Novel type of fimbriae encoded by the large plasmid of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H(-)

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H(-), pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed sfpA, sfpH, sfpC, sfpD, sfpJ, and sfpG, which mediate mannose-resistant hemagglutination and the expression of fimbriae. sfp genes are similar to the pap genes, encoding P-fimbriae of uropathogenic E. coli, but the sfp cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of Proteus mirabilis. The putative adhesin gene sfpG, responsible for the hemagglutination phenotype, shows significant homology neither to papG nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the sfp gene cluster is a characteristic of SF EHEC O157:H(-) strains and is not present in other EHEC isolates, diarrheagenic E. coli, or other Enterobacteriaceae. The sfp gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H(-).     

Effects of fluorodeoxyuridine on the developing inner ear of the rat

The inner ear in rats develops from the surface ectoderm on day 8 of a 22-day gestational period. Labeled thymidine incorporation studies have indicated that in the developing inner ear most of the cells undergo terminal mitosis between gestational days 13 and 15. During this period the developing inner ear would be particularly vulnerable to environmental hazards. To test this hypothesis, pregnant rats were given a single intraperitoneal injection of 5-fluoro-2'-deoxyuridine (FUdR), an antimitotic substance, on gestational days 12 to 16. The rats also received one injection of 3H-thymidine 1 h prior to the removal of the fetuses. The animals were killed after various time intervals following the treatment, and the otocysts or inner ears were prepared for morphologic observations and biochemical assays. The cells in the inner ear of rats exposed to FUdR exhibited pyknotic nuclei and chromatolytic degeneration, and they eventually died. By 4 h after the administration of FUdR, pyknotic nuclei were seen in the antiluminal zone of the otic epithelium, and there was a substantial decrease in the number of the otic cells. This decline in cell number was seen until 24 h after treatment. However, the inner ears from the fetuses exposed to FUdR during gestational days 12--15 showed complete recovery from the toxic effects of the drug when examined on day 21 of gestation. The phenomenon of programmed cell death observed in the developing inner ear of the rat indicates that more cells are produced during the earlier stages of development than are required for the definitive adult structures. This phenomenon may represent an important protective feature. The redundant production of cells perhaps allows the developing otocysts to respond to an environmental stress by subtotal destruction of cells from the pool of undifferentiated cells, resulting in relatively fewer congenital anomalies of the inner ear.     

Expression of BCL-2 and p53 in oncocytic (Hürthle cell) tumors of the thyroid: An immunohistochemical study

Previous studies have established that thyroid follicular neoplasms of higher malignant potential show a high p53 and low bc1-2 expression. This however has not been well studied in Oncocytic (Hürthle cell) neoplasms, the management of which remains controversial. We therefore studied the expression of p53 and bc1-2 in 18 Hürthle cell adenomas (HCA) and 8 Hürthle cell carcinomas (HCC) and compared them with their benign and malignant counterparts, respectively, including 16 follicular adenomas (FA) and 68 papillary carcinomas (PC). All 16 FA were bc1-2 positive, 4 were 2+, and 12 were 3+. On the other hand, 14/18 (78%) HCA showed bc1-2 expression, 5 were 1+, 6 were 2+, and only 3 were 3_. Similarly, HCC showed a weaker bc1-2 staining pattern compared to PC. Only 1 FA showed grade 1, p53 staining, the remaining 15 were negative, and 15/18 HCA showed p53 expression of varying grades. This difference in p53 staining was statistically significant (p=0.005). A significant p53 overexpression was also seen in HCC compared to PC (p=0.005). In conclusion, there appears to be an inverse relationship between p53 and bc1-2 expression in thyroid follicular neoplasms. A higher expression of p53 and lower levels bc1-2 in Hürthle cell neoplasms may have biological and clinical implications. This may support a more aggressive surgical treatment for HCA compared to FA.     

Evidence that prostate gonadotropin-releasing hormone receptors mediate an anti-tumourigenic response to analogue therapy in hormone refractory prostate cancer

Gonadotropin-releasing hormone analogue (GnRHa) therapy is an established method of androgen withdrawal in the treatment of prostate cancer. The present study investigated if the expression of prostate GnRH receptors (GnRHRs) might influence the response to GnRHa. GnRHR protein expression was first studied in a panel of prostate cancer cell lines. In androgen-dependent cells, GnRHR expression was unchanged following acute or chronic androgen withdrawal. In these cells, GnRHa significantly inhibited androgen-induced cell proliferation (p = 0.01). In contrast, GnRHa was unable to further suppress basal levels of cell proliferation induced by androgen withdrawal. In androgen-independent prostate cancer cells, variable levels of GnRHR expression were observed. In these cells, GnRHa treatment blocked cell proliferation (p = 0.001) and invasion (up to 70%) induced by fibroblast growth factor stimulation. Crucially, this effect was only evident in cells that expressed high levels of the GnRHR. GnRHa treatment also significantly inhibited the ability of these cells to recover from a cytotoxic insult (50% inhibition). The clinical significance of prostate GnRHR was tested by immunohistochemistry in a preliminary cohort of patients treated with GnRHa or surgical castration. There was no association between GnRHR expression and pathological grade, clinical stage, time to PSA nadir (p = 0.82) (n = 35) or progression to hormone refractory disease (p = 0.22) (n = 21), irrespective of the treatment method. GnRHa therapy in the presence of high GnRHR expression however, was found to be associated with longer disease-specific survival (mean survival 85 months, p = 0.002). In contrast, high GnRHR expression was not associated with survival among surgically castrated patients (mean survival 50 months, p = 0.7). Taken together, these data support the notion of a functional interaction between GnRHa and the GnRHR, which results in an anti-tumourigenic effect on prostate cancer cells. Findings from this report have direct implications for the use of GnRHR as a novel therapeutic target in hormone refractory prostate cancer.     

Enzyme immunoassay detection of antigen-specific immunoglobulin g antibodies in longitudinal serum samples from patients with cryptosporidiosis

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of Cryptosporidium infection in the population.     

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine

Comparative mutagenic and genotoxic effects of three antimalarialdrugs, chloroquine, primaquine and amodiaquine, were assessedin the Ames mutagenicity assay (in strains TA97a, TA100, TA102and TA104) and in vivo sister chromatid exchange (SCE) and chromosomeaberration (CA) assays in bone marrow cells of mice. These arethe most commonly used antimalarial drugs available at presentthroughout the world. The results of the bacterial mutagenicityassays showed a very weak mutagenic effect of all three drugsin Salmonella strains TA97a and TA100 both with and withoutS9 mix and in TA104 only with S9 mix. The results of the invivo SCE and CA assays indicate that these three drugs are genotoxicin bone marrow cells of mice. ³To whom correspodence should be addressed. Tel: +91 33 473 3491; Fax: +91 33 473 5197; Email: iichbio{at}giascl01.vsnl.net.in     

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

BACKGROUND: There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). METHODS: This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. RESULTS: Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1-2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52-665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. CONCLUSION: Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors.     

Boleodorus similis n. sp. (Nematoda: Nothotylenchinae) from India

Summary A new species, B. similis, is described from male and female specimens collected from soil around roots of Plumeria acutifolia Poir from North India. It is distinguished by a short and robust body, a broad lateral field, a more posteriorly located vulva in female and the position of phasmid and the posterior extension of lateral field in male.With 8 Figures in the Text