Prediction of key milk biomarkers in dairy cows through milk mid-infrared spectra and international collaborations.

At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (ß-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-ß-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects. Benefiting from this international collaboration, the dataset comprised a total of 9,143 milk samples from 3,758 cows located in 589 herds across 10 countries and represented 7 breeds. The samples were analyzed by reference chemistry for biomarker contents, whereas the MIR analyses were performed on 30 instruments from different models and brands, with spectra harmonized into a common format. Four quantitative methodologies were evaluated to address the strongly skewed distribution of some biomarkers. Partial least squares regression was used as the reference basis, and compared with a random modification of distribution associated with PLS (random-downsampling-PLS), an optimized modification of distribution associated with PLS (KennardStone-downsampling-PLS), and support vector machine (SVM). When the ability of MIR to predict biomarkers was too low for quantification, different qualitative methodologies were tested to discriminate low versus high values of biomarkers. For each biomarker, 20% of the herds were randomly removed within all countries to be used as the validation dataset. The remaining 80% of herds were used as the calibration dataset. In calibration, the 3 alternative methodologies outperform the PLS performances for the majority of biomarkers. However, in the external herd validation, PLS provided the best results for isocitrate, glucose-6P, free glucose, and lactate dehydrogenase (coefficient of determination in external herd validation [R2v] = 0.48, 0.58, 0.28, and 0.24, respectively). For other molecules, PLS-random-downsampling and PLS-KennardStone-downsampling outperformed PLS in the majority of cases, but the best results were provided by SVM for citrate, BHB, acetone, NAGase, and progesterone (R2v = 0.94, 0.58, 0.76, 0.68, and 0.15, respectively). Hence, PLS and SVM based on the entire dataset provided the best results for normal and skewed distributions, respectively. Complementary to the quantitative methods, the qualitative discriminant models enabled the discrimination of high and low values for BHB, acetone, and NAGase with a global accuracy around 90%, and glucose-6P with an accuracy of 83%. In conclusion, MIR spectra of milk can enable quantitative screening of citrate as a biomarker of energy deficit and discrimination of low and high values of BHB, acetone, and NAGase, as biomarkers of ketosis and mastitis. Finally, progesterone could not be predicted with sufficient accuracy from milk MIR spectra to be further considered. Consequently, MIR spectrometry can bring valuable information regarding the occurrence of energy deficit, ketosis, and mastitis in dairy cows, which in turn have major influences on their fertility and survival.

Usability of bacteriological milk analyses for genetic improvement of udder health in Austrian Fleckvieh cows.

In addition to somatic cell count records and clinical mastitis diagnoses, results of bacteriological milk analyses provide valuable information regarding udder health. The pathogen causing an udder infection is currently not considered in Austria as part of the information used for estimation of routine breeding values for mastitis resistance. Therefore the objective of this study was to estimate heritabilities for, and genetic correlations between, udder traits of bacterial infection (bacterial infection, gram-positive and gram-negative bacterial infection) and routinely recorded udder health traits [acute mastitis, chronic mastitis, culling due to udder health problems, and somatic cell score (SCS)] in Austrian Fleckvieh cows. The basis for the genetic analyses was a data set with results from bacteriological milk analyses collected from 237 dairy farms and 6,822 cows over a period of 1 yr. Traits were defined as binary, apart from SCS, for which measures were available continuously. Multivariate analyses using a linear animal model were applied for estimating genetic parameters. The heritabilities for the occurrence of bacterial udder infection traits were 0.01. Heritabilities were 0.04 for acute mastitis, 0.02 for chronic mastitis, 0.02 for culling due to udder health problems, and 0.20 for SCS. Genetic correlations between bacteriological infection and the routinely recorded udder health traits were positive and ranged from 0.62 to 0.96. The genetic correlation between gram-positive and gram-negative bacterial infection was -0.20. The genetic correlation between acute and chronic mastitis was also close to zero. These results show that mastitis caused by different pathogens may be seen as different traits. As analyses were based on a relatively small data set and results were associated with rather high standard errors, further research with a larger data set should be carried out to confirm these results.

Use of benchmarking to monitor and analyze effects of herd size and herd milk yield on cattle health and welfare in Austrian dairy farms.

The modernization and intensification of the dairy industry has led to larger herd sizes and higher milk production, both globally and in Austria. Benchmarking allows the monitoring of animal health and welfare as well as the identification of potential for improvement by comparing certain parameters with other farms with similar management environments. Using data from the Austrian routine recording system of various traits of milk production, fertility, and health, farmers and their veterinarians (with the consent of the farmer) can compare farm parameters with detailed data available from their district or state and ensure more efficient herd management. The aim of the present study was to provide an overview of dairy milk production in Austria based on the annual herd health reports and to examine the effects of herd size and milk production on fertility and health parameters. Annual herd health reports from all farms participating in the health monitoring system were considered, and analyses were conducted across breeds. A large variation between farms was observed. The results showed that, based on parameters of milk yield and herd size for the range of farms within this study, it cannot be concluded that these circumstances automatically lead to poor animal health. Farms with very small herd sizes differed significantly from those with larger herd sizes. Overall herd size effects were however small in Austria. Higher milk production based on a single farm does not necessarily cause more health and fertility problems; however, we detected a tendency for an increased risk of fertility, udder, and metabolic diagnoses. An active health management program might result in higher incidence rates for fertility or udder diagnoses, as a veterinary treatment might be economically superior if, for example, the calving interval can be shortened or the somatic cell count can be reduced. The results of the present study showed that it is advisable to use different benchmarks in combination for monitoring health, as well as for deciding on strategies to improve overall herd health management. Animal health reports on Austrian dairy cows are continuously being developed and new parameters integrated.

Short communication: Use of lameness scoring to genetically improve claw health in Austrian Fleckvieh, Brown Swiss, and Holstein cattle.

The specific objective of this study was to evaluate the use of lameness scoring to genetically improve claw health in Austrian Fleckvieh, Brown Swiss, and Holstein cows based on data from the "Efficient Cow" project. In 2014, a 1-yr data collection was carried out. Data from 6,519 cows kept on 161 farms were recorded. At each time of milk recording, lameness scores were assessed by trained staff of the milk recording organizations. Hoof trimming on these farms was documented and recorded as well. Veterinarian diagnoses and culling due to foot and leg problems from these farms were available from the routine recording system. As repeated lameness records per cow and lactation were available, an overall lactation lameness score was calculated. Estimated heritabilities for lameness were 0.11, 0.05, and 0.09 for Fleckvieh, Brown Swiss, and Holstein, respectively; however, only heritability estimates for Fleckvieh were significantly different from zero. Breeding values for lameness were obtained, reversed in sign, and cows were ranked according to their breeding value. A low breeding value for lameness resistance (the bottom 10% of the cows) was associated with a significantly higher frequency of trimmed cows, which indicates that the cows selected by the farmer to be trimmed are not completely random. Additionally, a high breeding value for lameness resistance (the top 10% of the cows) was associated with lower frequencies of claw diseases recorded at trimming, claw and leg diagnoses, and culling due to foot and leg problems, which highlights the usefulness of lameness scoring for genetic improvement of claw health. Overall, selecting for a better lameness score has the potential to reduce claw diseases, especially the frequency of severe claw diseases that lead to culling.

Genetic analysis of efficiency traits in Austrian dairy cattle and their relationships with body condition score and lameness.

This study is part of a larger project whose overall objective was to evaluate the possibilities for genetic improvement of efficiency in Austrian dairy cattle. In 2014, a 1-yr data collection was carried out. Data from 6,519 cows kept on 161 farms were recorded. In addition to routinely recorded data (e.g., milk yield, fertility, disease data), data of novel traits [e.g., body weight (BW), body condition score (BCS), lameness score, body measurements] and individual feeding information and feed quality were recorded on each test-day. The specific objective of this study was to estimate genetic parameters for efficiency (related) traits and to investigate their relationships with BCS and lameness in Austrian Fleckvieh, Brown Swiss, and Holstein cows. The following efficiency (related) traits were considered: energy-corrected milk (ECM), BW, dry matter intake (DMI), energy intake (INEL), ratio of milk output to metabolic BW (ECM/BW0.75), ratio of milk output to DMI (ECM/DMI), and ratio of milk energy output to total energy intake (LE/INEL, LE = energy in milk). For Fleckvieh, the heritability estimates of the efficiency (related) traits ranged from 0.11 for LE/INEL to 0.44 for BW. Heritabilities for BCS and lameness were 0.19 and 0.07, respectively. Repeatabilities were high and ranged from 0.30 for LE/INEL to 0.83 for BW. Heritability estimates were generally lower for Brown Swiss and Holstein, but repeatabilities were in the same range as for Fleckvieh. In all 3 breeds, more-efficient cows were found to have a higher milk yield, lower BW, slightly higher DMI, and lower BCS. Higher efficiency was associated with slightly fewer lameness problems, most likely due to the lower BW (especially in Fleckvieh) and higher DMI of the more-efficient cows. Body weight and BCS were positively correlated. Therefore, when selecting for a lower BW, BCS is required as additional information because, otherwise, no distinction between large animals with low BCS and smaller animals with normal BCS would be possible.

Design, implementation, and evaluation of a mobile application for patient empowerment and management of long-term follow-up after childhood cancer.

In Germany, about 1,800 new cases of childhood cancer are diagnosed every year. The chances of survival have increased significantly over the last 40 years due to the continuous improvement of treatment strategies. The number of childhood cancer survivors in Germany thus ranges around 30,000 nowadays. But their treatment with surgery, chemotherapy, and radiation has certain side-effects. In addition to the acute effects during the treatment phase, the disease- and treatment-related late effects can occur even decades after the end of therapy. These late effects draw attention as the survival rate constantly increases. Two-thirds of the former patients retain long-term consequences, nearly a fifth with a resulting diminished quality of life. Early detection of these late effects can help to reduce or even to prevent serious health damage. Therefore, the study group LESS supplies long-term follow-up recommendations for former patients. The project described in this paper was to design and implement a mobile application to increase the compliance for this aftercare program. This application provides information about the patient's individual aftercare plan and supports appointment management as well as a reminding functionality. A prototype for former osteosarcoma patients was tested and evaluated in two university hospitals. First results show the application's very high potential for patient empowerment.

Hind feet position score: A novel trait to genetically reduce lameness incidence.

Lameness is an important health and welfare issue that causes considerable economic losses in dairy herds. The objective of this study was to investigate whether the hind feet position score (HFPS) can be used as an auxiliary trait for genetic evaluation of lameness. The HFPS is evaluated by visual scoring of the position of both the hind-digits to the mid-line of the cow's body. The higher the heel height of the lateral claw, the higher is the HFPS, and the higher is the risk for development of lameness. In total, 3,478 records from 1,064 Fleckvieh cows from 35 farms were obtained between September 1, 2021, and March 5, 2022. Data collection was carried out by the regional milk recording organizations. Hind feet position was scored visually by trained personnel during routine milk performance testing in the milking parlor using a 3-class scoring system: score 1 = 0° to <17° indicating a balanced heel height of both the medial and the lateral claw; score 2 = angle of 17° to 24°; score 3 = angle of >24°. After all cows had been milked, locomotion scoring was performed for each animal using a 5-class scoring system with locomotion scores ranging between 1 (normal) and 5 (severely lame). Using HFPS, sensitivity and specificity were 69.5% and 66.8%, respectively, for detecting lameness defined by locomotion score ≥2. For genetic analyses, a bivariate linear animal model was fitted with fixed effects of herd, parity, lactation stage, and classifier, and random effects of animal and permanent environment. Heritabilities for HFPS and locomotion score were 0.07 and 0.10, respectively, and the genetic correlation between the 2 traits studied was 0.80. These results suggest that the HFPS could be used for genetic evaluations to reduce lameness incidence in dairy cattle.

Emerging challenges of the impacts of pharmaceuticals on aquatic ecosystems: A diatom perspective.

Pharmaceuticals are a ubiquitous group of emerging pollutants of considerable importance due to their biological potency and potential to elicit effects in wildlife and humans. Pharmaceuticals have been quantified in terrestrial, marine, fresh, and transitional waters, as well as the fauna and macro-flora that inhabit them. Pharmaceuticals can enter water ways through different human and veterinary pathways with traditional wastewater treatment, unable to completely remove pharmaceuticals, discharging often unknown quantities to aquatic ecosystems. However, there is a paucity of available information regarding the effects of pharmaceuticals on species at the base of aquatic food webs, especially on phytoplankton, with research typically focussing on fish and aquatic invertebrates. Diatoms are one of the main classes of phytoplankton and are some of the most abundant and important organisms in aquatic systems. As primary producers, diatoms generate ∼40 % of the world's oxygen and are a vital food source for primary consumers. Diatoms can also be used for bioremediation of polluted water bodies but perhaps are best known as bio-indicators for water quality studies. However, this keystone, non-target group is often ignored during ecotoxicological studies to assess the effects of pollutants of concern. Observed effects of pharmaceuticals on diatoms have the potential to be used as an indicator of pharmaceutical-induced impacts on higher trophic level organisms and wider ecosystem effects. The aim of this review is to present a synthesis of research on pharmaceutical exposure to diatoms, considering ecotoxicity, bioremediation and the role of diatoms as bio-indicators. We highlight significant omissions and knowledge gaps which need addressing to realise the potential role of diatoms in future risk assessment approaches and help evaluate the impacts of pharmaceuticals in the aquatic environment at local and global scales.

Mast cells promote lung vascular remodelling in pulmonary hypertension.

Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to non-cardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH.

Characterization of a monoclonal antibody directed against the biologically active site of human interleukin 1.

Human IL-1 was successfully used to produce an anti-IL-1 mAb. Anti-IL-1 (IgG2a) blocked IL-1-mediated thymocyte and fibroblast proliferation, but did not interfere with the biological effects of other lymphokines, such as IL-2 or IL-3. The antibody immunoprecipitated biosynthetically radiolabeled 33, 17, and 4 kD IL-1. An immunoadsorbent column yielded 20% of initial activity, and upon HPLC size-exclusion chromatography, affinity-purified IL-1 had a molecular mass of approximately 4 kD. These results provide first evidence of a monoclonal anti-IL-1 that reacts with different species of IL-1 and apparently binds to an epitope close to the active site of IL-1. Thus, anti-IL-1 IgG may be very helpful for further investigations of the molecular as well as biological characteristics of IL-1 and related mediators.

Human keratinocytes are a source for tumor necrosis factor alpha: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light.

Tumor necrosis factor alpha (TNF-alpha), in addition to being cytotoxic for certain tumor cells, has turned out as a multifunctional cytokine that is involved in the regulation of immunity and inflammation. Since human keratinocytes have been demonstrated to be a potent source of various cytokines, it was investigated whether epidermal cells synthesize and release TNF-alpha. Supernatants derived from normal human keratinocytes (HNK) and human epidermoid carcinoma cell lines (KB, A431) were tested both in a TNF-alpha-specific ELISA and a bioassay. In supernatants of untreated epidermal cells, no or minimal TNF-alpha activity was found, while after stimulation with lipopolysaccharide (LPS) or ultraviolet (UV) light, significant amounts were detected. Western blot analysis using an antibody directed against human TNF-alpha revealed a molecular mass of 17 kD for keratinocyte-derived TNF-alpha. These biological and biochemical data were also confirmed by Northern blot analysis revealing mRNA specific for TNF-alpha in LPS- or ultraviolet B (UVB)-treated HNK and KB cells. In addition, increased TNF-alpha levels were detected in the serum obtained from human volunteers 12 and 24 h after a single total body UVB exposure, which caused a severe sunburn reaction. These findings indicate that keratinocytes upon stimulation are able to synthesize and release TNF-alpha, which may gain access to the circulation. Thus, TNF-alpha in concert with other epidermal cell-derived cytokines may mediate local and systemic inflammatory reactions during host defense against injurious events caused by microbial agents or UV irradiation.

Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes.

Proopiomelanocortin (POMC), the precursor for melanotropic, corticotropic, and opioid peptides such as alpha-melanocyte-stimulating hormone (alpha MSH), ACTH, and other related peptides, was originally identified as a product of the pituitary gland. However, recent evidence shows that POMC products can also be produced by nonpituitary tissues. Because keratinocytes, the major constituent of the epidermis exhibit the capacity to release a variety of proinflammatory and immunomodulatory mediators, the present study was performed to investigate whether human keratinocytes are able to produce POMC-derived peptides. Supernatants of human normal keratinocytes and an epidermal carcinoma cell line (A431) contained significant levels of immunoreactive alpha MSH and ACTH. Upon immuneprecipitation and size-exclusion chromatography, keratinocyte-derived alpha MSH exhibited a molecular mass of approximately 1 kD and was biologically active as demonstrated in a tyrosinase bioassay. Northern blot analysis revealed the expression of POMC-specific transcripts (1.3 kb) in both normal keratinocytes and A431 cells. The production of alpha MSH and ACTH could be significantly upregulated both at the protein and mRNA level upon treatment with phorbol myristate acetate, ultraviolet light, or interleukin 1. These data provide first evidence that human keratinocytes produce POMC-derived peptides such as alpha MSH and ACTH. Because POMC-derived peptides recently have been recognized as potent immunomodulatory mediators, their presence in the epidermis may have a major impact on the skin immune system.

Home-based versus clinic-based self-sampling and testing for sexually transmitted infections in Gugulethu, South Africa: randomised controlled trial.

OBJECTIVES: To test whether more women are screened for sexually transmitted infections when offered home-based versus clinic-based testing and to evaluate the feasibility and acceptability of self-sampling and self-testing in home and clinic settings in a resource-poor community. METHODS: Women aged 14-25 were randomised to receive a home kit with a pre-paid addressed envelope for mailing specimens or a clinic appointment, in Gugulethu, South Africa. Self-collected vaginal swabs were tested for gonorrhoea, chlamydia and trichomoniasis using PCR and self-tested for trichomoniasis using a rapid dipstick test. All women were interviewed at enrollment on sociodemographic and sexual history, and at the 6-week follow-up on feasibility and acceptability. RESULTS: 626 women were enrolled in the study, with 313 in each group; 569 (91%) completed their 6-week follow-up visit. Forty-seven per cent of the women in the home group successfully mailed their packages, and 13% reported performing the rapid test and/or mailing the kit (partial responders), versus 42% of women in the clinic group who kept their appointment. Excluding partial responders, women in the home group were 1.3 (95% CI 1.1 to 1.5) times as likely to respond to the initiative as women in the clinic group. Among the 44% who were tested, 22% tested positive for chlamydia, 10% for trichomoniasis, and 8% for gonorrhoea. CONCLUSIONS: Self-sampling and self-testing are feasible and acceptable options in low-income communities such as Gugulethu. As rapid diagnostic tests become available and laboratory infrastructure improves, these methodologies should be integrated into services, especially services aimed at young women.

A comparison of two methods of partner notification for sexually transmitted infections in South Africa: patient-delivered partner medication and patient-based partner referral.

The objective of this study was to determine acceptability and feasibility of patient-based partner referral (PBPR) and patient-delivered partner medication (PDPM) among female sexually transmitted infection (STI) patients in a community-based STI screening study. Women were randomized to STI screening at home or at a clinic. STI patients could choose between PBPR and PDPM. Six-week follow-up interviews, and in-depth interviews, were conducted. STI prevalence was high. Most of the 106 women with an STI chose PDPM, mainly because partners would not have time or would not want to attend a clinic, and to ensure that partners received treatment. Nearly all partners reportedly took medication (94; 89% took it in front of the woman) or went to a clinic for treatment (92%). No adverse events were reported. Good communication emerged as the key to successful partner notification. In conclusion, PDPM could be used as a strategy to improve STI treatment coverage.

Prevalence of equine herpesvirus type 2 (EHV-2) DNA in ocular swabs and its cell tropism in equine conjunctiva.

Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased horses were positive. To define the main virus reservoir ocular tissue from 13 randomly selected horses without pathological evidence of ocular disease were analysed by nested PCR. In two horses optic nerve, lacrimal gland and conjunctiva, in further two cases lacrimal gland and conjunctiva and in four horses the conjunctiva only were EHV-2 PCR positive. For specifying the target cell we focused on conjunctivae and selected 3 out of 15 clinically healthy slaughterhouse horses positive for EHV-2 by PCR. In situ hybridisation on sections of these paraffin embedded conjunctivae localized viral genome in histiocyte-like cells of the submucosa. Immunohistochemical staining with an EHV-2 or S100 specific polyclonal antiserum demonstrated that Langerhans cells were co-localized in the same region of the sample section where virus positive cells were detected. Furthermore, we concluded that detection of viral antigen revealed a productive virus infection.

Expression and release of interleukin-1 by different human melanoma cell lines.

The influence of immunologic parameters on the clinical course of malignant melanoma is increasingly evident. However, it is not known which factors contribute to the immunologic host reaction against malignant melanoma. Because epidermal cells and, in particular, normal as well as transformed keratinocytes recently have been demonstrated to release various immunomodulating cytokines, the capacity of melanoma cells to produce interleukin-1 (IL-1) was examined. Accordingly, supernatants derived from different melanoma cell lines contained significant levels of IL-1 activity. Upon high-performance liquid chromatography (HPLC) gel filtration, melanoma cell-derived IL-1 (MEL-IL-1) exhibited molecular weight heterogeneity, and HPLC chromatofocusing revealed major activity at pH 5.0 and minor activity at pH 7.0. A monoclonal antibody directed against monocyte-derived IL-1 blocked MEL-IL-1 activity significantly and was able to precipitate four species of biosynthetically radiolabeled MEL-IL-1 (25, 17, 6, and 4 kilodaltons), suggesting that MEL-IL-1 is identical to monocyte-derived IL-1. This was also confirmed by Northern blot analysis detecting IL-1 alpha specific mRNA in melanoma cells by hybridization with a cDNA fragment encoding for IL-1 alpha. Thus, melanoma cells, like other epidermal cells, exhibit the capacity to release the immunomodulating cytokine IL-1 and, therefore, probably have the potency to influence host defense mechanisms directed against malignant melanoma.

Tumor necrosis factor alpha sensitizes low epidermal growth factor receptor (EGFR)-expressing carcinomas for anti-EGFR therapy.

Analysis of 1,060 xenotransplants derived from cancer cell lines as wel as spontaneously occurring tumors from the larynx, pharynx, mammary gland, uterine cervix, and vulva revealed that tumor regression induced by treatment with monoclonal antibodies (EMD 55900 and EMD 72000 against the epidermal growth factor receptor (EGFR) could be enhanced by tumor necrosis factor alpha (TNF-alpha) treatment in vivo. Moreover, tumor that primarily do not respond to antibody treatment can be made suscep tible by additional TNF-alpha treatment. To investigate the in vivo effects of monoclonal antibodies, we treated tumors derived from cell lines (A431 and Detroit 562) as well as spontaneously occurring squamous cell carci nomas and adenocarcinomas (transplanted on NMRI-nu/nu mice) gener ally with EMD 55900 (40 microg/g mouse) and its humanized version EMD 72000 (40 microg/g mouse). When treated with EMD 55900 and EMD 72000 carcinomas with an EGFR concentration of > or = 70 fmol/mg protein showed significant reduction in tumor size compared with untreated controls. The degree of tumor regression correlated with the EGFR concentration of the tumor. In mice treated with TNF-alpha (0.5 microg/g mouse) and EMD 55900 72000 simultaneously, we observed enhanced antitumor effects up to complete tumor eradication. Carcinomas with an EGFR concentration <70 fmol/mg protein could be made susceptible to treatment with EMD 55900 and EMD 72000 by simultaneous treatment with TNF-alpha, resulting in a significant reduction in tumor size.

Screening for calreticulin mutations in a cohort of patients suspected of having a myeloproliferative neoplasm.

BACKGROUND: The discovery of calreticulin (CALR) has shown it to be the second most frequent mutation after the Janus Kinase 2 (JAK2) mutation in myeloproliferative neoplasms (MPNs). Its structure indicates various functions, of which two are to ensure calcium homeostasis and proper folding of other target proteins. Over 36 types of CALR mutations have been identified, all causing a recurrent frameshift in the C-terminal domain affecting CALR's localisation and calcium-binding function. OBJECTIVE: To screen a cohort of 89 patients suspected of having an MPN for the CALR mutations. METHODS: Capillary and gel electrophoresis were used in conjunction as confirmatory tests to screen the cohort of patients. RESULTS: Of three samples containing a type 1 CALR mutation, two were heterozygous and one homozygous for a 52-base pair deletion in CALR. CONCLUSIONS: Most studies report CALR mutations to be present only in patients with primary myelofibrosis or essential thrombocythaemia, with mutual exclusivity to JAK2 mutations. The findings of this study indicate that JAK2 and CALR mutations are no longer considered mutually exclusive. Similarly, patients with a polycythaemia vera phenotype could also carry a CALR mutation.

The fibronectin/α3ß1 integrin axis serves as molecular basis for keratinocyte invasion induced by ßHPV.

Organ-transplant-recipients exhibit cancerization of the skin from which multiple human papillomavirus (HPV)-positive squamous cell carcinomas (SCCs) arise. However, the molecular basis for HPV-induced invasion of skin keratinocytes is not known. We generated a transgenic mouse model expressing the E7 oncoprotein of HPV8 in the murine epidermis under the control of the keratin-14 promoter and showed that E7 is carcinogenic in mice. We further showed that both, the E7-expressing keratinocyte and mesenchymal components of the extracellular matrix as critical in eliciting the invasive behavior. E7 expression in basal keratinocytes, grown on fibronectin, led to epithelial-mesenchymal transition mediated by a cadherin switch. E7-positive keratinocytes displayed enhanced EDA-fibronectin expression and secretion and stimulated dermal fibroblasts to express EDA-fibronectin. Deposition of fibronectin was also detected in the peritumoral stroma of HPV8-positive skin SCC. When grown on fibronectin, E7-positive keratinocytes, in particular stem cell-like cells, exhibited increased cell surface levels of the α3-integrin chain. Functional blocking confirmed α3 as a critical molecule sufficient to induce E7-mediated invasion. This mechanistic link is further supported by expression of an E7-mutant, impaired in targeting α3 to the cell surface. These findings highlight the importance of epithelial-extracellular matrix interaction required for keratinocyte invasion and provide further mechanistic evidence for a role of HPV in skin carcinogenesis.

Substance P primes the formation of hydrogen peroxide and nitric oxide in human neutrophils.

Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP-induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin-dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP-primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO.