ObjectiveTo explore the application value of probe-based confocal laser endomicroscopy (pCLE) in rapidly detecting and evaluating the morphological characteristics of digestive tract tissues in mice. MethodsTwelve male SPF Kunming mice aged 6 weeks were randomly divided into two groups. Six mice were subjected to gastric gavage with 52% Red Star Erguotou to establish the model, and six were given saline by gastric gavage as a control. After 28 days of modeling, 3 mice were randomly selected from each group. After deep anesthesia induced by inhalation of 3% isoflurane, the mice were sacrificed by cervical dislocation. The stomach, duodenum, jejunum, and rectum tissues were excised and immersed in 1% fluorescein sodium solution for staining. The microstructure of the mucosal surface of each tissue was observed using pCLE. The remaining mice in the model group and the control group were deeply anesthetized by inhaling 3% isoflurane, then cardiac perfusion was performed successively with saline and 4% paraformaldehyde. The stomach, duodenum, jejunum, and rectum tissues were excised for dehydration, section and hematoxylin-eosin (HE) staining, and the morphological changes of the tissues were observed under a microscope. ResultsUnder pCLE imaging, fluorescence staining on the surface of the gastrointestinal mucosa was uniform in the control group; the morphology of gastric pits, intestinal villi, and intestinal crypts was intact, arranged compactly, and had distinct boundaries. In the model group, the gastrointestinal mucosa exhibited mucosal swelling and deformation, with uneven fluorescence staining and fluorescein leakage. Furthermore, some tissues showed defects or cell shedding, and the boundaries between adjacent characteristic structures (e.g., gastric pits, intestinal crypts) were blurred. HE staining showed that the gastrointestinal tissue structure of the control group mice was normal and well-organized, with no structural defects. Moreover, submucosal glands were uniform in size, with no hyperplasia observed, and no obvious inflammatory cell infiltration. In the model group, some gastrointestinal mucosal structures were defective and sparsely arranged; submucosal glands showed atrophy, accompanied by obvious inflammatory cell infiltration. The histological characteristics detected by pCLE were consistent with those of HE staining. ConclusionpCLE can be used to obtain rapid, real-time, large-scale, and high-resolution microscopic imaging of the gastrointestinal mucosa, realistically and comprehensively displaying its physiological and microstructural characteristics. It shows promising prospects and practical utility in the histological evaluation of digestive system injuries in small animals.

Objective To assess the prevalence of mycoplasma genitalium(MG)in patients attending sexually transmitted disease(STD)clinic in Guangzhou,and to provide an epidemiological foundation for clinical treatment and laboratory diagnostics.Methods Utilizing real-time polymerase chain reaction(PCR),we analyzed MG DNA in 2,749 clinical specimens collected from 2,722 outpatients in the Dermatology Hospital of Southern Medical University from July 2019 to December 2021.Concurrent testing for MG,Chlamydia trachomatis(CT),and Neisseria gonorrhoeae(NG)was performed on 2,382 of these specimens.Patient data extracted from medical records were used to investigate the correlation between STD symptoms and MG infection.Results The investigation revealed that the overall prevalence of MG infection was 4.4%among the sampled patients(120 out of 2,722),with a higher prevalence in males(4.9%,or 87 out of 1,790)compared to in females(3.5%,or 33 out of 932).Notably,the prevalence decreased with increasing age.The highest incidence of MG infection was observed in females aged 18～25 years(6.4%,or 18 out of 281),while the lowest was in males aged 46 years and above(1.5%,or 5 out of 342),showing a statistically significant variation across age groups(P<0.05).Among males with urethritis symptoms,MG positive rate was significantly higher at 7.3%(42 out of 574).The rate of single MG infection was prominent,accounting for 89.9%(71 out of 79)in MG-positive male patients and 61.5%(16 out of 26)in MG-positive female patients.Co-infection rate of MG with CT was 1.2%in females and 0.3%in males,indicating a significant dif-ference(P<0.05).Conclusion The findings suggest a relatively high prevalence of MG infection and co-infection with CT among STD clinic attendees in Guangzhou,particularly in the younger demographic.The study underscores the need for early screening and vigilant surveillance of MG to mitigate its transmission among sexually active popula-tions at high risk.

Objective: To explore the clinical effect of free anterolateral thigh flap in repairing large annular soft tissue defect of lower leg after burn. Methods: From January 2014 to December 2018, 9 patients with large annular soft tissue defects of lower legs after burns were hospitalized in Zhengzhou First People′s Hospital, including 1 case with wounds on both legs. After debridement, area of wounds was 16 cm×11 cm-38 cm×21 cm, and the burn wounds were repaired with free anterolateral thigh flaps in the area of 18 cm×12 cm-32 cm×24 cm. End-to-end anastomosis of posterior tibial vessels or anterior tibial vessels with lateral circumflex femoral vessels was performed in manual way or by microvascular stapler. For the affected legs without condition for anastomosis, the sound medial lower leg flaps with areas of 10 cm×8 cm-15 cm×10 cm were excised and made into skin tubes, the posterior tibial vessels of the flaps were anastomosed with the vessels of free anterolateral femoral flaps, and the wounds of the injured lower legs were repaired by bridge-type cross-over free transplantation of anterolateral thigh flaps. The pedicles were broken 4 to 5 weeks later. The donor site was transplanted with autologous intermediate split-thickness skin graft from thigh. The outcome of the treatment, the number of perforators included in the flaps, and the anastomotic vessel in the recipient area of patients were recorded. The anastomosis time between manual way and microvascular staplers was recorded and compared. The patency of blood vessels, methods of free transplantation, and follow-up condition were recorded. Data were processed with Wilcoxon rank sum test for two independent samples. Results: All the 10 free flaps and skin grafts of 9 patients survived, and all the wounds were closed by primary operation. Seven flaps contained two perforators each, and three flaps contained three perforators each. The anastomotic vessels were posterior tibial vessels in 6 recipient areas and anterior tibial vessels in 4 recipient areas. Microvascular stapler was used to anastomose 12 veins, while 8 veins and 10 arteries were anstomosed manually. The time consumed by the former method was 4.00 (3.55, 4.38) min, significantly shorter than 12.80 (12.13, 13.40) min of the latter (W=78.00, P<0.01). The patency rates of veins and arteries were 100%. There was no vascular crisis due to vascular anastomosis. Three patients underwent bridge-type cross-over free transplantation, while the others underwent conventional free transplantation. Follow-up for 3 to 30 months showed that the donor site of the thigh had good motor function, without numbness or pain, but hypertrophy of scar could be seen. Four patients had slightly overstaffed flaps transplanted in the recipient area of the lower legs, while the other patients were satisfied with their appearance, and the walking function of the affected limbs gradually recovered. Conclusions Free anterolateral thigh flap transplantation is a safe and reliable clinical limb salvage method for the repair of large annular soft tissue defect of lower leg after burn. Intraoperative application of microvascular stapler for venous anastomosis can shorten the time of vascular anastomosis and has great clinical application value.

Objective: To explore the effects of free anterolateral femoral or medial calf flaps in the repair of severe facial burns. Methods: From January 2014 to October 2017, 18 patients with severe facial burns were admitted to Zhengzhou First People′s Hospital, including 12 males and 6 females, aged 15-78 years. Autologous intermediate split-thickness skin grafts were transplanted to replace oral mucosa in 4 patients with perforating cheek defects, and 8 patients underwent early vacuum sealing drainage and autologous intermediate split-thickness skin grafting to reduce the wound area to 14 cm×6 cm-22 cm×14 cm before flap transplantation. The wounds of 15 patients were repaired with free anterolateral femoral flaps, and the wounds of the other 3 patients were repaired with free medial calf flaps. The area of flaps ranged from 16 cm×7 cm to 24 cm×17 cm. The facial artery or superficial temporal artery was anastomosed end-to-end with lateral femoral circumflex artery or posterior tibial artery under microscope routinely and manually, and the two accompanying veins were anastomosed end-to-end by Coupler microvascular anastomat. The donor site was sutured or transplanted with autologous intermediate split-thickness skin graft. The anastomosis time of veins was recorded. The patency rate of vascular was calculated. The survival status of flaps were observed. The recovery of recipient area was observed during follow-up. Results: The anastomosis time of two veins in this group was 6-10 minutes, with an average of 8.5 minutes. The patency rates of veins and arteries were 100%. There was no vascular crisis due to the anastomosis problem. The free flaps survived well in 16 patients; one patient had hemorrhage under the flap 6 hours after operation, and the blood circulation of flaps turned well after hemostasis by surgical exploration; the other patient had 3 cm necrosis at the distal end of flap after operation, and the wound was closed after dressing change and autologous intermediate split-thickness skin grafting. The patients were followed up for 2 to 24 months after discharge. Most of the five senses function recovered. The color and texture of the flaps were not consistent with those of the normal facial skin. Some flaps were slightly swollen. Oral integrity was restored in 4 patients with perforating cheek defect with mouth opening of 2.2-3.5 cm. Conclusions Free anterolateral thigh flaps or medial calf flaps can repair severe facial burn wounds. It takes less time to anastomose venous vessels by microvascular anastomat during operation and can ensure the quality of venous anastomosis.

Objective To evaluate the regulatory role of azithromycin-induced persistent Chlamydia trachomatis (Ct) infection in the apoptosis of Hela229 cells.Methods Hela229 cells were firstly co-cultured with Ct for 22 hours,and then cultured with Dulbecco's modified Eagle's medium (DMEM) containing 0.08 mg/L azithromycin for 26 hours to establish a cell model of persistent Ct infection (persistent infection group).These infected Hela229 cells cultured with azithromycin-free DMEM served as a cell model of acute Ct infection (acute infection group).After 48-hour infection with Ct,azithromycin was removed,and infected Hela229 cells in the above 2 groups were successively cultured with DMEM for the resurgence of Ct.Immunofluorescence assay and electron microscopy were performed to verify the persistent Ct infection model.The Hela229 cells in the persistent infection group and acute infection group as well as uninfected Hela229 cells (control group) were treated with staurosporine (STS) for 4 hours to induce the apoptosis,and then cell apoptosis was detected by Hoechst 33258 staining,annexin V/propidium iodide staining and flow cytometry.Results After the treatment with azithromycin,atypical inclusions with aberrant reticulate bodies appeared in the Ct-infected cells.After removing azithromycin,cells were cultured until 96 hours after infection,and infectious elementary bodies reappeared in the Ct inclusions.After the treatment with STS,Hoechst staining showed that there was loose chromatin in the persistently infected cells,while chromatin condensation was observed in the uninfected cells.After 24-hour infection with Ct and 4-hour induction with STS,the apoptosis rate was significantly higher in the persistent infection group (45.567％ ± 2.631％) than in the acute infection group (38.567％ ± 1.701％,t =2.686,P =0.028),but significantly lower in the persistent infection group than in the uninfected group (69.800％ ± 2.835％,t =8.187,P ＜ 0.001).After 48-hour infection with Ct and 4-hour induction with STS,there was a significant difference in the apoptosis rate between the persistent infection group (46.700％ ± 5.257％) and acute infection group (61.767％ ± 1.815％,t =5.781,P ＜ 0.001),as well as between the persistent infection group and the uninfected group (68.667％ ± 3.156％,t =7.421,P ＜ 0.001).Conclusion This study showed that azithromycin-induced persistent Ct infection regulated the apoptosis of host cells,and this effect lasted 48 hours.

Objective To isolate and culture a clinical strain (GDI) of Treponema pallidum (Tp) in Guangdong province,and to investigate the difference in nonsynonymous single nucleotide polymorphisms (nsSNPs) between the GD1 strain and Tp Nichols strain.Methods The GD1 strain was isolated from the hard chancre in a patient with primary syphilis in Guangdong province,and continuously subcultured in the testes of New Zealand white rabbit.The serial subcultivation of GD1 was multi-verified by dark-field microscopy,polymerase chain reaction (PCR) for TP0548 gene,DNA sequencing and genotyping.Meglumine diatrizoate density gradient centrifugation was performed to isolate rabbit tissues and concentrate GD1,and DNA sequencing was used to verify the nsSNPs in the TP0443 and TP0584 genes.Results The GD1 strain was successfully isolated from the lesions of the patient with syphilis,and classified as a subtype f of TP0548.Compared with the American Tp (Nichols strain),there were nsSNP mutations in the GD1 strain.One mutation was located in the TP0443 gene,leading to the the substitution of threonine by alanine at amino acid position 120,and another one was located in the TP0584 gene,which caused a change from alanine to threonine at amino acid position 314.Conclusion The GD1 strain was successfully isolated firstly from the lesions in a patient with syphilis in Guangdong province,and nsSNP mutations were confirmed in the GD 1 strain on the etiology.

OBJECTIVETo observe the clinical effects of free musculo-cutaneous flap bridging with contralateral posterior tibial vessel on repair of lower extremity soft tissue defect.

METHODSFrom February 2006 to June 2013, 10 patients with soft tissue defect on lower shank and foot were included. The posterior tibial vessel on healthy lower extremity was chosen as recipient vessel and anastomosed with free latissimus dorsi musculo-cutaneous flap, or free latissimus dorsi musculo-cutaneous flap combined with thoracic-umbilical skin flap or anterolateral femoral musculo-cutaneous flap. The retrograde bridged flap was transposed to repair defect on contralateral lower shank and foot. The wound area ranged from 40 cm x 21 cm to 22 cm x 15 cm, with flap size from 48 cm x 26 cm to 25 cm x 18 cm. Meanwhile the defects on donor sites were covered with skin graft and both lower extremities were fixed with kirschner wires at middle tibia and calcaneus. The kirschner wires were removed at 4 weeks and pedicles were cut off 5-8 weeks postoperatively. Six patients received posterior tibial vessel reanastomosis at the same time of pedicle cutting.

RESULTSAll the 10 flaps survived and 3 patients received thinning of flaps due to excessive thickness. During the follow-up period of 3 months to 2 years follow up, the ambulatory function of injured legs recovered gradually with satisfactory appearance. The reanastomosed posterior tibial vessel on the healthy side was recovered.

CONCLUSIONSAppropriate bridged musculo-cutaneous flaps is suitable for extensive soft tissue defect of lower shank and foot. It is a safe and effective method for limb salvage.

Objective To trace changes in the transcript level of the Treponema pallidum(Tp)protein Tp0751 in skin lesions of a rabbit model of early syphilis. Methods Three New Zealand white rabbits were intracutaneously injected with 0.1 ml of Tp (Nichols Seattle strains)suspensions (107 treponemes/ml)at 10 sites on the shaved back to establish a model of early syphilis. All the rabbits received a single injection with the total amount of treponemes being 107. Then, skin changes at injection sites were observed, and the size of skin rashes was recorded on a daily basis. Skin specimens sized 0.4 cm × 0.4 cm were excised from an injection site and a non-injection site(negative control)separately every 3 days for the detection of Tp0751 and Tp0574 mRNAs. The whole experiment lasted 30 days, and a total of 11 skin biopsies were carried out. Fluorescence-based quantitative PCR was performed to measure the mRNA expressions of Tp0751 and Tp0574 continuously and dynamically during the development of chancre. Results After intracutaneous injection of Tp suspensions, red papules occurred on the back of rabbits on day 6, and reached maximum size on day 19 with the formation of ulcer and chancre. On day 25, disseminated secondary syphilides gradually appeared all over the body surface of the rabbits. The mRNA expression levels of Tp0574 and Tp0751 increased at the early stage, peaked onday 15 (compared with the other time points, all P < 0.05), thereafter rapidly declined, but rose slightly on day 27. The standardized expression level of Tp0751 mRNA increased gradually after day 15, and peaked on day 24 (compared with the other time points, all P < 0.05). Conclusion The transcript level of Tp0751 was high in rabbits at the late stage of Tp clearance when generalized disseminated secondary syphilides had not appeared, suggesting that Tp0751 may be involved in the systemic spread of Tp.

Objective To clone ,construction ,express and purify Tp47 of Treponema pallidum (Tp) ,and assess the immunoreac‐tivity by Western‐Blot .Methods Tp47 was amplified by polymerase chain reaction ,and then cloned to the vector pGEX‐6P‐1 .The correct sequence of the recombinant plasmids pGEX‐6P1‐Tp47 was transformed into Escherichia coli BL21 (DE3) and induced .The expression product was analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis and Western‐Blot .The expression protein was purified .Serum of different clinical stages of syphilis was used as the antibody to detect the immunoreactivity of the protein by Western‐Blot .Results A fusion protein with molecular weight about 71 × 103 was attained .Western‐Blot proved that the recombinant protein can react with Tp IgG positive sera .And the specificities and sensitivities of the diagnostic reagent detected by sera were 100% .Conclusion The recombinant protein Tp47 was expressed and purified with good antigen activity ,which could provide the basis of theory and practice for the development of early diagnostic kit applying to detect Tp infection .

Objective To determine the frequency of T helper type 22 (Th22) cells and expression level of interleukin-22 (IL-22) in peripheral blood of patients with psoriasis vulgaris,and to investigate their relationship with disease severity and clinical course.Methods Peripheral blood samples were obtained from 40 patients with psoriasis vulgaris and 30 healthy human controls.Five-color flow cytometry was performed to determine the percentage of Th22 cells in peripheral blood,and enzyme-linked immunosorbent assay (ELISA) to measure the expression of serum IL-22.Statistical analysis was carried out by t test and Pearson correlation analysis.Results Both the percentage of Th22 cells and serum level of IL-22 in peripheral blood were significantly higher in patients with psoriasis vulgaris than in healthy human controls (Th22 cells:0.65％ ± 0.48％ vs.0.33％ ± 0.15％,t =3.89,P ＜ 0.01; IL-22:(67.96 ± 14.32) vs.(40.59 ± 9.91) ng/L,t =9.45,P ＜ 0.01).Further more,Th22 cell percentage and IL-22 serum level in peripheral blood were both positively correlated with psoriasis area and severity index (PASI) in these patients (r =0.38,0.94,P ＜ 0.05 and 0.01,respectively),but neither of them correlated with clinical course of psoriasis vulgaris (r =0.20,0.10,respectively,both P ＞ 0.05).Conclusions The percentage of Th22 cells and level of IL-22 are increased in peripheral blood of patients with psoriasis vulgaris,and both of them are correlated with disease severity.