The variation in chemical composition of barley feed with or without enzyme supplementation influences nutrient digestibility and subsequently affects performance in piglets.

This study investigates the effect of dietary supplementation of a ß-glucanase and ß-xylanase enzyme mix to barley based diets, at two different chemical compositions achieved through different agronomical conditions on growth performance, coefficient of apparent total tract digestibility (CATTD) of nutrients, selected faecal microbial populations and faecal scores in piglets. Sixty-four piglets (11.7 kg (SD 0.96)) housed in pens of two were assigned to one of four dietary treatments (n = 8). The dietary treatments were as follows: (T1) low quality barley diet, (T2) low quality barley diet containing a ß-glucanase and ß-xylanase enzyme supplement, (T3) high quality barley diet and (T4) high quality barley diet containing a ß-glucanase and ß-xylanase enzyme supplement. Piglets offered the low quality barley-based diet had a higher (p < .05) average daily gain (ADG) (0.73 vs. 0.69 kg, SEM 0.001), gain:feed (G:F) ratio (0.61 vs. 0.58 kg, SEM 0.011) and a higher CATTD (p < .001) of dry matter (DM), organic matter (OM), nitrogen (N), ash, gross energy (GE) and neutral detergent fibre (NDF) compared with piglets offered the high quality barley diet. Piglets offered the high quality barley-based diet had reduced faecal scores compared to piglets offered the low quality barley-based diet (2.44 vs. 2.57, SEM 0.036) (p < .05). There was a higher population of Lactobacillus spp. (11.6 vs. 10.5 log gene copy number/g faeces, SEM 0.177) (p < .001) and total volatile fatty acid (VFA) concentration (185 vs. 165 mmol/g faeces, SEM 5.658) (p < .001) in the faeces of piglets offered the high quality barley-based diet compared to piglets offered the low quality barley-based diet. The inclusion of a ß-glucanase and ß-xylanase enzyme complex had no effect on any variable measured. In conclusion, the higher quality barley-based diet showed beneficial effects on the faecal Lactobacillus spp. population and faecal scores of the piglets; however, the higher level of ß-glucans in the diet decreased nutrient digestibility and subsequently decreased the performance.

IL-4 increases Simian immunodeficiency virus replication despite enhanced SIV immune responses in infected rhesus macaques.

It is widely believed that a Th1 type CD4 response is critical for enhancement of CD8 immunity and for controlling HIV-1 infection. Th2 type responses, such as what might be seen in a chronic parasitic infection, would sacrifice cellular immunity and thus benefit the virus at the expense of the host. However, there has been little direct examination of the hypothesis in a primate model system. Accordingly, the simian immunodeficiency virus (SIV) infected rhesus macaque model was used to investigate the impact of immunisation with SIV expressing DNA constructs and co-injection with IL-4 on the SIV specific immunological responses, lymphocyte cell counts, as well as the impact on viral load. IL-4 is a Th2 type cytokine, which enhances antibody production and inhibits a CD4 Th1 phenotype. Rhesus macaques were infected with 10 AID50 of SIVmac239 and treated with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) 9 weeks post-infection. During PMPA treatment, animals were immunised with plasmids that expressed the SIV proteins, env, rev, gag and pol. In addition, they were immunised with a construct that encoded the gene for IL-4. IL-4 co-immunisation increased the neutralizing antibody titres in this group. Importantly, the viral loads in animals vaccinated with IL-4 expressing plasmid increased during the immunisation regimens despite the higher neutralizing antibody titres. In addition, neutralizing antibodies did not correlate with viral set point prior to PMPA treatment, however, there was a correlation between viral loads and antibody titres following the treatment with PMPA. Antibody titres decreased following the suppression of viral load. Importantly, vaccination in the absence of IL-4 protected CD4 levels without increasing viral load. The data support the hypothesis that inappropriate immune bias toward a Th2 pathway would ultimately enhance disease progression.

Detection of cancer cells in peripheral blood of breast cancer patients using reverse transcription-polymerase chain reaction for epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) has been reported to be expressed in high levels in primary breast cancer by immunohistochemistry. In the present study, a reverse transcription (RT)-PCR assay using EGFR primers was developed and evaluated for the detection of circulating micrometastases in the blood of breast cancer patients. Total RNA was extracted from breast cancer cell lines and from the blood of 23 control individuals and 37 breast cancer patients. After reverse transcription, outer and nested primers for EGFR were used for cDNA amplification. RNA integrity was confirmed with parallel RT-PCR amplification using beta2-microglobulin primers. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized by UV photography. Southern blotting was used to confirm EGFR specificity. The nested EGFR RT-PCR assay was capable of detecting a lower limit of 100 fg of total RNA from the A431 cell line. EGFR RNA was identified from the blood of 4 of 18 (22%) metastatic breast cancer patients, 0 of 6 locally recurrent breast cancer patients, 0 of 13 adjuvant breast cancer patients, and 0 of 23 controls (P = 0.03, metastatic versus control). The 18 metastatic breast cancer patients all had progressive disease at the time of blood sampling. The identity of the four EGFR-positive bands was confirmed by Southern blotting. The presence of RT-PCR positivity for EGFR was not a treatment-related phenomenon, because three of the four EGFR-positive patients were not receiving treatment at the time of blood collection. RT-PCR for EGFR is a sensitive and specific method for the detection of circulating micrometastases in a proportion of patients with metastatic breast cancer.

Markers of bone resorption in patients treated with pamidronate.

Pyridinoline (PYD), deoxypyridinoline (DPD), and N-telopeptide (NTX) are markers of bone resorption. In cancer patients with bone metastases, NTX is more often elevated than either of the pyridinolines. Bisphosphonates inhibit osteoclasts and their treatment decreases skeletal complications of malignancy. The aim of this study was to correlate urinary PYD, DPD, and NTX levels with clinical events in patients receiving pamidronate. 25 cancer patients with lytic bone disease were treated with monthly pamidronate combined with endocrine or chemotherapy; 27 others were on placebo. Twenty-four hour urines were collected at baseline, 1, 3 and 6 months. NTX values were determined by enzyme-linked immunosorbent assay (ELISA); PYD and DPD values were determined by reverse phase high performance liquid chromatography (HPLC). Two hour urines were also collected weekly for 21 patients. The greatest difference as a result of pamidronate treatment was observed in NTX values. Maximum suppression was achieved 2 weeks after treatment. Of the 25 patients who received pamidronate, 21 had initially elevated NTX values. 12 of the 21 finished with normal NTX values, whilst 9/21 had NTX values which remained abnormally elevated. The proportions of patients with fractures between these two subgroups approached statistical significance (P = 0.07) while the proportions with bony disease progression were significant (P = 0.03, Fisher's exact test). Measuring NTX levels appears useful in monitoring bisphosphonate therapy of bone metastases. The goal of treatment should be to normalise NTX excretion.

Biochemical markers of bone turnover in patients with metastatic bone disease.

Several biochemical markers of bone formation and bone resorption have recently been developed. These markers have been evaluated for clinical utility in patients with metabolic bone disease, including Paget disease and osteoporosis, and for their potential use in cancer patients whose disease has metastasized to bone. We have evaluated seven markers of bone turnover in the plasma and urine of 94 patients with newly diagnosed or progressive malignancy with and without clinical evidence of bone metastases. As determined by a positive bone scan and (or) bone survey, 30 patients had metastases to bone; 51 patients had metastatic cancer without overt bony involvement; and 13 patients had local disease without bone metastases. To evaluate the predictive value of these markers in the metastatic population, we utilized a "Z-score" and logistic regression analysis to distinguish patients with documented bone metastatic disease from those patients without clinical evidence of bone metastases. The higher the Z-score, the better the marker predicts the presence of bone metastases. With this statistical approach, urine N-telopeptide measurements had the highest Z-score and the most significant association with the probability of bone metastases. Urine deoxypyridinoline was the second most predictive marker of bone metastases. Thus, biochemical markers of bone resorption might be of use to predict the presence of bone metastases in cancer patients and to monitor the efficacy of antiresorptive therapy in patients treated for metastatic bone disease.

Epidermal growth factor receptor ectodomain in the urine of patients with squamous cell carcinoma.

In this study we determined the levels of the epidermal growth factor receptor (EGFR) in the urine of patients with squamous cell carcinoma compared to levels in the urine of normal volunteers and patients with nonsquamous cell carcinoma. A 24-h urine specimen was collected from 50 normal volunteers, 50 patients with nonsquamous cell carcinoma, and 42 patients with squamous cell carcinoma. An ELISA using mAbs to the external domain of the EGFR was used to measure levels of the receptor in the urine samples. Measurement of the EGFR ectodomain in the 24-h urine specimens showed detectable levels in 15 (36%) of 42 squamous cell carcinoma patients compared to 3 (6%) of 50 controls and 8 (16%) of 50 nonsquamous patients. It was also observed that 10 (53%) of 19 patients with metastatic squamous cell carcinoma had detectable EGFR ectodomain levels compared to 5 (22%) of 23 squamous cell patients with localized disease. Thus, we concluded that the EGFR ectodomain was detectable in the urine in a significantly higher number of patients with squamous cell carcinoma than normal volunteers or patients with nonsquamous cell carcinoma.

Increased urinary excretion of pyridinium cross-links in cancer patients.

Pyridinoline (PYD) and deoxypyridinoline (DPD), two collagen-based cross-links found in bone, were measured by high-performance liquid chromatography in urine samples from 65 control subjects and 97 patients with either untreated or progressive cancer. Patients with cancer had significantly (P < 0.001) higher urine concentrations of PYD and DPD than did control subjects. Both cross-links were increased in cancer patients with and without clinically detectable bone metastases, although patients with bone and liver involvement had higher mean concentrations. The mean concentrations of both cross-links were also significantly higher in the urine samples of inpatients than in an outpatient ambulatory population. These findings suggest that the measurement of PYD and DPD in urine may be useful in assessing bone metastases and bone resorption in cancer patients.