Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

Rumen-protected choline supplementation in periparturient dairy goats: effects on liver and mammary gland

The current study investigated the effects of supplementing rumen-protected choline (RPC) on metabolic profile, selected liver constituents and transcript levels of selected enzymes, transcription factors and nuclear receptors involved in mammary lipid metabolism in dairy goats. Eight healthy lactating goats were studied: four received no choline supplementation (CTR group) and four received 4g RPC chloride/day (RPC group). The treatment was administered individually starting 4 weeks before expected kidding and continuing for 4 weeks after parturition. In the first month of lactation, milk yield and composition were measured weekly. On days 7, 14, 21 and 27 of lactation, blood samples were collected and analysed for glucose, beta-hydroxybutyrate, non-esterified fatty acids and cholesterol. On day 28 of lactation, samples of liver and mammary gland tissue were obtained. Liver tissue was analysed for total lipid and DNA content; mammary tissue was analysed for transcripts of lipoprotein lipase (LPL), fatty acid synthase (FAS), sterol regulatory binding proteins 1 and 2, peroxisome proliferator-activated receptor gamma and liver X receptor alpha. Milk yield was very similar in the two groups, but R PC goats had lower (P < 0.05) plasma beta-hydroxybutyrate. The total lipid content of liver was unaffected (P = 0.890), but the total lipid/DNA ratio was lower (both P < 0.05) in RPC than CTR animals. Choline had no effect on the expression of the mammary gland transcripts involved in lipid metabolism. The current plasma and liver data indicate that choline has a positive effect on liver lipid metabolism, whereas it appears to have little effect on transcript levels in mammary gland of various proteins involved in lipid metabolism. Nevertheless, the current results were obtained from a limited number of animals, and choline requirement and function in lactating dairy ruminants deserve further investigation.

Effects of nutrient restriction on mammary cell turnover and mammary gland remodeling in lactating dairy cows

The aim of this study was to investigate the effects of a severe nutrient restriction on mammary tissue morphology and remodeling, mammary epithelial cell (MEC) turnover and activity, and hormonal status in lactating dairy cows. We used 16 Holstein x Normande crossbred dairy cows, divided into 2 groups submitted to different feeding levels (basal and restricted) from 2 wk before calving to wk 11 postpartum. Restricted-diet cows had lower 11-wk average daily milk yield from calving to slaughter than did basal-diet cows (20.5 vs. 33.5 kg/d). Feed restriction decreased milk fat, protein, and lactose yields. Restriction also led to lower plasma insulin-like growth factor 1 and higher growth hormone concentrations. Restricted-diet cows had lighter mammary glands than did basal-diet cows. The total amount of DNA in the mammary gland and the size of the mammary acini were smaller in the restricted-diet group. Feed restriction had no significant effect on MEC proliferation at the time of slaughter but led to a higher level of apoptosis in the mammary gland. Gelatin zymography highlighted remodeling of the mammary extracellular matrix in restricted-diet cows. Udders from restricted-diet cows showed lower transcript expression of alpha-lactalbumin and kappa-casein. In conclusion, nutrient restriction resulted in lower milk yield in lactating dairy cows, partly due to modulation of MEC activity and a lower number of mammary cells. An association was found between feed restriction-induced changes in the growth hormone-insulin-like growth factor-1 axis and mammary epithelial cell dynamics.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas.

The clinical impact of p16 status in fine-needle aspirates of cervical lymph node metastasis of head and neck squamous cell carcinomas

Lymph node involvement is prognostically the most determinant clinical factor for patients with head and neck squamous cell carcinomas (HNSCCs). Ultrasound of the neck and fine-needle aspiration (FNA) cytology is one of the first diagnostic procedures and the most accurate diagnostic staging tool for the neck. Patients with HPV-positive oropharyngeal carcinomas (OPSCC) show a significantly better prognosis when compared with HPV-negative OPSCC. P16 overexpression is accepted as surrogate marker for HPV-positive in OPSCC. These HPV/p16-positive OPSCC are localized either in the palatal tonsils or the base of tongue and frequently present with lymph node metastases. We analyzed the correlation and reliability of p16 expression of the FNA of the lymph node metastasis with the immunohistochemical expression of p16 of the same lymph node metastasis and its corresponding primary tumor, as it could be of importance for determining the localization and different prognosis of the primary tumor. 54 HNSCC patients were evaluated, p16 expression of the primary tumors and their lymph node metastases correlated precisely. In 25 of the 54 HNSCC patients, a FNA of the lymph node metastases was taken before the treatment. The positive cytological and immunohistochemical p16 staining correlated exactly. Of the 17 histologically p16-negative lymph node metastases 15 FNA were p16-negative, whereas two samples were p16-positive. In our view, a cytological p16 analysis of cervical lymph node metastasis can facilitate the correct localization of the primary tumor and discriminate reliably HPV-positive OPSCC from HPV-negative HNSCC with their significantly diverse prognosis.

Facial basal cell carcinomas recurring after photodynamic therapy: a retrospective analysis of histological subtypes

Photodynamic therapy (PDT) is an established treatment for basal cell carcinomas (BCCs). Although recurrences are sometime observed, their histological patterns have never been specifically studied or compared with the one of the initial tumor.

Use and relevance of a bovine mammary gland explant model to study infection responses in bovine mammary tissue

Our aim was to develop an explant model to define more precisely the early response of bovine mammary epithelial cells to infection. Therefore we investigated the mRNA expression encoding for some soluble immunological factors in lipopolysaccharide (LPS)-treated bovine mammary gland explants. Explants were taken out from the mammary gland of eight lactating cows after slaughter then incubated with LPS (10 mug/ml) for 6 h. The mRNA expression of alpha-lactalbumin (alpha-la), various cytokines, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and two immunoglobulin receptors, the neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIGR), were assessed with qPCR before and after 3 h and 6 h of LPS challenge. Both immunoglobulin receptors and alpha-la increased at 3 h then recovered their initial level at 6 h whereas IL-1beta, IL-6 and IL-8 increased only after 6 h (P<0.05). Surprisingly, TNF-alpha transcripts did not show any regulation in response to the LPS treatment. We nevertheless concluded that our model was valid to examine the short-term response of mammary epithelial cell challenged with LPS.

Lactoferrin (Lf): Retinoid interactions in the mammary glands of transgenic mice overexpressing human Lf

Induction of protein expression in a tissue-specific manner by gene transfer over-expression techniques has been one means to define the function of a protein in a biological paradigm. Studies with retinoid reporter constructs transfected in mammary cell lines suggests that lactoferrin (Lf) affects retinoid signaling pathways and alters apoptosis. We tested the effects and interactions of over-expressed mammary-specific human lactoferrin (hLf) and dietary retinol palmitate on lactation and mammary gland development in mice. Increased retinol palmitate in the diet increased daily retinol equivalents (RE) to 2.6-fold over the normal mouse control diet. Transgene (Tg) expression in the dam fed control diet depressed pup weight gain. Severe depression of pup weight gain was observed when homozygote TgTg dams were fed the RE diet. Normal weight gain was restored when pups were placed with a wild type dam fed the RE diet; conversely, normal growing pups from the wild type dams showed declining weight gains when fostered to the TgTg RE-fed dams. Northern analysis of mammary tissue extracts showed a reduction in WAP and an increase in IGFBP-3 mRNA that was associated with the presence of the transgene. Histological evaluation of 3 days lactating mammary tissue showed mammary epithelial cells from TgTg animals contained excessive secretory products, suggesting a block in cellular secretion mechanisms. In addition, the mammary cells displayed a cellular apical membrane puckering that extended into the alveoli lumens. These studies demonstrate an in vivo interaction of Tg-hLf expression and dietary retinoids in mouse mammary glands. While normal mammary gland physiology may not be representative by these experiments because high Lf concentrations during early lactation are abnormal, the demonstrated biological interaction suggests that typical periods of high Lf concentrations may have impact upon developing and involuting mammary glands.

Radionecrosis or tumor recurrence after radiation of laryngeal and hypopharyngeal carcinomas

OBJECTIVE: To analyze the incidence and diagnostic difficulties of radionecrosis vs tumor recurrence of laryngeal and hypopharyngeal carcinomas. STUDY DESIGN AND SETTING: Retrospective study on 341 patients treated by radiation alone or radiochemotherapy. The clinicopathologic findings, work-up, treatment, and follow-up of 20 patients with symptoms suggestive but negative for tumor recurrence on initial imaging studies and endoscopy were analyzed. RESULTS: The incidence of chondroradionecrosis in 341 irradiated patients was 5%. Ten of 20 patients initially negative for tumor recurrence were treated by total laryngectomy; in all laryngectomy specimens, chondroradionecrosis was present, in six specimens associated with tumor recurrence. Ten patients were treated by tracheotomy and tumor recurrence was detected in one patient during follow-up. CONCLUSION: Chondroradionecrosis is a relatively rare treatment complication. Typical imaging findings suggestive of radionecrosis are often missing. Tumor recurrence may be present beneath an intact mucosa and missed by endoscopy.

Elective neck dissection for carcinomas of the oral cavity: occult metastases, neck recurrences, and adjuvant treatment of pathologically positive necks

BACKGROUND: Supraomohyoid neck dissection (SOHND) is currently performed in patients with carcinoma of the oral cavity with clinically negative neck. Most investigators consider SOHND as a staging procedure. METHODS: Records of 100 patients with cancer of the oral cavity and clinically negative neck undergoing SOHND were reviewed. The rate and significance of occult metastases are evaluated, the neck recurrences are analyzed and the indication of adjuvant radiation of pN+ necks is discussed. RESULTS: In 34 of 1814 of analyzed lymph nodes, metastatic disease was detected as follows: 30 macrometastases and 4 micrometastases. In 13 of 34 metastases (38%), extracapsular spread was observed. Twenty of 100 patients (20%) had to be upstaged. In 9 of 87 (10%) patients without local recurrence and with a minimal follow-up of 24 months, 5 ipsilateral (4 within the dissection field) and 5 contralateral neck recurrences were observed. Regional recurrence developed in 4% and 35% of patients with pN0 and pN+ necks, respectively. CONCLUSIONS: In 20% of patients with oral cavity tumors and pN0 neck, occult metastases were disclosed. Neck recurrences developed significantly more often in patients with pN+ than in those with pN0 necks. To evaluate the exact indication for an adjuvant treatment of patients with cN0/pN+ necks, prospective studies should be performed.

Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA

PURPOSE: To investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, (177)Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3. METHODS: In vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand (177)Lu-AMBA was used and compared with established bombesin radioligands such as (125)I-Tyr(4)-bombesin and (125)I-[D: -Tyr(6),beta-Ala(11),Phe(13),Nle(14)]-bombesin(6-14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour. RESULTS: (177)Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. (177)Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with (177)Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with (177)Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with (177)Lu-AMBA than with the conventional bombesin radioligands. CONCLUSION: The present in vitro study suggests that (177)Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessels.