Autologous blood salvage in the era of patient blood management.

Almost 150 years after the first autologous blood transfusion was reported, intraoperative blood salvage has become an important method of blood conservation. The primary goal of autologous transfusion is to reduce or avoid allogeneic red blood cell transfusion and the associated risks and costs. Autologous salvaged blood does not result in immunological challenge and its consequences, provides a higher quality red blood cell that has not been subjected to the adverse effects of blood storage, and can be more cost-effective than allogeneic blood when used for carefully selected surgical patients. Cardiac, orthopaedic and vascular surgery procedures with large anticipated blood loss can clearly benefit from the use of cell salvage. There are safety concerns in cases with gross bacterial contamination. There are theoretical safety concerns in obstetrical and cancer surgery; however, careful cell washing as well as leucoreduction filters makes for a safer autologous transfusion in these circumstances. Further studies are needed to determine whether oncologic outcomes are impacted by transfusing salvaged blood during cancer surgery. In this new era of patient blood management, where multimodal methods of reducing dependence on allogeneic blood are becoming commonplace, autologous blood salvage remains a valuable tool for perioperative blood conservation. Future studies will be needed to best determine how and when cell salvage should be utilized along with newer blood conservation measures.

Mutations associated with familial melanoma impair p16INK4 function.

Cell division is controlled by a series of positive and negative regulators which act at sequential points throughout the cell cycle. Disturbance of these checks could contribute to cancer by allowing excessive cell proliferation. The point in G1 at which cells irrevocably commit to DNA synthesis is controlled by protein complexes consisting of cyclin-dependent kinases (CDK4 or CDK6) and cyclins (D1, D2 or D3). These complexes are inhibited by low molecular weight proteins, such as p16INK4 (refs 1,2), p15INK4B (ref. 3) and p18 (ref. 4). Deletion or mutation of these CDK-inhibitors could lead to unchecked cell growth, suggesting that members of the p16INK4 family may be tumour suppressor genes. The recent detection of p16INK4 (MTS1) mutations in familial melanoma kindreds, many human tumour cell lines, and primary tumours is consistent with this idea. Previously, we described eight germline p16INK4 substitutions in 18 familial melanoma kindreds. Genetic analyses suggested that five mutations predisposed carriers to melanoma, whereas two missense mutations had no phenotypic effect. We now describe biochemical analyses of the missense germline mutations and a single somatic mutation detected in these families. Only the melanoma-predisposing mutants were impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 and cyclin D1/CDK6 complexes in vitro. Our data provide a biochemical rationale for the hypothesis that carriers of certain p16INK4 mutations are at increased risk of developing melanoma.

Synergistic anti-tumor effects between oncolytic vaccinia virus and paclitaxel are mediated by the IFN response and HMGB1.

Recent developments in the field of oncolytic or tumor-selective viruses have meant that the clinical applications of these agents are now being considered in more detail. Like most cancer therapies it is likely that they will be used primarily in combination with other therapeutics. Although several reports have shown that oncolytic viruses can synergize with chemotherapies within an infected cancer cell, it would be particularly important to determine whether factors released from infected cells could enhance the action of chemotherapies at a distance. Here, we demonstrate in vitro synergy between oncolytic vaccinia and taxanes. However, we also show, for the first time, that this synergy is at least partly due to the release of factors from the infected cells that are capable of sensitizing surrounding cells to chemotherapy. Several cellular factors were identified as being mediators of this bystander effect, including type I interferon released soon after infection and high-mobility group protein B1 (HMGB1) released after cell death. This represents the first description of these mechanisms for beneficial interactions between viral and traditional tumor therapies. These data may provide a direct basis for the design of clinical trials with agents currently in the clinic, as well as providing insight into the development of next generation viral vectors.

A phase II, multicenter, open-label randomized study of motesanib or bevacizumab in combination with paclitaxel and carboplatin for advanced nonsquamous non-small-cell lung cancer.

BACKGROUND: This phase II study estimated the difference in objective response rate (ORR) among patients with advanced nonsquamous non-small-cell lung cancer (NSCLC) receiving paclitaxel-carboplatin (CP) plus motesanib or bevacizumab. PATIENTS AND METHODS: Chemotherapy-naive patients (N = 186) were randomized 1:1:1 to receive CP plus motesanib 125 mg once daily (qd) (arm A), motesanib 75 mg twice daily (b.i.d.) 5 days on/2 days off (arm B), or bevacizumab 15 mg/kg every 3 weeks (q3w) (arm C). The primary end point was ORR (per RECIST). Other end points included progression-free survival (PFS), overall survival (OS), motesanib pharmacokinetics, and adverse events (AEs). RESULTS: ORRs in the three arms were as follows: arm A, 30% (95% confidence interval 18% to 43%); arm B, 23% (13% to 36%); and arm C, 37% (25% to 50%). Median PFS in arm A was 7.7 months, arm B 5.8 months, and arm C 8.3 months; median OS for arm A was 14.0 months, arm B 12.8 months, and arm C 14.0 months. Incidence of AEs was greater in arms A and B than in arm C. More grade 5 AEs not attributable to disease progression occurred in arm B (n = 10) than in arms A (n = 4) and C (n = 4). Motesanib plasma C(max) and C(min) values were consistent with its pharmacokinetic properties observed in previous studies. CONCLUSIONS: The efficacy of 125 mg qd motesanib or bevacizumab plus CP was estimated to be comparable. Toxicity was higher but manageable in both motesanib arms. Efficacy and tolerability of motesanib 125 mg qd plus CP in advanced nonsquamous NSCLC are being further investigated in a phase III study.

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae.

Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae. The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected. Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone. Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry. p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs. These results suggest that wild-type p53 has a growth-inhibitory activity in S. cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.

A p21(Waf1/Cip1)carboxyl-terminal peptide exhibited cyclin-dependent kinase-inhibitory activity and cytotoxicity when introduced into human cells.

In the present study, we report the cyclin-dependent kinase (Cdk)-inhibitory activity of a series of p21waf1/cip1 (p21) peptide fragments spanning the whole protein against the cyclin D1/Cdk4 and cyclin E/Cdk2 enzymes. The most potent p21 peptide tested in our initial peptide series, designated W10, spanned amino acids 139 to 164, a region of p21 that has been found independently to bind to proliferating cell nuclear antigen and also to inhibit Cdk activity. We go on to report the importance of putative beta-strand and 3(10)-helix motifs in the W10 peptide for cyclin-dependent kinase-inhibitory activity. We also describe the cellular activity of W10 and derivatives that were chemically linked to an antennapedia peptide, the latter segment acting as a cell membrane carrier. We found that the W10AP peptide exhibited growth inhibition that resulted from necrosis in human lymphoma CA46 cells. Furthermore, regions in the W10 peptide responsible for Cdk-inhibition were also important for the degree of this cellular activity. These studies provide insights that may eventually, through further design, yield agents for the therapy of cancer.

A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.

Allele shuffling: conjugational transfer, plasmid shuffling and suppressor analysis in Saccharomyces cerevisiae.

Trans-acting suppressor analysis represents a powerful genetic technique capable of revealing interactions among biochemical pathways in vivo. Suppressor characterization in Saccharomyces cerevisiae has traditionally utilized meiotic segregation for the requisite manipulation of strain genotypes. Meiotic segregation is not compatible with all yeast genotypes and can be prohibitively labor intensive when examining large collections of suppressors. To facilitate rapid phenotypic analysis of suppressor mutations, we have devised a novel genetic strategy called 'allele shuffling'. This plasmid-based method should in principle identify allele-specific, allele-dependent and bypass suppressors. A centromere vector (YCp) was developed that can be directly transferred from Escherichia coli to yeast via 'trans-kingdom' conjugation. Suppressors of a thermolabile cdc23 allele, cdc23-39, were isolated in the background of a yeast host strain harboring the mutant cdc23-39 gene positioned on a counterselectable plasmid. CDC23 or cdc23-39 genes cloned into a mobilizable YCp vector were then transferred directly from E. coli cultures to each suppressed yeast strain on the surfaces of agar plates. Plasmid shuffling of the cdc23-39 allele transconjugants segregated away the original cdc23-39 gene present during mutagenesis, allowing the intra- or extragenic nature of suppression to be determined. Phenotypes (if any) produced by suppressor mutations were revealed in those transconjugants receiving the wild-type CDC23-containing episome. The allele shuffling method should be generally applicable to the analysis of suppressors of any essential yeast gene.

Multifunctional yeast high-copy-number shuttle vectors.

A set of four yeast shuttle vectors that incorporate sequences from the Saccharomyces cerevisiae 2 mu endogenous plasmid has been constructed. These yeast episomal plasmid (YEp)-type vectors (pRS420 series) differ only in their yeast selectable markers, HIS3, TRP1, LEU2 or URA3. The pRS420 plasmids are based on the backbone of a multifunctional phagemid, pBluescript II SK+, and share its useful properties for growth in Escherichia coli and manipulation in vitro. The pRS420 plasmids have a copy number of about 20 per cell, equivalent to that of YEp24. During non-selective yeast growth, pRS420 plasmids are lost through mitotic segregation at rates similar to other YEp vectors and yeast centromeric plasmid (YCp) vectors, in the range of 1.5-5% of progeny per doubling. The pRS420 series provides high-copy-number counterparts to the current pRS vectors [Sikorski and Hieter, Genetics 122 (1989) 19-27].

Kynurenine aminotransferase I activity in human placenta.

The aim of the study was to detect and characterize placental kynurenine aminotransferase I (KAT I) activity in physiological pregnancy at term. Placental KAT I was inhibited by l -glutamine, l -tryptophan, and l -phenylalanine and reached optimum activity at pH 9.8. When pyruvate was used as a co-factor, the KAT I activity was significantly higher than the activity of this enzyme in the presence of 2-oxoglutarate. In the light of our findings placental KAT I seems to have biochemical characteristics of KAT I detected in human brain.

Zinc status in women with premature rupture of membranes at term.

Zinc concentrations were measured by atomic absorption spectrometry in whole blood, scalp hair, pubic hair, and colostrum from patients at term with and without premature rupture of membranes (PROM). A maternal zinc index was established for each patient, expressed as an average ranking of the four determinations. The mean +/- SD value of the maternal zinc index in patients with PROM was significantly lower than in patients without this complication (4.33 +/- 1.18 versus 5.97 +/- 1.39, respectively; P = .0002). The inverse relationship between maternal zinc index and parity was statistically significant (r = -0.61; P = .04). These results suggest that the subnormal tissue zinc content in pregnancy may play a role as a causative factor in PROM at term.