The hybrid spheroid clonogenic assay for the intrinsic radio- and chemo-sensitivities of human tumors.

The Hybrid Spheroid assay is based on packaging tumor cells into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates provide an in vivo-like environment for entrapped test cells. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of noncolony forming spheroids and the Poisson distribution, the average number of clonogens per spheroid can be calculated. The clonogenicity and radiation survival curves of cells derived from human tumors (of the maxilla, tongue, larynx, mouth floor, lung, breast, ovary, and colon) were so determined. Plating efficiency was increased in these normally poorly plating tumor cells, thus enabling survival measurements which are not practical using conventional methods. The Hybrid Spheroid assay has also been applied to determine the chemosensitivity of colon cancer cells.     

Assessment of chlorinated pesticide residues in cigarette tobacco based on supercritical fluid extraction and GC-ECD

It has been established that the organochlorinated compounds(OCC) DDT and DDE are xenoestrogens which influence both normaland neoplastic estrogen-responsive tissues. Therefore, it hasbeen hypothesized that OCC contribute to the risk for breastcancer. Although the food chain has been recognized as a majorsource of human exposure to these compounds, tobacco and tobaccosmoke were also considered as sources of exposure to OCC. Thisstudy was aimed at quantifying OCC in tobacco and cigarettesmoke and at documenting changes in the concentrations of thesepesticides in tobacco products since 1970 when OCC were bannedfor use on tobacco. To determine the levels of OCC residueson tobacco, we developed a new method, based on supercriticalfluid extraction, followed by clean-up on an alumina column,and analysis by gas chromatography with electron capture detection.The detection limit for an individual OCC is 1 ng/g tobacco,the relative SD is <10% for each analyte and the new methodcompares well with the standardized method that involves conventionalorganic solvent extraction. The major OCC determined in thetobaccos and in cigarette smoke of US commercial brands thatwere manufactured in the preceding three decades were p, p'-isomersof DDD (1540–20 220 ng/g tobacco), DDT (720–13 390ng) and DDE (58–730 ng). Since 1970, the concentrationsof individual OCC in tobacco have gradually decreased by >98%.The transfer rate from tobacco into mainstream smoke amountsto 22% for DDD, 19% for DDT and 27% for DDE. Today, the concentrationsof the OCC in US tobacco are below the maximum permissible limitsset by the Environmental Protection Agency. While until 1970the OCC in tobacco and tobacco smoke contributed significantlyto the bioaccumulation of the pesticides in smokers, at thistime tobacco and cigarette smoke are a minor source of humanexposure.     

Pelvifemoral external fixation for the treatment of open fractures of the proximal femur caused by firearms

Seventeen patients with open fractures of the upper third of the femur were treated using a pelvifemoral external fixation device. All of them had grade III open fractures resulting from high-velocity missile and explosive injuries with massive foreign body contamination. Sciatic nerve injury was present in five (29.4%); abdominal viscera and thoracic wall injuries were present in two patients (11.8%). There were no major arterial injuries. Full weight bearing was allowed after clinical and radiological bone healing (average 11.5 months). Chronic osteitis with fistula and sequestra developed in two (11.8%) patients. There were no nonunions and no refractures. Minor painless limitation of hip motion persisted in all patients. Upper-third femoral open fractures due to firearms are a unique type of open fractures. They are usually highly comminuted; therefore, stable fixation is difficult or impossible to achieve using external fixation with transfixation of the fracture site. On the other hand, the risk of infection is high following intramedullary nailing. Pelvifemoral external fixation allows adequate management of the soft tissue wounds, provides stable bone fixation and allows early patient mobilization.     

Potential hepatoprotective effects of fullerenol C60(OH)24 in doxorubicin-induced hepatotoxicity in rats with mammary carcinomas

The aim of this study was to investigate the potential protective role of fullerenol C60(OH)24 on doxorubicin-induced liver toxicity using in vivo (female Sprague-Dawley rats) and in vitro (human hepatocellular carcinoma - HepG2; colorectal adenocarcinoma cell lines - Caco-2) approaches. The first (healthy control) and second (control with chemically induced mammary carcinomas) group received saline only. The third, fourth and fifth group (all with breast cancer) were injected (i.p.) with a single dose of doxorubicin (8mg/kg), doxorubicin/fullerenol (100mg/kg of fullerenol 30min before administration of 8mg/kg doxorubicin) and fullerenol (100mg/kg), respectively. Two days after treatment, the rats were sacrificed. Results showed that treatment with doxorubicin alone caused significant changes in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), as well as in the levels of malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px), total antioxidant status (TAS), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) in the liver tissue. These effects were significantly reduced for all investigated parameters by pre-treatment with fullerenol but not for the MDA and GSH level. The HepG2 and Caco-2 cell lines were continuously treated with fullerenol for 12h, 24h, 48h and 96h at concentrations of 10microg/mL and 44microg/mL. With the aim of evaluating the modulating activity of fullerenol on doxorubicin-induced hepatotoxicity, the cell lines were simultaneously treated with doxorubicin (1microm; 5microm) and fullerenol (10microg/mL; 44microg/mL) in different combinations. When the cells are treated with 5microm doxorubicin along with the fullerenol, we can see a significant improvement of the cell capability during the entire time-line. We can conclude that fullerenol has cytotoxic effects on HepG2 by itself, but when the oxidative stress is too high the cytotoxic effects of fullerenol are overcome by its protective role as a strong antioxidant compound.     

Role of autologous fibrin tissue adhesive in abdominal surgery

Fibrin tissue adhesive (FTA) is an agent developed for achieving better hemostasis and adhesion of living tissue. FTA appears to have no tissue toxicity, promotes a firm seal in seconds to minutes, is reabsorbed in days to weeks following application, and appears to promote local tissue growth and repair. It can be used in various surgical procedures. It has been used preoperatively, perioperatively and postoperatively in abdominal surgical procedures. There were no side effects. Improvement of surgical hemostasis was obvious in all patients. Anastomotic leakages were closed in a shorter time without surgical intervention. Sero-lymphatic drainage after surgical procedures that include extensive lymph node dissections was less. Use of FTA in treatment of fistula in ano was successful. The data would indicate that use of FTA may be a good alternative in solving various conditions in every day clinical practice, although a bigger randomized series, and longer follow up is needed.     

Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources

The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (alpha1, alpha2, beta, gamma, kappa, epsilon, eta, iota, lambda, theta, and zeta) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int- micro, Int-nu, and Int-xi. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx(-)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(-) and ehxA-negative isolates. Int-beta, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-zeta (39 of 213 [18.3%] isolates; 11 serotypes), Int-theta (25 of 213 [11.7%] isolates; 15 serotypes), Int-gamma (19 of 213 [8.9%] isolates; 9 serotypes), and Int-epsilon (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes alpha1, alpha2, kappa, lambda, xi, micro, nu, and iota were infrequently identified; and Int-eta was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (alpha, beta-xi, gamma, kappa, epsilon-eta-nu, iota- micro, lambda, theta, and zeta). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.     

Cell surface antigens of Mycoplasma species bovine group 7 bind to and activate plasminogen

Mycoplasma species bovine group 7 bound plasminogen at the cell surface in a lysine-dependent manner. Cell-bound plasminogen was rapidly activated to plasmin by exogenous urokinase, and this activity was associated with plasminogen binding capacity. Binding assays using plasminogen modified with a trifunctional cross-linking agent revealed several binding proteins.     

Bovine non-O157 Shiga toxin 2-containing Escherichia coli isolates commonly possess stx2-EDL933 and/or stx2vhb subtypes

stx(2) genes from 138 Shiga toxin-producing Escherichia coli (STEC) isolates, of which 127 were of bovine origin (58 serotypes) and 11 of human origin (one serotype; O113:H21), were subtyped. The bovine STEC isolates from Australian cattle carried ehxA and/or eaeA and predominantly possessed stx(2-EDL933) (103 of 127; 81.1%) either in combination with stx(2vhb) (32 of 127; 25.2%) or on its own (52 of 127; 40.4%). Of 22 (90.9%) bovine isolates of serotype O113:H21, a serotype increasingly recovered from patients with hemolytic uremic syndrome (HUS) or hemorrhagic colitis, 20 contained both stx(2-EDL933) and stx(2vhb); 2 isolates contained stx(2vhb) only. Although 7 of 11 (63.6%) human O113:H21 isolates associated with diarrhea possessed stx(2-EDL933), the remaining 4 isolates possessed a combination of stx(2-EDL933) and stx(2vhb). Three of the four were from separate sporadic cases of HUS, and one was from an unknown source.     

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n = 10 per group) or Suvaxyn^® M. hyo (n = 12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n = 12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.     

Mixed phenotype acute leukemia of T/myeloid type with a prominent cellular heterogeneity and unique karyotypic aberration 45,XY, dic(11;17)

Introduction. A 26‐yr‐old male patient with mixed phenotype acute leukemia of T/myeloid type with prominent leukemic cell heterogeneity, and the presence of a so far unreported karyotype aberration in this type of acute leukemia 45,XY, dic(11;17)(11qter→11p11.2::17p11.2→17qter) is presented. Methods. Flow immunocytometry was performed by direct multicolor immunofluorescent technique on bone marrow aspirates. Cytogenetic analyses were performed using G‐banding method by direct preparation of unstimulated bone marrow cells and following 24 hours of culture in RPMI 1540 culture medium with 25% fetal calf serum at 37°C Results. The flow immunocytometry of bone marrow nucleated cells revealed the existance of three distinct blast cell populations with overlapping immunophenotypes. Predominant blast cell population had an early myeloid phenotype and aberrant expression of CD7 antigen (HLA‐DR⁺, CD34⁺, anti‐MPO⁺, CD117⁺, CD33⁺, CD13⁺, CD7^+low, cyCD3^?, TdT^?). The other two blast cell populations, smaller in cell diameter and less sizable in cell proportion, both shared the T‐lymphoid features. The patient was treated with ADE protocol (etoposide, cytarabine and doxorubicine). A complete remission was achieved and lasted 5 months. Conclusion. A case of MPAL with complex biological features, 45,XY, dic(11;17)(11qter→11p11.2::17p11.2→17qter) karyotype and an aggressive, therapy‐resistant clinical course, is presented.