Breast cancers related to BRCA mutations are associated with particular biological features. Here we report the clinical and pathological characteristics of breast cancer in Chinese women with and without BRCA mutations and of carriers of BRCA1 mutations compared to BRCA2 mutations. Two hundred and 26 high-risk Hong Kong Chinese women were tested for BRCA mutations, medical information was obtained from medical records, and risk and demographic information was obtained from personal interviews. In this cohort, 28 (12.4%) women were BRCA mutation carriers and among these carriers, 39.3% were BRCA1 and 60.7% were BRCA2 mutations. Mutation carriers were more likely to have a familial history of breast and ovarian cancer, high-grade cancers, and triple negative (TN) cancers. Prevalence of TN was 48.3% in BRCA carriers and 25.6% in non-carriers and was 67.7% in BRCA1 and 35.3% in BRCA2 carriers. Estrogen receptor (ER) negative cancer was significantly associated with BRCA1 mutations, especially in those under 40 years of age. BRCA-related breast cancer in this Chinese population is associated with family history and adverse pathological/prognostic features, with BRCA2 mutations being more prevalent but BRCA1 carriers having more aggressive and TN cancers. Compared to Caucasian populations, prevalence of BRCA2 mutations and TN cancer in BRCA2 mutation carriers in Chinese population are elevated.

Breast cancers related to BRCA mutations are associated with particular biological features. Here we report the clinical and pathological characteristics of breast cancer in Chinese women with and without BRCA mutations and of carriers of BRCA1 mutations compared to BRCA2 mutations. Two hundred and 26 high-risk Hong Kong Chinese women were tested for BRCA mutations, medical information was obtained from medical records, and risk and demographic information was obtained from personal interviews. In this cohort, 28 (12.4%) women were BRCA mutation carriers and among these carriers, 39.3% were BRCA1 and 60.7% were BRCA2 mutations. Mutation carriers were more likely to have a familial history of breast and ovarian cancer, high-grade cancers, and triple negative (TN) cancers. Prevalence of TN was 48.3% in BRCA carriers and 25.6% in non-carriers and was 67.7% in BRCA1 and 35.3% in BRCA2 carriers. Estrogen receptor (ER) negative cancer was significantly associated with BRCA1 mutations, especially in those under 40 years of age. BRCA-related breast cancer in this Chinese population is associated with family history and adverse pathological/prognostic features, with BRCA2 mutations being more prevalent but BRCA1 carriers having more aggressive and TN cancers. Compared to Caucasian populations, prevalence of BRCA2 mutations and TN cancer in BRCA2 mutation carriers in Chinese population are elevated.

Background and aims: Although peroxisome proliferator activated receptor γ (PPARγ) agonists have been implicated in differentiation and growth inhibition of cancer cells, the potential therapeutic and chemopreventive effects on gastric cancer are poorly defined. We examined the in vitro and in vivo effects of PPARγ ligands on growth of gastric cancer, and the effect of PPARγ activation on expression of cyclooxygenase 2 (COX-2) and cancer related genes.

Methods: Gastric cell lines (MKN28 and MKN45) were treated with two specific PPARγ ligands: ciglitazone and 15-deoxy-Δ12,14-prostaglandin J2. Cell growth was determined by bromodeoxyuridine incorporation assay and apoptosis was measured by DNA fragmentation. Expression of COX-2 was determined by western blot and real time quantitative polymerase chain reaction (PCR). Expression profiles of cancer related genes were screened with cDNA array. In vivo growth of implanted MKN45 cells in nude mice was monitored after oral treatment with rosiglitazone.

Results: PPARγ ligands suppressed the in vitro growth of MKN45 cells in a dose dependent manner whereas prostacyclin, a PPARδ agonist, had no growth inhibitory effect. Growth inhibition was more pronounced in MKN45 cells, which was accompanied by DNA fragmentation and downregulation of COX-2. Screening by cDNA microarray showed that PPARγ ligand treatment was associated with upregulation of bad and p53, and downregulation of bcl-2, bcl-xl, and cyclin E1 in MKN45 cells, which was confirmed by quantitative real time PCR. In contrast, MKN28 cells with lower PPARγ and COX-2 expression levels had lower growth inhibitory responses to PPARγ ligands. Microarray experiments only showed induction of the bad gene in MKN28 cells. In vivo growth of MKN45 cells in nude mice was retarded by rosiglitazone. Mean tumour volume in rosiglitazone treated mice was significantly lower than controls at six weeks (p = 0.019) and seven weeks (p = 0.001) after treatment.

Conclusions: PPARγ ligands suppress both in vitro and in vivo growth of gastric cancer and may play a major role in cancer therapy and prevention.

Allelic frequencies of RFLPs at loci closely linked to the HD gene, D4S95, D4S91, D4S141, and D4S90, were determined in 13 Huntington's disease (HD) patients from nine Chinese families and 129 normal subjects. These were similar for non-HD and HD chromosomes and the HD gene in Chinese is associated with multiple haplotypes. Hence the HD gene probably arose independently in the background haplotypes of the Chinese population. The heterozygosity rates for the two most useful RFLP sites are 0.659 for D4S95-AccI VNTR and 0.494 for D4S141-HindIII. (CAG)n repeat numbers ranged from 12 to 27 in 174 normal chromosomes. In 52 meiotic recombinations, the (CAG)n repeats were stably inherited in normal families. In HD families, 12 of 13 HD patients had expanded (CAG)n repeats of 40 to 58. Additionally, 10 asymptomatic family members had expanded (CAG)n repeats and the inheritance of the expanded repeat was unstable in these families.

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Purpose

Early events in colorectal tumorigenesis include mutation of the adenomatous polyposis coli (APC) gene and epigenetic hypermethylation with transcriptional silencing of the O6-methylguanine DNA methyltransferase (MGMT), human mut L homologue 1 (hMLH1), and P16/CDKN2A genes. Epigenetic alterations affect genetic events: Loss of MGMT via hypermethylation reportedly predisposes to guanine-to-adenine or cytosine-to-thymine (G:C→A:T) transition mutations in KRAS and P53, and silencing of hMLH1 leads to high levels of microsatellite instability (MSI-H)/mutator phenotype, suggesting that epigenetic-genetic subtypes exist.

Experimental Design

We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas.

Results

We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in non – MSI-H tumors, was associated with BRAF mutation (OR, 4.5; P <0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002).

Conclusions

Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.

Introduction

With evolutions in surgical techniques, minimally invasive surgical (MIS) repair with Achillon applicator has been introduced. However, there is still a lack of literature to investigate into the clinical merits of MIS over open surgery. This study aims to investigate the correlation between clinical outcome, gait analysis and biomechanical properties comparing both surgical methods.

Materials and methods

A single centre retrospective review on all the consecutive operated patients between January 2004 and December 2008 was performed. Twenty-six patients (19 male and 7 female; age 40.4 ± 9.2 years) had experienced a complete Achilles tendon rupture with operative repair. Nineteen of the patients, 10 MIS versus 9 open repairs (13 men with a mean age of 40.54 ± 10.43 (range 23-62 yrs) and 6 women with a mean age of 45.33 ± 7.71 (range 35-57 yrs) were further invited to attend a thorough clinical assessment using Holz's scale and biomechanical evaluation at a mean of 25.3 months after operation. This study utilized the Cybex II isokinetic dynamometer to assess the isokinetic peak force of plantar-flexion and dorsiflexion of both ankles. The patients were also invited to return to our Gait Laboratory for analysis. The eight-infrared camera motion capture system (VICON, UK) was utilized for the acquisition of kinematic variables. Their anthropometric data was measured according to the Davis and coworkers' standard.

Results

The mean operative time and length of hospital stay were shorter in the MIS group. The operative time was 54.55 ± 15.15 minutes versus 68.80 ± 18.23 minutes of the MIS group and Open group respectively (p = 0.045), whereas length of stay was 3.36 ± 1.21 days versus 6.40 ± 3.70 days respectively (p = 0.039). There is statistically significant decrease (p = 0.005) in incision length in MIS group than the open surgery group, 3.23 ± 1.10 cm versus 9.64 ± 2.55 cm respectively. Both groups attained similar Holz's scores, 11.70 ± 0.95 versus 12.0 ± 1.50 respectively (p = 0.262). The mean percentage stance time of the injured leg for MIS patient was 58.44% while the mean percentage stance time of the injured leg for patients with open repair was 56.57%. T-test has shown there were no significance differences between the results of the two groups of patients. The loss of peak torque and total work done with respect to the injured side were similar between the MIS and open group.

Discussion and conclusion

MIS using Achillon method can achieve smaller incisions, shorter operative time and hospital stay. There is no statistical significance difference in clinical outcome, the stance time to strike time ratio and biomechanical properties on the leg receiving Achilles tendon repair using MIS method and open surgery.

The 2009 pandemic influenza H1N1 (H1N1pdm) virus was generated by reassortment of swine influenza viruses of different lineages. This was the first influenza pandemic to emerge in over 4 decades and the first to occur after the realization that influenza pandemics arise from influenza viruses of animals. In order to understand the biological determinants of pandemic emergence, it is relevant to compare the tropism of different lineages of swine influenza viruses and reassortants derived from them with that of 2009 pandemic H1N1 (H1N1pdm) and seasonal influenza H1N1 viruses in ex vivo cultures of the human nasopharynx, bronchus, alveoli, and conjunctiva. We hypothesized that virus which can transmit efficiently between humans replicated well in the human upper airways. As previously reported, H1N1pdm and seasonal H1N1 viruses replicated efficiently in the nasopharyngeal, bronchial, and alveolar epithelium. In contrast, representative viruses from the classical swine (CS) (H1N1) lineage could not infect human respiratory epithelium; Eurasian avian-like swine (EA) (H1N1) viruses only infected alveolar epithelium and North American triple-reassortant (TRIG) viruses only infected the bronchial epithelium albeit inefficiently. Interestingly, a naturally occurring triple-reassortant swine virus, A/SW/HK/915/04 (H1N2), with a matrix gene segment of EA swine derivation (i.e., differing from H1N1pdm only in lacking a neuraminidase [NA] gene of EA derivation) readily infected and replicated in human nasopharyngeal and bronchial epithelia but not in the lung. A recombinant sw915 with the NA from H1N1pdm retained its tropism for the bronchus and acquired additional replication competence for alveolar epithelium. In contrast to H1N1pdm, none of the swine viruses tested nor seasonal H1N1 had tropism in human conjunctiva. Recombinant viruses generated by swapping the surface proteins (hemagglutinin and NA) of H1N1pdm and seasonal H1N1 virus demonstrated that these two gene segments together are key determinants of conjunctival tropism. Overall, these findings suggest that ex vivo cultures of the human respiratory tract provide a useful biological model for assessing the human health risk of swine influenza viruses.

The majority of patients with pandemic influenza H1N1 2009 had mild illness, but some, including those with no risk factors for severe disease, may succumb to this infection. Besides viral factors such as the D222/225G substitution of the hemagglutinin, host factors such as IgG2 subclass deficiency recently was reported to be associated with severe disease in a cohort of Australian patients besides other known risk factors, including underlying chronic illness, extremes of age, and pregnancy. We conducted a case-control study of 38 Asian patients with respiratory failure due to severe pandemic influenza and compared the results to those for 36 mild cases. None had selective IgG2 deficiency, but the level of IgG2 subclass was significantly lower in the severe cases (3.55 g/liter versus 4.75 g/liter; P = 0.002), whereas the levels of IgG1, IgG3, and IgG4 were not significantly different from those of the mild cases. Previous studies suggested that some IgHG2 and FcγRIIa genotypes were associated with IgG2 deficiency. The allelic frequency of the IgHG2 genotypes in our severe cases was not correlated with their levels of IgG2, while that of FcγRIIa was not significantly different from that of the general Han Chinese population (P = 0.216). Only the overall cytokine/chemokine profile (P = 0.029) and serum globulin level (P = 0.005) were found to be independently associated with the IgG2 level by multivariate analysis. The lower IgG2 level in our severe group might be related to cytokine dysregulation rather than being a significant risk factor for severe pandemic influenza. The importance of this finding for therapeutic intervention will require further studies of larger cohorts of patients.

Objectives

To present and discuss the epidemiological and clinical aspects, as well as therapeutic options and outcome of de novo renal cell carcinoma (RCC) of the native kidneys in a series of Chinese renal transplant recipients.

Patients and Methods

A retrospective, cohort study examining all renal transplant recipients with the diagnosis of RCC of native kidney followed up in two major regional hospitals in Hong Kong between January 2000 and December 2009. Clinical data included age, gender, cause of renal failure, symptoms at presentation, duration of transplantation, immunosuppressive therapy, and history of acquired cystic kidney disease (ACKD). Laboratory, radiographic, operative, and pathology reports were used to assess the tumor extent.

Results

Among the 1,003 renal transplant recipients recruited, 12 transplant recipients had a nephrectomy for a total of 13 RCC. The prevalence of de novo RCC was 1.3%. The mean age at diagnosis of RCC was 48.4 years, and the median time from transplantation to diagnosis was 6.1 years. ACKD was found in 6 (50%) of the patients. All patients except one were asymptomatic. pT1 disease was found in ten patients with a mean tumor size of 3.2 cm. All patients were treated successfully with radical nephrectomy. After a median follow-up of 38 months, two patients (16.7%) died. One died of sepsis, and the other died of metastatic carcinoma.

Conclusions

With increasing data showing a better prognosis if RCC is detected early by screening, it is time to consider screening all kidney transplant recipients for ACKD and RCC.

Background:

Id-1 is overexpressed in and correlated with metastatic potential of prostate cancer. The role of Id-1 in this metastatic process was further analysed.

Methods:

Conditioned media from prostate cancer cells, expressing various levels of Id-1, were used to stimulate pre-osteoclast differentiation and osteoblast mineralisation. Downstream effectors of Id-1 were identified. Expressions of Id-1 and its downstream effectors in prostate cancers were studied using immunohistochemistry in a prostate cancer patient cohort (N=110).

Results:

We found that conditioned media from LNCaP prostate cancer cells overexpressing Id-1 had a higher ability to drive osteoclast differentiation and a lower ability to stimulate osteoblast mineralisation than control, whereas conditioned media from PC3 prostate cancer cells with Id-1 knockdown were less able to stimulate osteoclast differentiation. Id-1 was found to negatively regulate TNF-β and this correlation was confirmed in human prostate cancer specimens (P=0.03). Furthermore, addition of recombinant TNF-β to LNCaP Id-1 cell-derived media blocked the effect of Id-1 overexpression on osteoblast mineralisation.

Conclusion:

In prostate cancer cells, the ability of Id-1 to modulate bone cell differentiation favouring metastatic bone disease is partially mediated by TNF-β, and Id-1 could be a potential therapeutic target for prostate cancer to bone metastasis.

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.

Background

The antiapoptotic and epithelial–mesenchymal transition activities of Twist have been implicated in the neoplastic transformation and the development of metastasis, respectively. Upregulation of Twist, described in several types of human cancer, also acts as a prognostic marker of poor outcome.

Aim

To investigate Twist expression in oesophageal squamous cell carcinoma (SCC) and its prognostic value in a Chinese cohort of patients with oesophageal SCC.

Methods

Twist expression in primary oesophageal SCC of 87 Chinese patients was investigated by immunohistochemical staining. Twist protein level in one immortalised normal oesophageal epithelial cell line and six oesophageal SCC cell lines was measured by western blot analysis. Twist mRNA level in 30 pairs of frozen specimens of primary oesophageal SCC and non‐neoplastic oesophageal epithelium from the upper resection margin of corresponding oesophagectomy specimen was also determined by semiquantitative reverse transcription‐PCR.

Results

It was found that Twist was upregulated in oesophageal SCC cell lines, and its mRNA and protein levels were both increased in oesophageal SCC and the non‐neoplastic oesophageal epithelium (p<0.001). In addition, a high level of Twist expression in oesophageal SCC was significantly associated with a greater risk for the patient of developing distant metastasis within 1 year of oesophagectomy (OR 3.462, 95% CI 1.201 to 9.978; p = 0.022).

Conclusions

Our results suggest that upregulation of Twist plays a role in the neoplastic transformation to oesophageal SCC and subsequent development of distant metastasis. Twist may serve as a useful prognostic marker for predicting the development of distant metastasis in oesophageal SCC.

Objective

To explore the left ventricular (LV) electrical activation pattern in heart failure (HF) and its implication to cardiac resynchronization therapy (CRT).

Design and setting

Observational study at the University Teaching Hospital.

Patients

23 optimally treated patients with HF with New York Heart Association class III, QRS duration >120 ms and LV ejection fraction <35%.

Interventions

The LV endocardial activation pattern and total activation time (Tat) was determined by non‐contact mapping and the LV mechanical dys‐synchrony was determined by standard deviation (Ts‐SD) and maximal difference (Ts‐diff) of time to peak systolic contraction (Ts) among 12 LV segments using tissue Doppler imaging before receiving CRT.

Main outcome measures

Correlation between electrical and mechanical dys‐synchrony; volumetric responder to CRT at 3 months; HF hospitalisation or death by Kaplan–Meier analysis.

Results

Homogenous (type I, n = 8) and presence of conduction block (type II, n = 15) patterns were identified. Significant correlation between Tat and Ts‐SD/Ts‐diff was noted only in type II (r = 0.73/0.56, p = 0.002/0.03). Ts‐SD and Ts‐diff in type II were significantly longer than type I. 12 patients in type II and 2 in type I were CRT responders (p = 0.01). After 487 (447) days, patients with type II pattern had significantly lower risk of HF hospitalisation or death than those with type I (log rank χ2 = 5.25; p = 0.02).

Conclusion

Patients with type II LV endocardial activation pattern had a more favourable echocardiographic and clinical response to CRT than those with type I pattern.

Background

The BRCA1 gene is an important breast cancer susceptibility gene. Promoter polymorphisms can alter the binding affinity of transcription factors, changing transcriptional activity and may affect susceptibility to disease.

Methods and Results

By direct sequencing of the BRCA1 promoter region, we identified four polymorphisms c.-2804T>C (rs799908:T>C), c.-2265C>T (rs11655505:C>T), c.-2004A>G (rs799906:A>G) and c.-1896(ACA)1/(ACA)2 (rs8176071:(ACA)1/(ACA)2) present in Hong Kong Chinese. Each was studied independently and in combination by functional assays. While all four variants significantly altered promoter activity, the c.-2265T allele most clearly provided stronger binding than the C allele and the most common mutant haplotype which contains the c.-2265T allele increased promoter activity by 70%. Risk association first tested in breast cancer cases and age-matched controls of Hong Kong Chinese women and replicated in a large population-based study of Shanghai Chinese, altogether totaling over 3,000 subjects, demonstrated the c.-2265T allele carriers had a reduced risk for breast cancer (combined ORs=0.80, 95%CI=0.69–0.93; p=0.003) which was more evident among women aged ≥45 years at first diagnosis of breast cancer and without family history of breast cancer (combined ORs=0.75, 95%CI=0.61–0.91; p=0.004). The most common haplotype containing the c.-2265T allele also showed significant risk association for women aged ≥45 years without family history of breast cancer (ORs=0.64, 95%CI=0.46–0.89; p=0.008).

Conclusion

Our comprehensive study of BRCA1 promoter polymorphisms demonstrated four variants which altered promoter activity, and with the most significant contribution from c.-2265C>T, which could affect susceptibility to breast cancer in the Chinese population. Its significance in other populations remains to be investigated.

Objective

To compare the values of three different forms of tissue Doppler imaging (TDI) processing in predicting left ventricular (LV) reverse remodelling—namely, tissue velocity, displacement and strain mapping.

Design

Standard echocardiography with TDI was performed before and 3 months after cardiac resynchronisation therapy (CRT).

Setting

University teaching hospital.

Patients

55 patients with heart failure who received CRT and were followed up for at least 3 months were recruited.

Interventions

During off‐line analysis, the time to peak systolic velocity in the ejection phase, time to peak positive displacement and time to peak negative strain were measured in the six basal, six mid‐segmental model. Parameters of systolic asynchrony derived by velocity, displacement and strain mapping were correlated with percentage reduction in LV end systolic volume (LVESV) and absolute gain in ejection fraction (EF).

Results

Among the three TDI processing technologies, all parameters of tissue velocity correlated with LV reverse remodelling (r  = −0.49 to r  =  −0.76, all p < 0.001), but the predictive value was strongest in models with 12 LV segments. For displacement mapping, only the two parameters that included 12 LV segments correlated modestly with reduction in LVESV (r  =  −0.36, p < 0.05) and gain in EF. However, none of the strain mapping parameters predicted a favourable echocardiographic response. The receiver operating characteristic (ROC) curve areas were higher for parameters of tissue velocity based on 12 LV segments (ROC areas 0.88 and 0.94) than the corresponding areas derived from displacement mapping (ROC areas 0.72 and 0.71).

Conclusion

Tissue velocity parameters of systolic asynchrony are superior to those of displacement and strain mapping in predicting LV reverse remodelling response after CRT.

Id protein family consists of four members namely Id-1 to Id-4. Different from other basic helix–loop–helix transcription factors, they lack the DNA binding domain. Id proteins have been shown to be dysregulated in many different cancer types and their prognostic value has also been demonstrated. Recently, Id-1 has been shown to be upregulated in oesophageal squamous cell carcinoma (ESCC). However, the prognostic implications of Id proteins in ESCC have not been reported. We examined the expression of the Id proteins in ESCC cell lines and clinical ESCC specimens and found that Id protein expressions were dysregulated in both the ESCC cell lines and specimens. By correlating the expression levels of Id proteins and the clinicopathological data of our patient cohort, we found that M1 stage tumours had significantly higher nuclear Id-1 expression (P=0.012) while high nuclear Id-1 expression could predict development of distant metastasis within 1 year of oesophagectomy (P=0.005). In addition, high levels of Id-2 expression in both cytoplasmic and nuclear regions predicted longer patient survival (P=0.041). Multivariate analysis showed that high-level expression of Id-2 in both cytoplasmic and nuclear regions and lower level of nuclear Id-1 expression were independent favourable predictors of survival in our ESCC patients. Our results suggest that Id-1 may promote distant metastasis in ESCC, and both Id-1 and Id-2 may be used for prognostication for ESCC patients.

Purpose

We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR).

Methods

A case-control study was performed to study the genetic association between DR and polymorphic variants of EDN1 (Lys198Asn), LTA (IVS1–80C>A, IVS1–206G>C, IVS1–252A>G), eNOS (Glu298Asp), and ITGA2 (BgI II) in a Chinese population with type 2 diabetes mellitus. A well defined population with type 2 diabetes, consisting of 127 controls and 216 DR patients, was recruited.

Results

A higher frequency of the Asn/Asn genotype of EDN1 was found in individuals with at least 10 years of diabetes and no retinopathy (controls) compared with DR patients with any duration of diabetes (DR: 2.3%; control: 11.0%; p=0.0002). The Asn allele was also more frequent in controls than DR patients (DR: 16.4%; control: 29.5%; p=0.007). Multiple logistic regression analysis showed that the Asn/Asn genotype was the factor most significantly associated with reduced risk of DR (odds ratio=0.19; 95% CI: 0.07-0.53; p=0.002) and with late onset of diabetes (Asn/Asn: 59 years; Lys/Lys + Lys/Asn: 53 years; p=0.02). Moreover, the Lys/Lys genotype was more common among patients with nonproliferative (75.7%) than proliferative DR (56.9%; p=0.008). The distributions of Lys198Asn alleles in hypertension did not differ from normotensive subjects. No associations between DR and polymorphisms of LTA, eNOS, or ITGA2 were detected, and there were no detectable gene-gene or gene-environmental interactions among the polymorphisms.

Conclusions

The Asn/Asn genotype of EDN1 was associated with a reduced risk of DR and with delayed onset of type 2 diabetes.

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded α1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

Author Summary

The assembly and folding of secreted proteins in the endoplasmic reticulum (ER) is exquisitely regulated by a complex mechanism that maintains an equilibrium between folded and unfolded proteins. Perturbation of this homeostasis induces ER stress, which, if not alleviated through ER stress signaling (ERSS), ultimately triggers cell death. Normal bone growth occurs through a highly coordinated differentiation program that yields specialized cartilage cells (chondrocytes); when this program is disrupted, chondrodysplasia, or malformed skeletons, can result. Chondrodysplasias caused by mutations that affect protein assembly and secretion are characterized by a disorganization of bony growth plates and distension of the ER. We tested whether these chondrodysplasia characteristics were linked to ERSS. By investigating the impact of ER stress on the cell fate of hypertrophic chondrocytes (HCs) in transgenic mice expressing mutations in collagen that prevent proper folding, we revealed a novel adaptive mechanism that helps alleviate the unfolded protein load. Instead of undergoing apoptosis, the HCs undergoing ER stress adapt, re-enter the cell cycle, and revert to a less-mature state in which expression of the mutant collagen is reduced. Our findings have broad implications for adaptive mechanisms to ER stress in vivo and for the pathophysiology underlying chondrodysplasias caused by mutations that impact on protein assembly and secretion.

When subjected to ER stress (by expression of misfolded or unfolded proteins), hypertrophic chondrocytes undergo alterations to their developmental program that may be part of an adaptive response.

Estimates of seropositivity to a new infectious agent in a community are useful to public health. For severe acute respiratory syndrome (SARS), the figures are conflicting. Herein, we screened 12,000 people in a community stricken by SARS 10 months previously and found 53 individuals (0.44%) who had immunoglobulin G antibodies to the SARS coronavirus (SARS-CoV) nucleocapsid (N) produced in bacteria. However, only seven of these (group 1) had sera which also reacted with the native N antigen expressed in SARS-CoV-infected Vero cells, N-transfected 293T cells, and tissues of infected SARS patients. Of these, six individuals had had SARS previously. The remaining person, as well as the 46 other individuals (group 2), were healthy and had no history of SARS. Group 1 antibodies recognized epitopes located slightly differently in N from those of group 2 antibodies, and a mouse hybridoma antibody resembling the former type was generated. Unusually, group 2 antibodies appeared to recognize cross-reactive bacterial epitopes that presumably were posttranslationally modified in eukaryotes and hence were probably not induced by SARS-CoV or related coronaviruses but rather by bacteria. The N antigen is thus highly unique. The extremely low rate (0.008%) of asymptomatic SARS infection found attests to the high virulence of the SARS-CoV virus.

Background

Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.

Methods

We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.

Results

We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.

Conclusion

The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.

Owing to the difficulties in isolating the virus and the lack of routine surveillance, the clinical significance of human parainfluenza virus 4 (HPIV-4) is less well defined than that of the other human parainfluenza viruses. We describe the first outbreak of HPIV-4 infection in a developmental disabilities unit, involving 38 institutionalized children and three staff members, during a 3-week period in autumn 2004. Most subjects had upper respiratory tract infections (URTI), while lower respiratory tract infections (LRTI) occurred in three children (7%), one complicated by respiratory failure requiring ventilation support. All patients recovered. Nasopharyngeal aspirates tested for HPIV-4 were positive by reverse transcriptase PCR (RT-PCR) in all 41 cases (100%), by direct immunofluorescence in 29 of 39 tested cases (74%), and by cell cultures in 6 of 37 cases (16%), and serum was positive for antibodies against HPIV-4 in all 35 cases (100%) with serum samples available. In addition, RT-PCR detected HPIV-4 in four children (three LRTI and one URTI) out of 115 patients with community-acquired respiratory tract infection. Molecular analysis of the 1,198-bp phosphoprotein sequences showed that HPIV-4 isolates among the cases were genetically similar, whereas the community controls were more genetically distant, supporting nosocomial transmission of a single HPIV-4 genotype during the outbreak. Moreover, the HPIV-4 causing the outbreak is more closely related to HPIV-4A than HPIV-4B. HPIV-4 may be an important cause of more severe respiratory illness in children. The present RT-PCR assay is a sensitive, specific, and rapid method for the diagnosing HPIV-4 infection. To better define the epidemiology and clinical spectrum of disease of HPIV-4 infections, HPIV-4 should be included in the routine panels of respiratory virus detection on respiratory specimens.

Background

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus. It may progress to respiratory failure, and a significant proportion of patients die. Preliminary data suggest that a high viral load of the SARS coronavirus is associated with adverse outcomes in the intensive care unit, but the relation of viral load to survival is unclear.

Methods

We prospectively studied an inception cohort of 133 patients with virologically confirmed SARS who were admitted to 2 general acute care hospitals in Hong Kong from Mar. 24 to May 4, 2003. The patients were followed until death or for a minimum of 90 days. We used Cox proportional hazard modelling to analyze potential predictors of survival recorded at the time of presentation, including viral load from nasopharyngeal specimens (measured by quantitative reverse transcriptase polymerase chain reaction [PCR] of the SARS-associated coronavirus).

Results

Thirty-two patients (24.1%) met the criteria for acute respiratory distress syndrome, and 24 patients (18.0%) died. The following baseline factors were independently associated with worse survival: older age (61–80 years) (adjusted hazard ratio [HR] 5.24, 95% confidence interval [CI] 2.03–13.53), presence of an active comorbid condition (adjusted HR 3.36, 95% CI 1.44–7.82) and higher initial viral load of SARS coronavirus, according to quantitative PCR of nasopharyngeal specimens (adjusted HR 1.21 per log10 increase in number of RNA copies per millilitre, 95% CI 1.06–1.39).

Interpretation

We found preliminary evidence that higher initial viral load is independently associated with worse prognosis in SARS. Mortality data for patients with SARS should be interpreted in light of age, comorbidity and viral load. These considerations will be important in future studies of SARS.

Background/Aim—Human papillomaviruses (HPVs) are important, but not sufficient, for the development of cervical cancer. All three human ß-herpesviruses—cytomegalovirus (CMV) and human herpesviruses (HHV) types 6 and 7—have been detected in the cervix. In addition, CMV and HHV-6 can interact with HPVs in vivo. This study examined the possible role of ß-herpesviruses in cervical cancer development.

Methods—HPV, CMV, HHV-6, and HHV-7 were detected by the polymerase chain reaction using cervical scrapes taken at colposcopy from 388 women. HPV types were identified using restriction fragment length polymorphisms. Colposcopy guided biopsies were taken from abnormal areas, and the histological findings were regarded as the final diagnoses. The associations between herpesvirus infection and the degree of cervical lesion were analysed with respect to HPV status.

Results—Of the 388 women, 51.8% had a normal cervix, 14.4% had cervical intraepithelial neoplasia grade 1 (CIN1), 8.2% had CIN2, 19.3% had CIN3, and 6.2% had invasive carcinoma. Overall, the positive rates for high, intermediate, and low risk HPVs were 18.8%, 21.4%, and 5.2%, respectively. Fifteen patients harboured HPVs for which the genotype could not be identified. Positive rates for CMV, HHV-6, and HHV-7 were 9.5%, 3.6%, and 3.4%, respectively. HPV positive patients carried a higher risk for high grade lesions (CIN2/3 or carcinoma) (odds ratio (OR), 5.24; 95% confidence interval (CI), 3.19 to 8.62; χ2 = 51.79; p < 0.001), whereas those positive for CMV, HHV-6, or HHV-7 did not. Thirteen of 131 patients with high grade lesions had HPV/herpesvirus coinfections, but no association with the cervical lesion was noted. Furthermore, positive rates for herpesviruses among HPV negative, high/intermediate risk HPV negative, and high risk HPV negative subgroups were similarly low and without a significant association.

Conclusions—The ubiquitous nature of herpesviruses may pose difficulty in elucidating their pathogenic role. These results indicate that CMV, HHV-6, and HHV-7 are bystanders rather than cofactors in the oncogenesis of cervical cancer.

Key Words: human papillomavirus • human herpesvirus 6 • human herpesvirus 7