in Atlanta, GA, USA between January 2002 and June 2008. We determined the sensitivity, specificity, and positive and negative predictive value of a commercially available and FDA-approved NAAT (amplified MTD, Gen-Probe) compared to the gold standard of culture. A cost analysis was performed and included costs related to laboratory tests, hospital charges, anti-TB medications, and contact investigations. Average cost per patient was calculated under two conditions: (1) using a NAAT on all AFB smear-postive respiratory specimens and (2) not using a NAAT. One-way sensitivity analyses were conducted to determine sensitivity of cost difference to reasonable ranges of model inputs. Results During a 6 1/2 year study period, there were 1,009 patients with an AFB smear-positive respiratory specimen at our public urban hospital. We found the NAAT to be highly sensitive (99.6%) and specific (99.1%) on AFB smear-positive specimens compared to culture. Overall, the positive predictive value (PPV) of an AFB smear-positive respiratory specimen for culture-confirmed TB was 27%. The PPV of an AFB smear-positive respiratory specimen for culture-confirmed TB was significantly higher for HIV-uninfected persons compared to those who were HIV-seropositive (152/271 [56%] vs. 85/445 [19%]; RR = 2.94, 95% CI 2.36–3.65, p

Objectives Nosocomial pathogens such as Acinetobacter baumannii are a growing public health threat, due in part to their increasing resistance to antibiotics. Since some strains are resistant to all available antibiotics, novel therapies are urgently needed. Plasmablasts are short-lived B cells found in the blood that can be collected and harnessed to produce therapeutic antibodies. We set out to determine whether plasmablasts are induced during infection with A. baumannii and other nosocomial pathogens. Methods We obtained blood samples from patients infected with antibiotic-resistant nosocomial pathogens, and analysed their plasmablast response by flow cytometry. Results We observed a strong induction of plasmablasts in patients with antibiotic-resistant A. baumannii infection. Furthermore, plasmablasts were also induced in response to other drug-resistant nosocomial pathogens. Conclusions These data suggest that plasmablasts may be broadly harnessed to develop therapeutic antibodies to combat otherwise untreatable antibiotic-resistant infections.

1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3 +/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3 +/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3 +/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3 +/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3 +/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development.

Clinical efforts to repair cartilage defects delivering cells or engineered cartilage implants into the lesions have met with limited success. This study used a critical-size chondral defect model in immunocompromised rat xiphoid cartilage to test whether endogenous chondrogenesis could be achieved using human bone matrix scaffolds to deliver human cartilage particles and/or a variant isoform of fibroblast growth factor-2 (FGF2-variant). Seventy-two male athymic RNU rats were enrolled in this study with eight rats per experimental group. Decellularized and demineralized human bone matrix scaffolds loaded with human articular cartilage particles or heat-inactivated cartilage particles were combined with different doses of the FGF2-variant. Scaffolds were implanted into 3-mm-diameter critical-size defects prepared using a biopsy punch through the center of the xiphoid. The samples were evaluated 28 days postsurgery using X-ray, equilibrium partitioning of ionic contrast microcomputed tomography, and safranin O-stained histological sagittal sections. Scaffolds containing cartilage particles plus the FGF2-variant induced dose-dependent increases in the formation of neocartilage (p0.05), which was distributed homogeneously throughout the defects in comparison to scaffolds containing only the FGF2-variant. These effects were less pronounced when scaffolds with heat-inactivated cartilage particles were used. These results demonstrate that endogenous repair of chondral defects can be achieved in the absence of exogenous cells or bone marrow, suggesting that a similar approach may be successful for treating chondral lesions clinically.

Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)(2)D(3). In intestinal epithelium and chondrocytes, 1,25(OH)(2)D(3) stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)(2)D(3) stimulates phospholipase A(2) (PLA(2))-dependent rapid release of prostaglandin E(2) (PGE(2)), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3. Pdia3 was present in caveolae based on co-localization with lipid rafts and caveolin-1. In Pdia3-silenced (Sh-Pdia3) cells, 1,25(OH)(2)D(3) failed to stimulate PKC and PGE(2) responses; in Pdia3-overexpressing cells (Ov-Pdia3), responses to 1,25(OH)(2)D(3) were augmented. Downstream mediators of Pdia3, PLA(2)-activating protein (PLAA) and arachidonic acid, stimulated similar PKC activation in wild-type, Sh-Pdia3, and Ov-Pdia3 cells supporting the hypothesis that Pdia3 mediates the membrane action of 1,25(OH)(2)D(3). Treatment of MC3T3-E1 cells with 1,25(OH)(2)D(3) for 9 min stimulated rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased expression of alkaline phosphatase, MMP-13, and osteopontin but decreased expression of osteocalcin, osteoprotegerin (mRNA and protein), and smad2. These effects were attenuated in Sh-Pdia3 cells. Sh-Pdia3 cells produced higher numbers of von Kossa-positive nodules and alizarin red-positive nodules compared with WT cells with or without 1,25(OH)(2)D(3) treatment whereas Ov-Pdia3 did not show any mineralization. Our data suggest Pdia3 is an important initiator of 1,25(OH)(2)D(3)-stimulated membrane signaling pathways, which have both genomic and non genomic effects during osteoblast maturation.

1,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] is a critical regulator of bone development. Protein disulfide isomerase A3 (Pdia3) is a multifunctional protein that has been associated with rapid membrane-initiated signalling by 1alpha,25(OH)2D3 in several cell types. To identify the physiological roles of Pdia3 in skeletal development, we generated Pdia3-deficient mice. No homozygous mice were observed at birth, indicating that the targeted disruption of the Pdia3 gene resulted in embryonic lethality. Pdia3 deficiency also resulted in skeletal manifestations as revealed by muCT analysis of femurs from 15-week-old heterozygous mice. The Pdia3+/- mice had increased metaphyseal bone volume and trabeculae compared to Pdia3+/+ mice. In contrast, the area and thickness of cortical bone at the femoral mid-diaphysis of Pdia3+/+ mice significantly exceeded that of Pdia3+/- mice. In vitro studies in osteoblast-like MC3T3-E1 cells showed that silencing of Pdia3 abolished 1alpha,25(OH)2D3-induced rapid activation of protein kinase C (PKC) while overexpression of Pdia3 resulted in augmentation of PKC activity by 1alpha,25(OH)2D3. Taken together, these data indicated that Pdia3 plays a crucial role in 1alpha,25(OH)2D3-regulated bone formation and the Pdia3-PKC signalling pathway might be involved in this process.

Autologous cell-based tissue engineering using three-dimensional porous scaffolds has provided a good option for the repair of cartilage defects. Silk fibroin-based scaffolds are naturally degradable materials with excellent biocompatibility and robust mechanical properties, indicating potential applications in cartilage tissue engineering. In this study, silk fibroin scaffolds prepared by freeze-drying (FD) and salt-leaching (SL300 and SL500) were fully characterized and used to study the effects of silk fibroin scaffold properties on chondrocyte attachment, proliferation and differentiation. The synergistic effects of scaffold properties and hydrodynamic environment generated by in vitro rocking culture were also investigated using static cultures as control. FD scaffolds with small pore size and lower porosity increased cell attachment but inhibited cell penetration and limited cell proliferation and differentiation. In contrast, SL scaffolds displaying a bigger pore size, higher porosity and crystallinity resulted in homogenous cell distribution, increasing cell proliferation and advanced chondrocyte differentiation in terms of their spherical morphology, predominant chondrogenic gene expression and abundant cartilaginous extracellular matrix production. A hydrodynamic environment was beneficial to chondrocyte proliferation, differentiation, and integrin gene expression in a pore size dependent manner with superior cartilage matrix production but limited hypertrophic differentiation obtained using chondrocyte-seeded SL500 scaffolds. Integrin alpha5beta1 might mediate these effects. Chondrocyte/SL500 silk fibroin constructs obtained under in vitro rocking culture might serve as an excellent implant for in vivo cartilage defect reparation.

Background: The etiology and outcomes of blood-stream infections (BSI) among paediatric patients is not well described in resource-limited countries including Georgia. Methodology: Patients with positive blood cultures at the largest paediatric hospital in the country of Georgia were identified by review of the medical and laboratory records of patients who had blood for cultures drawn between January 2004 and June 2006. Results: Of 1,693 blood cultures obtained during the study period, 338 (20%) were positive; of these, 299 were included in our analysis. The median age was 14 days from a range of 2 days to 14 years of which 178 (60%) were male; 53% of the patients with a positive culture were admitted to the Neonatal Intensive Care Unit (NICU). Gram-negative bacilli (GNB) represented 165 (55%) of 299 cultures. Further speciation of 135 (82%) of 165 Gram-negative rod (GNR) was not possible because of lack of laboratory capacity. Overall, mortality was 30% (90 of 299). Among the 90 children who died, 80 (89%) were neonates and 68 (76%) had BSI caused by Gram-negative organisms. In multivariate analysis, independent risk factors for in-hospital mortality included an age of less than 30 days (OR=4.00, 95% CI 1.89-8.46) and having a positive blood culture for a Gram-negative BSI (OR = 2.38, 95% CI 1.32-4.29). Conclusions: A high mortality rate was seen among children, particularly neonates, with positive blood cultures at the largest paediatric hospital in Georgia. Because of limited laboratory capacity, microbiological identification of common organisms known to cause BSI in children was not possible and susceptibility testing was not performed. Improving the infrastructure of diagnostic microbiology laboratories in countries with limited resources is critical in order to improve patient care and clinical outcomes, and from a public health standpoint, to improve surveillance activities.

Background.A cluster of patients with respiratory cultures positive for Pseudomonas aeruginosa with a unique antibiogram was observed during June and July 2007 at a 1,000-bed urban teaching hospital in Atlanta, Georgia. These P. aeruginosa isolates were recovered from bronchoscopically obtained specimens.Methods.A cross-sectional study was performed to assess whether the cluster was associated with exposure to a particular bronchoscope (B1); cultures from specimens from the bronchoscopes and the environment were obtained, and the P. aeruginosa isolate type was determined using pulsed-field gel electrophoresis (PFGE). Records of patients exposed to B1 during the cluster period were reviewed.Results.Twelve patients with a culture positive for P. aeruginosa with the unique susceptibility pattern were identified in June-July 2007. No cases were documented from March 1 through May 31, 2007. Culture specimens obtained from B1 after high-level disinfection revealed P. aeruginosa, prompting removal of B1 from service on July 23, 2007. No cases occurred after that date. Eleven (55%) of 20 patients who were exposed to Bl during the cluster period had a culture positive for P. aeruginosa, compared with 1 (2%) of 53 patients who were exposed to other bronchoscopes (P < .001). PFGE patterns for P. aeruginosa isolates obtained from case patients and from B1 were identical. An engineering evaluation of B1 documented several internal damages. Two (10.5%) of 19 patients exposed to Bl during the cluster period may have developed P. aeruginosa infection following exposure to B1.Conclusions.An outbreak or pseudo-outbreak of P. aeruginosa infection occurred in association with use of a damaged bronchoscope. Periodic engineering maintenance may be needed to prevent bronchoscope contamination that is resistant to high-level disinfection.

In addition to basic microbiological methods, such as microscopy and culture, to detect pathologic organisms, antigen or antibody detection methods by immunoassay and nucleic acid detection by amplification technology have been developed, are commonly used, and will be expanded further for rapid and accurate diagnosis of the common or newly emerging infection-causing agents, such as viruses, in clinical as well as public health laboratories. Since the first competitive radioimmunoassay was developed more than 40 years ago for human insulin detection (Yalow and Berson, 1960), immunoassays have been developed with emphasis on fast and sensitive detection technologies and automated systems. Due to the demand of large-scale screening for epidemiology, blood bank, prenatal care, and diagnosis of HIV and hepatitis, more immunodiagnostic procedures are performed using instruments and reagents similar to traditional immunochemistry platforms, including tests for oncology, toxicology, cardiology, and endocrinology. Immunoassays for detection of host-produced antibodies directed against microorganisms, particularly viruses, is now one of the most widely used analytical techniques in laboratory medicine (Andreotti et al., 2003; Peruski and Peruski, 2003). This chapter will review the antibody detection assays, limitations for detection and identification of infectious agents, and look into the application of those technologies with emphasis on development of detection methods such as chemiluminescent and multianalyte profile (xMAP), automation and their clinical application in the areas of diagnosing HIV, hepatitis, and other viral infections.

After Hurricane Katrina, 50 patients were evacuated to Grady Memorial Hospital in Atlanta, Georgia, with limited medical records. The infection control department ordered contact precautions for 16 Patients. Surveillance cultures performed on admission identified colonization with multidrug-resistant (MDR) bacteria in 9 patients (18%). Presence of a wound was the strongest predictor for MDR colonization. More data are needed to reliably predict MDR bacterial colonization.

Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells.We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions.Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.

Background African American and Latina women in the United States suffer from sexually transmitted infections at higher rates than white women. It is particularly important to prevent HPV in these groups as they also suffer disproportionately from cervical cancer. From 2006, a prophylactic HPV vaccine has been approved for use in girls and women aged 9–26 years. However, public health focus has been on young girls and teens. There are limited options for young women over 19 years of age who have aged out of the Vaccines for Children entitlement program. The objectives of this study were to assess a sample of minority women who attend neighbourhood health centers to determine predictors of vaccination, and to assess for the presence of HPV infection in these women. Those without evidence of prior HPV infection may benefit from a prophylactic vaccine. Methods Between April 2009 and April 2010, we enrolled a convenience sample of 100 African American and 100 Latina women who completed a computer-assisted personal interview. Participants were queried regarding: demographics, risk for sexually transmitted infections including drug or alcohol use, HPV vaccine willingness, knowledge, attitudes and beliefs, and vaccination status. Frequencies were calculated using SAS, version 9.2. Self-collected vaginal swab samples from 118 participants were tested for HPV using line probe assay. Results Participants were poor with 113 (57%) having a household income of Conclusions Three years after vaccine approval, the majority in a sample of vulnerable women had not been vaccinated despite their willingness. Public health campaigns have been successful at raising awareness and making vaccine acceptable, but may be less successful at providing the vaccine to vulnerable women. Strategies should focus on delivering vaccine to African American and Latina women in order to decrease cervical cancer disparities.

Background Foreign-born, HIV-infected persons are at risk for sub-clinical parasitic infections acquired in their countries of origin. The long-term consequences of co-infections can be severe, yet few data exist on parasitic infection prevalence in this population. Methodology/Principal Findings This cross-sectional study evaluated 128 foreign-born persons at one HIV clinic. We performed stool studies and serologic testing for strongyloidiasis, schistosomiasis, filarial infection, and Chagas disease based on the patient's country of birth. Eosinophilia and symptoms were examined as predictors of helminthic infection. Of the 128 participants, 86 (67%) were male, and the median age was 40 years; 70 were Mexican/Latin American, 40 African, and 18 from other countries or regions. Strongyloides stercoralis antibodies were detected in 33/128 (26%) individuals. Of the 52 persons from schistosomiasis-endemic countries, 15 (29%) had antibodies to schistosome antigens; 7 (47%) had antibodies to S. haematobium, 5 (33%) to S. mansoni, and 3 (20%) to both species. Stool ova and parasite studies detected helminths in 5/85 (6%) persons. None of the patients tested had evidence of Chagas disease (n = 77) or filarial infection (n = 52). Eosinophilia >400 cells/mm3 was associated with a positive schistosome antibody test (OR 4.5, 95% CI 1.1–19.0). The only symptom significantly associated with strongyloidiasis was weight loss (OR 3.1, 95% CI 1.4–7.2). Conclusions/Significance Given the high prevalence of certain helminths and the potential lack of suggestive symptoms and signs, selected screening for strongyloidiasis and schistosomiasis or use of empiric antiparasitic therapy may be appropriate among foreign-born, HIV-infected patients. Identifying and treating helminth infections could prevent long-term complications., Author Summary Undiagnosed and untreated parasitic infections can have severe consequences for human immunodeficiency virus (HIV)-infected persons. An estimated 2 billion people worldwide are infected with soil-transmitted helminths and schistosomiasis, yet there are few data on the prevalence in HIV-infected immigrants to more developed countries. This information could help clinicians determine what testing is needed and what signs or symptoms to expect. We performed serologic, stool, and urine testing for selected parasites in 128 foreign-born persons receiving care at an HIV clinic in Atlanta, Georgia. We found that 26% had serologic evidence of infection with Strongyloides stercoralis and 29% had serologic evidence of schistosomiasis. Because these were likely chronic processes, symptoms and signs were often absent; only weight loss was significantly associated with strongyloidiasis. High eosinophil counts were also associated with parasitic infection. This study suggests the need for targeted screening of foreign-born, HIV-infected persons for parasitic infections (mainly strongyloidiasis and schistosomiasis) or the use of empiric antiparasitic therapy, particularly among those with unexplained eosinophilia. Although there are established guidelines for screening of refugees, health care providers should consider the risk of these organisms in patients who have entered the United States through other pathways.