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.

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.

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.

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.

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.

Leptin is present in human cord blood.

It has recently been reported that the ob gene receptor was expressed on human and murine hematopoietic stem cells and that the ob gene product leptin stimulated hemato- and lymphopoiesis at the stem cell level. These findings suggest a role for leptin in hemato- and lymphopoiesis during fetal development. There is at present no evidence, however, that leptin is synthesized and released by the fetus. To investigate this possibility, we have measured plasma leptin concentrations in the cord blood of 78 newborn infants. We found that leptin was present in all 78 infants in concentrations comparable with those found in adults (0.6-55.7 ng/ml). Overall, plasma leptin concentrations in the cord blood of infants correlated with birth weight (r = 0.74, P < 0.001). These observations show that leptin is synthesized and released by fetal fat cells. In addition, they are compatible with the concept that leptin may play a role in human fetal hematopoiesis.

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.

Loss of heterozygosity and mutational analysis of the PTEN/MMAC1 gene in synchronous endometrial and ovarian carcinomas.

Mutations of the human putative protein tyrosine phosphatase (PTEN/MMAC1) gene at chromosome 10q23 have been found frequently in type I endometrial carcinomas. Endometrioid adenocarcinoma is the most frequent histology seen in patients with clinically determined synchronous endometrial and ovarian carcinomas. We report a high incidence of PTEN/MMAC1 mutations and 10q23 loss of heterozygosity (LOH) in patients with synchronous endometrial and ovarian carcinomas. Paraffin-embedded precision microdissected tumors were analyzed for 10 matched synchronous endometrial and ovarian cancers and 11 matched control metastatic endometrial cancers. Single-stranded conformation polymorphism analysis was used to screen for mutations in all tumors and corresponding normal lymphocyte DNA. LOH was determined using a panel of four microsatellite markers within the PTEN/MMAC1 locus. PTEN/MMAC1 mutations were found in 43% (9 of 21) of the endometrial cancers studied, similarly represented in the clinically synchronous group (5 of 10 or 50%) and the advanced metastatic group (4 of 11; 36%; P = 0.53). In two of the five cases of clinically synchronous cancers, identical or progressive PTEN mutations were found in both the endometrial and ovarian cancers, suggesting that the ovarian tumor is a metastasis from the endometrial primary. PTEN/MMAC1 mutations in the advanced endometrial cancers were similar in the corresponding metastases. In one case, the mutation was seen in only one of two metastatic lymph nodes. The LOH analysis demonstrated 55% LOH in at least one PTEN/MMAC1 marker. These findings suggest that the putative tumor suppressor gene PTEN/MMAC1 may be a viable molecular marker to differentiate synchronous versus metastatic disease in a subset of clinically synchronous endometrial and ovarian carcinomas.

Modulation of keratinocyte proliferation by skin innervation.

Several lines of evidence suggest that sensory nerves ending at the skin have profound influences on their target, the epidermis. To test the hypothesis, we examined the consequences of denervation on the paw skin of rats by eliminating its innervation. We investigated temporal changes of nerve degeneration, keratinocyte proliferation and differentiation, gene expression, and epidermal thickness. Nerve terminals in the epidermis began to degenerate within 24 h after denervation. All epidermal nerves were completely degenerated by 2 d. During the interval of nerve degeneration, there was a significant reduction of bromodeoxyuridine incorporation from 24 h of nerve injury (39 +/- 7% of the control side, p 0.01). By 2 d, there was a further reduction of bromodeoxyuridine labeling (11 +/- 8%, p < 0. 0001). The incorporation of bromodeoxyuridine remained depressed when the skin was denervated (35 +/- 11%, p < 0.01). Four days after eliminating skin innervation, the denervated epidermis became thinner than the control epidermis (70 +/- 8% of the control, p < 0. 01). Epidermal thinning was associated with a significant decrease in expression of glyceraldehyde-3-phosphate dehydrogenase and beta-actin transcripts (33 +/- 8% of the control epidermis from postoperative day 4, p < 0.001). Other aspects of keratinocyte differentiation, including the patterns of keratin expression, and programmed cell death, were unaltered by skin denervation. These data indicate that skin denervation is sufficient to influence keratinocyte proliferation and therefore epidermal thickness.

Pathology of nerve terminal degeneration in the skin.

To characterize the pathology of epidermal nerve degeneration and regeneration, we investigated temporal and spatial changes in skin innervation of the mouse footpad. Within 24 hours after sciatic nerve axotomy, terminals of epidermal nerves appeared swollen and there was a mild reduction in epidermal nerve density (5.7 +/- 2.8 vs 12.7 +/- 2.2 fibers/mm, p < 0.04). Epidermal nerves completely disappeared by 48 hours (0.2 +/- 0.2 vs 14.2 +/- 0.9 fibers/mm, p < 0.001). Concomitant with the disappearance of epidermal nerves, the immunocytochemical pattern of the subepidermal nerve plexus became fragmented. At the electron microscopic level, the axoplasm of degenerating dermal nerves was distended with organelles and later became amorphous. Beginning from day 28 after axotomy, collateral sprouts from the adjacent saphenous nerve territory extended into the denervated area with a beaded appearance. They never penetrated the epidermal-dermal junction to innervate the epidermis. In contrast, 3 months after nerve crushing, the epidermis on the surgery side resumed a normal innervation pattern as the epidermis on the control side (10.3 +/- 3.9 vs 10.6 +/- 1.5 fibers/mm, p = 0.1). This study demonstrates the characteristics of degenerating and regenerating nerves, and suggests that successful reinnervation mainly originates from regenerating nerves of the original nerve trunks. All these findings provide qualitative and quantitative information for interpreting the pathology of cutaneous nerves.

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.

Influence of cutaneous nerves on keratinocyte proliferation and epidermal thickness in mice.

We evaluated the influence of skin innervation on the epidermis in mice. The rich innervation of skin was demonstrated by immunocytochemistry with protein gene product 9.5, a ubiquitin carboxy hydrolase. Protein gene product-immunoreactive nerve fibers were in the epidermis, subepidermal plexus, dermal nerve trunks, and nerve terminals around sweat glands. Effects of denervation on the plantar surface of the hind foot was assessed by comparing the thickness of the epidermis, which was innervated by the sciatic nerve. Within 48 h after sectioning of the sciatic nerve, protein gene product (+)-nerves in the territory of the sciatic nerve were completely degenerated. There was a significant thinning of the denervated epidermis 72 h post-transection (30.5+/-1.1 vs 41.4+/-2.9 microm, 74+/-4% of the control side). The reduction in epidermal thickness persisted when skin remained denervated (69-75% of the control side). Incorporation of bromodeoxyuridine was reduced 24 h after denervation (71+/-6% of the control side). Reduction in bromodeoxyuridine-incorporation was most pronounced within 48 h after denervation (19+/-6% of the control side). Therefore, the reduction in bromodeoxyuridine-labeling followed a similar temporal course as the thinning of the epidermis (25-50%). Both epidermal thinning and reduced bromodeoxyuridine-labeling were reversed by epidermal reinnervation three months after denervation. Patterns of keratinocyte differentiation and programmed cell death were unaffected by skin denervation. These findings are consistent with the notion that skin innervation exerts influence on the proliferation of keratinocytes and the thickness of the epidermis, and offers a new look at the interaction between nociceptive nerves and their innervated targets.

Skin Innervation and Its Effects on the Epidermis.

Sensory innervation of the skin subserves protective sensations for the body to prevent thermal and noxious injuries. Neurophysiologically, they belong to the categories of Adelta and C fibers, usually with caliber less than one micro m in diameter. Morphological demonstration of the terminals of these nerves in the epidermis has been recognized recently by sensitive immunocytochemistry and an axonal marker, the protein gene product 9.5 (PGP). PGP is a ubiquitin C-terminal hydrolase, which is abundantly present in the nervous system, and particularly enriched in the unmyelinated nerves. Sensory nerves positive for PGP arise from the dorsal root ganglion, pass through the dermis, parallel the epidermis-dermis border, penetrate the basement membrane, move vertically and upwards in the epidermis with tortuous course and knobby appearance, and finally terminate at the granular layers of the epidermis. In rodents, denervation of the skin results in degeneration of epidermal nerves within 48 h of nerve transection, and thinning of the epidermis. In humans, application of this technique to evaluate disorders of the peripheral nervous system makes study of the degeneration of sensory nerve terminals possible. Patients with sensory neuropathy had fewer epidermal nerves than normal subjects, consistent with the notion of distal axonopathy. This approach has the potential to evaluate human sensory neuropathy in temporal and spatial domains. In addition, the influences of epidermal denervation open a new field to explore the interactions between sensory nerves and keratinocytes.

Ultrastructural localization and regulation of protein gene product 9.5.

Protein gene product 9.5 (PGP), a ubiquitin hydrolase, is abundant in the nervous system. To investigate the ultrastructural localization of PGP and the regulation of its expression, we performed electron microscopic immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR) on normal and transected rat sciatic nerves. In normal nerves, strong PGP-immunoreactivity was localized in the myelinated and unmyelinated axons with virtually no staining in the Schwann cells. After nerve degeneration, denervated Schwann cells exhibited intense staining for PGP, corroborated with up-regulation of PGP transcripts by RT-PCR. The present data suggest that the pattern of expression of PGP is more complicated than was expected previously, and reflects the integrity of nerves and status of axon-Schwann cell interactions.

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.

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).

Detection of genes for heat-stable enterotoxin in Escherichia coli by biotinylated ST-DNA probes.

Reference strains of enterotoxigenic Escherichia coli (ETEC), non-enterotoxigenic Escherichia coli (non-ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), and other enteropathogenic bacteria were used to prove the reliability of BIO-ST-DNA probe hybridization. In addition, 417 strains of E. coli isolated from children with diarrheal diseases in Shanxi Children's Hospital were examined for BIO-ST-DNA probe hybridization. In the test, BIO-ST-DNA hybridization was compared with suckling mouse assay in identifying ST-ETEC. The results obtained by both methods showed no significant difference. It was found that identification of ST-ETEC using hybridization is a simple, sensitive and more practical method.

Comparative evaluation of nine different methods for detecting enterotoxin of Escherichia coli.

Nine different methods for detecting enterotoxin of Escherichia coli were studied and compared. We found rabbit ileal-loop test and suckling mouse assay were both quite accurate and reliable for detecting heat labile toxin (LT) and heat stable toxin (ST) of enterotoxigenic Escherichia coli (ETEC). Mouse ileal-loop test was simple, but its sensitivity and specificity were comparatively low. CHO cell-culture assay might be more sensitive and specific. LT-DNA probe was the most sensitive and specific method. In practical application, PIHT (plate immunohemolytic test), Biken's, SPA-CoA and ELISA methods are recognized as simple, rapid, sensitive and specific methods for detecting ETEC-LT. These methods can be selected for use in clinical laboratory.

Study of the identification of enterotoxigenic Escherichia coli by LT-DNA gene hybridization.

Reference strains of enterotoxigenic Escherichia coli (ETEC), non-ETEC, and other enteropathogenic bacteria were used to prove the reliability of LT-DNA gene hybridization. In the test, LT-DNA gene hybridization was compared with plate immunohemolytic test (PIHT) in identifying LT-ETEC. The results obtained by both methods showed no significant differences. 791 strains of E. coli isolated from 1,875 children with acute diarrhea in Taiyuan Children's Hospital were examined for LT-ETEC by LT-DNA gene hybridization and PIHT. 289 strains examined by LT-DNA gene hybridization were LT positive, while 205 strains examined by PIHT were LT positive. Three different assays were done: colony hybridization, PIHT and fecal direct blot hybridization on each of 74 fecal specimens from children with acute diarrhea. It was found that identification of LT-ETEC using fecal direct blot hybridization is a simple, sensitive and more practical method.