Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor.

The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells.

Differences between soluble complement-fixing antigens derived from P3HR-1, RAJI, and Hk-Ly-28 cell lines.

Soluble complement-fixing (CF) antigens of the virus-producing and virus-nonproducing P3HR-1 and RAJI Burkitt lymphoma cell lines and Hk-Ly-28 nasopharyngeal cancer cell line, identified by use of viral capsid antigen-positive human sera, could readily be distinguished by differences in their stability to chemical and physical conditions. The P3HR-1 soluble CF antigen lost titer when heated or exposed to acid perchlorate under conditions in which RAJI and Hk-Ly-28 soluble antigens were stable. Differences in solubility were also found. These results contribute not only to the interpretation of the relationship of Epstein-Barr virus (EBV)-associated components of RAJI to P3HR-1 and Hk-Ly-28 cell lysates but also to the selection of conditions for the development of identification tests and purification procedures for CF antigens and antibodies of Burkitt's lymphoma, nasopharyngeal cancer, and other EBV-associated tumors.

Stability and dissociation of P3H4-1 Burkitt's lymphoma cell soluble complement-fixing antigen identified with human serum.

The soluble complement-fixing antigen of the P3HR-1 Burkitt's lymphoma cell line, identified with human serum containing antibody against Epstein-Barr virus viral capsid antigen, loses activity under a variety of conditions. The major characteristic reported here is retention of activity on exposure to specific physical or chemical conditions followed by loss of activity after subsequent neutral dialysis. Antigen of untreated cell lysates, which retains activity after dialysis against large volumes of neutral buffers or 0.14 M NaCl, will lose activity if the lysate is first heated to 56 degrees or if the lysate is exposed to acid perchlorate, and the resulting precipitate and supernatant are redialyzed separately to neutral pH. The P3HR-1 soluble complement-fixing antigen appears to be an aggregate including a labile, dissociable component.

A type I ovine interferon with limited similarity to IFN-alpha, IFN-omega and IFN-tau: gene structure, biological properties and unusual species specificity.

A gene encoding a 195 amino-acid (a.a.) polypeptide with a putative 23 a.a. signal sequence that had about 60% a.a. sequence identity to ovine interferon-omega (OvIFN-omega) and 55% or less identity to BoIFN-tau, OvIFN-tau and all known IFN-alpha and -beta has been identified from an ovine genomic DNA library. Surprisingly, it shared almost complete identity to genes for rabbit IFN-omega within its coding sequence and proximal promoter region, although the two were different in their 3'-ends. This IFN (tentatively termed ovine IFN-omega variant, OvIFN-omegav), purified in recombinant form from E. coli, had normal antiviral activity when tested on sheep fetal tongue and brain cells and rabbit kidney cells, but very low activity towards bovine, goat and human cells. It competed with 125I-labeled BoIFN-tau for binding to IFN receptors on ovine cells. Expression of OvIFN-omegav was not detected by reverse transcription-PCR either in ovine peripheral blood leukocytes infected with Sendai virus, or in any other tissues examined. OvIFN-omegav may represent a previously unrecognized, non-virally inducible type I subtype distinct from IFN-alpha, -beta, -omega and -tau. The presence of a conserved gene in rabbit and sheep could reflect a recent interspecies transfer.

Operator radiation exposure during percutaneous transluminal coronary angioplasty.

Operator radiation exposure during percutaneous transluminal coronary angioplasty as compared with that during routine coronary angiography is unknown. Therefore, cumulative radiation exposure at operator eye level was measured in two physicians (operators 1 and 2) during performance of coronary angioplasty and routine coronary angiography. The physicians participated together during angioplasty in eight patients; they performed routine angiography separately in eight patients each. Cumulative radiation exposure for eight angioplasty procedures was 140 mrads for operator 1 and 130 mrads for operator 2. In contrast, exposure during eight routine angiograms was 80 mrads for operator 1 and 60 mrads for operator 2. Mean cineangiographic time per case was similar (p = NS) during angioplasty (44.1 +/- 14.0 seconds for both operators) and angiography (49.7 +/- 6.1 seconds for operator 1, 47.6 +/- 16.1 seconds for operator 2). In contrast, fluoroscopy time was longer (p less than 0.01) for angioplasty (34.5 +/- 17.7 min) compared with angiography (13.1 +/- 5.1 min for operator 1, 13.7 +/- 8.2 min for operator 2). Thus, operator radiation exposure during percutaneous transluminal coronary angioplasty was, on average, 93% greater than during routine coronary angiography and was related to the duration of fluoroscopy rather than cineangiography.

Unique features of the trophoblast interferons.

The trophoblast interferons (IFN) are Type I IFN with about 50% amino acid sequence identity to the leukocyte IFN (IFN-alpha). They are the major secretory products of the trophoblast of ruminant ungulate species during pregnancy in the period immediately preceding attachment and implantation when they have been implicated in the phenomenon known as maternal recognition of pregnancy. The trophoblast IFN have antiviral and antiproliferative activities typical of other Type I IFN, but unlike IFN-alpha, -beta and -omega are poorly responsive to viral induction and have a highly restricted pattern of expression. Nevertheless, a recombinant bovine IFN-alpha can mimic many of the properties of the trophoblast IFN and has been used pharmacologically to improve pregnancy success in sheep. It still remains unclear, however, whether the trophoblast IFN have unique biological properties or whether they are unusual merely by virtue of the location, magnitude and temporal nature of their expression at a critical time during pregnancy.

Preferential induction of apoptosis by interferon (IFN)-beta compared with IFN-alpha2: correlation with TRAIL/Apo2L induction in melanoma cell lines.

On the basis of in vitro inhibition of tumor cell growth, IFNs have been generally considered to be antiproliferative proteins. To probe further the potential mechanisms of the antitumor effects of IFNs, we have assessed apoptosis in response to IFN-alpha2 and IFN-beta in cell lines of varied histologies, with a focus on melanomas. Many of the cell lines tested underwent apoptosis in response to IFN-beta, as assessed both by Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. In general, IFN-beta had greater growth inhibitory and proapoptotic effects than IFN-alpha2 on all cell lines. The melanoma cell line WM9, sensitive to growth inhibition by IFNs, had a greater degree of apoptosis than A375 melanoma cells, which were largely resistant to antigrowth effects of IFNs. IFN-beta-induced apoptosis was dependent on activation of the caspase cascade with cleavage of caspases 3, 8, and 9 and of the caspase 3 substrate, poly(ADP-ribose) polymerase. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl keton or benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl keton, inhibited IFN-beta-induced apoptosis. Other changes associated with apoptosis, including the movement of cytochrome c from mitochondria to cytoplasm and DNA fragmentation, were also identified in response to IFN-beta. Apo2L ligand [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)] was one of the early genes induced by IFN-beta in apoptosis-sensitive WM9 cells. Other sensitive melanoma cell lines had a similar IFN-beta-specific induction of TRAIL. Neutralizing antibody to TRAIL inhibited IFN-beta-induced apoptosis in WM9 cells. In resistant A375 cells, IFN-beta did not induce TRAIL/Apo2L expression. Thus, induction of TRAIL by IFNs in some tumor types may initiate the apoptotic cascade. This study offers another mechanism for the antitumor effects of IFNs.

Multiple regulatory elements are required to direct trophoblast interferon gene expression in choriocarcinoma cells and trophectoderm.

Interferon-tau (IFN tau) is produced exclusively by the trophectoderm during the peri-implantation stage of pregnancy in ruminant ungulate species. Human choriocarcinoma cells (Jar) stably transfected with 1.8 kilobases of promoter from a bovine IFN tau gene ahead of a human GH (hGH) reporter gene constitutively synthesize hGH, but expression is not increased further by exposure to Newcastle disease virus. This and earlier experiments suggest that the transcriptional cues regulating IFN tau expression are distinct from those operating on other type I IFN genes. Transient transfection experiments reveal that two distinct promoter regions are required for full constitutive expression: one proximal (to position -126), which directs basal expression, and a more distal promoter region (positions -280 to -400), which acts as an enhancer. Nuclear extracts prepared from ovine conceptuses during the period of IFN tau expression interact with the proximal promoter region (positions -34 to -126) to form several complexes of high electrophoretic mobility. Although nucleotide sequence motifs potentially capable of binding the transcription factor IRF-1 are present in this region, IRF-1 does not transactivate the IFN tau gene. The distal part of the promoter contains only one region (-322 to -358) that forms a complex with these conceptus nuclear extracts. Both proximal and distal gel shift patterns become dramatically different when IFN tau gene expression ceases, perhaps reflecting the appearance of transcriptional repressors. Together these experiments support the conclusion that the control of IFN tau gene expression is very different from that of other type I IFN genes and that trophoblast-specific expression depends upon distal as well as proximal promoter regulatory elements.

Silencing of the gene for the alpha-subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4.

CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG beta-subunit (hCGbeta) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGbeta gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG alpha-subunit (hCGalpha) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the -170 hCGalpha promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the -148 hCGalpha or the -99 hCGalpha promoter. Unexpectedly, no Oct-3/ 4-binding site was identified within the -170 to -148 region of the hCGalpha promoter, although one was found around position -115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGalpha gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGalpha mRNA and protein by 70-80%. Oct-3/4 is therefore capable of silencing both hCGalpha and hCGbeta gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGalpha and hCGbeta genes.

Insulin-like growth factor-I and -II messenger RNA expression in muscle, heart, and liver of streptozotocin-diabetic swine.

We have investigated the effects of streptozotocin-diabetes and fasting in juvenile swine by monitoring IGF-I and -II gene expression in muscle, heart, and liver tissues. In diabetic pigs, IGF-I messenger RNAs (mRNA) were decreased by 50% in muscle and liver tissues, and by 70% in heart. The imposition of fasting on diabetic animals tended to further decrease IGF-I mRNA levels, and fasting alone also decreased IGF-I mRNA abundance in the three tissues (P less than 0.05). Insulin therapy restored IGF-I mRNA levels to normal in muscle and livers but was less effective in hearts of diabetic pigs. Relative IGF-I mRNA expression in heart and muscle tissues was 2-fold and 4-fold higher, respectively, than in liver tissues under normal conditions in these animals. Serum IGF-I concentrations and tissue extractable immunoreactive IGF-I levels were also measured. Serum IGF-I was markedly decreased in the diabetic state, dropping to 70% below control levels (P less than 0.01). Extractable IGF-I in liver declined by 50% with diabetes (P less than 0.01), and by 30% in muscle with diabetes and fasting (P less than 0.05), but no significant changes in heart levels of IGF-I protein were detected. Expression levels of IGF-II mRNAs in the three tissues were unaffected by diabetes or fasting. These results are consistent with earlier observations in rat liver and further demonstrate that IGF-I expression in muscle and heart is altered by diabetes and fasting, whereas IGF-II mRNAs do not change.

The interferon-tau genes of the giraffe, a nonbovid species.

Genes for interferon-tau (IFNT) have been cloned from several Bovidae, and the IFNT lineage has been predicted to have arisen from the IFNW, as the divergence of the Ruminantia from other artiodactyls. Southern genomic blotting with probes specific for IFNT identified a gene in the giraffe, a nonbovid member of the Ruminantia. Here this gene has been cloned, sequenced, and expressed. It encodes a 172-amino acid mature protein with antiviral activity on bovine cells. Giraffe IFN-tau lacks the normally conserved Cys99 but has a pair of other cysteines (Cys64 and Cys86) that could provide a disulfide bridge. The giraffe IFNT gene possesses the highly conserved promoter region that distinguishes IFNT from IFNW. It seems likely that IFNT emerged before the divergence of the Giraffidae and Bovidae, which occurred some 24 million years ago.

The evolution of the type I interferons.

There are five recognized subtypes within the type I interferons (IFN), IFN-alpha, IFN-beta, IFN-delta, IFN-omega, and IFN-tau, although others may remain to be described, and the IFN-omega may have to be subdivided further because of their evident structural complexity. Together, they constitute an ancient family of intronless genes, possibly present in all vertebrates. THe IFNA/IFNB genes originated by duplication of a progenitor after the divergence of birds, most probably about 250 million years ago (MYA). The avian gene itself proceeded to duplicate to form a series of independent subtypes. The IFND, to date described only in the pig, arose from the IFNA lineage before the emergence of mammals about 180 MYA and might, therefore, be generally distributed in present day species. The IFNB, which occurs as a single gene in primates and rodents, have been duplicated in some other orders. Recent events have produced 10 or more genes in bovid species. The IFNA, which are clustered with the IFNW in humans and cattle, exist as multiple genes in all mammals so far examined as a result of a series of duplication events, some of which occurred recently and, therefore, independently in separate mammalian lineages. The IFNW diverged from the IFNA approximately 130 MYA, just prior to the emergence of mammals, and have continued to duplicate since then. The IFNT, which play a role in reproduction of ruminants, arose from an IFNW within the Artiodactyla suborder about 36 MYA and are found only in the suborder Ruminantia. These genes have also continued to duplicate to form an extensive family. Consequently, their involvement in early pregnancy is a feature of ruminants and not of other mammalian species.

The antiproliferative and antiviral activities of IFN-tau variants in human cells.

The IFN-tau are type I IFN expressed by the early trophoblast of cattle and sheep but have activity on human cells and have been predicted to have potential therapeutic value. We have compared a series of mutant bovine and ovine IFN-tau with regard to their ability to inhibit the proliferation of Daudi cells and to evoke an antiviral (AV) response in WISH cells. Whereas Daudi cell growth was inhibited by Bo-IFN-tau1 in the 1 nM range, WISH cells were much less responsive, requiring exposure to 150 nM for protection against vesicular stomatitis virus. Replacement of lysines at positions 34, 107, 121, and 132 in Bo-IFN-tau, which are in regions predicted to interact with the type I receptor, led to modest but significant alterations in antiproliferative (AP) and AV activities. Replacement of the lysine residues at 160 and 164 had marked effects on biopotency, with K160 being particularly important. The different IFN-tau were able to activate the transcription factors ISGF3 and AAF (GAF) in Daudi cells at concentrations that correlated reasonably well with their AP potencies. Stat activation occurred in WISH cells in response to approximately 2 nM Bo-IFN-tau1, but ISGF3 formation could not be demonstrated even at the 100-fold higher IFN-tau concentrations that gave viral protection. Pretreatment of WISH cells with Hu-IFN-gamma allowed ISGF3 formation to be observed in response to subsequent treatment with Bo-IFN-tau1 or type I human IFN but did not increase the AV responsiveness of the cells. No evidence was found that IFN-tau elicit uniquely different responses on human cells than type I Hu-IFN, except they are much less potent. The data emphasize the importance of a region near the carboxyl terminus for the functional activity of type I IFN, and that although ISFG3 formation may be necessary, its mere presence is not sufficient to provide an antiviral response.

Traditionally-used antimalarials from the Meliaceae.

A quantitative ethnobotanical approach to antimalarial drug discovery led to the identification of Lansium domesticum Corr. Ser. (Meliaceae) as an important antimalarial used by Kenyah Dyak healers in Indonesian Borneo. Triterpenoid lansiolides with antimalarial activity were isolated from the bark and shown to have activity in both in vitro bioassays with Plasmodium falciparum, and in mice infected with P. berghei. A survey of African and tropical American Meliaceae led to further development of the limonoid gedunin from the traditionally used medicinal plants, tropical cedar, Cedrela odorata L., and neem, Azadirachta indica A. Juss. Gedunin has significant in vitro activity but initially showed poor in vivo activity. In vivo activity was improved by (1) incorporation into an easy to absorb suspension, (2) preparation of a more stable compound, 7-methoxygedunin; and (3) synergism with dillapiol, a cytochrome P450 3A4 inhibitor. The results show the potential for both antimalarial drug and phytomedicine development from traditionally used plants.

Effects of morphine sulfate on human coronary blood flow.

Because morphine causes coronary vasoconstriction in conscious dogs, human coronary blood flow was measured with the thermodilution technique before and after administration of morphine sulfate, 0.2 mg/kg body weight (maximum 15 mg) intravenously, in 10 patients to determine if the canine experience is clinically applicable. Coronary blood flow increased from a baseline value of 104.4 +/- 13.4 (mean +/- standard error of the mean) to 113.0 +/- 17.4 ml/min (difference not significant) 15 minutes after the administration of morphine. Baseline coronary vascular resistance was 1.14 +/- 0.19 mm Hg/ml/min; 15 minutes after morphine administration the resistance value was 1.02 +/- 0.17 (P less than 0.025). There was no significant change between baseline values and values 15 minutes after morphine administration in systemic mean arterial pressure (98.2 +/- 5.3 to 92.8 +/- 4.7 mm Hg); heart rate (69.5 +/- 3.5 to 72.6 +/- 3.4 beats/min), left ventricular ejection time (0.345 +/- 0.009 to 0.342 +/- 0.007 second) or tension-time index (2,324 +/- 128 to 2,291 +/- 149 mm Hg/sec per min). The slight coronary vasodilation noted after morphine administration in this study is in marked contrast to the significant coronary vasoconstriction demonstrated in the unanesthetized dog.

Prolapsed left atrium behind the left ventricular posterior wall: two dimensional echocardiographic and angiographic features.

Left atrial enlargement can usually be detected accurately using M mode echocardiography. However, in the presence of heart disease, asymmetric enlargement may lead to inaccurate assessment of left atrial size and shape. Pericardial effusion can usually be diagnosed on the basis of characteristic M mode echocardiographic findings. However, false positive patterns sometimes occur with the use of this single dimensional technique. Three patients with a greatly enlarged left atrium are described whose M mode echocardiogram suggested significant posterior pericardial fluid accumulation. In each patient, two dimensional echocardiography detected portions of a huge left atrium that prolapsed behind the left ventricular posterior wall and mimicked an isolated posterior pericardial effusion. In one case a right anterior oblique left ventricular cineangiogram suggested the presence of a ventricular septal defect or a false aneurysm of the left ventricle due to the prolapsed left atrium. Because two dimensional echocardiography can provide accurate spatial orientation with visualization of intracardiac structures in relation to one another in real time, it can identify the presence of left atrial prolapse and play an important role in the differential diagnosis of isolated echo-free spaces behind the left ventricle detected with M mode echocardiography.

Enhanced metabolic vasodilation secondary to diuretic therapy in decompensated congestive heart failure secondary to coronary artery disease.

Since sodium and water retention have been implicated as major factors limiting maximal metabolic vasodilation in congestive heart failure (CHF), the effect of rigorous diuresis on maximal vasodilatory capacity was studied systematically in 9 subjects hospitalized with decompensated CHF. Peak reactive hyperemic blood flow, measured by strain-gauge plethysmography, was used as an index of maximal vasodilatory capacity. After 24 hours of diuresis and a 2.2-kg weight loss, maximal flow increased from 19.9 to 26.1 ml/min X 100 ml (p less than 0.05). Despite a further 1.4-kg weight loss between 24 and 48 hours, maximal blood flow increased no more (26.1 to 25.8 ml/min X 100 ml). Since blood pressure did not change significantly, minimal forearm resistance and maximal conductance showed similar improvements. It is unlikely that vasoconstrictor hormone changes could account for this effect since a marked decrease in plasma norepinephrine occurred in only 2 of 8 subjects and plasma renin activity decreased in only 1 subject. As a group there was no significant change in norepinephrine level, which remained substantially above normal (1,525 to 1,148 pg/ml), or in plasma renin activity (12.3 to 18.9 ng/ml/hour). Because the improvement in vasodilator capacity reached a plateau by 24 hours despite continued diuresis, and because peak reactive hyperemic blood flow was still 32% below normal, it is suggested that a second mechanism besides sodium and water retention is responsible for a significant portion of the impaired peripheral vasodilation in CHF.

Propranolol rebound--a retrospective study.

To assess the effects of sudden withdrawal of propranolol on inpatients with coronary artery disease, 102 patients admitted for cardiac catheterization were evaluated. Criteria for inclusion in the study were angiographically documented coronary artery disease, propranolol therapy at a mean daily dose of at least 80 mg and abrupt discontinuation of propranolol therapy before catheterization. There were 55 patients (mean age 52.5) who discontinued propranolol therapy (mean daily dose 127 mg) and a control group of 47 patients (mean age 53) who continued to receive propranolol (mean daily dose 143 mg). The criteria for morbidity were death, myocardial infarction or change in pain pattern. In the withdrawal group there were no deaths, one myocardial infarction judged to be related to catheterization and only one instance of a change in pain pattern. Thus, propranolol rebound appears to occur infrequently among hospitalized patients with reduced activity.

Coronary artery bypass surgery; a stimulus to modify existing risk factors?

Coronary arterial atherosclerosis is known to be associated with the risk factors of a positive family history, smoking, systemic hypertension, diabetes mellitus, and elevated serum cholesterol levels. Modification of these risk factors, where possible, is prudent. The risk factor data of 226 (212 men, 14 women) subjects who underwent coronary artery bypass surgery for symptomatic obstructive coronary artery disease are presented. Prior to surgery, an attempt was made to educate the subjects in regard to the risk factors and they were urged to modify these factors. All underwent repeat evaluation one year after operation. Although over half of the subjects had had a prior myocardial infarction and all had had aortocoronary bypass surgery, strong stimuli to modify risk factors, there was little modification of the risk factors of smoking and serum cholesterol. There was some modification of the hypertension risk factor. This study documents the need for a very early approach in life to prevent acquiring risk factors and the need for more research into better methods of behavior modification in the adult population.

Persistence of biologic activity after disappearance of propranolol from the serum.

In order to evaluate the duration of the biologic effects of propranolol after the drug was discontinued, we evaluated a variety of noninvasively determined hemodynamic parameters. Significant depression was found in the heart rate (18 per cent), cardiac output (13 per cent) (determined echocardiographically), and the triple product of blood pressure, heart rate, and systolic ejection time (16 per per cent) during administration or propranolol (200 mg. per day) to 9 normal volunteers. Significant depression of these parameters was present 12 hours after discontinuing the drug. By 12 hours, serum propranolol levels had returned 90 per cent toward their base line; however, at the same time, the heart rate and cardiac output had returned only 19.4 and 14.3 per cent toward their base-line values, and the triple product had returned 41 per cent toward its baseline. By 36 hours no biologic effect was seen. Thus if propranolol were discontinued 2 days prior to cardiac surgery, no significant biologic effect would remain to complicate the patient's postoperative course.