Synergistic effect of IL-4 and TNF-alpha in the induction of monocytic differentiation of a mouse myeloid leukaemic cell line (WEHI-3B JCS).

We have previously shown that non-cytotoxic concentrations (600-1200 U/ml) of recombinant mouse tumour necrosis factor-alpha (TNF-alpha) can induce differentiation of a subclone (JCS) of the WEHI-3B myelomonocytic leukaemia cell line into mature cells with the characteristics of macrophages. In the present study, the effects of recombinant mouse interleukin-4 (IL-4), either alone or in combination with mouse TNF-alpha, on the growth and differentiation of JCS cells were examined. IL-4 alone (20-5000 U/ml) inhibited the growth of JCS cells in a dose-dependent manner but did not induce cell differentiation. However, combinations of IL-4 and TNF-alpha acted in synergy to inhibit cell proliferation and induce monocytic differentiation of JCS cells, as shown by increased expression of the macrophage differentiation antigens (F4/80, Mac-1), stimulation of phagocytic activity, induction of non-specific esterase and NBT-reducing activities, increased plastic adherence and morphological criteria. Similar synergistic interactions were also shown by human TNF-alpha and mouse IL-4, indicating that TNF-alpha might exert its effects through the low-affinity (p55) TNF receptors. Moreover, the clonogenicity of JCS cells in vitro and their tumorigenicity in vivo were significantly reduced by combined TNF-alpha and IL-4 treatment. Our results indicate that TNF-alpha can act as a differential signal for JCS cells and that its effects are modulated by IL-4. Therefore, the combination of TNF-alpha and IL-4 may be useful in the treatment of some forms of myelomonocytic leukaemia.     

重组MHCⅠ类分子K^b-EGFP分子的构建及表达

目的 构建绿色荧光蛋白标记的MHCⅠ类分子K^b(K^b-EGFP融合蛋白)，并在哺乳动物细胞中获得功能性表达。方法 RT-PCR的方法获得小鼠MHCⅠ类分子K^b cDNA，克隆入真核表达载体pEGFP-N1中EGFP分子的上游，脂质体转染COS-7细胞，G418加压筛选获得抗性重组细胞。荧光显微镜下观测重组分子的表达及胞内定位情况。结果 分子克隆的方法获得了预期的重组质粒，质粒经DNA序列测定证实阅读框无误，转染COS-7细胞后的抗性克隆胞浆内可以观察到明亮的绿色荧光，特征性地分布于细胞质膜，及核周、膜下的囊泡结构中。结论 所构建的K^b-EGFP融合蛋白在细胞内表达后具有MHCⅠ类分子的特征性分布，提示该重组分子具有野生型MHCⅠ类分子的生物学特性和胞内分布特征，为进一步深入研究MHCⅠ类分子胞内动态分布及分子功能提供了良好的细胞和分子模型。     

Immune modulatory effects of Prunella vulgaris L. on monocytes/macrophages

Prunella vulgaris L. (Labiatae), a popular Western and Chinese herbal medicine, has long been associated with anti-viral and anti-bacterial effects. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects remain unknown. As a biological response modifier (BRM), the polysaccharides isolated from P. vulgaris have been shown to up-regulate the immune responses of monocytes/macrophages. However, the immune stimulatory effects seem to contradict its well-known anti-inflammatory properties. We hypothesized that the anti-microbial effects exhibited by the polysaccharides isolated from P. vulgaris encompass both anti-inflammatory and immune stimulatory effects. One of the polysaccharide fractions PV2IV markedly stimulated the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2IV stimulation was as high as that stimulated by bacterial endotoxin lipopolysaccharide (LPS) in a dose-dependent manner. In addition, PV2IV also increased cellular protein levels of inducible nitric oxide synthase (iNOS) and mRNA for tumor necrosis factor-alpha (TNFalpha). Similar to the effects of a high dose of LPS, the fraction PV2 could trigger activation-induced cell death (AICD) by stimulating caspase-3 activity and reduction of MTT uptake in monocytes/macrophages. These results may help our understanding of the molecular mechanism of P. vulgaris, which exhibited both immune stimulatory and anti-inflammatory effects against microbial invasion.     

Comparison of screening methods for detection of extended-spectrum beta-lactamases and their prevalence among Escherichia coli and Klebsiella species in Hong Kong

Three tests, the disk diffusion test, the double-disc synergy test and the inhibitor-potentiated disc diffusion test, were compared for their abilities to detect production of extended-spectrum beta-lactamases (ESBL) in 702 Escherichia coli and 472 Klebsiella spp. strains from four hospitals. Eleven percent E. coli and 13% Klebsiella spp. were found to produce ESBL. As an indicator of ESBL activity, the sensitivities of the five extended-spectrum beta-lactams were as follows: cefotaxime (100%), cefpodoxime (99.3%), ceftriaxone (98.6%), aztreonam (93%) and ceftazidime (57.7%) when interpreted using the National Committee for Clinical Laboratory Standards criteria. Their positive predictive values ranged from 67.8-83.8%. Both the inhibitor-potentiated disc diffusion test and the double-disc synergy test (at three inter-disc widths of 20, 25 and 30 mm) were capable of identifying all the ESBL-producers. However, at a single inter-disc width of 30 mm, the double-disc synergy test has limited sensitivity (83.8%). As a second test for confirming ESBL activity in strains with reduced susceptibility to beta-lactams, the inhibitor-potentiated disc diffusion test is therefore a simple and reliable option.     

Interleukin-6 induced at mucosal surfaces by gram-negative bacterial infection.

Interleukin-6 (IL-6) was produced in response to mucosal and systemic infection of mice with gram-negative bacteria. The IL-6 response was controlled by the lipopolysaccharide gene, Lps; in C3H/HeN mice (Lpsn/Lpsn), the urinary IL-6 levels increased within 30 min after challenge with Escherichia coli, but no response occurred in C3H/HeJ mice (Lpsd/Lpsd). In lipopolysaccharide-responder mice, the levels of local and systemic IL-6 were related to the degree of infection. The urinary response dominated after intravesical challenge, and the serum response dominated after intraperitoneal challenge. The results demonstrate that IL-6 is activated as part of lipopolysaccharide-induced mucosal and systemic responses to gram-negative bacterial infections.     

Expression of c-myc and c-fms oncogenes in trophoblastic cells in hydatidiform mole and normal human placenta.

AIMS: To compare the expression of c-myc and c-fms proto-oncogenes in the placenta and hydatidiform mole. METHODS: Twelve hydatidiform moles and six induced abortion cases were collected. c-myc and c-fms proto-oncogene expression was analysed by northern blot hybridisation and immunohistochemical staining. RESULTS: The results of northern blot hybridisation analysis showed that c-fms was expressed more strongly in hydatidiform moles compared with normal placenta of similar gestational age. Moreover, c-fms mRNA concentrations increased with more advanced gestational age in moles but not in normal placentas. c-myc expression was very low in hydatidiform moles and normal placentas. Both oncogenes, however, had no direct correlation with the clinical course of the molar pregnancies. CONCLUSION: The difference in c-fms expression between hydatidiform moles and normal placentas suggests that c-fms may have a role in the development of molar pregnancies.     

Therapeutic Vaccines for Cervical Cancer

Human papillomavirus (HPV) infection is associated with transformation and clonal expansion of infected epithelial cells, resulting in the production of a benign growth, i.e. a wart. Recently, however, HPV has emerged as the primary causative agent of cervical carcinoma, malignancy being associated with the presence of the viral genome (predominantly genotypes 16 and 18) in cancerous cells. The only HPV proteins reliably expressed in neoplastic lesions are the 'oncogenic' E6 and E7 proteins, that serve both as tumour-specific markers and potential targets for immunotherapeutic intervention. As intracellular (nuclear) proteins, the E6 and E7 gene products may be hidden from the humoral immune response. Attention has thus focused on the generation of a vaccine capable of inducing or stimulating a cellular immune response to HPV 16 and HPV 18 E6 and E7. Vaccine development has been constrained by the absence of an appropriate animal model, the oncogenic nature of E6 and E7 and technical difficulties associated with detection of cytotoxic T cell responses to these antigens. Despite these difficulties, vaccine strategies have now been devised based on immunisation with synthetic peptide, whole protein and a vaccinia virus recombinant. Phase I/II human clinical trials have been initiated, and preliminary results have demonstrated the induction of specific cellular immune responses after immunisation. The HPV-associated neoplasia in cervical cancer represents an excellent target for therapeutic intervention because the tumour-associated antigens are so clearly defined. As such, it provides an appropriate model for establishing the general principles of cancer immunotherapy in humans.     

Gemella bacteraemia characterised by 16S ribosomal RNA gene sequencing

AIMS: To define epidemiology, clinical disease, and outcome of gemella bacteraemia by 16S rRNA gene sequencing. To examine the usefulness of the Vitek, API, and ATB systems in identifying two gemella species. METHODS: All alpha haemolytic streptococci other than Streptococcus pneumoniae isolated from blood cultures during a six year period were identified by conventional biochemical methods, the Vitek system, and the API system. 16S rRNA gene sequencing was performed on all isolates identified by both kits as gemella with >or= 95% confidence or by either kit as any bacterial species with < 95% confidence. The ATB expression system was used to identify the two isolates that were defined as gemella species by 16S rRNA gene sequencing. RESULTS: Of the 302 alpha haemolytic streptococci other than S pneumoniae isolated, one was identified as Gemella morbillorum, and another as Gemella haemolysans by 16S rRNA gene sequencing. The patient with monomicrobial G morbillorum bacteraemia was a 66 year old man with community acquired infective endocarditis with septic thromboemboli. The patient with G haemolysans bacteraemia was a 41 year old woman with hospital acquired polymicrobial bacteraemia during the neutropenic period of an autologous bone marrow transplant for non-Hodgkin's lymphoma, the first case of its kind in the English literature. The API and ATB expression systems only identified the second strain as G haemolysans at 94% and 99% confidence, respectively, whereas the Vitek system identified none of the two strains correctly at > 70% confidence. CONCLUSIONS: Gemella bacteraemia is uncommon. 16S rRNA gene sequencing is the method of choice for identification of gemella and gemella-like isolates.     

Is there an effect of monocular deprivation on the proportions of X and Y cells in the cat lateral geniculate nucleus?

Summary The proportions and receptive field properties of X and Y cells in the A and A1 layers of the lateral geniculate nucleus (LGN) were studied in monocularly deprived cats. Contrary to previous reports, we found that there was no change in the relative number of Y cells in the geniculate layers driven by the deprived eye. There was also no marked change in the spatial resolution of X or Y cells driven from the deprived eye as compared to the cells driven from the normally experienced eye. In these same cats, the visual evoked potential from stimulation of the deprived eye with grating patterns was markedly reduced in amplitude. Furthermore, the cell bodies of the cells in the LGN driven by the deprived eye had shrunk. Therefore, these usual consequences of monocular deprivation are not necessarily associated with a loss of geniculate Y cells.     

The syed temporary interstitial iridium gynaecological implant: an inverse planning system

Patients with advanced gynaecological cancer are often treated with a temporary interstitial implant using the Syed template and Ir- 192 ribbons at the Memorial Sloan-Kettering Cancer Center. Urgency in planning is great. We created a computerized inverse planning system for the Syed temporary gynaecological implant, which optimized the ribbon strengths a few seconds after catheter digitization. Inverse planning was achieved with simulated annealing. We discovered that hand-drawn target volumes had drawbacks; hence instead of producing a grid of points based on target volume, the optimization points were generated directly from the catheter positions without requiring an explicit target volume. Since all seeds in the same ribbon had the same strength, the minimum doses were located at both ends of the implant. Optimization points generated at both ends ensured coverage of the whole implant. Inverse planning took only a few seconds, and generated plans that provide a good starting point for manual improvement.