Diversity of genomic breakpoints in TFG-ALK translocations in anaplastic large cell lymphomas: identification of a new TFG-ALK(XL) chimeric gene with transforming activity

Anaplastic large cell lymphomas are associated with chromosomal aberrations involving the anaplastic lymphoma kinase (ALK) gene at 2p23 that result in the expression of novel chimeric ALK proteins with transforming properties. In most of these tumors, the t(2;5)(p23;q35) generates the NPM-ALK fusion gene. However, several studies have now demonstrated that genes other than NPM may be fused to the ALK gene. We have recently described two different ALK rearrangements involving the TRK-fused gene (TFG) in which the same portion of ALK was fused to different length fragments of the 5' TFG region. These two rearrangements encoded chimeric proteins of 85 kd (TFG-ALK(S)) and 97 kd (TFG-ALK(L)), respectively. In this study, we have identified a new ALK rearrangement in which the catalytic domain of ALK was fused to a larger fragment of the TFG gene (TFG-ALK(XL)), encoding for a fusion protein of 113 kd. Genomic analysis of these three TFG-ALK rearrangements revealed that the TFG breakpoints occur at introns 3, 4, and 5, respectively, whereas the ALK breakpoints always occur in the same intron. No homologous regions or known recombination sequences were found in these regions. Transfection experiments using NIH-3T3 fibroblasts showed a similar transforming efficiency of TFG-ALK variants compared with NPM-ALK. In addition, in common with NPM-ALK, the TFG-ALK proteins formed stable complexes with the signaling proteins Grb2, Shc, and PLC-gamma. In conclusion, these findings indicate that the TFG may use a variety of intronic breakpoints in ALK rearrangements generating fusion proteins of different molecular weights, but with similar transforming potential than NPM-ALK.     

Survey of rotavirus G and P types associated with human gastroenteritis in São Paulo, Brazil, from 1986 to 1992.

Rotavirus strains causing gastroenteritis in Brazilian children were characterized by PCR-based typing assays. In addition to strains bearing the major human G and P types, large numbers of strains bearing P3 (M37-like), P6 (HCR3-like), untypeable P and G types, and complex mixtures of P and G types not previously recognized were present in the community.     

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector

Summary. We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4µg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.Authors contributed equally to the results presented in this report.     

Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa

In 25 patients (14 suffering from obstructive azoospermia, sixfrom non-obstructive azoospermia, three from astheno-azoospermiaand two from absence of ejaculation) spermatozoa were extractedfrom testicular biopsies. Intracytoplasmic sperm injection (ICSI)with fresh testicular spermatozoa was performed in 18 cases;spermatozoa in excess were cryopreserved in pills. No pregnancieswere achieved. In the remaining seven patients, testicular spermatozoawere retrieved and cryopreserved during a diagnostic testicularbiopsy. After thawing, sperm motility was assessed in 17 cases(68%), and 18 ICSI with cryopreserved testicular spermatozoawere performed. The mean two-pronuclear (2PN) fertilizationrate was 59%, the mean cleavage rate was 92%, and six clinicalpregnancies were achieved, all of them still ongoing (pregnancyrate 33%). A comparison of the results of ICSI carried out withfresh or cryopreserved testicular spermatozoa showed that themean 2PN fertilization rates per cycle (53 compared with 55%),mean cleavage rates per cycle (99 compared with 96%) and embryoquality were not significantly different In conclusion, cryopreservationof testicular spermatozoa is feasible, even in patients withnon-obstructive azoospermia, and the results of ICSI with frozen-thawedtesticular spermatozoa are similar to those obtained using freshtesticular spermatozoa. Cryopreservation of testicular spermatozoamay avoid repetition of testicular biopsies to retrieve spermatozoafor successive ICSI cycles in patients in whom the only sourceof motile spermatozoa is the testicle.     

Effect of cyclosporine on the response of normal human lymphocytes to cytomegalovirus in vitro.

Lymphocytes from healthy volunteers seropositive for cytomegalovirus (CMV) demonstrated strong lymphoproliferative responses to CMV-infected and glutaraldehyde-fixed foreskin fibroblasts (CMVFFx) when cocultured for 6 days. Addition of the immunosuppressive drug cyclosporine (CsA) to the cultures resulted in a 10-fold reduction (P less than 0.001) in counts per minute of [3H]thymidine uptake. The proliferative response to noninfected fixed fibroblasts was also reduced 10-fold. Kinetic studies showed an inhibition of the lymphoproliferative response and not an alteration in the time course kinetics in CsA-treated cultures. Cytotoxicity to CMV-infected and unfixed fibroblasts by lymphocytes primed by CMVFFx in the presence of 0.5 micrograms of CsA per ml was significantly reduced (P less than 0.01) as compared with untreated cultures but remained significantly above background level (P less than 0.01). The cytotoxic response was still present but reduced at concentrations of greater than or equal to 1.0 micrograms/ml. Cytotoxicity to noninfected fibroblasts by CMVFFx-primed lymphocytes could be eliminated by as little as 0.5 micrograms of CsA per ml. Stimulation of lymphocytes by CMVFFx but not by noninfected fixed fibroblasts resulted in the in vitro generation of suppressor cells. CsA at a final concentration of 1.0 or 2.5 micrograms/ml did not significantly impair the induction of cells capable of suppressing the lymphoproliferative response of fresh autologous mononuclear cells to CMVFFx. The findings described above may have important clinical implications in that a degree of protective immunity to CMV-infected cells is maintained even in the presence of CsA.     

Accuracy of a commercial enzyme-linked immunosorbent assay for CagA in patients from Brazil with and without gastric carcinoma

We validated a commercial enzyme-linked immunosorbent assay for the detection of anti-CagA antibodies in Brazilian patients with Helicobacter pylori infection. The test presented high sensitivity (97.4%) and specificity (88.9%) when employed in patients without gastric carcinoma. However, in gastric carcinoma patients, the test was neither sensitive nor specific enough to detect cagA-positive H. pylori infection.     

Effects of a feed additive blend on broilers challenged with heat stress

ABSTRACT

We evaluated a blend of medium-chain fatty acids (MCFA), organic acids, and a polyphenol antioxidant on gut integrity. Eighty Ross Broilers were exposed to 20–22°C (control – normothermic) or to 35–39.5°C (heat stress) for eight hours a day for a period of 1 or 5 days. Birds were fed a standard diet, or a diet supplemented with the test blend. Thereafter, birds were euthanized, and intestinal sections were excised for morphological, morphometric and gene expression analyses. Blood samples were collected for glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px) activity and trolox equivalent antioxidant capacity (TEAC) determination. Heart and liver tissues were used to quantify the expression of heat shock proteins 60 and 70 (HSP60 and HSP70, respectively) and inhibitor of kappa light chain gene enhancer in B cells alpha (IKBA). The jejunum was the most sensitive intestinal section, where heat stress modulated the expression of HSP70, of the inflammatory markers IKBA, interleukin 8 (IL-8), interferon gamma (IFNγ), and toll-like receptor 4 (TLR4). Moreover, expression of tight junctions (CLDN1, ZO1 and ZO2) and nutrient transporters (PEPT1 and EAAT3) was modulated especially in the jejunum. In conclusion, the feed additive blend protected intestines during heat stress from the decrease in villus height and crypt depth, and from the increase in villus width. Especially in the jejunum, heat stress played an important role by modulating oxidative stress and inflammation, impairing gut integrity and nutrient transport, and such deleterious effects were alleviated by the feed additive blend.

RESEARCH HIGHLIGHTS

• Jejunum is the most sensitive intestinal segment during heat stress.

• Heat stress affects the expression of tight junctions and nutrient transporters.

• Feed management helps to alleviate the disturbances caused by heat stress.

• A blend of MCFA, organic acids and a polyphenol protects broilers under heat stress.

     

An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish

A hitherto unrecognised lethal toxin from the extracellular products (ECP) of Aeromonas hydrophila is described. The pure toxin was 300 times more toxic than the crude ECP and is the most toxic substance so far described from this bacterium, with a minimum lethal dose of 0.05 micrograms g-1 fish. The toxin had high acetylcholinesterase activity and occurred in native ECP as a monomeric 15.5 kDa polypeptide. The purified toxin had five isoelectric focusing forms ranging from pl 4.45 to 4.70. The ECP of each of six strains of A. hydrophila isolated from fish possessed acetylcholinesterase activity suggesting that the toxin is common in this species. The toxin was not a cytolysin and produced no gross pathology in injected fish. Its enzymic nature, low lethal dose, lack of tissue pathology and its apparent narcotic effect suggest that this toxin may act upon the central nervous system of the fish.     

Frequent recovery of a single clonal type of multidrug-resistant Staphylococcus aureus from patients in two hospitals in Taiwan and China

One hundred thirty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from patients with S. aureus infections between January 1998 and February 1999 in two hospitals, one located in Taipei, Taiwan, and another in Nanjing, People's Republic of China, were examined for antibiotic susceptibility and for clonal type by a combination of three methods: hybridization of ClaI restriction digests with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis of chromosomal SmaI digests. Selected isolates representing each clonal type were also analyzed by spaA typing, multilocus sequence typing, and a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. The overwhelming majority of isolates (126 of 132 or 95%) belonged to minor variants of a single clonal type resembling the Brazilian and Hungarian epidemic MRSA clones, which showed a common spaA type and which were either sequence type 239 (ST239) or ST241 (a single-locus variant of ST239) in association with SCCmec type III or IIIA.