Short communication: Effects of mammary biopsy in the dry period on activity and feeding behavior of dairy cows.

In dairy cattle, mammary biopsies are commonly used to study development and function of the mammary gland. The objective of this study was to investigate changes in activity and feeding patterns following the mammary biopsy procedure. Pregnant, nonlactating Holstein dairy cows (20 d before expected calving date) were exposed to either (1) a biopsy procedure, in which a mammary tissue sample (60 × 4 mm in diameter) was obtained from cows (n = 9) using a biopsy tool from the rear left quarter, following administration of a sedative (xylazine, 20 µg/kg of body weight) and local anesthetic (3 mL of lidocaine), or (2) a sham procedure, in which cows (n = 8) were removed from the pen and restrained for a similar duration of time as for the biopsy procedure. Behavior of cows was monitored for 5 d, beginning on the day following biopsy (approximately 14 h after the procedure). Cows were fitted with accelerometers to record daily lying time, lying bout frequency, and lying side. Daily individual feed intake was recorded using the Calan Broadbent feeding system, and feeding time and meal characteristics were determined from a subset of cows (n = 6 per treatment) using a change-of-state data logger to record the times the cows were accessing the feed bunk. Total daily lying time did not differ between treatments [13.9 h/d; standard error (SE) = 0.56], but biopsied cows had more frequent, shorter lying bouts on the biopsied side on d 1 following the procedure (6.67 vs. 4.25 bouts/d, SE = 1.03, and 70.0 vs. 97.0 min/bout, SE = 8.6; left vs. right side), whereas control cows had no side preference. We found no effects of treatment on feed intake and feeding time but, on the first day after treatment, biopsied cows had meals that were more frequent (7.2 vs. 4.6 meals/d; SE = 0.93) and tended to be shorter (28.2 vs. 60.9 min/meal; SE = 11.8) than control cows. In conclusion, we did not detect effects of mammary biopsy on feed intake or lying time during our time frame of observation, but activity patterns were altered, which could be indicative of increased overall restlessness and specific pain in the biopsied quarter.

Short communication: Repeated mammary tissue collections during lactation do not alter subsequent milk yield or composition.

Mammary biopsy collection (MB) is a valuable approach for studying mammary gland biology, but it is unclear if repeated MB impair the performance of lactating dairy cows. The objective of this trial was to examine the effect of repeated MB during lactation on udder health, dry matter intake (DMI), and lactation performance of lactating dairy cows. Sixty-four multiparous, mid-lactation Holstein cows were enrolled in a 29-wk trial, and 32 cows were randomly selected for repeated MB. The MB and non-MB (NMB) cows had similar parity (2.6 ± 0.9) and days in milk (96.5 ± 56.3 d) at enrollment. All animals were housed in the same barn and managed in the same manner. Cows were milked 3 times daily with milk yield recorded at each milking. Milk composition was measured weekly and DMI recorded daily. Three MB were performed per cow: 1 wk after enrollment and at 15 and 24 wk. The first and third MB were performed on the left rear quarter, whereas the second MB was on the right rear quarter. The MB were performed based on previously described procedures using a rotating stainless steel cannula with a retractable blade connected to a cordless drill, with appropriate sedation and antiseptic treatment after each MB. After MB, udder health, surgical wound healing, and presence of blood in milk were visually examined at each milking. Blood was cleared from milk 3.86 ± 2.0 d after MB. During the experiment, 4 rear quarters of MB cows and 5 rear quarters from NMB cows were diagnosed and treated for clinical mastitis. No differences were observed in DMI, milk yield, somatic cell score, or milk concentration and yields of fat, protein, lactose, and solids-not-fat between MB and NMB. In conclusion, lactating cows recover rapidly from MB, and repeated MB have no long-term effects on DMI, milk yield and composition, or udder health of lactating dairy cows.

Effects of feeding betaine-containing liquid supplement to transition dairy cows.

Betaine is a natural compound found in sugar beets that serves as a methyl donor and organic osmolyte when fed to animals. The objective was to evaluate the effect of feeding betaine-containing molasses on performance of transition dairy cows during late summer in 2 trials. In early September, cows were randomly assigned to betaine (BET) or control (CON) groups either shortly after dry off (trial 1; n = 10 per treatment) or 24 d before calving (trial 2; n = 8 per treatment) based on parity and previous mature equivalent milk yield. Cows were fed common diets supplemented either with a liquid supplement made of molasses from sugar cane and condensed beet solubles containing betaine [BET, 89.1 g/kg of dry matter (DM)] or a sugar cane molasses-based liquid supplement without betaine (CON) until 8 wk postpartum. The liquid supplements had similar nutrient contents and were fed at a rate of 1.1 and 1.4 kg DM/d for pre- and postpartum cows, respectively. Starting at their entry in the studies, cows were housed in the same freestall barn without a cooling system. After calving, all cows were housed in the same barn cooled by misters and fans and milked thrice daily. Intake was recorded daily and body weight and body condition score were assessed every 2 wk. Milk yield was recorded at each milking and composition was analyzed weekly. Blood samples were collected weekly from a subset of cows to assess concentrations of metabolites and AA. No treatment effects were apparent for DM intake and body weight in the prepartum and postpartum periods. For cows enrolled at dry off, BET supported higher milk yield (45.1 vs. 41.9 kg/d) and fat content (4.78 vs. 4.34%) and elevated plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate in early lactation compared to CON. However, no differences were observed for milk yield, most milk component contents and yields, and blood metabolites between treatments for cows enrolled during the close-up period. Compared to cows in the CON group, BET cows enrolled during the far-off period tended to have lower plasma concentrations of Met, Thr, and Trp during the pre- and postpartum periods. They also had lower plasma concentrations of Lys and Phe before calving but higher plasma Gly concentration after parturition. In conclusion, feeding a betaine-containing liquid supplement from far-off through early lactation improves lactation performance but increases adipose tissue mobilization and production of ketone bodies in early lactation.

Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells.

The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

Short communication: Effect of heat stress during the dry period on gene expression in mammary tissue and peripheral blood mononuclear cells.

Heat stress (HT) during the dry period compromises mammary gland development, decreases future milk production, and impairs the immune status of dairy cows. Our objective was to evaluate the effect of cooling HT cows during the dry period on gene expression of the mammary gland and peripheral blood mononuclear cells (PBMC). Cows were dried off 46 d before their expected calving and assigned to 2 treatments, HT or cooling (CL). Cows in the CL group were cooled with sprinklers and fans whereas HT cows were not. After parturition, all cows were housed in a freestall barn with cooling. The PBMC were isolated at dry-off and at -20, 2, and 20 d relative to calving from a subset of cows (HT, n=9; CL, n=10), and mammary biopsies were taken at the same intervals (HT, n=7; CL, n=6) for RNA extraction. Gene expression was assessed using a custom multiplex gene expression assay based on traditional reverse transcription-PCR. Genes involved in prolactin (PRL) signaling [PRL receptor long form, PRL receptor short form, suppressor of cytokine signaling (SOCS)2, SOCS3, IGF2, IGF binding protein 5, and cyclin D1], fatty acid metabolism (acetyl-CoA carboxylase α (ACACA) and lipoprotein lipase (LPL)], and IGF1 were evaluated in mammary tissue, and genes related to fatty acid metabolism [ACACA, fatty acid synthase (FASN), and LPL], cytokine production [IL6, IL8, and tumor necrosis factor (TNF)], and IGF1 were evaluated in PBMC. No differences were observed in PRL signaling or fatty acid metabolism gene expression in the mammary gland. In PBMC, HT cows had greater mRNA expression of IGF1 and TNF during the transition period relative to CL and upregulated IL8 and downregulated FASN mRNA expression at 2 d relative to calving. We conclude that cooling HT cows during the dry period alters expression of genes involved in cytokine production and lipid metabolism in PBMC.

Correlation of rectal temperature and peripheral temperature from implantable radio-frequency microchips in Holstein steers challenged with lipopolysaccharide under thermoneutral and high ambient temperatures.

Early detection of disease can speed treatment, slow spread of disease in a herd, and improve health status of animals. Immune stimulation increases rectal temperature (RT). Injectable radio-frequency implants (RFI) can provide temperature at the site of implantation. The fidelity of peripheral site temperature, determined by RFI, relative to RT is unknown in cattle. We hypothesized that during lipopolysaccharide (LPS) challenge, temperature at 3 peripheral sites would be similar to RT in steers (n = 4; BW 77 ± 2.1 kg). The 3 sites were 1) subcutaneous (SC) at the base of the ear (ET); 2) SC posterior to the poll (PT); and 3) SC beneath the umbilical fold (UT). Steers were housed in controlled temperature (CT) rooms (between 18 and 21°C; n = 2/room). Rectal temperature, ET, PT, and UT were recorded every 8 h daily. On d 7, 21, 22, 36, and 37, RT and RFI were taken every 5 min for 6 h, every 15 min for 3 h, and every 30 min for 15 h. To test RFI during a simulated immune challenge, LPS (E. coli 055:B5) was injected intravenously (i.v.) at 1000 h on d 22 and 37. Basal temperatures (°C) were RT (38.7 ± 0.20), ET (37.1 ± 0.86), PT (36.7 ± 0.57), and UT (36.3 ± 0.97). Rectal temperature increased to 39.9 ± 0.30°C after LPS, but ET, PT, and UT decreased. Heat stress also increases RT, which makes it difficult to identify sick animals using RT. The second hypothesis tested was that ET positively correlates to RT and negatively correlates to RT during LPS under heat stress. Four steers (127 ± 7.3 kg) were housed in CT chambers (n = 2/chamber), implanted with a RFI, and allowed 2 wk to acclimate. One chamber remained at 20°C, the other was increased to 34°C starting at 0800 h for a period of 48 h. The LPS was administered i.v. to all steers at 1000 h on d 2. After a 2-wk recovery at 20°C, the temperature was increased in the other chamber, resulting in a crossover design with each steer serving as its own control. Pearson's correlation coefficients for ET and RT were 0.30 (P < 0.01) during heat stress, 0.20 (P < 0.05) during heat stress with LPS challenge, 0.34 (P < 0.01) during thermoneutrality, and -0.42 (P < 0.01) during thermoneutrality with LPS. These data refute the hypothesis that RT and peripheral temperature move in synchrony after LPS challenge. These data suggest that individual response be considered when identifying models for use of ET, but these RFI have potential for use in the early detection of diseases that alter basal temperature.

Bovine luteal prolactin receptor expression: potential involvement in regulation of progesterone during the estrous cycle and pregnancy.

In the present study, we performed quantitative reverse-transcription PCR (qPCR) to examine changes in gene expression of prolactin receptor (long form: l-PRLR; short form: s-PRLR) and 20α-hydroxysteroid dehydrogenase (20α-HSD; EC 1.1.1.149) in the bovine corpus luteum (CL) throughout the estrous cycle and pregnancy. Western blotting was used to determine protein abundance. Bovine CL were collected and luteal stages (n = 6/stage) were classified by macroscopic observation as early (d 1 to 4 after ovulation), mid (d 5 to 10), late (d 11 to 17), and regressing (d 18 to 20). A CL of pregnancy (n = 6) was determined by the presence of conceptus (d 28 to term). The mRNA for both forms of PRLR were expressed at all the luteal stages. Expression of s-PRLR and l-PRLR mRNA was less (P < 0.01) during early and regressing luteal stages compared with mid and late stages. Expression of s-PRLR mRNA in CL of pregnancy was greater (P < 0.01) than early, mid, and regressing CL and did not differ from late luteal stage expression. A greater (P < 0.01) expression of l-PRLR mRNA was observed in pregnant vs. early and regressing CL. In addition, qPCR showed the presence of 20α-HSD mRNA during all luteal stages of the estrous cycle, with the greatest (P < 0.01) expression observed in the regressing luteal stage. Western blotting revealed protein abundance of both PRLR isoforms during all luteal stages and pregnancy, with a predominance of the s-PRLR protein. Densitometry analysis indicated that protein abundances of s-PRLR were greater (P < 0.05) than l-PRLR during early, mid, and late luteal stages and did not differ during the regressing luteal stage. Protein abundances of 20α-HSD were least (P < 0.05) during the early luteal stage. In conclusion, results of the current study suggest a possible involvement of PRLR, especially s-PRLR, in the regulation of progesterone secretion and metabolism during the bovine estrous cycle and pregnancy.

Heat stress abatement during the dry period influences metabolic gene expression and improves immune status in the transition period of dairy cows.

Heat stress (HT) and photoperiod affect milk production and immune status of dairy cows. The objective was to evaluate the effects of HT abatement prepartum under controlled photoperiod on hepatic metabolic gene expression and cellular immune function of periparturient Holstein cows (n=21). Cows were dried off 46 d before expected calving date and assigned to treatments by mature equivalent milk production. The treatments were 1) HT and 2) cooling (CL), both imposed during a photoperiod of 14L:10D. Rectal temperature was measured twice daily, whereas respiration rate was measured 3 times/wk at 1500 h during the entire dry period. After calving, cows were housed in a freestall barn with cooling, and milk yield was recorded daily up to 140 d in milk. Liver samples were taken at dry off, -20, 2, and 20 d relative to calving by biopsy. Under a similar schedule, neutrophil function was determined in blood of cows on HT (n=12) and CL (n=9). Blood samples were taken on -46, -32, -18, 0, 14, 28, and 42 d relative to calving for measurement of metabolites and were collected twice daily from -7 to 2 d relative to calving for prolactin (PRL) analysis. The HT cows had greater concentrations of PRL at 0 d relative to calving (150 vs. 93; SEM=11 ng/mL) and had higher afternoon rectal temperatures (39.4 vs. 39.0; SEM=0.04°C) and elevated respiration rates (78 vs. 56; SEM=2 breaths/min) during the prepartum period compared with CL cows. Relative to HT cows, CL cows had greater hepatic expression of PRL-R, SOCS-3, and CAV-1 mRNA. Neutrophil oxidative burst was greater in CL cows relative to HT cows at 2 d (61 vs. 42; SEM=6%) and at 20 d (62 vs. 49; SEM=5%) relative to calving, and phagocytosis was greater in CL cows at 20 d (47 vs. 33; SEM=4%) relative to calving compared with HT cows. Humoral response, as measured by IgG secretion against ovalbumin challenge, was greater for CL cows at -32 d (0.44 vs. 0.33; SEM=0.05 OD) and -21 d (0.60 vs. 0.50±0.04 OD) relative to calving compared with HT cows. These results suggest that HT abatement during the dry period improved innate and acquired immune status as measured by neutrophil function and immunoglobulin secretion against ovalbumin challenge, and altered hepatic gene expression related to PRL signaling in the periparturient period or subsequent lactation.

Heat stress abatement during the dry period influences prolactin signaling in lymphocytes.

Heat stress perturbs prolactin (PRL) release and affects dairy cow lactational performance and immune cell function. We hypothesized that greater PRL concentration in plasma of heat-stressed cows relative to cooled cows would decrease expression of prolactin receptor (PRL-R) mRNA and increase mRNA expression of suppressors of cytokine signaling (SOCS) in lymphocytes, altering their cytokine production. To test this hypothesis, multiparous Holstein cows were dried off 46 d before their expected calving date and assigned randomly to heat stress (HT; n=9) or cooling (CL; n=7) during the entire dry period. A second study was conducted the following year with an additional 21 cows (12 HT; 9 CL). Lymphocytes were isolated from cows at -46, -20, +2, and +20 d relative to expected calving date and mRNA expression of PRL-R, SOCS-1, SOCS-2, SOCS-3, cytokine-inducible SH2-containing protein (CIS), and heat shock protein 70 KDa A5 (HSPA5), and housekeeping genes hydroxymethylbilane synthase (HMBS), ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit (ATP5B), and ribosomal protein S9 (RPS9) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Cows exposed to HT had greater PRL concentration in plasma compared with CL cows. Measurement of lymphocyte proliferation indicated that lymphocytes of CL cows proliferated more than those from HT cows and exressed more PRL-R mRNA and less SOCS-1 and SOCS-3 mRNA relative to HT cows. Further, lymphocytes from CL cows produced more tumor necrosis factor-alpha (TNF-alpha) than those from HT cows. These results suggest that changes in PRL-signaling pathway genes during heat stress are associated with differential cytokine secretion by lymphocytes and may regulate lymphocyte proliferation in dairy cows.

Flow differences between endometrial polyps and cancer: a prospective study using intravenous contrast-enhanced transvaginal color flow Doppler and three-dimensional power Doppler ultrasound.

OBJECTIVES: To evaluate whether assessment of blood flow by transvaginal color Doppler and three-dimensional power Doppler imaging, enhanced by intravenous contrast, may be useful in the differentiation between benign endometrial polyps and endometrial cancer. METHODS: A prospective study was performed comparing 17 women with benign endometrial polyps and 17 women with endometrial cancer. Transvaginal color Doppler and three-dimensional power Doppler angiography were performed before and after injection of intravenous contrast. The pulsatility index (PI) and the resistance index (RI) of the polyp feeding vessel or central vessel in malignant lesions, as well as the power Doppler endometrial flow indices vascularization index (VI), flow index (FI) and vascularization flow index (VFI), were calculated before and after enhancement by contrast, and compared between the two groups of women. RESULTS: PI (mean difference +/- SD, 0.68 +/- 0.22; 95% CI, 0.23-1.13; P = 0.004) and RI (mean difference +/- SD, 0.16 +/- 0.08; 95% CI, 0.00-0.32; P = 0.045) were significantly lower in vessels of malignant tumors than in those of benign endometrial polyps after enhancement by intravenous contrast. No significant differences in PI, RI, VI, FI or VFI before enhancement by contrast, or in VI, FI or VFI after enhancement by contrast, were detected between women with endometrial polyps and those with endometrial cancer. CONCLUSIONS: Transvaginal color Doppler examination enhanced by intravenous contrast may help to discriminate between benign endometrial polyps and cancer. Larger studies are required to confirm these findings.

P2X7 receptor-Pannexin1 complex: pharmacology and signaling.

Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 microM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.

Mammary response to exogenous prolactin or frequent milking during early lactation in dairy cows.

Frequent milking of dairy cows during early lactation results in a persistent increase in milk yield; however, the mechanism underlying this effect is unknown. We hypothesized that increased exposure of the mammary gland to prolactin (PRL) mediates the milk yield response. Fifteen multiparous Holstein cows were assigned to 3 treatments for the first 3 wk of lactation: twice daily milking with (2x + PRL) or without (2x) supplemental exogenous PRL, or 4 times daily milking (4x). Mammary biopsies were obtained at 7 DIM, and rates of [(3)H]thymidine incorporation into DNA in vitro were determined. Mammary expression of suppressors of cytokine signaling (SOCS)-1, -2, and -3; the long form of PRL-receptor; and alpha-lactalbumin mRNA was measured by real-time reverse-transcription PCR. Incorporation of [(3)H]thymidine into DNA was not affected by frequent milking or PRL treatment; however, analysis of autoradiograms revealed that stromal cell proliferation was greater in 4x cows. Mammary expression of SOCS-1 was not affected by milking frequency or PRL treatment. Expression of SOCS-2 mRNA was increased with frequent milking or PRL treatment, whereas expression of SOCS-3 mRNA was reduced by frequent milking or exogenous PRL. Abundance of PRL-receptor mRNA was reduced, whereas alpha-lactalbumin mRNA was increased with PRL treatment. These results demonstrate that the bovine mammary gland is responsive to exogenous PRL during early lactation. In addition, differences in the response to frequent milking or exogenous PRL during early lactation indicate distinct effects of PRL and milk removal on the mammary function of dairy cows.

Major advances associated with environmental effects on dairy cattle.

It has long been known that season of the year has major impacts on dairy animal performance measures including growth, reproduction, and lactation. Additionally, as average production per cow has doubled, the metabolic heat output per animal has increased substantially rendering animals more susceptible to heat stress. This, in turn, has altered cooling and housing requirements for cattle. Substantial progress has been made in the last quarter-century in delineating the mechanisms by which thermal stress and photoperiod influence performance of dairy animals. Acclimation to thermal stress is now identified as a homeorhetic process under endocrine control. The process of acclimation occurs in 2 phases (acute and chronic) and involves changes in secretion rate of hormones as well as receptor populations in target tissues. The time required to complete both phases is weeks rather than days. The opportunity may exist to modify endocrine status of animals and improve their resistance to heat and cold stress. New estimates of genotype x environment interactions support use of recently available molecular and genomics tools to identify the genetic basis of heat-stress sensitivity and tolerance. Improved understanding of environmental effects on nutrient requirements has resulted in diets for dairy animals during different weather conditions. Demonstration that estrus behavior is adversely affected by heat stress has led to increased use of timed insemination schemes during the warm summer months to improve conception rates by discarding the need to detect estrus. Studies evaluating the effects of heat stress on embryonic survival support use of cooling during the immediate postbreeding period and use of embryo transfer to improve pregnancy rates. Successful cooling strategies for lactating dairy cows are based on maximizing available routes of heat exchange, convection, conduction, radiation, and evaporation. Areas in dairy operations in which cooling systems have been used to enhance cow comfort, improve milk production, reproductive efficiency, and profit include both housing and milking facilities. Currently, air movement (fans), wetting (soaking) the cow's body surface, high pressure mist (evaporation) to cool the air in the cows' environment, and facilities designed to minimize the transfer of solar radiation are used for heat abatement. Finally, improved understanding of photoperiod effects on cattle has allowed producers to maximize beneficial effects of photoperiod length while minimizing negative effects.

Low or absent SPARC expression in acute myeloid leukemia with MLL rearrangements is associated with sensitivity to growth inhibition by exogenous SPARC protein.

Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular glycoprotein with growth-inhibitory and antiangiogenic functions. Although SPARC has been implicated as a tumor suppressor in humans, its function in normal or malignant hematopoiesis has not previously been studied. We found that the leukemic cells of AML patients with MLL gene rearrangements express low to undetectable amounts of SPARC whereas normal hematopoietic progenitors and most AML patients express this gene. SPARC RNA and protein levels were also low or undetectable in AML cell lines with MLL translocations. Consistent with its tumor suppressive effects in various solid tumor models, exogenous SPARC protein selectively reduced the growth of cell lines with MLL rearrangements by inhibiting cell cycle progression from G1 to S phase. The lack of SPARC expression in MLL-rearranged cell lines was associated with dense promoter methylation. However, we found no evidence of methylation-based silencing of SPARC in primary patient samples. Our results suggest that low or absent SPARC expression is a consistent feature of AML cells with MLL rearrangements and that SPARC may function as a tumor suppressor in this subset of patients. A potential role of exogenous SPARC in the therapy of MLL-rearranged AML warrants further investigation.

Pediatric Oncology Group (POG) studies of acute myeloid leukemia (AML): a review of four consecutive childhood AML trials conducted between 1981 and 2000.

From 1981 to 2000, a total of 1823 children with acute myeloid leukemia (AML) enrolled on four consecutive Pediatric Oncology Group (POG) clinical trials. POG 8101 demonstrated that the induction rate associated with the 3+7+7 combination of daunorubicin, Ara-C, and 6-thioguanine (DAT) was greater than that associated with an induction regimen used to treat acute lymphoblastic leukemia (82 vs 61%; P=0.02). Designed as a pilot study to determine the feasibility of administration of noncross-resistant drug pairs and later modified to assess the effect of dose intensification of Ara-C during the second induction course, POG 8498 confirmed the high initial rate of response to DAT (84.2%) and showed that dose intensification of Ara-C during the second induction course resulted in a trend toward higher event-free survival (EFS) estimates than did standard-dose DAT (2+5) during the second induction course (5 year EFS estimates, 22 vs 27%; P=0.33). Age <2 years and leukocyte count <100 000/mm3 emerged as significantly good prognostic factors. The most significant observation made in the POG 8498 study was the markedly superior outcome of children with Down's syndrome who were treated on the high-dose Ara-C regimen. POG 8821 compared the efficacy of autologous bone marrow transplantation (BMT) with that of intensive consolidation chemotherapy. Intent-to-treat analysis revealed similar 5-year EFS estimates for the group that underwent autologous BMT (36+/-4.7%) and for the group that received only intensive chemotherapy (35+/-4.5%) (P=0.25). There was a high rate of treatment-related mortality in the autologous transplantation group. The study demonstrated superior results of allogeneic BMT for patients with histocompatible related donors (5-year EFS estimate 63+/-5.4%) and of children with Down's syndrome (5-year EFS estimate, 66+/-8.6%). The POG 9421 AML study evaluated high-dose Ara-C as part of the first induction course and the use of the multidrug resistance modulator cyclosporine. Preliminary results showed that patients receiving both high-dose Ara-C for remission induction and the MDR modulator for consolidation had a superior outcome (5-year EFS estimate, 42+/-8.2%) than did patients receiving other treatment; however, the difference was not statistically significant. These four studies demonstrate the importance of dose intensification of Ara-C in the treatment of childhood AML; cytogenetics as the single most prognostic factor and the unique curability of AML in children with Down's syndrome.

Proteomic analysis of childhood leukemia.

Childhood acute lymphoblastic and myeloid leukemias are stratified into molecular and cytogenetic subgroups important for prognosis and therapy. Studies have shown that gene expression profiles can discriminate between leukemia subtypes. Thus, proteome analysis similarly holds the potential for characterizing different subtypes of childhood leukemia. We used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to analyze cell lysates from childhood leukemia cell lines as well as pretreatment leukemic bone marrow derived from childhood leukemia cases. Comparison of the acute myeloid leukemia (AML) cell line, Kasumi, and the biphenotypic myelomonocytic cell line, MV4;11, with the acute lymphoblastic leukemia (ALL) cell lines, 697 and REH, revealed many differentially expressed proteins. In particular, one 8.3 kDa protein has been identified as a C-terminal truncated ubiquitin. Analysis of childhood leukemia bone marrow showed differentially expressed proteins between AML and ALL, including a similar peak at 8.3 kDa, as well as several proteins that differentiate between the ALL t(12;21) and hyperdiploid subtypes. These results demonstrate the potential for proteome analysis to distinguish between various forms of childhood leukemia. Future analyses are warranted to validate these findings and to investigate the role of the C-terminal truncated ubiquitin in the etiology of ALL.

Exposure to short day photoperiod during the dry period enhances mammary growth in dairy cows.

Exposure to short day photoperiod (SD; 8 h light:16 h dark) during the dry period increased milk yield of cows in the subsequent lactation. We hypothesized that this effect is due to increased growth of mammary cells in response to enhanced prolactin signaling to influence the insulin-like growth factor (IGF) axis. Multiparous Holstein cows were dried off 60 d before parturition and assigned to long day photoperiod (LD; 16 h light:8 h dark) or SD during the dry period. Mammary biopsies were obtained at approximately -40, -20, -10 and +10 d relative to expected calving. Expression of IGF-I, IGF-II, and IGF binding protein-5 mRNA was assessed by real time reverse transcription-polymerase chain reaction. In SD cows, incorporation of [3H]-thymidine in vitro increased from -40 d to -20 d and was greater at -20 d than in LD cows. A later increase in proliferation was observed at -10 d in LD cows. For both groups, cell proliferation decreased during lactation. Analysis by terminal deoxynucleotidyl transferase dUTP nick end labeling revealed that the apoptotic index in mammary epithelial cells was less in SD cows than in LD cows. Expression of IGF-II mRNA increased during the dry period and into lactation and was greater in SD cows. Expression of IGF binding protein-5 mRNA increased during lactation, but was unaffected by day length. Expression of IGF-I did not differ over time or between treatments. We concluded that exposure to SD during the dry period enhanced mammary growth relative to LD, and this may be related to increased expression of IGF-II. Treatment differences in the temporal pattern of proliferation indicated the existence of a critical period wherein photoperiod affects mammary gland development during the dry period.

Phase I trial of intrathecal liposomal cytarabine in children with neoplastic meningitis.

PURPOSE: We performed a phase I trial of intrathecal (IT) liposomal cytarabine (DepoCyt; Enzon Pharmaceuticals, Piscataway, NJ and SkyePharma Inc, San Diego, CA) to determine the maximum-tolerated dose, the dose-limiting toxicities, and the plasma and CSF pharmacokinetics of IT lipsomal cytarabine in children >/= 3 years of age with advanced meningeal malignancies. PATIENTS AND METHODS: Eighteen assessable patients received IT liposomal cytarabine through either an indwelling ventricular access device or via lumbar puncture. Liposomal cytarabine was given once every 2 weeks during induction, once every 4 weeks during consolidation, and once every 8 weeks during the maintenance phase of treatment. The initial dose was 25 mg, with subsequent escalations to 35 and 50 mg. CSF pharmacokinetic samples were obtained in a subset of patients. RESULTS: Arachnoiditis, characterized by fever, headache, nausea, vomiting, and back pain was noted in the first two patients at the 25 mg dose level. Therefore, subsequent patients were treated with dexamethasone, beginning the day of liposomal cytarabine administration and continuing for 5 days. Headache (grade 3) was dose limiting in two of eight patients enrolled at the 50 mg dose level. Eight of the 14 patients assessable for response demonstrated evidence of benefit manifest as prolonged disease stabilization or response. CONCLUSION: The maximum-tolerated dose and recommended phase II dose of liposomal cytarabine in patients between the ages of 3 and 21 years is 35 mg, administered with dexamethasone (0.15 mg/kg/dose, twice a day for 5 days). A phase II trial of IT liposomal cytarabine in children with CNS leukemia in second or higher relapse is in development.