The outcome of 12-week corticosteroid therapy in COVID-19-related diffuse interstitial lung abnormalities.

INTRODUCTION: The efficacy of long-course corticosteroid therapy in treating COVID-19-related diffuse interstitial lung abnormalities (DILA) needs to be better understood. We aimed to investigate the benefits of 12-week corticosteroid treatment in COVID-19-related DILA by evaluating computed tomography (CT) lung severity scores. MATERIALS AND METHODS: This retrospective, single-centre observational study included patients aged 18 years or older admitted with moderate to severe COVID-19 pneumonia who received 12 weeks of oral prednisolone between January 2021 and December 2021. We recorded clinical parameters, baseline CT scores and post-treatment, modified Medical Research Council (mMRC) dyspnoea scale and pulmonary function tests. RESULTS: A total of 330 patients were analysed. The mean (standard deviation, SD) age was 54.6 (14.2) years, and 43% were females. Three-point nine per cent (3.9%) require noninvasive ventilation (NIV), while 14.6% require mechanical ventilation (MV). On follow-up at 12 weeks, the CT patterns showed improvement in ground-glass opacities, perilobular density and consolidation. There was an improvement in the mean (SD) CT score before and after prednisolone therapy, with values of 17.3 (5.3) and 8.6 (5.5), respectively (p<0.001). The median mMRC was 1 (IQR 0-1), and 98.8% had a radiological response. The common side effects of prednisolone therapy were weight gain (13.9%), hyperglycaemia (1.8%) and cushingoid habitus (0.6%). CONCLUSION: A 12-week treatment with prednisolone showed significant improvement in CT scores with minimal residual dyspnoea and was relatively safe. Longer duration of steroids may be beneficial in moderate to severe COVID-19- related DILA.

Predictive risk factors for pneumothorax following fluoroscopic-guided transbronchial lung biopsy.

INTRODUCTION: Fluoroscopic-guided transbronchial lung biopsy (FG-TBLB) is routinely performed via bronchoscopy to diagnose focal peripheral lesions and diffuse lung disease. Identifying the risk factors of FG-TBLB-related pneumothorax can assist the operator in taking pre-emptive measures to prepare for this potential complication. MATERIALS AND METHODS: We retrospectively analysed data from 157 patients who underwent FG-TBLB, with the primary outcome being procedure-related pneumothorax. We assessed several risk factors for pneumothorax following FG-TBLB: patient characteristics, location of biopsy, number of biopsies and computed tomography pattern. Univariate and multivariate logistic regression analyses were performed. RESULTS: One-hundred fifty-seven patients were included [mean (SD) age 57.9 (16.2) years; 60.5% male]. The most common location for FG-TBLB was the right upper lobe (n=45, 28.7%). The mean (SD) number of biopsy samples was 6.7 (2.1). Radiographic evidence of pneumothorax was reported in 12 (7.6%) patients, with 11 of those requiring intercostal chest tube intervention (mean air leak time: 5.7 days and 1 had persistent air leak requiring autologous blood patch pleurodesis. None experienced pneumothorax recurrence. Female gender and upper lobe location of the biopsy were identified as predisposing factors for pneumothorax. In the multivariable analysis, upper lobe biopsies were associated with a higher risk of pneumothorax (OR 0.120; 95% CI 0.015-0.963; p = 0.046). CONCLUSION: The overall rate of pneumothorax is low. We recognise the increased risk of pneumothorax associated with upper lobe biopsy. These findings suggest that clinicians should exercise caution when performing FGTBLB in this region and consider alternative biopsy locations whenever feasible. We suggest adequate planning and preparation should be implemented to minimise the risk of pneumothorax following FG-TBLB.

A case series of pulmonary alveolar proteinosis: Response differently to whole lung lavage.

Pulmonary alveolar proteinosis (PAP) is a rare disease and its prognosis can be improved by whole lung lavage (WLL). Herein, we present three cases with idiopathic PAP treated successfully with either single or double WLL in the same setting. All three of them presented with exertional dyspnoea with radiographic findings of pulmonary infiltrates. They showed a marked clinical and physiologic improvement post WLL. Two of them were in remission. These three cases were diagnosed using different lung biopsy modalities, including video-assisted thoracoscopic lung biopsy, computed tomography-guided percutaneous transthoracic tru-cut needle lung biopsy, and transbronchial forceps lung biopsy (TBLB), respectively. The current cases have shown that TBLB may provide adequate diagnostic yield, and the invasive surgical lung biopsy may not be necessary to achieve a definitive diagnosis.

CT and MRI findings of acute calculous cholecystitis and its complications in Singapore: A pictorial review.

INTRODUCTION: Acute cholecystitis (AC) is a common problem encountered in surgical practice. This occurs due to obstruction of the cystic duct by calculi resulting in inflammation of the gallbladder. Increasingly, contrast enhanced computed tomography (CECT) and Magnetic Resonance Imaging (MRI) scans are being used for assessment. While the imaging features of AC are well recognized and extensively described in the literature, radiological features of the rarer complications related to AC such as pseudoaneurysm formation and gallbladder volvulus are less well known. We aim to describe these rarer findings in our pictorial review, to better educate the clinician and radiologist, such that timely diagnoses can be reached, and relevant management can be affected. METHODS: A collection of cases showing the common acute gallbladder pathologies and complications such as acute cholecystitis, gangrenous cholecystitis, emphysematous cholecystitis, haemorrhagic cholecystitis, Mirizzi's syndrome, gallbladder perforation and abscess formation, were collected between July 2016 and March 2018 at two different medical institutions in Singapore. In addition, rarer cases of gallbladder volvulus and vascular complications such as cystic artery pseudoaneurysms and vessel erosions, were also followed up. RESULTS: The CT and MRI imaging features of these conditions were discussed, with key diagnostic imaging features emphasized. CONCLUSION: Acute gallbladder pathologies are commonly encountered in day-to-day radiology practice. Knowledge of the rarer gallbladder pathologies and their key imaging features will help the radiologist, in particular, the on call radiologist in training, improve diagnostic accuracy and allow for timely management.

Clots in tuberculosis.

Tuberculosis (TB) is a common communicable disease. Active TB infection may be complicated by both venous and arterial thrombosis which are often under-recognised. We report two patients with incidental TB associated thrombosis involving different venous systems. Both responded to anticoagulant and anti-tuberculous therapy (ATT). Patients with tuberculosis are at risk of VTE and careful monitoring for venous thromboembolism (VTE) is needed during ATT. Our case illustrates the importance of having a high index of suspicion for silent VTE as it may complicate active TB infection.

A case of xanthogranulomatous appendicitis in the female pelvis.

Xanthogranulomatous inflammation is an uncommon form of chronic inflammatory process. Only a few isolated case reports of xanthogranulomatous appendicitis (XA) have been published. XA has nonspecific imaging findings and cannot be reliably differentiated on imaging from locally advanced malignancy. XA however follows a benign course and can potentially be treated with surgical resection.

Understanding of Coronavirus Disease 2019 (COVID-19) and the practice of preventive measures among doctors and nurses in a university teaching hospital- A cross-sectional study.

INTRODUCTION: Knowledge and adequate practice of preventive measures among health care workers (HCWs) are important to reduce the risk of COVID-19 transmission. METHODS: A cross-sectional study was conducted among doctors and nurses in the medical department in Pusat Perubatan Universiti Kebangsaan Malaysia between November 18, 2020 and December 18, 2020 during the third wave of COVID-19 epidemic in Malaysia. We studied the knowledge and practice of preventive measures of COVID-19 among doctors and nurses in the COVID-19 or sudden acute respiratory infection (SARI) wards and general medical wards. Data was collected using a validated self-designed google form online-questionnaire. RESULTS: A total of 407 subjects completed the study and 80.8% were females; 55.8% were aged between 30-39 years; 46.4% were medical doctors. The main source of COVID-19 knowledge was the Ministry of Health Malaysia (MOH) website (35.1%). Majority (97%) had sufficient knowledge and 82% practiced proper preventive measures. Doctors had a higher mean knowledge score compared to nurses (p < 0.001). HCWs working in COVID-19 or SARI wards scored higher in knowledge questions compared to those in the general medical wards (p = 0.020). Nurses practiced better preventive measures (p < 0.001). Good knowledge could not be predicted based on professions (OR: 0.222, 95% CI: 0.048 - 1.028, p = 0.054). Majority were unable to recall the proper steps of donning (85.8%) and doffing (98.5%). CONCLUSIONS: Although majority had good knowledge and practiced proper preventive measures, there was a poor recall in donning and doffing steps regardless of place of practice. The MOH website is a useful platform for tailored continuous medical education and regular updates on COVID-19. Regular training and retraining on donning and doffing of PPE is needed to bridge this gap.

A descriptive study of the psychological experience of health care workers in close contact with a person with COVID-19.

BACKGROUND: COVID-19 has the potential to affect the mental health of health care workers (HCWs). It is known that HCWs who serve as front-liners during the COVID-19 pandemic experience stress and have the fear of contracting the infection. Little is known of how being a positive contact affects HCWs. OBJECTIVE: We examined the experience of HCWs who were quarantined following a close unprotected contact with a COVID-19 positive colleague and explore the psychological impact especially as the timing of the quarantine coincided with the Eid (annual Muslim festival) celebration in Malaysia. METHODS: This was a cross-sectional on-line questionnaire study, involving HCWs exposed to a COVID-19 positive colleague in Universiti Kebangsaan Malaysia Medical Centre, a teaching hospital. Data on demographics, levels of depression, anxiety and stress using a validated depression, anxiety, and stress scale (DASS-21) questionnaire, aspects of quarantine, wearing of masks, hand hygiene practice and swab experience were collected. RESULTS: Twenty-two HCWs participated. Eighteen (81.8%) were between 30-39 years and 17 (77.3%) were women. Majority 19 (86.3%) were Malays. There were twelve (54.5%) medical officers, 5 (22.7%) specialists and 5(22.7%) allied health staff. Eighteen out of 22 (81.8%) felt they were able to do home quarantine adequately. All tested negative with a mean (Standard Deviation) hour of contact of 2.56±2.38 hours. Eighteen reported their biggest concern was infecting their families. CONCLUSION: HCWs undergoing contact swabbing and quarantine are vulnerable to depression, anxiety and stress. The ability of the HCW to adequately home quarantine should not be taken for granted. Psychological support should be offered to HCWs who are positive contacts.

Lung computed tomography patterns of a cluster of asymptomatic young males with COVID-19 admitted to a teaching hospital in Kuala Lumpur.

BACKGROUND AND OBJECTIVE: Coronavirus Disease 2019 (COVID- 19) was first reported in Malaysia in March 2020. We describe here the clinical characteristics and computed tomography (CT) patterns in asymptomatic young patients who had laboratory-confirmed COVID-19. METHODS: This is a retrospective observational study where 25 male in-patients with laboratory-confirmed COVID-19 in Hospital Canselor Tuanku Muhriz. Demographics, clinical data and CT images of these patients were reviewed by 2 senior radiologists. RESULTS: In total there were 25 patients (all males; mean age [±SD], 21.64±2.40 years; range, 18-27 years). Patients with abnormal chest CT showed a relatively low normal absolute lymphocytes count (median: 2.2 x 109/L) and absolute monocyte count (median: 0.5 x 109/L). Lactate dehydrogenase was elevated in 5 (20%) of the patients. The procalcitonin level was normal while elevated levels of alanine aminotransferase, total bilirubin, platelet and C-reactive protein were common. Baseline chest CT showed abnormalities in 6 patients. The distribution of the lesions were; upper lobe 3 (12%) lower lobe 3 (12%) with peripheral distribution 4 (16%). Of the 25 patients included, 4 (16%) had ground glass opacification (GGO), 1 (4%) had a small peripheral subpleural nodule, and 1 (4%) had a dense solitary granuloma. Four patients had typical CT features of COVID-19. CONCLUSION: We found that the CT imaging showed peripheral GGO in our patients. They remained clinically stable with no deterioration of their respiratory symptoms suggesting stability in lung involvement. We postulate that rapid changes in CT imaging may not be present in young, asymptomatic, non-smoking COVID-19 patients. Thus the use of CT thoraxfor early diagnosis may be reserved for patients in the older agegroups, and not in younger patients.

Tactile acuity and predominance of central sensitization in subjects with non-specific persistent low back pain.

Aim: This study aimed to evaluate differences in tactile acuity (TA) in people with non-specific persistent low back pain (NSPLBP) with and without predominant central sensitisation (CS).Method: An analytical cross-sectional study was conducted with 45 participants divided into three groups: (i) subjects with NSPLBP with predominant CS (n = 14), (ii) subjects with NSPLBP without predominant CS (n = 16) and (iii) subjects without low back pain (n = 15). Using an analogue calliper, TA was measured using the two-point discrimination threshold (TPD) in the three groups, both horizontally and vertically in the painful region. The analysis was based on the comparison of median discrimination thresholds between groups using the Kruskal-Wallis test.Result: A higher median TPD value was observed in the group with NSPLBP with predominant CS (vertical measurement 37.5 mm; horizontal measurement 52.5 mm) compared to the group with NSPLBP without predominant CS (vertical measurement 32.5 mm; horizontal measurement 33.8 mm) and the group without low back pain (vertical measurement 30 mm; horizontal measurement 27.5 mm) (p < 0.0001), both in vertical and horizontal measurement.Conclusion: The findings found in this study highlight the need to differentiate patients with NSPLBP with predominant CS when considering therapeutic evaluation as an indirect mechanism for assessing the perceptual function of the primary somatosensory cortex.

Role of IL-13Rα2 in modulating IL-13-induced MUC5AC and ciliary changes in healthy and CRSwNP mucosa.

BACKGROUND: The IL-13 receptor α2 (IL-13Rα2) is a receptor for IL-13 which has conflicting roles in mediating IL-13 responses in the lower airway, with little known about its impact on upper airway diseases. We sought to investigate the expression of IL-13 receptors, IL-13Rα1 and IL-13Rα2, in chronically inflamed nasal epithelium, and explore IL-13-induced signaling pathways in an in vitro model of human nasal epithelial cells (hNECs). METHODS: The protein and mRNA expression levels of IL-13 and its receptors in nasal biopsies of patients with nasal polyps (NP) and healthy controls were evaluated. We investigated goblet cell stimulation with mucus hypersecretion induced by IL-13 (10 ng/mL, 72 hours) treatment in hNECs using a pseudostratified epithelium in air-liquid interface (ALI) culture. RESULTS: There were significant increases in IL-13, IL-13Rα1, and IL-13Rα2 mRNA and protein levels in NP epithelium with healthy controls as baseline. MUC5AC mRNA positively correlated with IL-13Rα2 (r = .5886, P = .002) but not with IL-13Rα1 in primary hNECs. IL-13 treatment resulted in a significant increase in mRNA and protein levels of IL-13Rα2 only in hNECs. IL-13 treatment induced an activation of extracellular signal-regulated kinases (ERK)1/2 and an upregulation of C-JUN, where the IL-13-induced effects on hNECs could be attenuated by ERK1/2 inhibitor (50 µmol/L) or dexamethasone (10-4 -10-7  mol/L) treatment. CONCLUSIONS: IL-13Rα2 has a potential role in IL-13-induced MUC5AC and ciliary changes through ERK1/2 signal pathway in the nasal epithelium. IL-13Rα2 may contribute to airway inflammation and aberrant remodeling which are the main pathological features of CRSwNP.

Predicting early recurrence of hepatocellular carcinoma with texture analysis of preoperative MRI: a radiomics study.

AIM: To investigate the feasibility of using texture analysis in preoperative magnetic resonance imaging (MRI) to predict early recurrence (ER) in hepatocellular carcinoma (HCC) post-curative surgery. MATERIAL AND METHODS: Institutional review board was obtained. A retrospective review of all patients who underwent hepatectomy between 1 January 2007 and 31 December 2015 was performed. Inclusion criteria included preoperative MRI, tumour size ≥1 cm, new cases of HCC. Exclusion criteria included loss to follow-up, ruptured HCCs, movement artefacts, and previous hepatectomy or interval adjuvant therapy. Patients were divided into ER and late or no recurrence (LNR) groups. ER was defined as new foci of HCC within 730 days of curative surgery. Radiomics feature extraction was performed on T2, diffusion-weighted imaging (DWI), T1 arterial, and T1 portovenous acquisitions on MATLAB (Mathworks, Matick, MA, USA). The MaZda software was used to analyse 290 texture parameters and PRTools was used for feature selection. RESULTS: Fifty patients (43 male, mean age 67 years) were divided into ER (n=20) and LNR (n=30) groups. Serum alpha-fetoprotein level (p=0.026), serum É£-glutamyltranspeptidase (p=0.014), Child-Pugh score (p=0.02) and the presence of vascular invasion (gross and/or microvascular, p=0.025) were found to be statistically significant different between the two groups. Parameters S(4,0)SumVarnc, S(0,3)SumOfSqs, and S(1,1)DifVarnc of the equilibrium phase were most accurate, achieving 84%, 82%, and 78% accuracy, respectively. CONCLUSION: Texture analysis of preoperative MRI has the potential to predict ER of HCC with up to 84% accuracy using an appropriate, single texture analysis parameter. Future studies are needed to validate these findings.

Scrotal emergencies: An imaging perspective.

Acute scrotal pain is a common complaint in emergency or primary care practice. A myriad of pathologies need to be considered, and while the clinical history often leads the clinician to the correct diagnosis, radiologists are often called upon to provide further diagnostic information through ultrasonography. Here, we present the sonographic features of various scrotal emergencies, as well as a few pitfalls. Through this, we explore the breadth of urgent pathologies that may be encountered, both traumatic and non-traumatic. For the clinician who performs point-of-care ultrasound, whether in clinic or in the emergency department, we hope that this pictorial article will lay a good foundation for confident and accurate image interpretation.

Human papillomavirus 16 (HPV16) and HPV52 E6-specific immunity in HIV-infected adults on combination antiretroviral therapy.

OBJECTIVES: Human papillomavirus (HPV)-associated cancers disproportionately affect those infected with HIV despite effective combination antiretroviral therapy (cART). The primary aim of this study was to quantify HPV16 and HPV52 E6-specific interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) T-cell responses, a correlate of protective immunity, in the first year following cART initiation and subsequently in those patients with suboptimal (sIR) and optimal (oIR) immune reconstitution. METHODS: Ninety-four HIV-infected patients were recruited to the study; a longitudinal cohort of patients recruited just prior to commencing cART and followed up for 48 weeks (n = 27), and a cross-sectional cohort (n = 67) consisting of patients with sIR (CD4 T-cell count < 350 cells/µL) and oIR (CD4 T-cell count > 500 cells/µL) after a minimum of 2 years on cART. Controls (n = 29) consisted of HIV-negative individuals. IFN-γ ELISPOT responses against HPV16 and HPV52 E6 were correlated to clinical characteristics, anal and oral HPV carriage, T-cell maturational subsets, markers of activation, senescence and T-regulatory cells. RESULTS: HPV16 and HPV52 E6-specific T-cell responses were detected in only one of 27 patients (3.7%) during the initial phase of immune recovery. After at least 2 years of cART, those who achieved oIR had significantly higher E6-specific responses (9 of 34; 26.5%) compared with those with sIR (2 of 32; 6.3%) (P = 0.029). Apart from higher CD4 T-cell counts and lower CD4 T-cell activation, no other immunological correlates were associated with the detection of HPV16 and HPV52 E6-specific responses. CONCLUSIONS: HPV16 and HPV52 E6-specific IFN-γ T-cell responses, a correlate of protective immunity, were detected more frequently among HIV-infected patients who achieved optimal immune recovery on cART (26.5%) compared with those with suboptimal recovery (6.3%).

HLA-B*13:01 and the dapsone hypersensitivity syndrome.

BACKGROUND: Dapsone is used in the treatment of infections and inflammatory diseases. The dapsone hypersensitivity syndrome, which is associated with a reported mortality of 9.9%, develops in about 0.5 to 3.6% of persons treated with the drug. Currently, no tests are available to predict the risk of the dapsone hypersensitivity syndrome. METHODS: We performed a genomewide association study involving 872 participants who had received dapsone as part of multidrug therapy for leprosy (39 participants with the dapsone hypersensitivity syndrome and 833 controls), using log-additive tests of single-nucleotide polymorphisms (SNPs) and imputed HLA molecules. For a replication analysis, we genotyped 24 SNPs in an additional 31 participants with the dapsone hypersensitivity syndrome and 1089 controls and performed next-generation sequencing for HLA-B and HLA-C typing at four-digit resolution in an independent series of 37 participants with the dapsone hypersensitivity syndrome and 201 controls. RESULTS: Genomewide association analysis showed that SNP rs2844573, located between the HLA-B and MICA loci, was significantly associated with the dapsone hypersensitivity syndrome among patients with leprosy (odds ratio, 6.18; P=3.84×10(-13)). HLA-B*13:01 was confirmed to be a risk factor for the dapsone hypersensitivity syndrome (odds ratio, 20.53; P=6.84×10(-25)). The presence of HLA-B*13:01 had a sensitivity of 85.5% and a specificity of 85.7% as a predictor of the dapsone hypersensitivity syndrome, and its absence was associated with a reduction in risk by a factor of 7 (from 1.4% to 0.2%). HLA-B*13:01 is present in about 2 to 20% of Chinese persons, 1.5% of Japanese persons, 1 to 12% of Indians, and 2 to 4% of Southeast Asians but is largely absent in Europeans and Africans. CONCLUSIONS: HLA-B*13:01 was associated with the development of the dapsone hypersensitivity syndrome among patients with leprosy. (Funded by the National Natural Science Foundation of China and others.).

Thymic carcinoma presenting with an unusual and delayed metastasis to the neural foramen, mimicking thoracic spinal radiculopathy.

We report a case of metastatic thymic carcinoma which presented as an enhancing mass located in the neural foramen of the thoracic spine. More common tumours which arise in the neural foramen would include a neurogenic tumour or developmental anomalies such as a foregut duplication cyst. This case is singular firstly because the lesion present as radiculopathy which mimics a neurogenic tumour. Secondly, the presentation was unusually delayed as the patient presented to our centre more than a decade after the resection of the primary tumour in another institution.

The timing of deep brain stimulation for Parkinson disease in the UK from 1997 to 2012.

BACKGROUND AND PURPOSE: Deep brain stimulation (DBS) for Parkinson disease (PD) has traditionally been reserved for the late stages of the disease. There is evidence that DBS is also effective if applied earlier in the disease course. Changes in the frequency of DBS procedures in the UK over a 15-year period were investigated. METHODS: A retrospective review was performed of patient age and disease duration for DBS surgery for PD in UK neurosurgical units from 1997 to 2012 using departmental databases. RESULTS: The number of DBS procedures in the UK increased from three in 1997 to over 80 per year during this period. The mean age at the time of surgery (60 years) and the mean duration of PD at the time of DBS (11 years) remained unchanged over 15 years. CONCLUSIONS: The age and disease duration at which DBS is performed for PD in the UK has been static over a 15-year period and DBS appears to remain a therapy for PD applied late in its course. This may change in the light of clinical evidence suggesting a benefit for earlier DBS.

Laboratory detection of strongyloidiasis: IgG-, IgG4 - and IgE-ELISAs and cross-reactivity with lymphatic filariasis.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG, IgG4 and IgE antibodies against Strongyloides stercoralis. A commercial ELISA (IVD Research, USA) was also used, and the sensitivities and specificities of the four assays were determined. Serum samples from 26 patients with S. stercoralis infection and 55 patients with other infections or no infection were analysed. Sensitivities of the IgG4 , IgG, IgE and IgG (IVD) assays were 76.9%, 84.6%, 7.7% and 84.6%, respectively, while the specificities were 92.7%, 81.8%, 100% and 83.6%, respectively. If filariasis samples were excluded, the specificities of the IgG4 -ELISA and both IgG-ELISAs increased to 100% and 98%, respectively. A significant positive correlation was observed between IgG- and IgG4 -ELISAs (r = 0.4828; P = 0.0125). IgG- and IgG- (IVD) ELISAs (r = 0.309) were positively correlated, but was not significant (P = 0.124). Meanwhile there was no correlation between IgG4 - and IgG- (IVD) ELISAs (r = 0.0042; P = 0.8294). Sera from brugian filariasis patients showed weak, positive correlation between the titres of antifilarial IgG4 and the optical densities of anti-Strongyloides IgG4 -ELISA (r = 0.4544, P = 0.0294). In conclusion, the detection of both anti-Strongyloides IgG4 and IgG antibodies could improve the serodiagnosis of human strongyloidiasis. Furthermore, patients from lymphatic filariasis endemic areas who are serologically diagnosed with strongyloidiasis should also be tested for filariasis.

Engineering three-dimensional constructs of the periodontal ligament in hyaluronan-gelatin hydrogel films and a mechanically active environment.

BACKGROUND AND OBJECTIVE: Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan-gelatin hydrogel films (80-100 µm) in a format capable of being mechanically deformed. MATERIAL AND METHODS: Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1-3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin. RESULTS: Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1-3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures. CONCLUSION: Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell-cell and cell-matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment.

Advanced glycation end-products (AGEs) and functionality of reverse cholesterol transport in patients with type 2 diabetes and in mouse models.

AIMS/HYPOTHESIS: We investigated the contribution of AGEs to the impairment of reverse cholesterol transport (RCT) variables in diabetic individuals and in two animal models of diabetic obesity and of renal impairment. METHODS: The capacity of plasma and HDL from 26 individuals with moderately controlled type 2 diabetes to support cholesterol efflux was compared with 26 age- and sex-matched individuals without diabetes. We also compared the rates of RCT in vivo in two animal models: db/db mice and mice with chronic renal failure. RESULTS: Diabetic individuals had characteristic dyslipidaemia and higher levels of plasma AGEs. The capacity of whole plasma, ApoB-depleted plasma and isolated HDL to support cholesterol efflux was greater for diabetic patients compared with controls despite their lower HDL-cholesterol levels. The capacity of plasma to support cholesterol efflux correlated with plasma levels of cholesteryl ester transfer protein and levels of ApoB, but not with levels of AGE. RCT was severely impaired in db/db mice despite elevated HDL-cholesterol levels and no change in AGE concentration, whereas RCT in uraemic mice was unaffected despite elevated AGE levels. CONCLUSIONS/INTERPRETATION: AGEs are unlikely to contribute significantly to the impairment of RCT in type 2 diabetes.