Cholangiography during laparoscopic cholecystectomy--cumulative sum analysis of an institutional learning curve.

The ability to perform intraoperative cholangiography during laparoscopic cholecystectomy is an essential skill for the laparoscopic biliary surgeon. The volume of experience required to be able to consistently obtain a cholangiogram during laparoscopic cholecystectomy has not been determined. Cumulative sum analysis is a statistical technique which generates a graphical display that identifies periods of performance that fall below a predetermined standard for a given task. The cumulative sum (S(n)) for a series of observations is defined as: S(n)= summation operatorX(I) - X(o), where X(I) = 0 for a success, X(I) = 1 for a failure, and X(o) is the acceptable failure rate for the process under study. This function is plotted against the number of observations to create a curve. When the curve has a positive slope, the acceptable failure rate is being exceeded. When it reaches a plateau, the observed failure rate is equal to the acceptable failure rate. When the curve has a negative slope, the observed failure rate is lower than the acceptable failure rate. We performed a cumulative sum analysis of the first 97 intraoperative cholangiograms attempted during lap-aroscopic cholecystectomy at our institution. The results demonstrated that 46 cases were required to reach a level of proficiency where a cholangiogram could be obtained in 95% of attempts. Success rates of 85% and 90% were achieved at 16 and 25 cases, respectively. This form of analysis is a useful tool for estimating the number of attempts required to achieve a desired success rate when learning new procedures.

Patterns of morbidity and resource consumption associated with laparoscopic cholecystectomy in a VA medical center.

BACKGROUND: The generally low incidence of morbidity and reduced rate of health care resource consumption commonly associated with laparoscopic cholecystectomy (LC) have been established from studies of patient populations which are distinct from that served by the Department of Veterans Affairs (VA) health care system. We sought to assess the outcomes of this procedure when performed on VA beneficiaries. MATERIALS AND METHODS: Demographic and perioperative data for all patients undergoing attempted LC in our facility were recorded in a prospective database beginning 1 January 1993. The information in this registry was analyzed to determine the demographics of the treated population, the spectrum of biliary tract disease encountered, and patterns of morbidity and resource consumption. RESULTS: LC was attempted in 141 cases. Median patient age was 62 years. The indication for surgery was either acute cholecystitis or biliary pancreatitis in 63 cases (45%). Thirteen patients (9%) developed major complications. These patients were significantly older (mean age 68 vs 59 years) than patients whose course was uncomplicated. Twenty-seven cases (19%) required conversion to an open procedure, most commonly for acute cholecystitis. Progressive cholecystitis was associated with a conversion rate of 64%. Both conversion and the development of a major complication produced significant increases in length of stay. CONCLUSIONS: The population undergoing attempted LC in the VA system is characterized by relatively advanced age and high incidences of comorbid illness and complicated biliary tract disease. These attributes increase the frequency of major morbidity and of conversion to open cholecystectomy, which in turn increase resource consumption. Comparisons between the outcomes of attempted LC in VA centers and "benchmark" results obtained in other settings should be controlled for these factors.

Activation of the IL-1 gene in UV-irradiated mouse skin: association with inflammatory sequelae and pharmacologic intervention.

The relationship between ultraviolet irradiation, interleukin-1 production, and inflammatory sequelae and the pharmacologic inhibition of these events was investigated in Balb/c mice exposed to ultraviolet irradiation from a bank of six Westinghouse FS40 sunlamps. The resulting edema (66% increase), inflammatory cell infiltration, and rise in the acute-phase reactant (fourfold) serum amyloid P component was preceded by the activation of the interleukin-1 beta gene and enhanced product formation. Administration of dexamethasone, which is known to inhibit interleukin-1 production, inhibited the inflammatory response to ultraviolet irradiation. Thus, production of interleukin-1 may be one of the initial events leading to the consequences of ultraviolet irradiation exposure.

Interleukin-1 release by rat synovial cells is dependent on sequential treatment with gamma-interferon and lipopolysaccharide.

To determine the potential regulatory mechanisms involved in synovial cell interleukin-1 (IL-1) release, the ability of gamma-interferon (gamma-IFN) to influence IL-1 release was assessed. Rat synovial cells cultured in the presence of a variety of stimuli, including lipopolysaccharide (LPS), failed to release IL-1. However, pretreatment of synovial cells with gamma-IFN, followed by LPS stimulation, resulted in increased levels of intracellular IL-1 as well as release of IL-1 from the cell. The level of IL-1 release was dependent on the concentration of both gamma-IFN and LPS, and on length of exposure to the gamma-IFN. The kinetic and dose requirements for gamma-IFN-dependent IL-1 release were similar to those for Ia antigen expression, but LPS was necessary for IL-1 messenger RNA induction, intracellular IL-1 accumulation, and IL-1 release. In addition, sequential treatment, i.e., gamma-IFN followed by LPS, was essential for IL-1 induction. Substitution of phorbol ester or calcium ionophore for gamma-IFN did not result in similar IL-1 release. In addition, induction of IL-1 messenger RNA by another stimulus was not sufficient to result in IL-1 release following LPS treatment. These results suggest that release of IL-1 by rat synovial cells requires the production of a regulatory signal, which is inducible by gamma-IFN.

The influence of interferon alpha and gamma, singly or in combination on human natural cell mediated cytotoxicity.

The influence of interferon alpha and gamma alone or in combination on the augmentation of human natural cytotoxicity was studied. Treatment of peripheral blood lymphocytes with IFN- alpha led to a rapid augmentation of NK activity, in contrast to IFN-gamma where target cell killing was observed only following 18 hrs exposure of lymphocytes to IFN-gamma. The results of the single cell assay paralleled those obtained using the Chromium release test, but neither interferon type caused an increase in the number of target binding lymphocytes. The combined effect of IFN-alpha and IFN-gamma in stimulating human natural cytotoxicity demonstrated individual lymphocyte responses to be variable. Exposure of lymphocytes to IFN-alpha and IFN-gamma for 18 hrs prior to assay for cytotoxicity usually decreased the level of cytotoxicity compared with control values, whereas other treatment regimes gave an additive and sometimes synergistic effect. Only treatment with IFN-alpha for 18 hrs and IFN-gamma for one hr produced a synergistic response in the majority of individuals tested. We conclude from this study that individual responses to IFN-alpha and IFN-gamma alone or in combination are variable and dependent upon timing of exposure of lymphocytes to individual interferon types, and possibly reflects the donor status at the time of sampling.

Inhibition of IFN-gamma-induced Ia antigen expression on synovial fibroblasts by IL-1.

Naturally occurring substances capable of the negative regulation of class II molecules on synovial fibroblasts may play an important role in controlling the sustained immune processes ongoing in the rheumatoid joint. We report here that rIL-1 is capable of such a negative regulatory process. The simultaneous addition of rIL-1 and rIFN-gamma to rat synovial fibroblasts resulted in decreased Ia Ag and mRNA expression when compared with synovial fibroblasts treated with IFN-gamma alone. Both rIL-1 alpha and rIL-beta inhibited to a similar degree with the level of inhibition being dependent on both the concentration of IL-1 and IFN-gamma. Other cytokines, including IFN-alpha/beta, IL-2, and TNF, had no antagonistic effect on IFN-gamma-induced Ia expression. Time course experiments showed that IL-1 inhibited when present immediately before addition of IFN-gamma or when added during the first 24 h of IFN-gamma stimulation but not at later time points. Indomethacin failed to reverse the IL-1-mediated inhibition, despite the fact that exogenously added PGE2 also inhibited IFN-gamma-induced Ia expression. IL-1 treatment of synovial cells did not alter the ability of IFN-gamma to bind to the cells. These findings provide evidence for a negative regulatory role for IL-1 on synovial fibroblasts independent of PGE2 production and thus suggest that IL-1 is capable of both pro- and antiinflammatory actions within the rheumatoid joint.

Growth-inhibitory activity of interferon-beta against human colorectal carcinoma cell lines.

Recombinant human interferon beta (rIFN-beta) inhibited in a time- and dose-dependent manner the proliferation of 18/18 human colon carcinoma cell lines in monolayer culture and 8/9 lines in a soft agar assay but had no effect on 4 human fibroblast cell lines. Maximal inhibition of cell proliferation by rIFN-beta required repetitive treatment (every 2 days) with lymphokine (50 units/ml). Furthermore, the inhibitory activity of rIFN-beta was neutralized by polyclonal antibodies against natural IFN-beta. In contrast to rIFN-beta, rIFN-alpha was inactive against all colon cell lines tested, and rIFN-gamma, with the exception of HT-29 cells, was similarly ineffective. These data demonstrate that rIFN-beta is a potent growth inhibitor of colon carcinoma cells in vitro, and suggest that studies on its mechanism of action may lead to a better understanding of the regulation of colon tumor cell proliferation.

Interleukin-1 induces interleukin-1 alpha and interleukin-1 beta gene expression in synovial fibroblasts and peripheral blood monocytes.

Cellular interactions involved in the chronic inflammatory response, characteristic of those found in the joints of rheumatoid arthritis patients, were investigated by examining the effect of interleukin-1 (IL-1), tumor necrosis factor alpha, and gamma-interferon on the regulation of IL-1 gene expression and production by synovial fibroblasts. Biologically active IL-1 was detected in lysates of IL-1-treated rat and human fibroblasts that had been isolated from synovial tissue by collagenase digestion. Northern blot analysis of RNA isolated from these cells revealed the expression of IL-1 alpha and IL-1 beta transcripts. Neither the IL-1 transcripts nor the biologic activity of IL-1 was found in untreated synovial fibroblasts. The messenger RNA induction in synovial cells was followed by a time- and dose-dependent expression of intracellular IL-1 activity. Human monocytes and human skin fibroblasts also responded to IL-1 treatment by producing IL-1-specific transcripts. These observations suggest that IL-1 plays a key role in stimulating immune and inflammatory responses and in sustaining those responses through continued production at sites of inflammation.

Differential expression of murine macrophage-mediated tumor cytotoxicity induced by interferons.

The requirements for interferon (IFN)-induced priming of murine peritoneal macrophages for cytolysis of tumor cell lines of distinct histological origin were investigated. Lysis of B16 melanoma targets required exposure of elicited macrophages to recombinant murine gamma interferon plus lipopolysaccharide (LPS) together, while sequential treatment of macrophages with IFN-gamma then LPS resulted in lysis of P815 mastocytoma targets. The kinetics of macrophage activation by IFN-gamma and LPS for lysis of P815 and B16 melanoma targets varied considerably, 8 h being sufficient for P815 targets but 24 h being required for B16 targets. Pretreatment of the macrophages with the antibiotic polymyxin B was able to inhibit completely the induction of tumor lysis of B16 targets but not of P815 targets. In addition, IFN-alpha/beta was able to prime macrophages for lysis of P815 targets but not of B16. Finally, the kinetics of priming macrophages with IFN-gamma for lysis of B16 targets had a profound effect on the subsequent exposure time requirement for LPS. The results indicate that the induction of murine macrophage-mediated tumor cytotoxicity can vary considerably depending on the amount and type of interferon used, the presence of a second signal, and the type of tumor target used.

Augmentation of human natural killer cell activity by influenza virus antigens produced in Escherichia coli.

A series of Escherichia coli cloned influenza viral gene products were assayed for their ability to augment human natural cytotoxicity in overnight cultures (18 h) at 37 degrees C. Nylon wool nonadherent peripheral blood mononuclear cells (PBMC) proved responsive to stimulation by a number of cloned viral proteins, the most effective being the nonstructural (NS1) protein (but not NS2 protein) and haemagglutinin and matrix antigen components fused to the N-terminal 81 amino acid sequence of NS1. Furthermore, interferon (IFN) was generated in cultures in which enhanced cytotoxicity was detected and was identified as mostly IFN alpha (greater than 90%) with less than 10% IFN gamma contamination. The cell type responding to antigen stimulation was present in Percoll fractions enriched for large granular lymphocytes (LGLs); furthermore PBMC activated by NS1 protein fractionated in the low density Percoll fractions (LGL enriched). Using specific anti-IFN antisera, it was shown that IFN alpha but not IFN gamma was responsible for the enhancement of cytotoxicity. Interferon induction and activation of cytotoxicity could not be ascribed to the presence of contaminating bacterial products. These results suggest that a particular NS1 protein configuration is capable of activating human natural killer cells via the induction of IFN alpha.

Lymphokine production by mitogen and antigen activated mouse intraepithelial lymphocytes.

Although most intraepithelial lymphocytes (IEL) in mouse small intestine bear surface markers classically associated with T lymphocytes, the T-cell nature of these cells remains controversial. In the present study IEL from normal mice, or from mice infected with the gut nematode Trichinella spiralis, were therefore tested for their ability to produce T-cell-derived lymphokines in response to in vitro stimulation with concanavalin A (Con A) or with specific worm antigens. The data show that Con A-stimulated IEL produce minimal amounts of IL-2, and intermediate levels of IFN-gamma and IL-3 in comparison to the levels produced by spleen T cells. The FDC-P2 cell line, which proliferates in response to both IL-3 and GM-CSF, was identified as the most sensitive and reproducible indicator of lymphokine activity in supernatants from mitogen-stimulated IEL from normal mice. IEL isolated from mice infected with T. spiralis also produced high levels of FDC-P2 growth factors when challenged in vitro with Trichinella-derived antigens; however, normal IEL did not respond to this stimulus. The data thus provide evidence that antigen-sensitive T cells can arise in (or migrate to) the gut epithelium during gut infection.

Antigenic cross-reaction between the alpha types of human and mouse interferon.

Cross-neutralization of the alpha and beta types of human and mouse interferons was tested using antibodies directed against the heterologous types of interferons. The alpha type of mouse interferon (MuIFN-alpha) prepared from L cells was found to be completely neutralizable by high-titered antibody against human alpha-type interferon (HuIFN-alpha), although the titers were much lower than those obtained in neutralization reactions with the homologous interferon. MuIFN-alpha from virus-induced lymphocytes, as well as non-glycosylated MuIFN-alpha from L cells, reacted similarly. The cross-reaction was also observed by the binding of anti-HuIFN-alpha antibody to a column of immobilized MuIFN-alpha. The binding experiment indicated that only a small fraction of the anti-HuIFN-alpha antibody population is heterologous reactive. Reciprocally, HuIFN-alpha from L cells, again with relatively low antibody titers, and the antibody responsible for the heterologous reaction was shown to be the anti-MuIFN-alpha and not anti-MuIFN-beta type. It is concluded that the alpha types of human and mouse interferon bear an antigenic homology. On the other hand, no significant antigenic cross-reactivity has been detected between the beta types of human and mouse interferons.

Biological properties of human interferon fractions obtained by blue sepharose chromatography.

Human interferons of lymphoid and nonlymphoid origins were segregated into several fractions by virtue of their elution properties from Blue Sepharose. According to desorption of the main portion of antiviral activity, three major classes could be discerned: 1. Leukocyte interferons eluting predominantly in 0.5 M NaCl buffer, 2. Lymphoblastoid interferons eluting mostly in 1 M NaCl buffer, and 3. Cell culture-derived fibroblast and bladder carcinoma interferons requiring ethylene glycol in addition to 1 M NaCl for elution. Desorption behavior from Blue Sepharose did not necessarily correlate with the presence of specific antigenic markers and no consistent segregation of Le and F antigens in individual fractions was observed. All fractions exhibited comparable activity in heterologous sheep and homologous human cells. Therefore, no distinctive biological features could be associated with multiple interferon species isolated by Blue Sepharose chromatography.

Antigenic properties of human lymphoblastoid interferons.

Most virus-induced human lymphoblastoid interferons examined contained variable proportions of the Le and F antigenic species described for human leukocyte interferon. The F species was not detectable in interferons liberated spontaneously from human lymphoblastoid cells in culture. Lymphoblastoid interferons differed considerably in their interaction with the same anti-interferon serum. Spontaneous interferons required approximately ten times less antibody for neutralization than interferon induced by virus in the same cultures or in Namalva cells. The findings suggest that either spontaneous interferons contain fewer inactive antibody-binding molecules than virus-induced lymphoblastoid interferons or the number and distribution of antibody-combining sites, and possibly other surface properties of the interferon molecule, may be influenced by the manner in which spontaneous and induced interferons egress from the cells.

Production of antibodies to human interferons in mice.

Neutralizing antibodies were raised in mice that had been inoculated repeatedly with moderate quantities of human leukocyte interferon highly purified by affinity chromatography on immobilized anti-interferon globulins. Interferon preparations of lesser purity sensitized the mice to subsequent inoculations of interferon and almost invariably caused death before anti-interferon titers developed. Antibody-purified interferon stabilized by sodium dodecyl sulfate was a superior antigen to interferon that had received mouse serum albumin as an additive. The amount of antibody could be augmented by experimental induction of ascites. The antibodies specifically neutralized leukocyte and lymphoblastoid interferons but not those interferons obtained cultures of human foreskin fibroblasts, embryonic kidney cells, and amnion cells.

Antigenic properties and heterospecific antiviral activities of human leukocyte interferon species.

Human interferon obtained in peripheral leukocytes was purified approximately 1000-fold by affinity chromatography on anti-leukocyte interferon globulins coupled to Sepharose 4B, and by filtration on SDS-Sephadex G-100. The interferon was subsequently resolved into two molecular species by adsorption chromatography on SDS-hydroxylapatite. The two species which were eluted at different phosphate molarities from hydroxylapatite, could also be distinguished on the basis of electric charge properties and they migrated at different rates in SDS-polyacrylamide gels. Crossneutralization tests with monospecific rabbit anti-leukocyte and anti-fibroblast interferon sera revealed that the two species possessed leukocyte interferon-specific antigenic determinants. Both were immunogenic in mice and they were neutralized to a comparable degree by antisera against either component. A variable degree of antiviral activity was expressed by both interferon components in bovine, porcine and murine cells. However, the two interferon species were equally active in this respect, and the protective effects exhibited in homologous and heterologous cell cultures were similarly susceptible to reduction by beta-mercapto-ethanol. We conclude that the two molecular species of human leukocyte interferon are biologically similar.

Biological properties of human leukocyte interferon components.

Human leukocyte interferon, purified approximately 1000-fold by affinity chromatography on immobilized anti-interferon globulins and SDS-Sephadex filtration, was resolved into one major and one minor component by adsorption chromatography on hydroxylapatite and electrophoresis in polyacrylamide gels. These components were indistinguishable in their capacity to protect bovine, porcine and murine cells, and the antiviral activities of both were equally susceptible to reduction by beta-mercaptoethanol. They were neutralized to the same degree of rabbit anti-leukocyte interferon but were not neutralized by rabbit antifibroblast interferon serum. Mice immunized with either component developed antibodies to both but failed to form antibodies against human fibroblast interferon. Our present evidence indicates that the two components posses at most only minor structural and antigenic dissimilarities.