Microbiological quality and somatic cell count in bulk milk of dromedary camels (Camelus dromedarius): descriptive statistics, correlations, and factors of variation.

The objectives of the present study were to monitor the microbiological quality and somatic cell count (SCC) of bulk tank milk at the world's first large-scale camel dairy farm for a 2-yr period, to compare the results of 2 methods for the enumeration of SCC, to evaluate correlation among milk quality indicators, and to determine the effect of specific factors (year, season, stage of lactation, and level of production) on milk quality indicators. The study was conducted from January 2008 to January 2010. Total viable count (TVC), coliform count (CC), California Mastitis Test (CMT) score, and SCC were determined from daily bulk milk samples. Somatic cell count was measured by using a direct microscopic method and with an automatic cell counter. In addition, production parameters [total daily milk production (TDM, kg), number of milking camels (NMC), average milk per camel (AMC, kg)] and stage of lactation (average postpartum days, PPD) were recorded for each test day. A strong correlation (r=0.33) was found between the 2 methods for SCC enumeration; however, values derived using the microscopic method were higher. The geometric means of SCC and TVC were 394×10(3) cells/mL and 5,157 cfu/mL during the observation period, respectively. Somatic cell count was >500×10(3) cells/mL on 14.6% (106/725) and TVC was >10×10(3) cfu/mL on 4.0% (30/742) of the test days. Both milk quality indicators had a distinct seasonal pattern. For log SCC, the mean was lowest in summer and highest in autumn. The seasonal pattern of log TVC was slightly different, with the lowest values being recorded during the spring. The monthly mean TVC pattern showed a clear difference between years. Coliform count was <10 cfu/mL in most of the samples (709/742, 95.6%). A positive correlation was found between log SCC and log TVC (r=0.32), between log SCC and CMT score (r=0.26), and between log TVC and CC in yr 1 (r=0.30). All production parameters and stage of lactation showed strong seasonal variation. Log SCC was negatively correlated with TDM (r=-0.35), AMC (r=-0.37), and NMC (r=-0.15) and positively correlated with PPD (r=0.40). Log TVC had a negative correlation with AMC (r=-0.40) but a positive correlation with NMC (r=0.32), TDM (r=0.16), and PPD (r=0.45). The linear mixed model with stepwise variable selection showed that the main sources of log SCC variation were PPD, TDM, PPD × season, and season. For log TVC, the same factors and year contributed to the variation.

Growth regulation of human melanocytes: mitogenic factors in extracts of melanoma, astrocytoma, and fibroblast cell lines.

Melanocytes derived from fetal or adult skin do not propagate in vitro unless cultured in the presence of factors such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In a search for physiological factors regulating the growth of melanocytes, extracts of various cultured cell types were tested. Factors produced by melanoma and astrocytoma cell lines support continued proliferation of melanocytes in the absence of TPA. WI-38, a fibroblast cell line derived from human embryonic lung, was the most active source of melanocyte growth factors. No melanocyte growth-promoting activity was found in extracts of cultured neuroblastoma, renal cancer, normal keratinocytes, or renal epithelium. Nerve growth factor, epidermal growth factor, melanocyte-stimulating hormone, transforming growth factor-beta, and platelet-derived growth factor did not have growth-promoting activity for melanocytes. The presence of melanocyte growth factors and TPA together resulted in the strongest mitogenic activity for melanocytes, permitting the recovery (at 20 days) of 4 to 20 times as many cells as in growth factor or TPA alone.

Isolation of a preadipocyte cell line from rat bone marrow and differentiation to adipocytes.

A unique population of rat adipocyte precursor cells was derived from normal rat bone marrow. The epitheloid-like preadipocytes were isolated from a mixed culture of bone marrow cells by a combination of differential trypsinization, enrichment by Ficoll gradient centrifugation, and differential seeding. This cell line, designated RBM-Ad, can be fully differentiated into multilocular adipocytes morphologically resembling brown adipose tissue. No changes in the differentiation pattern are observed during propagation of these cells, and they have been successfully carried and differentiated up to passage 49. Histological staining of differentiated cells with Sudan black, Sudan IV, and oil red O indicates the presence of lipids in intracellular vesicles. The nonselective beta-adrenergic agonist isoproterenol stimulates adenylyl cyclase activity in both preadipocytes and differentiated adipocytes. In contrast, BRL-37344, a beta 3-adrenergic receptor-specific agonist, stimulates adenylyl cyclase activity and glycerol release in differentiated adipocytes, but not preadipocytes. In addition, differentiated adipocytes contain messenger RNA encoding the brown adipose-specific protein, thermogenin. Thus, this rat preadipocyte cell line can be differentiated into adipocytes that histologically and functionally resemble brown adipose tissue.

Characterization of the binding of [125I-iodo-histidyl, methyl-Phe7] neurokinin B to the neurokinin-3 receptor.

We have characterized the binding of [125I-iodo-histidyl, methyl Phe7]neurokinin B (125I-NKB) to the human neurokinin-3 (NK3) receptor. 125I-NKB specifically binds to the NK3 receptor expressed in CHO cells with a Kd of 0.2 nM. The ligand displays little crossreactivity with the human NK1 and NK2 receptors. The binding of 125I-NKB to the human NK3 receptor and to rat cortex membranes is inhibited by neurokinin B with IC50 of 1.5 nM and 4 nM, respectively. In contrast, 350- to 500-fold higher concentrations of substance P and neurokinin A are required to inhibit binding to either receptor preparation. The data suggest that 125I-NKB is a high affinity, selective ligand for the human and rat NK3 receptor.

L-750355, a human beta3-adrenoceptor agonist; in vitro pharmacology and profile of activity in vivo in the rhesus monkey.

The profile of in vitro and in vivo biology of a human beta3-adrenoceptor agonist, (S)-N-[4-[2-[[3[(2-amino-5-pyridinyl)oxy]-2-hydroxy-propyl]amino]-eth yl]-phenyl]-4-isopropylbenzenesulfonamide, L-750355, is described. Using cloned human and rhesus beta1-, beta2- and beta3-adrenoceptors, expressed in Chinese hamster ovary (CHO) cells, L-750355 was shown to be a potent, albeit partial, agonist for the human (EC(50)=10 nM; % maximal receptor activation=49%) and rhesus (EC(50)=28 nM; % maximal receptor activation=34%) beta3-adrenoceptors. Furthermore, L-750355 stimulates lipolysis in rhesus adipocytes in vitro. L-750355 is a weak partial agonist (EC(50)=3.2 microM; % maximal receptor activation=33% ) for the human beta1-adrenoceptor but exhibits no agonist activity for rhesus beta1- or beta2-adrenoceptors of either human or rhesus origin. Administration of L-750355 to anesthetized rhesus monkeys, as a series of rising dose intravenous infusions, evokes dose-dependent glycerolemia and tachycardia with no change in mean arterial blood pressure or plasma potassium. The dose-response curve for L-750355-induced glycerolemia lies to the left of that for tachycardia. Propranolol, at a dose (0.3 mg/kg, i.v. ) that attenuates isoproterenol-induced changes in heart rate and glycerolemia, abolished L-750355-induced tachycardia but had no effect on L-750355-induced glycerolemia.

Regulation of cell-based therapeutic products intended for human applications in the EU.

AIMS: Recent developments in the field of cell-based therapeutic products (CBTPs) have forced the EU to revise its legislation on therapeutic products by enacting several new legal instruments. In this study, we investigate how CBTPs are regulated and what determines their regulatory classification. Furthermore, we compare the regulatory burden between CBTPs in different product categories. MATERIALS & METHODS: Product categories covering CBTPs were identified and characteristics critical for the regulatory classification of a CBTP were determined in each category. The effect of the critical characteristics on the classification was evaluated by constructing a decision tree that covers all possible combinations of the critical characteristics. Differences in the regulatory burden between CBTPs were evaluated by comparing regulations crucial for placing a therapeutic product on the EU market between the product categories. RESULTS: Regulation of CBTPs has been divided between the main product categories of the EU legal framework for therapeutic products on the basis of the characteristics of the cells that the CBTPs contain. The regulatory burden is lowest for CBTPs regulated as blood, cells or tissues, and highest for CBTPs regulated as medicinal products. CONCLUSION: CBTPs exist in all product categories of the EU legal framework for therapeutic products. However, the current framework does not cover all possible CBTPs. Furthermore, our results indicate that the regulatory burden of a CBTP is related to the risk it may pose to the health and safety of recipients.

Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin.

Cultures consisting almost entirely of human melanocytes were obtained from epidermal single-cell suspensions by using phorbol 12-myristate 13-acetate (10 ng/ml) in the culture medium. At this concentration, phorbol ester is toxic to human keratinocytes but not to melanocytes. When the seeding density was optimal (0.8-2 x 10(4)/cm2) and the medium contained both phorbol ester and cholera toxin, melanocytes proliferated extensively. Under these conditions, human melanocytes could be passaged serially in vitro and grown in quantity. This cell culture system can thus be used to answer basic questions related to pigment cell biology and may serve as a control for studies of malignant melanocytes.