Characterization of human breast cancer tissues by infrared imaging.

Fourier Transform InfraRed (FTIR) spectroscopy coupled to microscopy (IR imaging) has shown unique advantages in detecting morphological and molecular pathologic alterations in biological tissues. The aim of this study was to evaluate the potential of IR imaging as a diagnostic tool to identify characteristics of breast epithelial cells and the stroma. In this study a total of 19 breast tissue samples were obtained from 13 patients. For 6 of the patients, we also obtained Non-Adjacent Non-Tumor tissue samples. Infrared images were recorded on the main cell/tissue types identified in all breast tissue samples. Unsupervised Principal Component Analyses and supervised Partial Least Square Discriminant Analyses (PLS-DA) were used to discriminate spectra. Leave-one-out cross-validation was used to evaluate the performance of PLS-DA models. Our results show that IR imaging coupled with PLS-DA can efficiently identify the main cell types present in FFPE breast tissue sections, i.e. epithelial cells, lymphocytes, connective tissue, vascular tissue and erythrocytes. A second PLS-DA model could distinguish normal and tumor breast epithelial cells in the breast tissue sections. A patient-specific model reached particularly high sensitivity, specificity and MCC rates. Finally, we showed that the stroma located close or at distance from the tumor exhibits distinct spectral characteristics. In conclusion FTIR imaging combined with computational algorithms could be an accurate, rapid and objective tool to identify/quantify breast epithelial cells and differentiate tumor from normal breast tissue as well as normal from tumor-associated stroma, paving the way to the establishment of a potential complementary tool to ensure safe tumor margins.

Label-free phenotyping of peripheral blood lymphocytes by infrared imaging.

It is now widely accepted that the immune microenvironment of tumors and more precisely Tumor Infiltrating Lymphocytes (TIL) play an important role in cancer development and outcome. TILs are considered to be important prognostic and predictive factors based on a growing body of clinical evidence; however, their presence at the tumor site is not currently assessed routinely. FTIR (Fourier transform infrared) imaging has proven it has value in studying a range of tumors, particularly for characterizing tumor cells. Currently, very little is known about the potential for FTIR imaging to characterize TIL. The present proof of concept study investigates the ability of FTIR imaging to identify the principal lymphocyte subpopulations present in human peripheral blood (PB). A negative cell isolation method was employed to select pure, label-free, helper T cells (CD4(+)), cytotoxic T cells (CD8(+)) and B cells (CD19(+)) from six healthy donors PB by Fluorescence Activated Cell Sorting (FACS). Cells were centrifuged onto Barium Fluoride windows and ten infrared images were recorded for each lymphocyte subpopulation from all six donors. After spectral pre-treatment, statistical analyses were performed. Unsupervised Principal Component Analyses (PCA) revealed that in the absence of donor variability, CD4(+) T cells, CD8(+) T cells and B cells each display distinct IR spectral features. Supervised Partial Least Square Discriminant Analyses (PLS-DA) demonstrated that the differences between the three lymphocyte subpopulations are reflected in their IR spectra, permitting their individual identification even when significant donor variability is present. Our results also show that a distinct spectral signature is associated with antibody binding. To our knowledge this is the first study reporting that FTIR imaging can effectively identify T and B lymphocytes and differentiate helper T cells from cytotoxic T cells. This proof of concept study demonstrates that FTIR imaging is a reliable tool for the identification of lymphocyte subpopulations and has the potential for use in characterizing TIL.

Breast cancer and melanoma cell line identification by FTIR imaging after formalin-fixation and paraffin-embedding.

Over the past few decades, Fourier transform infrared (FTIR) spectroscopy coupled to microscopy has been recognized as an emerging and potentially powerful tool in cancer research and diagnosis. For this purpose, histological analyses performed by pathologists are mostly carried out on biopsied tissue that undergoes the formalin-fixation and paraffin-embedding (FFPE) procedure. This processing method ensures an optimal and permanent preservation of the samples, making FFPE-archived tissue an extremely valuable source for retrospective studies. Nevertheless, as highlighted by previous studies, this fixation procedure significantly changes the principal constituents of cells, resulting in important effects on their infrared (IR) spectrum. Despite the chemical and spectral influence of FFPE processing, some studies demonstrate that FTIR imaging allows precise identification of the different cell types present in biopsied tissue, indicating that the FFPE process preserves spectral differences between distinct cell types. In this study, we investigated whether this is also the case for closely related cell lines. We analyzed spectra from 8 cancerous epithelial cell lines: 4 breast cancer cell lines and 4 melanoma cell lines. For each cell line, we harvested cells at subconfluence and divided them into two sets. We first tested the "original" capability of FTIR imaging to identify these closely related cell lines on cells just dried on BaF2 slides. We then repeated the test after submitting the cells to the FFPE procedure. Our results show that the IR spectra of FFPE processed cancerous cell lines undergo small but significant changes due to the treatment. The spectral modifications were interpreted as a potential decrease in the phospholipid content and protein denaturation, in line with the scientific literature on the topic. Nevertheless, unsupervised analyses showed that spectral proximities and distances between closely related cell lines were mostly, but not entirely, conserved after FFPE processing. Finally, PLS-DA statistical analyses highlighted that closely related cell lines are still successfully identified and efficiently distinguished by FTIR spectroscopy after FFPE treatment. This last result paves the way towards identification and characterization of cellular subtypes on FFPE tissue sections by FTIR imaging, indicating that this analysis technique could become a potential useful tool in cancer research.

Seroprevalence of Actinobacillus pleuropneumoniae serovars 2, 3 and 9 in slaughter pigs from Belgian fattening farms.

Fifty randomly selected fattening pig herds were studied to investigate the epidemiological characteristics of infections with Actinobacillus pleuropneumoniae serovars 2, 3 and 9, and to identify risk factors for their within-herd seroprevalences. Information about 13 farm characteristics was obtained by means of a questionnaire and used to assess potential risk factors for the percentage of slaughter pigs with antibodies against each of the three serovars. The presence of antibodies was measured with an indirect ELISA. The median within-herd seroprevalence for serovar 2 was 58 per cent (range 0 to 100 per cent), for serovar 3, 53 per cent (range 10 to 95 per cent), and for serovar 9, 35 per cent (range 5 to 100 per cent). All but one farm tested positive for A pleuropneumoniae serovar 2, and all the farms were positive for A pleuropneumoniae serovars 3 and 9. There was a positive association (P < 0.05) between each pair of serovars. The within-herd seroprevalence of serovar 2 was significantly associated with the density of pig herds in the municipality (odds ratio [OR] = 1.60; P < 0.05) and with the absence of preventive medication at the start of the fattening period (OR = 2.77; P < 0.10). No significant risk factors were found for serovar 3. The percentage of pigs positive for serovar 9 was significantly associated with a slaughter date in June (OR = 2.30; P < 0.10) and with herds in which the finishing houses were not divided into separate compartments (OR = 2.99; P < 0.05).

Effectiveness of treatment with lincomycin hydrochloride and/or vaccination against Mycoplasma hyopneumoniae for controlling chronic respiratory disease in a herd of pigs.

A herd of pigs infected with Mycoplasma hyopneumoniae was used in a double-blind randomised trial to assess the effectiveness of three control strategies against chronic respiratory disease in growing-finishing pigs. One group of 61 pigs received 220 ppm lincomycin hydrochloride in the feed from day 71 to day 91, a second group was vaccinated against M. hyopneumoniae at four and 28 days of age, and a third group received both treatments; a fourth group was left untreated as a control. Throughout the nursery-finishing period (day 29 to slaughter) the average daily weight gain and feed conversion rate of all the treated groups were slightly better than in the controls, but there were no significant differences between them. There were no significant differences between the treated groups in terms of clinical signs, serology, pathology or mortality, which was very low throughout the trial.

Herd factors associated with the seroprevalences of Actinobacillus pleuropneumoniae serovars 2, 3 and 9 in slaughter pigs from farrow-to-finish pig herds.

This cross-sectional epidemiologic study was conducted in 150 randomly selected farrow-to-finish pig herds to investigate descriptive epidemiological characteristics of infections with three different serovars of Actinobacillus pleuropneumoniae, and to identify risk factors for the within-herd seroprevalences of these serovars. Different farm characteristics (n = 28) were examined as potential risk factors for the percentage of pigs with antibodies against serovars 2, 3 and 9. The presence of antibodies was measured using an indirect ELISA. Logistic regression analyses were used to assess the associations between the potential risk factors and the proportion of seropositive pigs. The median within-herd seroprevalences were 95% (range: 0-100%), 100% (range: 10-100%), and 35% (range: 0-100%) for serovars 2, 3, and 9, respectively. There was a positive association (P < 0.001) between each of these serovars. The within-herd seroprevalence of serovar 2 was significantly higher in farms that purchased gilts from > or = 2 origin herds (OR = 2.33; P < 0.05) and in farms with poor biosecurity measures (OR = 4.62; P < 0.05). The proportion of pigs seropositive for serovar 3 was significantly higher when tested pigs were slaughtered in May-August and in November-December (OR = 5.96; P < 0.001), in herds without a growing unit (OR = 2.63; P < 0.01), and in herds with a direct air-entry into the finishing unit (OR = 1.92; P < 0.05). The within-herd seroprevalence of serovar 9 increased significantly in herds with poor biosecurity measures (OR = 1.76; P < 0.05). The study documented that infections with A. pleuropneumoniae serovars 2, 3, and 9 were very common in the selected herds, and that the sero-epidemiological characteristics and risk factors showed some variation depending on the serovar. The purchase policy of gilts and biosecurity measures are risk factors that can be improved fairly easily on pig farms.

Non-infectious factors associated with macroscopic and microscopic lung lesions in slaughter pigs from farrow-to-finish herds.

A cross-sectional epidemiological study was conducted in 150 randomly selected farrow-to-finish herds to investigate which non-infectious factors might act as risk indicators for the prevalence and severity of macroscopic and microscopic lung lesions in slaughter pigs. Data were collected during herd visits through inspections of the pigs and through interviews with the farmers. Macroscopic lung lesions of pneumonia and pleuritis were recorded at slaughter from 25 pigs per herd, and microscopic lung lesions of lymphohistiocytic infiltration were recorded from 10 pigs per herd. The median herd level prevalences were 24 per cent for pneumonia, 16 per cent for pleuritis and 60 per cent for lymphohistiocytic infiltration. Pneumonia lesions were negatively associated with pleuritis lesions and positively associated with lymphohistiocytic infiltration. Pleuritis lesions were negatively associated with lymphohistiocytic infiltration. The prevalence and the severity of pneumonia lesions were increased by a high frequency of purchasing gilts and by a slaughter date in January to February. The presence of a growing unit also increased the severity of pneumonia. The prevalence and the severity of pleuritis lesions were higher when there were more pig herds in the municipality, and when there were poor biosecurity measures, and their prevalence was increased by a slaughter date in January to February, and their severity by a slaughter date in March to April. An increase in the airspace stocking density in the finishing unit also increased the prevalence of pleuritis. The prevalence and the severity of lymphohistiocytic infiltration in the lung tissue were higher in herds purchasing gilts. Pigs raised in pens with slatted floors were also at higher risk of more severe lesions of lymphohistiocytic infiltration.

Efficacy of tilmicosin phosphate (Pulmotil premix) in feed for the treatment of a clinical outbreak of Actinobacillus pleuropneumoniae infection in growing-finishing pigs.

A double-blind randomized clinical trial was carried out to investigate the efficacy of tilmicosin (Pulmotil premix) for the treatment of a clinical outbreak of Actinobacillus pleuropneumoniae infection in growing-finishing pigs. The effects of tilmicosin administration in the feed at 400 mg/kg and an injection therapy of clinically diseased pigs with long-acting oxytetracycline (Terramycine LA) at 20 mg/kg bodyweight were compared. Both groups, totalling 147 pigs, were compared during a medication period of 15 days and a post-medication period of 11 days by means of different clinical and performance parameters. During the medication period, the tilmicosin group showed a significant advantage with respect to the number of new disease cases (P < 0.01), and a non-significant advantage regarding the number of removed pigs (P = 0.16), the number of sick pigs that recovered (P = 0.27) and the time to recovery (P = 0.42). During the post-medication period, the pigs of the tilmicosin group showed numerical non-significant benefits (P > 0.05) with respect to the clinical parameters. During the overall study period (26 days), the average daily gain and the feed conversion ratio were both significantly (P < 0.01) better in pigs from the tilmicosin group compared with pigs from the oxytetracycline group. This study demonstrated that in-feed medication of tilmicosin at a dosage of 400 mg/kg is efficacious for the treatment of a clinical respiratory disease outbreak of A. pleuropneumoniae infection in growing-finishing pigs. Compared with oxytetracycline injection of clinically diseased pigs, the tilmicosin treatment is particularly beneficial in the prevention of new disease cases while increasing or maintaining the performance of the pigs.

A comparative study of the preventive use of tilmicosin phosphate (Pulmotil premix) and Mycoplasma hyopneumoniae vaccination in a pig herd with chronic respiratory disease.

This study was conducted to compare the effects of a preventive in-feed medication programme using tilmicosin (Pulmotil 200 premix, Elanco Animal Health) at 200 p.p.m. with those of Mycoplasma hyopneumoniae (Mh) vaccination programme (Stellamune Mycoplasma, Pfizer Animal Health). A pig herd with chronic respiratory disease in which infection with Mh played an important role was selected, and a total of 204 piglets were randomly allocated to either the medication (P) or the vaccination (V) group. Pigs in the P group received medicated feed for 3 weeks after weaning (days 34-55), and for 2 weeks late in the nursery period (days 77-98). The piglets in the V group were vaccinated twice intramuscularly, at 4 and 22 days of age. The two groups were compared on the basis of average daily gain (ADG), feed conversion rate (FCR), additional curative medication days (CMD), overall mortality (major variables), a coughing index, pneumonia lesions, and serology against Mh, influenza H1N1 and influenza H3N2 viruses, Actinobacillus pleuropneumoniae (App) and porcine reproductive and respirator, syndrome virus (PRRSV) (minor variables). No significant differences (P > 0.05) were observed for ADG (555 g/day in P group; 567 g/day in V group), FCR (2.64 in P group; 2.41 in V group) and mortality rate (11% in P group; 7% in V group). The average number of additional curative medication days (CMD) per pig was significantly higher (P < 0.01) in the P group (1.5) than in the V group (0.58). At slaughter age, the serological results and the prevalence of macroscopic lung lesions were comparable in the two groups (P > 0.05). With the exception of CMD, the preventive use of tilmicosin at this swine farm was found to confer similar beneficial effects to Mh vaccination.

Herd factors associated with the seroprevalences of four major respiratory pathogens in slaughter pigs from farrow-to-finish pig herds.

The objective of this study was to investigate sero-epidemiological aspects of Mycoplasma hyopneumoniae (Mh), influenza H1N1 and H3N2 viruses and Aujeszky disease virus (ADV) in fattening pigs from 150 randomly selected farrow-to-finish pig herds. Different herd factors were examined as potential risk indicators for the percentage of pigs with antibodies against the 4 pathogens. The median within-herd seroprevalences of the pathogens were: Mh 76%, H1N1 100%, H3N2 40% and ADV 53%. There was a positive association between the seroprevalences of both influenza viruses, and a negative association between the seroprevalences of ADV and H1N1. The percentage of pigs seropositive for Mh increased with the purchase of gilts and with the season (slaughter date in March-April). The within-herd seroprevalences of both influenza viruses were higher in the case of a higher density of pig herds in the municipality. A higher number of fattening pigs per pen additionally increased the risk of being seropositive for H3N2. The percentage of pigs with anti-gE-antibodies against the wild type ADV increased with higher airspace stocking density in the finishing unit, increasing herd size, increasing number of pig herds in the municipality and slaughter date in March-April. Increased seroprevalences for these 4 respiratory pathogens were mostly associated with pig density in the herd and its vicinity, the winter period, and with the purchase of gilts. Purchase of gilts, number of fattening pigs per pen and airspace stocking density are risk factors that can be managed directly by farmers striving to attain a high respiratory health status of pigs.

Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine influenza viruses and Aujeszky's disease virus in slaughter pigs from fattening pig herds.

Epidemiological aspects of Mycoplasma hyopneumoniae (Mh), influenza H1N1 and H3N2 viruses, and Aujeszky's disease virus (ADV) were investigated in slaughter pigs from 50 fattening pig herds. Herd factors as potential risk indicators for respiratory disease were obtained by means of a questionnaire. At slaughter, blood samples were collected from each herd, and the proportion of seropositive pigs per herd was assessed for each of these pathogens. The median herd-level seroprevalence of the agents were: Mh 88%, H1N1 100%, H3N2 60% and ADV 90%. The percentage of herds in which all investigated fattening pigs were seronegative for these agents was: Mh 0%, H1N1 0%, H3N2 12% and ADV 18%. The percentage of herds in which all investigated fattening pigs were seropositive for these agents was: Mh 8%, H1N1 71%, H3N2 22% and ADV 40%. A positive association was found between influenza H1N1 and H3N2 viruses, and a negative association between influenza H3N2 virus and ADV. There were no risk indicators for the seroprevalence of Mh. Three risk indicators were associated with the seroprevalence of influenza H1N1 virus: a fully slatted floor, an increasing number of pigs in the municipality and dry feeding. Three risk indicators were found for the seroprevalence of influenza H3N2 virus: purchase of pigs from > or = two herds, an increasing number of pigs in the municipality and natural ventilation. The seroprevalence of ADV was influenced by two risk indicators: an increasing number of pig herds in the municipality and an increasing number of pigs per pen.

Effect of vaccination against Mycoplasma hyopneumoniae in pig herds with an all-in/all-out production system.

A multi-site field study was conducted to evaluate an inactivated Mycoplasma hyopneumoniae (Mh) vaccine in 14 pig herds infected by Mh and practising an all-in/all-out production system. In each herd, a vaccinated and control group of 250 pigs each were compared during the growing/finishing period with respect to performance parameters (major variables) and by means of clinical, serological and pathological parameters (ancillary variables). Mh vaccination significantly (P < 0.05) improved daily weight gain (+22 g), feed conversion ratio (-0.07), medication costs (-0.476 ECU/pig) (1 ECU = US$1.0269542), prevalence of pneumonia lesions (-14%) and severity of pneumonia lesions (-3%). Mortality rate, severity of coughing and carcass quality were not significantly influenced by Mh vaccination. Serological results of Mh and other respiratory pathogens are presented and discussed. A cost-benefit analysis based on significantly improved performance parameters demonstrated that Mh vaccination was economically attractive as it resulted in an increase of the net return to labour with 1.300 ECU per finishing pig sold. The sensitivity of the economic benefit was illustrated towards fluctuations in pig finishing prices.

The effect of vaccination against Mycoplasma hypopneumoniae in pig herds with a continuous production system.

An inactivated Mycoplasma hyopneumoniae vaccine was evaluated in five pig herds clinically infected with enzootic pneumonia and practising a continuous production system in the growing/finishing unit. In each herd, a vaccinated and control group of approximately 47 pigs each were individually monitored from birth until slaughter. Vaccinated pigs received the first dose at about 1 week of age and the second approximately 3 weeks later. During all production stages, an equal number of vaccinated and control pigs was present in the same pen. Both groups were compared with respect to zootechnical parameters (major variables) and by means of serological, pathological, and bacteriological parameters (ancillary variables). Daily weight gain was improved by 14 gr/day during the period from 8 days of age until slaughter (P = 0.0486) and by 25 gr/day during the growing/finishing period (P = 0.0067). Mortality rate, and the costs for curative medication were not significantly improved by vaccination. The results of the ancillary variables are presented and discussed.

Enzootic pneumonia in pigs.

This article reviews current knowledge concerning enzootic pneumonia. Enzootic pneumonia, caused by Mycoplasma hyopneumoniae and exacerbated by secondary invaders, occurs worldwide and has been known for many years. The organism, with its typical characteristics, invades the respiratory tract in several successive steps. Clinical symptoms and lesion development are the result of the pathogenic capacity of Mycoplasma hyopneumoniae and the defence reactions in the lung. The economic relevance of pneumonia is influenced to a large extent by common secondary infections which follow an initial M. hyopneumoniae infection. Different tests for the diagnosis of pneumonia in individual pigs and in groups are available. Treatment and control is not simple since enzootic pneumonia is a multi-factorial disease. Some epidemiological aspects and the most important measures for prevention of the disease are described.

Influence of the oestrous cycle on experimental intrauterine E. coli infection in the sow.

The influence of the oestrous cycle on the onset of endometritis in the sow was studied. Ten pubertal, unmated gilts of the Belgian Negative Landrace were used. Nine gilts were inoculated into the uterus by laparotomy with a suspension of an E. coli strain isolated from the uterus of a discharging sow from a herd having many problems with vaginal discharge and a lowered fertility. One gilt was as a control inoculated with 2 ml of a PBS-solution. All sows inoculated during dioestrus developed clinical symptoms, but only 1 of the 5 gilts inoculated at standing oestrus developed a vaginal discharge. These data confirm the hypothesis that the stage of the oestrous cycle has an important influence on the onset of endometritis. The resistance to E. coli infections was higher when the gilts were inoculated during oestrus.

Nedocromil sodium treats symptoms of perennial allergic conjunctivitis not fully controlled by sodium cromoglycate A double-masked placebo controlled group comparative study.

In a double-masked group comparative study, 20 patients received 2% nedocromil sodium four times daily and 23 placebo eye drops, for treatment of perennial allergic conjunctivitis (PAC). All had at least a one-year diagnosis of bilateral PAC and remained symptomatic despite using 2% sodium cromoglycate eye drops four times daily for at least 14 days Symptom severity (0-4) during sodium cromoglycate monotherapy was then recorded in a one-week baseline prior to randomisation, a minimum total score of 11 being mandatory. During the trial, no eye medication was allowed other than the test treatment. Clinic examinations were made before and after baseline and after one, three and six weeks' treatment, and patients kept daily diary cards of eye symptom severity. Compared to placebo, nedocromil sodium significantly (p<0.05) improved diary scores for itching (weeks 3, 4, 5, 6), total symptoms (weeks 5, 6) and general eye condition (week 6). Mean scores over the final four weeks, allowing a two-week washout, showed similar improvements in itching (p = 0.01), total symptoms (p = 0.05) and general eye condition (p = 0.04). Clinical assessments again favoured nedocromil sodium, which improved itching (week 3, p = 0.002), burning (week 6, p = 0.007), overall eye condition (weeks 3-6, p<0.05). and conjunctival thickening and hyperaemia (weeks 3-6, p<0.05). Finally, both patient (p = 0.02) and clinician (p = 0.0015) opinions of efficacy favoured nedocromil sodium over placebo. These results show nedocromil sodium to be effective in controlling symptoms of perennial allergic conjunctivitis which persisted during treatment with sodium cromoglycate.

25 years of a unique chromosome-breakage system. I. Principal features and comparison to other systems.

The principal features of the D. robusta chromosome-breakage system are reviewed and compared to intrinsic chromosome-breakage systems in other Drosophila species, particularly the 'hybrid dysgenesis' systems of D. melanogaster. The data indicate that the D. robusta system is unique in many respects, especially in its combination of a reciprocal cross effect, traceable to its maternal or cytoplasmic inheritance, exclusive susceptibility of the paternal chromosomes, and the randomness of the breaks on the chromosomes.