Image-based determination of chord lengths in air-dried lungs.

An image-based method was developed in order to determine chord lengths in sections of dog and sheep lungs air-dried at 25 cm H2O transpulmonary pressure. To facilitate image processing, optical contrast in the sections was optimized with respect to section thickness, stain type, stain concentration, staining temperature, staining time and clearing method. Digital processing of images used standard procedures, e.g. thresholding, dilation and thinning, as well as algorithms written to subtract background, delete spots and measure chord lengths. Correlation of image-based vs. manual determination of mean chord length in 17 sections from a sheep lung, stained for optimal contrast yielded an R2 of 0.82 (P < 0.0001). For 95 sections from three dog lungs, stained with lower contrast, R2 was 0.65 (P < 0.0001). Weaker correlations were observed between image-based and manual determinations of the standard deviation, the geometric standard deviation, and the 95th percentile of chord lengths (P < 0.05). The results show that image-based stereology of inflated air-dried lungs can provide valid measures of mean chord length and other statistics of chord length distribution.

Characterization of metalworking fluid aerosols in bearing grinding operations.

The concentrations and size distribution of metalworking fluid aerosols were investigated in grinding operations in the bearing manufacturing industry. Fifteen paired open- and closed-face cassette samples and five cascade impactor samples were obtained in each of three types of grinding machinery (face, microcentric, and progressive). Aerosol mass concentration as measured by open-face filter sampling ranged from 0.34 to 2.43 mg/m3. As measured by closed-face sampling the range was 0.14 to 2.01 mg/m3. For each grinding process, open-face concentration was significantly higher than the closed-face concentration (paired t-test, p <0.05). Mass median aerodynamic diameter (MMAD) ranged from 3.33 to 6.26 microm. The percentage of mass greater than 9 microm ranged from 8.0 to 45.3. The MMAD and fraction greater than 9 microm were significantly greater for the aerosol produced by the face grinder compared with the other two processes. The results indicate that (1) closed-face sampling results in a lower aerosol mass concentration, as compared with open-face sampling, with the degree of difference being somewhat dependent on grinding process; and (2) the particle size distribution and concentration of metalworking fluid aerosols may vary with the type of grinding operation sampled.

Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease.

OBJECTIVE: To evaluate the association among clinical signs, results of cytologic evaluation of bronchoalveolar lavage (BAL) fluid, and measures of pulmonary function in horses with inflammatory respiratory disease. ANIMALS: 9 healthy horses, 5 horses with inflammatory airway disease (IAD), and 9 horses with chronic obstructive pulmonary disease (COPD). PROCEDURES: Clinical examination, lung function tests, and BAL were performed on each horse. RESULTS: Standard lung mechanics of horses with exacerbated COPD differed significantly from those of healthy horses; however, there were few differences among horses with IAD, horses with COPD during remission, and healthy horses. Most variables for forced expiration (FE) in horses with COPD or IAD differed significantly from those for healthy horses. Results of clinical examination had low to moderate sensitivity and predictive values for a diagnosis of COPD (range, 67 to 80%). Results of FE tests had high sensitivity, specificity, and predictive values for a diagnosis of COPD (79 to 100%), and results of standard lung mechanics tests had low sensitivity and predictive values (22 to 69%). Percentage of neutrophils in BAL fluid was highly sensitive (100%) but moderately specific (64%) for a diagnosis of COPD. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical examination is moderately accurate for establishing a diagnosis of COPD. Forced expiration tests can specifically detect early signs of airway obstruction in horses with COPD and IAD that may otherwise be inapparent. Cytologic evaluation of BAL fluid allows early detection of inflammatory respiratory disease, but it is not specific for COPD.

[The Tarihü'l-Etibba by Ishak bin Huneyn]. / Ishâk B. Huneyn'in Târîhü'l-Etibbâ'si.

This is a translation of Rosenthal's article on Ishak b. Hunayn's Târîh al-Atibbâ, into Turkish. It also includes Ioannes Philoponos's (John Philoponos or Yahyâ en-Nahvî chronology of physicians. The article deals with the chronology of ancient physicians from the beginning to the year 290 of the Hegira and their views on the sources of medicine. The translator contributes by means of foot notes.

Production of stem-cell transplants according to good manufacturing practice.

Peripheral blood stem cells (PBSCs) are used for transplantation to reconstitute the hematopoietic system after high-dose chemotherapy. They are harvested from peripheral blood after mobilization by cytokines and/or chemotherapy. Further ex vivo manipulation steps (e.g., selection of CD34+ PBSCs, purging, expansion, and differentiation or gene transfer) can be performed. In 1997, more than 12,000 PBSC preparations were transplanted in Europe and the total number is steadily increasing [1]. To ensure quality and safety of the final cell products intended for clinical use, national and international guidelines and regulations have been issued. The implementation of a quality assurance (QA) program including the principles of good manufacturing practice (GMP) and a quality control system is a major requirement. GMP regulations apply to all phases of cell collection, processing, and storage, and to documentation, training of personnel, and equipment of the cell processing laboratory. They have to be followed by pharmaceutical companies and medical doctors who are involved in PBSC processing at academic institutions. The complicated regulatory network for the manufacturing of cell products will help to standardize these procedures and ensure consistent quality and safety in the long term. This will be in the interest of patients and reduce risks of application of individual cell preparations.

Forced expiration: a test for airflow obstruction in horses.

The purpose of this study was to assess whether our method of inducing forced expiration detects small airway obstruction in horses. Parameters derived from forced expiratory flow-volume (FEFV) curves were compared with lung mechanics data obtained during spontaneous breathing in nine healthy horses, in three after histamine challenge, and in two with chronic obstructive pulmonary disease (COPD) pre- and posttherapy with prednisone. Parameters measured in the healthy horses included forced vital capacity (FVC = 41.6 +/- 5.8 liters; means +/- SD) and forced expiratory flow (FEF) at various percentages of FVC (range of 20.4-29.7 l/s). Histamine challenge induced a dose-dependent decrease in FVC and FEF at low lung volume. After therapy, lung function of the two COPD horses improved to a point where one horse had normal lung mechanics during tidal breathing; however, FEF at 95% of FVC (4.9 l/s) was still decreased. We concluded that FEFV curve analysis allowed the detection of induced or naturally occurring airway obstruction.

Crucial role of TNF-alpha in CD8 T cell-mediated elimination of 3LL-A9 Lewis lung carcinoma cells in vivo.

The role of perforin, IFN-gamma, and TNF-alpha in anti-tumor CD8 T cell immunity was examined in a new tumor model using a CD8 T cell epitope (GP33) derived from lymphocytic choriomeningitis virus as a tumor-associated Ag. In contrast with parental 3LL-A9 (A9) Lewis lung carcinoma cells that progressively grow in C57BL/6 mice, s.c. injection of GP33-transfected A9GP33 tumor cells induced a protective GP33-specific CD8 T cell response that led to complete tumor cell elimination. Tumor regression was dependent on perforin, IFN-gamma, or TNF-alpha, because A9GP33 tumors developed in mice deficient in one of these genes. A9GP33 tumors arising in perforin- and IFN-gamma-deficient mice represented GP33 Ag-loss variants, demonstrating that GP33-specific CD8 T cells from these mice were able to exert an Ag selection pressure. In contrast, tumor cells growing in TNF-alpha knock-out mice still expressed the tumor-associated GP33 peptide despite the presence of activated GP33-specific CD8 T cells. These findings provide evidence for a crucial role of TNF-alpha in A9 tumor cell elimination by CD8 T cells in vivo.

The distribution of inhaled particles in aerosol measurements of pulmonary airspace size.

When the deposition of aerosol boluses is used to estimate mean pulmonary airspace size, an implicit assumption is made that the inhaled particles are distributed uniformly among normal and diseased lung regions. This assumption was examined in a series of dogs in which emphysema was experimentally induced by exposure to papain. After the experimental disease had developed for several weeks, boluses of fluorescent particles were inhaled, using a breathing pattern similar to that used for aerosol measurements of airspace size. The lungs were then excised and 18-20 tissue blocks were obtained from each lung. A section from each tissue block was analyzed to determine the mean liner intercept (Lm), which was considered as an index of lung injury. In the same sections, the density of particles was determined by counting particles in a number of microscopic fields and dividing the particle count by the number of fields sampled. Correlation analysis generally revealed a negative correlation of particle density with Lm, indicating fewer particles being delivered to diseased regions. One lung, however, showed a positive correlation between particle density and Lm. Correction for the fractional deposition of aerosol in the lung regions weakened but did not reverse the relationship between particle density and Lm. A model calculation of the effect of the observed nonuniform distribution of aerosol on the determination of airspace size found a negligible effect of uneven ventilation on mean airspace size determination in this experimental preparation.

Aerosol measurement of airspace diameter correlates with morphometry in normal and papain-exposed air dried lungs.

Aerosol measures of effective airspace diameter (EAD) were correlated with morphometric parameters in a series of 8 canine lungs, 5 of which had been exposed to papain in order to cause experimental emphysema. In an effort to preserve alveolar dimensions without shrinkage, the lungs were fixed by air drying at total lung capacity. EAD was measured with 400-cm2 boluses, with mean penetration index (Pen), defined as volumetric bolus penetration/total lung capacity, equal to 0.34 (EAD400), and with 800-cm2 aerosol boluses, with mean Pen equal to 0.59 (EADdeep). Morphometric analysis, following measurement of EADs, determined the mean linear intercept and other parameters of the chordlength distribution, including the 90th, 95th, and 99th percentiles (p90, p95, p99), the standard deviation, and the geometric standard deviation (GSD). EAD400 was significantly correlated with Lm (r2 = .68, P < .05), p95, (r2 = .85, P < .01), p99 (r2 = .94, P < .001), and GSD (r2 = .94, P < .001). Similar results were found with EADdeep. In general, correlations were stronger between EAD and p95, p99, or GSD than between EAD and Lm. A comparison with a previous similar study relating EAD to morphometry in a series of lungs fixed by formalin fixation found closer agreement between EAD and morphometry when lungs were fixed by air drying. Overall, the data support the validity of EAD as an in vivo method of determining airspace size at total lung capacity.

Cmdr1, a chicken P-glycoprotein, confers multidrug resistance and interacts with estradiol.

We have cloned from a chicken intestinal cDNA library Cmdr1, the first avian P-glycoprotein. Cmdr1 is 67% and 69% identical to proteins encoded by the human MDR1 and MDR2 genes, respectively. Functional expression of Cmdr1 in both mouse NIH 3T3 and yeast cells demonstrated that Cmdr1 represents the avian ortholog of human Mdr1, since it confers resistance to several anticancer drugs and the fluorescent dye rhodamine 6G. Northern and immunoblot analysis showed that CMDR1 is highly expressed throughout the intestine and in the liver, and to a considerable extent in kidney, brain, lung, heart, eye and follicles. In situ hybridization revealed a cell type-specific expression of CMDR1 in the intestinal epithelium, with high levels in the villi of the small and large intestine as well as crypt cells. These data suggest that Cmdr1 could play a role in intestinal detoxification. Most interestingly, immunoblotting showed that Cmdr1 is also expressed in ovarian tissues, particularly in theca cells, the major site for ovarian estrogen production in birds. Indeed, competition experiments indicated that Cmdr1 interacts with estradiol, since rhodamine 6G efflux was efficiently blocked by estradiol in NIH 3T3 cells expressing Cmdr1. Rhodamine efflux was also blocked by PSC-833, a specific inhibitor of steroid-transporting P-glycoproteins from mammalian cells. We propose that Cmdr1 in ovarian cells could be involved in the cell type-specific transport or release of estrogen that is essential for avian follicular development.

Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep.

Seven experiments including a total of 47 pigs, 11 wild boars, 26 rabbits, 10 hares and 16 sheep were carried out to assess the efficacy, safety and transmission of the Chinese vaccine strain of the classical swine fever virus (CSFV) administrated by the oral route. Within 3 weeks after oral vaccination, a clear seroconversion occurred in the pigs. Six weeks after vaccination, vaccinated pigs were fully protected against a virulent challenge. The C-strain was not isolated from tonsils, spleen, lymph nodes, thymus, saliva, urine and faeces of pigs within 4 days after oral vaccination. In one experiment, susceptible pigs were placed in direct contact with vaccinated pigs. None of these contact-exposed pigs became serologically positive for CSFV antibodies. It is concluded that the C-strain induces protection in pigs when administrated by the oral route and is not shed by vaccinated pigs. Serum anti-CSFV antibodies developed in seven out of eight wild boars vaccinated by the oral route. No vaccine virus was detected in the spleen and tonsils of these animals. The results in wild boar were in accordance with those obtained in domestic pigs. Sheep did not show any clinical signs after oral vaccination while rabbits had moderate hyperthermia and growth retardation. No clinical response to oral immunisation in hares was detected. At the end of the experiment, no sheep had detectable serum antibodies against CSFV, whereas a few vaccinated rabbits and hares became seropositive. None of the contact-exposed rabbits and hares seroconverted. These data indicate that the C-strain is safe for sheep and as expected, moderately or not pathogenic for rabbits and hares. These efficacy and safety studies on oral vaccination with the C-strain under experimental conditions provide essential information for further studies in wild boars under experimental and field conditions, including assays with baits to control a CSF epidemic.

Aerosol probes of lung injury in a 28-wk longitudinal study of mild experimental emphysema in dogs.

After baseline measurements of lung mechanics, effective air space diameter (EAD), and aerosol dispersion (AD), three dogs were exposed to two treatments of aerosolized papain (3 ml of a 4% solution), and measurements were repeated during a 28-wk follow-up period. EAD and AD were measured with boluses of 0.7-micrometer particles of di-2-ethylhexl sebacate, with Pen (i.e., volumetric bolus penetration/total lung capacity) between 0.1 and 0.4. After papain exposure, EAD increased a mean of 28% (P < 0.0001) and AD (Pen = 0.3, 0.4) increased 4-7% (P < 0.03). The progression of injury was indicated by increasing trends in total lung capacity (P < 0.05), residual volume (P < 0.05), and EAD (P = 0.06) through week 18. There was no evidence of disease progression between weeks 18 and 28, whereas some of the data for individual dogs suggested partial recovery from lung injury at week 28. The results show that aerosol probes can detect and characterize mild lung injury in experimental emphysema.

[Continuous delivery of epidermal growth factor to wounds in vivo by genetically modified fibroblasts transfected with a novel chimeric construct].

OBJECTIVE: This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS: It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS: Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION: A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.

Changes in medical education: the beliefs of medical students.

This study aimed to explore medical students' beliefs about medical education in the light of recent General Medical Council (GMC) recommendations for change and to examine how these beliefs relate to the students' year of training. All undergraduate medical students at a London medical school were given a questionnaire concerning basic sociodemographic information and levels of agreement with a number of statements relating to medical education. The response rate was 75.4% (n = 383). Most students agreed with the majority of recommendations for change. However, two key recommendations, 'more community-based teaching' and 'more optional courses', were supported by only 50.2 and 46.3% of respondents, respectively. Using factor analysis, students' responses were classified into five educational belief orientations relating to 'psychosocial' (e.g. communication skills), 'scientific' (e.g. new technologies), 'active' (e.g. optional courses), 'reform' (e.g. decreasing factual load) and 'group' (e.g. small-group teaching) educational belief orientations. The results showed variations in students' belief orientations across the 5 years of training. The results are discussed in terms of their implications for implementing the GMC recommendations and the impact of medical education on students' belief systems.

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo.

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.

Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium.

Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34+ and CD34+/HLA-DRlo peripheral blood progenitor "stem" cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34+, CD34+/HLA-DRlo, and CD34+/CD38lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy.

Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter.

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

Cytokine gene transfer in cancer therapy.

New strategies based on gene transfer technology are employed in cancer therapy. Cytokines are polypeptides involved in immunity and inflammation, and essentially control the magnitude of the immune response. Genetically modified tumor cells releasing various cytokines have been shown to enhance tumor immunogenicity and to induce the regression of preexisting tumors. In some instances, immunological memory has been generated to resist the subsequent challenge with unmodified, parental tumor cells. Cytokine gene transfer into antitumor effector cells, as well as antigen presenting cells, is also being investigated to augment antitumor immune responses.

Oncogene and HSP-70 expression in primary tumor cell cultures of renal cell carcinoma compared to their corresponding cell line.

BACKGROUND: Tumor progression in renal cell carcinoma (RCC) can be explained by a multistep model, in which the activation of certain oncogenes such as c-neu and c-fos appear to be early events in tumorigenesis, while the expression of p53 and pan-ras are found in advanced stages. MATERIAL AND METHODS: The expression of oncogenes and growth factors was examined in 29 primary tumor cell cultures (PTCC) of RCC using immunocytochemistry. RESULTS: In PTCC high expression of c-neu and c-fos was present in all tumors, whereas mdr, TGF-alpha, EGF, c-myc, pan-ras, p53 and HSP-70 was detected at low expression levels. In 27% (8/29) of PTCC, cell lines (CL) were established. Oncogene expression was increased in CL compared to PTCC. CONCLUSION: The pattern of oncogene expressions found in CL is similar to findings described in highly malignant tumors in vivo. Therefore, the establishment of CL seems to depend on a selective recruitment of tumor cells with an upregulated oncogene expression.

Cloning and sequence analysis of the immediate promoter region and cDNA of porcine granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage. Recombinant G-CSF (rG-CSF) is routinely used in the prevention of chemotherapy-induced neutropenia and in the setting of bone marrow transplantation. Chronic idiopathic and congenital neutropenic disorders also show improvement after rG-CSF injections. Applications of either rG-CSF or G-CSF gene transfected cells into mice give rise to leukocytosis, which can be measured easily. This makes G-CSF a versatile tool for studying systemic effects of therapeutic proteins delivered by genetically modified cells in vivo. Although the biological activity of G-CSF is not species-specific, studies on long-term expression would require the use of species-identical proteins in order to avoid host immune reactions against the foreign gene product. Because of the physiological and immunological similarity of pigs and human, the pig has become an important large-animal model for biomedical research. We have therefore cloned porcine G-CSF cDNA from RNA isolated from pig PBLs. Pig G-CSF is a 195-amino-acid polypeptide that shares a high degree of homology to human (78%), murine (71%) as well as rat (68%) G-CSF. In contrast to human and murine, but not to rat G-CSF, a different ATG translation start codon is used, resulting in a shorter, but still functional signal sequence.