Short communication: relationships between α-lactalbumin and quality traits in bulk milk.

The main objective of this study was to investigate whether the α-lactalbumin (α-LA) content of bulk milk is related with some known inflammatory markers and milk quality traits. An additional objective was to study whether combining α-LA, haptoglobin (Hp), and serum amyloid A (SAA) in an acute phase index (API) could be useful as an alternative marker for bulk milk quality. For the dairy industry, it is of great importance to receive high quality bulk milk for manufacture of liquid milk and dairy products. The somatic cell count (SCC) is currently used as an indirect marker for bulk milk quality, but because it is somewhat insensitive and unspecific, interest exists in alternative markers. Bulk milk samples were analyzed for α-LA, SCC, polymorphonuclear leukocyte count, Hp, SAA, fat, lactose, total protein and casein contents, casein number, protein composition, proteolysis, and coagulating properties. No significant differences were found in SCC, polymorphonuclear leukocyte count, Hp, or SAA between milk samples containing low, medium, or high concentrations of α-LA. Differences between α-LA groups were, however, found in some milk quality traits because high α-LA concentration was related to low concentrations of α(S1)-, α(S2)-, and ß-caseins and high concentrations of lactose and ß-lactoglobulin. A high API was related to low lactose content and casein number. Samples with high SCC contained less casein and had a lower casein number than milk with a low SCC, and proteolysis was significantly higher in high SCC milk than in low SCC milk. Neither α-LA nor API proved to be a better marker than SCC for the quality traits investigated, and α-LA was not considered to be a useful inflammatory marker in bulk milk.

Antisense c-myc and immunostimulatory oligonucleotide inhibition of tumorigenesis in a murine B-cell lymphoma transplant model.

BACKGROUND: Because the development of drug-resistant cells can lead to relapses in patients with lymphoma treated with chemotherapy, new approaches are needed for effective disease management, such as those targeting the c-MYC proto-oncogene with antisense oligonucleotides. Our goal was to investigate whether antisense c-myc oligonucleotides could prevent tumorigenesis in a B-cell lymphoma model. METHODS: Immunocompetent mice received subcutaneous injections of tumor cells from a transgenic mouse model of Burkitt's lymphoma. For 7 consecutive days, beginning 1 day after tumor cell transplantation, the mice were given either a DNA phosphorothioate oligonucleotide complementary to c-myc codons 1-5 (myc6) or other c-myc-related oligonucleotides at a dose of 0.76 mg per day subcutaneously. Myc protein expression, normalized to beta-actin expression, was measured by western blotting of tumor and splenic proteins. To determine whether tumor inhibition by myc6 could be a result of B-cell activation, we compared the activity of myc6 with that of an immunostimulatory oligonucleotide, mcg. RESULTS: In comparison with control treatments (saline vehicle, scrambled-sequence oligonucleotide, or double-mismatch oligonucleotide), treatment with myc6 delayed tumor onset by 3 days, decreased total tumor mass at sacrifice (i.e., 17 days after tumor cell transplantation) by 40% +/- 16% (mean +/- standard error), and decreased the splenic Myc-to-actin ratio. Inhibition of tumors by myc6 and mcg (both of which share a dACGTT motif) was comparable. Administration of an oligonucleotide sequence complementary to c-myc codons 384-388 (myc55) delayed tumor onset by 5-6 days, decreased total tumor mass at sacrifice by 65% +/- 6%, and reduced the splenic Myc-to-actin ratio to below that produced by myc6. A 14-day treatment regimen of myc55 alternating with mcg completely inhibited tumor formation during the therapeutic schedule. CONCLUSIONS: A combined oligonucleotide regimen, based on antisense c-MYC and immunostimulatory oligonucleotides, should be investigated to increase the number and duration of complete remissions obtained after standard chemotherapy for B-cell lymphoma.

Down-regulation of c-MYC antigen expression in lymphocytes of Emu-c-myc transgenic mice treated with anti-c-myc DNA methylphosphonates.

In transgenic mice bearing a murine immunoglobulin enhancer/c-myc fusion transgene (Emu-myc), it was found that antisense DNA methylphosphonates targeted against c-myc mRNA inhibited production of c-MYC protein in peripheral lymphocytes. The decrease in protein was measured 3-4 h after i.v. administration of a 300-nmol dose. c-MYC was detected by immunofluorescence of fixed cells stained with an anti-c-MYC antiserum. In addition, DNA methylphosphonates did not induce acute toxicity following i.v. administration of a 300-nmol dose. An identically administered scrambled sequence oligomer did not decrease c-MYC protein or induce toxicity. Finally, recovery of DNA methylphosphonates from the blood plasma of treated mice indicated that the oligomers remained intact up to 3 h, while their concentrations decreased rapidly for the first h, then slowly decreased over the next 2 h. This is the first demonstration of sequence-specific antisense DNA methylphosphonate inhibition of gene expression in the bloodstream of an animal model.

Antisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice.

Antisense DNA has shown an ability to target specific oncogene transcripts and inhibit their expression in cells, but the degree to which sustained treatment can suppress total levels of an oncogenic product and alter tumorigenesis in vivo remains to be determined. In this study, NIH-3T3 cells transformed by the activated c-Ha-ras oncogene from T24 human bladder cancer cells were treated for 3 consecutive days in vitro with an antisense DNA pentadecamer complementary to a target in the 5'-flanking region of the c-Ha-ras RNA transcript. Following antisense DNA treatment, a portion of the cells was lysed for measurement of RAS p21 while the remaining cells were evaluated for tumorigeneity by injection s.c. into athymic nude mice at a dose of 5 x 10(5) cells/mouse. The 3 days of treatment with the anti-c-Ha-ras DNA reduced RAS p21 cellular levels by more than 90% while a nonspecific control DNA reduced p21 levels by approximately 20%. Tumor growth of cells treated with anti-c-Ha-ras DNA was significantly reduced for up to 14 days following the end of treatment and implantation into the mice whereas the nonspecific control DNA had no significant effect. These effects on tumor growth were evident in two different strains of nude mice and in both males and females. It is suggested that the pronounced decrease in RAS p21 levels produced by anti-c-Ha-ras DNA resulted in a reversal of the transformed phenotype, and it is this reversal which accounts for the prolonged inhibition of tumorigenesis following antisense DNA treatment.

Escherichia coli ribosomal protein S1 does not protect the 49-nucleotide 3' terminal cloacin fragment of 16 S rRNA from nuclease S1.

Footprinting of ribosomal protein S1 on the 49-nucleotide 3' terminal cloacin DF13 fragment of 16 S rRNA at physiological ionic strength, pH and temperature yielded no detectable protection of any nucleotides from subsequent attack by the single strand specific nuclease S1, even at large excesses of ribosomal protein S1.

Escherichia coli initiation factor IF3 binding to AUG and AUG-containing single strands and hairpin loops, and nonspecific binding to polymers.

Nitrocellulose filter binding and equilibrium dialysis detected the binding of Escherichia coli initiation factor IF3 to AUG, An UGUm single strands and hairpin loops, poly(A,U,G), poly(U), and f2 RNA. No binding was detected for GUA, A8 U, or the hairpin loop A5 GC5 U5. AUG-specific binding, per nucleotide, is strong; nonspecific binding, per nucleotide, is weak.

Inhibition of deacylation and improvement in N-hydroxysuccinimide ester modification of phenylalanyl-tRNA by dimethyl sulfoxide.

We have found that dimethyl sulfoxide significantly inhibits deacylation of Phe-tRNA. This allows a high pH in N-hydroxysuccinimide ester reactions while maintaining a high level of aminoacylated tRNA, improving the overall yield of the Phe-tRNA modification reaction.

Synthesis and intramolecular crosslinking of chlorambucilyl (prolyl)n [3H]phenylalanyl-tRNAPhe (yeast), n = 0, 5, 11 and 15.

Rigid, variable-length oligoproline crosslinking reagents, which we call molecular rulers, are a potentially powerful tool for probing the solution structures of tRNA and other biological macromolecules. We wish to demonstrate the feasibility of molecular rulers on a well-studied model system, yeast phenylalanine tRNA, before applying them to less well understood structures. We have found chlorambucil (4-(4-(bis(2-chloroethyl)amino)phenyl)-butanoic acid) to be suitable for use as an alkylating function attached to the imino end of oligo-L-proline spacers which are peptide bonded at their carboxyl ends to the alpha-amine of [3H]Phe-tRNAPhe (yeast). Chlorambucil and chlorambucilyl oligoprolines may be readily and sensitively assayed by their alkylation kinetics with aqueous pyridine as measured by optical absorbance of the product. The pyridine reaction seemed to be the first order in chlorambucil, k1 = (5.4 +/- 1.0) X 10(-3) min-1, zero order in pyridine, and was strongly inhibited by Me2SO. Filter assays of tRNA alkylation by chlorambucilyl [3H]prolyl proline suggested that this reaction is also first order in alkylation reagent, but somewhat dependent on tRNA concentration, and also strongly inhibited by Me2SO. Full alkylation activity was regained upon removal of Me2SO. Modification of [3H]Phe-tRNAPhe (yeast) with the N-hydroxysuccinimide esters of chlorambucilyl (prolyl)n was accomplished with yields of 100% for n = 0, 92% for n = 5, 94% for n = 11 and 44% for n = 15, in 80% Me2SO/CHCl3 at pH 9, 37 degrees C, conditions under which chlorambucil alkylation of tRNA is strongly inhibited. The rates of intramolecular crosslinking of chlorambucilyl (prolyl)n [3H]Phe-tRNAPhe (yeast) were measured assuming a first-order process, giving K1 = (5.3 +/- 0.2) X 10(-3) min-1 for n = 0, (3.2 +/- 0.4) X 10(-4) min-1 for n = 5, (6.8 +/- 0.8) X 10(-5) min-1 for n = 11 and (1.6 +/- 0.4) X 10(-4) min-1 for n = 15. Yields of intramolecularly crosslinked tRNA were 80% for n = 0 after 4 h in 10 mM NH4OAc (pH 6)/1 mM Mg(OAc)2 at 37 degrees C, 7% for n = 5, 3% for n = 11, and 5% for n = 15.

Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs.

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99sTc] chelator-PNA-D (CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.

Trophoblast expression of the minor histocompatibility antigen HA-1 is regulated by oxygen and is increased in placentas from preeclamptic women.

INTRODUCTION: Maternal T-cells reactive towards paternally inherited fetal minor histocompatibility antigens are expanded during pregnancy. Placental trophoblast cells express at least four fetal antigens, including human minor histocompatibility antigen 1 (HA-1). We investigated oxygen as a potential regulator of HA-1 and whether HA-1 expression is altered in preeclamptic placentas. METHODS: Expression and regulation of HA-1 mRNA and protein were examined by qRT-PCR and immunohistochemistry, using first, second, and third trimester placentas, first trimester placental explant cultures, and term purified cytotrophoblast cells. Low oxygen conditions were achieved by varying ambient oxygen, and were mimicked using cobalt chloride. HA-1 mRNA and protein expression levels were evaluated in preeclamptic and control placentas. RESULTS: HA-1 protein expression was higher in the syncytiotrophoblast of first trimester as compared to second trimester and term placentas (P<0.01). HA-1 mRNA was increased in cobalt chloride-treated placental explants and purified cytotrophoblast cells (P = 0.04 and P<0.01, respectively) and in purified cytotrophoblast cells cultured under 2% as compared to 8% and 21% oxygen (P<0.01). HA-1 mRNA expression in preeclamptic vs. control placentas was increased 3.3-fold (P = 0.015). HA-1 protein expression was increased in syncytial nuclear aggregates and the syncytiotrophoblast of preeclamptic vs. control placentas (P = 0.02 and 0.03, respectively). DISCUSSION: Placental HA-1 expression is regulated by oxygen and is increased in the syncytial nuclear aggregates and syncytiotrophoblast of preeclamptic as compared to control placentas. Increased HA-1 expression, combined with increased preeclamptic syncytiotrophoblast deportation, provides a novel potential mechanism for exposure of the maternal immune system to increased fetal antigenic load during preeclampsia.

Strategies for administering targeted therapeutic oligodeoxynucleotides.

Antisense or antigene DNA therapy of diseases that are due to aberrant gene expression is an exciting possibility. A variety of synthetic DNA derivatives has been applied in attempts to regulate the expression of many different genes, both in cell culture and in intact organisms (e.g. mice). Realistic design of human oligodeoxynucleotide-based therapeutic strategies requires many aspects of a candidate disease to be considered (including the disease prevalence, the number and nature of genes and mutations involved, and the tissues which must be targeted). For each DNA derivative intended for therapy, methods of targeting, modes of administration, pharmacokinetics, tissue distribution, toxicity, degradation and excretion must all be considered.

Inducible high expression of the Escherichia coli infC gene subcloned behind a bacteriophage T7 promoter.

The gene for Escherichia coli translational initiation factor 3 (infC) has been inserted into an overexpression plasmid under the control of the bacteriophage T7 promoter. The infC plasmid was then used to transform a host with a chromosomal T7 RNA polymerase gene controlled by the lacUV5 promoter. Induction of T7 RNA polymerase expression in the host cells resulted in a 200-fold overexpression of infC mRNA and a 100-fold overproduction of initiation factor 3. Rapid batch purification of biologically active IF3 yielded predominantly the long form of IF3, implying that the short form is an artifact of purification by traditional methods.

Transposon Tn7 gene insertion into an evolutionarily conserved human homolog of Escherichia coli attTn7.

Escherichia coli transposon Tn7 can integrate into its target DNA sequence, attTn7 at the 3' end of glmS, with high specificity and efficiency. Remarkably, the insertional recognition sequence in the E. coli genome displays a high degree of identity with the corresponding region at the 3' end of the corresponding human gene for glutamine-fructose-6-phosphate transaminase (GFPT), located at 2p13. It was therefore of interest to determine whether Tn7 could recognize the corresponding human sequence, and transpose at that site. Strains of E. coli DH5alpha were prepared carrying the tnsA-E genes on one plasmid, and attTn7 or the human equivalent on a second recipient plasmid within the alpha-complementation fragment of the lacZ gene. Each strain was transformed with a donor plasmid carrying a gentamycin resistance gene within the Tn7L and Tn7R cassettes. Restriction mapping and sequence analysis of recipient plasmids isolated from white colonies demonstrated that Tn7 inserted the gentamycin resistance gene both into the E. coli attTn7 sequence, and into its human counterpart. No nonspecific insertion was observed in a control plasmid containing only the lacZ fragment. These results provide a basis to investigate whether TnsA-D proteins can mediate gene insertion into comparably conserved sites in eukaryotic chromosomes.

Increased risk of recurrent venous thromboembolism during hormone replacement therapy--results of the randomized, double-blind, placebo-controlled estrogen in venous thromboembolism trial (EVTET).

Recent observational studies suggest a 2-4 fold increased risk of venous thromboembolism (VTE) in women taking hormone replacement therapy (HRT). The present study was started before publication of these studies, and the aim was to determine if HRT alters the risk of VTE in high risk women. The study was a randomized. double-blind, and placebo-controlled clinical trial with a double-triangular sequential design. Females with previously verified VTE were randomized to 2 mg estradiol plus 1 mg norethisterone acetate, 1 tablet daily (n = 71) or placebo (n = 69). The primary outcome was recurrent deep venous thrombosis (DVT) or pulmonary embolism (PE). Between 1996 and 1998 a total of 140 women were included. The study was terminated prematurely based on the results of circumstantial evidence emerging during the trial. Eight women in the HRT group and one woman in the placebo group developed VTE. The incidence of VTE was 10.7% in the HRT group and 2.3% in the placebo group. In the HRT group, all events happened within 261 days after inclusion. The sequential design did not stop the study, but strongly indicated a difference between the two groups. Our data strongly suggests that women who have previously suffered a VTE have an increased risk of recurrence on HRT. This treatment should therefore be avoided in this patient group if possible. The results also support those of recent epidemiological studies, which also indicate increased risk of VTE in non-selected female populations during HRT.

Transformed and immortalized cellular uptake of oligodeoxynucleoside phosphorothioates, 3'-alkylamino oligodeoxynucleotides, 2'-O-methyl oligoribonucleotides, oligodeoxynucleoside methylphosphonates, and peptide nucleic acids.

Direct quantitative comparisons of cellular uptake across a wide variety of analogs and cell types are necessary for the design of oligonucleotide diagnostic and therapeutic applications. This work reports quantitative cellular uptake and nuclear localization of [14C]oligodeoxynucleoside phosphorothioates (PS), 3'-alkylamino oligodeoxynucleoside phosphodiesters (PO-NH2), 2'-O-methyl oligoribonucleoside phosphodiesters (2OM), peptide nucleic acids (PNA), and oligodeoxynucleoside methylphosphonates (MP) in several transformed or immortalized cell lines. All analogs demonstrated active cellular uptake in that intracellular concentrations greatly exceeded the extracellular 1 microM concentration within 1-3 hr. However, by 9-24 hr, cellular accumulations of PS exceeded those of PO-NH2 and 2OM by 3- to 5-fold, PNA by 6- to 7-fold, and MP by 8- to 10-fold. Similar results were observed in two transformed cell lines, HL-60 leukocytes and H-ras transformed fibroblasts, using three different heterogeneous sequences. H-ras and IGF-1R transformed fibroblasts had a 2- to 5-fold higher uptake of all analogs than non-transformed immortalized fibroblasts. Nuclear levels of the PO-NH2, PS, and MP analogs were approximately 25% of total cellular uptake, while nuclear percentages of 2OM and PNA were less than 20%, suggesting some differences in nuclear localization among the analogs. These observations provide a direct quantitative comparison of cellular uptake as a function of oligonucleotide modification, and imply that transformation enhances cellular uptake. From the perspective of therapy and diagnosis, clear trade-offs were apparent between efficiency of uptake on the one hand, and nuclease resistance and hybridization strength on the other.

A prospective study of the association between isolated fetal pyelectasis and chromosomal abnormality.

OBJECTIVE: To determine the incidence of chromosomal abnormalities among fetuses with isolated pyelectasis. METHODS: Between March 1991 and March 1994, 121 cases of isolated fetal pyelectasis were identified at our institution. Pyelectasis was defined as a renal pelvis anteroposterior diameter of at least 4 mm before 33 weeks' gestation, and at least 7 mm at 33 weeks or thereafter. Once identified, women were offered antenatal genetic testing; if they declined, consent was sought for umbilical cord blood studies at delivery. RESULTS: Chromosomal evaluation was available in 99 women. Two chromosomal abnormalities were identified: one trisomy 21 and one mosaic 46, XY/47, XYY. The ages of the women were 32 and 28 years, respectively. Calculation of adjusted risks for Down syndrome and all chromosomal abnormalities indicated a 3.9-fold increase in Down syndrome risk and a 3.3-fold increase in risk for all chromosomal abnormalities in the presence of isolated fetal pyelectasis. CONCLUSION: Isolated fetal pyelectasis is associated with increased risk, over that related to age, for both Down syndrome and all chromosomal abnormalities. These factors may be valuable in counseling individual patients regarding the appropriateness of amniocentesis.

A dose-response study in situational insomnia with zopiclone, a new tranquilizer.

Patients scheduled for next-day elective surgery were included in a randomized double-blind trial that compared the hypnotic and adverse effects of a placebo and four doses (3,5,6,9,13.5 mg) of a new compound, zopliclone. Sleep patterns were evaluated using a questionnaire; side effects were noted, and tests evaluating coordination and alertness were performed. Except for dreams and morning responses, there were significant differences in sleep indices at all dose levels. The 3.5-mg dose produced some significant differences compared with the placebo. All responses to the higher three doses were statistically significant; however, there was only a slight increase in effectiveness between 6 and 9 mg. Alertness, coordination tests, blood pressure, and adverse effects did not differ significantly among doses, except for the incidence of drowsiness and a bitter after-taste in the mouth.

Oligonucleotide treatment of ras-induced tumors in nude mice.

Oligonucleotides have shown an ability to target specific oncogene transcripts and inhibit their expression in cells, but the degree to which sustained treatment can suppress the levels of an oncogenic protein enough to benefit a patient remains to be determined. This question has been studied in several ways. First, the relationship of antisense DNA inhibition to the predicted secondary structure of human H-RAS oncogene mRNA was examined in transformed mouse cells that form solid tumors. Inhibition of H-Ras expression was sequence-specific, dose-dependent, and correlated with inhibition of focus formation. The efficacy of the first intron antisense sequence in reducing H-Ras expression was greater than that of the initiation codon target. Second, H-RAS transformed solid tumor cells were pretreated in vitro with normal oligonucleotides, after which tumor growth from the treated cells was tested in nude mice. The three days of treatment with the first intron antisense DNA reduced H-Ras cellular levels by more than 90% whereas a nonspecific control DNA reduced H-Ras levels by approx 20%. Tumor growth of cells treated with H-RAS antisense oligonucleotide was significantly reduced for up to 14 d following the end of treatment and implantation into the mice, whereas the nonspecific control DNA had no significant effect. Third, H-RAS transformed bladder cancer cells were implanted into nude mice, after which the mice were treated for 31 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with H-RAS 12th codon antisense oligonucleotide was reduced by about 80% throughout the treatment period, reiterating the sustained effect seen in pretreated tumor cells. However, the scrambled phosphorothioate control inhibited tumor growth by about 60%, illustrating some nonspecific inhibition. Fourth, K-RAS transformed pancreatic cancer cells were treated in culture and in nude mice. Inhibition of K-Ras expression with a phosphorothioate oligonucleotide directed against a 5'-UTR sequence was sequence-specific and dose-dependent. K-RAS transformed pancreatic cancer cells were implanted into nude mice, after which the mice were treated for 14 d with oligonucleotide phosphorothioates. Tumor growth in mice treated with K-RAS 5'-UTR antisense oligonucleotide was reduced by about 50% throughout the treatment period, reiterating the sustained effect seen with H-RAS transformed cells. In this case, the sense phosphorothioate control did not inhibit tumor growth, demonstrating that nonspecific inhibition is not a characteristic of all phosphorothioate sequences. The next logical steps include testing oligonucleotide efficacy against other tumor types, toxicological testing in higher species, and clinical trials in human subjects.

Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Risk factors for morbidity and mortality in mitral valve replacement.

Risk factors of operative mortality and long term survival were identified in 219 patients who underwent mitral valve replacement (MVR) using Bjørk-Shiley mechanical prostheses. Early mortality was 7.3%. The accumulated follow-up time was 1134 patient-years, and the 5-year survival for the total cohort was 78 +/- 3%. Independent prognostic factors of early mortality were poor NYHA class, which carried a relative risk (RR) of 3.2, and ischaemic aetiology, with a RR of 2.2. Ischaemic aetiology was the sole predictor of heart pump failure requiring intra-aortic balloon pump support (RR = 2.7). Independent risk factors of total mortality (early and late) were male sex (RR = 2.3), NYHA class III-IV (RR = 2.4), presence of mitral regurgitation (RR = 3.2) and relative heart volume (RR = 1.6 for a 800 ml/m2 size compared to a heart of 550 ml/m2). Our results underline the importance of patient-related factors in MVR, and indicate that care is needed in comparing the quality of MVR from different institutions with respect to mortality and morbidity. The results of MVR are palliative rather than curative except in female patients with NYHA class II function and mitral stenosis, in whom cure was attained.