Sensory nerve degeneration in a mouse model mimicking early manifestations of familial amyloid polyneuropathy due to transthyretin Ala97Ser.

AIMS: Sensory nerve degeneration and consequent abnormal sensations are the earliest and most prevalent manifestations of familial amyloid polyneuropathy (FAP) due to amyloidogenic transthyretin (TTR). FAP is a relentlessly progressive degenerative disease of the peripheral nervous system. However, there is a lack of mouse models to replicate the early neuropathic manifestations of FAP. METHODS: We established human TTR knock-in mice by replacing one allele of the mouse Ttr locus with human wild-type TTR (hTTRwt ) or human TTR with the A97S mutation (hTTRA97S ). Given the late onset of neuropathic manifestations in A97S-FAP, we investigated nerve pathology, physiology, and behavioural tests in these mice at two age points: the adult group (8 - 56 weeks) and the ageing group (> 104 weeks). RESULTS: In the adult group, nerve profiles, neurophysiology and behaviour were similar between hTTRwt and hTTRA97S mice. By contrast, ageing hTTRA97S mice showed small fibre neuropathy with decreased intraepidermal nerve fibre density and behavioural signs of mechanical allodynia. Furthermore, significant reductions in sural nerve myelinated nerve fibre density and sensory nerve action potential amplitudes in these mice indicated degeneration of large sensory fibres. The unaffected motor nerve physiology replicated the early symptoms of FAP patients, that is, sensory nerves were more vulnerable to mutant TTR than motor nerves. CONCLUSIONS: These results demonstrate that the hTTRA97S mouse model develops sensory nerve pathology and corresponding physiology mimicking A97S-FAP and provides a platform to develop new therapies for the early stage of A97S-FAP.

Blockage of cisplatin-induced autophagy sensitizes cervical cancer cells to cisplatin.

Development of chemoresistance is a major obstacle that leads to the recurrence and progression of cervical cancer (CC). Autophagy, meaning, "eating of self", has shown paradoxical functions in tumors. In this study, we first investigated the process of autophagy induction by cisplatin in CC cells. Next, we investigated the role of autophagy in cisplatin-sensitivity of CC cells via blockage of cisplatin-induced autophagy. The results demonstrated that cisplatin induces autophagy in CC HeLa cells via upregulating the formation of autophagic vesicle, promoting the conversion of LC-I to LC-II, and increasing the expression of autophagy-related gene 7 (Atg-7). On the other hand, the autophagy inhibitor, 3MA, downregulated cisplatin-induced formation of autophagic vesicles, reduced the conversion of LC-I to LC-II, and decreased Atg-7 expression. Moreover, 3MA reversed the reduction in cellular viability and induction of apoptosis by cisplatin in HeLa cells. Our results imply that autophagy blockage may play a key role in the chemosensitivity of cervical cancers.

Correlation between use of simvastatin and lovastatin and female lung cancer risk: a nationwide case-control study.

BACKGROUND: The objective of this study was to determine the association between statin use and female lung cancer in Taiwan. METHODS: In this case-control study, we used information from the Taiwan National Health Institute Research Database on 17,329 patients (cases) aged 20 years or older recently diagnosed with lung cancer between 2005 and 2010 and 17,329 patients without lung cancer to assess the association between female lung cancer and statin use, even adjustment for its comorbidities. RESULTS: After adjusting for age and associated risk factors, we determined that women who engaged in long-term use of simvastatin at a defined daily dose (DDD) of over 150 have a reduced risk of lung cancer compared with those who did not use statins (odds ratio: 0.77, 95% confidence interval: 0.62-0.97) in women. However, lovastatin was not significantly associated with lung cancer in women. Among female patients with pre-existing comorbidities of respiratory diseases such as chronic obstructive pulmonary disease, hypertension, stroke and pulmonary tuberculosis, statins reduced the risk of lung cancer. CONCLUSIONS: Simvastatin use at a DDD of more than 150 is correlated with an approximately 20% reduction in the risk of lung cancer in women.

Increased risk of chronic fatigue syndrome following herpes zoster: a population-based study.

Chronic fatigue syndrome (CFS) is a complex disorder accompanied by unexplainable persistent fatigue, in which several etiological factors exist, such as viral infections. Using the National Health Insurance Research Database (NHIRD) of Taiwan, this study evaluated the association between herpes zoster (HZ) infection and the risk of CFS, and examined the possibility of patients developing postviral fatigue effects, including the possibility of developing other unexplainable chronic fatigue conditions. In this prospective cohort study using the NHIRD, we identified 9,205 patients with HZ infection [ICD-9 (International Classification of Disease, Ninth Revision), code 053] and 36,820 patients without HZ infection (non-HZ) from 2005 to 2007, and followed up to the end of 2010. The incidence rate of CFS was higher in the HZ cohort than in the non-HZ cohort (4.56 vs. 3.44 per 1,000 person-years), with an adjusted hazard ratio of 1.29 [95 % confidence interval (CI) = 1.09-1.53]. It was shown that the risk of CFS without comorbidity for each patient increased from 1.25- to 1.36-fold between the CFS and non-CFS cohorts; with long-term follow-up, the HZ cohort showed a significantly higher cumulative incidence rate of developing CFS than the non-HZ patients. We propose that patients with chronic fatigue might exist in a subset of patients that would be associated with HZ infection. The actual mechanism of development of CFS that is attributed to HZ infection remains unclear. The findings of this population cohort study provide pivotal evidence of postviral fatigue among patients with HZ infection.

Chronic fatigue syndrome is associated with the risk of fracture: a nationwide cohort study.

PURPOSE: Chronic fatigue syndrome (CFS) is a complex disorder that is associated with unreasonable persistent fatigue. CFS has also been reported to be a possible risk factor for osteopathy. We propose that CFS might be associated with an increased risk of fracture. METHODS: We used the National Health Insurance Research Database to conduct a prospective cohort study, identifying 3744 patients with a CFS diagnosis (International Classification of Diseases, Ninth Revision, Clinical Modification code 780.71) and 14 976 patients without CFS until 2006, with follow-up observed until the end of 2010. RESULTS: The incidence rate of fracture was higher in the CFS cohort than in the non-CFS cohort (17.44 vs. 14.53 per 1000 person-year, respectively), with an adjusted hazard ratio of 1.14 (95% confidence interval = 1.00-1.30). The risks of fracture between CFS and non-CFS were shown without comorbidity for each would be elevated than with other comorbidities, particularly in osteoporosis. The patients without osteoporosis in the CFS cohort exhibited a 1.16-fold higher risk of fracture than did those in the non-CFS cohort. CONCLUSIONS: We propose that CFS-related fracture might not be associated with osteoporosis. The mechanism for developing CFS-related fracture remains unclear; however, we recommend noticing the prevention of fracture for CFS patients before clarifying the aetiology of CFS-related fracture.

Clinical presentations and skin denervation in amyloid neuropathy due to transthyretin Ala97Ser.

OBJECTIVE: Familial amyloid polyneuropathy (FAP) due to amyloidogenic transthyretin (TTR) is often associated with impairment of thermonociceptive functions. This study investigated skin innervation and its clinical significance in genetically defined FAP due to a hot-spot Ala97Ser TTR mutation (Ala97Ser). METHODS: Skin biopsies were performed on the distal leg of patients with Ala97Ser, and intraepidermal nerve fiber (IENF) densities were quantified. RESULTS: There were 19 unrelated patients with Ala97Ser manifesting a late-onset (59.47 +/- 5.70 years) generalized neuropathy with disabling motor, sensory, and autonomic symptoms. Against a background of a slowly progressive course, 7 patients (36.8%) exhibited additional rapid declines in neurologic deficits, which were associated with elevation of the protein content in the CSF (p < 0.001). The IENF density was markedly reduced in Ala97Ser patients compared to age- and gender-matched controls (0.99 +/- 1.11 vs 8.31 +/- 2.87 fibers/mm, p < 0.001). Skin denervation was present in all patients and was lower in patients with a higher disability grade (0.17 +/- 0.26 vs 1.37 +/- 1.16 fibers/mm, p = 0.003). Albuminocytologic dissociation in the CSF was observed in 14 patients (73.7%), and the IENF density was negatively correlated with the CSF protein concentration (p = 0.015). CONCLUSIONS: Skin denervation was common in Ala97Ser, and degeneration of cutaneous nerve terminals was correlated with the severity of clinical phenotypes and the level of CSF protein.

Peptide mimicrying between SARS coronavirus spike protein and human proteins reacts with SARS patient serum.

Molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells. Severe acute respiratory syndrome (SARS) is a new human respiratory infectious disease caused by SARS coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV plays an important role in the virus entry into a cell. In this study, eleven synthetic peptides from the S protein were selected based on its sequence homology with human proteins. Two of the peptides D07 (residues 927-937) and D08 (residues 942-951) were recognized by the sera of SARS patients. Murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells A549. Another peptide D10 (residues 490-502) stimulated A549 to proliferate and secrete IL-8. The present results suggest that the selected S protein regions, which share sequence homology with human proteins, may play important roles in SARS-CoV infection.

Ginsenoside Rd attenuates neuroinflammation of dopaminergic cells in culture.

In Parkinson's disease clinical and experimental evidence suggest that neuroinflammatory changes in cytokines caused by microglial activation contribute to neuronal death. Experimentally, neuroinflammation of dopaminergic neurons can be evoked by lipopolysaccharide (LPS) exposure. In mesencephalic primary cultures LPS (100 microg/ml) resulted in 30-50% loss of dendritic processes, changes in the perikarya, cellular atrophy and neuronal cell loss of TH-immunoreactive (TH+) cells. iNOS activity was increased dose dependently as well as prostaglandin E2 concentrations. Ginsenosides, as the active compounds responsible for ginseng action, are reported to have antioxidant and anti-inflammatory effects. Here ginsenoside Rd was used to counteract LPS neurodegeneration. Partial reduction of LPS neurotoxic action was seen in dopaminergic neurons. Cell death by LPS as well as neuroprotective action by ginsenoside Rd was not selective for dopaminergic neurons. Neuronal losses as well as cytoprotective effects were similar when counting NeuN identified neurons. The anti-inflammatory effect of ginsenoside Rd could equally be demonstrated by a reduction of NO-formation and PGE2 synthesis. Thus, protective mechanisms of ginsenoside Rd may involve interference with iNOS and COX-2 expression.

Regional blood flow occlusion during extensive pelvic procedures for ovarian cancer: a randomized trial.

The objective of this study was to determine the effects of intraoperative aortic occlusion on blood loss and operative time when used during en bloc resection of internal reproductive organs, pelvic peritoneum, and rectosigmoid colon [modified posterior exenteration (MPE)] for primary cytoreduction of ovarian cancer. Patients undergoing MPE, without palpable distal aortic plaque or calcification, were randomized to: (a) complete distal aortic occlusion (

Improved method for measurement of human plasma xanthine oxidoreductase activity.

The XOR activity in human plasma was measured by quantifying the XOR-derived uric acid (UA) in plasma using the high-performance liquid chromatography (HPLC) equipped with a UV detector. Chromatographic separation consisted of the mobile phase (a mixture of 0.1% trifluoroacetic acid in Milli-Q water and 0.085% trifluoroacetic acid in acetonitrile in a mix ratio of 99:1) running through a Zorbax StableBond SB-C(18) column at a flow-rate of 1 ml/min. Deproteinization with heat-treatment of plasma samples after the reaction was used in the assay to avoid splitting of the UA and xanthine peaks caused by acid deproteinization that could interfere the accurate determination of human plasma XOR activity in our case. Based on the examination of the dependence of XOR activity on added amounts of xanthine and reaction times, the amount of xanthine and reaction time for XOR activity assay were determined to prevent the errors caused by the limiting effect of substrates and plateau phase of the reaction. Using this method, human plasma XOR activities of 25 healthy people were measured. The average human plasma XOR activity was 2.1+/-0.8 (x10(-3) U/ml).

Cutaneous innervation in chronic inflammatory demyelinating polyneuropathy.

The authors evaluated epidermal nerve density (END) and thermal thresholds in 18 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). END of patients with CIDP were lower than those of controls (4.5 +/- 2.9 vs 10.5 +/- 3.9 fibers/mm, p < 0.001). Reduced END were associated with autonomic symptoms. Thermal thresholds of patients with CIDP were elevated (88.2% for warm stimuli and 70.6% for cold stimuli). Patients with CIDP have small-fiber sensory and autonomic neuropathies.

Quantitative pathology of cutaneous nerve terminal degeneration in the human skin.

Pathological diagnosis of neuropathy has traditionally depended on ultrastructural examinations of nerve biopsy specimens, particularly for sensory neuropathies affecting unmyelinated and small-myelinated nociceptive nerves. These sensory nerves terminate in the epidermis of the skin, and the pathology of neuropathy usually begins from nerve terminals. We investigated the feasibility of diagnosing small-fiber sensory neuropathy by evaluating cutaneous innervation. Skin biopsy specimens of 3-mm in diameter were obtained from the distal leg and the distal forearm of 55 healthy controls and 35 patients with sensory neuropathy. In the healthy controls, conventional intraepidermal nerve fiber densities (IENF densities) as measured using the image analysis system in the distal forearm and in the distal leg were correlated (r=0.55, P<0.0001), with significantly higher values in the distal forearm than in the distal leg (17.07+/-6.51 vs 12.92+/-5.33 fibers/mm, P<0.001). Compared to IENF densities of healthy controls, these values of neuropathic patients were significantly reduced in the distal forearm (5.82+/-6.50 fibers/mm, P<0.01) and in the distal leg (2.40+/-2.30, P<0.001). We further explored the possibility of quantifying skin innervation by counting "ocular intraepidermal nerve fiber density" (ocular nerve fiber density) with no aid of an image analysis system. This was based on the fact that the epidermal length on specifically defined sections was very close to the predicted epidermal length of 3 mm, the diameter of skin punches (P=0.14). Ocular nerve fiber densities were significantly correlated with IENF densities as measured by the image analysis system (r=0.99, P<0.0001). Dermal nerve fibers of neuropathic patients either disappeared or became degenerated. These findings were consistent with the notion of early terminal degeneration in neuropathy, and will facilitate quantitative interpretation of epidermal innervation in human neuropathy.

Cutaneous nerve terminal degeneration in painful mononeuropathy.

Nociceptive nerves innervate the skin and play an important role in the generation of neuropathic pain. However, it remains elusive whether and how nociceptive nerve terminals degenerate in neuropathic pain conditions. To address this issue, we investigated cutaneous innervation in a model of painful mononeuropathy, the chronic constriction injury (CCI). The hind paws of rats were immunocytochemically stained with a pan-axonal marker, protein gene product 9.5 (PGP 9.5). Within 2 days after CCI, rats exhibited thermal hyperalgesia, and there was a partial depletion of epidermal nerves. The extent of reduction in epidermal nerves after CCI was variable with an epidermal nerve density of 3.65 +/- 1.97 fibers/mm (compared to 15.39 +/- 1.58 fibers/mm on the control side, P < 0.02). There was a mild but concomitant increase in PGP 9.5 (+) Langerhans cells in the epidermis of the skin with CCI (10.19 +/- 1.99 vs 7.75 +/- 1.36 cells/mm, P < 0.05). In the skin denervated by tight ligation of the sciatic nerve, epidermal nerves were completely depleted (0 fibers/mm vs. 12.26 +/- 1.44 fibers/mm on the control side, P < 0.001). Animals with tight ligation of the sciatic nerve exhibited thermal anesthesia. These findings suggest that the epidermis is partially denervated in CCI, and that a partial injury of nerves is correlated with the development of neuropathic pain.

Degeneration of nociceptive nerve terminals in human peripheral neuropathy.

Patients with peripheral neuropathy have symptoms involving small-diameter nociceptive nerves and elevated thermal thresholds. Nociceptive nerves terminate in the epidermis of the skin and are readily demonstrated with the neuronal marker, protein gene product 9.5 (PGP 9.5). To investigate the pathological characteristics of elevated thermal thresholds, we performed PGP 9.5 immunocytochemistry on 3 mm punch skin biopsies (the forearm and the leg) from 55 normal subjects and 35 neuropathic patients. Skin innervation was evaluated by quantifying epidermal nerve densities. Epidermal nerve densities were reduced in neuropathic patients compared to normal subjects. Epidermal nerve densities were variably correlated with thermal thresholds. The proportion of neuropathic patients with reduced epidermal nerve densities was larger than the proportion of neuropathic patients with elevated thermal thresholds. These results indicated that degeneration of epidermal nerve terminals preceded the elevation of thermal thresholds. Skin biopsy together with immunocytochemical demonstration of epidermal innervation offers a new approach to evaluate small-fiber sensory neuropathy.

[Cloning and efficient expression of cytokine human MK in E. coli].

For cloning the cytokine human Midkine (MK) gene, we designed by PCgene program and synthesized a pair of PCR specific primers according to the reported human MK cDNA sequence. Total cellular RNA was extracted from a human hepatoblastoma cell line HepG2, and then the target DNA fragment was obtained by RT-PCR and subcloned into plasmid pUC118. Checked with radioisotope sequencing and ABI 377A sequencer, the nucleotide sequence of the cloned MK cDNA was identical with the reported one. A prokaryotic expression vector, named pBV220, was used to express the MK protein efficiently in E. coli strain TG1 and a predicted band of 16.5 kD in Mr by 15% SDS-PAGE was found. The expressed recombinant protein was found in insoluble aggregated form and accounted for about 31.21% of the total cellular proteins. The first 15 N-terminal amino acid sequence analysis of this protein by Edman degradation method showed that it was accordant with that predicted from the cDNA sequence. The activity of neurite outgrowth-promoting of the MK crude samples was tested with brain cells isolated from 18-day embryos of SD rat.

Chromosome 4 deletions are frequent in invasive cervical cancer and differ between histologic variants.

OBJECTIVE: Patterns of discontinuous deletion of chromosome 4 have been described in histologic variants of lung carcinomas and may represent different "hotspot" targets for gene-environment interactions. Since similar environmental risks exist for cervical cancer, we investigated patterns of discontinuous deletion in two major histologic variants. METHODS: Thirteen archival cases of squamous cell cancer (SCCA) and 11 cases of adenocarcinoma (AC) were precisely microdissected. Matched normal and tumor DNA were used for polymerase chain reaction (PCR) based loss of heterozygosity (LOH) analyses using 19 polymorphic markers spanning chromosome 4. Human papillomavirus (HPV) detection was determined by PCR using general and type-specific primers (HPV 16, 18). Differences in LOH between histologic tumor types and chromosomal regions were determined using Fisher's exact test. RESULTS: Loss at any chromosome 4 locus occurred in 92% of all tumors studied, with the majority of deletions occurring on the long arm of the chromosome. Four discrete minimal regions of discontinuous deletion (R) were identified. For these regions, LOH frequencies were 76% (R1, 4q34-q35), 48% (R2, 4q25-q26), 36% (R3, 4p15.1-p15.3), and 26% (R4, 4p16). Loss in SCCA predominated at 4q (4q34-q35; 83%) and in AC at 4p (4p15.3; 50%). Overall LOH on the p arm was significant in AC (82%) compared to SCCA (31%) (P = 0.02). HPV detection was similar in SCCA (85%) and AC (73%), and HPV 16/18 subtypes were similarly represented in both histologies. CONCLUSIONS: Chromosome 4 deletions are frequent in cervical carcinomas. Different patterns of deletion between SCCA and AC may represent gene regions targeted by different gene-environment interactions in these tumor subtypes.

Molecular Papanicolaou tests in the twenty-first century: molecular analyses with fluid-based Papanicolaou technology.

OBJECTIVE: This study was undertaken to demonstrate the feasibility of performing molecular analyses at the deoxyribonucleic acid, ribonucleic acid and protein levels of cervical cytologic examination with a methanol fluid-based Papanicolaou specimen collection system. STUDY DESIGN: Genomic deoxyribonucleic acid and total ribonucleic acid were extracted from cell pellets obtained from the residual fluid-based Papanicolaou specimen collection buffer after clinical processing. Genomic and human papillomavirus deoxyribonucleic acid polymerase chain reaction and reverse transcriptase-polymerase chain reaction were performed. Messenger ribonucleic acid transcript analysis and human papillomavirus 16 E6 mutational analysis were also performed. Methylation-specific polymerase chain reaction was used to evaluate hypermethylation status of the p16 gene and the gene for E-cadherin. Immunohistochemical staining for protein expression was performed on the processed monolayer slides. RESULTS: Cell pellets from the residual fluid-based cytologic specimen yielded good quality deoxyribonucleic acid and ribonucleic acid. Molecular analyses of genomic deoxyribonucleic acid were successful for the identification of human papillomavirus E6 and p53 polymorphism status by means of restriction enzyme digestion and direct sequencing. Methylation status of the promotor regions of the p16 tumor suppressor gene and the gene for E-cadherin were also successfully identified. Ribonucleic acid was used as the template for transcript analysis and mutational analysis of the corresponding complementary deoxyribonucleic acid of the p53 gene. Protein expression analysis was demonstrated by immunohistochemical staining for carcinoembryonic antigen. CONCLUSION: It is feasible to conduct multiple molecular analyses at the deoxyribonucleic acid, ribonucleic acid, and protein levels of the cervicovaginal cell pellets from the residual fluid-based Papanicolaou cytologic specimen. This relatively simple and widely used collection system will allow significant advances in molecular epidemiology and eventual development of a molecular Papanicolaou test.

Allelic loss and microsatellite alterations of chromosome 3p14.2 are more frequent in recurrent cervical dysplasias.

Epidemiological studies have documented the unpredictable clinical progression or recurrence of cervical dysplasia. Recent studies have shown several molecular changes in cervical cancers and their associated dysplasia. We conducted molecular analyses on a retrospectively ascertained cohort of recurrent and nonrecurrent cervical dysplasia cases in an attempt to define molecular biomarkers to predict progressive or recurrent disease. Cases were chosen if long-term follow-up (3-5 years after conization) and biopsy confirmation were available. Paraffin-embedded, postconization cervical tissues from 19 recurrent and 18 nonrecurrent dysplasias were analyzed. Human papillomavirus (HPV) was identified by PCR for general and type-specific (HPV-16 and HPV-18) primers. Allelotyping analysis was performed by multiplex PCR using a panel of 16 microsatellite markers targeting putative tumor suppressor gene regions on chromosomes 3p, 5p, 6p, 9p, 11q, and 17p. The overall rate of HPV infection was similar in both groups. In the allelotyping analysis, loss of heterozygosity at the fragile histidine triad region in 3p14.2 was significantly higher in the recurrent group than in the nonrecurrent group (P = 0.005). Furthermore, microsatellite alterations (MAs) were more frequent in the recurrent group (mean MA index, 0.254) as compared with the nonrecurrent group (mean MA index, 0.085; P = 0.0025). These findings suggest that HPV status alone does not predict recurrence and that loss of heterozygos. ity at the fragile histidine triad region may represent a potential biomarker in predicting recurrence. Frequent MAs in the recurrent group may represent an underlying genomic instability that creates susceptibility for allelic loss, thus increasing the risk for recurrence or progression.