Factors affecting resolution of oculomotor nerve palsy following endovascular embolization of posterior communicating artery aneurysms.

PURPOSE: To investigate the effect of endovascular embolization of posterior communicating artery (Pcom) aneurysms on concomitant oculomotor nerve palsy (OMNP) and factors affecting the effect of treatment. MATERIALS AND METHODS: Patients with the Pcom aneurysms concomitant with OMNP were retrospectively enrolled for endovascular treatment of the aneurysms. All patients had the endovascular management. The clinical effect, degree of OMNP, size of the aneurysm, type of treatment, subarachnoid hemorrhage (SAH), and time from onset to treatment were analyzed on the resolution of OMNP. RESULTS: Ninety-six patients with 99 Pcom aneurysms were enrolled and treated endovascularly, with the success rate of 100%. Immediately after endovascular treatment, 75 aneurysms (75.75%) got complete occlusion, and 24 (24.24%) nearly complete occlusion. Followed up for 3-18 (mean 8.52±0.56) months, complete resolution of the OMNP was achieved in 63 patients (65.63%), partial resolution in 21 (21.88%), and non-recovery in the other 12 (12.50%). The degree of OMNP at onset, SAH, and time from onset to treatment were significantly (P<0.05) correlated with the resolution of OMNP. Univariate analysis revealed that younger age of the patient, degree of OMNP at onset, presence of subarachnoid hemorrhage, and time from disease onset to treatment were significantly (P<0.05) associated with the recovery of OMNP. Multivariate analysis revealed that the younger age, degree of OMNP at onset, and time from disease onset to treatment were significantly (P<0.05) associated with the recovery of OMNP. CONCLUSION: Endovascular embolization of Pcom aneurysms concomitant with OMNP can effectively improve the OMNP symptoms, especially for patients with moderate and a shorter history of OMNP. Younger age, degree of oculomotor nerve palsy at onset, and time from onset to treatment may significantly affect recovery of oculomotor nerve palsy.

[Evaluation of the performance of InnowaveDX MTB/RIF for the detection of Mycobacterium tuberculosis complex and rifampicin resistance].

Objective: To evaluate the performance of Mycobacterium tuberculosis and rifampicin resistance mutation detection kit (InnowaveDX MTB/RIF, referred to as "InnowaveDX") in diagnosing tuberculosis and rifampicin resistance using sputum samples. Methods: From June 19, 2020 to May 16, 2022, patients with suspected tuberculosis were prospectively and consecutively enrolled in Hunan Provincial Tuberculosis Prevention and Control Institute, Henan Provincial Hospital of Infectious Diseases and Wuhan Jinyintan Hospital. A total of 1 328 patients with suspected tuberculosis were finally included. According to the inclusion and exclusion criteria, 1 035 pulmonary tuberculosis patients (357 were confirmed tuberculosis cases and 678 were clinically diagnosed tuberculosis cases) and 180 non-tuberculosis patients were finally included. Sputum samples were collected from all patients for routine sputum smear acid-fastness tests, mycobacterial culture and drug susceptibility testing. Moreover, the diagnostic value of Xpert®MTB/RIF (referred to as "Xpert") and InnowaveDXin detecting tuberculosis and rifampicin resistance was evaluated. Clinical diagnosis and culture results of Mycobacterium tuberculosis were used as reference standards to assess tuberculosis diagnosis, and phenotypic drug sensitivity and Xpert were used as reference standards to assess rifampicin resistance. The sensitivity, specificity, positive predictive value and negative predictive value of the two methods for tuberculosis diagnosis and rifampicin resistance were analyzed. The consistency of the two techniques was analyzed usingkappa test. Results: Taking clinical diagnosis as the reference standard, the detection sensitivity of InnowaveDX [58.0% (600/1 035)] was higher than that of Xpert [51.7% (535/1 035)] in 1035 patients with pulmonary tuberculosis, and the difference was statistically significant (P<0.001). In 270 pulmonary tuberculosis patients with culture-positive pulmonary tuberculosis identified as M.tuberculosis-complex, the positive rates of InnowaveDX and Xpert were both high [99.6%(269/270)and 98.2%(265/270), respectively] and there was no statistical difference. In culture-negative patients with pulmonary tuberculosis, the sensitivity of InnowaveDX was 38.8% (198/511), which was higher than that of Xpert (29.4%, 150/511), and the difference was statistically significant (P<0.001). Taking phenotypic drug-susceptibility testing (DST) as reference, the sensitivity of InnowaveDX to rifampicin resistance was 99.0% (95%CI: 94.7%-100.0%) and the specificity was 94.0%(95%CI: 88.5%-97.4%). With Xpert as the reference, the sensitivity and specificity of InnowaveDX were 97.1% (95%CI: 93.4%-99.1%) and 99.7% (95%CI: 98.4%-100.0%), respectively, and the kappa value was 0.97 (P<0.001). Conclusions: InnowaveDX show a high sensitivity for detecting Mycobacterium tuberculosis, especially in pulmonary tuberculosis patients with a clinical diagnosis and negative culture results. It also showed high sensitivity in detecting rifampicin resistance with DST and Xpert as reference respectively. InnowaveDX is an early and accurate diagnostic tool for TB and drug-resistant TB, particularly suitable for application in low- and middle-income countries.

[Clinical value of routine contrast esophagram in the diagnosis of anastomotic leakage for three-incision esophagectomy with cervical anastomosis].

Objective: To examine the clinical value of routine contrast esophagram (RCE) for the diagnosis of anastomotic leakage (AL) after three-incision esophagectomy with cervical anastomosis. Methods: Clinical data of 1 022 patients with esophageal cancer who underwent McKeown three-incision esophagectomy with cervical anastomosis from January 2015 to December 2019 at Department of Minimally Invasive Esophageal Surgery, Tianjin Medical University Cancer Hospital and Institute were analyzed retrospectively. There were 876 males and 146 females, aging(M(IQR)) 48(16) years (range: 36 to 84 years). There were 253 patients (24.8%) with neoadjuvant therapy, and 817 patients (79.9%) with minimally invasive esophagectomy. According to the diagnosis and treatment habits of the attending surgeons, 333 patients were included in the RCE group, and RCE was performed on the 7th day postoperative, while 689 patients were included in the non-RCE group, and RCE was performed when the patients had suspicious symptoms. Taking clinical symptoms, RCE, CT, endoscopy and other methods as reference to the diagnosis of AL, the sensitivity and specificity were used to analyze and evaluate the efficacy of RCE for the diagnosis of AL. The data were compared by U test or χ² test between groups. Results: The incidence rate of AL after three-incision esophagectomy was 7.34% (75/1 022), including 30 cases in the RCE group and 45 cases in the non-RCE group (9.0%(30/333) vs. 6.5%(45/689), χ²=2.027, P=0.155). The diagnostic time of AL was 9(5) days postoperative (range: 4 to 30 days). Among them, 23 cases showed cervical leakages, 50 cases showed intro-thoracic leakages, and 2 cases both cervical and intro-thoracic leakages. The diagnostic time of patients with intro-thoracic leakages was longer than that of cervical leakages (10(4) days vs. 6(3) days, Z=-2.517, P=0.012). Among the 333 patients in the RCE group, 16 cases of RCE indicated leakages including 11 cases of true positive and 5 cases determined to be false positive, while 317 cases indicated no abnormalities including 19 cases developed leakages. The sensitivity and specificity of RCE to detect AL were 36.7%(11/30) and 98.3%(298/333), respectively. The Youden-index was 0.35, and the diagnostic accuracy was 92.8%(309/333). The positive and negative predictive value were 11/16 and 94.0%(298/317), respectively. Conclusions: Routine contrast esophagram after three-incision esophagectomy with cervical anastomosis has low sensitivity and high specificity in the diagnosis of AL. The diagnostic time of AL is the 9th day after surgery. It is necessary to prolong the observation time clinically, and combine RCE with CT, endoscopy and other inspection methods for diagnosis.

Factors affecting resolution of oculomotor nerve palsy following endovascular embolization of posterior communicating artery aneurysms.

PURPOSE: To investigate the effect of endovascular embolization of posterior communicating artery (Pcom) aneurysms on concomitant oculomotor nerve palsy (OMNP) and factors affecting the effect of treatment. MATERIALS AND METHODS: Patients with the Pcom aneurysms concomitant with OMNP were retrospectively enrolled for endovascular treatment of the aneurysms. All patients had the endovascular management. The clinical effect, degree of OMNP, size of the aneurysm, type of treatment, subarachnoid hemorrhage (SAH), and time from onset to treatment were analyzed on the resolution of OMNP. RESULTS: Ninety-six patients with 99 Pcom aneurysms were enrolled and treated endovascularly, with the success rate of 100%. Immediately after endovascular treatment, 75 aneurysms (75.75%) got complete occlusion, and 24 (24.24%) nearly complete occlusion. Followed up for 3-18 (mean 8.52±0.56) months, complete resolution of the OMNP was achieved in 63 patients (65.63%), partial resolution in 21 (21.88%), and non-recovery in the other 12 (12.50%). The degree of OMNP at onset, SAH, and time from onset to treatment were significantly (P<0.05) correlated with the resolution of OMNP. Univariate analysis revealed that younger age of the patient, degree of OMNP at onset, presence of subarachnoid hemorrhage, and time from disease onset to treatment were significantly (P<0.05) associated with the recovery of OMNP. Multivariate analysis revealed that the younger age, degree of OMNP at onset, and time from disease onset to treatment were significantly (P<0.05) associated with the recovery of OMNP. CONCLUSION: Endovascular embolization of Pcom aneurysms concomitant with OMNP can effectively improve the OMNP symptoms, especially for patients with moderate and a shorter history of OMNP. Younger age, degree of oculomotor nerve palsy at onset, and time from onset to treatment may significantly affect recovery of oculomotor nerve palsy.

Potential role of miR-425, miR-155 and miR-33 in Streptococcus pneumoniae pneumonia by using bioinformatics analysis and experimental validation.

Streptococcus pneumoniae (S. pneumoniae) pneumonia is the most common cause of community-acquired pneumonia (CAP). Previous studies have suggested the diagnostic potential of microRNAs (miRNAs) in infectious diseases. In the present study, we aimed to evaluate the potential role of miRNAs in S. pneumoniae pneumonia by using bioinformatics analysis and experimental validation. Gene Expression Omnibus (GEO) datasets including GSE97922 and GSE83615 were analyzed for identifying the differentially expressed miRNAs; the miRNA-target genes network was constructed by using miRNet and the targeted genes were subject to Gene Ontology enrichment, Kyoto Encyclopedia of Genes and Genomes and REACTOME pathway analysis; the miRNA and mRNA expression levels were determined by quantitative real-time PCR; protein concentrations were determined by enzyme-linked immunosorbent assay. Our results showed that miR-425, miR-155 and miR-33 were up-regulated in the serum from CAP patients when compared to healthy controls; whereas there was no significant difference in serum miR-222, miR-149, miR-186 and miR-132 expression levels between healthy controls and CAP patients. In vitro functional studies showed that lipopolysaccharides (LPS) induced the up-regulation of miR-425, miR-155 and miR-33 in RAW264.7 cells, and miR-425, miR-155 and miR-33 inhibition attenuated LPS-induced inflammatory responses in RAW264.7 cells. In conclusion, our results showed that miR-425, miR-155 and miR-33 were up-regulated in the serum from CAP patients by using bioinformatics analysis and experimental validation; furthermore, miR-425, miR-155 and miR-33 inhibition attenuated LPS-induced inflammatory responses in RAW264.7 cells. Nevertheless, our studies are still at the preliminary stages, and the detailed roles of miR-425, miR-155 and miR-33 in S. pneumoniae pneumonia still require further investigation.

[Comparison of the prognostic value of the seventh and eighth edition of The AJCC Esophageal Cancer Staging System for the patients with stage â ¡ and â ¢esophageal squamous cell carcinoma].

Objective: To compare and evaluate the prognostic value of the 7(th) and 8(th) edition of The AJCC Esophageal Cancer Staging System for patients with stage Ⅱ and Ⅲ esophageal squamous cell carcinoma. Methods: The clinical data of 328 esophageal cancer patients who received operation at Department of Esophageal Cancer, Tianjin Tumour Hospital from January 2006 to December 2010 were restrospectively analyzed. There were 63 female and 265 male patients. The mean age was 65 (range: 33 to 87) years. Univariate and multivariate analysis were performed to identify the prognosis factors. Results: The five years overall survival rates among patients with stage Ⅱ and Ⅲ were both significantly different (χ(2)=87.035, 84.730, all P=0.000) according to the 7(th) and 8(th) editions of the TNM staging systems. The five years overall survival rate among patients with stage ⅡB and ⅢA were significantly different (39.6% vs 23.4%, P=0.001) according to the 7(th) edition of the esophageal cancer staging systems.According to the 8(th) edition of the esophageal cancer staging system, the 5 years survival rate of patients with stage ⅡA and ⅡB, ⅢB and Ⅳ was statistically significant (58.5% vs. 35.5%, P=0.040; 18.9% vs. 0, P=0.000). In multivariate analysis, tumor size, T staging, N staging and tumor differentiation (HR=1.592, 95%CI: 1.185 to 2.139, P=0.002; HR=1.519, 95% CI: 1.236 to 1.867, P=0.000; HR=1.647, 95% CI: 1.448 to 1.874, P=0.000; HR=1.404, 95% CI: 1.059 to 1.861, P=0.018) were the main independent prognosis factors affecting the prognosis of esophageal squamous cell carcinoma patients. Conclusions: Both the 7(th) and the 8(th) editions of TNM staging systems are able to reflect the clinical prognosis of patients receiving radical resection of esophageal cancer, and the factors of tumor size, differentiaton, invasion depth and lymph node metastases are the independent predictors of prognosis. The 8(th) edition provides a more detailed and more reasonable for the staging of stage Ⅱ and Ⅲ for esophageal cancer patients than the 7(th) edition, and it is more accurate for the prognosis of patients with esophageal cancer after surgery.

Clinical significance of preoperative and postoperative cytokeratin 19 messenger RNA level in peripheral blood of esophageal cancer patients.

The purpose of this study is to analyze the correlation between preoperative/postoperative Cytokeratin 19 (CK19) messenger RNA (mRNA) level in peripheral blood (PB) and the clinical significance in esophageal cancer patients with different clinicopathological factors. We detected the preoperative and postoperative CK19 mRNA level in the PB of 139 esophageal cancer patients who underwent complete resection and evaluated its clinical significance. We found that both the preoperative and postoperative CK19 mRNA level increased in the esophageal cancer patients with lymph node metastasis, relapse or distant metastasis compared with that in cancers without lymph node metastasis, relapse or distant metastasis. High postoperative CK19 mRNA levels indicate a short disease-free survival (DFS) for the whole cohort esophageal cancer patients, whereas the high preoperative CK19 mRNA levels only indicate a short DFS for the esophageal cancer patients with squamous cell carcinoma, TNM III stage, and lymph node metastasis. The dynamic change of CK19 mRNA levels could indicate the prognosis of esophageal cancer patients. The patients with decreasing CK19 mRNA level after surgery had good prognosis, and the patients with changeless CK19 mRNA level had poor prognosis. Taken together, CK19 mRNA levels could be a promising marker in assessing prognosis or assigning treatment for the esophageal cancer patients according to different clinicopathological factors.

B cells facilitate platelet production mediated by cytokines in patients with essential thrombocythaemia.

We investigated the role of activated B cells in thrombopoiesis through the production of interleukin (IL)-1beta and IL-6 in patients with essential thrombocythaemia. The number of B cells did not differ between essential thrombocythaemia patients, irrespective of the presence of Janus activated kinase-2 V617F mutation or wild type, and age-matched healthy adults. However, the number of IL-1beta/IL-6-producing B cells was significantly higher in essential thrombocythaemia patients than that in healthy controls. The relatively high level of IL-1beta/IL-6 production by B cells was associated with serum B cell-activating factor and expression of Toll-like receptor 4 on B cells. A high level of B cell-activating factor was present in essential thrombocythaemia patients with both Janus activated kinase-2 genotypes. Incubation with B cell-activating factor enhanced the expression of Toll-like receptor 4 on B cells. IL-1beta and IL-6 production was not stimulated by B cell-activating factor alone; Toll-like receptor 4 was activated by lipopolysaccharide or patients' sera to produce IL-1beta and IL-6 in B cells. Moreover, essential thrombocythaemia patient B cells facilitated megakaryocyte differentiation when co-cultured with CD34+ haematopoietic stem cells. Antibody neutralisation of IL-1beta and IL-6 attenuated megakaryocyte differentiation. These data suggest that B cells play a crucial role in thrombopoiesis in essential thrombocythaemia patients.

Primary esophageal adenosquamous carcinoma: a retrospective analysis of 24 cases.

Primary adenosquamous carcinoma (ASC) of the esophagus is a rare kind of malignancy characterized by mixed glandular and squamous differentiation as well as a propensity for aggressive clinical behavior. Data on the evaluation of the clinicopathological features and the prognosis of patients suffering from this malignancy are few because of the rarity of this disease. We conducted a retrospective review of 24 patients with primary esophageal ASC among 6546 esophageal cancer patients who underwent transthoracic esophagectomy in our hospital. The clinicopathological presentation, diagnosis, treatment, and prognostic factors of the patients were respectively investigated. The Kaplan-Meier method and the log rank test were used to calculate and compare overall survival (OS). The Cox proportional hazards model was employed to identify independent prognostic factors. There were 18 males and 6 females with a median age of 60 years (range: 40-78 years). The clinical symptoms, macroscopic type, as well as the radiological and endoscopic features of esophageal ASC were similar to those of esophageal squamous cell carcinoma. Sixteen (88.9%) of the 18 cases who underwent preoperative esophagoscopic biopsy were misdiagnosed as adenocarcinoma or squamous cell carcinoma. The overall median follow-up period was 36 months, and the median survival time was 32 months. The 1, 3, 5-year OS rates were 75.0%, 48.5%, and 19.4%, respectively. Univariate analysis showed that gender (P=0.047), lymph node metastasis (P=0.007), and TNM stage (P=0.037) were important factors associated with OS of the 22 patients who underwent radical resection. Multivariate analysis showed that the pathological N stage was the only independent prognostic factor (P=0.031, hazard ratio [HR], 5.369, 95% confidence interval [CI], 1.167-24.700). These results suggest that esophageal ASC is an uncommon disease prone to be misdiagnosed by endoscopic biopsy. Surgical resection is the primary treatment, but the prognosis of ASC is usually poorer than conventional squamous cell carcinoma. Lymph node metastasis is an independent prognostic factor after radical resection.

5-aminoimidazole-4-carboxamide ribonucleoside and AMP-activated protein kinase inhibit signalling through NF-κB.

Activation of nuclear factor-kappa B (NF-κB) is one of the most important pro-inflammatory mechanisms in disease. In this study, we show that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an intermediate in nucleoside metabolism, inhibits signalling by NF-κB in three cell types, including bovine aortic endothelial cells (BAEC). The block in the NF-κB signalling pathway occurred beyond degradation of IκB-α and movement of p65 into the nucleus of BAEC. There was, however, reduced binding of NF-κB from AICAR-treated cells to a κB-consensus oligonucleotide, suggesting that part of the mechanism was a reduction in NF-κB DNA-binding activity. Although AICAR is metabolized to ZMP and then adenosine, adenosine had no effect on activation of an NF-κB reporter. ZMP, however, activates the metabolic stress-sensing AMP-activated protein kinase (AMPK). Transfection of active AMPK into BAEC reduced NF-κB reporter activity compared with a kinase-dead mutant, suggesting that part of the ability of AICAR to inhibit NF-κB signalling is due to activation of AMPK. Inhibition of NF-κB signalling may be important in the anti-inflammatory action of drugs such as sulfasalazine and methotrexate, which led to the accumulation of AICAR within target cells.

Detection of cytomegalovirus reactivation in cancer patients receiving chemotherapy.

While increasing numbers of cytomegalovirus (CMV)-associated diseases are occurring in patients undergoing conventional chemotherapy, information regarding CMV reactivation is limited. This pilot study was conducted to investigate CMV reactivation induced by chemotherapy. Seven blood samples were collected from each of 15 patients with newly diagnosed malignant disease, at baseline before chemotherapy, and once every month after chemotherapy was commenced. CMV viral loads in leukocytes were determined by real-time PCR. Host responses to changes in viral loads were assessed by assaying CMV-specific IgG titres and tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma levels in each of the blood samples, and by scoring the number of CMV-associated clinical symptoms that developed. All except one patient experienced CMV reactivation during the course of chemotherapy, with the average viral load peaking after the third course of treatment. Titres of CMV-specific IgG increased in line with the increase in viral load. Plasma levels of TNF-alpha and IFN-gamma initially decreased from baseline, and then rose to peak levels at the same time as, or shortly after, the highest viral loads were recorded. Clinical symptoms potentially attributable to CMV infection appeared as the viral load increased. It was concluded that the incidence of CMV reactivation in patients receiving conventional chemotherapy is high. Reactivation is not asymptomatic, but was self-limiting in most of these cases. Increases in plasma TNF-alpha and IFN-gamma occur after reactivation, but not before.

Non-lethal Candida albicans cph1/cph1 efg1/efg1 transcription factor mutant establishing restricted zone of infection in a mouse model of systemic infection.

The cph1/cph1 efg1/efg1 Candida albicans mutant cells were non-lethal in a mouse model of systemic infection. We investigated in vivo proliferation and invasion of C. albicans cells in infected mice to elucidate the interaction between the host and the pathogen. Homogenates of kidneys from the mice infected with the wild-type and the mutant C. albicans cells yielded a mean of 2.1 x 10 7 CFU/g and 2.2 x 10 6 CFU/g, respectively. The kidneys from the mice infected with the wild-type cells showed extensive renal cortical necrosis associated with neutrophilic infiltration. There were also wild-type hyphal cells present in abundance. Hence, tubular necrosis leading to renal failure in the mice may be the cause of death. Although the cph1/cph1 efg1/efg1 mutant cells were not lethal, they were capable of establishing restricted zones of infection and colonization near the renal pelvis instead of simply being cleared by the immune system in mice.

Sublytic complement injury does not activate NF-kappa B, or induce mitogenesis in rat mesangial cells.

Sublytic complement injury to glomerular mesangial cells, mediated by the terminal membrane attack complex of complement (C5b-9), is a potential initiating mechanism in IgA nephropathy. Sublytic complement injury has been reported to result in the production of a variety of pro-inflammatory molecules and growth factors, including many regulated by the transcription factor NF-kappa B. To determine the importance of complement injury in the pro-inflammatory signalling which occurs in IgA nephropathy, we investigated NF-kappa B activation following sublytic complement injury to cultured rat glomerular mesangial cells (RMCs). A sublytic dose of rabbit anti-Thy 1.1 (THY) serum and normal human serum was selected based upon flow cytometry, chromium-release assay, and induction of superoxide production. No significant C5b-9-induced NF-kappa B activation was detected by electrophoretic mobility shift assays, luciferase activity of RMCs transfected with a NF-kappa B-driven luciferase reporter construct, nor by Northern blots for the NF-kappa B-responsive mRNA species monocyte chemoattractant protein-1 or I kappa B alpha. Furthermore, measurements of (3)H incorporation following sublytic complement injury showed inhibition of mesangial cell mitogenesis in comparison to the heat-inactivated serum treatment and to THY alone. The results of this study suggest that sublytic complement injury to RMC does not directly activate NF-kappa B nor induce mesangial cell proliferation in mesangial cells. Other mechanisms such as IgA immune complex formation must be required to produce these events in IgA nephropathy.

Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons.

BACKGROUND: The genetic modification of pigs is a powerful strategy that may ultimately enable successful xenotransplantation of porcine organs into humans. METHODS: Transgenic pigs were produced by microinjection of gene constructs for human complement regulatory proteins CD55 and CD59 and the enzyme alpha1,2-fucosyltransferase (H-transferase, HT), which reduces expression of the major xenoepitope galactose-alpha1,3-galactose (alphaGal). Kidneys from CD55/HT and CD55/CD59/HT transgenic pigs were transplanted into nephrectomised, nonimmunosuppressed adult baboons. RESULTS: In several lines of transgenic pigs, CD55 and CD59 were expressed strongly in all tissues examined, whereas HT expression was relatively weak and did not significantly reduce alphaGal. Control nontransgenic kidneys (n=4) grafted into baboons were hyperacutely rejected within 1 hr. In contrast, kidneys from CD55/HT pigs (n=2) were rejected after 30 hr, although kidneys from CD55/CD59/HT pigs (n=6) maintained function for up to 5 days. In the latter grafts, infiltration by macrophages, T cells, and B cells was observed at days 3 and 5 posttransplantation. The recipients developed thrombocytopenia and abnormalities in coagulation, manifested in increased clotting times and an elevation in the plasma level of the fibrin degradation product D-dimer, within 2 days of transplantation. Treatment with low molecular weight heparin prevented profound thrombocytopenia but not the other aspects of coagulopathy. CONCLUSIONS: Strong expression of CD55 and CD59 completely protected porcine kidneys from hyperacute rejection and allowed a detailed analysis of xenograft rejection in the absence of immunosuppression. Coagulopathy appears to be a common feature of pig-to-baboon renal transplantation and represents yet another major barrier to its clinical application.

Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.

Anti-xenograft immune responses in alpha 1,3-galactosyltransferase knock-out mice.

Although originally generated to test the effect of eliminating the alpha-Gal epitope on HAR, it is becoming increasingly clear that GalT KO mice offer a convenient and inexpensive model to investigate many aspects of the anti-xenorgraft immune response. Clearly, not all aspects of anti-xenograft rejection responses are identical in mice and primates, which should be kept in mind when interpreting results of GalT KO mouse studies. However, with this and other mouse models it is possible to test a large number of variables, which is impractical for both logistical and financial reasons with primates. Furthermore the short gestation time and large litter size of mice means that genetic strategies targeting different aspects of the anti-xenograft immune response can be combined and subsequently tested to identify the optimal combination of genetic and therapeutic approaches to achieve long term xenograft survival. In this regard the GalT KO mouse has been and will continue to be a valuable small animal model for the study of all facets of xenograft rejection involving anti-Gal antibodies.

The intracellular parasite Theileria parva protects infected T cells from apoptosis.

Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells.

Regulation of nuclear factor kappaB by corticosteroids in rat mesangial cells.

Nuclear factor kappaB (NF-kappaB) is one of the most important proinflammatory transcription factors. The anti-inflammatory activity of steroids in leukocytes is partly due to inhibition of signaling by NF-kappaB, but it is not known whether steroids inhibit NF-kappaB in kidney cells. Since the mesangial cell is important in several kidney diseases, especially mesangial proliferative glomerulonephritis, the aims of this study were: (1) to define the mechanism of NF-kappaB activation in rat glomerular mesangial cells; and (2) to determine whether steroids inhibit activation of NF-kappaB in these cells. Electrophoretic mobility shift assays (EMSA) showed that interleukin-1beta and tumor necrosis factor-alpha activated NF-kappaB from 15 min to 48 h after stimulation. Supershift EMSA demonstrated that p65 and p50 were the predominant subunits involved. Degradation of the inhibitory subunit IkappaB-alpha was first observed 15 min after stimulation by Western blot, was maximal at 15 to 30 min (>90% by densitometry), and had returned to near normal levels at 90 min. In contrast, IkappaB-beta was maximally degraded at 60 to 120 min and was still reduced at 48 h (<50% of the untreated level). Although treatment of mesangial cells with dexamethasone increased IkappaB-alpha mRNA by 1.92x and protein by 1.45x over controls, pretreatment did not inhibit degradation of IkappaB-alpha or -beta in response to stimulation, or prevent the increase in NF-kappaB binding activity shown by EMSA. However, dexamethasone significantly inhibited the increase in monocyte chemoattractant protein-1 mRNA seen after stimulation with interleukin 1beta, although this was not complete. It did not reduce transcription of an NF-kappaB reporter. In comparison, the pyrrolidine derivative of dithiocarnamate (PDTC), a known inhibitor of NF-kappaB, prevented the increase in NF-kappaB binding activity and significantly reduced transcription of the NF-kappaB reporter. These studies suggest that steroids can partially inhibit transcriptional activation by NF-kappaB in mesangial cells but not through an increase in IkappaB-alpha protein alone. Their effect must occur at the promoter and may be restricted to some NF-kappaB-responsive genes. Therapies that block NF-kappaB more effectively than steroids in mesangial cells, therefore, may be useful in the treatment of mesangial proliferative glomerulonephritis.

Knock out of alpha1,3-galactosyltransferase or expression of alpha1,2-fucosyltransferase further protects CD55- and CD59-expressing mouse hearts in an ex vivo model of xenograft rejection.

BACKGROUND: Organs from transgenic animals with high-level endothelial expression of the human complement regulatory factors CD55 and CD59 are significantly protected from human complement-mediated injury. Elimination or reduction of the major xenoepitope alphaGal, achieved by knocking out the alpha1,3-galactosyltransferase gene (Gal KO) or expressing human alpha1,2-fucosyltransferase (H transferase or HTF), also affords protection, although to a lesser degree. In this study, we examined whether the protection provided by strong CD55 and CD59 expression can be augmented by the Gal KO or HTF modifications. METHODS: Hearts from four groups of mice (wild type, CD55/CD59, CD55/CD59/HTF, and CD55/CD59/Gal KO) were perfused ex vivo with 40% human plasma. Mean heart work for each group was compared over a 60-min period. RESULTS: Wild-type hearts ceased to function effectively within 15 min of plasma addition. CD55/CD59 hearts displayed prolonged survival and maintained approximately 10% maximum work at the end of perfusion. Introduction of Gal KO or HTF onto the CD55/CD59 background resulted in a further prolongation, with work maintained at 20-30% of the maximum level. CONCLUSIONS: We used an ex vivo model to demonstrate that eliminating alphaGal expression further prolongs the function of mouse hearts expressing high levels of CD55 and CD59. In addition, we showed that reducing alphaGal by expressing HTF is equally as effective in prolonging CD55/CD59 heart function as knocking out Gal transferase, thus providing a feasible strategy for translating these advances to the pig.

Molecular characterization of Gluconobacter oxydans recA gene and its inhibitory effect on the function of the host wild-type recA gene.

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.