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Introduction: Benign prostatic hyperplasia (BPH) is common in the ageing male. Clinical manifestations like retention impact on a patient’s quality of life. Alterations in androgen activity at the androgen receptor complex level in the prostate contribute to prostatic hyperplasia with the highest incidence occurring in males in their 70’s. There remains a paucity of cases in young males who develop acute urinary retention secondary to BPH. We present a case of a 27-year-old male who developed acute urinary retention secondary to BPH who required a Holmium Laser Enucleation of his Prostate (HOLEP). Case description: A 27 year old man was admitted in acute urinary retention. BPH was diagnosed via way of radiological imaging and histological assessment. After pre-operative sperm banking and suprapubic catheterisation, the patient underwent a HOLEP. He had biochemically confirmed hypogonadotrophic hypogonadism which was at odds with his muscular, physical appearance. Total testosterone levels had fluctuated following admission suggesting an exogenous substance was interfering with the hypothalamic-pituitary-gonadal axis but he denied exogenous steroid use. Result: The patient successfully passed his voiding trial on the second post-operative day and remained catheter free. Post-operative uroflowmetry and sexual function remain unknown as patient disengaged with follow up. Conclusion: HOLEP prostatectomy is a safe and effective way of managing BPH in younger patients following sperm banking and assessment by endocrinology.

The diagonal band of Broca (DBB) contains the second largest cholinergic cell group in the human brain, known as the nucleus of the vertical limb of the DBB (nvlDBB). It has major projections to the hippocampus, but it is often underinvestigated, partly due to its ill-defined anatomical boundaries and hence the difficulty of reliable sampling. In this chapter, we have reviewed the historical literature to reestablish the anatomy of the nvlDBB, distinguishing it from neighboring basal forebrain cholinergic nuclei. Although varying degrees of neuronal loss in the nvlDBB have been reported in a range of neurological disorders, and in the aged brain, the significant nvlDBB cholinergic neuronal loss reported in Lewy body dementias is of particular interest. Retrograde tracer study in rodents has demonstrated reciprocal connections between the DBB and the hippocampal CA2 subfield, an area particularly susceptible to Lewy pathologies. Previous functional studies have demonstrated that the nvlDBB is particularly involved in memory retrieval, a cognitive domain severely affected in Lewy body disorders. Based on these observations, we propose an anatomical and functional connection between the cholinergic component of the nvlDBB (Ch2) and the hippocampal CA2.

Although the precise neuropathological substrates of cognitive decline in Parkinson’s disease (PD) remain elusive, it has long been regarded that pathology in the CA2 hippocampal subfield is characteristic of Lewy body dementias, including dementia in PD (PDD). Early non-human primate tracer studies demonstrated connections from the nucleus of the vertical limb of the diagonal band of Broca (nvlDBB, Ch2) to the hippocampus. However, the relationship between Lewy pathology of the CA2 subfield and cholinergic fibres has not been explored. Therefore, in this study, we investigated the burden of pathology in the CA2 subsector of PD cases with varying degrees of cognitive impairment and correlated this with the extent of septohippocampal cholinergic deficit. Hippocampal sections from 67 PD, 34 PD with mild cognitive impairment and 96 PDD cases were immunostained for tau and alpha-synuclein, and the respective pathology burden was assessed semi-quantitatively. In a subset of cases, the degree of CA2 cholinergic depletion was quantified using confocal microscopy and correlated with cholinergic neuronal loss in Ch2. We found that only cases with dementia have a significantly greater Lewy pathology, whereas cholinergic fibre depletion was evident in cases with mild cognitive impairment and this was significantly correlated with loss of cholinergic neurons in Ch2. In addition, multiple antigen immunofluorescence demonstrated colocalisation between cholinergic fibres and alpha-synuclein but not tau pathology. Such specific Lewy pathology targeting the cholinergic system within the CA2 subfield may contribute to the unique memory retrieval deficit seen in patients with Lewy body disorders, as distinct from the memory storage deficit seen in Alzheimer’s disease.

Aims Galanin is a highly inducible neuroprotective neuropeptide and in Alzheimer's disease (AD), a network of galaninergic fibres has been reported to hypertrophy and hyperinnervate the surviving cholinergic neurons in the basal forebrain. We aimed to determine (i) the extent of galanin hyperinnervation in patients with AD and Lewy body disease and (ii) whether galanin expression relates to the neuropathological burden and cholinergic losses. Methods Galanin immunohistochemistry was carried out in the anterior nucleus basalis of Meynert of 27 Parkinson's disease (PD) cases without cognitive impairment (mild cognitive impairment [MCI]), 15 with PD with MCI, 42 with Parkinson's disease dementia (PDD), 12 with Dementia with Lewy bodies (DLB), 19 with AD, 12 mixed AD/DLB and 16 controls. Galaninergic innervation of cholinergic neurons was scored semiquantitatively. For a subgroup of cases (n = 60), cholinergic losses were determined from maximum densities of choline acetyltransferase positive (ChAT+ve) neurons and their projection fibres. Quantitative data for α-synuclein, amyloid beta and tau pathology were obtained from tissue microarrays covering cortical/subcortical regions. Results Significant losses of cholinergic neurons and their projection fibres were observed across all diseases. Galaninergic hyperinnervation was infrequent and particularly uncommon in established AD and DLB. We found that hyperinnervation frequencies are significantly higher in the transition between PD without MCI to PDD and that higher burdens of co-existent AD pathology impair this galaninergic response. Conclusions Our results suggest that galanin upregulation represents an intrinsic response early in Lewy body diseases but which fails with increasing burdens of AD related pathology.

Although the nucleus of the vertical limb of the diagonal band of Broca (nvlDBB) is the second largest cholinergic nucleus in the basal forebrain, after the nucleus basalis of Meynert (nbM), it has not generally been a focus for studies of neurodegenerative disorders. However, the nvlDBB does have an important projection to the hippocampus and discrete lesions of the rostral basal forebrain have been shown to disrupt retrieval memory function, a major deficit seen in patients with Lewy body disorders. One reason for its neglect is that the anatomical boundaries of the nvlDBB are ill defined and this area of the brain is not part of routine diagnostic sampling protocols. We have reviewed the history and anatomy of the nvlDBB and now propose guidelines for distinguishing nvlDBB from other neighbouring cholinergic cell groups for standardising future clinicopathological work. Thorough review of the literature regarding neurodegenerative conditions reveals inconsistent results in terms of cholinergic neuronal loss within the nvlDBB. This is likely to be due to the use of variable neuronal inclusion criteria and omission of cholinergic immunohistochemical markers. Extrapolating from those studies showing significant nvlDBB neuronal loss in Lewy body dementia, we propose an anatomical and functional connection between the cholinergic component of the nvlDBB (Ch2) and the CA2 subfield in the hippocampus which may be especially vulnerable in Lewy body disorders. This article is protected by copyright. All rights reserved.

Current tissue‐clearing protocols for imaging in three dimensions (3D) are typically applied to optimally fixed, small‐volume rodent brain tissue – which is not representative of the tissue found in diagnostic neuropathology laboratories. We present a method to visualise the cerebral cortical vasculature in 3D in human post‐mortem brain tissue which had been preserved in formalin for many years. Tissue blocks of cerebral cortex from two control cases, two Alzheimer's brains and two cases from Alzheimer's patients immunised against Aβ(42) were stained with fluorescent Lycopersicon esculentum agglutinin (Tomato lectin), dehydrated and cleared using an adapted three‐dimensional imaging of solvent cleared organs (3DISCO) protocol to visualise the vascular endothelium. Tissue was imaged using light sheet and confocal microscopy and reconstructed in 3D using amira software. The method permits visualisation of the arrangement of the parallel penetrating cortical vasculature in the human brain. The presence of four vascular features including anastomosis, U‐shaped vessels, spiralling and loops were revealed. In summary, we present a low cost and simple method to visualise the human cerebral vasculature in 3D compatible with prolonged fixation times (years), allowing study of vascular involvement in a range of normative and pathological states.

Different neurodegenerative conditions can have complex, overlapping clinical presentations that make accurate diagnosis during life very challenging. For this reason, confirmation of the clinical diagnosis still requires postmortem verification. This is particularly relevant for clinical trials of novel therapeutics where it is important to ascertain what disease and/or pathology modifying effects the therapeutics have had. Furthermore, it is important to confirm that patients in the trial actually had the correct clinical diagnosis as this will have a major bearing on the interpretation of trial results. Here we present a simple protocol for pathological assessment of neurodegenerative changes.

Purpose To report our early experiences with failure mode and effects analysis (FMEA) in a community practice setting. Methods The FMEA facilitator received extensive training at the AAPM Summer School. Early efforts focused on department education and emphasized the need for process evaluation in the context of high profile radiation therapy accidents. A multidisciplinary team was assembled with representation from each of the major department disciplines. Stereotactic radiosurgery (SRS) was identified as the most appropriate treatment technique for the first FMEA evaluation, as it is largely self-contained and has the potential to produce high impact failure modes. Process mapping was completed using breakout sessions, and then compiled into a simple electronic format. Weekly sessions were used to complete the FMEA evaluation. Risk priority number (RPN) values > 100 or severity scores of 9 or 10 were considered high risk. The overall time commitment was also tracked. Results The final SRS process map contained 15 major process steps and 183 subprocess steps. Splitting the process map into individual assignments was a successful strategy for our group. The process map was designed to contain enough detail such that another radiation oncology team would be able to perform our procedures. Continuous facilitator involvement helped maintain consistent scoring during FMEA. Practice changes were made responding to the highest RPN scores, and new resulting RPN scores were below our high-risk threshold. The estimated person-hour equivalent for project completion was 258 hr. Conclusions This report provides important details on the initial steps we took to complete our first FMEA, providing guidance for community practices seeking to incorporate this process into their quality assurance (QA) program. Determining the feasibility of implementing complex QA processes into different practice settings will take on increasing significance as the field of radiation oncology transitions into the new TG-100 QA paradigm.

Idiopathic granulomatous mastitis (IGM) is a rare, benign inflammatory disorder of the breast. Clinical features may include painful breasts, erythema, subcutaneous nodules, and ulcerative lesions. It can mimic various other breast pathologies, and it is a diagnosis of exclusion after infection, malignancy, and other inflammatory conditions have been ruled out. In this article, we present a case of IGM developing in a 40-year-old female 3 months after hospitalization for myxedema coma. A contrast-enhanced magnetic resonance imaging of the breasts showed bilateral edema, and a biopsy was negative for malignancy or infection. She was started on prednisone and had noticeable improvement of ulcerations within several weeks. IGM is a rare condition that requires a multimodal treatment approach. Often recalcitrant disease is encountered and requires surgical intervention, immunosuppression, and antimicrobial therapy. The diagnosis should be entertained in patients with bilateral breast inflammation to avoid unnecessary surgical resection early on.

IntroductionRecent studies have highlighted the accumulated use of anticholinergic drugs is associated with an increased risk of dementia, and many of those are used for symptomatic management in Parkinson’s disease (PD). Unfortunately, the only available post-mortem study investigating the effect of anticholinergic agents on Alzheimer’s pathology burden suffers from several methodological limitations. Therefore, we aimed to validate if cumulative use of anticholinergic medication will increase Alzheimer’s pathologies in the post-mortem PD brains.Material and methodsRetrospective case note analysis was carried out on 54 selected PD cases from the Parkinson’s UK Brain Bank and cases were stratified into no, low and high anticholinergic burden using literature-based anticholinergic scores. Association of anticholinergic burden and Alzheimer’s pathology in post-mortem tissues was analysed with logistic regression modelling.ResultsA higher anticholinergic burden is associated with a greater tau burden in entorhinal cortex in PD cases (OR=2.21). Interestingly, a high anticholinergic burden decreases the odds of amyloid-beta presence in the anterior hippocampus (OR=0.127), entorhinal cortex (OR=0.124) and frontal cortex (OR=0.113) of PD cases.ConclusionWe have demonstrated that anticholinergic drugs have varying effect on Alzheimer’s pathology in post-mortem PD brain tissues. Complex mechanism is probably involved and require further studies.

The meteoric rise in prominence of the Islamic State of Iraq and Syria (IS), also known pejoratively by the Arabic term Daesh, since late 2013 provides a unique opportunity to analyze the evolution of a distinctive brand of extremism that draws on distorted interpretations of Islam in the heart of the region where that religion was born. The deceptively rapid advance of IS in Iraq surprised many around the globe, including Americans, but the current situation was predictable and in fact should have been expected. The US intelligence community (IC), for example, warned that a full troop pullout from Iraq in December 2011 would cause unrest in the country and require the United States (US) to commit military forces in the future.1

Three-dimensional visualization of intact tissues is now being achieved by turning tissues transparent. CLARITY is a unique tissue clearing technique, which features the use of detergents to remove lipids from fixed tissues to achieve optical transparency. To preserve tissue integrity, an acrylamide-based hydrogel has been proposed to embed the tissue. In this study, we examined the rationale behind the use of acrylamide in CLARITY, and presented evidence to suggest that the omission of acrylamide-hydrogel embedding in CLARITY does not alter the preservation of tissue morphology and molecular information in fixed tissues. We therefore propose a novel and simplified workflow for formaldehyde-fixed tissue clearing, which will facilitate the laboratory implementation of this technique. Furthermore, we have investigated the basic tissue clearing process in detail and have highlighted some areas for targeted improvement of technologies essential for the emerging subject of three-dimensional histology.

Despite its potency, the wider use of immunotherapy for B cell malignancies is hampered by the lack of well-defined tumor-specific Ags. In this study, we demonstrate that an evolutionarily conserved 37-kDa immature laminin receptor protein (OFA-iLRP), a nonimmunogenic embryonic Ag expressed by a variety of tumors, is rendered immunogenic if targeted to the APCs using the CCR6 ligands MIP3α/CCL20 and mDF2β. The CCR6 targeting facilitated efficient Ag cross-presentation and induction of tumor-neutralizing CTLs. Although the Ag targeting alone, without activation of dendritic cells (DCs), is proposed to induce tolerance, and MIP3α does not directly activate DCs, the MIP3α-based vaccine efficiently induced protective and therapeutic antitumor responses. The responses were as strong as those elicited by the OFA-iLRP fusions with moieties that activated DCs and Th1-type cytokine responses, mDF2β, or mycobacterial Hsp70 Ag. Although the same cDNA encodes the dimerized high-affinity mature 67-kDa mLRP that is expressed in normal tissues to stabilize the binding of laminin to cell surface integrins, the vaccines expressing OFA-iLRP elicited long-term protective CD8+ T cell-mediated memory responses against syngeneic B cell lymphoma, indicating the potential application of these simple vaccines as preventive and therapeutic formulations for human use.

Treatment of osteoporosis with high-dose fluoride alone does not reduce fracture risk. We hypothesized that the antifracture efficacy of fluoride could be optimized by its use in low doses combined with an antiresorptive agent.Subjects included 80 women with postmenopausal osteoporosis who had been taking estrogen for at least 1 yr.Subjects were randomized to receive monofluorophosphate (MFP) (fluoride content of 20 mg/d) or placebo over 4 yr in a double-blind trial.There were progressive increases in lumbar spine bone density over the duration of the study (MFP, 22%; placebo, 6%; P0.0001). In the trabecular bone of L3, these increases were even greater (MFP, 49%; placebo, 2.5%; P0.0001). In the proximal femur, there were smaller but significant treatment effects (P = 0.015). Total body scans and their subregions also showed significantly greater increases in the MFP group. Bone formation markers increased significantly in the MFP group at yr 1. Hyperosteoidosis was present in biopsies from five of seven MFP subjects, with osteomalacia in two of seven. The hazards ratio for vertebral fractures was 0.20 (95% confidence interval, 0.05-1.30), and the incidence rate ratio was 0.12 (95% confidence interval, 0.06-0.23; P0.01). The hazards ratio for nonvertebral fractures was 3.3 (95% confidence interval, 0.8-12.0).We conclude that fluoride at 20 mg/d produces substantial increases in bone mineral density but still interferes with bone mineralization. This indicates that most previous studies with this ion have used toxic doses and that much lower doses should be assessed to find a safe dose window for the use of this powerful anabolic agent.

Aims CLARITY is a novel technique which enables three-dimensional visualization of immunostained tissue for the study of circuitry and spatial interactions between cells and molecules in the brain. In this study, we aimed to compare methodological differences in the application of CLARITY between rodent and large human post mortem brain samples. In addition, we aimed to investigate if this technique could be used to visualize Lewy pathology in a post mortem Parkinson's brain. Methods Rodent and human brain samples were clarified and immunostained using the passive version of the CLARITY technique. Samples were then immersed in different refractive index matching media before mounting and visualizing under a confocal microscope. Results We found that tissue clearing speed using passive CLARITY differs according to species (human vs. rodents), brain region and degree of fixation (fresh vs. formalin-fixed tissues). Furthermore, there were advantages to using specific refractive index matching media. We have applied this technique and have successfully visualized Lewy body inclusions in three dimensions within the nucleus basalis of Meynert, and the spatial relationship between monoaminergic fibres and Lewy pathologies among nigrostriatal fibres in the midbrain without the need for physical serial sectioning of brain tissue. Conclusions The effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.

Recently it has become clear that more potent methods for DNA vaccine delivery need to be developed to enhance the efficacy of DNA vaccines. In vivo electroporation has emerged as a potent method for DNA vaccine delivery. In a mouse model, we evaluated the CD8(+) T lymphocyte response to a prostate cancer DNA vaccine encoding prostate-specific antigen (PSA) after intradermal electroporation. A significantly increased gene expression (100- to 1000-fold) and higher levels of PSA-specific T cells, compared to DNA delivery without electroporation, was demonstrated. Interestingly, investigation of a panel of different electroporation conditions showed that only some conditions that induce high levels of gene expression additionally induced cellular immunity. This suggests that electroporation parameters should be carefully optimized, not only to enhance transfection efficiency, but also to enhance the immune response to the vaccine. This study demonstrates the applicability of intradermal electroporation as a delivery method for genetic cancer vaccines and other DNA vaccines relying on antigen-specific T cell induction. Recently it has become clear that more potent methods for DNA vaccine delivery need to be developed to enhance the efficacy of DNA vaccines. In vivo electroporation has emerged as a potent method for DNA vaccine delivery. In a mouse model, we evaluated the CD8(+) T lymphocyte response to a prostate cancer DNA vaccine encoding prostate-specific antigen (PSA) after intradermal electroporation. A significantly increased gene expression (100- to 1000-fold) and higher levels of PSA-specific T cells, compared to DNA delivery without electroporation, was demonstrated. Interestingly, investigation of a panel of different electroporation conditions showed that only some conditions that induce high levels of gene expression additionally induced cellular immunity. This suggests that electroporation parameters should be carefully optimized, not only to enhance transfection efficiency, but also to enhance the immune response to the vaccine. This study demonstrates the applicability of intradermal electroporation as a delivery method for genetic cancer vaccines and other DNA vaccines relying on antigen-specific T cell induction.

It has been well established that neuronal loss within the cholinergic nucleus basalis of Meynert (nbM) correlates with cognitive decline in dementing disorders such as Alzheimer’s disease (AD). Friedrich Lewy first observed his eponymous inclusion bodies in the nbM of postmortem brain tissue from patients with Parkinson’s disease (PD) and cell loss in this area can be at least as extensive as that seen in AD. There has been confusion with regard to the terminology and exact localisation of the nbM within the human basal forebrain for decades due to the diffuse and broad structure of this “nucleus”. Also, while topographical projections from the nbM have been mapped out in subhuman primates, no direct clinicopathological correlations between subregional nbM and cortical pathology and specific cognitive profile decline have been performed in human tissue. Here, we review the evolution of the term nbM and the importance of standardised nbM sampling for neuropathological studies. Extensive review of the literature suggests that there is a caudorostral pattern of neuronal loss within the nbM in AD brains. However, the findings in PD are less clear due to the limited number of studies performed. Given the differing neuropsychiatric and cognitive deficits in Lewy body-associated dementias (PD dementia and dementia with Lewy bodies) as compared to AD, we hypothesise that a different pattern of neuronal loss will be found in the nbM of Lewy body disease brains. Understanding the functional significance of the subregions of the nbM could prove important in elucidating the pathogenesis of dementia in PD.

Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by deformities of long bones, kyphosis and acetabular protrusion, increasing in severity as affected children pass through adolescence. Biochemical and histological evidence indicate that there is extremely rapid bone turnover, with indices of both bone resorption and formation greatly increased. A genome-wide search, in a family with three children affected by idiopathic hyperphosphatasia, suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within this locus, was an obvious candidate, given the critical role of OPG in regulating osteoclast development. All three affected siblings were homozygous for a 3 bp inframe deletion in exon 3 of the TNFRSF11B gene, resulting in the loss of an aspartate residue. Their parents (who were first cousins) were heterozygous for the mutation. Recombinant wild-type and mutant OPG cDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected from the conditioned medium. In vitro measurements of bone resorption showed that wild-type OPG suppressed bone resorption, whereas the mutant form did not, confirming this to be an inactivating mutation. This description of abnormal OPG function in humans expands the spectrum of genetic bone diseases arising from perturbations of the OPG/RANK-L/RANK system that regulates osteoclastogenesis.

Objective: A DNA disc chip assay, based on comparative genomic hybridization, was designed to measure changes in sperm DNA intensities. The objective was to analyze the DNA integrity of hyperactive sperm cells after mild heat treatment. Design: The assay based on a multiple cell comet assay was used to analyze changes in genomic DNA. Washed sperm DNA were tested on the assay and images stored in a microarray design. Setting: Clinical and academic research environment. Patient(s): Frozen-thawed washed sperm from different donors (n = 7). Intervention(s): Discarded sperm leftover from trial washes carried out at 37° and 40°C were frozen and processed for the DNA disc chip assay. Main Outcome Measure(s): Fluorescent intensities of DNA disc chips and sperm variables. Result(s): Heat treatment resulted in more than eightfold increase in sperm hyperactive motility with little degradation in DNA integrity. Sperm with low hyperactivation was associated with alterations in DNA after heat treatment. Conclusion(s): The DNA disc chip assay was simple, inexpensive, and permitted assisted reproduction technologies laboratories to use comparative genomic hybridization for cytogenotoxicity testing. However, the assay required manual processing, a fluorescent microscope, and computer. The data showed an association between sperm hyperactivation and DNA integrity suggesting that the hyperactivation marker may be used for selecting quality sperm for intracytoplasmic sperm injection. More studies are needed to examine temperature effects on ejaculated human sperm.

Purpose : The effect of human papillomavirus (HPV) DNA from the E6-E7 region on the integrity of DNA in blastocyst stage embryonic cells was studied. The study design paralleled the event whereby HPV DNA from the infecting virus would target host cell DNA. The objectives were (a) to determine if the DNA of blastocysts were disrupted by the presence of HPV DNA and (b) to determine if the intensity of DNA damage was associated with the type of HPV. Methods : This study involved superovulating female mice, mating, collecting one-cell embryos, and culturing to the expanded blastocyst stage. The blastocysts were infected with PCR-synthesized DNA fragments from either HPV type 16, 18, 31, or 33. The blastocyst DNA were analyzed by comet assay after 24 h of incubation. The fluorescent images were digitized and the pixel intensity of each blastocyst was measured. Results : Only the DNA of HPV type 16 was associated with significant DNA fragmentation in comparison with the other HPV types. There was no relationship between HPV DNA fragment size and the intensity of DNA fragmentation. Conclusions : The data suggested that one mode of action of HPV type 16 was to initiate apoptosis of embryonic cells through DNA fragmentation. The effect of HPV 16 occurred rapidly within 24 h. The intensity of DNA damage was not linked to the specific type of HPV. However, the results do not rule out the other HPV types affecting embryos under conditions different from this study.

Purpose:C-myc was studied in cyclooxygenase (COX)-2 associated granulosa cell apoptosis. Methods: Granulosa cells (N = 5 cases) were incubated for 24 h in either 1 or 50 M COX-2 inhibitor, 1 or 50 M COX-1/COX-2 inhibitor, negative or positive controls. Single primer polymerase chain reaction of c-myc exon 1 were performed. Bisbenzimide-stained control single-stranded (ssDNA) were hybridized to SYBR Gold-stained ssDNA and fluorescent images analyzed. Results:C-myc was disrupted by the high-dose COX-2 inhibitor. Cell viability decreased with COX-1 and COX-2 inhibition. However, cell viability was similar for the positive control and at low-dose COX-2 inhibition. Conclusions: Inhibition of both COX-1 and COX-2 initiated apoptosis without disrupting c-myc suggesting a protective effect on c-myc. The low dosage of the COX-2 inhibitor did not disrupt c-myc and cell viability. C-myc sensitization was not part of apoptosis.

WE have been working with the camelid industry, the AHVLA and Defra, as well as colleagues in the British Veterinary Camelid Society, to help create a voluntary health scheme for bovine TB in alpacas. We are pleased to be able to report that working in collaboration with SureTest and Enfer Scientific, we can now bring a test and statistical analysis system for bovine TB to the UK. This will be available to all camelids initially. The Enferplex test we are using has been validated in alpacas (Rhodes and others 2012). It is a serological assay that identifies the presence of antibodies to Mycobacterium bovis by use of seven different bovine TB antigens, which are placed separately as individual spots on the surface of the test well. If antibodies to bovine TB are present in the blood sample, they will bind to the relevant antigen and the resultant reaction produces a luminescent reaction, the light from which can be measured and quantified as a number. Thresholds are set for each individual antigen and if the level of light is above this threshold, a positive reaction is deemed to have occurred. A positive reaction, which indicates that the animal is …

Purpose: Despite advances in assisted reproduction, there is no progress in quality control bioassays. The objectives were to develop a comet assay to measure DNA fragmentation in thawed cryopreserved oocytes and compare this assay with one-cell mouse embryo bioassay.

Purpose: Serum factors in patients with recurrent spontaneous abortions (RSA) inhibit mouse embryo development in vitro. Serum factors affecting DNA integrity remain to be tested. The null hypothesis was that patient sera do not affect DNA integrity. The objectives were (a) to use the oocyte comet assay to assess DNA damage after exposure to patient sera and (b) to determine the effect of sera from gravidity 0 parity 0 patients to induce DNA apoptosis. Methods: Luteal phase sera were drawn 1 week after embryo transfer following assisted reproductive procedures. Frozen–thawed hamster zona intact oocytes at metaphase II were incubated in groups of eight in either control medium or medium supplemented with 50% patient serum for 1.5 h at 37°C in room air. The oocytes were fixed, stained in acridine orange, embedded in agarose, lysed, and alkaline electrophoresis performed. The intensities of the digitized fluorescent images were analyzed. Results: The sera of nonpregnant patients (64%) caused significant fragmentation of hamster oocyte DNA when compared with pregnant patient sera. This difference was also observed when adjusted for patient age. Sera of patients that had never been pregnant also resulted in oocyte DNA fragmentation. Conclusions: The results suggested that sera from patients that did not conceive contained factors that did not support cell growth by causing DNA fragmentation and apoptosis. The level of the apoptotic factors varied from cycle to cycle. However, more studies are needed to determine if the sera factors actually reach the uterine environment to cause the undesirable effects.

To study the association between low percentages of intact sperm acrosomes and fertilization failures in conventional IVF procedures.A retrospective study.Clinical and academic research environment.Patients undergoing treatment of infertility.Sperm cells were fixed and stained using the Spermac stain.Percentages of intact acrosomes and fertilization.There was a significant association between specimens with40% intact acrosomes and failed conventional IVF procedures. Among the 29 cases with40% intact acrosomes, 9 cases (31%) resulted in zero penetration of the oocytes. The mean (+/-SEM) percentage of fertilization was lower in the abnormal acrosome group (43.3% +/- 6.5%) than in the normal acrosome group (64.1% +/- 5.6%). The status of the sperm acrosome was not correlated with the results of fertilization in intracytoplasmic sperm injection procedures.Sperm with low percentages of intact acrosomes were associated with failed fertilization. The Spermac stain was useful for assessing acrosomes and identifying possible male factor infertility problems. The results suggested that a minimum percentage of sperm with intact acrosomes are needed for fertilization to occur in vitro.

Background Some critically ill patients have difficulty in mobilizing their respiratory secretions. These patients can develop pulmonary atelectasis that may result in hypoxemia. There are some data to show that atelectasis may be prevented by turning a patient from side to side utilizing special beds. Study objectives To determine the role of kinetic therapy (KT) combined with mechanical percussion (P) in the resolution of established atelectasis of the lungs and hypoxemia in critically ill, hospitalized patients. (KT was defined as rotation of a patient along the longitudinal axis of ≥ 40° to each side continuously.) Design Prospective and randomized study (2:1 test to control group). Patients Twenty-four patients with respiratory failure, either mechanically ventilated or spontaneously breathing, who demonstrated segmental, lobar, or unilateral entire lung atelectasis were studied. Setting Medical ICU and adult respiratory ward in a county hospital in New York. Interventions Seventeen patients were treated with KT combined with mechanical P using a KT system (Triadyne Kinetic Therapy System; KCI; San Antonio, TX). Seven patients received manual repositioning and manual P every 2 h. Both groups received similar conventional therapy with inhaled bronchodilators and suctioning. Results Partial or complete resolution of atelectasis was seen in 14 of 17 patients (82.3%) in the test group as compared with 1 of 7 patient (14.3%) in the control group. The median duration to resolution of atelectasis was 4 days in the test group. Bronchoscopy was performed in 3 of 7 patients in the control group, but in none of the patients in the test group. A cost of $720 was incurred per patient for utilizing the specialty beds for a mean duration of 4 days. An improvement in oxygenation index occurred in the test group (change in baseline Pa o 2 /fraction of inspired oxygen from 207.4 ± 106.7 mm Hg to 318 ± 100.7 mm Hg) at the end of therapy, while the control group showed a reduction over a similar duration of time (181.3 ± 96.3 mm Hg to 112 ± 21.2 mm Hg). Conclusions KT and mechanical P therapy resulted in significantly greater partial or complete resolution of atelectasis as compared with conventional therapy. There was a generalized trend toward statistical significance in the improvement of oxygenation and a reduced need for bronchoscopy in the group receiving KT and P therapy.

Objective: To analyze the effects of certain herbs on sperm DNA and on the fertilization process. Design: Prospective comparative study. Setting: Clinical and academic research environment. Patient(s): Donor sperm specimens. Intervention(s): Zona-free hamster oocytes were incubated for 1 hour in saw palmetto ( Serenoa repens ), echinacea purpura, ginkgo biloba, St. John's wort ( Hypericum perforatum ), or control medium before sperm-oocyte interaction. The DNA of herb-treated sperm was analyzed with denaturing gradient gel electrophoresis. Main Outcome Measure(s): Oocyte penetration and integrity of the sperm BRCA1 exon 11 gene. Result(s): Pretreatment of oocytes with 0.6 mg/mL of St. John's wort resulted in zero penetration. A lower concentration (0.06 mg/mL) had no effect. High concentrations of echinacea and ginkgo also resulted in reduced oocyte penetration. Exposure of sperm to echinacea purpura and St. John's wort resulted in DNA denaturation. In contrast, saw palmetto and ginkgo had no effect. Sperm exposed to 0.6 mg/mL of St. John's wort showed mutation of the BRCA1 exon 11 gene. Conclusion(s): High concentrations of St. John's wort, echinacea, and ginkgo had adverse effects on oocytes. Saw palmetto had no effect. The data suggested that St. John's wort, ginkgo, and echinacea at high concentrations damage reproductive cells. St. John's wort was mutagenic to sperm cells.

Purpose:Biological vectors for cell transfection are mainly viral in origin, with inherent shortcomings. Mycoplasmas are ubiquitous organisms that traverse cells easily. The objective was to determine if Ureaplasma urealyticum (T-mycoplasma) would vector exogenous BRCA1 DNA into blastocysts.

Amylin is a peptide hormone cosecreted with insulin from the pancreatic beta-cells that can act as an osteoblast mitogen and as an inhibitor of bone resorption. The effects on bone of its systemic administration are uncertain. The present study addresses this question in adult male mice that were given daily subcutaneous injections of amylin (10.5 microgram) or vehicle (n = 20 in each group) for 4 wk. Histomorphometric indices of bone formation increased 30-100% in the amylin-treated group, whereas resorption indices were reduced by approximately 70% (P < 0.005 for all indices). Total bone volume in the proximal tibia was 13.5 +/- 1.4% in control animals and 23.0 +/- 2.0% in those receiving amylin (P = 0.0005). Cortical width, tibial growth plate width, tibial length, body weight, and fat mass were all increased in the amylin-treated group. It is concluded that systemic administration of amylin increases skeletal mass and linear bone growth. This peptide has potential as a therapy for osteoporosis if its bone effects can be dissociated from those on soft tissue mass.

SUMMARY Image-guided large-core needle biopsy (LCNB) of the breast is becoming widely accepted as an accurate, minimally invasive and economical way to obtain a tissue diagnosis of breast lesions. However, much of this work has been done with expensive dedicated equipment, often favouring stereotaxic guidance. Image-guided LCNB was introduced to Middlemore Hospital based around existing inexpensive equipment, and stereotaxic or ultrasound guidance was chosen depending on which showed the lesion best. Multidisciplinary clinical, radiological and pathological assessment of each case was performed. The results of 213 biopsies (112 stereotaxic and 101 ultrasound guidance) are reported here. Malignancy was shown or suspected in 85 biopsies (40%). The absolute sensitivity for malignancy was 97% with complete sensitivity of 100%. The absolute specificity was 100% and the complete specificity 98.5%. Large-core needle biopsy can be successfully implemented in a large institution without investment in expensive equipment while maintaining high ratios of malignant/benign biopsies. Attention to technique and careful multidisciplinary review are important to the success of such a programme.

This study explored the interpersonal consequences of narcissistic and borderline personality disorders. One hundred forty-nine undergraduate males and females were instructed to watch one of four videotapes depicting male or female narcissists or borderlines. A modified version of the First Impressions Questionnaire (FIQ: Bryan, Coleman, Ganong, & Bryan, 1986) was used to measure participants' perceptions of each personality type. Results suggested that the interpersonal consequences of the borderline personality style are more negative than those associated with narcissism. The male and female exhibiting borderline personality disorder were perceived as more insecure, more unstable, more unpredictable, more aggressive, and less powerful than the male and female exhibiting narcissistic personality disorder. A sequential time analysis failed to produce negative carry-over effects following presentation of personality disorders, suggesting that an initial neutral impression may override a subsequent negative impression of disordered personality when evaluated in a second neutral segment.

Purpose:The objectives of this study were (1) to determine the sperm hyperactivation and related kinematic parameters at 40°C after using four sperm wash procedures and (2) to correlate the heat-induced hyperactivation data with cases of clinical pregnancies from either artificial insemination or standard in vitro fertilization (IVF).

Objective: To analyze the motility and fertilizing capacity of sperm treated with different antibiotics. Design: Prospective comparative study. Setting: Clinical and academic research environment. Patient(s): Pooled cryopreserved donor sperm (n = 14). Intervention(s): Sperm were washed with Percoll and resuspended in HEPES-buffered human tubal fluid medium containing either amoxicillin, of ioxacin, ciprofloxacin hydrochloride, nitrofurantoin monohydrate, doxycycline hyclate, cefuroxime axetil, or control medium. Main Outcome Measure(s): Sperm kinematic and fertilizing parameters. Result(s): Sperm hyperactivation was decreased in physiologic concentrations of ciprofloxacin hydrochloride and doxycycline hyclate over the course of 48 hours. At pharmacologic concentrations, ciprofloxacin hydrochloride, cefuroxime axetil, and nitrofurantoin monohydrate adversely affected motility with decreased rapid progression. Cessation of motility occurred in cefuroxime axetil and nitrofurantoin monohydrate. Sperm hyperactivation was also absent. Cefuroxime axetil decreased the percentage of intact acrosomes. In contrast, physiologic doses of ciprofloxacin hydrochloride or of ioxacin enhanced sperm fertilizing capacity. Conclusion(s): Ciprofloxacin affected hyperactivation by altering membrane properties, whereas doxycycline inhibited the capacitation process. Cessation of motility in cefuroxime axetil was linked to disrupted sperm head membranes. Sperm motility and fertilizing capacity were decreased in nitrofurantoin because of decreased metabolism. The positive effect of of ioxacin on fertilizing capacity did not involve changes in acrosome.

Alzheimers disease (AD) is the most common neurodegenerative disease affecting millions of people in the world. Cognitive impairments such as progressive memory loss are devastating manifestations from this disease. Current pharmacological treatment has limited efficacy and only provides symptomatic relief without long-term cure. As a result, cell-replacement therapy using stem cells is an emerging potential treatment to AD. In the last decade, there have been animal trials using stem cells to treat and modulate cognitive impairment in AD models via three different mechanisms - replacing the damaged or dead cholinergic neurons; protecting neurons by reducing toxic amyloid protein aggregates or insoluble tau neurofibrillary tangles and promoting neurogenesis in hippocampus by neurotrophic secretions from stem cells. All of the trials showed promising results and improved our understandings about the mechanism of dementia in AD. With the continued improvement in safety profile of stem cell therapy and the creation of a better animal AD model in which to test them, it is feasible that stem cells could be trialled in humans for AD treatment in the next 5-10 years. © 2013 The Author 2013. Published by Oxford University Press.

Objective To determine the prevalence of mycoplasmas in washed sperm and to compare the penetration of zona-free hamster oocytes by sperm with and without mycoplasmas. Design Prospective comparative study. Setting Clinical and academic research environment. Patient(s) Semen from 34 male patients. Intervention(s) Specimens were divided, Percoll washed, and scanned for differences in kinematic parameters. Sperm DNA was extracted and assayed for mycoplasma DNA using the polymerase chain reaction-ELISA method targeting the consensus gene of 15 mycoplasma species. Remaining sperm were processed by centrifuge, Percoll, or TEST (TES and Tris) Yolk Buffer (TYB) and assessed for penetration capacity. Main Outcome Measure(s) Detection of mycoplasma DNA. Result(s) Mycoplasma DNA was detected in 29.4% of the Percoll-washed sperm. The penetration of oocytes by mycoplasma-positive sperm (59.5%±17.3%; mean±SEM) was less than mycoplasma-negative sperm (86.8%±5.4%) in the TYB-processed group. Conclusion(s) Mycoplasma DNA is demonstrated in almost a third of the Percoll-washed sperm. Because there were no other cell types except sperm, the results suggest that the mycoplasmas were either internalized or attached to the membranes. The reduced penetration by mycoplasma-positive sperm after 48-hour TYB suggest mycoplasmas required time to affect sperm function. Similarities between hypo-osmotic swelling and between kinematic parameters suggest that the mechanism does not involve differences in membrane integrity and in motility patterns.

A high-performance liquid chromatography method for analyzing the phenolic diterpenes present in rosemary (Rosmarinus officinalis L.) and commercial rosemary extracts is reported. Carnosic acid was the major phenolic diterpene present in rosemary leaves, with lesser amounts of 12-methoxycarnosic acid and carnosol. Several commercial rosemary extracts also were analyzed by this method, and in addition to these three compounds other phenolic diterpenes, such as 7-methoxyrosmanol, 7-methoxy-epirosmanol, and rosmanol, were found in some samples. These latter three compounds seem to be artifacts, produced from carnosic acid by oxidation and cyclization. The major phenolic diterpenes were isolated, and their relative antioxidatn activities in soybean oil were measured by the Rancimat. The potency of carnosic acid was more than twice that of any other compound. The antioxidant activity of pure carnosic acid was compared to butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butylhydroquinone (TBHQ) and was several times greater than BHT and BHA but less than TBHQ. Nuclear magnetic resonance data for several of the compounds that were incompletely characterized in previous literature are reported.

Background. Interest in combined modality treatment and in quality of life issues may affect the choice of radical vulvectomy as the treatment of choice in many vulvar carcinomas. To evaluate the potential role of combined radiation and chemotherapy with or without local excision as primary treatment for squamous cell carcinoma of the vulva, the outcomes of 19 patients with this disease treated with combination therapy were reviewed. Methods. Nineteen patients were treated between September 1987 and October 1992. Fifteen patients had American Joint Committee on Cancer Stage III disease ; 4 had Stage II. All had clinically negative inguinal lymph nodes with the exception of two patients who had positive ipsilateral inguinal nodes that were removed before treatment. The patients received 45-50 Gy to the pelvis and inguinal nodes with concurrent chemotherapy that consisted of 5-fluorouracil given as a 96-hour continuous infusion (1000 mg/m 2 /d) during weeks 1 and 5 of radiation. A single dose of mitomycin-C (10 mg/m 2 ) during the first day of chemotherapy has been used since November 1991. Ten patients were boosted with implants or electrons and 6 others underwent local excision. Results. The median follow-up was 34 months. Responses were determined clinically 1 month after completion of the radiation and chemotherapy. Clinically, complete responses were obtained in 10 patients (53%), partial responses in 7 (37%), and no response in 1 ; 1 patient progressed during treatment. The combined modality therapy (radiation/chemotherapy/with or without wide local excision) resulted in a local control rate of 74% (14/19). All five treatment failures occurred within 6 months of treatment. Four of these patients were rendered disease free by radical vulvectomy and/or exenteration, for an overall local control rate of 95% (18/19). Conclusion. Concurrent radiation therapy and chemotherapy with local excision performed as needed, appears to be a reasonable alternative to radical vulvectomy in patients with primary squamous cell carcinoma of the vulva. Radical surgery remains a viable option for patients in whom primary therapy has failed. Cancer 1995 ;75 :2289-94.

Objective To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. Design Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. Setting Clinical and academic research environment. Main Outcome Measure Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. Results Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. Conclusion The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

Background The use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans. Methods Herein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP). Results Neither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of DNA. After division of a defined DNA dose into multiple skin sites, the humoral response was particularly enhanced by Biojector while cellular responses were particularly enhanced by EP. Furthermore, a close correlation between in vivo antigen expression and cell-mediated as well as humoral immune responses was observed. Conclusions These results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.