Optimising the biocompatibility of 3D printed photopolymer constructs in vitro and in vivo.

3D printing is a rapid and accessible fabrication technology that engenders creative custom design solutions for cell scaffolds, perfusion systems and cell culture systems for tissue engineering. Critical to its success is the biocompatibility of the materials used, which should allow long-term tissue culture without affecting cell viability or inducing an inflammatory response for in vitro and in vivo applications. Polyjet 3D printers offer arguably the highest resolution with the fewest design constraints of any commercially available 3D printing systems. Although widely used for rapid-prototyping of medical devices and 3D anatomical modelling, polyjet printing has not been adopted by the tissue engineering field, largely due to the cytotoxicity of leachates from the printed parts. Biocompatibility in the context of cell culture is not commonly addressed for polyjet materials, as they tend to be optimised for their ability to fabricate complex structures. In order to study the potential issues surrounding the leaching of toxins, we prepared cell culture substrates using the commercially available MED610 photopolymer. The substrates were cleaned using either the manufacturer-specified 'biocompatible' washing procedures, or a novel protocol incorporating a sonication in isopropanol and water step. We then compared the effectiveness of these both in vitro and in vivo. Using primary mouse myoblast cultures, the manufacturer's protocol led to inconsistent and poorer cell viability when compared to the sonication protocol (p = 0.0002 at 48 h after indirect exposure). Subdermal implantation of MED610 into nude rats demonstrated a significant foreign body response with a greater number of giant cells (p = 0.0161) and foreign bodies (p = 0.0368) when compared to the sonication protocol, which was comparable to the control (sham) groups. These results present an improved, cytocompatible cleaning protocol of printable photopolymers to facilitate creative 3D-printed custom designs for cell culture systems for both in vitro and in vivo tissue engineering applications.

Utility of temporal artery biopsy samples for genome-wide analysis of giant cell arteritis.

Giant Cell Arteritis (GCA) is the most common vasculitis affecting the elderly. Archived formalin-fixed paraffin-embedded (FFPE) temporal artery biopsy (TAB) specimens potentially represent a valuable resource for large-scale genetic analysis of this disease. FFPE TAB samples were obtained from 12 patients with GCA. Extracted TAB DNA was assessed by real time PCR before restoration using the Illumina HD FFPE Restore Kit. Paired FFPE-blood samples were genotyped on the Illumina OmniExpress FFPE microarray. The FFPE samples that passed stringent quality control measures had a mean genotyping success of >97%. When compared with their matching peripheral blood DNA, the mean discordant heterozygote and homozygote single nucleotide polymorphisms calls were 0.0028 and 0.0003, respectively, which is within the accepted tolerance of reproducibility. This work demonstrates that it is possible to successfully obtain high-quality microarray-based genotypes FFPE TAB samples and that this data is similar to that obtained from peripheral blood.

A case of myelopathy, myopathy, peripheral neuropathy and subcortical grey matter degeneration associated with recessive compound heterozygous POLG1 mutations.

This 54year old woman presented with symptoms of sensory ataxic neuropathy, with cerebellar features. She developed further weakness, visual disturbances with diplopia, dysarthria and dysphasia. After her death at 66years, she was found to have compound heterozygous mutations of POLG1 gene in muscle, and Southern blot showed low levels of multiple deletions of mitochondrial DNA. Neuropathological examination showed profound dorsal column and dorsal spinocerebellar tract degeneration, degeneration of dorsal root ganglia and Clarke's nucleus in spinal cord and severe predominantly sensory peripheral neuropathy. The brain showed severe neuronal loss and gliosis in substantia nigra, medial posterior thalamus and head of caudate. Excess numbers of COX-negative fibres and "ragged-red" fibres were found in five skeletal muscles sampled.

Cystic lesion of the ventriculus terminalis in an adult.

We present a 40-year-old man with conus medullaris syndrome secondary to a cystic lesion of the ventriculus terminalis (CLVT) and review the relevant literature. The patient presented with 4 years of worsening right leg weakness, and examination showed bilateral fasciculations and hyporeflexia. MRI showed a cystic lesion at T11-12. He was managed with a T11-12 laminectomy and fenestration of an intramedullary cyst. A total of 32 patients, including ours, have been described since 1968: 24 were female with a mean age of 46.6 years. All patients presented symptomatically: five were managed conservatively, four using percutaneous aspiration under MRI guidance, and 22 with open surgery. We conclude that symptomatic patients are best managed surgically, although percutaneous aspiration is an emerging technique.

The four horsemen: clinicopathological correlation in 407 hospital autopsies.

BACKGROUND: Few previous papers comparing clinical diagnoses with autopsy findings present sensitivities and positive predictive values for individual conditions. The aim of this study is to determine the sensitivity and positive predictive value of current clinical diagnosis both overall and for individual conditions. METHODS: Retrospective review of hospital records of a consecutive series of patients undergoing hospital autopsy at two metropolitan teaching hospitals over a 7-year period (407 patients). Comparison was made with autopsy reports to allocate one of three outcome measures (true-positive, false-positive, false-negative) to each condition. RESULTS: The overall sensitivity and positive predictive value of clinical diagnosis were 0.74 and 0.93 respectively. Pneumonia, acute myocardial infarction, bowel ischaemia and pulmonary embolism were each present in greater than 10% of patients and had sensitivities below 0.70 and positive predictive values below 0.90. CONCLUSION: There exists a large burden of clinically undiagnosed and incorrectly diagnosed disease in hospital. Pneumonia, acute myocardial infarction, bowel ischaemia and pulmonary embolism represent important and difficult diagnostic challenges.

Diagnostic errors in patients dying in hospital: radiology's contribution.

We propose a comprehensive taxonomy of diagnostic errors in radiology that incorporates requests, image acquisition, radiological reports and evidence of communication to the treating team, and is retrospectively applicable to a given set of radiological episodes using pre-existing standard hospital databases. The taxonomy applies four binary tests to each diagnostic error using widely available hospital records, such as radiological requests, images in Picture Archiving and Communication System, radiological reports and hospital patient records. The taxonomy classifies errors into seven mutually exclusive patterns: no relevant imaging, consistent error (technical non-demonstration), consistent error (human error), ignored correct dissenting radiology result, de novo radiology error (technical non-demonstration), de novo radiology error (human error), and ignored correct confirmatory radiology result. The taxonomy was validated against a set of 250 diagnostic errors identified from an audit of clinical and radiological diagnoses with autopsy as the reference standard. All errors were successfully classified by the taxonomy, and the point of initiation of the error assigned. Of a total of 250 diagnostic errors, 138 (55%) had no relevant imaging performed. Ninety percent of all errors (226) were due to human error only, whether at the stage of clinical suspicion, the radiologist's diagnosis, or afterwards. Of the 112 imaged errors, only 12 (11%) were initiated at diagnostic imaging. The taxonomy of diagnostic error we present is comprehensive, allows retrospective audit of error with commonly available data, and provides clinically useful identification of the point of error initiation.

Sporadic encephalitis lethargica.

Three women (aged 21-36 years) developed acute illnesses that were similar to epidemic encephalitis lethargica. Each presented with a neuropsychiatric disturbance that was succeeded by pyrexia, a fluctuating conscious state and involuntary movements including oculogyria. Cerebrospinal fluid examination showed a predominantly lymphocytic pleocytosis (64-120x10(6) cells/L) and oligoclonal bands were detected in two cases. Two patients died, while the third made a gradual recovery. Post-mortem examination in the two fatal cases showed changes of lymphocytic meningitis and focal diencephalic lymphocytic infiltration, although these changes were mild relative to the effects of the clinical illness. The diagnosis of sporadic encephalitis lethargica relies on identifying shared clinical features with the past epidemic disease plus circumstantial evidence of immunological activity from laboratory investigations and some tests of exclusion of other disorders.

Trauma as a precipitant of haemorrhage in synovial cysts.

Synovial cysts have been well reported as a cause of sciatica, with a sudden acute exacerbation being attributed to haemorrhage and subsequent enlargement of the cyst. Cyst formation is attributed to facet joint degeneration associated with a defect or rupture of the joint capsule. The mechanisms of haemorrhage have not been well described previously. Two cases of haemorrhagic synovial cysts causing acute exacerbation of sciatica are described. Both cases were directly attributable to manipulation of degenerate spines. The MRI and histopathological findings are discussed and we propose a mechanism whereby excessive stress on a degenerate revascularized synovium leads to haemorrhage within synovial cysts.

Intraocular lymphoma: a series of 14 patients with clinicopathological features and treatment outcomes.

AIMS: To assess the clinical features, pathology, mortality (systemic outcome) and ocular complications (visual outcome) of a cohort of patients treated for intraocular lymphoma. METHODS: Retrospective case analysis of medical records and review of pathology of a consecutive series of patients presenting with intraocular lymphoma in Melbourne over 11 years between 1990 and 2000. Categorical factors influencing survival were examined by the Kaplan-Meier estimator and groups compared with the log rank test. RESULTS: A total of 14 patients were included. The median age of onset of symptoms was 62.5 years. Most were male (64%) and had bilateral eye involvement (64%). The commonest presentation was vitritis in 12 patients, with a median delay of 4 months before diagnosis. In all, 10 patients had B-cell lymphoma, three patients T-cell lymphoma and one null-cell. Four patients had prior systemic lymphoma. Eight patients had primary central nervous system non-Hodgkin's lymphoma (PCNSL). Treatment included combined radiation to the eye and chemotherapy in 10 patients. Complications of radiotherapy included cataract in five (50%), dry eyes in four (40%), punctate keratopathy in two (20%), radiation retinopathy in two (20%), and optic atrophy in one (10%). A total of 11 patients died of lymphoma (79%). One has residual ocular disease, while two have survived for more than 5 years from initial presentation. Although currently disease free, one of these has a poor visual outcome with acuity less than 6/60 secondary to ocular complications of treatment. CONCLUSIONS: Our study had 29% with prior systemic lymphoma, 57% associated with PCNSL and 14% with intraocular disease only. Overall survival is low (21%) and relapses common in those surviving beyond 12 months. Visual outcome in survivors is very poor due, in large part, to significant complications from radiotherapy.

Pathology of cyclodiode laser: a series of nine enucleated eyes.

AIM: To study the histological effects of cyclodiode laser treatment in humans, and to compare these findings with the clinical course, treatment response, complications, and indications for enucleation. METHOD: Detailed histological examination of nine enucleation specimens was undertaken in conjunction with a retrospective review of patient case notes. RESULTS: Retreatments had been undertaken in three cases. Although all globes showed damage to pars plicata, intact ciliary processes within the treatment zone were present in all cases. Pars plana injury was also noted in two thirds of cases. Inflammation was mild. Ciliary epithelial proliferation was seen in most cases with increasing time following treatment, in a disorganised pattern, without replication of the ciliary epithelial bilayer. No regeneration of the ciliary processes with fibrovascular cores was found. The three patients with good IOP control at enucleation had all had multiple diode treatments. Neither phthisis nor sympathetic ophthalmia was seen. CONCLUSIONS: Diode laser cyclophotocoagulation produces very characteristic injury to pars plicata, which frequently extends into pars plana, but with only mild persisting inflammation. Ciliary processes are, however, frequently spared within the treatment zone and may account for early or late treatment failure.

Presymptomatic motor neuron loss and reactive astrocytosis in the SOD1 mouse model of amyotrophic lateral sclerosis.

In familial amyotrophic lateral sclerosis (fALS), there is a need to establish more precisely the progression of the disease, particularly whether there is gradual presymptomatic neuronal loss or an abrupt loss coinciding with the symptomatic stage. To elucidate this, we investigated the progression of motor neuron loss through morphological techniques, reactive astrocytosis, and expression of ubiquitin and neurofilament proteins, by immunohistochemistry, in SOD1 G93A mice with a protracted disease course and control mice. Loss of motor neurons in SOD1 G93A mice followed a biphasic progression, with an initial loss at 126 days of age, followed by a gradual loss from onset of symptoms through to end-stage disease. Reactive astrocytosis was first observed at 70 days of age and showed a gradual increase through to end-stage disease. This suggests that there is a need for early detection of fALS cases, and potential therapeutic treatments may be more beneficial if administered at an early stage.

Scedosporium endophthalmitis: two fatal disseminated cases of Scedosporium infection presenting with endophthalmitis.

The incidence of disseminated infection with Scedosporium species is increasing in patients with haematological malignancy. Two fatal cases are reported of patients with acute myeloid leukaemia and neutropenia who presented with Scedosporium endophthalmitis. Diagnosis of fungal infection was delayed as blood and vitreous cultures were positive only after 3 days in patient 1 and blood culture was positive at 7 days in patient 2. Despite antifungal therapy with amphotericin B and additional fluconazole in patient 2, both patients died of overwhelming fungal septicaemia. Post-mortem examination of the right globe in patient 1 showed haemorrhagic necrotizing chorioretinitis with numerous fungal hyphae in choroidal vessels, choroid, retina and vitreous. Scedosporium species are often resistant to conventional antifungal therapy including amphotericin B. Diagnosis is difficult and mortality in disseminated infection is high.

Controlled study of the use of autologous serum in dry eye patients.

PURPOSE: To determine the efficacy and safety of topical autologous serum as a treatment of dry eye patients. METHODS: A 2-month, prospective, single-masked, placebo-controlled study was conducted in patients with bilateral severe dry eye. One eye was randomized to receive the patient's own serum as a tear substitute, and the fellow eye received unpreserved normal saline solution as a placebo. Subjective symptoms and clinical parameters of dry eye including conjunctival impression cytology were assessed at baseline and 1 week, 1 month, and 2 months after treatment. RESULTS: Twelve dry eye patients were enrolled. Both subjective symptoms (discomfort, foreign-body sensation, dryness, and photophobia), objective signs (fluorescein and rose bengal staining and conjunctival impression cytology) improved significantly in treated eyes compared with baseline. Control eyes also had improvement in symptoms, signs, and rose bengal staining compared with baseline. Neither Schirmer test results nor tear break-up time improved in either group. The means score of all parameters were improved in both groups, and the results of conjunctival impression cytology were better in treated eyes; however, these results are not significantly different. There were no serious adverse effects observed in this study. CONCLUSIONS: There was a trend toward improvement in symptoms and signs of dry eye including cytologic changes after application of autologous serum in severe dry eye patients. However, this trend was not statistically significant. A larger scale study is warranted.

Impression cytology following mitomycin C therapy for ocular surface squamous neoplasia.

BACKGROUND/AIMS: Topical mitomycin C (MMC) therapy has been used for treatment of ocular surface squamous neoplasia (OSSN) since 1994. Relatively few studies have reported the cellular changes in ocular surface following MMC. METHODS: Impression cytology was studied in four patients with ocular surface squamous neoplasia, either primary or recurrence after previous excisional biopsy. The authors studied samples obtained using Millipore filters at intervals between 4 and 17 weeks after commencement of MMC, and compared them with pretreatment cytology. RESULTS: MMC induced changes of cytomegaly, cytoplasmic vacuolation, nucleomegaly with nuclear wrinkling, and binucleation or multinucleation were seen in some cells in all samples. However, nuclear/cytoplasmic (N/C) ratio in these enlarged cells was normal. These changes mimicked those seen following radiation therapy in uterine cervix. Changes of increased nuclear and cell size with increased N/C ratio were seen in some dysplastic cells. The predominant form of cell death was apoptosis with fewer cells showing necrosis. CONCLUSIONS: MMC appears to produce cell death in OSSN by apoptosis and necrosis. Cellular changes related to MMC mimic those caused by radiation-cytomegaly, nucleomegaly, and vacuolation. MMC related changes may persist in ocular surface epithelium for at least 8 months following MMC therapy.

Reliability of impression cytology for the diagnosis of ocular surface squamous neoplasia employing the Biopore membrane.

AIM: To evaluate the accuracy of impression cytology employing a Biopore membrane device in the diagnosis of ocular surface squamous neoplasia (OSSN). METHODS: The histology of patients undergoing excision biopsy for the suspected diagnosis of OSSN over a 20 month period was compared with the reported cytology of impression cytology specimens obtained preoperatively using the Biopore membrane device. RESULTS: 25 excision biopsies were performed for suspected OSSN. There was accurate correlation in 20 out of the 25 cases (80%). In three cases (12%), there was poor correlation in that only a few dysplastic cells and hyperkeratosis were noted on cytology, whereas histology showed keratinising dysplasia in two cases and a moderately differentiated keratinising squamous cell carcinoma in the third. There were two cases (8%) of non-correlation--one keratinising dysplasia on histology, but anucleate keratin, squamous metaplasia, and rare atypical cells on cytology, and another case with very mild focal dysplasia on histology but squamous metaplasia on cytology. Review of the cytology showed that dysplastic cells were either rare or absent from the cytology specimens in these two cases; it was felt that these represented sampling problems rather than true false negatives. There were no false positives on cytology. Final diagnosis on histology was squamous cell carcinoma in one; carcinoma in situ in two; keratinising dysplasia in 15, non-keratinising dysplasia in seven. CONCLUSION: Impression cytology employing the Biopore membrane accurately predicts the histological diagnosis of OSSN. The presence of hyperkeratosis, inflammatory cells with only a few dysplastic cells, may indicate high grade keratinising dysplasia or squamous cell carcinoma. The method is both rapid and easy to perform in routine clinical practice. These findings suggest that there may be a role for its use in the initial assessment and follow up of patients with suspected OSSN.

Ocular adnexal lymphoproliferative disease: a series of 73 cases.

BACKGROUND: This study involved 73 patients with lymphoproliferative lesions of the ocular adnexa. The lesions were categorized using the Revised European American Lymphoma classification of lymphoid tissues and analysed to determine the frequency and prognostic impact of tumour type, location, stage and patient's age and sex. METHODS: The clinical, histopathological, immunohistochemical and phenotypic analysis by flow cytometry and follow-up data were studied. RESULTS: The ocular adnexal lymphoproliferative lesions included 70 lymphomas and six reactive lymphoid hyperplasia. Seventy-nine per cent had stage IE disease, 4% stage II, 1.5% stage III and 15.5% stage IV. Five patients (7%) had a past history of systemic lymphoma. Major histological types were extranodal marginal zone lymphoma (MZL) in 44 (63%), follicular (FL) in 12 (17%), diffuse large B-cell (DLBCL) in eight (11%), mantle cell (MCL) in two (3%), B-cell chronic lymphocytic leukaemia (CLL)/small lymphocytic lymphoma in two (3%), peripheral T-cell lymphoma (PTCL) one (1.5%) and natural killer cell lymphoma (NKCL) in one (1.5%). Longest survival was seen in those with low-grade lymphomas (MZL and FL) and worst in PTCL and NKCL. Lymphoma-related mortality was 2% for MZL, 33% for FL, 38% for DLBCL, and 100% for MCL, PTCL and NKCL. Systemic lymphoma was present prior to, at presentation or at subsequent follow up in 26/68 (39%) of all lymphoma patients, 17% for MZL, 38% for DLBCL, 83% for FL, and 100% for MCL, CLL, PTCL and NKCL. CONCLUSION: The majority of ocular adnexal lymphomas were low-grade B-cell lymphomas (MZL). Multivariate analysis showed that the only significant independent predictors of all causes of mortality were the histological type of lymphoma and the stage of disease at presentation.

Multifocal chorioretinal atrophy associated with herpes zoster ophthalmicus.

A 73-year-old woman developed multiple depigmented lesions in the fundus 4-6 months after an episode of acute Herpes zoster ophthalmicus. Post-mortem examination of the globe 15 years after this acute episode confirmed multiple old chorioretinal scars probably due to vasculitis of the short posterior ciliary arteries and branches. Patchy old infarcts were also noted in the iris.

Evaluation of risk factors for degenerative joint disease associated with hip dysplasia in German Shepherd Dogs, Golden Retrievers, Labrador Retrievers, and Rottweilers.

OBJECTIVE: To determine whether age, breed, sex, weight, or distraction index (DI) was associated with the risk that dogs of 4 common breeds (German Shepherd Dog, Golden Retriever, Labrador Retriever, Rottweiler) would have radiographic evidence of degenerative joint disease (DJD) associated with hip dysplasia. DESIGN: Cross-sectional prevalence study. ANIMALS: 15,742 dogs. PROCEDURE: Hips of dogs were evaluated radiographically by use of the ventrodorsal hip-extended view, the compression v ew, and the distraction view. The ventrodorsal hip-extended view was examined to determine whether dogs had DJD. For each breed, a multiple logistic regression model incorporating age, sex, weight, and DI was created. For each breed, disease-susceptibility curves were produced, using all dogs, regardless of age, and dogs grouped on the basis of age. RESULTS: Weight and DI were significant risk factors for DJD in all breeds. For German Shepherd Dogs, the risk of having DJD was 4.95 times the risk for dogs of the other 3 breeds combined. In all breeds, the probability of having DJD increased with age. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the probability of having hip DJD increased with hip joint laxity as measured by use of DI. This association was breed-specific, indicating that breed-specific information on disease susceptibility should be incorporated when making breeding decisions and when deciding on possible surgical treatment of hip dysplasia.

Ocular-central nervous system lymphoma mimicking posterior scleritis with exudative retinal detachment.

OBJECTIVE: We describe an unusual ocular presentation of ocular-central nervous system lymphoma in a young patient. DESIGN: Interventional case report and literature review. METHODS: A previously well 24-year-old white woman presented with left eye pain and reduced vision. Episcleral injection, globe tenderness, an afferent pupil defect, and exudative retinal detachment were present. Computed tomographic scan of the head and orbits demonstrated scleral thickening, retinal detachment, and no other abnormality. A provisional diagnosis of posterior scleritis with exudative retinal detachment was made. Investigation for underlying connective tissue diseases was negative. There was an initial prompt response to corticosteroid therapy. The patient's symptoms and signs then recurred, and a left third cranial nerve palsy developed. Systemic investigations including lumbar puncture ultimately led to the diagnosis of primary T-cell central nervous system (CNS) lymphoma. Serologic tests for human immunodeficiency virus were negative. MAIN OUTCOME MEASURES AND RESULTS: The patient underwent orbital and cranial irradiation and intrathecal and systemic chemotherapy. Despite an initial response to treatment, she returned with a recurrence of the lymphoma in the anterior segment of the left eye. Her systemic disease progressed rapidly, and she died shortly thereafter. CONCLUSIONS: This patient's young age and initial presentation mimicking posterior scleritis with unilateral exudative retinal detachment, without evidence of vitreous involvement, are highly unusual for ocular involvement in primary CNS lymphoma. A review of the literature highlights the atypical nature of this presentation.