Unraveling methylation changes of host macrophages in Mycobacterium tuberculosis infection.

OBJECTIVES: To characterize at high resolution DNA methylation changes of cytokines which occur in the genome of macrophages in association with Mycobacterium tuberculosis (MTB) infection METHODS: We studied the methylation profiles of THP-1 derived macrophage cells infected with clinical MTB strains [Beijing/W & non-Beijing/W lineage, sensitive (INH(S), RIF(S)) & resistant (INH(R), RIF(R)) strains] and of host macrophages from MTB infected cohorts (active & latent patients) with the human methylation CpG islands microarrays. RESULTS: Methylated modification on the promoter sequences of cytokines and their receptors were found to be associated with MTB infection in a strain- and host-dependent manner. Our epigenetic analyses revealed that infection with Beijing/W MTB strains enhanced IL6R, IL4R and IL17R hyper-methylations in infected macrophages. Validation of IL6R methylated sequence confirmed that MTB infection induced DNA methylation of CpG67 region in the IL6R promoter. In addition, studies on the human macrophage methylation profiles from the patient cohorts indicated that the methylation rate of IL17 family members and related genes were significantly altered in patients with active MTB infections. CONCLUSIONS: Our study offered novel insights into the epigenetic changes in the interaction of host macrophages in MTB infections and warrant further explorations into these changes in modulating the immune response in active and latent MTB infections.

Endothelial chimerism in chronic sclerotic-type chronic graft-versus-host disease (GVHD) and GVHD-associated angiomatosis.

Graft-versus-host disease-associated angiomatosis (GVHD-AA) is an uncommon manifestation of chronic GVHD consisting of friable vascular proliferations. Using fluorescence in situ hybridization, we demonstrate the presence of donor-derived endothelial cells within areas of GVHD-AA. This is the first documented occurrence of a benign neoplastic growth in relationship to a form of chronic GVHD.

Differential CTLA-4 expression in human CD4+ versus CD8+ T cells is associated with increased NFAT1 and inhibition of CD4+ proliferation.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a costimulatory molecule that negatively regulates T-cell activation. Originally identified in murine CD8(+) T cells, it has been found to be rapidly induced on human T cells. Furthermore, CTLA-4 is expressed on regulatory T cells. Clinically, targeting CTLA-4 has clinical utility in the treatment of melanoma. Whether the expression of CTLA-4 is differentially regulated in CD8(+) vs CD4(+) human T cells is unclear. Here, we analyzed CTLA-4 in normal human CD4(+) and CD8(+) T-cell subsets and show for the first time that CTLA-4 is expressed significantly higher in the CD4(+) T cells than in CD8(+) T cells. CTLA-4 is higher at the protein and the transcriptional levels in CD4(+) T cells. This increase is due to the activation of the CTLA-4 promoter, which undergoes acetylation at the proximal promoter. Furthermore, we show that blocking CTLA-4 on CD4(+) T cells permits greater proliferation in CD4(+) vs CD8(+) cells. These findings demonstrate a differential regulation of CTLA-4 on CD4(+) and CD8(+) T-cell subsets, which is likely important to the clinical efficacy for anti-CTLA-4 therapies. The findings hint to strategies to modulate CTLA-4 expression by targeting epigenetic transcription to alter the immune response.

Affinity-selected heparan sulfate for bone repair.

Bone morphogenetic protein (BMP)-2 is a potent bone healing compound produced at sites of bone trauma. Here we present a therapeutic strategy to harness the activity of endogenously produced BMP-2 by delivery of an affinity-matched heparan sulfate (HS) glycos aminoglycan biomaterial that increases the bioavailability, bioactivity and half-life of this growth factor. We have developed a robust, cost effective, peptide-based affinity platform to isolate a unique BMP-2 binding HS variant from commercially available preparations of HS, so removing the manufacturing bottleneck for their translation into the clinic. This affinity-matched HS enhanced BMP-2-induced osteogenesis through improved BMP-2 kinetics and receptor modulation, prolonged pSMAD signaling and reduced interactions with its antagonist noggin. When co-delivered with a collagen implant, the HS was as potent as exogenous BMP-2 for the healing of critical-sized bone defects in rabbits. This affinity platform can be readily tuned to isolate HS variants targeted ata range of clinically-relevant growth and adhesive factors.

Blood loss in spinal tumour surgery and surgery for metastatic spinal disease: a meta-analysis.

There is currently no consensus about the mean volume of blood lost during spinal tumour surgery and surgery for metastatic spinal disease. We conducted a systematic review of papers published in the English language between 31 January 1992 and 31 January 2012. Only papers that clearly presented blood loss data in spinal surgery for metastatic disease were included. The random effects model was used to obtain the pooled estimate of mean blood loss. We selected 18 papers, including six case series, ten retrospective reviews and two prospective studies. Altogether, there were 760 patients who had undergone spinal tumour surgery and surgery for metastatic spinal disease. The pooled estimate of peri-operative blood loss was 2180 ml (95% confidence interval 1805 to 2554) with catastrophic blood loss as high as 5000 ml, which is rare. Aside from two studies that reported large amounts of mean blood loss (> 5500 ml), the resulting funnel plot suggested an absence of publication bias. This was confirmed by Egger's test, which did not show any small-study effects (p = 0.119). However, there was strong evidence of heterogeneity between studies (I(2) = 90%; p < 0.001). Spinal surgery for metastatic disease is associated with significant blood loss and the possibility of catastrophic blood loss. There is a need to establish standardised methods of calculating and reporting this blood loss. Analysis should include assessment by area of the spine, primary pathology and nature of surgery so that the amount of blood loss can be predicted. Consideration should be given to autotransfusion in these patients.

Reduction in vasovagal reaction rate in young first-time blood donors by collecting 350 mL rather than 450 mL.

BACKGROUND: There is a paucity of studies on the magnitude of reduction of vasovagal reaction by reduced collection volume. This study was thus conducted to determine the difference in reaction rates between two collection volumes among the young first-time donors who are at particular risk of reaction. STUDY DESIGN AND METHODS: This retrospective study analyzed 38,436 whole blood donations made by young (aged 16 to 18 years) first-time donors. The effect of collection volume on vasovagal reaction was compared among different weight subgroups for both sexes by chi-square test. RESULTS: For females in all weight subgroups and two of the male lower-weight subgroups, the reduction percentages ranged from 35% to 58% (p < 0.05). It was also noted that, among the females, a higher weight was associated with a higher percent reduction in the reaction rate. CONCLUSION: With reduced collection volume, this study detected large and significant reduction in reaction rates among all females, as well as lower-weight males.

Long-term clinical outcomes after Streptococcus bovis isolation in asymptomatic blood donors in Hong Kong.

BACKGROUND: Risk of transfusion-transmitted bacterial sepsis has been substantially reduced by a bacterial surveillance program (BST). However, new problems emerge as asymptomatic bacteremia is detected in blood donors. Streptococcus bovis bacteremia, which is known to associate with infective endocarditis and colonic carcinoma, is an example. STUDY DESIGN AND METHODS: A retrospective study was conducted to examine the demographic and clinical outcome of this group of donors. All confirmed culture-positive cases under the BST were retrieved and those donors with S. bovis bacteremia were contacted for follow-up. Viable culture samples were sent for detailed microbiologic analysis. RESULTS: From 1998 to 2012, a total of 16 donors were found to have S. bovis bacteremia, giving an estimated prevalence of 1 in 110,800 donations. They consisted of nine men and seven women with median age of 43.5 years. Eight donors had undergone colonoscopy with colonic carcinoma detected in two and benign adenoma in four. Five of the 16 isolates could be retrieved for 16S DNA sequencing. Four were identified as S. gallolyticus ssp. pasteurianus and one as S. gallolyticus ssp. gallolyticus. The two patients with colonic carcinoma had one each of subspecies pasteurianus and gallolyticus. CONCLUSION: The findings highlight a close association of S. bovis and colonic carcinoma. We recommend prompt donor follow-up if S. bovis bacteremia is detected. Besides, all confirmed S. bovis should be fully characterized by molecular technique.

A study of the predonation hemoglobin and iron status among Hong Kong Chinese blood donors.

BACKGROUND: Predonation hemoglobin (PDH) is used to safeguard donors' welfare, and low hemoglobin (Hb) is known to be the most frequent reason for donor deferral. A study was initiated to assess the PDH and iron status of blood donors in Hong Kong. STUDY DESIGN AND METHODS: This observational study was designed with four groups of whole blood donors invited (group 1-eligible first time donors, group 2-eligible repeat donors with zero or one donation in preceding 12 months, group 3-eligible repeat donors with at least two donations in preceding 12 months, group 4-repeat donors being deferred for low PDH). Predonation blood samples were obtained for blood counts and iron status. Mann-Whitney test, Kruskal-Wallis test, and chi-square test for trend were applied for statistical analysis. RESULTS: A total of 836 donors were recruited, of which 35 were excluded because of hemoglobinopathy. An inverse relationship between serum ferritin level and number of donations in the preceding 12 months was observed in both sexes. Iron deficiency was significantly seen in 35.1% of male and 65.3% of female deferred donors. More importantly, up to 7.2, 5.8, and 29.5% of the female donors in groups 1, 2, and 3 were found to be iron deficient despite having a high enough PDH. CONCLUSION: This is the first study to assess PDH and iron status in Chinese blood donors. Iron depletion is noted with increasing number of blood donations in the preceding 12 months. Advice on iron repletion is a necessary step for donor welfare and strategies should be developed to ensure that donors have adequate PDH.

Effects of implantation of bone marrow mesenchymal stem cells, disc distraction and combined therapy on reversing degeneration of the intervertebral disc.

Although success has been achieved with implantation of bone marrow mesenchymal stem cells (bMSCs) in degenerative discs, its full potential may not be achieved if the harsh environment of the degenerative disc remains. Axial distraction has been shown to increase hydration and nutrition. Combining both therapies may have a synergistic effect in reversing degenerative disc disease. In order to evaluate the effect of bMSC implantation, axial distraction and combination therapy in stimulating regeneration and retarding degeneration in degenerative discs, we first induced disc degeneration by axial loading in a rabbit model. The rabbits in the intervention groups performed better with respect to disc height, morphological grading, histological scoring and average dead cell count. The groups with distraction performed better than those without on all criteria except the average dead cell count. Our findings suggest that bMSC implantation and distraction stimulate regenerative changes in degenerative discs in a rabbit model.

Allelic imbalance at 13q31 is associated with reduced GPC6 in Chinese with sporadic retinoblastoma.

BACKGROUND/AIMS: Loss of heterozygosity (LOH) has been discovered in retinoblastoma (RB) in previous studies. In this study, we aimed to discover potential tumour suppressor genes through investigation of the incidence of allelic loss in chromosome 1, 6, 9, 13, 19, 20, 21, 22 and X in Chinese sporadic retinoblastoma patients and to study the expression of genes flanking LOH region 13q31. METHODS: Twenty-five microdissected RB samples were analysed to investigate the LOH in 140 microsatellite markers. Expression of genes flanking D13S265 was investigated by real-time quantitative-PCR on available frozen samples. The promoter and entire coding region of GPC6 were examined for sequence changes in an extended batch of 29 RB samples. RESULTS: Allele losses were found in 92% (23/25) of the tumours. We identified a new LOH locus at 13q31 (D13S265) with a high occurrence rate (67%, 14/21) apart from the RB1 locus (68%, 17/25). Expression study detected the reduced expression of Glypican 6 (GPC6) transcript significantly associated with the LOH at 13q31 (p=0.024). Furthermore, mutation screening revealed no remarkable sequence alteration in GPC6 that could affect its expression. CONCLUSION: Results suggest that a reduction in GPC6 mRNA in retinoblastoma is associated with the non-random allelic loss at 13q31 that could contribute to RB development.

A novel mechanism: Erxian Decoction, a Chinese medicine formula, for relieving menopausal syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Many clinical and experimental reports demonstrated that Erxian Decoction (EXD) was effective in relieving menopausal syndrome. AIM OF THE STUDY: The mechanisms of action of EXD were explored on the endocrine and antioxidant regimen. MATERIALS AND METHODS: Menopause causes a decline in both endocrine function and activities of antioxidant enzymes. In this study, 12-month-old female Sprague-Dawley-rats (SD-rats) with a low serum estradiol level were employed. Their endocrine functions after treatment with EXD were assessed by the determination of their serum estradiol level and ovarian mRNA levels of aromatase, which is a key enzyme for biosynthesis of estradiol. Meanwhile, superoxide dismutase-1 (SOD), catalase (CAT) and glutathione peroxidase (GPx-1) in the liver were also determined to assess the effect of EXD on the antioxidant regimen. RESULTS: Results revealed a significant elevation in serum estradiol level and the mRNA level of ovarian aromatase and liver CAT in the EXD-treated menopausal rat model. CONCLUSIONS: The results obtained from mRNA and estradiol level of the present investigation revealed that the EXD relieves the menopausal syndrome involved an increase of endocrine and antioxidant function through, at least, the activation of aromatase and CAT detoxifying pathways.

Adipose-derived stem cells from pregnant women show higher proliferation rate unrelated to estrogen.

BACKGROUND: Adipose tissue contains an abundant population of multipotent adipose-derived stem cells (ASCs) and has been an excellent source of mesenchymal stem cells for cell therapy and tissue engineering. To ensure successful cell therapies, consistency of stem cell performance across donors is critical. However, the effect of the donor's reproductive status on ASC proliferation rate and differentiation capacity is undefined. METHODS: We investigated whether the yield and function of ASCs are affected by the woman's reproductive status: pregnancy, premenopause or menopause. ASCs were isolated from the abdomen of 15 women and their proliferation rates and differentiation capacities were compared by cell count. The capacity of ASCs to differentiate into the chondrogenic lineage was investigated by quantitative real-time polymerase chain reaction and immunohistochemistry. RESULTS: There was no significant difference in the differentiation capacity between the three groups, whereas the proliferation rate of ASCs from pregnant women was significantly higher than from the other two groups (P < 0.05). The proliferation rate of ASCs after estrogen treatment remained unchanged. CONCLUSIONS: Despite the higher proliferation rate in pregnant women, ASCs showed consistency in cell differentiation capacity and were unaffected by donor status. This suggests that factors other than estrogen are responsible for the difference in proliferation.

Treatment of psoriasis with etanercept in a patient with a history of primary B-cell lymphoma.

The use of immunobiologicals that suppress an overly active immune system in psoriasis carries with it the possibility of cancer development as a result of immunosuppression. Patients with a history of malignancy may be at risk for recurrence when treated with immunosuppressive agents. Moreover, autoimmune diseases, such as psoriasis, have been associated with an increased risk of lymphoma. Therefore, risk-benefit assessments must take into account the clinical severity and treatment of psoriasis. We describe a 59-year-old white man with a history of primary B-cell lymphoma, severe recalcitrant plaque-type psoriasis and psoriatic arthritis, who was started on etanercept for treatment of his psoriasis and psoriatic arthritis. The patient has a long history of remission of his lymphoma. After treatment, the patient experienced significant global improvement with essentially complete remission of the cutaneous lesions and arthritis, and had no recurrence of his lymphoma or other systemic complications while on etanercept after follow-up for > 3 years.

Immunopathogenesis of mycosis fungoides/Sézary syndrome (cutaneous T-cell lymphoma).

T cells are critical effectors of the adaptive immune response and play an important role in cutaneous immunity. In the skin, various cell types cooperate together, from components of both the innate immunity and adaptive immunity, provide sentinel function to mediate the immune response. However, when T cell function becomes abnormal, there is a loss of normal effector immune function, and the abnormal T cells become a cause of disease as well. Mycosis fungoides (MF) is a cutaneous T cell lymphoma (CTCL) that preferentially travels to the epidermis. When skin homing T cells become malignant, the clinical consequences reflect not only the presence of the malignant cells, but likely from a complex reaction of the immune response to the malignant cell. The clinical presentation is the evolving manifestation of the steps in cancer immunosurveillance. Analysis of gene expression in MF/CTCL patients has provided support for the role of the immune response in the early phase of the disease and a loss of immune response in advance stages of MF/CTCL. This review will focus on cytokine gene expression abnormalities in the clinical stages of the disease and discuss the relationship between the clinical and immunologic abnormalities to gain a better understanding of mechanisms important in the evolution of this disease. A better understanding of the immunopathogenesis of MF/CTCL would support innovative strategies for the development of novel therapies to treat this T cell malignancy.

Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.