[Clinical characteristics of patients with multiple myeloma harboring 6q deletion].

Objective: To study the clinical and cytogenetic characteristics of patients with multiple myeloma harboring 6q deletion, with the aim to determine the impact of 6q deletion on survival. Methods: This study included the retrospective analysis of 382 newly diagnosed patients with multiple myeloma in our hospital from 2014 to 2017 and compared the clinical and cytogenetic characteristics between patients with and without 6q deletion. The log-rank test and the Cox proportional hazards regression model were used to analyze prognostic factors for progression-free survival (PFS) and overall survival (OS) . Results: Compared to those without 6q, the patients with 6q deletion were older (median age, 63 vs 58 years, P=0.039) , had higher incidence of t (4; 14) (30.4% vs 16.4% , P=0.020) , and higher proportion of complex karyotypes (22.2% vs 5.3% , P=0.001) . Univariate survival analysis using the log-rank test revealed that 6q deletion was associated with shorter PFS. However, by the Cox multivariate proportional hazards regression model, 6q deletion was not an independent prognostic factor and its effect on survival was affected by age, t (4; 14) , and other risk factors. Conclusions: 6q deletion was common in elderly patients with multiple myeloma and was often accompanied by t (4;14) and complex karyotypes. However, 6q deletion was not an independent prognostic factor for multiple myeloma.

MiRNA-485-5p, inhibits esophageal cancer cells proliferation and invasion by down-regulating O-linked N-acetylglucosamine transferase.

OBJECTIVE: Previous reports suggest that miRNA-485-5p is dysregulated and contributes to tumorigenesis in some cancer types. Nevertheless, the biological role of miRNA-485-5p in esophageal cancer (EC) is not well understood. Additionally, we found that the expression of miR-485-5p in EC tissues was aberrant. PATIENTS AND METHODS: Quantitative RT-PCR (qRT-PCR) was used to demonstrate the expression of miRNA-485-5p in EC cell lines. Cell counting kit-8 (CCK-8) assay and transwell assay indicated that miRNA-485-5p overexpression inhibited cell proliferation, migration, and invasion in EC cell lines. Additionally, Western blotting, dual-luciferase reporter assay, and rescue assay predicted that O-linked N-acetylglucosamine transferase (OGT) was a direct target of miRNA-485-5p. Moreover, we showed that miRNA-485-5p regulated EC tumorigenesis by down-regulating OGT expression in vitro and in vivo. RESULTS: The upregulation of miR-485-5p (fold change = 44 and 26 in ECA109 and TE-1, respectively; p<0.001) was showed by qRT-PCR. Compared with the control groups, the expression miR-485-5p significantly suppressed the proliferation, migration, and invasion of EC cells. The bioinformatic analysis predicted that the 3' untranslated region (UTR) of OGT contains one miR-485-5p target sequences. Western blotting and dual-luciferase reporter assay showed that activation of OGT 3'UTR was increased by co-transfection with miR-485-5p. Finally, CCK-8 assay predicted that the rescue effects of OGT expression on miR-485-5p induced inhibition of cell growth and tumor weight in Eca109 and TE1 cells. CONCLUSIONS: Our results suggest that miRNA-485-5p is a suppressor of EC tumorigenesis and could serve as a novel candidate for therapeutic applications in EC treatment.

[The coping styles and its relationship with quality of life among 1 968 female breast cancer patients in Shanghai in 2014].

Objective: To study the coping styles and its relationship with quality of life among part of the female breast cancer patients in Shanghai in 2014. Methods: In August of 2014, 1-3 block groups affiliated to Shanghai Cancer Rehabilitation Club were selected from each of the 17 districts of Shanghai by convenient sampling method. Respondents were recruited from these block groups via putting up posters in the community or top three hospitals nearby. The inclusion criteria were as follows: female, aged from 18 to 80 years old; the initial diagnosis or primary tumor was breast cancer, and active treatments including surgery, radiotherapy and chemotherapy were finished; capable of basic reading and comprehension, and there was no communication disorder; daily activities were not limited, and with no mental disorder or dysgnosia. Totally 2 205 respondents were included. Questionnaire survey was conducted to collect the information of demographic characteristics, disease characteristics, result of European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30), and result of the Ways of Coping Inventory-Cancer Version (WOC-CA). 1 968 valid questionnaires were withdrawn. The results of respondent characteristics and WOC-CA were under descriptive analysis, and the differences of coping styles among groups of different demographic characteristics were compared using t test. In addition, we analyzed the correlation between respondents' coping styles and quality of life using partial correlation analysis. Results: The average age of the 1 968 respondents was (58.7±7.4) years old, and BMI was (24.4±5.4) kg/m(2). The score of positive coping styles was 2.04±0.41, and the score of negative coping styles was 1.79±0.33. The scores of Physical Functioning (PF), Role Functioning (RF), Cognitive Functioning (CF), Emotional Functioning (EF), Social Functioning (SF), and Global Health (GH) were 83.40±12.18, 90.80±15.92, 80.72±16.29, 84.67±15.19, 80.99±20.91, 65.27±21.21, respectively. Positive coping styles correlated with PF, EF, and GH positively, and the correlation coefficients were separately 0.581 (P=0.046), 0.593 (P=0.045), 0.770 (P<0.001). Negative coping styles correlated with RF, CF, EF, and SF negatively, and the correlation coefficients were separately-0.672 (P=0.021),-0.815 (P=0.005),-0.121 (P<0.001),-0.123 (P<0.001). Conclusion: Part of the female breast cancer patients in Shanghai in 2014 mainly adopted positive coping styles, and in general the positive coping styles correlated with quality of life positively and negative coping styles correlated with quality of life negatively.

The effect of percutaneous endoscopic lumbar discectomy under different anesthesia on pain and immunity of patients with prolapse of lumbar intervertebral disc.

OBJECTIVE: To explore the effect of percutaneous transforaminal endoscopic discectomy under different anesthesia on pain and immunity of patients with lumbar disc herniation. PATIENTS AND METHODS: 92 cases of patients with lumbar disc herniation in the Affiliated Hospital of Qingdao University from February 2015 to January 2016 were collected. These patients were randomly divided into control group and observation group (n = 46). Patients in the control group underwent percutaneous transforaminal endoscopic discectomy with the use of local anesthesia, while patients in the observation group used continuous epidural anesthesia. Oswestry Disability Index (ODI) and Visual Analogue Scale of Pain (VAS) were used to compare the surgical effect and the degree of pain of patients in the two groups. Adverse reactions (nausea, vomiting, dizziness, drowsiness) of patients in two groups were compared. T lymphocytes subset level (CD4+, CD8+) and inflammatory cytokines (IL-2, TNF) in the immune system were compared on the 1st, 3rd, and 10th day post-operatively. RESULTS: The pain degree of patients in the two groups had no significant difference before their operations (p > 0.05). The intraoperative pain rate of patients in the observation group was significantly lower than the control group (p < 0.05). Patients in both groups achieved a remarkable decrease of pain intensity on month 1 and month 3 post-operatively (p < 0.05). There is no significant difference between the two groups (p > 0.05). ODI scores of patients in the two groups had no significant difference pre-operatively (p > 0.05). Patients in both groups achieved a remarkable decrease of ODI scores after surgery (p < 0.05), and there is no significant difference between the two groups (p > 0.05). The occurrence of adverse reactions in the observation group was significantly lower than the control group (p < 0.05). On day 1 and 3 post-operatively, CD4+ and CD8+ levels of patients in both groups were lower than before operation, and data in the control group decreased more than the observation group (p < 0.05). IL-2 and TNF-α levels of patients in the two groups were significantly higher than pre-operatively, and data in the control group was higher than the observation group (p < 0.05). On day 10 post-operatively, all the indexes returned to the preoperative level. CONCLUSIONS: Both continuous epidural anesthesia and local anesthesia can reduce or avoid perioperative pain, but continuous epidural anesthesia has more advantages than local anesthesia, and it can improve the immune function for patients undergoing PTED for LDH.

Identification of a novel cosavirus species in faeces of children and its relationship with acute gastroenteritis in China.

OBJECTIVES: To assess the prevalence of human cosavirus (HCosV) in China and to determine the association of a novel HCosV (Cosa-CHN) with acute gastroenteritis (AGE). METHODS: A case-control study with 461 paired stool samples from diarrhoea and healthy children was conducted. Real-time PCR and nested PCR were used to detect the HCosVs. Rapid amplification of cDNA ends was used to obtain the ends of the Cosa-CHN. RESULTS: Known HCosVs were detected in two control samples, while Cosa-CHN was detected in eight (1.7%) and six (1.3%) of the case and control samples respectively. The complete genome of Cosa-CHN comprises 7213 bp. The P1 and P2 regions of the Cosa-CHN were closely related to those of HCosV B, while the P3 region was most similar to that of HCosV D, albeit with low amino acid identities (66 and 67% respectively). Phylogenetic analyses of the polyprotein and partial VP3/VP1 regions indicated that Cosa-CHN could be classified as a novel species (tentatively named HCosV G) in cosavirus. There was no significant difference in detection rate (p 0.59) or mean virus load (p 0.43) of Cosa-CHN between the cases and controls. Statistical analysis revealed no association between Cosa-CHN and AGE (p 0.76), and the virus did not exacerbate clinical symptoms. CONCLUSIONS: A low prevalence of HCosV was detected, but a novel Cosavirus species was found in children with and without gastroenteritis in this study. The evidence did not support a causative role for the novel virus in paediatric AGE.

[Effects and its mechanism of Nimotuzumab on radiosensitivity of esophageal carcinoma ECA-109 and TE-13 cell lines].

Objective: To investigate the effects of nimotuzumab on radiosensitivity of ECA-109 and TE-13 esophageal carcinoma cell lines and explore its possible mechanism. Methods: The ECA-109 and TE-13 cells were divided into control group, irradiation group, medicine group, and combined group (irradiation + medicine). In the combined group, ECA-109 and TE-13 cells were treated with nimotuzumab for 24 h before irradiation, and the cells were collected 2 h after irradiation. The radiosensitizing effects of nimotuzumab on ECA-109 and TE-13 cells were evaluated by clone formation assay. Cell apoptosis was detected by flow cytometry. Western blotting was used to evaluate the expression of EGFR, p-EGFR, DNA-PKcs, p-DNA-PKcs and γH2AX. Results: The values of Dq (quasithreshold dose), D0(mean lethal dose)and SF2 (surviving fraction at 2 Gy) of ECA-109 and TE-13 cells in the combined group were significantly lower than those of the radiation group (for ECA-109 cells, 1.11 vs. 1.72, 1.40 vs. 2.14, 0.42 vs. 0.66, respectively; for TE-13 cells, 0.41 vs. 0.46, 0.43 vs. 0.65, 0.40 vs. 0.71, respectively (all P<0.05). The sensitivity enhancement ratio (SER) of ECA-109 and TE-13 cells were 1.35 and 1.43, respectively. Flow cytometry showed that the apoptosis rate of ECA-109 and TE-13 cells in the combined group were significantly higher than those of the radiation group [for ECA-109 cells, (41.31±1.52)% vs. (9.54±0.52)%; for TE-13 cells, (46.28±0.28)% vs. (11.32±0.31)%, both P<0.01]. Western blotting showed that the expression levels of EGFR and DNA-PKcs were not significantly different in all groups (all P>0.05). Compared with those of the control group, p-EGFR and p-DNA-PKcs of the radiation group were significantly higher in both cell lines (P<0.05), and the γH2AX levels in the radiation group and medicine group were significantly higher than that of the control group (P<0.05). Compared with those of the radiation group and medicine group, p-EGFR and p-DNA-PKcs protein expression in the combined group were decreased significantly (P<0.05), while γH2AX protein expression was significantly increased (P<0.05). Conclusions: Nimotuzumab can enhance the radiosensitivity of esophageal cancer ECA-109 and TE-13 cells. The potential mechanism may be related to the inhibition of EGFR phosphorylation and down-regulation of DNA damage repair proteins. The radiosensitizing effect of nimotuzumab is greater on poorly differentiated esophageal cancer cells.

Aetiology of diarrhoeal disease and evaluation of viral-bacterial coinfection in children under 5 years old in China: a matched case-control study.

Globally, diarrhoeal diseases are the second leading cause of death among children under 5 years old. Few case-control studies on the aetiology of diarrhoea have been conducted in China. A case-control study on 922 children under 5 years old who presented with diarrhoea and individually matched controls was conducted in China between May 2011 and January 2013. Quantitative PCR was used to analyze stool samples for 10 diarrhoeal pathogens. Potential enteric pathogens were detected in 377 (81.8%) of 461 children with diarrhoea and 215 controls (46.6%, p <0.001). Rotavirus, norovirus GII, Shigella and adenovirus were qualitatively associated with diarrhoea. Using receiver operating characteristic curves, the optimal cutoff threshold for defining a symptomatic individual was 72, 5840, and 10(4) copies per reaction for rotavirus (odds ratio 259), norovirus GII (odds ratio 10.6) and Shigella (odds ratio 5.1). The attributable fractions were 0.18 for rotavirus, 0.08 for norovirus GII, 0.01 for Shigella and 0.04 for adenovirus. Coinfections between pathogens were common. Two pairs, rotavirus and adenovirus, and norovirus GII and Salmonella were positively associated. The co-occurrence of rotavirus and sapovirus, astrovirus, enterotoxigenic Escherichia coli or Campylobacter jejuni only occurred in children with disease. Coinfection was not correlated with clinical symptoms. Quantitative data are critical. Our results indicate that increased pathogen loads increase the OR between diarrhoea and rotavirus, norovirus GII and Shigella. Coinfections with rotavirus and norovirus GII are common and occur in a nonrandom distribution. Despite testing for ten diarrhoeal pathogens, over two-thirds of cases do not have a recognized attributable cause.

Do physical exercise and reading reduce the risk of Parkinson's disease? a cross-sectional study on factors associated with Parkinson's disease in elderly Chinese veterans.

BACKGROUND: The purpose of this study was to investigate risk factors for and factors protecting against Parkinson's disease (PD) in elderly Chinese veterans. METHODS: Using a database containing detailed information on the health status of the nervous system in elderly Chinese veterans, univariate and multivariate analyses of factors that may be associated with PD were performed. Univariate analysis of qualitative data was done using the Pearson Chi-square and Fisher's exact tests, and the Mann-Whitney U nonparametric test was used for univariate analysis of quantitative data. Multivariate logistic regression analysis was used to identify independent risk factors for and factors protecting against PD in elderly Chinese veterans. RESULTS: A total of 9,676 elderly Chinese veterans were enrolled, including 228 cases with PD and 183 cases with Parkinson's syndrome, with 9,265 non-PD subjects serving as controls. Age (odds ratio [OR] 1.343, 95% confidence interval [CI] 1.028-1.755) and medical history of essential tremor (OR 1.228, 95% CI 1.081-1.396) were identified as independent risk factors for PD, with age being the most important risk factor. Physical exercise (OR 0.478, 95% CI 0.355-0.643) and reading (OR 0.513, 95% CI 0.357-0.735) were identified as independent factors protecting against PD, and physical exercise showed better protection against PD relative to reading. Smoking, alcohol use, anemia, cerebral trauma, education level, and electromagnetic field exposure showed no association with PD. CONCLUSION: Physical exercise and reading may be independent factors that protect against PD among elderly Chinese veterans, while advancing age and medical history of essential tremor may be independent risk factors for PD. This study was cross-sectional, so further research is needed to confirm its results.

Association between p53 codon 72 polymorphisms and clinical outcome of nasopharyngeal carcinoma.

We conducted a cohort study to investigate whether polymorphisms in p53 at codon 72 are associated with tumor response and survival time of advanced nasopharyngeal carcinoma (NPC) patients treated with radiotherapy. The study population included 127 subjects with NPC who were enrolled at Binzhou Medical University between September 2008 and December 2009. Cox proportional hazard regression was used to assess the association between polymorphisms in the p53 gene and progression-free survival (PFS) and overall survival (OS) of NPC patients. During the follow-up period, 42 patients died and 72 patients showed progression at the end of the study. Of the 127 patients, median PFS was 22.5 ± 1.2 months (1-36 months), and the median OS time was 28.2 ± 1.1 months (2-36 months). The p53 codon 72 Pro/Pro genotype was associated with a longer median PFS time of 30.3 months compared with 18.2 months for patients with Arg/Arg variants. Moreover, the p53 codon 72 Pro/ Pro genotype was associated with a longer median OS time of 31.6 months compared with 25.8 months for those with Arg/Arg variants; the P value was marginally significant. We showed that variants in p53 codon 72 may be an independent predictor for PFS and OS of NPC patients.

Accelerated hyperfractionation field-involved re-irradiation combined with concurrent capecitabine chemotherapy for locally recurrent and irresectable rectal cancer.

OBJECTIVES: To evaluate the efficacy and treatment-related toxicity of accelerated hyperfractionation field-involved re-irradiation combined with concurrent capecitabine chemotherapy for locally recurrent and irresectable rectal cancer (LRIRC). METHODS: 72 patients with LRIRC who underwent the treatment were studied. Three-dimensional conformal accelerated hyperfractionation radiotherapy (3D-CAHRT) was performed and the dose was delivered with a schedule of 1.2 Gy twice daily, with an interval of at least 6 h between fractions, 5 days a week. Concurrent capecitabine chemotherapy was administered twice daily. After 36 Gy in 30 fractions over 3 weeks, patients were evaluated to define the resectability of the disease. If resection was not feasible irradiation was resumed until the total dose administered to the tumour reached 51.6-56.4 Gy. RESULTS: Two patients temporarily interrupted concurrent chemoradiation because of Grade IV diarrhoea. The remaining 70 patients completed the planned concurrent chemoradiation. In all patients, the complete response rate was 8.3% and the partial response rate was 51.4%. The overall response rate was 59.7% and clinical benefit rate was 93.1%. Symptomatic responses proved to be obvious and tumour resection was performed in 18 patients. The overall median survival time and median progression-free survival time were 32 and 17 months, respectively. 3 year overall survival and progression-free survival were 45.12% and 31.19%, respectively. Severely acute toxicities included Grade III-IV diarrhoea and granulocytopenia with 9.7% and 8.3% incidence respectively. Small bowel obstruction was severely late toxicity, and the incidence was 1.4%. CONCLUSION: Three-dimensional conformal accelerated hyperfractionation field-involved re-irradiation combined with concurrent capecitabine chemotherapy might be an effective and well-tolerated regimen for patients with LRIRC.

Phase I study of concurrent selective lymph node late course accelerated hyper-fractionated radiotherapy and pemetrexed, cisplatin for locally advanced esophageal squamous cell carcinoma.

The optimized concurrent chemoradiotherapy has not been established for patients with advanced esophageal squamous cell carcinoma (SCC). The aim of the present study was to evaluate the safety and efficacy of concurrent chemotherapy and selective lymph node (SLN) late course accelerated hyperfractionated (LCAF) intensity modulated radiotherapy (IMRT) for the patients with thoracic SCC. Twelve patients with T3-4N0-1M0-1a thoracic esophageal SCC were included. The total dose of SLN LCAF IMRT was 59.6 Gy/34 fractions in 5.4 weeks. The concurrent chemotherapy protocol was as following: cisplatin 10 mg/m(2) on days 1-5 and 22-26, pemetrexed in escalating doses, from the base level of 500 mg/m(2) once every 21 days. The primary objectives were to determine the maximum tolerated dose (MTD), recommended dose (RD), and dose limiting toxicities (DLTs). Secondary end point included determination of preliminary radiographic response rates. As a result, three patients were enrolled in dose level 1 with pemetrexed 500 mg/m(2) and nine patients in dose level 0 with 400 mg/m(2) , respectively. At dose level 1, DLTs occurred in two of three patients. However, only two of nine patients in Level 0 developed DLTs. The complete response and partial response were observed in eight and four patients, respectively. Furthermore, no patient experienced cancer progression with a median follow-up of 9 months. In conclusion, the concurrent SLN LCAF IMRT and chemotherapy is feasible. The MTD of pemetrexed in this regimen was 500 mg/m(2) and RD was 400 mg/m(2) . Although toxicities were common, the protocol was safe, well tolerated, and achieved an encouraging outcome.

Prognostic value of 18F-FDG PET/CT before and after radiotherapy for locally advanced nasopharyngeal carcinoma.

BACKGROUND: The purpose of this study was to evaluate the prognostic value of maximal standard uptake values (SUVs(max)) from serial fluor-18-fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG PET/CT) in patients with locally advanced nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: From October 2002 to January 2004, 62 patients with locally advanced NPC who underwent (18)F-FDG PET/CT scan before and after radiotherapy were reviewed retrospectively. We examined the association of SUV(max) and the results of long-term follow-up of the patients. RESULTS: Patients having tumors with a lower SUV(max) had significantly better 5-year overall survival (OS) (P= 0.0187) and disease-free survival (DFS) (P = 0.0163) than patients with a greater SUV(max). The patients who showed with metabolic complete response had a significantly higher 5-year OS (P = 0.0237) and DFS (P = 0.0186) than patients with metabolic partial response. Poor prognosis was found in patients with the SUV(max) of neck nodes larger than that at the primary tumor site (SUV(max-N) > SUV(max-P)) (P = 0.0440). CONCLUSIONS: (18)F-FDG uptake, as measured by the SUV(max) before radiotherapy and metabolic response after radiotherapy, may predict the prognosis in locally advanced NPC. High (18)F-FDG uptake before and after radiotherapy may be useful for identifying patients requiring more aggressive treatment.

Risk factors of peripheral arterial disease and relationship between low ankle brachial index and mortality from all-cause and cardiovascular disease in Chinese patients with hypertension.

The aim of this study was to evaluate the risk factors of peripheral arterial disease (PAD) and the relationship between ankle brachial index (ABI) and mortality from all-cause and cardiovascular disease (CVD) in Chinese patients with hypertension. The ABI cohort Study was designed to investigate risk factors of PAD and the relationship between ABI and mortality from all-cause and CVD in Chinese patients. ABI was identified at baseline by measuring systolic pressure at bilateral brachial and tibial arteries. Mortality surveillance was completed from November 2005 to January 2006. Among 3047 participants with hypertension at baseline, 839 (27.5%) were in the low-ABI group. Older age, female gender, higher serum level of triglycerides, lower serum level of high-density lipoprotein, a history of diabetes and a history of smoking were associated with low ABI. During the 13-month follow-up, there were 252 deaths, of which 100 died of CVD. Low ABI was associated with mortality from all-cause and CVD, whose adjusted relative risk was 1.619 (95% confidence interval 1.190-2.203) and 2.454 (1.531-3.933), respectively, in Cox regression models. The survival rate was significantly lower in the low-ABI group than in the normal-ABI group. This study demonstrated that low ABI was independently associated with a high risk of all-cause and CVD mortality in Chinese patients with hypertension. ABI should be promoted as an ideal tool to predict mortality in diabetic patients.

[Treatment of cervical disc herniation with percutaneous arthroscopic microdisectomy].

OBJECTIVE: To study the indications and efficacy of percutaneous cervical arthroscopic microdiscectomy (PCAD). METHODS: Twenty eight patients with cervical discs herniation or disorders received endoscopic spine surgery, 37 intervertebral cervical discs underwent partial nuclectomy or total nucleotomy; the efficacy and complication were evaluated in three months after operation. RELUT: Good/excellent results were obtained in 23/28(82.2%) cases according to MacNab criteria; no serious complication was found; the cervical stability did not decrease in most cases. CONCLUSION: PCAD is a safe, accuracy and rational method in diagnosing and treating herniated cervical disc and discogenic disorders.

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins, subunits, and unrelated adjuvants.

We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to intact skin. While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens. The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens. Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 microg. Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin. Consistent with the latter finding, non-ADP-ribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1beta [IL-1beta] fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice. These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants. This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application.

Expression of interferon-gamma by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity.

Several lines of evidence suggest that overexpression of interferon gamma (IFN-gamma) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-gamma by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-gamma. The effect of IFN-gamma on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-gamma vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-gamma-expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-gamma or control vector. Marrow cells transduced with either vector were transplanted into W/Wv recipient mice. The percentage of vector-containing cells in the mIFN-gamma mice was significantly lower than in the control mice, suggesting that mIFN-gamma-transduced primitive cells may not have survived culture, or that mIFN-gamma directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-gamma-transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-gamma group. These data indicate that ectopic or overexpression of mIFN-gamma, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo.

Murine bone marrow expressing the neomycin resistance gene has no competitive disadvantage assessed in vivo.

The neomycin phosphotransferase (neo) gene is one of the most common marker genes used in gene transfer experimentation, but potential effects of neo gene expression in vivo have not been systematically investigated. Several early clinical retroviral gene transfer studies have suggested that neo gene expression could have deleterious effects on hematopoiesis, owing to a discrepancy between the level of neo-marked transduced marrow progenitor cells compared with mature circulating progeny cells posttransplantation (Brenner et al., 1993; Kohn et al., 1995; Brenner, 1996b). We examined the long-term in vivo repopulating ability of bone marrow from transgenic mice expressing neo from a strong constitutive promoter using a competitive repopulation assay. Different ratios of neo transgenic and wild-type congenic marrow cells were cotransplanted into W/Wv recipient mice. The percentages of blood cells containing the neo transgene in each group of recipient mice monitored for 4 months posttransplantation closely matched the input ratios of neo transgenic to congenic control marrow cells. Similar concordances of engraftment with input ratios of neo transgenic cells were also found in spleen, thymus, and whole marrow of recipient mice at 4 months posttransplantation. Analysis of the beta-hemoglobin phenotype (beta(single) for the neo transgenic and C57 control cells and beta(diffuse) for the congenic competitor HW80 cells) in recipients confirmed erythroid repopulation from neo transgenic marrow cells at levels matching the input ratios. We conclude that hematopoietic cells expressing neo had no engraftment or maturation defects detectable in vivo. These results suggest that the low-level contribution of vector-marked cells to circulating populations in clinical trials is not due to direct deleterious effects of neo gene expression on hematopoiesis.

Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective disadvantage.

Green fluorescent protein (GFP) has been used as a reporter molecule for gene expression because it fluoresces green after blue-light excitation. Inclusion of this gene in a vector could allow rapid, nontoxic selection of successfully transduced cells. However, many attempts by our laboratory to isolate stable retroviral producer cell clones secreting biologically active vectors containing either the highly fluorescent S65T-GFP mutant or humanized GFP have failed. Vector plasmids containing various forms of GFP and the neomycin resistance gene were transfected into three different packaging cell lines and fluorescence was observed for several days, but stable clones selected with G418 no longer fluoresced. Using confocal microscopy, the brightest cells were observed to contract and die within a matter of days. RNA slot-blot analysis of retroviral producer supernatants showed no viral production from the GFP plasmid-transfected clones, although all clones derived after transfection with an identical retroviral construct not containing GFP produced virus. Genomic Southern analysis of the GFP-transduced clones showed a much higher probability of rearrangement of the priviral sequences than in the control non-GFP clones. Overall, 18/34 S65T-GFP clones and 17/33 humanized-GFP clones had rearrangements, whereas 2/15 control non-GFP clones had rearrangements. Hence, producer cells expressing high levels of these GFP genes seem to be selected against, with stable clones undergoing major rearrangements or other mutations that both abrogate GFP expression and prevent vector production. These observations indicate that GFP may not be an appropriate reporter gene for gene transfer applications in our vector/packaging system.