Unique CRF01_AE Gag CTL epitopes associated with lower HIV-viral load and delayed disease progression in a cohort of HIV-infected Thais

Cytotoxic T Lymphocytes (CTLs) play a central role in controlling HIV-replication. Although numerous CTL epitopes have been described, most are in subtype B or C infection. Little is known about CTL responses in CRF01_AE infection. Gag CTL responses were investigated in a cohort of 137 treatment-naïve HIV-1 infected Thai patients with high CD4+ T cell counts, using gIFN Enzyme-Linked Immunospot (ELISpot) assays with 15-mer overlapping peptides (OLPs) derived from locally dominant CRF01_AE Gag sequences. 44 OLPs were recognized in 112 (81.8%) individuals. Both the breadth and magnitude of the CTL response, particularly against the p24 region, positively correlated with CD4+ T cell count and inversely correlated with HIV viral load. The breadth of OLP response was also associated with slower progression to antiretroviral therapy initiation. Statistical analysis and single peptide ELISpot assay identified at least 17 significant associations between reactive OLP and HLA in 12 OLP regions; 6 OLP-HLA associations (35.3%) were not compatible with previously reported CTL epitopes, suggesting that these contained new CTL Gag epitopes. A substantial proportion of CTL epitopes in CRF01_AE infection differ from subtype B or C. However, the pattern of protective CTL responses is similar; Gag CTL responses, particularly against p24, control viral replication and slow clinical progression.     

Predictors,Neuroimaging Characteristics and Long-Term Outcome of Severe European Tick-Borne Encephalitis: A Prospective Cohort Study

MethodsA cohort of 111 TBE patients (median age 51, range 17–75 years; 42% females) was analysed prospectively. Data were acquired from the department of neurology, University Hospital Heidelberg, and the infectious diseases registry of the Robert-Koch institute Berlin. Neurological status was ascertained by protocol at admission and discharge and the degree of disability was scored using the modified RANKIN Scale (mRS; clinical score addressing neurological disability, range from 0, healthy to 6, dead) at admission and at follow-up. Follow-up examination was conducted by means of a telephone interview. To identify independent predictors for severe TBE and functional outcome, modelled logistic regression was performed. MRI changes were correlated with infection variants. To assess alpha-motor neuron injury patterns, we used high-resolution magnetic resonance neurography (hrMRN). Analyses were performed at the Department of Neurology, University Hospital, University of Heidelberg from April 2004 through September 2014ResultsAcute course: 3.6% of patients died during the acute infection. All patients with a lethal course suffered from meningoencephaloradiculitis (MER, 14.4% of the cohort), which is associated with a significantly higher risk of requiring intensive care (p = 0.004) and mechanical ventilation (p<0.001) than menigoencephalitis (ME, 27.9% of the cohort). At admission, both MER and ME groups were severely affected, with the MER group having a statistically higher mRS score (median of 5 in the MER groups versus 4 in the ME group; p<0.001). Long-term outcome: outcome for MER was considerably worse (median mRS = 4) than for ME (mRS = 1, p<0.0001) and meningitis (mRS = 0, 57.7% of the cohort). Risk factors: advanced age (p<0.001) and male gender (p = 0.043) are independent risk factors for a severe infection course. Furthermore, we identified pre-existing diabetes mellitus (p = 0.024) as an independent risk factor for MER. In MER, alpha-motor neuron injury accounts for the poor prognosis confirmed by hrMRN.

Conclusion and Relevance

These data provide critical information for neurologists and other health professionals to use in evaluating TBEV patients who live in or travel to endemic areas. This information can be used to classify clinical presentation and estimate infection-associated complications and individual prognosis. Furthermore, the risk for severe, disabling infections in older patients should prompt general practitioners to recommend and encourage vaccination to those patients living in or travelling to endemic areas.     

Tumour necrosis factor -308 and -238 promoter polymorphisms are predictors of a null virological response in the treatment of Brazilian hepatitis C patients

Certain host single nucleotide polymorphisms (SNPs) affect the likelihood of a sustained virological response (SVR) to treatment in subjects infected with hepatitis C virus (HCV). SNPs in the promoters of interleukin (IL)-10 (-1082 A/G, rs1800896), myxovirus resistance protein 1 (-123 C/A, rs17000900 and -88 G/T, rs2071430) and tumour necrosis factor (TNF) (-308 G/A, rs1800629 and -238 G/A, rs361525) genes and the outcome of PEGylated α-interferon plus ribavirin therapy were investigated. This analysis was performed in 114 Brazilian, HCV genotype 1-infected patients who had a SVR and in 85 non-responders and 64 relapsers. A significantly increased risk of having a null virological response was observed in patients carrying at least one A allele at positions -308 [odds ratios (OR) = 2.58, 95% confidence intervals (CI) = 1.44-4.63, p = 0.001] or -238 (OR = 7.33, 95% CI = 3.59-14.93, p < 0.001) in the TNF promoter. The risk of relapsing was also elevated (-308: OR = 2.87, 95% CI = 1.51-5.44, p = 0.001; -238: OR = 4.20, 95% CI = 1.93-9.10, p < 0.001). Multiple logistic regression of TNF diplotypes showed that patients with at least two copies of the A allele had an even higher risk of having a null virological response (OR = 16.43, 95% CI = 5.70-47.34, p < 0.001) or relapsing (OR = 6.71, 95% CI = 2.18-20.66, p = 0.001). No statistically significant association was found between the other SNPs under study and anti-HCV therapy response.     

CDC28 protein kinase regulatory subunit 1B (CKS1B) expression and genetic status analysis in oral squamous cell carcinoma

CKS1B is a member of the highly conserved cyclin kinase subunit 1 (CKS1) protein family which interacts with cyclin-dependent kinases and plays a critical role in cell cycle progression. In oral squamous cell carcinoma (OSCC), as in other malignancies, CKS1B overexpression has been correlated with reduced survival. To our knowledge, no studies evaluating the genetic status of CKS1B gene in OSCC have been reported. Herein, genetic and protein status of CKS1B were analyzed by immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) techniques in a series of primary OSCC (n=51) and lymph node OSCC metastases samples (n=14). The observed results were compared with those obtained in either inflammatory (oral lichen planus [OLP]) (n=13) and premalignant oral mucosal lesions (oral leukoplakia) (n=16). A significant CKS1B overexpression was observed in OSCC and lymph node metastases samples than in OLP and oral leukoplakia (mean 70% vs 35%, p<0.001). CKS1B overexpression correlated with p27 loss of expression (p=0.0013) and SKP2 overexpression (p<0.00). FISH study disclosed statistical differences in both gene amplifications and gains between samples corresponding to OSCC and metastases from those of OLP and leukoplakia (p<0.001). Amplifications were present in 53% of OSCC samples and 33% of lymph node metastases vs 14% of oral leukoplakia and 0% of OLP biopsy specimens (p=0.002). Polysomies of chromosome 1 were seen in 46% of OSCC, 33% of ganglionar metastases, 14% of oral leukoplakia and 10% of OLP (p=0.036). Correlation of CKS1B over-expression and gains (both polysomies and amplifications) determined by FISH was statistically significant (p<0.001). Our results indicate that a high CKS1B expression is a common finding in primary OSCC which correlates with p27 low expression and SKP2 overexpression. This phenomenon may be due either to numerical (chromosome 1 polysomy) or structural (amplifications) CKS1B genetic abnormalities. This phenotypical and cytogenetic profile is not observed in premalignant or inflammatory oral mucosal lesions.     

The relationship of the ESR1 gene polymorphisms with the presence of coronary artery disease determined by coronary angiography

Effects of estrogen on the cardiovascular system, mediated mainly by estrogen receptor type alpha (ER alpha), have been well-defined and specific polymorphisms in the ER alpha gene (ESR1) have been associated with several coronary heart diseases including coronary artery disease (CAD) in studies covering different populations. In the present study, we aimed to investigate whether there is an association between two of the known polymorphisms in the ESR1, named c.454-397T>C and c.454-351A>G, and CAD in a Turkish population. One hundred sixty eight patients with CAD and 99 patients without CAD were included in the study. The ESR1 c.454-397T>C and c.454-351A>G polymorphisms were studied by the conventional polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. While no association was found between the c.454-351A>G polymorphism and CAD, the c.454-397T>C genotype distributions were statistically significant independent of known risk factors between CAD-positive (CAD+) and CAD-negative (CAD-) groups (p = 0.001). TT genotype was more frequent in CAD- group than in CAD+ group, 22.2% and 4.8%, respectively. CC genotype was associated with increased risk of CAD (p = 0.001) compared to the TT genotype. When comparing the distribution of CC + TC genotypes to that of TT genotype in CAD+ and CAD- groups, the frequency of CC + TC genotypes showed a significant increase independent of known CAD risk factors in CAD+ subjects (p = 0.001). As a conclusion, a statistically significant relationship between the ESR1 c.454-397T>C polymorphism and CAD were found independent of known CAD risk factors in a Turkish population.     

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2′-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20–24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50 mg/kg) treatment induced a meiotic delay of about 24 h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50 mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.     

Study on the characteristics of contrast-enhanced ultrasound and its utility in assessing the microvessel density in ovarian tumors or tumor-like lesions

Angiogenesis is a critical factor in tumor growth and metastasis, and microvessel density (MVD) was an important parameter for assessing vessels in tumors. However, radiologic assessment of tumor vascularity is not yet well established. In our study, we aimed at investigating the efficacy of contrast-enhanced ultrasonography (CEUS) in exploring the vascularity of the ovarian tumors or tumor-like lesions to assess the relationship between the parameters of the peak intensity (PI) and area under curve (AUC) on CEUS and MVD in ovarian masses. Compared to the contrast-enhanced ultrasound technique, conventional ultrasound shows limitation in differentiating benign and malignant ovarian tumors. The former is promising in improving the sensitivity of detecting small vessels and blood flow in ovarian tumors. Our results showed clear differences in enhancement patterns between benign and malignant ovary tumors or tumor-like lesions. The PI and AUC in the malignant tumors were significantly higher than those in the benign tumors or tumor-like lesions (p=0.001 and =0.01, respectively). The MVD was 43.1 ± 20.4 in the benign tumors or tumor-like lesions and was 65.3 ± 22.3 in the malignant ones (p= 0.01). In both the benign and malignant groups, the PI and AUC were correlated significantly with the MVD (r=0.595, p = 0.001; r =0.533, p = 0.003, respectively). The PI and AUC in CEUS can reflect the MVD in ovarin tumors. The PI and AUC of the ovarian masses in the contrast transvaginal sonography show significant correlation with the angiogenesis and may help in assessing tumor vascularity in ovarian masses.     

The Relationship Between Lesion Size and Load to Failure After Stabilization of Simulated Metastatic Lesions of the Proximal Femur

BackgroundAs overall cancer survival continues to improve, the incidence of metastatic lesions to the bone continues to increase. The subsequent skeletal related events that can occur with osseous metastasis can be debilitating. Complete and impending pathologic femur fractures are common with patients often requiring operative fixation. However, the efficacy of an intramedullary nail construct, on providing stability, continue to be debated. Therefore, the purpose of this study was to utilize a synthetic femur model to determine 1) how proximal femur defect size and cortical breach impact femur load to failure (strength) and stiffness, and 2) and how the utilization of an IMN, in a prophylactic fashion, subsequently alters the overall strength and stiffness of the proximal femur.MethodsA total of 21 synthetic femur models were divided into four groups: 1) intact (no defect), 2) 2 cm defect, 3) 2.5 cm defect, and 4) 4 cm defect. An IMN was inserted in half of the femur specimens that had a defect present. This procedure was performed using standard antegrade technique. Specimens were mechanically tested in offset torsion. Force-displacement curves were utilized to determine each constructs load to failure and overall torsional stiffness. The ultimate load to failure and construct stiffness of the synthetic femurs with defects were compared to the intact synthetic femur, while the femurs with the placement of the IMN were directly compared to the synthetic femurs with matching defect size.ResultsThe size of the defect invertedly correlated with the load the failure and overall stiffness. There was no difference in load to failure or overall stiffness when comparing intact models with no defect and the 2 cm defect group (p=0.98, p=0.43). The 2.5 cm, and 4.5 cm defect groups demonstrated significant difference in both load to failure and overall stiffness when compared to intact models with results demonstrating 1313 N (95% CI: 874-1752 N; p<0.001) and 104 N/mm (95% CI: 98-110 N/mm; p=0.03) in the 2.5 cm defect models, and 512 N (95% CI: 390-634 N, p<0.001) and 21 N/mm (95% CI: 9-33 N/mm, p<0.001) in the models with a 4 cm defect. Compared to the groups with defects, the placement an IMN increased overall stiffness in the 2.5 cm defect group (125 N/mm; 95% CI:114-136 N/mm; p=0.003), but not load to failure (p=0.91). In the 4 cm defect group, there was a significant increase in load to failure (1067 N; 95% CI: 835-1300 N; p=0.002) and overall stiffness (57 N/mm; 95% CI:46-69 N/mm; p=0.001).ConclusionProphylactic IMN fixation significantly improved failure load and overall stiffness in the group with the largest cortical defects, but still demonstrated a failure loads less than 50% of the intact model. This investigation suggests that a cortical breach causes a loss of strength that is not completely restored by intramedullary fixation. Level of Evidence: II     

Altered expression of the cell cycle regulatory molecules pRb, p53 and MDM2 exert a synergetic effect on tumor growth and chromosomal instability in non-small cell lung carcinomas (NSCLCs)

BACKGROUND: Recent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors. MATERIAL AND METHODS: A total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods. RESULTS: Aberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this "full abnormal" phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the "normal" phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the "normal" and "full abnormal" phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when the expression of two components was altered (p = 0.055). CONCLUSIONS: Our findings suggest that simultaneous deregulation of all members of the pRb-p53-MDM2 network confers an additive effect on tumor growth. The apoptotic pathway seems to be more susceptible to its defects than the cell proliferation machinery. The findings of the ploidy analysis, which are in parallel with those regarding the proliferative activity and the apoptotic rate study, further support the concept that these molecules constitute a tightly regulated network participating in cell cycle control and chromosomal stability.     

Establishment of neuronal connectivity during development of the Drosophila larval visual system

We used confocal microscopy in conjunction with specific antibodies and enhancer trap strains to investigate the development of specific neuronal connections in a simple model system, the larval visual system of Drosophila. We find that the establishment of axonal projections from the larval photoreceptor neurons to their central nervous system targets involves a series of discrete steps. During embryogenesis, the larval optic nerve contacts several different cell types, including optic lobe pioneer (OLP) neurons and a number of glial cells. We demonstrate that OLP neurons are present and project normally in glass (gl) mutant embryos in which the larval optic nerve fails to develop, suggesting that they do not depend on interactions with the larval optic nerve for differentiation and proper axonal projection. The OLPs fail to differentiate properly in disconnected (disco) mutant embryos, where appropriate connections between the larval optic nerve and its targets in the brain are not formed. The disco gene is expressed in the OLPs and may therefore act autonomously to direct the differentiation of these cells. Taken together, our results suggest that the OLPs act as an intermediate target required for the establishment of normal optic nerve projection and connectivity. © 1995 John Wiley & Sons, Inc.     

Genetic alterations in gastric precancerous lesions

To investigate the occurrence of 17p (p53) loss of heterozygosity (LOH) and increased 4N or aneuploidy in gastric precancerous lesions (GPL), and their association with Helicobacter pylori (H pylori) infection. A total of 78 gastric mucosal biopsy specimens, including 10 normal mucosa and 68 gastric precancerous lesions [chronic atrophic gastritis (CAG, n = 20), intestinal metaplasia (IM, n = 12), low grade dysplasia (LGD, n = 15), and high grade dysplasia (HGD, n = 21)] were studied using PCR and flow cytometry. A modified Giemsa staining technique was used to detect H pylori. The study was performed in Erzurum Numune Hospital between 2007 and 2009. 17p (p53) LOH was observed in (1/20) 5% of CAG, in (2/12) 16% of IM, in (3/15) 20% of LGD and in (11/21) 53% of HGD. There was correlation between prevalence of 17p (p53) LOH and histological type of GPL (P = 0.004). Similarly, increased 4N or aneuploidy was detected in (1/20) 5% of CAG, in (1/12) 8% of IM, in (2/15) 13% of LGD and in (9/21) 43% of HGD. The correlation was found between aneuploidy and histological type of GPL (P = 0.009). However, there was no correlation between presence of H pylori infection in histological type of GPL (P = 0.921). On the other hand, a significant association was found between increased 4N or aneuploidy and 17p (p53) LOH in all of GPL (P = 0.0001). However, there was no statistically significant association between H pylori infection and 17p (p53) LOH or increased 4N/aneuplody in GPL. 17p (p53) LOH and increased 4N or aneuploidy are closely related to the early stages of gastric carcinogenesis.     

Quantification of Estrogen Receptor-Alpha Expression in Human Breast Carcinomas With a Miniaturized,Low-Cost Digital Microscope: A Comparison with a High-End Whole Slide-Scanner

Introduction

A significant barrier to medical diagnostics in low-resource environments is the lack of medical care and equipment. Here we present a low-cost, cloud-connected digital microscope for applications at the point-of-care. We evaluate the performance of the device in the digital assessment of estrogen receptor-alpha (ER) expression in breast cancer samples. Studies suggest computer-assisted analysis of tumor samples digitized with whole slide-scanners may be comparable to manual scoring, here we study whether similar results can be obtained with the device presented.

Materials and Methods

A total of 170 samples of human breast carcinoma, immunostained for ER expression, were digitized with a high-end slide-scanner and the point-of-care microscope. Corresponding regions from the samples were extracted, and ER status was determined visually and digitally. Samples were classified as ER negative (<1% ER positivity) or positive, and further into weakly (1–10% positivity) and strongly positive. Interobserver agreement (Cohen’s kappa) was measured and correlation coefficients (Pearson’s product-momentum) were calculated for comparison of the methods.

Results

Correlation and interobserver agreement (r = 0.98, p < 0.001, kappa = 0.84, CI95% = 0.75–0.94) were strong in the results from both devices. Concordance of the point-of-care microscope and the manual scoring was good (r = 0.94, p < 0.001, kappa = 0.71, CI95% = 0.61–0.80), and comparable to the concordance between the slide scanner and manual scoring (r = 0.93, p < 0.001, kappa = 0.69, CI95% = 0.60–0.78). Fourteen (8%) discrepant cases between manual and device-based scoring were present with the slide scanner, and 16 (9%) with the point-of-care microscope, all representing samples of low ER expression.

Conclusions

Tumor ER status can be accurately quantified with a low-cost imaging device and digital image-analysis, with results comparable to conventional computer-assisted or manual scoring. This technology could potentially be expanded for other histopathological applications at the point-of-care.