Banana peel culture as an indigenous medium for easy identification of late-sporulation human fungal pathogens

Aim: Fungi are increasing in incidence as human pathogens and newer and rarer species are continuously being encountered. Identifying these species from growth on regular culture media may be challenging due to the absence of typical features. An indigenous and cheap medium, similar to the natural substrate of these fungi, was standardised in our laboratory as an aid to species identification in a conventional laboratory setting. Materials and Methods: Ripe banana peel pieces, sterilised in an autoclave at 121°C temperature and 15 lbs pressure for 15 min promoted good growth of hyphae and pycnidia or acervuli in coelomycetes, flabelliform and medusoid fruiting bodies of basidiomycetes and fruit bodies such as cleistothecium in ascomycetes. The growth from the primary isolation medium was taken and inoculated onto the pieces of double-autoclaved ripe banana peel pieces in a sterile glass Petri dish with some moisture (sprinkles of sterile distilled water). A few sterile coverslips were placed randomly inside the Petri dish for the growing fungus to stick on to it. The plates were kept at room temperature and left undisturbed for 15–20 days. At a time, one coverslip was taken out and placed on a slide with lactophenol cotton blue and focused under the microscope to look for fruit bodies. Results: Lasiodiplodia theobromae, Macrophomina phaseolina, Nigrospora sphaerica, Chaetomium murorum, Nattrassia mangiferae and Schizophyllum commune were identified by characteristic features from growth on banana peel culture. Conclusions: Banana peel culture is a cheap and effective medium resembling the natural substrate of fungi and is useful for promoting characteristic reproductive structures that aid identification.     

Pathogenesis of Rushton bodies: A novel hypothesis

Rushton bodies (RBs) are one of the characteristic features seen in the epithelial lining of odontogenic cysts mainly radicular, dentigerous and odontogenic keratocyst. It has two different histo-morphological appearances; granular and homogeneous. Although widely investigated, the exact pathogenesis and histogenesis of RBs is still an enigma. Many hypotheses were made in the literature but none has explained conceivably the two histo-morphological appearances of RBs and their association with inflammation. In the present paper the various pathogenesis for the formation of RBs proposed till date are discussed along with proposal for a novel hypothesis. The given hypothesis is mainly related to inflammation and its effect on pore size of basement membrane of odontogenic cystic epithelium. It explains RBs association with inflammation as well as existence of two histo-morphological appearances. The proposed hypothesis also justifies the RB’s presence inside the lining epithelium of odontogenic cyst despite its hematogenous origin. Future studies are advocated for isolating RBs using laser capture microdissection and subsequent biochemical, histochemical and electron microscopic analysis to substantiate the proposed hypothesis.     

Psammoma bodies in fine needle aspiration cytology of the breast: A clinicopathological study of 30 cases

Psammoma bodies (PBs) in breast lesions are rare and little is known about the role of these structures in breast pathology. This study has looked in to the diagnostic significance of PBs in fine needle aspiration (FNAC) of breast lesions. Over a 5‐year period, FNACs of the breast were done in 4,563 subjects, of which 1,678 were diagnosed to be malignant. On review of all breast aspirates including non‐neoplastic lesions, 30 cases showed PBs to be associated with breast carcinoma (BC). Cytological features were correlated with clinical, radiological, histological, and immunohistochemical findings. All 30 aspirates and their corresponding histological sections showed varying number of PBs and nonpsammomatous bodies (NPBs). For comparison, 31 cases of age‐matched BC without PBs and NPBs in both aspirates and sections were studied. Statistical analysis using Chi‐square test was done to compare BC with and without PBs. BC with PBs was characterized by papillary pattern of malignant cells, mucin in the background, infiltration by macrophages, cellular degeneration, overexpression of estrogen receptor (ER), and progesterone receptor (PR) and moderate positivity (2+) for Her2/neu. Calcium deposition has long been implicated in the pathogenesis of many degenerative diseases; hence the formation of PBs may be relevant in breast oncology. The presence of PBs in FNAC of clinically suspected breast lesions which are cytologically negative for malignancy warrants further histological confirmation. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

Adherence of Staphylococcus epidermidis to extracellular matrix proteins and effects of fibrinogen-bound bacteria on oxidase activity and apoptosis in neutrophils

Staphylococcus epidermidis often causes foreign-body infections such as those associated with hip prostheses, but the underlying pathogenic mechanisms are not fully understood. We performed spectrophotometry to study the ability of S. epidermidis to bind to immobilised fibrinogen, fibronectin, vitronectin, and collagen. The strains were isolated from infected hip prostheses or from normal flora and the well-known protein-binding strain Staphylococcus aureus Cowan was used as positive control. We also analysed the interaction between neutrophils and a fibrinogen-bound prosthesis-derived strain of S. epidermidisby measuring chemiluminescence to determine the neutrophil oxidative response and binding of annexin V to indicate neutrophil apoptosis. We found that binding of S. epidermidis to extracellular matrix proteins varied under different growth conditions, and that prosthesis isolates adhered better to vitronectin than did strains from normal flora. The oxidative response caused by fibrinogen-bound S. epidermidis was not above the background level, which was in marked contrast to the distinct response induced by fibrinogen-associated S. aureus Cowan. Furthermore, fibrinogen-adhering S. epidermidis retarded neutrophil apoptosis. We conclude that surface-bound S. epidermidis induces only a weak inflammatory response, which in combination with the ability of the adherent bacteria to retard neutrophil apoptosis may contribute to low-grade inflammation and loosening of prostheses.     

重组人Flt3配体在大肠杆菌中的高效表达和生物学活性的鉴定

根据天然的人Flt3配体(Flt3ligand,FL)基因和遵循适合大肠杆菌BL21(DE3)表达体系表达条件及不改变FL天然蛋白氨基酸序列的原则,对其进行改造,所获得的FL功能片段(FL134)克隆入PET30a表达载体,在C端融合了6个His,继而在大肠杆菌中诱导表达。结果表明重组蛋白rhFL134以包涵体形式在大肠杆菌(BL21)中可得到较高水平的表达。通过蛋白的变性、复性和HiTrapTM亲和层析获得了纯度达92%以上的rhFL134重组蛋白。体外的活性实验显示,重组蛋白rhFL134具有显著的促小鼠骨髓细胞的集落形成和扩增人脐带血中的CD34+细胞的生物学活性。     

Lipid bodies: structural markers of inflammatory macrophages in innate immunity

Objective and design The correlation between innate immune responses and formation of cytoplasmic lipid bodies (LBs) was investigated in vivo in inflammatory macrophages from rats infected with Trypanosoma cruzi, the intracellular parasite which causes Chagas’ disease. Material and methods We used an experimental model of high-dose irradiation prior to infection, which depletes the humoral and cellular immune responses except for the phagocytic activity of macrophages. Rats, irradiated or not, were infected with T. cruzi and macrophages from different origins (peritoneum, heart, uterus) were studied by transmission electron microscopy (TEM). Results As documented by quantitative TEM, innate immune responses induced prominent formation, structural changes and intracellular interactions of LBs. LBs significantly increased their size and changed their osmiophilia in response to both infection alone and when macrophages were challenged with irradiation-induced increased parasite load. Remarkably, a consistent LB-phagolysosome association was identified. LBs were surrounding, attached to or internalized by phagolysosomes. Conclusions We demonstrated that LBs are dynamic organelles notably involved in the host response to acute T. cruzi infection, an event that may be important for pathogen control during innate immunity. Our findings highlight LBs as structural markers of the innate immune responses in phagocytic cells. Received 14 December 2005; returned for revision 12 March 2006; accepted by G. Wallace 21 March 2006     

Marginal matters: Pregnancy loss as a social event

Studies on fertility in Africa have known a major paradigm shift when demographic concerns about ‘overpopulation’ came to be replaced by new ideas about reproductive health, rights, and choices during the 1994 International Conference on Population and Development (ICPD). Whereas this shift has allowed for more recognition of losses during pregnancy which had been virtually absent in previous demographic accounts of high fertility rates, the new discourse on rights and choices turns most of its attention to induced loss. Losses that spontaneously occur remain merely bound to the medical realm. Yet this paper shows that for many women in Africa and elsewhere, spontaneous pregnancy loss is a daily life reality which is inherently related to many social affairs, i.e. life and death, illness and suffering, marriage and kinship, the body and personhood. The rather reductionist biomedical discourse prevalent in the global health arena largely ignores these themes and social complexities – thus causing a gap between health policies and daily life realities for women. Drawing on eleven months of anthropological fieldwork in Cameroon in 2004 and 2008, this article explores the way in which socio-cultural insights could contribute to a better understanding of the experiences of women coping with pregnancy loss. The notions of ‘vital conjunctures’ and ‘social bodies’ will form an alternative approach to decision-making in case of reproductive mishaps. By applying these concepts to the personal story of an informant, their relevance and contribution to an interdisciplinary discussion on the topic become clear. The author argues for an integration of anthropological expertise in international reproductive health debates and explores how interdisciplinary work could make health policies on reproductive loss less marginal than is the case at present.     

Novel insights into changes in biochemical properties of keratins 8 and 18 in griseofulvin-induced toxic liver injury

Keratins 8 and 18 (K8/18) intermediate filament proteins are believed to play an essential role in the protection of hepatocytes against mechanical and toxic stress. This assertion is mainly based on increased hepatocyte fragility observed in transgenic mice deficient in K8/18, or carrying mutations on K8/18. The molecular mechanism by which keratins accomplish their protective functions has not been totally elucidated. Liver diseases such as alcoholic hepatitis and copper metabolism diseases are associated with modifications, in hepatocytes, of intermediate filament organisation and the formation of K8/18 containing aggregates named Mallory-Denk bodies. Treatment of mice with a diet containing griseofulvin induces the formation of Mallory-Denk bodies in hepatocytes. This provides a reliable animal model for assessing the molecular mechanism by which keratins accomplish their protective role in the response of hepatocytes to chemical injuries. In this study, we found that griseofulvin intoxication induced changes in keratin solubility and that there was a 5% to 25% increase in the relative amounts of soluble keratin. Keratin phosphorylation on specific sites (K8 pS79, K8 pS436 and K18 pS33) was increased and prominent in the insoluble protein fractions. Since at least six K8 phosphoepitopes were detected after GF treatment, phosphorylation sites other than the ones studied need to be accounted for. Immunofluorescence staining showed that K8 pS79 epitope was present in clusters of hepatocytes that surrounded apoptotic cells. Activated p38 MAPK was associated with, but not present in K8 pS79-positive cells. These results indicate that griseofulvin intoxication mediates changes in the physicochemical properties of keratin, which result in the remodelling of keratin intermediate filaments which in turn could modulate the signalling pathways in which they are involved by modifying their binding to signalling proteins.     

大肠杆菌表达的人源性抗CTLA4单链抗体三种复性方法比较

比较表达于大肠杆菌中的人源性抗细胞毒性T淋巴细胞抗原4单链抗体(A n ti-CTLA 4 scFv)的三种体外复性方法的复性效率。稀释、透析和亲和柱上三种复性方式复性A n ti-CTLA 4 scFv,采用B rad ford法分析蛋白复性得率,采用间接细胞EL ISA法检测所得蛋白的活性。透析复性蛋白得率最高,稀释复性蛋白得率其次,亲和柱上复性蛋白得率最低;透析复性所得蛋白结合活性是稀释复性的1.95倍,是亲和柱上复性所得蛋白活性的4.13倍(无谷胱甘肽氧化还原对G SH/G SSH)及3.63倍(有G SH/G SSH)。结论:采用含0.15 m o l/L N aC、l1 mm o l/L G SSH和3 mm o l/L G SH的50 mm o l/L T ris-HC l(pH 8.0)缓冲液作为A n ti-CTLA 4 scFv的复性液,4℃下透析复性48 h,可获得较高复性得率和结合活性。     

Identification of oleosins as major allergens in sesame seed allergic patients

BACKGROUND: The prevalence of sesame allergy is increasing in European countries. Cases of severe allergy lack any evidence of specific immunoglobulin (Ig)Es by prick tests and CAPSystem-FEIA. The reasons for this negativity are unknown. METHODS: In 32 patients displaying immediate symptoms such as anaphylactic shock, asthma, urticaria, angioedema, sesame allergy was diagnosed by double-blind placebo-controlled food challenge (DBPCFC) or convincing clinical history. However, 10 patients had negative prick tests and CapSystem-FEIA. The specificity of IgEs was further investigated by enzyme-linked immunosorbent assay (ELISA), isoelectrofocalisation (IEF)-blotting, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) blotting using total sesame extracts and purified fraction of oil bodies. Monospecific rabbit antibodies directed to two oleosin isoforms (15 and 17 kDa) were used. RESULTS: By ELISA, white sesame seed extract allowed the detection of higher levels of IgE than brown sesame extract. In all sera, numerous bands binding IgEs were detected by IEF or SDS-PAGE. In reducing conditions, two bands (15-17 kDa), could be separated from 2S albumin. Oleosins, present in oil bodies fractions, were recognized by IgEs from all sera. CONCLUSION: Oleosins are major allergens of sesame seeds and may be relevant to severe anaphylaxis. Falsely negative prick tests could be due to the lack of oleosins in presently available extracts, or to the fact that epitopes might be buried in the inner molecule. Detection tests currently used to identify sesame allergens based on sesame vicillins or other storage proteins could be insufficient for the detection of sesame seed contamination. Oleosins have been named Ses i 4 (17 kDa) and Ses i 5 (15 kDa), in accordance with the IUIS Nomenclature Committee.