Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity.

Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross-linking by the Abs; (c) occurred in HeLa cells, but not in A9 cells transfected with human p75-R or in HeLa cells expressing cytoplasmically truncated p75-R mutants, indicating that it involves cell-specific activities of the intracellular domain of the receptor; (d) was synergistic with the cytocidal effect of Abs against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling activity of p75-R. These effects: (a) could be observed also in cells in which only p55-R signaled for the cytocidal effect; (b) were not dependent on receptor cross-linking by the Abs; (c) varied according to the site at which the Abs bound to the receptor; and (d) were correlated inversely with the effects of the Abs on TNF binding to p75-R. That is, Abs binding to the membrane-distal part of the receptor's extracellular domain displaced TNF from the p75 receptor and enhanced cytocidal effect, whereas Abs that bind to the membrane-proximal part of the extracellular domain--a region at which a conformational change seems to take place upon TNF binding--decreased the dissociation of TNF from p75-R and inhibited its cytocidal effect. The above findings suggest that p75-R contributes to the cytocidal effect of TNF both by its own signaling and by regulating the access of TNF to p55-R.

Assessment of usefulness exhibited by different tacks in laparoscopic ventral hernia repair.

BACKGROUND: Laparoscopic ventral hernia repair is becoming a popular technique with good results and fast postoperative recovery. The mesh is placed directly under the peritoneum and anchored with transabdominal sutures and tacks. However, the ideal size of the mesh covering the hernia orifice is know, nor the ideal type or amount of tacks has to be described. METHODS: To assess the forces acting on a single tack, a mathematical model of the ventral hernia was created. The force was described in reference to the surface of the hernia orifice and the pressure in the abdominal cavity. The following different types of mesh were examined in vitro: Proceed (knitted mesh), Dual Mesh (expanded polytetrafluoroethylene [ePTFE] flat mesh), and Shelhigh (biologic flat mesh). The following different tacks also were examined: Protac, Anchor, and EMS. A pig model was used to measure the forces needed to destroy the connection between mesh and tissue and to describe the place of destruction (mesh, tissue, or tack) and the force needed. RESULTS: The force acting on a single tack proportionally depends on the surface of the hernia orifice and the pressure in the abdominal cavity. The force needed to disconnect the tissue and mesh reached 8.97 +/- 0.11 N for ProTac, 2.67 +/- 0.22 N for Anchor, and 6.67 +/- 1.32 N for EMS. These values do not allow the mesh to be held in the right position when the orifice exceeds 10 cm for Protac and EMS. The disconnection of the EMS and Protac junction damages the tissue. Anchor tacks are insufficient to hold the mesh and stay in the tissue CONCLUSIONS: In the case of small hernias (diameter<10 cm) EMS or ProTac used alone are not enough to hold the mesh. Anchor is not recommended alone in any hernia.

Is there a need for a mesh plug in inguinal hernia repair? Randomized, prospective study of the use of Hertra 1 mesh compared to PerFix Plug.

Although the mesh plug procedure is an effective operation, sutureless implantation of a single onlay mesh, if successful, would avoid the risks of plug-related complications. One hundred patients with primary inguinal hernias were randomized to undergo PerFix Plug or Hertra 1 implantation. Mean duration of the operation was similar, 38 versus 35 min for plug and onlay mesh implantation, respectively (nonsignificant, NS). The level of postoperative pain and early complication rate was similarly low in both groups (NS). Recurrence rate was the same-2 patients in each group (4%)-all following large hernia repair. The level of long-term discomfort was low after each type of operation (NS). Implantation of the onlay mesh-Hertra 1 as compared to the use of PerFix Plug is simple, safe, and equally effective in small and medium inguinal hernia repair, suggesting that a plug device is not necessary for successful hernia surgery.

Management of inguinal hernia on peritoneal dialysis: an audit of current Polish practice and call for a standard.

Peritoneal dialysis is becoming more and more common as a method of treating patients at the final stage of renal failure. In the year 2002 the number of patients treated with this method in Poland amounted to 1324. Studies have demonstrated that inguinal and abdominal hernias develop more frequently in chronic renal failure patients. The percentage of patients with hernia diagnosed within the first five years of dialysis is about 10%. Continuation of dialysis with the hernia condition left untreated may result in severe complications which are the third most frequent cause of converting treatment method into hemodialysis in PD patients. Currently in Poland there is no national standard in existence as to the management of hernias, and the only British standard from 1998 does not reflect the present expertise of either surgical treatment or dialysis methods. The aim of the current questionnaire based study investigating Polish peritoneal dialysis centers was to assess the treatment when hernia had been diagnosed in the PD patient. Of 49 dialysis centers in Poland, 39 do have protocols on managing the patient before and after the operation. A considerable diversity has been found as to surgical techniques used and the ways the patients are managed in hospital. Following the need expressed by 33 dialysis centers in Poland for some standard for relevant procedures, the authors formulated principles of modern hernia treatment in PD patients. Accordingly, the main principles include: 1) consulting a surgeon collaborating with the center before qualifying for peritoneal dialysis and when hernia symptoms have manifested; 2) Tension-free methods used in a treatment of choice (recommended by PHS); 3) Application of antibiotic prophylaxis (preferably first generation cephalosporin); 4) Induction of local or epidural anesthesia; 5) Peritoneal dialysis programme does not need to be discontinued but low volume dwells or preferably intermittent APD is recommended immediately after surgery.

Factors influencing surgeons' choice of method for hernia repair technique.

Tension-free hernioplasty is performed using prosthetic material in one-half of hernia repair procedures in Poland but in 85% of those in the region of Pomerania. This questionnaire study of surgeons in Pomerania examined their sources of knowledge about and the factors influencing their choice of groin hernia surgery. The questionnaire was sent to surgeons from 19 hospitals and was answered by 109 (83% of hernia surgeons in the region). We analyzed their reported knowledge of particular operative techniques, factors important in selecting the technique (personal experience, trends in surgical center), and the available sources of information (e.g., medical literature, internet, information from teachers, sales representatives). All respondents reported being familiar with and able to perform tension-free techniques, but only 44% are influenced by their individual professional skills in selecting the technique. Another 44% base their decision on trends in their hospital, and only 22% consider the patient's preferences. The most frequently quoted sources of scientific information are articles in the medical literature and conference reports (90%). Only 8% of the respondents are governed in their professional work by information from pharmaceutical company representatives. Most surgeons (70%) would prefer to make a decision about using a new surgical technique after practical training sessions or workshops led by experienced colleagues. In contrast to common opinion, the information from sales representatives are of only minor importance compared to that of evidence-based data and attendance at workshops and courses.

Natural killer sensitivity of tumor cells isolated from primary and metastatic lesions of four Bomirski melanoma variants.

Natural killer (NK) sensitivity of melanoma cells isolated from primary and metastatic lesions of four Bomirski melanoma variants was compared. The hamster melanomas differed in their growth rate and metastatic pattern. We found that during tumor growth of all the variants tested, NK sensitivity of melanoma cells at the metastasis formation stage was significantly lower in both primary and metastatic tumors than in cells isolated from primary tumors at transplantation. In the case of Ma, Ab and Ab-455, NK sensitivity of primary tumor cells was higher than that of the cells isolated from metastatic deposits. These data obtained from a spontaneous metastasis tumor model argue for the role of NK cells in preventing metastatic spread of Bomirski melanomas studied.

Genetic determinants of plasma ACE and renin activity in young normotensive twins.

OBJECTIVE: We investigated the determinants of plasma renin activity (PRA) and plasma levels of angiotensin-converting enzyme (pACE), including the effect of the D/I polymorphism of the angiotensin-converting enzyme (ACE) gene, in monozygotic (MZ) and dizygotic (DZ) twins. METHODS: Sixty-nine pairs of twins underwent measurements of blood pressure, pACE and ACE D/I genotyping. In addition, in 30 pairs ambulatory blood pressure (ABP) monitoring was carried out. To ascertain twin's zygosity, some highly discriminating variable number of tandem repeats micro- and mini-satellite systems were analysed by polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis and silver staining. The D/I polymorphism was assessed by PCR; pACE was measured in triplicate with a colorimetric assay, and PRA by a commercial kit. In DZ twins, identity by descent of the D/I alleles was examined by PCR amplification of a highly polymorphic simple sequence repeat at the human growth hormone gene. RESULTS: pACE levels were significantly (P < 0.01) higher in DD (9.27 +/- 2.60 IU/l, mean +/-SD) than in II (6.68 +/- 3.0), with DI having intermediate levels (7.93 +/- 2.7). No difference of PRA between different D/I genotypes was found. Twin data analysis showed a statistically significant heritability of pACE, but not of PRA. No differences between MZ and DZ twins in PRA, pACE and the relationship of the D/I genotype with pACE was found. Besides showing that the D/I genotype was the most important predictor of pACE, a multivariate analysis demonstrated that identity by descent of the D/I allele, as assessed by growth hormone (GH) genotyping, also significantly affected pACE. CONCLUSIONS: In this study of normotensive twins, pACE and not PRA showed significant heritability, the former being tightly associated with the D/I ACE gene polymorphism, and/or with a quantitative trait locus in linkage disequilibrium with it.

Natural killer sensitivity of four Bomirski melanoma variants.

The sensitivity of four hamster melanoma variant cells to natural killer (NK) cell lysis mediated by hamster blood and spleen mononuclear cells is examined. The melanoma variants differed in their differentiation level, growth rate, and frequency and localization of metastases. The cells of the melanoma variants were found to differ in their sensitivity to NK cell mediated lysis. A consistent relationship between high sensitivity to lysis and such tumor characteristics as low differentiation level, slow growth rate, low metastatic potential was not observed.

Serum p53 antibodies in small cell lung cancer: the lack of prognostic relevance.

Prognostic relevance of serum p53 antibodies was assessed in 96 patients with microscopically proven small cell lung cancer (SCLC). The study group included 67 males and 29 females; mean age 58 years; range 35--86 years; 60 with limited disease (LD), and 36 with extensive disease (ED). The control group consisted of 41 patients with non-malignant diseases. The presence of p53 antibodies was assayed by the immunoenzymatic method (P53 ELISA kit, PharmaCell, France). Antibodies were present in 26 SCLC cases (27%); 15 (25%) in LD and 11 (31%) in ED. Antibodies were also found in one out of 41 control subjects (2%). There was no correlation between the level of antibodies and clinical characteristics of SCLC patients including age, gender and extent of disease. The median follow-up for the entire group was 30 months (range: 11--39 months). By the time of analysis, 78 patients (82%) had deceased. Median survival in SCLC patients with and without antibodies was 42 and 39 weeks, respectively (log rank, P=0.81). These results indicate the lack of clinical relevance of serum p53 antibodies in SCLC.

No effect of genetic variation at the angiotensinogen locus on ambulatory blood pressure level in normotensive subjects.

The aim of the study was to evaluate the potential association between ambulatory blood pressure and the molecular variants T174M and M235T of the angiotensinogen gene in a random sample of young normotensive men (n = 145). The two point mutations were detected using restriction digests of a mispairing polymerase chain reaction product. Twenty-four-hour ambulatory blood pressure monitoring was performed with a SpaceLabs 90207 device. Ambulatory blood pressure levels did not vary according to T174M and M235T genotypes. When the subjects were grouped according to their blood pressure level (as indicated by tertiles of their 24-h ambulatory blood pressure), no significant differences in allele frequencies between the three groups were found. Our results indicate that the T174M and M235T molecular variants of the angiotensinogen gene have no major influence on ambulatory blood pressure in young normotensive subjects.

Interleukin 12 augments natural killer-cell mediated cytotoxicity in hairy cell leukemia.

Interleukin 12 (IL12, NKSF-natural killer stimulatory factor) was found to stimulate natural killer (NK) cell activity of hairy cell leukemia (HCL) patients. Two patients not responding to IL12 stimulation were also resistant to interferon alpha (IFN alpha)-mediated augmentation of NK activity. IL12 also enhanced slightly the interleukin 2 (IL2)-induced cytotoxicity of HCL patients, while IFN alpha reduced the stimulatory effect of IL2. These data suggest that interleukin 12 has NK modulatory properties in HCL leukemia patients, which may be different from those of IFN alpha.

Interleukin 2- and interferon alpha induced natural killer cell activity as a marker of progression in hairy cell leukemia.

Hairy cell leukemia (HCL), a rare B-cell chronic lymphoproliferative disorder, is often accompanied by immune abnormalities. A marked impairment of the natural killer cell-mediated cytotoxicity (NK activity) has been reported in most patients at diagnosis. In the present report a long-term follow-up study of NK activity of splenectomized HCL patients is recorded. Among patients who persisted with stable disease two groups, one with normal NK activity, and another with low NK activity, could be recognized. Patients with progressive stage were characterized by a low NK cytotoxic activity. In vitro tests showed that interferon alpha (IFN-alpha) and interleukin 2 (IL2) could increase the NK activity to normal levels only in HCL patients with stable disease, while in progressive HCL these cytokines showed a significantly decreased effect. These results indicate that cytokine-induced NK cytotoxicity appears to be a valuable parameter in assessing the stage of HCL.

Indomethacin inhibits kidney metastasis in bomirski melanoma-bearing hamsters, and modulates natural killer cytotoxic activity of tumor hosts in vivo and in vitro.

Natural killer (NK) cells are thought to play an important role in the control of metastatic dissemination. Therefore, stimulation of cytotoxic activity of NK cells against neoplastic cells could be preventive for metastatic spread. Bomirski amelanotic (Ab) melanoma of Syrian hamster is a transplantable tumor metastasizing preferably to the kidneys. During growth of the melanoma a significant depression of cytotoxic activity of NK cells of tumor hosts is observed. Treatment of melanoma-bearing hamsters with indomethacin provided in drinking water resulted in the increase of NK cytotoxic activity of blood cells and in the lower occurrence of kidney metastasis. Spleen cells obtained from healthy and melanoma-bearing hamsters were cultured in vitro with agents influencing NK activity. We found an augmentative effect of human interleukin 2 (IL2) and human tumor necrosis factor (TNF). We also observed the synergistic effect of IL2/TNF combination, which was present in both groups of animals. The stimulatory effects of cytokines could be potentiated by the additional supplementation of cultures with indomethacin. Similar experiments were performed on spleen cells isolated from the healthy and tumor-bearing animals treated in vivo with indomethacin. Also, in this group of hamsters in vitro stimulation of NK cell activity by the cytokines was effective. The studies presented may give insight into the pathogenesis of immune abnormalities seen in advanced stages of progression of Ab melanoma, and can provide an experimental basis for immunomodulation in this tumor model of spontaneous metastasis.

TNF receptors--how they function and interact.

Tumor necrosis factor (TNF) is a pleiotropic cytokine regulating various immune and inflammatory reactions. It induces cellular responses upon binding to specific cell surface receptors of two distinct molecular species--p55 and p75 TNF receptors (TNF-Rs). Cloning of the respective cDNAs and obtaining the receptor-specific molecular tools such a agonistic and blocking monoclonal antibodies, TNF muteins with exclusive ability to bind to only one receptor type, as well as generation of TNF-R mutants by site-directed mutagenesis enabled researchers to answer key questions of the biology of initial steps in the cascade of TNF signal transduction. The presented mini-review describes the mode of function of the two receptors and discusses the possible ways of interaction between them.

Synergistic effect of the angiogenesis inhibitor TNP-470 and tumor necrosis factor (TNF) on Bomirski Ab melanoma in hamsters.

Bomirski Ab transplantable melanoma bearing hamsters were treated with angiogenesis inhibitor TNP-470 or with rat tumor necrosis factor (TNF) at doses of 20 micrograms and 40 micrograms or with both TNP-470 and TNF at a dose of 20 micrograms each. The size of the growing tumor was measured everyday from the day when it was palpable. After the death of the animals the final size of their tumors was measured and the number and localisation of metastasis were determined. It was found that the rate of tumor growth was lowest in the group of animals treated with TNF at a dose of 40 micrograms and in the group of animals treated with TNP-470 and TNF at a dose of 20 micrograms. The frequency of metastasis was different in the experimental groups although their location was similar. The longest living animals belonged to the group treated with both TNP-470 and TNF. The results of the investigation indicate that TNP-470 and TNF act in a synergistic way that allows a decrease in the effective dose of TNF thus diminishing its noxious side effects.

Effect of tumor excision on NK cytotoxic activity and formation of metastases of Bomirski melanoma in hamsters.

The growth of transplanted Bomirski melanoma in hamsters is accompanied by the decrease of natural killer cytotoxic activity and the formation of metastases. The excision of primary tumors was carried out to examine what was the effect of the growing tumor and its metastases on the host's NK activity. It was found that the excision of primary tumor caused increased NK cytotoxic activity in comparison to that of nonoperated animals although it was still lower than that of healthy hamsters. It is concluded that both, a growing tumor and metastases, exert suppressive effects on NK activity and those effects add up. The pattern of metastases in operated animals was different to that observed in nontreated hamsters.

Cloning, expression, purification of rat tumor necrosis factor and demonstration of its antitumor activity in Bomirski amelanotic melanoma.

In order to analyze the effect of tumor necrosis factor (TNF) on the growth of Bomirski melanomas of hamster, we cloned, expressed and purified to homogeneity the rat tumor necrosis factor. The detailed procedure of chromatographic purification of the cytokine is reported. Polyclonal antibodies generated against the recombinant rat preparation were able to neutralize the rat and hamster TNF. We have found that the preparation of rat TNF is active in vivo and inhibits the growth of a Bomirski (Ab) amelanotic melanoma. Thus, a novel model system has been developed, which should facilitate analysis of antitumor and immunomodulatory function of TNF in cancer hosts.

Complete remission of Bomirski Ab amelanotic melanoma in hamsters treated with the angiogenesis inhibitor TNP-470.

The growth of solid tumors and their metastasis is dependent on the development of new blood vessels (angiogenesis). In our previous studies it was found that angiogenesis inhibitor TNP-470 acting systematically can decrease the rate of growth of transplantable Bomirski Abmelanoma in hamsters. In this study we applied TNP-470 (30 mg/kg) peritumorally from the day when tumor was palpable over 10 days, once daily. Animals were killed 6 months later and examination by autopsy and histological preparations showed the complete remission of transplanted tumor and the lack of metastasis. Thus, Ab melanoma can be effectively cured with TNP-470 angiogenesis inhibitor when the substance is applied locally.

Changes of natural killer cytotoxic activity and natural killer sensitivity during growth of Bomirski melanotic (Ma) and amelanotic (Ab) melanomas.

Natural killer (NK) cytotoxic activity during the growth of melanotic (Ma) and amelanotic (Ab) variants of Bomirski hamster melanoma, in the blood and the spleen, was examined. The melanoma variants differed in their growth rate and metastatic pattern. Ability to form conjugates by effectors with targets and cytotoxic effects were compared. As targets K562 cells and tumor cells were used. It was found that during the growth of Ma melanoma activity of NK cells was not changing but the sensitivity of tumor cells was decreasing. However, during growth of Ab melanoma both NK activity and NK sensitivity were reduced.

Activity of acid phosphatase in target binding cells expressing NK cytotoxic potential.

Activity of acid phosphatase was analysed in the NK-effectors from human blood forming conjugates with K 562 cells. Presence of activity of acid phosphatase was associated with the cytolytic potential of the NK-effectors. The non-lytic target-binders did not express activity of this enzyme. The results suggest that activity of acid phosphatase in the effector cells of NK reaction is important in the lethal hit stage.