Human tumor necrosis factor receptor (p55) and interleukin 10 gene transfer in the mouse reduces mortality to lethal endotoxemia and also attenuates local inflammatory responses.

Anticytokine therapies have been promulgated in gram-negative sepsis as a means of preventing or neutralizing excessive production of proinflammatory cytokines. However, systemic administration of cytokine inhibitors is an inefficient means of targeting excessive production in individual tissue compartments. In the present study, human gene transfer was used to deliver to organs of the reticuloendothelial system antagonists that either inhibit tumor necrosis factor-alpha (TNF-alpha) synthesis or block its interactions with cellular receptors. Mice were treated intraperitoneally with cationic liposomes containing 200 micrograms of either a pCMV (cytomegalovirus)/p55 expression plasmid that contains the extracellular domain and transmembrane region of the human p55 TNF receptor, or a pcD-SR-alpha/hIL-10 expression plasmid containing the DNA for human interleukin 10. 48 h later, mice were challenged with lipopolysaccharide (LPS) and D-galactosamine. Pretreatment of mice with p55 or IL-10 cDNA-liposome complexes improved survival (p < 0.01) to LPS-D-galactosamine. In additional studies, intratracheal administration of IL-10 DNA-liposome complexes 48 h before an intratracheal LPS challenge reduced pulmonary TNF-alpha levels by 62% and decreased neutrophil infiltration in the lung by 55% as measured by myeloperoxidase activity (both p < 0.05). Gene transfer with cytokine inhibitors is a promising option for the treatment of both the systemic and local sequelae of septic shock.

Interleukin 10 inhibits growth and granulocyte/macrophage colony-stimulating factor production in chronic myelomonocytic leukemia cells.

Autonomous release of hematopoietic growth factors may play a crucial role in the pathogenesis of certain hematological malignancies. Because of its cytokine synthesis-inhibiting action, interleukin 10 (IL-10) could be a potentially useful molecule to affect leukemic cell growth in such disorders. Chronic myelomonocytic leukemia (CMML) cells spontaneously form myeloid colonies (colony-forming units-granulocyte/macrophage) in methylcellulose, suggesting an autocrine growth factor-mediated mechanism. We studied the effect of recombinant human IL-10 (rhIL-10) on the in vitro growth of mononuclear cells obtained from peripheral blood or bone marrow of patients with CMML. IL-10 specifically binding to leukemic cells had a profound and dose-dependent inhibitory effect on autonomous in vitro growth of CMML cells. IL-10 significantly inhibited the spontaneous growth of myeloid colonies in methylcellulose in 10/11 patients, and autonomous CMML cell growth in suspension in 5/5 patients tested. Spontaneous colony growth from CMML cells was also markedly reduced by addition of antigranulocyte/macrophage colony-stimulating factor (GM-CSF) antibodies, but not by addition of antibodies against G-CSF, IL-3, or IL-6, IL-10-induced suppression of CMML cell growth was reversed by the addition of exogenous GM-CSF and correlated with a substantial decrease in GM-CSF production by leukemic cells, both at the mRNA and protein levels. Our data indicate that IL-10 profoundly inhibits the autonomous growth of CMML cells in vitro most likely through suppression of endogenous GM-CSF release. This observation suggests therapeutic evaluation of rhIL-10 in patients with CMML.

A human tumor necrosis factor (TNF) alpha mutant that binds exclusively to the p55 TNF receptor produces toxicity in the baboon.

A number of recent studies have demonstrated that cellular responses to tumor necrosis factor (TNF) mediated by the p55 and the p75 TNF receptors are distinct. To evaluate the relative in vivo toxicities of wild-type TNF alpha (wtTNF alpha) and a novel p55 TNF selective receptor agonist, healthy, anesthetized baboons (Papio sp.) were infused with a near-lethal dose of either wtTNF alpha or a TNF alpha double mutant (dmTNF alpha) that binds specifically to the p55, but not to the p75, TNF receptor. Both wtTNF alpha and dmTNF alpha produced comparable acute hypotension, tachycardia, increased plasma lactate, and organ dysfunction in Papio. However, administration of wtTNF alpha produced a marked granulocytosis and loss of granulocyte TNF receptors, whereas little if any changes in neutrophil number or cell surface TNF receptor density were seen after dmTNF alpha mutant administration. Infusion of dmTNF alpha resulted in a plasma endogenous TNF alpha response that peaked after 90-120 min. We conclude that selective p55 TNF receptor activation is associated with early hemodynamic changes and the autocrine release of endogenous TNF alpha. Significant systemic toxicity results from p55 TNF receptor activation, but the role of the p75 TNF receptor in systemic TNF toxicity requires further study.

A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.

Transfer of interleukin-4 and interleukin-10 in patients with severe inflammatory bowel disease of the rectum.

Inflammatory bowel disease (IBD) comprises the two disorders ulcerative colitis (UC) and Crohn's disease (CD). Although the etiology is still unclear, initiation and aggravation of the inflammatory processes seem to be due to a massive local mucosal immune response. An increased number of greatly activated macrophages seems to contribute to the onset of IBD by expressing upregulated costimulatory molecules (e.g., CD80/CD86) and a cytokine profile favouring a type I proinflammatory response. The release of interleukin 2 (IL-2) and Interferon-gamma (IFN-gamma) by naive T lymphocytes predominantly stimulates cytotoxic T lymphocytes, macrophages, and natural killer (NK) cells and increases the antigen-presenting potential of all these cell types. Opposite this proinflammatory immune reaction a compensatory type II antiinflammatory response has been suggested in the inflamed mucosa, involving mainly interleukin 4 and interleukin 10. Both cytokines are able to down-regulate inflammatory mediators including tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 and favor a humoral immune response. The main goal of this clinical trial is the local liposome-mediated gene transfer of these two antiinflammatory cytokines, interleukin 4 and interleukin 10, in patients with severe IBD of the rectum. This local administration of antiinflammatory cytokines will avoid toxic systemic side effects, prevents blocking of the beneficial effects of proinflammatory cytokines, e.g., TNF-alpha in other tissue compartments and increases the local concentration of interleukin 4 and interleukin 10 over a prolonged period of time. The combined effects of IL-4 and IL-10 have been shown to shift the Th1/Th2 cell activation in favor of a Th2 immune response which seems to be essential for fighting against the inflammation and ultimative healing.

Combined radiochemotherapy of locally advanced unresectable pancreatic adenocarcinoma with mitomycin C plus 24-hour continuous infusional gemcitabine.

BACKGROUND: In locally advanced pancreatic cancer, the combination of chemotherapy with radiotherapy is gaining increasing importance; although, in view of the reported long-term results of several contemporary trials, further improvements are certainly warranted. The aim of the present study was to evaluate the effectiveness and safety of a combined-treatment modality consisting of systemic chemotherapy with 24-h continuous infusional gemcitabine and mitomycin C, plus external beam radiotherapy in patients with localized unresectable adenocarcinoma of the pancreas. METHODS AND MATERIALS: Systemic chemotherapy consisted of mitomycin C 8 mg/m2 given as i.v. bolus injection on day 1 and gemcitabine administered as a 24-h continous infusion once weekly for 3 of 4 weeks. The starting dose of gemcitabine was 100 mg/m2 and dose levels were escalated in consecutive cohorts of 3-6 patients to 130 and 160 mg/m2, utilizing an escalating-dose Phase I trial design. Radiation therapy using megavolt irradiation (total dose, 45 Gy, 1.8 Gy/day) of 6 MV photons or greater with a 3- or 4-field technique was delivered concurrently for 5-6 weeks. RESULTS: Between January 1997 and August 1998, a total of 15 patients were enrolled in this trial, all of whom were assessable for toxicity, response, and survival. The dose-limiting toxicities at the 160 mg/m2 gemcitabine level were myelosuppression, specifically neutropenia +/- thrombocytopenia, and gastrointestinal symptoms, including stomatitis, vomiting, and diarrhea. Only 1 partial response was observed (7%), and disease was stabilized in 10 additional patients (67%). The median time to progression was 5.5 months (range, 2-12 months). Whereas all patients developed distant metastases, locoregional failure occurred in only 3. The median survival time was 8.3 months (range, 2.5 to 22.0+ months), and the 1-year survival rate was 13.3%. CONCLUSION: The MTD of gemcitabine when given as prolonged infusion in combination with mitomycin C and radiation therapy was 130 mg/m2/week. Therapeutic results suggest that combined chemoradiation with this regimen is feasible and effective for local control of pancreatic cancer, but essentially ineffective in counteracting metastatic tumor growth.

The metabolic effects of platelet-activating factor antagonism in endotoxemic man.

OBJECTIVE: To determine if the inflammatory phospholipid platelet-activating factor (PAF) participated in the symptomatologic, metabolic, and counterregulatory hormonal responses of human endotoxemia. DESIGN: In a double-blind, placebo-controlled study, five subjects received 10 mg of the PAF antagonist Ro 24-4736 orally, while five control subjects received a placebo. Eighteen hours later, all subjects were administered 4 ng/kg of endotoxin (lipopolysaccharide) intravenously. SETTING: The Clinical Research Center of The New York Hospital-Cornell Medical Center. PARTICIPANTS: Healthy male volunteers. MAIN OUTCOME MEASURES: Repeated measurements of vital signs, symptoms, cytokine and hormone levels, resting energy expenditure, platelet aggregation, and bleeding times were performed during a 24-hour period. RESULTS: Subjects who were pretreated with the PAF antagonist experienced fewer symptoms, including rigors at 1 hour (P < .05) and myalgias at 1 through 4 hours (P < .05) after administration of lipopolysaccharide. This was in concert with a diminished peak cortisol level (668 +/- 107 vs 959 +/- 159 nmol/L in controls; P < .05), epinephrine secretion (1057 +/- 165 vs 2029 +/- 431 nmol/L in controls; P < .05), and almost complete inhibition of PAF-induced platelet aggregation ex vivo. CONCLUSIONS: These findings in the face of unaltered circulating cytokines tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6, as well as the tumor necrosis factor receptor-I s, suggest that PAF may influence some endotoxin-induced, counterregulatory hormonal responses and symptoms through cytokine-independent mechanisms. This study further supports the role of PAF antagonists as an adjunct to cytokine blockade in the treatment of gram-negative sepsis.

Persistently elevated soluble tumor necrosis factor receptor and interleukin-1 receptor antagonist levels in critically ill patients.

The appearance of endogenously produced inhibitors against tumor necrosis factor (TNF) (soluble TNF-receptor type I, sTNFR-I) and interleukin-1 (IL-1 receptor antagonist, IL-1ra) was evaluated acutely in five normal patients after experimental endotoxemia lipopolysaccharide (LPS) and prospectively during a one to 11 week period in 12 septic, critically ill patients. Increased levels of both factors remained detectable in the circulation for up to 24 hours after LPS (2 nanograms per kilogram body weight) administration in normal patients. Despite free TNF-a activity being detected only sporadically (3 percent of the samples) and that IL-1 beta was never detectable in the patients in the intensive care unit, IL-6 bioactivity was present in 90 percent of initial samples. Circulating sTNFR-I levels up to 62,000 picograms per milliliter and IL-1ra levels of 14,800 picograms per milliliter were noted in the critically ill patients and remained consistently detectable throughout the extended period of evaluation. While there was no difference in IL-1ra levels between patients who survived or ultimately died, sTNFR-I levels were significantly (p < 0.001) lower in survivors compared with nonsurvivors. A correlation between circulating sTNFR-I and concurrent cortisol levels (r = 0.64; p < 0.002) was also noted. Furthermore, a correlation between sTNFR-I and the severity of initial insult, as assessed by APACHE II scores (r = 0.54; p < 0.01) was demonstrable. These naturally occurring cytokine antagonists likely represent additional indicators of the presence of an infectious or other inflammatory process and seem to persist in the circulation even during conditions in which their respective proinflammatory cytokines are not demonstrable.

The role of cytokines in cancer cachexia.

There is, at present, considerable interest in the possible role for the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1, interleukin-6, and interferon-gamma in the pathogenesis of cancer cachexia. Indirect evidence for such a role is based on the observation that chronic administration of many of these cytokines, either alone or in combination, can reproduce the myriad of host responses seen in experimental and human cancer cachexia. Elevated plasma levels of tumor necrosis factor-alpha, interleukin-2, and interferon-gamma have rarely been detected in patients or experimental animals with cancer, although interleukin-6 levels appear to correlate with tumor progression in animal models. The strongest evidence for a causal role for cytokines has come from rodent studies in which tumor-bearing animals have been passively immunized with antibodies directed against individual cytokines. Several groups have shown modest but significant improvements in food intake and lean tissue retention with antibodies directed against tumor necrosis factor-alpha, interleukin-1, interleukin-6, and interferon-gamma. However, there has been no consistent finding that one cytokine is universally involved in cancer cachexia in histologically distinct tumor models. One ominous finding in several tumor models has been that the endogenous production of cytokines appears to support tumor growth. Such findings raise the intriguing possibility that these cytokines, although contributors to tissue wasting and anorexia, may also serve the tumor as either direct or indirect cell growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Results of transverse tracheal resection in post-intubation tracheal stenoses]. / Ergebnisse mit der queren Trachealresektion bei Postintubations-Trachealstenosen.

Tracheal problems in form of stenosis and malacia are a calculated risk of long-term tracheal intubation. Results with conservative treatment of such problems by bougienage, laser therapy, biopsy, cryotherapy, local steroids, tracheal stenting, and tracheostomy are not satisfactory in a higher percentage of cases. Resectional therapy of benign tracheal lesions has become an established technique, which combines excellent functional results with a low complication incidence. We have treated 40 patients of 17 to 76 years of age with postintubation tracheal lesions by cross resection of the affected segment. Of these patients 40% had received conservative therapeutical steps preoperatively. The mean resection length was 3.0 cm (1.5 to 6.5 cm). The perioperative morbidity was 7.8%, mortality was 2.5%. 85% of the patients operated between 1970 and 1989 were reached for a follow-up examination with x-ray, pulmonary function test and endoscopy. The patients subjective satisfaction with the operative result was good in 85%, minor in 12% and less in 3%. The objective investigations proved very good results in 90%. Our experience confirm the good results of other authors and recommend the resection treatment for cases of postintubation tracheal lesions.

[The value of 2 distinct prognosis scores in patients with peritonitis. The Mannheim Peritonitis Index versus the Apache II score]. / Unterschiedliche Aussagekraft von zwei verschiedenen Prognose-Scores bei Patienten mit Peritonitis. Mannheimer Peritonitis-Index versus Apache II-Score.

Seventy patients suffering from purulent peritonitis entered this study, 31 of them were taken in prospectively, to contrast two different prognostic scores, the Mannheim Peritonitis Index (MPI) vs. the Apache II (APS II). The MPI is shown as a prognostic index for peritonitis with high accuracy in individual prognosis. The simultaneous use of both scores, the MPI as well as the APS II, leads to a negligible improvement of prognostic accuracy. Moreover, sensitivity and specificity with the MPI are of higher accuracy than calculated with the APS II.

[Validation study of the Mannheim Peritonitis Index]. / Validierungsstudie zum Mannheimer Peritonitis-Index.

113 patients suffering from purulent peritonitis entered this retrospective study for evaluation of the prognostic value of the Mannheim Peritonitis-Index. There was no lethality below an index x = 21, between x = 21 and x = 29, it was 29% and lethality increased to 100% in patients with an index x greater than or equal to 30. Statistical validation showed that prognosis was correct in 93% for the index x = 27, with a sensitivity and specificity of also 93%. Between x = 21 and x = 29 prognosis of the MPI was correct in at least 65%. The MPI is shown as a prognostic index for peritonitis with high accuracy in individual prognosis, that could be easy routinely documented.

[Etiology and consequences of postoperative wound infection]. / Atiologie und Konsequenz der postoperativen Wundinfektion.

The incidence of postoperative wound infection ranges between 4.6% and 36% after gastrointestinal operations respectively. To evaluate the factors which influence the postoperative wound infection we prospectively analyzed our patients between 1/1989 and 1/1990. 444 patients from three general surgical units of our clinic entered this study. The overall wound infection rate was 6.3%. We classified the patients into 3 operative groups: Group I: subcutaneous operations; Group II: intraabdominal operations without opening the GI-tract; Group III: gastrointestinal operations. Wound infection rate in group I was 1.8%, in group II 7.3% and in group III 13.7%. The differences were highly significant. Both univariate (chi 2-test) as well as a multivariate (Cox-Model) analysis were done. We figured out that classification of patients (p = 0.000), operation time (p = 0.009), operating room (p = 0.000), intensive care unit (p = 0.026), long-term antibiotic prophylaxis (p = 0.001), subcutaneous haematoma (p = 0.000) and length of closed drainage time (p = 0.001) are of significant value. In the Cox model the classification of patients into 3 groups surpassed all the other factors. Postoperative hospital stay was lengthened in patients with wound infection significantly (p = 0.0025).

Reoperation after cholecystectomy. The role of the cystic duct stump.

The so-called "Postcholecystectomy Syndrome" may be due to various pathological biliary causes. The aim of this study was to evaluate the significance of the cystic duct stump syndrome and if so, how often a long (greater than 1.5 cm) cystic duct stump was an indication for reoperation on the bile ducts after cholecystectomy in our patients. Three hundred and twenty two patients underwent a second operation on the bile ducts after cholecystectomy in the last ten years. In 35 patients (10.8%) a striking findings was a long cystic duct stump (greater than 1.5 cm). In 24 of these patients, a pathological finding, in addition to the long cystic duct stump, was found on exploration. Out of these 24 patients there were 14 with common bile duct stones; 6 with stenosis of the sphincter of Oddi; 3 with chronic pancreatitis and in one patient hepatitis was the cause of the symptoms. From the remaining 11 patients 8 had a stone in a partial gall bladder or cystic duct stump. One patient had a fistula between the cystic duct stump and duodenum and one a suture granuloma. There was only one patient where a 1.5 cm long cystic duct stump remnant was the only pathological finding. Four years after reoperation this patient is still suffering from the same intermittent gastrointestinal symptoms. We conclude that the cystic duct stump is hardly ever a cause for recurrent symptoms in itself. Total excision of the cystic duct does not eliminate the existence of postcholecystectomy symptoms.

[Liposome mediated gene transfer - the future therapy for sepsis and intraabdominal infection?] / Liposomen-mediierter Gentransfer ­ die zukünftige Therapieform bei Sepsis und intraabdomineller Infektion?

BACKGROUND: It is now generally accepted that over-production of pro-inflammatory cytokines (TNF-α, IL-1ß) produced by inflammatory cells contributes to the pathological consequences of septic shock. Neutralizing this exaggerated immune response by monoclonal antibodies (anti-TNF-α), receptor antagonists (IL-1rα) and anti-inflammatory cytokines (IL-10) did not result in a better outcome in septic patients. Firstly, this is due to the short biological half-lives of these natural antagonists or inhibitors of pro-inflammatory cytokines. Secondly, exaggerated pro-inflammatory cytokine production may contribute to pathology in one body compartment while, simultaneously, the same mediators may have beneficial effects in another compartment. Thus, systemic administration of cytokine inhibitors at levels sufficient to neutralize exaggerated cytokine production in one organ may also block the presumably beneficial aspects of cytokine production in another. METHODS: Our own results of animal experiments of the liposome mediated gene transfer are presented. RESULTS: Liposome mediated gene transfer seems to be a promising low-risk alternative to systemic anti-inflammatory therapies as it ensures the local delivery of high doses of cytokine inhibitors and antagonists over a prolonged period of time. CONCLUSIONS: The pathophysiological mechanism of sepsis and septic shock are well established. The concept of local intervention or compartimental blockade of overwhelming mediator production by gene transfer will be a new challenge in the future.

Combined sclerotherapy and operation for the treatment of bleeding oesophageal varices.

OBJECTIVE: To identify prognostic factors in a consecutive series of patients with bleeding oesophageal varices and develop an optimum regimen of treatment. DESIGN: Retrospective review. SETTING: I Department of Surgery, University Hospital, Vienna, Austria. PATIENTS: 301 consecutive patients with bleeding oesophageal varices. OUTCOME MEASURES: Median survival and survival at one year after sclerotherapy alone (n = 213), or sclerotherapy with portosystemic shunt (n = 54), Hassab's devascularisation (n = 29), or liver transplantation (n = 5). RESULTS: Prognosis was dependent on the severity of liver damage at the start of treatment. Median survival for Child's class A was 47 months, for Child's class B 54 months, and for Child's class C 2 months. The overall one year survival for patients in Child's class C was 33%, for sclerotherapy alone 28%, and for sclerotherapy and portosystemic shunt 42%, Hassab's devascularisation 50%, and liver transplantation 80%. CONCLUSION: Despite the small number of patients who underwent liver transplantation and their poor initial prognosis (Child's class C, n = 4; class B, n = 1) our results suggest that liver transplantation should be considered for the treatment of patients with end stage cirrhosis and bleeding varices.

Submucous large-bowel lipomas--presentation and management. An 18-year study.

Gastrointestinal lipomas are rare, but commonest in the colon and rectum, characteristically submucosal and seldom subserosal. An 18-year analysis revealed 17 cases of large-bowel lipoma, 13 presenting with colicky pain, abdominal discomfort, blood-stained feces or rectal bleeding and altered bowel habits and four asymptomatic. The 17 patients had totally 21 lipomas, all submucosal. No patients with multiple lipoma had evidence of lipoma at other sites. The ileocecal valve and cecum were most commonly affected, followed by the rectum, sigmoid colon and descending colon. Tumor size (largest diameter) was 0.5-10 cm, averaging 3.1 cm (3.5 cm in symptomatic, and 1.8 cm in asymptomatic patients). The primary diagnosis (with barium enema, colonoscopy and CT) was lipoma in only five cases, but CT gave the correct diagnosis in all three cases in which it was used. Two lipomas were found in surgical specimens from colorectal malignancy, while nine were misinterpreted as polyps and one as angiodysplasia. In symptomatic patients unnecessary colotomy or colonic resection may be avoidable by colonscopic removal of lipoma.

Open approach in pancreatic and infected pancreatic necrosis: laparostomies and preplanned revisions.

One hundred and two patients with acute necrotizing pancreatitis were treated in accordance with a combined regimen of necrosectomy, open drainage by laparostomies, and repeated re-explorations. The severity of pancreatitis was assessed by the APACHE II score (median 15 on admission). Eighty-seven (85%) patients were classified as having infected pancreatic necrosis and only 15 (15%) as having pancreatic necrosis. Overall, 36 (35%) patients died, most of multiple organ failure. Survival was significantly impaired by bacterial contamination of pancreatic necrosis (p = 0.008), bacteremia (p = 0.0001) and infected bronchial secretions (p = 0.05). The mortality rate was reduced from 53% to 28% by changing the regimen of re-explorations from on demand to regular 48 hour intervals. Despite the fact that open packing was associated with a high frequency of gastrointestinal fistulas (30%), this concept seems to be a successful and recommendable approach in the therapy of pancreatic and infected pancreatic necrosis.