Breeding and preliminarily phenotyping of a congenic mouse model with alopecia areata.

In the current study, the alopecia areata gene was introduced into the C57BL/6 (B6) mouse through repeated backcrossing/intercrossing, and the allelic homozygosity of congenic AA(tj)mice (named B6.KM-AA) was verified using microsatellites. The gross appearance, growth characteristics, pathological changes in skin, and major organs of B6.KM-AA mice were observed. Counts and proportions of CD4⁺ and CD8⁺ T lymphocytes in peripheral blood were determined by flow cytometry. Results show that congenic B6.KM-AA mice were obtained after 10 generations of backcrossing/intercrossing. B6.KM-AA mice grew slower than B6 control mice and AA skin lesions were developed by four weeks of age. The number of hair follicles was reduced, but hair structures were normal. Loss of hair during disease progression was associated with CD4⁺ and CD8⁺ T lymphocytes infiltration peri-and intra-hair follicles. No pathological changes were found in other organs except for the skin. In the peripheral blood of B6.KM-AA mice, the percentage of CD4⁺ T cells was lower and percentage of CD8⁺ T cells higher than in control mice. These findings indicate that B6.KM-AA mice are characterized by a dysfunctional immune system, retarded development and T-cell infiltration mediated hair loss, making them a promising new animal model for human alopecia areata.

Abnormal hypermethylation and clinicopathological significance of FBLN1 gene in cutaneous melanoma.

Fibulin-1 (FBLN1) is involved in the progression of some types of cancer. However, the role of FBLN1 in cutaneous melanoma (CM) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of FBLN1 inactivation in CM. The expression of FBLN1 mRNA in CM tissues and adjacent normal skin tissues was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Methylation-specific polymerase chain reaction was performed to examine the methylation status of the FBLN1 gene promoter. Furthermore, the methylation status of FBLN1 was analyzed with the clinicopathological characteristics and overall survival. qRT-PCR showed FBLN1 mRNA levels in cancerous tissues to be significantly decreased compared with that in adjacent normal skin tissues. The rate of FBLN1 promoter methylation was significantly higher in CM tissues than in adjacent normal skin tissues (P < 0.001). Downregulation of FBLN1 was strongly correlated with promoter methylation (P = 0.021). Promoter hypermethylation of FBLN1 was significantly associated with tumor stage (P = 0.019). In addition, FBLN1 methylation status was associated with significantly shorter survival time and was an independent predictor of overall survival. In conclusion, our results indicated that FBLN1 is a novel candidate of tumor suppressor gene and that promoter hypermethylation of FBLN1 is associated with tumor progression in CM.

Serum soluble MICB (sMICB) correlates with disease progression and survival in melanoma patients.

Recently, it was reported that soluble MICB (sMICB) may impair tumor immunogenicity by reducing natural killer group 2D ligand densities on malignant cells. The aim of this study was to elucidate the role of sMICB in melanoma patients. In the present study, we determined sMICB serum concentration in 125 melanoma patients of different clinical stages of disease compared with 30 healthy controls using an ELISA. The correlations between sMICB serum concentration and clinicopathologic variables were analyzed. sMICB serum level was significantly elevated (P < 0.0005) in melanoma patients (mean ± SE = 8.60 ± 0.26 ng/ml) compared with healthy controls (mean ± SE = 6.27 ± 0.25 ng/ml). Univariate analysis revealed a correlation of sMICB serum concentration with advanced stages of disease (P = 0.009). Only a slight increase in sMICB serum level (P = 0.057) could be observed in regard to the tumor burden. Patients undergoing current treatment with cytostatics (n = 18) revealed a strong increase in sMICB serum level (P < 0.0005), whereas treatment with IFN-α alone or combined with cytostatics (n = 19) showed no change in serum sMICB concentration. According to Kaplan-Meier analysis, elevated sMICB serum levels were associated with a poor overall and a progression-free survival. Multivariate analysis revealed sMICB serum concentration as an independent predictive factor for progression-free and overall survival. Our results show a prognostic relevance of serum sMICB in melanoma patients, indicating that the evaluation of sMICB serum level may be important for the selection of therapeutic strategies.

Increased serum level of thymidine kinase 1 correlates with metastatic site in patients with malignant melanoma.

Thymidine kinase 1 (TK1) is involved in cancer progression. Metastatic site is a prognostic factor in melanoma. We assessed whether serum levels of TK1 are associated with metastatic sites or prognosis in patients treated for stage IV melanoma. The study included 64 patients, who received dacarbazine or four-drug chemotherapy (dacarbazine, vincristine, bleomycin, and lomustine) both combined with interferon-alfa. Serum samples for TK1 were analyzed by ELISA. The patients (n = 22) with only skin and subcutaneous metastases had significantly lower mean TK1 levels (1,639 pg/ml) than the patients (n = 42) with other distant metastases (2,586 pg/ml, Mann-Whitney, p = 0.031). TK1 levels above the median (1,590 pg/ml) were significantly related to deep lymph node involvement (odds ratios 3.672; 95 % confidence intervals 1.024-12.843, p = 0.036). There were no other significant associations between TK1 levels and tumor burden nor were the levels significantly related to the response to therapy or survival. Those eight patients who had received previous adjuvant IFN-alfa therapy had lower mean TK1 levels (1,735 pg/ml) as compared to those 56 patients without previous IFN-alfa therapy (2,338 pg/ml, analysis of variance, p = 0.026). This is the first study exploring serum TK1 in melanoma. TK1 might be involved in the deep lymphatic dissemination and progression of melanoma metastasis.

[Phenotype analysis and mutant gene location of ventral yellow mouse (VY(Slac))].

The ventri-yellow pigmentation mouse (temporarily named VY(Slac)) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VY(Slac) presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VY(Slac) and B(6) for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F(2) 48 mice (VY(Slac)D(2)F(1)×D(2)) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F(2) mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene.

[Morphologic observation of the regenerated nerve in reconstructed penis with sensory nerve implantation in rabbit].

OBJECTIVE: To investigate the mechanism of sensory nerve regeneration of the reconstructed penis with sensory nerve implantation and to explore a new surgical technique to improve the postoperative sensory function in phallic reconstruction. METHODS: Adult male New Zealand rabbits were randomly divided into experimental group (n = 20, with sensory nerve implantation) and control group (n = 20, without sensory nerve implantation), which were both performed phalloplasty with a superficial epigastric faciovascular pedicle flap. Postoperatively, the nerve regeneration process of the reconstructed penis was observed histologically. RESULTS: In experimental group, the amount of CGRP positive nerve fibers increased markedly with the time prolonged, whereas merely a few CGRP positive fibers scattered in deep dermis 6 months later in the other group. The cutaneous sensory nerve regeneration of the reconstructed penis in experimental group shows the procedure that the myelinated axon began to exist within 3 months, thereafter the myelinated axon and unmyelinated axon were both observed under the electron microscope. CONCLUSION: These findings show that the rabbit model of phalloplasty with sensory nerve implantation can acquire well sensory reinnervation, and bring a light to clinical application for restoration of sensory function in reconstructed penis.

[A long-term study of regeneration of mechanical sensory fibers after free nerve transplantation to the rabbit reconstructed penis].

OBJECTIVE: To explore the regeneration of mechanical sensory fibers after free nerve transplantation. METHOD: Neuroelectrophysiological technique (single nerve fiber recording) was used to test the regeneration rate of mechanical sensory fibers, the proportion of rapidly and slowly adapting receptors, the stimulating thresholds of regenerated mechanoreceptors and conduction velocity of regenerated fibers. The regeneration pattern of the mechanoreceptors after free nerve transplantation to the rabbit reconstructed penis was also analyzed. RESULTS: 9 months after operation, the number of regenerated mechanical sensory fiber was almost normal. The regenerated rapidly adapting receptors had a higher proportion with higher mature degree than the regenerated slowly adapting receptors. 9 months after nerve transplantation the stimulating thresholds of regenerated mechanoreceptors and conduction velocity of regenerated fibers remained below normal. CONCLUSION: After free nerve transplantation to the rabbit reconstructed penis, the function of both rapidly and slowly adapting sensory nerve fiber partially recovered, but in different extent.

[Electrophysiology research on the spinal nerve source of rabbit penis cutaneous sensation].

OBJECTIVE: To explicate the spinal nerve source of the rabbit penis cutaneous sensation. METHODS: Twelve adult male rabbits were randomly divided into two groups of equal number. While mechanical stimuli were given to the penis by different von Frey hairs, single fiber activities were recorded in vivo in the left (Group A) and right (Group B) S1-S4 spinal nerves, respectively. The mechanical threshold, adaptability and conduction velocity of the fibers were analyzed. RESULTS: When the ipsilateral penis was mechanically stimulated, discharges were detected in S2 and S3 spinal nerve fibers, but not in S1 and S4. The discharge fibers were 39.67 +/- 3.14 (S2) and 21.00 +/- 2.19 (S3) in the left spinal nerve and 40.00 +/- 3.16 (S2) and 19.67 +/- 2.58 (S3) in the right. There was no obvious difference between the numbers of the left spinal nerves and the right ones (P > 0.05). CONCLUSION: The rabbit penis cutaneous sensation originates from S2 and S3 spinal nerves.

[Location of somatic sensory neurons of the skin and dorsal nerve of the penis in rabbits].

OBJECTIVE: To trace the segmental distribution of somatic sensory neurons of the skin and dorsal nerve in the rabbits penis. METHODS: The experiment was performed on 8 adult male rabbits with the nerve tracing method of retrograde axonal transport of horseradish peroxidase (HRP), which was injected into the dermis around the penis and the dorsal nerve of the penis. The rabbits were sacrificed five days later to harvest the spinal cord segments and the dorsal root ganglia of lumbosacral segments for histological study. RESULTS: The HRP tracing showed that a number of labeled HRP positive neurons appeared in spinal ganglia (S2 - S4) in all the rabbits, and distributed segmentally. The counts of the positive neurons different segments were: S2 (215.0 +/- 10.2) , S3 (242.2 +/- 8.3) and S4 (109.7 +/- 8.4) respectively, with statistically significant difference between the two groups. CONCLUSION: The rabbit's sensory nerve fibers in both the skin and the dorsal nerve of the penis are rooted in the S2-S4 segments of spinal ganglia, which distribute regularly.

[Transfection of agrin gene on the recovery of muscle function after free neurovascular muscle transfer].

OBJECTIVE: To investigate the effects of transfection of agrin gene on the recovery of muscle function after a free neurovascular muscle transfer. METHODS: The electrical gene transfection was performed when the gracilis muscle of the SD rat was completed free neurovascular transfer. The experimental group was treated with pCS2+ -agrin, the group with plasmid pCS2+ as the negative control and the group with normal saline as the frank control. The muscle function, expression of neural agrin and the junctional nAChR number was measured after the operation. RESULTS: At 4, 5 and 10 weeks postoperatively, the pCS2+ -agrin group was significantly better than the control groups in muscle function (P < 0.05 ). The immunohistochemical staining showed an increasing deposition of the agrin protein near the endplate at 1 and 5 weeks after the operation, but decreasing remarkably to the level of control groups at 10 weeks postoperatively. The pCS2+ -agrin group was significantly more than the control groups in junctional nAChR number at every points of the time postoperatively. CONCLUSIONS: Transfection of agrin gene in the transferred muscle may increase the early recovery of muscle function.

[Changes of acetylcholine receptor distribution at the motor end-plates following muscle transfer].

OBJECTIVE: To investigate the changes of acetylcholine receptor (AChR) distribution at the neuromuscular junction (i.e. motor end-plate) following the free neurovascular muscle transfer. METHODS: AChR in the gracilis muscle of the Wistar rat following free neurovascular transfer were labeled by fluorescent alpha-bungarotoxin and radioiodinated alpha-bungarotoxin. Then confocal microscope and gamma-counting were estimated to ACHR, qualitatively and quantitatively. RESULTS: The junctional AChR numbers decreased to a minimum at the fourth week postoperatively, whereas the extrajunctional receptor numbers increased. From the fifth week postoperatively, the number of junctional AChR's increased. Even at 30 weeks after transfer, the morphology of the neuromuscular junction failed to return to the preoperative style. The number of acetylcholine receptors at the reinnervated neuromuscular junction also remained lower than the control. CONCLUSION: The persistent weakness following free neurovascular muscle transfer may be attributed to these qualitative and quantitative changes at the neuromuscular junction.

Two kinds of ENU-induced scant hair mice and mapping of the mutant genes.

OBJECTIVE: To establish mouse models for human diseases through N-ethyl-N-nitrosourea (ENU) mutagenesis, and to provide groundwork to clone genes and study their functions after mapping the mutant genes. METHODS: 18 male D2 mice (G0) at age of 8-10 weeks old were injected intraperitoneally with ENU (100 mg/kg) once a week for three consecutive weeks. The treated male mice were mated with females of the same strain, and their offspring (G1) were used to screen for dominant and recessive mutation. After breeding the mutant F2 (D2B6 F1 intercrossing) mice, 39 microsatellites that are equally distributed on the mouse genome and are different between B6 and D2 strains were used to scan the genome. According to the log odds score (LODS) we determined whether these microsatellites were linked to the mutant genes and calculated the location of mutant genes based on their recombination ratio. RESULTS: We screened 532 G1 mice, of which 14 exhibited mutation phenotypes. None was dominantly hereditable. Two cases of recessive inheritable scant hair mice were obtained through testing 30 G1 mice with normal phenotype and potential recessive mutant genes. All showed scant coat hair, grew slowly, and hyperkeratoses of epidermis and bollicular horn plug in histological sections. Their visceral organs were not markedly different from normal, and they were named scant hair 1 Baojin (symbol is snthr(-1Bao)) and scant hair 2 Baojin (symbol is snthr(-2Bao)). Through microsatellite screening we found that the LODS between snthr(-1Bao) and D9Mit243 was 7.73, and the linkage was determined. After analyzing the recombination ratio between snthr(-1Bao) and microsatellite D9Mit18 which was near snthr(-1Bao) based on a total number of 126 F2 mice with the scant hair phenotype, we determined that snthr(-1Bao) was located at chromosome 9 and was 71cM from centromere. Using the same technique, snthr(-2Bao) was mapped to the same position as snthr(-1Bao). CONCLUSION: In our research, two cases of scant hair mice provide good models for the study of dermatology, and the location of mutant genes provides a solid foundation for cloning new mice scant hair genes.

[One-stage reconstruction of postburn whole auricle defect with MEDPOR ear scaffold covered with superficial temporoparietal fascia flap].

OBJECTIVE: To study the effect and the key points in the operative procedure of the one-stage reconstruction of postburn whole auricle defect with medpor car scaffold covered with superficial temporoparietal fascia (TPF) flap. METHODS: Medpor car scaffold was embedded under the superficial temporal (TFP) fascia. Razor-thin skin was grafted onto the surface of the fascia flap. RESULTS: Fifteen patients with postburn whole auricle defect were treated by one-stage reconstruction with Medpor ear scaffold during the last four years. It was successful in all the patients with satisfactory appearance of the reconstructed ears. CONCLUSION: Medpor possessed friendly biological compatibility. The reconstruction gave satisfactory results, and its advantages consisted of short operational time, easy manipulation, less injury to patients and good auricular contour.