This paper reports 20 patients with cryptococcal meningitis,which were misdiagnosed before anti-fungal therapy,and the majority of them was diagnosed as tuberculous meningitis or viral meningitis.These patients were treated either with amphotericin B or fluconazale alone,or in combina- tion with flucytosine respectively,of the 15 patients could be evaluated,10 were cured(66.7%),2 im- proved(13.3%),3 died(20%),1 relapsed(10%).The mortality rate among the under age group and the aged group was significantly higher than that of the adult group(71.4% vs 23%).It is the authors' experience that in the early stage intrathecal amphotericin B combines with intravenous fluoconazole and changes to oral fluconazole or itraconazole in the later stage may be a valuable approach for cryptococcal meningitis.

Objective To investigate the DNA typing, observe relationship between DNA fingerprinting patterns and serotypes of Cryptococcus neoformans, and find a suitable genotyping standard for Cryptococcus neoformans. Methods Three primers, including CN 1(GTG) 5, CN 2(GACA) 4 and CN 3(GATA) 4, were used to distinguish variations among strains of C.neoformans. Results The distinguishable fingerprinting bands for serotype of C.neoformans were yielded by primer CN 2. Using this primer, of 24 clinical and environmental isolates of serotype A of C.neoformans and 8 standard strains investigated by PCR, 20 strains produced complete identical fingerprinting patterns, the other 4 strains had different fingerprinting patterns. 2 strains of serotype B and C yielded indistinguishable fingerprinting patterns. Conclusions ①The majority of strains of serotype A had similar and stable fingerprinting patterns. ②Some strains of serotype B and C had an indistinguishable fingerprinting patterns. ③The same serotype strains from different sources may produce different DNA fingerprinting patterns. ④The DNA fingerprinting is a rapid, simple and feasible method for identifying Cryptococcus neoformans.

The patient is an 11-year old boy, who was born with universe alopecia as well as dry and coarse skin. When he was 3 months old, photophobia was noticed, and since then, upper respiratory tract infection had occurred twice a month complicated by frequent diarrhea. He had short stature with slight conjunc- rival congestion, corneal vascularization, opacity, coarseness and poor vision. No abnormality was found in the teeth, sweating ability, or hearing. He had universal alopecia; his skin was dry and rough with generalized rhombus- or polygon-shaped scaly patches. Particularly thick brown scales were observed on the upper limbs. Moreover, there were spiny follicular papules on the abdomen and axillae, hyperkeratosis of palm and sole, and dystrophic nails. Hyperextensibility of proximal interphalangeal joints of the third, fourth and fifth fingers was noticed. He also suffered from mental retardation, the verbal intelligence quotient being 52, performance intelligence quotient lower than 40, full intelligence quotient lower than 40, but no abnormality was found in the heart, lung, liver or spleen. Histopathology of skin on the abdomen suggested a change characteristic of ichthyosis. Chromosome analysis revealed a karyotype of 46, XY. This is the first diagnosed case of ichthyosis follicularis with atrichia and photophobia syndrome in China.

Objective To make a genetic diagnosis of sporadic neurofibromatosis type 1 with café-au-lait spots as the only presentation in a child.Methods Blood samples were collected from an 8-year-old child patient,his parents,and 100 healthy human controls.The mutation of NF1 gene was detected by PCR and direct sequencing.Results No mutation was detected in the NF1 gene of the parents or the healthy controls.There was a de novo nonsense mutation c.3520C ＞ T (p.Q1174X) in the NF1 gene of the patient,which leaded to a premature termination codon.Conclusions The child with café-au-lait spots as the only manifestation is diagnosed with sporadic neurofibromatosis type 1 by genetic testing.The mutation c.3520C ＞ T (p.Q1174X) may be an underlying cause of neurofibromatosis type 1.

Objective To evaluate the efficiency of PCR-restriction fragment length polymorphism (PCR-RFLP)aiming at the structure gene g6341,versus PCR fingerprinting analysis in the genotyping of Cryptococcus neoformans MethodsEight reference strains and 68 clinical and environmental isolates of C.neoformans were genotyped by PCR-RFLP and PCR fingerprinfing.In PCR fingerprinting,the minisatel lite-specific core sequence of wild-type phage M13 was used as a single primer.The structure gene g6341 was selected for PCR-RFLP analysis by sequence alignments of multiple genes,a pair of pnmers were developed based on the conserved region of g6341 gene.PCR products were digested with the appropriate restriction endonucleases,and RFLP profiles were analyzed.Partial sequence analysis of g6341 gene was performed for different genotypes of C.Neoformans.Phylogenetic analysis was done to study the relatedness between these genotypes.Results As sequence homology analysis showed,g6341 gene was suitable for RFLP analysis.In the case of enotyping of 76 C. Neoformans strains,the results obtained from PCR-RFLP were consistent with those from PCR fingerprinting.Sequence analysis of g6341 gene revealed a homology of 84%-97%among the eight genotypes as well as a consistency of 99%-100%within a same genotype.In the phylogenetic tree,genotypes VNⅠ,VNⅡ,VNⅢand VNⅣ belonged to one cluster,and genotypes VGⅠ,VGⅡ,VGⅢ and VGⅣ to another cluster.Conclusions PCR-RFLP analysis aiming at the structure gene g6341 is a useful tool to genotype C.neoformans.Sequence analysis of g6341 gene can disclose the relatedness among different molecular types of C.neoformans.

Objective To investigate genetic relationships among five serotypes of two variants of Cryptococcus neoformans. Methods PCR mediated DGGE (denaturing gradient gel electrophoresis) and sequence analysis of 28S rDNA of C. neoformans were performed in ten reference strains, C. neoformans capsular-deficient strain CAP10, and nineteen clinical isolates from non-HIV patients. Results The results of DGGE and analysis of nucleotide sequences of 28S rDNA showed identical patterns and nucleotide sequences in the serotype A and D of C. neoformans var. neoformans, which were distinct from the serotye B and C of C. neoformans var. gattii. The patterns and sequences of serotype AD coincided with those of C. neoformans var. gattii. The patterns and nucleotide sequences of C. neoformans capsular-deficient strain CAP10 (serotype D) and serotype A and D were identical. Of the nineteen clinical isolates, seventeen had patterns of serotype A and D, and the others had patterns of serotype B and C. Conclusions PCR mediated DGGE integrated with sequence analysis of 28S rDNA is a valuable tool for the classification of C. neoformans. The clinical isolate of C. neoformans var. neoformans is predominant in Chinese non-HIV patients. Serotype AD is genetically close to C. neoformans var. gattii rather than C. neoformans var. neoformans. The data seem not to be in favor of previous study that serotype A, C. neoformans var. grubii, is a new variant of C. neoformans.

[Objective] To investigate the change of liver histology, proliferation of BDEC, and expression of TGF-β in different stages of liver chelestusis. [Methods] ①Rat cholestatic livers were induced by common bile duct ligation (BDL) and separated into 3 groups, namely control group (DO), 7 days after BDL group (DT), and 18 days after BDL group (D18). ② Histological changes of livers in different groups were evaluated based on Knodell HAI score. ③ Real-time PCR was used to detect the expression of TGF-β in liver tissue and isolated BDEC in different groups. ④ Statistically analyzing the correlation between Knedell HAl score and the levels of TGF-β_1 mRNA. ⑤ In vitro study was performed to investigate the effect of TGF-β_1 on an immortalized mouse intrahepatic bililary epithelial cell line (mIBEC). [Results] (I) Knedell HAI score and the proliferation of intrahepatic bile ducts increased as the liver cholestasis aggravated. ② The levels of TGF-β_1, TGF-β_2, and TGF-β_3 mRNA were significantly up-regulated in liver tissues and BDEC as the liver cholestasis aggravated. ③ Positive correlation was found between Knedell fibrotic score and the levels of TGF-β_1 mRNA in liver tissues and BDEC(r=0.9376, P<0.05 and r=0.9682, P < 0.01). ④ In vitro study showed that TGF-β_1 inhibited the proliferation of mIBEC. [Conclusion] ① Liver injury, biliary proliferation, and the levels of TGF-β mRNA expression increased as liver cholestasis aggravated. ② The interaction of TGF-β_1 and BDEC plays an important role in the pathogenesis of BDL induced cholestatic liver disease. ④ Up-regulated expression of TGF-β_1 mRNA in the proliferated BDEC participates in the formation of BDL induced cholestatic liver fibrosis.

Objective To assess the subgenotypes of pathogenic Cryptococcus gattii isolates from China and to elucidate the epidemiological links between these domestic isolates and those from other parts of the world. Methods DNA was extracted from 9 clinical isolates of Ctyptococcus gattii from China. The partially variable regions of the three unlinked loci, namely IGS1, PLB1 and GEF1, were amplified and sequenced, and the bioinformation at these loci was obtained from GenBank for multi-locus sequences alignment and phylogenetic analysis. Results Of these 9 clinical isolates, 8 were genotype VG Ⅰ and mating type α with the same sequences at the tested regions as the reference strain WM276, which was a representative isolate of an independent subgenotype; 1 was of genotype VG Ⅱ and mating type α, which was the first report in China, with the tested sequences consistent with those of the referrence strain R272. Sequencing and phylogenetic analysis of GEF1 gene, which was located at mating type locus, successfully identified the genotypes and mating types of all the Cryptococcus gattii isolates involved here. Conclusions Multi-locus sequence analysis shows that causative Cryptococcus gattii isolates of genotype VG Ⅰ in China carry similar sequences at the tested loci in IGS1, PLB1 and GEF1 genes, to a widely distributed subgenotype in the world, and the sequences of the first VG Ⅱ genotype isolate from China resemble the less virulent subgenotype VG Ⅱ b found in Vancouver islands.

Objective To investigate the efficacy of autologous peripheral hematopoietic stem cell transplantation in the treatment of adult dermatomyositis. Methods A 21-year-old patient with dermatomyositis received autologous peripheral hematopoietic stem cell transplantation and was followed up for 6 years. Autologous peripheral hematopoietic stem cells were mobilized by recombinant human granulocyte colony stimulating factor (rhG-CSF) before the transplantation, and the conditioning regimens consisted of cyclophosphamide,methylprednisolone and cyclosporin. Rabbit anti-human T lymphocyte immunoglobulin began to be applied on day 3 after retransfer of stem cells. The improvement in symptoms, physical signs and biochemical indicators was observed, and hematopoietic restructuration and immunity resurrection were evaluated after the transplantation. Results After the transplantation, skin eruption greatly improved and gradually subsided. The muscle force of extremities restored from level Ⅳ before transplantation to level Ⅴ. The level of creatine kinase declined sharply after transplantation, but gradually returned to previous level. Leucocyte count began to decrease on the day of retransfer, and returned to the normal level on day 8. Immune function remained normal before and after the transplantation. Conclusion Autologous peripheral hematopoietic stem cell transplantation is an alternative treatment for severe and refractory dermatomyositis.

Objective To investigate the expression of μ-opioid system in the epidermis of patients with atopic dermatitis and its role in the pathogenesis of atopic dermatitis. Methods Thirty-two mice were equally divided into 4 groups, negative control group, pre-treatment group, naloxone group, and physiological saline group. Ovalbumin was used to sensitize mice in pretreatment group, naloxone group, and physiological saline group for 7 weeks, then, mice in naloxone group and physiological saline group were treated with intracutaneous naloxone or physiological saline solution for 1 week, respectively. Mice were killed in negative control group and pre-treatment group at the end of sensitization, and in naloxone group and physiological saline group after 1-week injection with naloxone or physiological saline, skin tissues were obtained from the back of killed mice and subjected to histological examination with HE staining and quantitative fluorescent PCR for the detection of mRNA expression of μ-opioid receptor (MOR) and its ligand (β-endorphin) in epidermis. The atopic dermatitis severity index of lesions and histological changes were assessed before and after the treatment. Results In comparison with the negative control mice, the epidermal expression level of MOR was signifieantly decreased (t = 2.549, P ＜ 0.05 ) in pre-treatment group, but increased in naloxone group and showed no statistical difference from the negative control group (t = 0.671, P ＞ 0.05). No significant difference was observed in the epidermal β-endorphin mRNA expression between negative control group and pre-treatment group or naloxone group (both P ＞ 0.05 ). The improvement of lesions could be visualized after treatment with naloxone (t = 8.338, P ＜ 0.01 ), which was concordant with the histological changes in naloxone group. Conchusions As an antagonist of MOR, naloxone can restore the expression of epidermal MOR in mice model for atopic dermatitis, and shows a certain efficacy in the treatment of atopic dermatitis, which proves that μ-opioid system is somewhat associated with the pathogenesis of atopic dermatitis.