PRRSV-2 viral load in critical non-lymphoid tissues is associated with late gestation fetal compromise.

The impact of late gestation PRRSV-2 infection is highly variable within a litter, with a subset of fetuses displaying varying degrees of compromise following infection while others remain viable despite significant systemic viral load. To understand the underlying cause of this variation, we examined the susceptibility, distribution and impact of viral infection within non-lymphoid tissues. Samples of brain, heart, kidney, liver, lung, and skeletal muscle were obtained from fetuses of pregnant gilts at gestation day 86, and the presence and distribution of CD163+ cells within each tissue evaluated via immunohistofluorescence. Equivalent samples were collected from phenotypic extremes representing resistant, resilient and susceptible fetuses at 21 days following infection of pregnant gilts with PRRSV-2 at day 86 of gestation. Viral load and its impact in each tissue was evaluated by a combination of qPCR, in vitro viral recovery, and local expression of IFNG and CD163. Resting populations of CD163+ cells were observed in all six non-lymphoid tissues from healthy day 86 fetuses, though the apparent density and the morphology of positive cells varied between tissue. Viral RNA was detected in all six tissues derived from fetuses previously classified as highly infected, and infectious viral particles successfully recovered. Significantly more viral RNA was detected in heart, brain, lung and skeletal muscle of susceptible fetuses, relative to their viable counterparts. Infection was associated with an increase in the expression of CD163 in brain, kidney and lung. In addition, the presence of virus in each tissue coincided with a significant upregulation in the expression of IFNG, but the scale of this response was not associated with fetal susceptibility. Thus, PRRSV-2 is widely distributed across these susceptible non-lymphoid fetal tissues, and fetal outcome is associated with local viral load in critical fetal organs.

Apalutamide and overall survival in non-metastatic castration-resistant prostate cancer.

BACKGROUND: In the SPARTAN study, compared with placebo, apalutamide added to ongoing androgen deprivation therapy significantly prolonged metastasis-free survival (MFS) and time to symptomatic progression in patients with high-risk non-metastatic castration-resistant prostate cancer (nmCRPC). Overall survival (OS) results at the first interim analysis (IA1) were immature, with 104 of 427 (24%) events required for planned final OS analysis. Here, we report the results of a second pre-specified interim analysis (IA2). METHODS: One thousand two hundred and seven patients with nmCRPC were randomized 2 : 1 to apalutamide (240 mg daily) or placebo. The primary end point of the study was MFS. Subsequent therapy for metastatic CRPC was permitted. When the primary end point was met, the study was unblinded. Patients receiving placebo who had not yet developed metastases were offered open-label apalutamide. At IA2, pre-specified analysis of OS was undertaken, using a group-sequential testing procedure with O'Brien-Fleming-type alpha spending function. Safety and second progression-free survival (PFS2) were assessed. RESULTS: Median follow-up was 41 months. With 285 (67% of required) OS events, apalutamide was associated with an improved OS compared with placebo (HR 0.75; 95% CI 0.59-0.96; P = 0.0197), although the P-value did not cross the pre-specified O'Brien-Fleming boundary of 0.0121. Apalutamide improved PFS2 (HR 0.55; 95% CI 0.45-0.68). At IA2, 69% of placebo-treated and 40% of apalutamide-treated patients had received subsequent life-prolonging therapy for metastatic CRPC. No new safety signals were observed. CONCLUSION: In patients with nmCRPC, apalutamide was associated with a 25% reduction in risk of death compared with placebo. This OS benefit was observed despite crossover of placebo-treated patients and higher rates of subsequent life-prolonging therapy for the placebo group.

Author Correction: WNT-activated bone grafts repair osteonecrotic lesions in aged animals.

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WNT-activated bone grafts repair osteonecrotic lesions in aged animals.

The Wnt pathway is a new target in bone therapeutic space. WNT proteins are potent stem cell activators and pro-osteogenic agents. Here, we gained insights into the molecular and cellular mechanisms responsible for liposome-reconstituted recombinant human WNT3A protein (L-WNT3A) efficacy to treat osteonecrotic defects. Skeletal injuries were coupled with cryoablation to create non-healing osteonecrotic defects in the diaphysis of the murine long bones. To replicate clinical therapy, osteonecrotic defects were treated with autologous bone graft, which were simulated by using bone graft material from syngeneic ACTB-eGFP-expressing mice. Control osteonecrotic defects received autografts alone; test sites received autografts treated ex vivo with L-WNT3A. In vivo µCT monitored healing over time and immunohistochemistry were used to track the fate of donor cells and assess their capacity to repair osteonecrotic defects according to age and WNT activation status. Collectively, analyses demonstrated that cells from the autograft directly contributed to repair of an osteonecrotic lesion, but this contribution diminished as the age of the donor increased. Pre-treating autografts from aged animals with L-WNT3A restored osteogenic capacity to autografts back to levels observed in autografts from young animals. A WNT therapeutic approach may therefore have utility in the treatment of osteonecrosis, especially in aged patients.

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter.

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80(o) C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.

Plasmacytoid dendritic cell-derived IFNα modulates Th17 differentiation during early Bordetella pertussis infection in mice.

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis). T helper 17 (Th17) cells have a central role in the resolution of the infection. Emerging studies document that type I interferons (IFNs) suppress Th17 differentiation and interleukin (IL)-17 responses in models of infection and chronic inflammation. As plasmacytoid dendritic cells (pDCs) are a major source of type I IFNs, we hypothesize that during B. pertussis infection in mice, pDC-derived IFNα inhibits a rapid increase in Th17 cells. We found that IFNα-secreting pDCs appear in the lungs during the early stages of infection, while a robust rise of Th17 cells in the lungs is detected at 15 days post-infection or later. The presence of IFNα led to reduced Th17 differentiation and proliferation in vitro. Furthermore, in vivo blocking of IFNα produced by pDCs during infection with B. pertussis infection resulted in early increase of Th17 frequency, inflammation, and reduced bacterial loads in the airways of infected mice. Taken together, the experiments reported here describe an inhibitory role for pDCs and pDC-derived IFNα in modulating Th17 responses during the early stages of B. pertussis infection, which may explain the prolonged nature of whooping cough.

Multiscale analyses of the bone-implant interface.

Implants placed with high insertion torque (IT) typically exhibit primary stability, which enables early loading. Whether high IT has a negative impact on peri-implant bone health, however, remains to be determined. The purpose of this study was to ascertain how peri-implant bone responds to strains and stresses created when implants are placed with low and high IT. Titanium micro-implants were inserted into murine femurs with low and high IT using torque values that were scaled to approximate those used to place clinically sized implants. Torque created in peri-implant tissues a distribution and magnitude of strains, which were calculated through finite element modeling. Stiffness tests quantified primary and secondary implant stability. At multiple time points, molecular, cellular, and histomorphometric analyses were performed to quantitatively determine the effect of high and low strains on apoptosis, mineralization, resorption, and collagen matrix deposition in peri-implant bone. Preparation of an osteotomy results in a narrow zone of dead and dying osteocytes in peri-implant bone that is not significantly enlarged in response to implants placed with low IT. Placing implants with high IT more than doubles this zone of dead and dying osteocytes. As a result, peri-implant bone develops micro-fractures, bone resorption is increased, and bone formation is decreased. Using high IT to place an implant creates high interfacial stress and strain that are associated with damage to peri-implant bone and therefore should be avoided to best preserve the viability of this tissue.

Accuracy of ultrasonography performed by examiners with varied training and experience in predicting specific pathology of adnexal masses.

OBJECTIVES: To assess the diagnostic performance of subjective assessment by Level II ultrasound examiners in predicting the specific histology of adnexal masses. METHODS: The women included in this prospective multicenter cross-sectional study were older than 16 years of age and had at least one adnexal mass. They underwent transvaginal sonography (TVS) performed by Level II examiners, all of whom were familiar with the International Ovarian Tumor Analysis (IOTA) group definitions of ultrasound features of ovarian masses. The final outcome was histology. Specific diagnoses were categorized into 16 groups. Agreement between subjective assessment and final histology was measured using unweighted kappa coefficients. Sensitivities and specificities were obtained for subjective assessment. RESULTS: Of the 1279 women who underwent TVS, 313 were included in the final analysis. Overall agreement (16 × 16 table) between subjective assessment and histology was moderate, with a Cohen's kappa coefficient of 0.59 (95% CI, 0.53-0.65). The specificity of subjective assessment ranged between 91% and 100% for all histological subgroups. Highest sensitivities were achieved in the diagnosis of simple cysts (100% (95% CI, 61-100%)), hydrosalpinges (100% (95% CI, 34-100%)), mature teratomas (88% (95% CI, 74-96%)), endometriomas (75% (95% CI, 61-85%)), ovarian fibromas (88% (95% CI, 47-100%)), tubo-ovarian abscesses (88% (95% CI, 47-100%)) and serous cystadenocarcinomas (82% (95% CI, 66-93%)). Serous cystadenomas were misdiagnosed most commonly (40.5%). The sensitivity of subjective assessment in diagnosing adnexal torsion was 54% (95% CI, 25-81%); the 17 confirmed and/or suspected cases of adnexal torsion were not included in the 313 cases examined and analyzed for diagnostic performance. CONCLUSION: Overall, subjective assessment by Level II examiners was good for the detection of simple cysts, endometriomas, mature teratomas, hydrosalpinges, fibroma, tubo-ovarian abscess and serous cystadenocarcinomas.

Cell viability after osteotomy and bone harvesting: comparison of piezoelectric surgery and conventional bur.

The aim of this study was to evaluate and compare the influence of a piezoelectric device versus a conventional bur on osteocyte viability and osteoblast and osteoclast activity using an in vivo mouse model. Osteotomies were created and bone grafts were harvested using either a conventional bur or a piezoelectric device; the resulting injuries and bone grafts were evaluated over an extended time-course using molecular and cellular assays for cell death (TUNEL assay), cell viability (4',6-diamidino-2-phenylindole (DAPI) staining), the onset of mineralization (alkaline phosphatase activity), and bone remodelling (tartrate-resistant acid phosphatase activity). Osteotomies created with a piezoelectric device showed greater osteocyte viability and reduced cell death. Bone grafts harvested with a piezoelectric device exhibited greater short-term cell viability than those harvested with a bur, and exhibited slightly more new bone deposition and bone remodelling. The difference in response of osteocytes, osteoblasts, and osteoclasts to bone cutting via a bur and via a piezoelectric device is negligible in vivo. Given the improved visibility and the margin of safety afforded by a piezoelectric device, they are the instrument of choice when cutting or harvesting bone to preserve soft tissue.

Finding the best antibody dilution by repeated immunostaining of the same tissue section.

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.

Multicentre external validation of IOTA prediction models and RMI by operators with varied training.

BACKGROUND: Correct characterisation of ovarian tumours is critical to optimise patient care. The purpose of this study is to evaluate the diagnostic performance of the International Ovarian Tumour Analysis (IOTA) logistic regression model (LR2), ultrasound Simple Rules (SR), the Risk of Malignancy Index (RMI) and subjective assessment (SA) for preoperative characterisation of adnexal masses, when ultrasonography is performed by examiners with different background training and experience. METHODS: A 2-year prospective multicentre cross-sectional study. Thirty-five level II ultrasound examiners contributed in three UK hospitals. Transvaginal ultrasonography was performed using a standardised approach. The final outcome was the surgical findings and histological diagnosis. To characterise the adnexal masses, the six-variable prediction model (LR2) with a cutoff of 0.1, the RMI with cutoff of 200, ten SR (five rules for malignancy and five rules for benignity) and SA were applied. The area under the curves (AUCs) for performance of LR2 and RMI were calculated. Diagnostic performance measures for all models assessed were sensitivity, specificity, positive and negative likelihood ratios (LR+ and LR-), and the diagnostic odds ratio (DOR). RESULTS: Nine-hundred and sixty-two women with adnexal masses underwent transvaginal ultrasonography, whereas 255 had surgery. Prevalence of malignancy was 29% (49 primary invasive epithelial ovarian cancers, 18 borderline ovarian tumours, and 7 metastatic tumours). The AUCs for LR2 and RMI for all masses were 0.94 (95% confidence interval (CI): 0.89-0.97) and 0.90 (95% CI: 0.83-0.94), respectively. In premenopausal women, LR2-RMI difference was 0.09 (95% CI: 0.03-0.15) compared with -0.02 (95% CI: -0.08 to 0.04) in postmenopausal women. For all masses, the DORs for LR2, RMI, SR+SA (using SA when SR inapplicable), SR+MA (assuming malignancy when SR inapplicable), and SA were 62 (95% CI: 27-142), 43 (95% CI: 19-97), 109 (95% CI: 44-274), 66 (95% CI: 27-158), and 70 (95% CI: 30-163), respectively. CONCLUSION: Overall, the test performance of IOTA prediction models and rules as well as the RMI was maintained in examiners with varying levels of training and experience.

Wild bees visiting cucumber on midwestern U.S. organic farms benefit from near-farm semi-natural areas.

Wild bees that provide pollination services to vegetable crops depend on forage resources, nesting sites, and overwintering sites in the agricultural landscape. The scale at which crop-visiting bees use resources in the landscape can vary regionally, and has not been characterized in the Midwestern United States. We investigated the effects of seminatural land cover on wild bee visitation frequency to cucumber (Cucumis sativus L.) and on wild bee species richness on 10 organic farms in Indiana. We estimated the spatial scale at which the effects of land cover were strongest, and also examined the effects of nonlandscape factors on wild bees. The visitation frequency of wild bees to cucumber was positively related to the proportion of seminatural land in the surrounding landscape, and this relationship was strongest within 250 m of the cucumber patch. The species richness of wild cucumber visitors was not affected by land cover at any spatial scale, nor by any of the nonlandscape factors we considered. Our results indicate that wild, crop visiting bees benefit from seminatural areas in the agricultural landscape, and benefit most strongly from seminatural areas within 250 m of the crop field. This suggests that setting aside natural areas in the near vicinity of vegetable fields may be an effective way to support wild, crop-visiting bees and secure their pollination services.

De-staining and re-staining mucins in formalin fixed paraffin sections.

Re-staining of formalin fixed paraffin sections sometimes is required and this requires prior de-staining. Some simple and effective protocols for de-staining are described. Mucihematoxylin and mucicarmine can be removed with acid alcohol. Zirconyl hematoxylin can be removed with periodic acid or Sinha's fixative. Alcian blue can be removed with 5% trifluoroacetic acid in dichloromethane. Colloidal iron can be bleached in 1% household bleach in alcohol. PAS can be removed with hydrogen peroxide or ammonium hydroxide. With few exceptions, de-stained sections can be re-stained with mucihematoxylin, PAS or Gabe's trichrome.

Hematein chelates of unusual metal ions for tinctorial histochemistry.

Hematoxylin is oxidized easily to hematein, an excellent stain for metal ions. If it already is bound to a substrate, the metal ion becomes a mordant linking the dye to the substrate. Metal ions added to hematein in solution are chelated by the hematein to form a lake. Most of these chelates stain animal tissues. They usually are bound to the tissue by a combination of hydrogen bonding of the hematein and ionic bonding of the metal ion. When binding of the lake to the tissue occurs by way of the metal ion, the metal ion is a mordant. Mordant staining often is specific. Chromium hematoxylin binds to strong acids; it can be made selective for protein-bound sulfonic acids. Zirconyl hematoxylin is selective for acidic mucins. Mucihematein can be made selective for all acidic mucins or for sulfomucins alone. Bismuth hematoxylin appears to be selective for the guanido group of arginine and there is some evidence that the bonding is covalent. Although it is not a histochemical stain, copper-chrome hematoxylin is an excellent stain for organelles with double membranes, i.e., mitochondria and nuclei.

Trends in ixodid tick abundance and distribution in Great Britain.

The popular, but rarely documented, view in Britain is that ticks have increased in distribution and abundance over recent years. To assess this, we gathered evidence for changes in tick distribution and abundance by distributing a survey questionnaire throughout Britain and by analysing trends in the prevalence of tick infestation on red grouse chicks Lagopus lagopus scoticus Latham (Galliformes: Tetranoidae), gathered over 19 years at three Scottish sites, and on deer (Cetartiodactyla: Cervidae) culled over 11 years on 26 Ministry of Defence (MoD) estates. Based on the survey, the current known distribution of Ixodes ricinus Linnaeus (Acari: Ixodidae) has expanded by 17% in comparison with the previously known distribution. The survey indicated that people perceive there to be more ticks today than in the past at 73% of locations throughout Britain. Reported increases in tick numbers coincided spatially with perceived increases in deer numbers. At locations where both tick and deer numbers were reported to have increased, these perceived changes occurred at similar times, raising the possibility of a causal link. At other locations, tick numbers were perceived to have increased despite reported declines in deer numbers. The perceptions revealed by the survey were corroborated by quantitative data from red grouse chicks and culled deer. Tick infestation prevalence increased over time on all grouse moors and 77% of MoD estates and decreased at six locations.

Mucin immunohistochemistry in the diagnosis and mapping of extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare skin cancer of the genital region in which cancer cells with enlarged nuclei and pale cytoplasm are scattered singly in the affected epidermis. These cancer cells, called Paget cells, contain mucin, which is never found in normal epidermis. The oligosaccharide side chains of Paget cell mucin end with sialic acid. Sialic acid is easily detected by zirconyl haematoxylin or alcian blue. The other sugars in the oligosaccharide chains can be detected by the periodic acid-Shiff reaction. Rarely, the diagnosis of EMPD is complicated by the absence of mucin from the Paget cells. We have examined such an atypical case. The oligosaccharide side chains, including the sialic acids, are absent. In both this case and a typical case, the Paget cells contain epithelial membrane antigen mucin (MUC1) core protein and usually contain gastric surface-type mucin (MUC5AC) core protein, which can be stained by antibodies. Since neither core protein is found in normal epidermis, epithelial membrane antigen core protein may be the most reliable diagnostic marker for extramammary Paget's disease. In both the atypical case and the typical case of Paget's disease, some cells that look like keratinocytes contain mucin core proteins. These may be incipient Paget cells. We suggest that using th epithelial membrane antigen core protein as a marker for the true extent of extramammary Paget's disease could facilitate complete excision and reduce the rate of recurrence.

Theoretical analysis of the magnetic Fréedericksz transition in the presence of flexoelectricity and ionic contamination.

We have derived an approximate analytical expression for the static director distortion of a planar nematic layer subject to a magnetic field H immediately above the critical Fréedericksz transition H=H{c} . The layer contains a voltage-independent density of positively and negatively singly charged ionic species that interact with the flexoelectric and dielectric polarizations which appear when the director is distorted. The analytical solution is shown to correspond closely to a full numerical calculation when H/H{c}=1.01. The analytical approach allows a quantitative insight into how the mobile charge shields the polarization for different values of the elastic constants, the ionic density, the flexoelectric coefficients, and the layer thickness.

Intracranial germ cell tumors: a single institution experience and review of the literature.

There is little literature to guide therapy in children and young adults with intracranial germ cell tumors. We present 17 consecutively diagnosed intracranial germ cell tumors at The Children's Hospital, Denver, from 1995 to 2001. Of 17 patients, 3 had considerable delay in diagnosis. Two with suprasellar tumors presented with dementia, blindness and pan-hypopituitarism and another with recurrent subarachnoid hemorrhage. Seven had germinoma, three were metastatic at diagnosis. Ten had non-germinomatous germ cell tumors (NGGCT), 5/10 were alpha feto-protein (AFP) positive only, one beta-human chorionic growth (betaHCG) factor positive only, 3 positive for AFP and betaHCG, and 1 malignant teratoma. Therapy for metastatic patients consisted of chemotherapy followed by craniospinal radiation (CSI). Patients with localized disease received chemotherapy followed by focal radiation. Two patients received chemotherapy only, one because she died of sepsis while receiving chemotherapy and one because of neurologic injury incurred during surgery parents elected for no therapy. Three patients have died, one of tumor recurrence, one from a remote complication of surgery and one of sepsis. Twelve patients are alive without evidence of disease from 10 to 68 months (median 31.5 months). All five children with only AFP positivity, treated with chemotherapy and focal radiation are alive without evidence of disease at 10, 16, 22, 41 and 41 months. Thus, there is little evidence that CSI is necessary in non-metastatic germinomas and AFP positive NGGCTs when combined chemotherapy and radiation therapy is used. However, complications of delayed diagnosis, surgery and chemotherapy are important causes of mortality, with only one patient dying of tumor.

Rising burden of immature sheep ticks (Ixodes ricinus) on red grouse (Lagopus lagopus scoticus) chicks in the Scottish uplands.

The sheep tick Ixodes ricinus (L.) (Acari: Ixodidae) is an ectoparasite of major economic and pathogenic importance in Scotland. Its distribution in the Scottish uplands is assumed to be governed by the abundance and distribution of its definitive hosts (deer and sheep) and climatic variables such as temperature and rainfall. As the numbers of its major host in Scotland, red deer, have increased dramatically and climatic conditions have become more favourable, the level of parasitism could have been expected to rise. We use data gathered from tick counts on over 4000 red grouse chicks Lagopus lagopus scoticus Latham (Galliformes: Tetraonidae) in various experiments over the past 19 years to ascertain whether the intensity and prevalence of parasitism has been increasing. From 1985 to 2003 the average tick burden of a parasitized red grouse chick has grown from 2.60 +/- 1.12 ticks per chick to 12.71 +/- 1.44. Over this period the percentage of chicks of a given brood parasitized has also increased from 4 +/- 2% to 92 +/- 3%. The possible implications of this increase in parasitism for red grouse production are discussed.

An improved formulation of the zirconyl hematoxylin stain for acidic mucins.

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.