Volume 90  ·   Issue 36  ·  2010

In the 1950s, when the technique of organ transplantation was just emerging, it was found that the rejection caused by immune factors was a huge obstacle to the success of organ transplantation. The causes of rejection were seriously studied and even fiercely debated.

Objective To investigate the efficacy and safety of protein A immunoadsorption (IA) therapy in the prevention of highly sensitized renal transplant recipients with acute rejection. Methods A retrospective analysis of preoperative clinical data was carried out in 12 renal transplant patients in our hospital from March 2008 to October 2009 with a high level of panel reactive antibody (PRA). Serum PRA and IgG, IgM, IgA were detected at pre and post-treatment. The acute rejection responses and adverse effects were observed. Results At post-treatment, PRA of both Class Ⅰ and Ⅱ antibody [14% (4%,27% ) vs 86% (73% ,98% ) ,6% (0,23%) vs 68% (34% ,88% ) ,P ＜0. 01] and serum total IgG levels significantly decreased [(550 ±341 ) g/L vs ( 1301 ±393) g / L, P ＜0. 01]. IgA and IgM were also lower than those at pre-treatment [(144 ±78)g/L vs (185 ± 93 ) g/L, (103 ±48)g/L vs (131 ± 66) g/L, P ＜0. 01]. Five cases had acute rejection reversible by anti-thymocyte globulin (ATG) or combined IA (n=2). Within 6 months, 1 cases developed Aspergillus pneumonia, 2 cases of Pneumocystis carinii pneumonia were cured. Conclusion IA therapy can reduce antibody levels in highly sensitized renal transplant recipients. When supplemented with induction therapy, it can effectively prevent and reduce rejection after renal transplantation.

Objective To study the relationship between the expression of serum human leucocyte antigen-G5 (HLA-G5)/soluble CD30 (sCD30) and the fuction of renal graft in kidney transplant recipients and investigate the immune status of recipients with combined HLA-G5 and sCD30. Methods From January 2002 to November 2008, a total of 66 kidney transplant recipients in our centre were selected as subjects and divided into three groups: stable function of renal graft (n =38), acute rejection (n= 15) and chronic rejection (n = 13). The expressions of serum HLA-G5 and sCD30 were detected. There were two different immune conditions with acute/chronic allograft rejection and normal renal graft in kidney transplant recipients as evaluated by combined HLA-G5 and sCD30. The sensitivity, specificity and critical value of the method were analyzed by the curve of receiver operating characteristic. Results The levels of HLA-G5 and sCD30 were significantly correlated with serum creatinine ( r = - 0.493, 0.691, both P ＜ 0. 01 ). Within the first year post-transplantation, the sensitivity was 75. 6% and the specificity 85. 7% when HLA-G5critical value 82 μg/L and sCD30 critical value 12.2 μg/L. After one year post-transplantation: the sensitivity was 92. 3% and the specificity 84. 6% when HLA-G5 critical value 141 μg/L and sCD30 critical value 10. 3μg/L. Conclusion The immune state of recipients are evaluated by combine HLA-G5 and sCD30 which may be a simple and valid method.

Objective To study the feasibility of human lecocyte antigen-G (HLA-G) as a posttransplantation prognostic biomarker and discuss the correlation of its receptor expression and the mechanisms. Methods A total of 215 recipients in our centre from February 2006 to June 2008 were divided into stable kidney function group (n = 173) and acute rejection group (n = 42). The soluble human lecocyte antigen-G5 (sHLA-GS) level in peripheral plasma was detected by ELISA. And the HLA-G receptor ILT-2, KIR2DL4 on T, B, NK lymphocytes were analyzed by flow cytometry (FCM). The sHLA-G5 cutoff level by ROC curve was employed to predict the events of acute post-transplantation rejection. And regression analysis was used to determine the association of sHLA-G5 with acute rejection. Results An optimal cutoff value of 139.0 μg/L could be defined for sHLA-G5 (sensitivity: 63.6%, specificity: 82. 1%, AUC:0. 780). Binary regression analysis showed that sHLA-G5 played an independent role on acute rejection (P=0.019, OR=0.039, 95%CI:2.091 -5.661). The rate of HLA-G receptor ILT-2 on CD4+T cell,CD8 +T cell and B cell in acute rejection group was statistically lower than that in stable kidney function group (21% ±7% vs52% ±17%, 23% ±6% vs 39% ±16%, 21% ±7% vs 39% ±16%, all P＜0. 05). The expression of KIR2DL4 on NK cells in acute rejection group was statistically lower than that in stable kidney function group (31% ± 10%vs 57% ±21% ,P ＜0. 05). Conclusion sHLA-G5 level may be predicted for acute rejection with a high sensitivity and specificity. The up-regulated expression of ILT-2 and KIR2DLT may contribute to immunology tolerance in peripheral circulation.

Objective To discuss the feasibility and efficacy of one-stage anteroposterior hemivertebra resection and segmental internal fixation for young children with congenital scoliosis. Methods Thirty-five patients undergoing one-stage anteroposterior hemi-vertebra resection and segmental internal fixation were retrospectively studied. The mean followed-up period was 5.3 years ( range: 1.2 - 8.7). The Cobb's angle of scoliosis at pre and post-operation was compared. Results All children's parents were satisfied with the outcome. The Cobb' s angle of scoliosis was corrected from (42.5 ± 6.7 )° to ( 16.2 ±3.2)° at post-operation. The coronal correction rate was 64.7%. The angle of kyphosis improved from preoperative(33.5 ± 5.2)° to postoperative ( 13.3 ± 5.6)° in 14 cases. Operative duration was 210 - 280 minutes with an average of 240 minutes. The intra-operative blood loss was 80 -200 ml with an average of 120 ml. There was no significant correction loss at follow-up. No neurological complication, infection or pedicular fracture was reported. Conclusion The procedure of one-stage anteroposterior hemi-vertebra resection and segmental internal fixation is a safe and effective treatment for scoliosis by congenital hemivertebra in young children. A satisfactory correction may be achieved with a short fusion segment.

Objective To analyze the relationship between the expression of SM22α and the lymph node (LN) metastasis of breast cancer and to investigate its molecular mechanisms. Methods Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of SM22α in breast cancer tissue and adjacent normal breast tissue. RT-PCR and Western blot were employed to investigate the SM22α mRNA and protein level in tissues of breast fibroadenoma, breast cancer without LN metastasis and breast cancer with LN metastasis. RT-PCR and zymography were used to detect the MMP2 and MMP9 expression and activity and TIMP1 expression level in breast fibroadenoma, breast cancer samples without LN metastasis and those with LN metastasis respectively. Results The expression level of SM22α mRNA in breast cancer was significantly lower than that in breast fibroadenoma or adjacent normal breast tissue (5.1% ±2.4% vs 15.1% ±5.3% vs 30.1% ±5.1%, P＜0.01). The protein and mRNA expression level of SM22α in breast cancer samples with LN metastasis were significant lower than those of breast cancer without LN metastasis (6.2% ± 3.1% vs 10.1% ±4.1%, P ＜0.01 ). Both the expression and activity of MMP2 and MMP9 in breast cancer samples with LN metastasis were significant higher than those without LN metastasis (P ＜ 0.01 ). A strong negative correlation was found between SM22α protein level and MMP2 activity (r= -0.848; n=27; P＜0.01) or MMP9 activity (r= -0.916; n=27; P＜0.01) in breast cancer tissue. Conclusion A down-regulation of SM22α in breast cancer is correlated with LN metastasis. SM22α may inhibit the LN metastasis through a negative regulation of MMP2 and MMP9 in breast cancer.

Objective To investigate the role of a real-time fluorescence quantitative polymerase chain reaction (PCR) in measuring the expression levels of a proliferation-inducing ligand and its receptors expression levels in peripheral blood of children with lymphoma. Methods Real-time fluorescence quantitative PCR was used to detect the expression levels of a proliferation-inducing ligand and its receptors in patients with Hodgkin disease (n = 10) or non-Hodgkin lymphoma (n =37) and healthy control (n =40). The correlation between the mRNA levels of a proliferation-inducing ligand and its receptors and differential stage of malignant lymphoma was analyzed. Results The results of 47 samples showed that the levels of prolifevation-inducing ligand, B cell maturation antigen, transmembrane activator and CAML interactor in the peripheral blood of lymphoma in children were significantly higher than those in normal children (1.13 ±0.09 vs 0.41 ±0.09, 1.22 ±0.11 vs 0.43 ±0.10, 0.89 ±0.12 vs 0.35 ±0.08, all P＜0.05). The level of a proliferation-inducing ligand and its receptors had no significant difference between Hodgkin disease and non-Hodgkin lymphoma ( P ＞ 0.05). The level of a proliferation-inducing ligand in Ⅰ -Ⅱ stage (0.88 ± 0.06, 0.90 ± 0.08 ) of malignant lymphoma in children was significantly lower than that in Ⅲ-Ⅳ stage( 1.21 ± 0.09, 1.23 ± 0.09, P ＜ 0.05 ) while the level of its receptors between various stages showed no difference (P ＞ 0.05). Conclusion The real-time fluorescence quantitative PCR has a high sensitivity and reproducibility. A proliferation-inducing ligand may play a great role in the development and progression of malignant lymphoma in children through its receptors of B cell maturation antigen,transmembrane activator and CAML interactor. A proliferation-inducing ligand and its receptors can be established as target molecules for early diagnosis and anti-cancer therapy.

Objective To explore the application significance of zinc transporter 8 autoantibody (ZnT8A) in the diagnostic classification of acute-onset diabetics. Methods According to the status of glutamic acid decarboxylase antibody(GADA) and tyrosine phosphatase antibody( IA2-A), 453 acute-onset diabetics were divided into A + subgroup ( any antibody positive ) and A - subgroup ( both antibodies negative). A total of 555 type 2 diabetics and 405 healthy controls were recruited. The distribution and correlated factors of ZnT8A were analyzed in the acute-onset diabetic group and two subgroups( A + and A- ). The clinical characteristics were compared between the patients with ZnT8A positive alone and patients without any antibody. All these islet antibodies were measured by radioligand assay. Results The prevalence of ZnT8A in acute-onset diabetics was 24.3% and it was significantly higher than that in type 2 diabetics( 1.8% ) and healthy controls( 1.0% ) ( both P ＜0.01 ). The frequency of ZnT8A in A + subgroup was much higher than A - subgroup(29.7% vs 15.8%, P ＜ 0.01 ). The positive rates of ZnT8A were much higher in all the subgroups with age at onset of ＜30yr than those with ≥30yr(0 -9, 34.9%; 10 -19, 26.7%; 20 -29, 26.3% vs ≥30 yr, 18.3%; all P＜0.05); furthermore, the rates were also higher in BMI ＜21.0 kg/m2 and 21.0 -25.0 kg/m2 subgroups than in BMI ＞25.0 kg/m2 subgroup(25.5% and 25.9% vs 8.7%, both P ＜ 0.05 ). The ZnT8A level was only positively correlated with IA2-A titer( r =0.165, P=0.01), but not related to such factors as GADA titer, age at onset, duration, body mass index,HbAlc and CP levels( all P ＞0.05). As compared with Ab- patients, the patients with ZnT8A positive alone had much higher insulin injection dosage [( 35.5 ± 9.3 ) U/d vs ( 29.8 ± 14.7 ) U/d, P ＜ 0.05], and much lower systolic blood pressures [( 107 ± 15 ) mm Hg vs ( 113 ± 16 ) mm Hg, P ＜ 0.05] and diastolic blood pressures [(69 ± 12) mm Hg vs (73 ± 12) mm Hg, P ＜ 0.05]. Conclusion ZnT8A testing may be applied in the diagnostic classification of acute-onset diabetics, especially in those without an evidence of GADA and IA2-A since it helps to identify a clinical phenotype which is more similar to the classic type 1 diabetes.

Objective To investigate the regulation in vascular tension of diabetic coronary artery by large conductance Ca2+ -activated K + channel ( BK channel) and to elucidate the mechanisms of coronary dysfunctions due to diabetes. Methods Regulation of vascular tension in normal coronary artery was evaluated by videomicroscopy system. Streptozotocin-induced rat diabetic animal model was established successfully by intraperitoneal injection. Coronary smooth muscle cells were isolated by enzyme digestion.The BK currents in control and diabetic groups were recorded by patch clamp technique in whole cell configuration. Changes of vascular tension in normal and diabetic coronary arteries were assayed by multiwire myograph system. Results More than 50% was contracted in inner diameters of coronary arteries when 100 nmol/L IBTX, a specific BK channel blocker, was applied. In comparison with normal group, the BK current densities in diabetic group significantly decreased when test potentials were more than 60 mV (P ＜0.05). The BK current densities at 150 mV in normal group and diabetic group were (275 ±40) pA/pF and (70 ± 10) pA/pF respectively. When 100 mmol/L KCI was washed out, vascular tensions of normal and diabetic coronary artery were (398 ± 38 ) mg and ( 390 ± 35 ) mg respectively ( P ＞ 0. 05 ); however,when 100 nmol/L IBTX was added, the vascular tensions of normal and diabetic coronary artery were (395 ± 40 )mg and (50 ± 7) mg( P ＜ 0. 05 ). Conclusion BK channels play an important role in the regulation of coronary vascular tension, whereas BK channels in diabetic coronary artery are dysfunction, BK currents decrease and vascular tensions increase.

Objective To investigate the effect of glycosylation modification on secretion of intein spliced B-domain-deleted FⅧ (BDD-FⅧ). Methods A total of 226 amino acid residues of FⅧ B domain with six potential asparagines-linked glycosylation sites (N6) were incorporated into heavy chain of BDD-FⅧ.By dual-vector co-transfer of heavy and light chain genes with fused intein ( N6HCIntN and IntCLC) into cultured 293 cells, the amounts of spliced BDD-FⅧ protein and coagulation activity in culture supernatant were observed by ELISA and Coatest method respectively. Results The amounts of spliced BDD-F Ⅷ protein and activity were up to (123 ± 18) ng/ml and (0.94 ±0. 11 ) U/ml in supernatant from cells cotransfected with N6HCIntN and IntCLC genes. And they were higher than those of cells co-transfected with intein-fused heavy ( HCIntN ) and light chain (IntCLC) genes [( 86 ± 12) ng/ml and (0. 65 ± 0. 07 )U/ml, both P ＜ 0. 05]. Spliced BDD-FⅧ protein and activity could also be detected in the supernatant from mixed cells individually transfected with N6HCIntN and IntCLC genes [( 18 ±6) ng/ml and (0. 15 ±0. 05) U/ml]. Conclusion It demonstrated that the glycosylation modified heavy chain can improve the secretion of intein spliced BDD-FⅧ and the protein splicing can occur independent of cellular mechanism.

Objective To investigate the expression and location of caveolin-1 (Cav-1), aquaporin 1 ( AQP1 ) and AQP5 in the lung of acute pancreatitis-associated lung injury rats and to determine the role of these molecules in the pathologic progress and the potential therapeutic mechanisms of Qingyitang. Methods Forty Wistar rats were randomly divided into sham operation (SHAM) group, acute lung injury (ALI)group, dexamethasone (DEX) group and Qingyitang (QYT) group. ALI was induced by reverse injection of deoxycholate into biliopancreatic duct of rats. Blood and lung tissues were collected after 24 h. Serum amylase, lung wet/dry (W/D) ratio and pathological section were detected to evaluate the degree of lung injury. reverse transcription-polymerase chain reaction was taken to detect the mRNA levels of Cav-1, AQP1 and AQP5. Lipid rafts were prepared for detection of the distribution and expression level of Cav-1, AQP1 and AQP5 proteins by Western blot. Results The concentration of serum amylase, the value of W/D and the degree of pathological lung injury obviously increased in ALI rats. Cav-1, AQP1 and AQP5 were present in the lung while the mRNA level decreased in ALI rats. Cav-1 appeared mainly in lipid rafts and less in non-lipid rafts. AQP1 was localized to lipid rafts while AQP5 to non-lipid rafts. The localization of these three molecules in the lung of ALI rats did not change compared with SHAM rats while their protein levels decreased. Compared with ALI rats, the concentration of serum amylase, the value of W/D and the degree of pathological lung injury obviously decreased in DEX and QYT rats. The mRNA and the protein expression of Cay-1, AQP1 and AQP5 increased in various degrees by DEX or QYT treatment. Conclusion Cav-1 and AQP1 are enriched in lipid rafts while AQP5 in non-lipid rafts. The down-regulated expression of these three molecules may play important role in acute pancreatitis-associated lung injury. DEX and QYT may relieve lung injury effectively by up-regulating the expressions of Cav-1, AQP1 and AQP5.

Objective To explore whether there is an interaction between Helicobacter pylori (H. pylori), omeprazole and gastrin on the proliferation and apoptosis of gastric epithelial cells. Methods With omeprazole, H. pylori and gastrin as treatment factors, the in vitro changes of gastric epithelial cells AGS and GES-1 were detected by MTS, Hoechst33342 and flow cytometry. Results (1)For a single factor, the gastric mucosal cells ( AGS and GES-1 ) treated by H. pylori or omeprazole show significantly higher apoptosis than that of control group [H. pylori and 104 nmol/L omeprazole group: ( 17.20 ± 0. 90) %,(12. 81 ±0.78)% vs (7.96 ±1.25)%; (4.60 ±0.34)%, (6.60 ±0.76)% vs (3.52 ±0. 16)%; all P ＜ 0. 05]. The gastric mucosal cells treated by H. pylori or omeprazole show significantly lower proliferation than that of control group( H. pylori and 104 nmol/L omeprazole group: 13. 35 ± 0. 55 vs 37.78 ± 1.98,47.62 ± 2. 40 vs 62.44 ± 4. 46; 27. 15 ± 1.64 vs 32. 76 ± 1.57, 29. 42 ± 1.44 vs 48. 86 ± 4. 95; all P ＜0. 05 ). The differences were statistically significant and the effects were correlated with the concentration of omeprazole. Gastrin had no direct effect on cellular proliferation and apoptosis ( all P ＞ 0. 05 ). ( 2 ) For a combinations of factors, the GES-1 cells treated with H. pylori and 500 ng/L gastrin had a significantly higher ratio [(7.25 ±0. 54) % vs (4. 60 ±0. 34) %, P ＜0. 05], while the other groups just showed a rising trend in the proportion of apoptosis cells, but there was no statistical significance. Flow cytometry analysis showed that combinations of H. pylori, omeprazole and gastrin caused lower proliferation than that of H. pylori group,and all changes were statistically significant ( AGS: 12. 68 ±0. 09, 12. 28 ±0. 31 vs 13. 35 ±0. 55; GES-1:22.06 ± 1.61, 18. 59 ± 0. 09 vs 27.15 ± 1.64; all P ＜ 0. 05 ). And combinations of 104 nmol/L omeprazole and 500 ng/L gastrin caused lower proliferation than that of 104 nmol/L omeprazole group(33. 23 ±5.98 vs 47.62 ± 2. 40 and 25.97 ± 0. 74 vs 29. 42 ± 1.44, both P ＜ 0. 05 ). (3) When comparing the apoptosis and proliferation changes of two cell lines, gastric cancer cells AGS had a more marked changes than immortalized cells GES-1. Conclusion Gastrin significantly enhances the effects of omeprazole and H. pylori on gastric epithelial cells. It is speculated that the factors of omeprazole, H. pylori and gastrin may have a synergistic effect of decreased proliferation and increased apoptosis of gastric mucosal cells. Thus a long-term proton pump inhibitor treatment of H. pylori patients may carry a higher risk of atrophic gastritis. But it needs to be confirmed by further experiments.

Objective To study the change of airway inflammation induced by Th1/Tc1 and the expression of CD4 + CD25 + regulatory T cells (Treg) in smoking cessation rats. Methods Fifty healthy male Wistar rats were randomly divided into five groups: 12-week normal control (group A, n = 10), 24-week normal control (group B, n = 10), 12-week smoke exposure (group C, n = 10), 24-week smoke exposure (group D, n = 10) and smoking cessation (group E, n = 10). Groups C, D and E were exposed to cigarettes for 12 weeks. At Week 12, groups A and C were sacrificed. Group D continued smoke exposure and group E had smoking cessation for 12 weeks. At Week 24, groups B, D and E were sacrificed.Pathomorphological changes of small airway were analyzed. The cells in BALF (bronchoalveolar lavage fluid) were collected and analyzed by absolute and differential cell counts. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of IFN-γ, IL-4, IL-8 and TNF-α And flow cytometry was employed to determine the Foxp3 + Treg cell populations and reverse transcription-polymerase chain reaction (RT-PCR) to assay the mRNA expression for Foxp3. Results ( 1 ) Compared with groups A and B, the airway inflammation score of groups C, D and E increased significantly ( all P ＜ 0.01 ). Compared with group C, the airway inflammation score of groups D and E both increased (all P ＜ 0.01 ), especially group D; (2) compared with groups A and B, the levels of IFN-γ, TNF-α and IL-8 in groups C, D and E increased (all P ＜0.01 ) while those of IL-4 decreased. The levels of IFN-γ, TNF-α and IL-8 showed no difference between groups C and E. The levels of IFN-γ, TNF-α and IL-8 were higher in group D than those in groups C and E; (3) the ratio of Foxp3 + Treg cells in BALF was higher in group C(7.4% ±0.8% ),group D ( 7.8% ± 1.7%)and group E(7.0% ± 1.4%)than group A(4.8% ± 1.2%)and group B (4.7% ± 1.2% ) ( all P ＜0.01 ). There were no differences in the ratio of Foxp3 + Treg cells among groups C, D and E (all P ＜ 0.05 ); (4) there was an elevated expression of Foxp3 mRNA in group C (0.22 ±0.02), group D(0.23 ±0.03), group E(0.20 ±0.04) versus group A(0.13 ±0.01 )and group B(0.11 ±0.02)( all P ＜ 0.01 ). But there was no difference in the expression of Foxp3 mRNA among groups C, D and E (all P ＞ 0.05 ). Conclusions Airway inflammation induced by Th1/Tc1 and an elevated expression of Treg cells in BALF are found in smoke exposure rats. Upon smoking cessation, the above-mentioned airway inflammation still persists and the expression of Treg cells in BALF shows no decrease. It suggests that an immune imbalance may be involved in the progression of Th1/Tc1-induced airway inflammation upon smoking cessation.

A 42-year-old male was admitted to the hospital on August 21, 2007, mainly due to anorexia and fatigue for 5 years. The patient had intermittent anorexia and anorexia without obvious trigger 5 years ago. The blood sodium was 135 mmol/L, blood chloride was 94 mmol/L, and blood potassium was 3.8 mmol/L. The liver and kidney function were normal. The MRI of the head showed hydrocephalus and mesencephalic aqueduct obstruction.

Nonsmall cell lung cancer (NSCLC) accounts for about 80% of the total number of lung cancers as the leading cause of tumor death at present. Most of NSCLC are in the middle and advanced stage at the time of diagnosis, so systemic chemotherapy is the main treatment.

heme oxygenase (HO) and endogenous carbon monoxide (CO), as HO-CO system, have many biological effects such as anti-inflammatory mediator damage, anti-oxygen free radical, anti-apoptosis and anti-fibrosis. They play an important role in pathophysiological state of the body, and are one of the main regulators of hepatobiliary system.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive pulmonary disease with unknown cause. The pathological manifestation is common interrogative pneumonia. It usually does not exceed 5 years from the onset of clinical symptoms to end-stage respiratory failure and death. At present, there is no effective therapeutic drug to improve the survival rate and quality of life of IPF.

A 13-year-old male was seen for six months because of multiple rash on his limbs. Six months ago, he appeared with multiple rash on his lower limbs without obvious trigger, without obvious pruritus, desquamation, fear of cold and fever, without fatigue and night sweat, without weight loss, and did not pay attention to treatment. The rash persisted. On May 15, 2010, he came to the dermatology department of our hospital for treatment and biopsy of the skin lesions.

A 63-year-old female was seen on January 15, 2010 due to poor defecation for more than 1 year and aggravation for 1 month. Physical examination: No obvious abnormalities were found in the abdomen. Anal diagnosis: A mass was palpable about 2 cm away from the anus, located outside the left anterior wall of the rectum, and was fixed.

The patient was a 42-year-old male who developed mild abdominal pain and diarrhea (3-6 times a day) 30 days before admission, often accompanied by purulent and bloody stool, and was given general treatment. The abdominal pain was aggravated and severe 1 day before admission, accompanied by nausea, vomiting, fever, oliguria, etc. He was admitted to hospital on December 4, 2008. Physical examination at admission showed acute facial appearance, voice absence in both lungs, clear breathing sound, abdominal distension, abdominal muscle tension, total abdominal tenderness, rebound pain, liver turbidity and intestinal sound disappearance.